Dense granule protein 3 of Toxoplasma gondii plays a crucial role in the capability of the tissue cysts of the parasite to persist in the presence of anti-cyst CD8+ T cells during the chronic stage of infection.

Toxoplasma gondii establishes chronic infection by forming tissue cysts, and this chronic infection is one of the most common parasitic infections in humans. Our recent studies revealed that whereas CD8+ T cells of genetically resistant BALB/c mice have the capability to remove the tissue cysts of the parasite through their perforin-mediated activities, small portions of the cysts are capable of persisting in the presence of the anti-cyst CD8+ T cells. It is currently unknown how those small portions of the cysts resist or escape the T-cell immunity and persist in the hosts. In the present study, we discovered that the cysts, which persisted in the presence of the perforin-mediated CD8+ T-cell immunity, have significantly greater mRNA levels for four dense granule proteins, GRA1, GRA2, GRA3, and GRA7, and one rhoptry protein, ROP35, than the total population of the cysts present in the absence of the T cells. In addition, increased levels of mRNA for GRA1, GRA3, and ROP35 in the cysts significantly correlated with their successful persistence through the condition in which greater degrees of reduction of the cyst burden occurred through anti-cyst CD8+ T cells. In addition, GRA3-deficient T. gondii displayed significantly enhanced elimination of the cysts by anti-cyst CD8+ T cells when compared to the wild-type parasite. These results indicate that GRA3 is a key molecule that mediates in the capability of T. gondii cysts to persist by resisting or evading the anti-cyst activity of CD8+ T cells during the later stage of infection.

IFN-γ production by brain-resident cells activates cerebral mRNA expression of a wide spectrum of molecules critical for both innate and T cell-mediated protective immunity to control reactivation of chronic infection with Toxoplasma gondii.

We previously demonstrated that brain-resident cells produce IFN-γ in response to reactivation of cerebral infection with Toxoplasma gondii. To obtain an overall landscape view of the effects of IFN-γ from brain-resident cells on the cerebral protective immunity, in the present study we employed NanoString nCounter assay and quantified mRNA levels for 734 genes in myeloid immunity in the brains of T and B cell-deficient, bone marrow chimeric mice with and without IFN-γ production by brain-resident cells in response to reactivation of cerebral T. gondii infection. Our study revealed that IFN-γ produced by brain-resident cells amplified mRNA expression for the molecules to activate the protective innate immunity including 1) chemokines for recruitment of microglia and macrophages (CCL8 and CXCL12) and 2) the molecules for activating those phagocytes (IL-18, TLRs, NOD1, and CD40) for killing tachyzoites. Importantly, IFN-γ produced by brain-resident cells also upregulated cerebral expression of molecules for facilitating the protective T cell immunity, which include the molecules for 1) recruiting effector T cells (CXCL9, CXCL10, and CXCL11), 2) antigen processing (PA28αß, LMP2, and LMP7), transporting the processed peptides (TAP1 and TAP2), assembling the transported peptides to the MHC class I molecules (Tapasin), and the MHC class I (H2-K1 and H2-D1) and Ib molecules (H2-Q1, H-2Q2, and H2-M3) for presenting antigens to activate the recruited CD8+ T cells, 3) MHC class II molecules (H2-Aa, H2-Ab1, H2-Eb1, H2-Ea-ps, H2-DMa, H2-Ob, and CD74) to present antigens for CD4+ T cell activation, 4) co-stimulatory molecules (ICOSL) for T cell activation, and 5) cytokines (IL-12, IL-15, and IL-18) facilitating IFN-γ production by NK and T cells. Notably, the present study also revealed that IFN-γ production by brain-resident cells also upregulates cerebral expressions of mRNA for the downregulatory molecules (IL-10, STAT3, SOCS1, CD274 [PD-L1], IL-27, and CD36), which can prevent overly stimulated IFN-γ-mediated pro-inflammatory responses and tissue damages. Thus, the present study uncovered the previously unrecognized the capability of IFN-γ production by brain-resident cells to upregulate expressions of a wide spectrum of molecules for coordinating both innate and T cell-mediated protective immunity with a fine-tuning regulation system to effectively control cerebral infection with T. gondii.

Selective Upregulation of Transcripts for Six Molecules Related to T Cell Costimulation and Phagocyte Recruitment and Activation among 734 Immunity-Related Genes in the Brain during Perforin-Dependent, CD8+ T Cell-Mediated Elimination of Toxoplasma gondii Cysts.

We recently found that an invasion of CD8+ cytotoxic T cells into tissue cysts of Toxoplasma gondii initiates an elimination of the cysts in association with an accumulation of microglia and macrophages. In the present study, we compared mRNA levels for 734 immune-related genes in the brains of infected SCID mice that received perforin-sufficient or -deficient CD8+ immune T cells at 3 weeks after infection. At 7 days after the T cell transfer, mRNA levels for only six genes were identified to be greater in the recipients of the perforin-sufficient T cells than in the recipients of the perforin-deficient T cells. These six molecules included two T cell costimulatory molecules, inducible T cell costimulator receptor (ICOS) and its ligand (ICOSL); two chemokine receptors, C-X-C motif chemokine receptor 3 (CXCR3) and CXCR6; and two molecules related to an activation of microglia and macrophages, interleukin 18 receptor 1 (IL-18R1) and chitinase-like 3 (Chil3). Consistently, a marked reduction of cyst numbers and upregulation of ICOS, CXCR3, CXCR6, IL-18R1, and Chil3 mRNA levels were also detected when the perforin-sufficient CD8+ immune T cells were transferred to infected SCID mice at 6 weeks after infection, indicating that the CD8+ T cell-mediated protective immunity is capable of eliminating mature T. gondii cysts. These results together suggest that ICOS-ICOSL interactions are crucial for activating CD8+ cytotoxic immune T cells to initiate the destruction of T. gondii cysts and that CXCR3, CXCR6, and IL-18R are involved in recruitment and activation of microglia and macrophages to the T cell-attacked cysts for their elimination.IMPORTANCE T. gondii establishes a chronic infection by forming tissue cysts, which can grow into sizes greater than 50 µm in diameter as a consequence of containing hundreds to thousands of organisms surrounded by the cyst wall within infected cells. Our recent studies using murine models uncovered that CD8+ cytotoxic T cells penetrate into the cysts in a perforin-dependent manner and induce their elimination, which is accompanied with an accumulation of phagocytic cells to the T cell-attacked target. This is the first evidence of the ability of the T cells to invade into a large target for its elimination. However, the mechanisms involved in anticyst immunity remain unclear. Immune profiling analyses of 734 immune-related genes in the present study provided a valuable foundation to initiate elucidating detailed molecular mechanisms of the novel effector function of the immune system operated by perforin-mediated invasion of CD8+ T cells into large targets for their elimination.

Penetration of CD8+ Cytotoxic T Cells into Large Target, Tissue Cysts of Toxoplasma gondii, Leads to Its Elimination.

CD8+ cytotoxic T cells kill target cells through direct cell-cell contact. However, it remains unclear how these T cells eliminate a target of large mass. We investigated how CD8+ T cells remove tissue cysts of Toxoplasma gondii, which can grow to the size of >50 µm in diameter within infected cells. Notably, immunohistologic analyses in the brains of infected mice visualized the presence of numbers of CD8+ immune T cells that had migrated halfway through the cyst wall as well as T cells located fully within the cysts. Perforin was required for their invasion and cyst elimination. Cysts invaded by the T cells displayed morphologic deterioration and destruction. Within these deteriorated cysts, granular structures intensely positive for granzyme B were detected in association with T. gondii bradyzoites. Furthermore, the bradyzoites within the destroyed cysts were located within accumulated ionized calcium binding adaptor molecule 1 (Iba1)-positive microglia and Ly6C+ macrophages, suggesting that these phagocytes had phagocytosed those organisms for their eradication. The present study uncovered a previously unappreciated capability of CD8+ cytotoxic T cells to penetrate into a large target, T. gondii cysts, for their elimination. This invasive capability of CD8+ cytotoxic T cells in collaboration with phagocytes appears to be a powerful effector mechanism that functions against not only T. gondii cysts but also other large targets, including solid cancers.

Inducible nitric oxide synthase in innate immune cells is important for restricting cyst formation of Toxoplasma gondii in the brain but not required for the protective immune process to remove the cysts.

Significantly larger numbers of Toxoplasma gondii cysts were detected in the brains of RAG1-/-NOS2-/- than RAG1-/- mice following infection. In contrast, the cyst numbers markedly decreased in a same manner in both strains of mice after receiving CD8+ immune T cells. Thus, NOS2-mediated innate immunity is important for inhibiting formation of cysts in the brain but not required for the T cell-initiated cyst removal, which is associated with phagocyte accumulation. Treatment with chloroquine, an inhibitor of endolysosomal acidification, partially but significantly inhibited the T cell-mediated cyst removal, suggesting that phagosome-lysosome fusion could be involved in the T. gondii cyst elimination.

Determination of a Key Antigen for Immunological Intervention To Target the Latent Stage of Toxoplasma gondii.

Toxoplasma gondii, an obligate intracellular protozoan parasite, establishes a chronic infection by forming cysts preferentially in the brain. Up to one third of the human population worldwide is estimated to be chronically infected with this parasite. However, there is currently no drug effective against the cyst form of the parasite. In addition, the protective immunity against the cysts remains largely unknown. We analyzed the molecular mechanisms by which the immune system detects host cells harboring the cysts to eliminate the latent stage of the parasite using mice with the H-2d haplotype, which are genetically resistant to the infection. Our study revealed that CD8+ immune T cells bearing TCR Vß8.1, 8.2 chain have a potent activity to remove T. gondii cysts from the brain. Our studies also uncovered that H-2Ld is the major Ag-presenting molecule to CD8+ T cells for initiating cyst elimination, and that CD8+Vß8.1, 8.2+ immune T cells recognize the N-terminal region (aa 41-152) of dense granule protein 6 (GRA6Nt) of the parasite presented by the H-2Ld molecule. Furthermore, CD8+ immune T cells induced by immunization with recombinant GRA6Nt were eventually capable of removing the cysts from the brain when transferred to infected immunodeficient mice lacking T cells. Thus, GRA6Nt is a novel and potent Ag to activate CD8+ T cells capable of removing T. gondii cysts. These observations offer a basis for immunological intervention to combat chronic infection with T. gondii by targeting the persistent cysts of the parasite.

CD8(+) T cells remove cysts of Toxoplasma gondii from the brain mostly by recognizing epitopes commonly expressed by or cross-reactive between type II and type III strains of the parasite.

Our previous study demonstrated that CD8(+) T cells remove cysts of Toxoplasma gondii from the brain through perforin-mediated mechanisms. We here show that a transfer of CD8(+) immune T cells primed with a type II or a type III strain of T. gondii both efficiently removed cysts of a type II strain from infected SCID mice, although the former tended to be slightly more efficient than the latter. Similarly, a transfer of type II-primed CD8(+) T cells removed cysts of a type III strain. Therefore, CD8(+) T cells are capable of removing T. gondii cysts by recognizing epitopes commonly expressed in types II and III strains or cross-reactive between these two genotypes.

Cutting Edge: IFN-γ Produced by Brain-Resident Cells Is Crucial To Control Cerebral Infection with Toxoplasma gondii.

In vitro studies demonstrated that microglia and astrocytes produce IFN-γ in response to various stimulations, including LPS. However, the physiological role of IFN-γ production by brain-resident cells, including glial cells, in resistance against cerebral infections remains unknown. We analyzed the role of IFN-γ production by brain-resident cells in resistance to reactivation of cerebral infection with Toxoplasma gondii using a murine model. Our study using bone marrow chimeric mice revealed that IFN-γ production by brain-resident cells is essential for upregulating IFN-γ-mediated protective innate immune responses to restrict cerebral T. gondii growth. Studies using a transgenic strain that expresses IFN-γ only in CD11b(+) cells suggested that IFN-γ production by microglia, which is the only CD11b(+) cell population among brain-resident cells, is able to suppress the parasite growth. Furthermore, IFN-γ produced by brain-resident cells is pivotal for recruiting T cells into the brain to control the infection. These results indicate that IFN-γ produced by brain-resident cells is crucial for facilitating both the protective innate and T cell-mediated immune responses to control cerebral infection with T. gondii.

Secondary metabolite profiles and antifungal drug susceptibility of Aspergillus fumigatus and closely related species, Aspergillus lentulus, Aspergillus udagawae, and Aspergillus viridinutans.

The incidence of Aspergillus infection has been increasing in the past few years. Also, new Aspergillus fumigatus-related species, namely Aspergillus lentulus, Aspergillus udagawae, and Aspergillus viridinutans, were shown to infect humans. These fungi exhibit marked morphological similarities to A. fumigatus, albeit with different clinical courses and antifungal drug susceptibilities. The present study used liquid chromatography/time-of-flight mass spectrometry to identify the secondary metabolites secreted as virulence factors by these Aspergillus species and compared their antifungal susceptibility. The metabolite profiles varied widely among A. fumigatus, A. lentulus, A. udagawae, and A. viridinutans, producing 27, 13, 8, and 11 substances, respectively. Among the mycotoxins, fumifungin, fumiquinazoline A/B and D, fumitremorgin B, gliotoxin, sphingofungins, pseurotins, and verruculogen were only found in A. fumigatus, whereas auranthine was only found in A. lentulus. The amount of gliotoxin, one of the most abundant mycotoxins in A. fumigatus, was negligible in these related species. In addition, they had decreased susceptibility to antifungal agents such as itraconazole and voriconazole, even though metabolites that were shared in the isolates showing higher minimum inhibitory concentrations than epidemiological cutoff values were not detected. These strikingly different secondary metabolite profiles may lead to the development of more discriminative identification protocols for such closely related Aspergillus species as well as improved treatment outcomes.

N-acetylated α-linked acidic dipeptidase is identified as an antigen of Histoplasma capsulatum.

Histoplasmosis, one of the most important mycoses, needs to be diagnosed rapidly and accurately. The main method used to diagnose histoplasmosis is serological detection of antibodies to the Histoplasma capsulatum H and M antigens. Several other protein antigens have been reported in H. capsulatum; however, they have not been used for diagnosis. In this study, we explored novel antigens that were detected during H. capsulatum infection. We obtained a protein mixture from H. capsulatum yeast cells after vigorous mixing in a 0.1% Triton X-100 solution. From the resultant pool, we detected nine spots that reacted with sera from patients with histoplasmosis and identified eight seroactive proteins with mass spectrometry. The seroactive proteins were purified, and their antigenicities were tested with an enzyme-linked immunosorbent assay (ELISA). ELISA revealed that the titer of the patients' sera to N-acetylated α-linked acidic dipeptidase was significantly higher than those of healthy volunteers (P < 0.01). These data indicate that N-acetylated α-linked acidic dipeptidase of H. capsulatum is recognized as a major antigen during histoplasmosis.

CXCL9 is important for recruiting immune T cells into the brain and inducing an accumulation of the T cells to the areas of tachyzoite proliferation to prevent reactivation of chronic cerebral infection with Toxoplasma gondii.

T cells are required to maintain the latency of chronic infection with Toxoplasma gondii in the brain. Here, we examined the role of non-glutamic acid-leucine-arginine CXC chemokine CXCL9 for T-cell recruitment to prevent reactivation of infection with T. gondii. Severe combined immunodeficient (SCID) mice were infected and treated with sulfadiazine to establish a chronic infection. Immune T cells from infected wild-type mice were transferred into the SCID mice in combination with treatment with anti-CXCL9 or control sera. Three days later, sulfadiazine was discontinued to initiate reactivation of infection. Numbers of CD4(+) and CD8(+) T cells isolated from the brains were markedly less in mice treated with anti-CXCL9 serum than in mice treated with control serum at 3 days after sulfadiazine discontinuation. Amounts of tachyzoite (acute stage form of T. gondii)-specific SAG1 mRNA and numbers of foci associated with tachyzoites were significantly greater in the former than the latter at 5 days after sulfadiazine discontinuation. An accumulation of CD3(+) T cells into the areas of tachyzoite growth was significantly less frequent in the SCID mice treated with anti-CXCL9 serum than in mice treated with control serum. These results indicate that CXCL9 is crucial for recruiting immune T cells into the brain and inducing an accumulation of the T cells into the areas where tachyzoites proliferate to prevent reactivation of chronic T. gondii infection.

VCAM-1/α4ß1 integrin interaction is crucial for prompt recruitment of immune T cells into the brain during the early stage of reactivation of chronic infection with Toxoplasma gondii to prevent toxoplasmic encephalitis.

Reactivation of chronic infection with Toxoplasma gondii can cause life-threatening toxoplasmic encephalitis in immunocompromised individuals. We examined the role of VCAM-1/α4ß1 integrin interaction in T cell recruitment to prevent reactivation of the infection in the brain. SCID mice were infected and treated with sulfadiazine to establish a chronic infection. VCAM-1 and ICAM-1 were the endothelial adhesion molecules detected on cerebral vessels of the infected SCID and wild-type animals. Immune T cells from infected wild-type mice were treated with anti-α4 integrin or control antibodies and transferred into infected SCID or nude mice, and the animals received the same antibody every other day. Three days later, sulfadiazine was discontinued to initiate reactivation of infection. Expression of mRNAs for CD3δ, CD4, CD8ß, gamma interferon (IFN-γ), and inducible nitric oxide synthase (NOS2) (an effector molecule to inhibit T. gondii growth) and the numbers of CD4(+) and CD8(+) T cells in the brain were significantly less in mice treated with anti-α4 integrin antibody than in those treated with control antibody at 3 days after sulfadiazine discontinuation. At 6 days after sulfadiazine discontinuation, cerebral tachyzoite-specific SAG1 mRNA levels and numbers of inflammatory foci associated with tachyzoites were markedly greater in anti-α4 integrin antibody-treated than in control antibody-treated animals, even though IFN-γ and NOS2 mRNA levels were higher in the former than in the latter. These results indicate that VCAM-1/α4ß1 integrin interaction is crucial for prompt recruitment of immune T cells and induction of IFN-γ-mediated protective immune responses during the early stage of reactivation of chronic T. gondii infection to control tachyzoite growth.

Evidence for finely-regulated asynchronous growth of Toxoplasma gondii cysts based on data-driven model selection.

Toxoplasma gondii establishes a chronic infection by forming cysts preferentially in the brain. This chronic infection is one of the most common parasitic infections in humans and can be reactivated to develop life-threatening toxoplasmic encephalitis in immunocompromised patients. Host-pathogen interactions during the chronic infection include growth of the cysts and their removal by both natural rupture and elimination by the immune system. Analyzing these interactions is important for understanding the pathogenesis of this common infection. We developed a differential equation framework of cyst growth and employed Akaike Information Criteria (AIC) to determine the growth and removal functions that best describe the distribution of cyst sizes measured from the brains of chronically infected mice. The AIC strongly support models in which T. gondii cysts grow at a constant rate such that the per capita growth rate of the parasite is inversely proportional to the number of parasites within a cyst, suggesting finely-regulated asynchronous replication of the parasites. Our analyses were also able to reject the models where cyst removal rate increases linearly or quadratically in association with increase in cyst size. The modeling and analysis framework may provide a useful tool for understanding the pathogenesis of infections with other cyst producing parasites.

Gene expression analysis of a murine model with pulmonary vascular remodeling compared to end-stage IPAH lungs.

BACKGROUND: Idiopathic pulmonary arterial hypertension (IPAH) continues to be one of the most serious intractable diseases that might start with activation of several triggers representing the genetic susceptibility of a patient. To elucidate what essentially contributes to the onset and progression of IPAH, we investigated factors playing an important role in IPAH by searching discrepant or controversial expression patterns between our murine model and those previously published for human IPAH. We employed the mouse model, which induced muscularization of pulmonary artery leading to hypertension by repeated intratracheal injection of Stachybotrys chartarum, a member of nonpathogenic and ubiquitous fungus in our envelopment. METHODS: Microarray assays with ontology and pathway analyses were performed with the lungs of mice. A comparison was made of the expression patterns of biological pathways between our model and those published for IPAH. RESULTS: Some pathways in our model showed the same expression patterns in IPAH, which included bone morphogenetic protein (BMP) signaling with down-regulation of BMP receptor type 2, activin-like kinase type 1, and endoglin. On the other hand, both Wnt/planar cell polarity (PCP) signaling and its downstream Rho/ROCK signaling were found alone to be activated in IPAH and not in our model. CONCLUSIONS: Activation of Wnt/PCP signaling, in upstream positions of the pathway, found alone in lungs from end stage IPAH may play essential roles in the pathogenesis of the disease.

Interferon-gamma- and perforin-mediated immune responses for resistance against Toxoplasma gondii in the brain.

Toxoplasma gondii is an obligate intracellular protozoan parasite that causes various diseases, including lymphadenitis, congenital infection of fetuses and life-threatening toxoplasmic encephalitis in immunocompromised individuals. Interferon-gamma (IFN-γ)-mediated immune responses are essential for controlling tachyzoite proliferation during both acute acquired infection and reactivation of infection in the brain. Both CD4+ and CD8+ T cells produce this cytokine in response to infection, although the latter has more potent protective activity. IFN-γ can activate microglia, astrocytes and macrophages, and these activated cells control the proliferation of tachyzoites using different molecules, depending on cell type and host species. IFN-γ also has a crucial role in the recruitment of T cells into the brain after infection by inducing expression of the adhesion molecule VCAM-1 on cerebrovascular endothelial cells, and chemokines such as CXCL9, CXCL10 and CCL5. A recent study showed that CD8+ T cells are able to remove T. gondii cysts, which represent the stage of the parasite in chronic infection, from the brain through their perforin-mediated activity. Thus, the resistance to cerebral infection with T. gondii requires a coordinated network using both IFN-γ- and perforin-mediated immune responses. Elucidating how these two protective mechanisms function and collaborate in the brain against T. gondii will be crucial in developing a new method to prevent and eradicate this parasitic infection.

Inhalation of Stachybotrys chartarum evokes pulmonary arterial remodeling in mice, attenuated by Rho-kinase inhibitor.

Stachybotrys chartarum, a ubiquitous fungus in our environment, has been suspected of causing respiratory symptoms in humans, such as acute infant pulmonary hemorrhage and asthma. We previously established a mouse model in which repeated inhalation of Stachybotrys chartarum spores caused pulmonary hypertension. To further investigate the model, particularly in the pulmonary circulation, mice were intra-tracheally injected with spores, 18 times over 12 weeks. Severe muscularization was observed in the small- to medium-sized pulmonary arteries. Bronchoalveolar lavage fluid revealed an increase in eosinophils accompanied by high concentrations of Th2-associated cytokines, IL-4, IL-5, but not Th1-associated IFN-γ. The remodeling was temporary, resolving after cessation of spore inhalation. Chronic inhibition of the RhoA/Rho-kinase pathway by fasudil attenuated pulmonary arterial remodeling. These data suggest that Stachybotrys-mediated remodeling is caused by Th2-associated inflammation and can be resolved by Rho-kinase inhibition, either through direct effects on smooth muscle hypertrophy or through indirect effects on vascular inflammation. These data also show that extensive pulmonary vascular remodeling, often thought of as a fixed lesion, will spontaneously resolve in the absence of underlying molecular etiology.

[Candidates for virulence factors of Aspergillus fumigatus].

The number of patients with invasive fungal infection (IFI) has dramatically increased since the beginning of the 1980s. Aspergillus fumigatus, the most common species recovered from aspergillosis, is an important pathogen of IFI. Recently, new antifungal agents have become available in Japan, but mortality from aspergillosis is still high. Early initiation of therapy seems to improve the survival rate. Study of virulence factors of the fungus may lead to the development of novel diagnostic tools or advancements in therapy. Many candidates of the fungal virulence factors have been studied including proteases and mycotoxins. We previously discussed the influence of fungal secondary metabolites such as gliotoxin and other low molecular components on the virulence, and showed that A. fumigatus produces potent cytotoxic substances other than gliotoxin. Studies are in progress to clarify the significance of the unknown substances.

Inhalation of Stachybotrys chartarum causes pulmonary arterial hypertension in mice.

Inhalation of Stachybotrys chartarum, a ubiquitous fungus in our living environment, has been suspected as a cause of acute idiopathic pulmonary haemorrhage in infants, but its relation to human diseases is not yet known. The aim of present study was to investigate the effect of repeated intratracheal injection of the fungus into mice, paying special attention to the pulmonary vascular system. Spores of S. chartarum were injected into the trachea of mice from 6 to 18 times over 4-12 weeks, and the lungs were examined by histopathology, morphometrics and haemodynamics. When 1 x 10(4) spores/mouse were injected, histopathological examination showed the development of pulmonary arterial hypertension (PAH). Symmetrical thickening of the intima and media of the pulmonary arterial walls was seen after six injections over 4 weeks. Right ventricular hypertrophy was also evident after 12 injections. PAH was confirmed by the elevation of right ventricular systolic pressure (20.1 +/- 5.7 mmHg in the injected group vs. 12.0 +/- 2.4 mmHg in the control group, P < 0.01). This study showed that the inhalation of S. chartarum caused PAH in mice, suggesting a potential of S. chartarum as a cause of human health problem such as PAH.

Activator protein 1 is triggered by Aspergillus fumigatus beta-glucans surface-exposed during specific growth stages.

Aspergillus fumigatus has been shown to trigger the activation of nuclear factor-kappaB (NF-kappaB), but it remains unclear whether other transcription factors are also induced following infection by this organism. In this study, we demonstrated that A. fumigatus also triggers activator protein 1 (AP-1), a transcription factor that plays an important role during the production of cytokines and chemokines. Swollen conidia strongly induce the activation of AP-1, and more than 80% of these were stained positively with anti-beta-glucan antibodies by fluorescence microscopy. Hyphae were also stained with anti-beta-glucan antibodies, albeit significantly weaker compared with swollen conidia. Furthermore, our present findings also showed that A. fumigatus triggers the activation of AP-1 in a dectin-1 (receptor for beta-glucan)-dependent manner. These data thus suggest that AP-1 is triggered by beta-glucan recognition on the surface of A. fumigatus. We also showed that Syk tyrosine kinase is required for AP-1 induction in this pathway. We therefore speculate that the dectin-1/Syk/AP-1 signaling pathway plays an important role in the host defense response to fungal infection.

The Pathogenicity of Stachybotrys chartarum.

Stachybotrys chartarum is a dematiaceous fungus that is ubiquitous in our living environment. This fungus has long been regarded as non-pathogenic and its inhalation effect on humans has been scarcely studied. Recently, however, epidemiologic studies on acute idiopathic pulmonary hemorrhage in infants suggested that the fungus might be potentially pathogenic to humans. To determine the pathogenicity of this fungus, its interaction with the host defense system was studied using polymorphonuclear leukocytes (PMNs) and macrophages. Histopathological analysis of mice intratracheally injected with this fungus was also performed. The results disclosed that the conidia of S. chartarum were resistant to the antifungal activities of alveolar macrophages in terms of phagocytosis, killing and inhibition of germination. However, the conidia could not survive in the lungs of mice when injected intratracheally. Lavage fluid of mycelia that contained the dark slimy material coating the surface of conidia showed cytotoxic activity against macrophages and PMNs. Intratracheal injection of conidia in mice resulted in intraalveolar infiltration of PMNs. When using multiple injections during a 3-week period, strong eosinophilic infiltration into the proximal alveoli and perivascular tissues was observed. Our results suggest that inhalation of conidia may cause serious damage to the human lung, particularly when repeated.