A Structural In Silico Analysis of the Immunogenicity of L-Asparaginase from Penicillium cerradense.

L-asparaginase is an essential drug used to treat acute lymphoid leukemia (ALL), a cancer of high prevalence in children. Several adverse reactions associated with L-asparaginase have been observed, mainly caused by immunogenicity and allergenicity. Some strategies have been adopted, such as searching for new microorganisms that produce the enzyme and applying protein engineering. Therefore, this work aimed to elucidate the molecular structure and predict the immunogenic profile of L-asparaginase from Penicillium cerradense, recently revealed as a new fungus of the genus Penicillium and producer of the enzyme, as a motivation to search for alternatives to bacterial L-asparaginase. In the evolutionary relationship, L-asparaginase from P. cerradense closely matches Aspergillus species. Using in silico tools, we characterized the enzyme as a protein fragment of 378 amino acids (39 kDa), including a signal peptide containing 17 amino acids, and the isoelectric point at 5.13. The oligomeric state was predicted to be a homotetramer. Also, this L-asparaginase presented a similar immunogenicity response (T- and B-cell epitopes) compared to Escherichia coli and Dickeya chrysanthemi enzymes. These results suggest a potentially useful L-asparaginase, with insights that can drive strategies to improve enzyme production.

Molecular and Phenotypic Characterization of Nine Patients with STAT1 GOF Mutations in China.

PURPOSE: Signal transducer and activator of transcription 1 (STAT1) is a transcription factor that mediates cellular responses to interferons (IFNs) and other cytokines and growth factors in diverse cell types. STAT1 gain-of-function (GOF) mutations result in an unexpectedly wide range of clinical features. It remains unclear why STAT1 GOF mutations result in such a broad spectrum of phenotypes. METHODS: We analyzed the clinical, molecular, and phenotypic characteristics of nine Chinese patients with STAT1 GOF mutations. RESULTS: This study enrolled nine patients with STAT1 GOF mutations including five novel mutations. We discuss the molecular and phenotypic characterization such as unique Penicillium marneffei lymphadenitis. Patients with STAT1 GOF mutations had defects in both innate and adaptive immunity, including impaired T cell receptor (TCR) diversity; reduced numbers of naïve and effector memory CD4+ T cells, memory B cells, and NK cells; and defects in the production of IL-17A and IFN-γ. In addition, experiments with primary immune cells revealed that enhanced STAT1 phosphorylation resulted from not only lower rates of STAT1 dephosphorylation but also increased total STAT1 expression. CONCLUSIONS: Our report provides the first comprehensive overview of the molecular genetics, clinical heterogeneity, and underlying immunological abnormalities of patients with STAT1 GOF mutations in China. In further study, to find the relationship between different STAT1 GOF mutations and clinical phenotype as well as the mechanism of increased total STAT1 expression will be needed.

Relationship between mold exposure, specific IgE sensitization, and clinical asthma: A case-control study.

BACKGROUND: Most of the findings related to the noxious effect of mold sensitization on asthma come from investigations based on Alternaria alternata, Cladosporium herbarum, and Aspergillus fumigatus. However, species such as Penicillium spp, Cladosporium sphaerospermum, Cladosporium cladosporioides, or Aspergillus versicolor display a more pronounced indoor tropism, and their potential harmful respiratory effects cannot be neglected. OBJECTIVE: The goal of this work was to relate mold sensitizations with asthma severity and with the level of indoor mold contamination among mold-sensitized patients with asthma and nonsensitized patients with asthma. METHODS: A case-control study was conducted and several asthma severity markers were compared between patients with asthma with and without mold sensitization. Indoor contamination of patients' dwellings was also investigated. RESULTS: Our findings confirmed the association between sensitization to A fumigatus and severity for patients with asthma in contrast with sensitization to other species. Indoor mold contamination was detected in approximately 90% of dwellings. Overall mold exposure was not associated with asthma severity. However, regardless of the sensitization, exposure to A fumigatus and Penicillium spp in dust was linked to an increased risk of severe asthma. CONCLUSION: The harmful nature of mold sensitization and mold exposure for patients with asthma was not confirmed in this study. However, sensitization to A fumigatus was associated with an increased risk for severe asthma. A better investigation of the properties of Penicillium spp is recommended because its exposure was found to be associated with a more pronounced impairment of lung function.

Acute Penicillium marneffei infection stimulates host M1/M2a macrophages polarization in BALB/C mice.

BACKGROUND: Penicillium marneffei (P. marneffei) is a thermally dimorphic fungus pathogen that causes fatal infection. Alveolar macrophages are innate immune cells that have critical roles in protection against pulmonary fungal pathogens and the macrophage polarization state has the potential to be a deciding factor in disease progression or resolution. The aim of this study was to investigate mouse alveolar macrophage polarization states during P. marneffei infection. RESULTS: We used enzyme-linked immunosorbent (ELISA) assays, quantitative real-time PCR (qRT-PCR), and Griess, arginase activity to evaluate the phenotypic markers of alveolar macrophages from BALB/C mice infected with P. marneffei. We then treated alveolar macrophages from infected mice with P. marneffei cytoplasmic yeast antigen (CYA) and investigated alveolar macrophage phenotypic markers in order to identify macrophage polarization in response to P. marneffei antigens. Our results showed: i) P. marneffei infection significantly enhanced the expression of classically activated macrophage (M1)-phenotypic markers (inducible nitric oxide synthase [iNOS] mRNA, nitric oxide [NO], interleukin-12 [IL-12], tumor necrosis factor-alpha [TNF-α]) and alternatively activated macrophage (M2a)-phenotypic markers (arginase1 [Arg1] mRNA, urea) during the second week post-infection. This significantly decreased during the fourth week post-infection. ii) During P. marneffei infection, CYA stimulation also significantly enhanced the expression of M1 and M2a-phenotypic markers, consistent with the results for P. marneffei infection and CYA stimulation preferentially induced M1 subtype. CONCLUSIONS: The data from the current study demonstrated that alveolar macrophage M1/M2a subtypes were present in host defense against acute P. marneffei infection and that CYA could mimic P. marneffei to induce a host immune response with enhanced M1 subtype. This could be useful for investigating the enhancement of host anti-P. marneffei immune responses and to provide novel ideas for prevention of P. marneffei-infection.

The characteristics of indoor and outdoor fungi and their relation with allergic respiratory diseases in the southern region of Turkey.

Indoor and outdoor fungal exposure has been shown to be associated with the development of allergic respiratory diseases. The aim of the study was to investigate the types and concentrations of airborne fungi inside and outside homes and evaluate the association between fungal levels and allergic diseases in the southern region of Turkey. A total of 61 children admitted with respiratory complaints to the pediatric allergy clinic between September 2007 and November 2008 were included in this study. The air samples were obtained using the Air IDEAL volumetric air sampler longitudinally for 1 year. A comprehensive questionnaire was used for medical history and housing conditions. Skin prick test was performed to determine fungal sensitivity and spirometric indices were employed. The predominant indoor fungal species were Cladosporium (69.3 %), Penicillium (18.9 %), Aspergillus (6.5 %), and Alternaria (3.1 %). A strong correlation between indoor and outdoor fungal levels was detected for the Cladosporium species (p < 0.001, r = 0.72) throughout the year. Living in a detached home (p = 0.036) and the presence of cockroaches (p = 0.005) were associated with total indoor fungal levels. The presence of cockroaches (aOR 3.5; 95 % CI 0.95-13.10, p = 0.059) was also associated with fungal sensitization at the edge of significance. The statistical cutoff values of indoor and outdoor Cladosporium levels to predict symptomatic asthma were found to be >176 CFU/m(3) (p = 0.003, AUC 0.696; sensitivity 65.5 %; specificity 68.7 %) and >327 CFU/m(3) (p = 0.038; AUC 0.713; sensitivity 66.6 %; specificity 76.9 %), respectively. Children with respiratory symptoms are exposed to a considerable level of fungi inside and outside their homes. The prevention of fungal exposure may provide valuable intervention for respiratory diseases.

Allergic fungal airway disease

Fungi are ubiquitous and form their own kingdom. Up to 80 genera of fungi have been linked to type I allergic disease, and yet, commercial reagents to test for sensitization are available for relatively few species. In terms of asthma, it is important to distinguish between species unable to grow at body temperature and those that can (thermotolerant) and thereby have the potential to colonize the respiratory tract. The former, which include the commonly studied Alternaria and Cladosporium genera, can act as aeroallergens whose clinical effects are predictably related to exposure levels. In contrast, thermotolerant species, which include fungi from the Candida, Aspergillus, and Penicillium genera, can cause a persistent allergenic stimulus independent of their airborne concentrations. Moreover, their ability to germinate in the airways provides a more diverse allergenic stimulus, and may result in noninvasive infection, which enhances inflammation. The close association between IgE sensitization to thermotolerant filamentous fungi and fixed airflow obstruction, bronchiectasis, and lung fibrosis suggests a much more tissue-damaging process than that seen with aeroallergens. This review provides an overview of fungal allergens and the patterns of clinical disease associated with exposure. It clarifies the various terminologies associated with fungal allergy in asthma and makes the case for a new term (allergic fungal airway disease) to include all people with asthma at risk of developing lung damage as a result of their fungal allergy. Lastly, it discusses the management of fungirelated asthma (AU)

Los hongos son obicuos y forman su propio reino. Hasta 80 géneros de hongos han sido asociados con la enfermedad alérgica tipo I; sin embargo, los reactivos comerciales para testar las sensibilizaciones a los mismos, se encuentran disponibles para un número relativamente pequeño de especies. En cuanto al asma, es importante distinguir entre especies incapaces de crecer en temperatura ambiente y aquellas que son termotolerantes y que, por lo tanto, tienen una capacidad potencial para colonizar el tracto respiratorio. Los primeros, que incluyen los géneros comúnmente estudiados Alternaria y Cladosporium, pueden actuar como aeroalérgenos y sus efectos clínicos están relacionados con los niveles de exposición. En contraste, las especies termotolerantes, que incluyen hongos del género Candida, Aspergillus y Penicillium, pueden causar un estímulo alergénico persistente independiente de su concentración en el aire. Además, su capacidad para germinar en las vías aéreas da lugar a estímulos alergénicos más diversos y puede dar como resultado infecciones no invasivas que facilitan la inflamación. La estrecha asociación entre sensibilización dependiente de IgE a hongos filamentosos termotolerantes y la obstrucción del flujo aéreo, bronquiectasias y fibrosis pulmonar sugiere que esto lleva a un proceso de daño tisular mucho mayor que el producido por aeroalérgenos. Esta revisión ofrece una visión general de los alérgenos de los hongos y los patrones de las enfermedades clínicas asociadas a su exposición. Trata de aclarar la terminología variada asociada con la alergia a hongos en asma, ofreciendo un nuevo término (enfermedad alérgica de las vías aéreas por hongos) para englobar a todos los pacientes asmáticos con riesgo de desarrollar daños pulmonares como resultados de su alergia a hongos. Por último, se discute el manejo del asma relacionada con hongos (AU)

Changes in PH in exhaled breath condensate after specific bronchial challenge test in patients with chronic hypersensitivity pneumonitis: a prospective study.

BACKGROUND: The aim of this study was to investigate the influence of the specific inhalation challenge (SIC) on changes of pH values in exhaled breath condensate (EBC) in patients with hypersensitivity pneumonitis (HP). METHODS: A prospective study of 85 patients with suspected HP, of whom 63 were diagnosed with HP due to exposure to avian or fungal antigens. In all cases, EBC samples were collected before and after completion of the SIC and pH values were determined. RESULTS: Taken as a whole, patients with HP did not present changes in EBC pH after SIC. However, considering only patients with exposure to molds, those diagnosed with HP had a significantly more acid pH post-SIC than those with another diagnosis (p = 0.011). This fact is not observed in patients exposed to bird's antigens. A ROC curve showed that a reduction in EBC pH of 0.3 units or more after SIC in patients diagnosed with HP due to exposure to molds had a sensitivity of 30 % (CI: 12.8 to 54.3 %) and a specificity of 100 % (CI: 65.5 to 100 %). CONCLUSION: EBC pH may be useful in interpreting SIC results in patients with HP, especially in those patients exposed to molds. Further studies are now required to test the validity of these proposals.

The in vitro fungicidal activity of human macrophages against Penicillium marneffei is suppressed by dexamethasone.

Penicillium marneffei (P. marneffei) is a pathogenic fungus that can persist in macrophages and cause a life-threatening systemic mycosis in immunocompromised hosts. To elucidate the mechanisms underlying this opportunistic fungal infection, we established the co-culture system of P. marneffei conidia and human monocyte-derived macrophages (MDM) for investigating the interactions between them. And, we impaired the immune state of MDM by the addition of dexamethasone (DEX). Compared with immunocompetent MDM without DEX treatment in response to P. marneffei, DEX could damage MDM function in initiating the innate immune response through decreasing TNF-α production and the proportion of P. marneffei conidia in mature phagolysosomes, while the red pigment secretion by P. marneffei conidia was promoted by DEX following MDM lysis. Our data provide the evidence that DEX-treated MDM have a low fungicidal activity against P. marneffei that causes penicilliosis in immunocompromised hosts.

Role of intracellular free calcium in killing Penicillium marneffei within human macrophages.

Increases in cytosolic Ca(2+) concentration ([Ca(2+)]c) promote phagocyte antimicrobial responses. Here, we investigated macrophages stimulated by Penicillium marneffei (P. marneffei). [Ca(2+)]c was determined in macrophages loaded with the fluorescent calcium probe Fura 2/AM as they were stimulated by P. marneffei. We found that P. marneffei induced an increase in [Ca(2+)]c in human macrophages. Further, increased [Ca(2+)]c with the ionophore A23187 promoted phagosomal acidification and maturation and reduced intracellular replication of P. marneffei in P. marneffei-infected human macrophages, whereas decreased [Ca(2+)]c with the chelation MAPTAM decreased TNF-α production, inhibited phagosomal acidification and maturation and increased intracellular replication of P. marneffei. These data indicate that Ca(2+) signaling may play an important role in controlling the replication of P. marneffei within macrophages.

Early-life mold and tree sensitivity is associated with allergic eosinophilic rhinitis at 4 years of age.

BACKGROUND: Nasal eosinophils are a biomarker for allergic rhinitis (AR) and are associated with increased symptom severity. OBJECTIVE: To identify predictors of allergic eosinophilic rhinitis (AER) in early childhood in children at higher risk for chronic allergic respiratory disorders. METHODS: In the Cincinnati Childhood Allergy and Air Pollution Study, infants born to aeroallergen-sensitized and symptomatic parents were examined and underwent skin prick testing (SPT) annually to 15 aeroallergens from 1 to 4 years of age. Wheal circumferences were traced and scanned and areas were determined by computer planimetry. At 4 years, AER was defined as (1) at least 1 positive aeroallergen SPT result, (2) presence of sneezing and runny nose without a cold or influenza, and (3) nasal eosinophilia of at least 5%. Wheal areas at 1 to 3 years were analyzed for an association with AER compared with children without AR. RESULTS: At 4 years, 487 children completed rhinitis health histories, SPT, and nasal sampling. Ninety-nine children (22.8%) had AR. Thirty-eight children had AER (8.8% of total sample and 38.4% of AR sample, respectively). At 3 years, for every 1-mm(2) increase in Penicillium species (adjusted odds ratio 1.18, 95% confidence interval 1.06-1.32, P = .002) and maple (adjusted odds ratio 1.07, 95% confidence interval 1.01-1.13, P = .02), wheal area significantly increased the risk of AER at 4 years of age. CONCLUSION: Allergic eosinophilic rhinitis was identified in 8.8% of children at 4 years of age. Age 3 years was the earliest that aeroallergen SPT wheal areas were predictive of AER. Skin testing at 3 years identifies children at risk for an AR phenotype with nasal eosinophilia.

Effect of Jun N-terminal kinase 1 and 2 on the replication of Penicillium marneffei in human macrophages.

Penicillium marneffei (P. marneffei) is a human pathogen which persists in macrophages and threatens the immunocompromised patients. To clarify the mechanisms involved, we evaluated the effect of c-Jun N-terminal kinase 1 and 2 (JNK1/2) on cytokine expression, phagosomal maturation and multiplication of P. marneffei in P. marneffei-stimulated human macrophages. P. marneffei induced the rapid phosphorylation of JNK1/2. Using the specific inhibitor of JNK1/2 (SP600125), we found that the inhibition of JNK1/2 suppressed P. marneffei-induced tumor necrosis factor-α and IL-10 production. In addition, the presence of SP600125 increased phagosomal acidification and maturation and decreased intracellular replication. These data suggest that JNK1/2 may play an important role in promoting the replication of P. marneffei. Our findings further indicate that the pathogen through the JNK1/2 pathway may attenuate the immune response and macrophage antifungal function.

Measurement of aeroallergens from furnace filters.

BACKGROUND: Exposure assessment is an important component of allergic disease diagnosis and management. Analysis for allergen content in vacuumed dust has been used traditionally. OBJECTIVE: To study allergen levels of dust taken from high-efficiency furnace filters in Midwestern homes. METHODS: Furnace filters used were FQT12 1-inch disposable filters with high-efficiency media placed in homes enrolled in the Kansas City Safe and Healthy Homes Project. Dust was removed from the filters by vacuuming. Fungal culture was used to obtain counts of viable spores. Aeroallergens Fel d1, Can f1, Mus m1, Der f1, Der p1, and Bla g2 and antigenic material from Alternaria, Aspergillus, Cladosporium, and Penicillium species were measured using commercially available immunoassay materials. RESULTS: Sixty filters were recovered from 56 homes after an average 135 days in situ. Mean weight of dust recovered was 2.43 g and correlated well with the time the filter was in place. Viable spore counts ranged to 4.8 × 10(7) per gram of dust. Mean fungal antigenic material ranged to 42 µg per gram for Cladosporium species. Mean aeroallergen material ranged to 7 µg per gram for Fel d1. Aeroallergen measurements were above the level of detection in 100% of houses for Fel d1 and 89% of houses for Bla g2. Levels of Fel d1 and Can f1 were strongly positively correlated. CONCLUSION: Allergens from 5 common aeroallergen species and antigenic material from 4 common fungal taxa can be measured in dust taken from high-efficiency furnace filters.

Traffic pollution is associated with early childhood aeroallergen sensitization.

BACKGROUND: No large, prospective, epidemiologic study has investigated the association between diesel exhaust particle (DEP) exposure and early aeroallergen sensitization and allergic rhinitis (AR) at 4 years of age. OBJECTIVE: To determine how exposure to traffic exhaust during infancy is associated with aeroallergen sensitization and AR at 4 years of age and the predictive utility of the wheal area at 1 to 3 years of age on AR at 4 years of age. METHODS: Infants born to aeroallergen sensitized parents were evaluated annually with skin prick tests to 15 aeroallergens with measurement of wheal areas. At 4 years of age, AR was defined as at least one positive aeroallergen skin prick test result and the presence of sneezing and a runny nose without a cold or flu. Infant (DEP) exposure was estimated using data from 27 air sampling monitors and a land use regression model. RESULTS: Complete data were available for 634 children at 4 years of age. Prevalence of AR increased annually from 6.9% to 21.9%. A positive trend was observed for high DEP exposure and aeroallergen sensitization at 2 and 3 years of age (odds ratio, 1.40; 95% confidence interval, 0.97-2.0) and (odds ratio, 1.35; 95% confidence interval, 0.98-1.85) but not with AR. At 2 years of age, every 1-mm(2) increase in the wheal area of timothy and Alternaria significantly increased the odds of AR at 4 years of age. At 3 years of age, every 1-mm(2) increase in the wheal area of fescue, dog, and Penicillium significantly increased the odds of AR at 4 years of age. CONCLUSION: DEP exposure enhances the risk of early aeroallergen sensitization. Aeroallergen wheal area at 2 and 3 years of age is associated with AR at 4 years of age.

Role of extracellular signal-regulated kinases 1 and 2 and p38 mitogen-activated protein kinase pathways in regulating replication of Penicillium marneffei in human macrophages.

Penicillium marneffei (P. marneffei) is a human pathogen which persists in macrophages and threatens the immunocompromised patients. To elucidate the mechanisms involved, we investigated the role of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 mitogen-activated protein kinase (p38) pathways in cytokine expression, phagosome-lysosome fusion and replication of P. marneffei in P. marneffei-infected human macrophages. Analysis of both ERK1/2 and p38 showed rapid phosphorylation in response to P. marneffei. Using specific inhibitors of p38 (SB203580) and MAP kinase kinase-1 (PD98059), we found that ERK1/2 and p38 were essential for P. marneffei-induced tumor necrosis factor-α production, whereas p38, but not that of ERK, was essential for IL-10 production. Furthermore, the presence of PD98059 always decreased phagosomal acidification and maturation and increased intracellular multiplication of P. marneffei, whereas the use of SB203580 always increased phagosomal acidification and maturation and decreased intracellular replication. These data suggest that a proper balance of between ERK1/2 and p38 may play an important role in controlling the replication of P. marneffei. Our findings further indicate a novel therapeutic avenue for treating P. marneffei by stimulating ERK1/2 or activating ERK1/2-dependent mechanisms.

Isolation, expression and characterization of a minor allergen from Penicillium crustosum.

A ribosomal P1 protein, Pen b 26 from Penicillium brevicompactum, was previously identified as a major allergen. A homolog protein was isolated and characterized from Penicillium crustosum which is not known to be allergenic mold. A cDNA library of P. crustosum was constructed and screened using a probe based on the DNA sequence of Pen b 26. A positive clone was isolated, expressed in Escherichia coli, purified and characterized by comparing its immunological and physical properties to Pen b 26. It was designated as Pen cr 26 and had a 321 nt ORF corresponding to 107 amino acids with a MW of 11 kDa. Pen cr 26 had strong sequence homology to Pen b 26 (92% for nucleotides and 86% for amino acids) and its physical and predicted structural properties were similar to the latter. The level of expression of Pen cr 26 was much lower than that of Pen b 26 in the same expression vector. Both proteins were recognized equally well by the IgG class specific antibodies, but Pen cr 26 was poorly recognized by Penicillium-sensitive atopic sera (IgE), suggesting striking antigenic difference in IgE epitopes, i.e., 87% were positive for Pen b 26 while only 23% were positive for Pen cr 26. The allergenicity of Pen cr 26 seems to be minor in nature and it could be a hypoallergenic variant of Pen b 26.

Wheeze in infancy: protection associated with yeasts in house dust contrasts with increased risk associated with yeasts in indoor air and other fungal taxa.

BACKGROUND: While fungal exposures are assumed to provoke wheeze through irritant or allergenic mechanisms, little is known about the differential effects of indoor and outdoor fungi on early-life wheeze. METHODS: In a Boston prospective birth cohort of 499 at-risk infants, culturable fungi in bedroom air and dust and outdoor air were measured at the age of 2-3 months. Wheeze was determined using bimonthly telephone questionnaires. Odds ratios were estimated for an interquartile increase in fungal natural log-transformed concentrations, adjusting for predictors of wheeze and potential confounders. RESULTS: Increased odds of 'any wheeze' (≥1 vs 0 episodes) by age one were positively associated with indoor dust Alternaria [odds ratio (OR) = 1.83; 95% confidence interval (CI), 1.07-3.14], Penicillium [OR = 1.18; (0.98-1.43)], and Cladosporium [OR = 1.47; (1.16-1.85)]; indoor air Penicillium [OR = 1.26; (0.92-1.74)]; and outdoor air Cladosporium [OR = 1.68; (1.04-2.72)]. In contrast, indoor dust yeasts were protective [OR = 0.78; (0.66-0.93)]. 'Frequent wheeze' (≥2 vs <2 episodes) by age one was borderline associated with dust yeasts [OR = 0.86; (0.70-1.04)] and indoor air yeasts [OR = 1.53; (0.93-2.53)]. Alternaria concentration was associated with any wheeze for children with maternal mold sensitization [OR = 9.16; (1.37-61.22)], but not for those without maternal mold sensitization [OR = 1.32; (0.79-2.20)]. CONCLUSIONS: While wheeze rates were higher with exposures to fungal taxa considered to be irritant or allergenic in sensitive subjects, yeasts in the home had a strong protective association with wheeze in infancy. Molecular microbiologic studies may elucidate specific components of innate microbiologic stimulants that lead to contrasting effects on wheeze development.

Antioxidative and immunogenic properties of catalase-peroxidase protein in Penicillium marneffei.

Penicillium marneffei is a significant opportunistic fungal pathogen in Southeast Asia and its ability to survive inside the host macrophages is believed to be important in the establishment of infection. Previously, we isolated a gene encoding a catalase- peroxidase (cpeA) from P. marneffei and showed that the cpeA transcript is specifically upregulated during yeast phase growth at 37 °C. In this study, the cpeA transcript was found to be induced during the mycelium to yeast phase transition and during stress conditions induced by hydrogen peroxide treatment. Null mutation of cpeA reduced the fungal tolerance to hydrogen peroxide but not to heat stress. These results indicated that the CpeA plays a crucial role in this fungus' oxidative stress response. Western blot analysis demonstrated that the CpeA induced antibody production in P. marneffei-infected patients, including highly exposed-healthy people. This is the first report that the catalase-peroxidase possesses an immunogenic property in fungi.

Development of in vitro macrophage system to evaluate phagocytosis and intracellular fate of Penicillium marneffei conidia.

Penicillium marneffei is a pathogenic fungus that can cause a life-threatening systemic mycosis in the immunocompromised hosts. We established the model for the phagocytosis of P. marneffei conidia by RAW264.7 murine macrophages and designated the fate of P. marneffei in RAW264.7 cells with respect to persistence, phagosome-lysosome-fusion. And we impaired the immune status of mouse and compared the fate and phagosome-lysosome-fusion of P. marneffei in immunocompetent and immunosuppressed mouse peritoneal macrophages cells. We found that conidia could germinate and survive in macrophages. Within 30 min and up to 2 h of heat-killed conidia internalization, the majority of all phagosome types were labeled for the EEA1 (endosomal markers) and LAMP-1 (lysosomal markers), respectively. But both the percentages of LAMP-1 and EEA1 that associated with live conidia were significantly lower than that with heat-killed conidia. Administration of cyclophosphamide resulted in a significant suppression of macrophages function (phagocytic and fungicidal) against P. marneffei that were not apparently seen. Our data provide the evidence that (i) intracellular conversion of P. marneffei conidia into yeast cells still could be observed in macrophages. (ii) Phagosomes containing live Penicillium marneffei conidia might inhibit the phagosome-lysosome-fusion and result to no acidification surrounding the organisms. (iii) Immunity impaired by cyclophosphamide could not influence the function, including phagocytosis, fungicidal activity and phagosome-lysosome-fusion, of macrophages against P. marneffei.