Neoformans Infection Research Articles

Immune stimulating and therapeutic potential of tuftsin-incorporated nystatin liposomes against Cryptococcus neoformans in leukopenic BALB/C mice

Cryptococcus neoformans infection is a common fungal infection in persons infected with human immune deficiency virus (HIV) or those with defective cell-mediated immunity. Since treatment of cryptococcal meningitis poses a big challenge, the present study aimed to develop a novel liposomal therapeutic formulation against cryptococcosis. Treatment with tuftsin-incorporated liposomes increased the anti-cryptococcal activity of murine peritoneal macrophages. Prophylactic treatment of mice with tuftsin-incorporated liposomes reduced the dissemination of C. neoformans to brain tissues. Moreover, the co-administration of tuftsin with nystatin liposomes augmented the anti-cryptococcal activity of nystatin, as mice treated with tuftsin-incorporated nystatin liposomes showed the highest survival and least fungal burden in their brain tissues. The results of the present study favour the use of immune-stimulating molecules along with antifungal agents in the treatment of opportunistic fungal infections.

Molecular characterization of antigen-specific B1 and B2 B-cells populations in Cryptococcus neoformans infection in mice (43.1)

Abstract We examined B1 and B2 B-cell repertoires in Cryptococcus neoformans infection in mice. Flow-cytometry based antigen-specific staining for B-cell populations generated in response to pulmonary infection was performed using labeled un-encapsulated and encapsulated C. neoformans strains. FACS sorting and single-cell nested PCR was employed to characterize the IgH repertoire. An increase in total peritoneal B1a, B1b and B2 cell numbers was observed as early as day 3 post-infection. Frequencies of both acapsular and capsular antigen-specific cells were increased in the B1a subpopulation (18%, 5%) in infected compared to naïve mice (11%, 2%). The heavy chains of antigen-specific B2 and B1b cells recognizing acapsular and capsular cryptococcus belonged predominantly to the VH1 family. Interestingly, increased VH11 (22%) and VH12 (33%) usage was observed in antigen-specific B1a cells recognizing acapsular and encapsulated cryptococcus, respectively, compared to the conventional B1a population. Although all acapsular (VH11+) and capsular (VH12+) B1a cells used the same VH11.2.53 and VH12.1.78 genes, 60% and 80% rearrangements were unique, respectively. VH genes in both cases were essentially germline, however 40% of acapsular specific B1a cells had 4 or more N-nucleotide additions compared to capsular specific B1a cells (20%). These results indicate that naturally occurring phosphatidyl choline reactive B1a cells have broad specificities and can bind to encapsulated cryptococcus.

Atypical sphenoid bone osteomyelitis in a maltanese dog caused by cryptococcosis: a case report

This article describes osteomyelitis of the sphenoid skull bone in a maltanese dog due to Cryptococcus neoformans var. neoformans infection. The affected dog was subjected to physical and neurological examinations. Complete blood count (CBC), biochemistry profile, lymph node biopsy, cerebrospinal fluid (CSF) examination were also performed. This case report describes abnormalities in magnetic resonance imaging (MRI) examination as well as the histhopathologic lesions of the skull bones and neurological symptoms of the dog.  

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

To develop an in vivo system that could quantitatively evaluate the therapeutic effects of antifungal drugs using a silkworm infection model with Cryptococcus neoformans. 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 (LD(50) 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. The silkworm-C. neoformans infection model is useful for evaluating the therapeutic effects of antifungal drugs. 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.

Contributions of the MyD88-Dependent Receptors IL-18R, IL-1R, and TLR9 to Host Defenses following Pulmonary Challenge with Cryptococcus neoformans

Signaling via the adapter protein, MyD88, is important in the host defense against Cryptococcus neoformans infection. While certain Toll-like receptors (TLRs) can enhance the clearance of Cryptococcus, the contributions of MyD88-dependent, TLR-independent pathways have not been fully investigated. We examined the roles of IL-1R and IL-18R in vivo by challenging C57BL/6 mice with a lethal strain of Cryptococcus. We found that the absence of IL-18R, but not IL-1R, causes a shift in the survival curve following pulmonary delivery of a virulent strain of C. neoformans (H99). Specifically, IL-18R-deficient mice have significantly shorter median survival times compared to wild-type mice following infection. Cytokine analysis of lung homogenates revealed that deficiency of IL-IR, IL-18R, or MyD88 is associated with diminished lung levels of IL-1β. In order to compare these findings with those related to TLR-deficiency, we studied the effects of TLR9-deficiency and found that deficiency of TLR9 also affects the survival curve of mice following challenge with C. neoformans. Yet the lungs from infected TLR9-deficient mice have robust levels of IL-1β. In summary, we found that multiple signaling components can contribute the MyD88-dependent host responses to cryptococcal infection in vivo and each drives distinct pulmonary responses.

Treatment of Early and Established Cryptococcus neoformans Infection with Radiolabeled Antibodies in Immunocompetent Mice

We investigated the utility of radioimmunotherapy (RIT) in early and established cryptococcal infection in immunocompetent mice. RIT with (213)Bi-18B7 antibody completely eliminated fungus from mouse lungs and brains for early infection, while (188)Re-18B7 significantly reduced CFU in the lungs or both lungs and brains during early and established infection, respectively. The results point to the independence of RIT of the immune status of the host, which is encouraging for translation of this strategy into the clinic.

Eosinophils elicit proliferation of naive and fungal-specific cells in vivo so enhancing a T helper type 1 cytokine profile in favour of a protective immune response against Cryptococcus neoformans infection.

Experimental Cryptococcus neoformans infection in rats has been shown to have similarities with human cryptococcosis, because as in healthy humans, rats can effectively contain cryptococcal infection. Moreover, it has been shown that eosinophils are components of the immune response to C.neoformans infections. In a previous in vitro study, we demonstrated that rat peritoneal eosinophils phagocytose opsonized live yeasts of C.neoformans, thereby triggering their activation, as indicated by the up-regulation of MHC and co-stimulatory molecules and the increase in interleukin-12, tumour necrosis factor-α and interferon-γ production. Furthermore, this work demonstrated that C.neoformans-specific CD4(+) and CD8(+) T lymphocytes cultured with these activated C.neoformans-pulsed eosinophils proliferated, and produced important amounts of T helper type 1 (Th1) cytokines in the absence of Th2 cytokine synthesis. In the present in vivo study, we have shown that C.neoformans-pulsed eosinophils are also able to migrate into lymphoid organs to present C.neoformans antigens, thereby priming naive and re-stimulating infected rats to induce T-cell and B-cell responses against infection with the fungus. Furthermore, the antigen-specific immune response induced by C.neoformans-pulsed eosinophils, which is characterized by the development of a Th1 microenvironment with increased levels of NO synthesis and C.neoformans-specific immunoglobulin production, was demonstrated to be able to protect rats against subsequent infection with fungus. In summary, the present work demonstrates that eosinophils act as antigen-presenting cells for the fungal antigen, hence initiating and modulating a C.neoformans-specific immune response. Finally, we suggest that C.neoformans-loaded eosinophils might participate in the protective immune response against these fungi.

A sensitive high-throughput assay for evaluating host-pathogen interactions in Cryptococcus neoformans infection.

Background Cryptococcus neoformans causes serious disease in immunocompromised individuals, leading to over 600,000 deaths per year worldwide. Part of this impact is due to the organism's ability to thwart what should be the mammalian hosts' first line of defense against cryptococcal infection: internalization by macrophages. Even when C. neoformans is engulfed by host phagocytes, it can survive and replicate within them rather than being destroyed; this ability is central in cryptococcal virulence. It is therefore critical to elucidate the interactions of this facultative intracellular pathogen with phagocytic cells of its mammalian host.Methodology/Principal FindingsTo accurately assess initial interactions between human phagocytic cells and fungi, we have developed a method using high-throughput microscopy to efficiently distinguish adherent and engulfed cryptococci and quantitate each population. This method offers significant advantages over currently available means of assaying host-fungal cell interactions, and remains statistically robust when implemented in an automated fashion appropriate for screening. It was used to demonstrate the sensitivity of human phagocytes to subtle changes in the cryptococcal capsule, a major virulence factor of this pathogen.Conclusions/SignificanceOur high-throughput method for characterizing interactions between C. neoformans and mammalian phagocytic cells offers a powerful tool for elucidating the relationship between these cell types during pathogenesis. This approach will be useful for screens of this organism and has potentially broad applications for investigating host-pathogen interactions.

Cryptococcus neoformans infection in ulcerative colitis with immunosuppressants

Cryptococcus neoformans infection in ulcerative colitis with immunosuppressants

Surface localization of glucosylceramide during Cryptococcus neoformans infection allows targeting as a potential antifungal.

Cryptococcus neoformans (Cn) is a significant human pathogen that, despite current treatments, continues to have a high morbidity rate especially in sub-Saharan Africa. The need for more tolerable and specific therapies has been clearly shown. In the search for novel drug targets, the gene for glucosylceramide synthase (GCS1) was deleted in Cn, resulting in a strain (Δgcs1) that does not produce glucosylceramide (GlcCer) and is avirulent in mouse models of infection. To understand the biology behind the connection between virulence and GlcCer, the production and localization of GlcCer must be characterized in conditions that are prohibitive to the growth of Δgcs1 (neutral pH and high CO2). These prohibitive conditions are physiologically similar to those found in the extracellular spaces of the lung during infection. Here, using immunofluorescence, we have shown that GlcCer localization to the cell surface is significantly increased during growth in these conditions and during infection. We further seek to exploit this localization by treatment with Cerezyme (Cz), a recombinant enzyme that metabolizes GlcCer, as a potential treatment for Cn. Cz treatment was found to reduce the amount of GlcCer in vitro, in cultures, and in Cn cells inhabiting the mouse lung. Treatment with Cz induced a membrane integrity defect in wild type Cn cells similar to Δgcs1. Cz treatment also reduced the in vitro growth of Cn in a dose and condition dependent manner. Finally, Cz treatment was shown to have a protective effect on survival in mice infected with Cn. Taken together, these studies have established the legitimacy of targeting the GlcCer and other related sphingolipid systems in the development of novel therapeutics.

Rat eosinophils stimulate the expansion of Cryptococcus neoformans-specific CD4+ and CD8+ T cells with a T-helper 1 profile

Experimental Cryptococcus neoformans infection in rats has been shown to have similarities with human cryptococcosis, revealing a strong granulomatous response and a low susceptibility to dissemination. Moreover, it has been shown that eosinophils are components of the inflammatory response to C.neoformans infections. In this in vitro study, we demonstrated that rat peritoneal eosinophils phagocytose opsonized live yeasts of C.neoformans, and that the phenomenon involves the engagement of FcγRII and CD18. Moreover, our results showed that the phagocytosis of opsonized C.neoformans triggers eosinophil activation, as indicated by (i) the up-regulation of major histocompatibility complex (MHC) class I, MHC class II and costimulatory molecules, and (ii) an increase in interleukin (IL)-12, tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) production. However, nitric oxide (NO) and hydrogen peroxide (H(2) O(2) ) synthesis by eosinophils was down-regulated after interaction with C.neoformans. Furthermore, this work demonstrated that CD4(+) and CD8(+) T lymphocytes isolated from spleens of infected rats and cultured with C.neoformans-pulsed eosinophils proliferate in an MHC class II- and class I-dependent manner, respectively, and produce important amounts of T-helper 1 (Th1) type cytokines, such as TNF-α and IFN-γ, in the absence of T-helper 2 (Th2) cytokine synthesis. In summary, the present study demonstrates that eosinophils act as fungal antigen-presenting cells and suggests that C.neoformans-loaded eosinophils might participate in the adaptive immune response.

Disseminated Cryptococcus neoformans infection and Crohn's disease in an immunocompetent patient

Cryptococcus neoformans is a human pathogen ubiquitously present in the environment. It primarily affects immunocompromised patients, but individuals with no underlying disease or immunodeficiency can also be affected. We herein describe the case of a patient found to have Crohn's disease and disseminated cryptococcosis simultaneously. She had no predisposing underlying cause for impaired immunity. Our patient showed signs that would have make it hard to discriminate between an inflammatory bowel disease and an infection if bowel only would have been involved. The patient underwent surgical intervention; medical therapy was effective against Cryptococcus. She is at now being followed-up for Crohn's disease. When dealing with patient affected with inflammatory bowel diseases, careful history taking, objective and instrumental examination are demandable in order not to overlook associated conditions or infectious diseases. Diagnosis and therapy of cryptococcosis infection in patient with Crohn's disease are herein discussed.

Pulmonary cryptococcosis misdiagnosed as smear-negative pulmonary tuberculosis with fatal consequences

HIV-associated pulmonary cryptococcosis is under diagnosed, and may progress to fatal meningoencephalitis. We present a case of HIV-associated pulmonary Cryptococcus neoformans infection, initially mis-diagnosed as smear-negative pulmonary TB, which progressed to fatal cryptococcal meningitis. Autopsy series suggest that pulmonary cryptococcosis is common in African AIDS patients, and, due to limited diagnostic capacity, often mis-diagnosed as smear negative TB. Serum cryptococcal antigen testing may facilitate diagnosis in such cases.

Immune reconstitution inflammatory syndrome (IRIS) associated with Cryptococcus neoformans infection in AIDS patients

Cryptococcosis is frequently associated to the immune reconstitution inflammatory syndrome (IRIS) in AIDS patients on highly active antiretroviral therapy (HAART). This study aimed to evaluate clinical and evolutive features of IRIS associated cryptococcosis patients in Uberaba, Brazil. Eighty-one AIDS individuals admitted at the teaching hospital with cryptococcal meningitis were evaluated and from these, 40 were prospectively followed. Of 40 patients with cryptococcosis, nine (22.5%) presented clinical and laboratory features of IRIS. Six (66.6%) were male, with a mean age of 37.2. Five (55.5%) presented cryptococcosis as first AIDS defining condition. In seven (77.9%) IRIS was characterised as a relapse of meningeal symptoms after 10 weeks, mean time of 72 days, of starting HAART whereas, two asymptomatic patients developed the syndrome as an unmasked cryptococcosis after 10 and 12 weeks on HAART. Lymphadenitis as isolated finding associated with IRIS was evidenced in three cases. All patients presented low CD4(+) and high RNA viral load baseline values. Cultures of cerebrospinal fluid and lymph-node fragments tissues of these cases were negative. Six of nine individuals developed high intracranial pressure requiring a daily relief lumbar puncture. No deaths occurred during the evolution of these patients. The incidence and clinical evolutive profile observed in this case series are in accordance with other reports elsewhere.

Insights into the Mechanisms of Protective Immunity against Cryptococcus neoformans Infection Using a Mouse Model of Pulmonary Cryptococcosis

Cryptococcus neoformans is an opportunistic fungal pathogen that causes life-threatening pneumonia and meningoencephalitis in immune compromised individuals. Previous studies have shown that immunization of BALB/c mice with an IFN-γ-producing C. neoformans strain, H99γ, results in complete protection against a second pulmonary challenge with an otherwise lethal cryptococcal strain. The current study evaluated local anamnestic cell-mediated immune responses against pulmonary cryptococcosis in mice immunized with C. neoformans strain H99γ compared to mice immunized with heat-killed C. neoformans (HKC.n.). Mice immunized with C. neoformans strain H99γ had significantly reduced pulmonary fungal burden post-secondary challenge compared to mice immunized with HKC.n. Protection against pulmonary cryptococcosis was associated with increased pulmonary granulomatous formation and leukocyte infiltration followed by a rapid resolution of pulmonary inflammation, which protected the lungs from severe allergic bronchopulmonary mycosis (ABPM)-pathology that developed in the lungs of mice immunized with HKC.n. Pulmonary challenge of interleukin (IL)-4 receptor, IL-12p40, IL-12p35, IFN-γ, T cell and B cell deficient mice with C. neoformans strain H99γ demonstrated a requirement for Th1-type T cell-mediated immunity, but not B cell-mediated immunity, for the induction of H99γ-mediated protective immune responses against pulmonary C. neoformans infection. CD4+ T cells, CD11c+ cells, and Gr-1+ cells were increased in both proportion and absolute number in protected mice. In addition, significantly increased production of Th1-type/pro-inflammatory cytokines and chemokines, and conversely, reduced Th2-type cytokine production was observed in the lungs of protected mice. Interestingly, protection was not associated with increased production of cytokines IFN-γ or TNF-α in lungs of protected mice. In conclusion, immunization with C. neoformans strain H99γ results in the development of protective anti-cryptococcal immune responses that may be measured and subsequently used in the development of immune-based therapies to combat pulmonary cryptococcosis.

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-gamma-producing C. neoformans strain (H99gamma) 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 H99gamma 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.

Nasal cryptococcosis in two dogs in New Zealand

CASE HISTORY: Two young, large-breed female dogs were presented with an acute onset of sneezing and nasal discharge. One patient had concurrent epistaxis and facial deformity. CLINICAL FINDINGS: Decreased airflow was noted through the left nostril in Case 1, while Case 2 showed facial deformity. Nasal radiographs from Case 1 showed a soft tissue opacity in the left nasal cavity and frontal sinus. Rhinoscopy revealed roughened, erythematous nasal turbinates in both patients, and a mass in the left caudal nasal cavity of Case 1. Cryptococcus spp. were demonstrated histopathologically on a nasal biopsy. Tissue culture and serum antigen titres were positive for Cryptococcus spp. DIAGNOSIS: Chronic rhinitis secondary to Cryptococcus gattii infection in Case 1, and Cryptococcus neoformans infection in Case 2. CLINICAL RELEVANCE: These are the first reported cases of Cryptococcus spp. rhinitis in dogs in New Zealand.

Atypical Micromorphology and Uncommon Location of Cryptococcosis: A Histopathologic Study Using Special Histochemical Techniques (One Case Report)

Here we report an unusual case of disseminated cryptococcosis in a patient with AIDS. Although typical Cryptococcus neoformans micromorphology was observed in tongue biopsy, cervical lymph node examination revealed atypical histopathologic findings. These included pseudohyphae, chains of budding yeasts and structures resembling germ tubes. Cryptococcus neoformans infection in supraclavicular lymph nodes was also confirmed by culture. The importance of using special histochemical techniques-Mayer's mucicarmine stain for mucicarminophilic capsule and Grocott's silver stain-in the diagnosis of cryptococcosis is reinforced.

Cryptococcus neoformans glucuronoxylomannan induces macrophage apoptosis mediated by nitric oxide in a caspase-independent pathway

Glucuronoxylomannan (GXM) is the major component of Cryptococcus capsular polysaccharide, which represents an essential virulence factor for this yeast. Cryptococcus neoformans infections in immunocompetent rats are associated with inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production by macrophages. This study demonstrates in vitro and in vivo that GXM promotes iNOS expression with NO production in rat macrophages. GXM also induced macrophage apoptosis after 48 h of culture, with this phenomenon being prevented by the iNOS inhibitor, aminoguanidine. The NO-induced macrophage apoptosis triggered by GXM was dependent on interactions with CD18, Fcgamma receptor II and protein kinase C activation, without participation of tyrosine kinases or mitogen-activated protein kinases. Furthermore, this study reveals that GXM down-regulates the macrophage caspase-3 activity, induces a caspase-independent cell death and promotes depolarization of mitochondria membrane potential with increased cytosolic expression of the apoptosis-inducing factor. Taken together, this study describes the pathways and mechanisms involved in the macrophage apoptosis promoted by GXM through NO generation. These findings indicate new mechanisms of immunomodulation for the main capsular polysaccharide of C. neoformans.

Duodenal ulcer with cryptococcus neoformans infection: report of a case

患者男,41岁.因上腹部隐痛6个月于2007年12月10日就诊.患者曾多次在外院诊治,胃镜取黏膜活检示消化性溃疡,给予抗幽门螺杆菌、抑酸及保护胃黏膜治疗,虽有所缓解但病情反复,且近期症状加重.体检:营养中等,腹平软,剑突下偏左轻压痛,无反跳痛,未扪及明显包块.胃镜检查示十二指肠球部前壁可见一不规则深凹溃疡,约1.2 cm×2.0 cm,底部被覆黄白厚苔,质韧,周围黏膜充血肿胀(图1).胃镜诊断:十二指肠球部溃疡(A1期)。