Global intercomparison of polyurethane foam passive air samplers evaluating sources of variability in SVOC measurements.

Polyurethane foam passive air samplers (PUF-PAS) are the most common type of passive air sampler used for a range of semi-volatile organic compounds (SVOCs), including regulated persistent organic pollutants (POPs) and polycyclic aromatic hydrocarbons (PAHs), and emerging contaminants (e.g., novel flame retardants, phthalates, current-use pesticides). Data from PUF-PAS are key indicators of effectiveness of global regulatory actions on SVOCs, such as the Global Monitoring Plan of the Stockholm Convention on Persistent Organic Pollutants. While most PUF-PAS use similar double-dome metal shielding, there is no standardized dome size, shape, or deployment configuration, with many different PUF-PAS designs used in regional and global monitoring. Yet, no information is available on the comparability of data from studies using different PUF-PAS designs. We brought together 12 types of PUF-PAS used by different research groups around the world and deployed them in a multi-part intercomparison to evaluate the variability in reported concentrations introduced by different elements of PAS monitoring. PUF-PAS were deployed for 3 months in outdoor air in Kjeller, Norway in 2015-2016 in three phases to capture (1) the influence of sampler design on data comparability, (2) the influence of analytical variability when samplers are analyzed at different laboratories, and (3) the overall variability in global monitoring data introduced by differences in sampler configurations and analytical methods. Results indicate that while differences in sampler design (in particular, the spacing between the upper and lower sampler bowls) account for up to 50 % differences in masses collected by samplers, the variability introduced by analysis in different laboratories far exceeds this amount, resulting in differences spanning orders of magnitude for POPs and PAHs. The high level of variability due to analysis in different laboratories indicates that current SVOC air sampling data (i.e., not just for PUF-PAS but likely also for active air sampling) are not directly comparable between laboratories/monitoring programs. To support on-going efforts to mobilize more SVOC data to contribute to effectiveness evaluation, intercalibration exercises to account for uncertainties in air sampling, repeated at regular intervals, must be established to ensure analytical comparability and avoid biases in global-scale assessments of SVOCs in air caused by differences in laboratory performance.

Impact of polycyclic aromatic hydrocarbons in mangroves from the Colombian pacific coast: Evaluation in sediments and bivalves.

Bivalves and sediments were sampled from mangroves in the Pacific Coast of Colombia to evaluate the concentrations of the 16 polycyclic aromatic hydrocarbons (PAHs) prioritized by U.S.EPA. Mangroves are highly vulnerable to anthropogenic activities, such as oil spills, which affect sediments and the organisms that depend on that ecosystem. Twelve samples of mangrove and non-mangrove (sandy) sediments and 20 samples of Anadara tuberculosa from mangrove were collected in marine and estuarine areas. In sediments and A. tuberculosa, the highest concentration of Æ©16PAHs was found in estuarine mangroves close to the Rosario River mouth, ranging from 171.4 to 564.0 ng g-1 and 31.0 to 169.0 ng g-1, respectively. For the bivalve, the concentrations showed less variability than sediment, with 25% and 20% of bivalve samples exceeding the limits established by the European Regulatory Commission and Ministry of Health and Social Protection of Colombia, respectively, which can cause effects on people's health. The PAHs isomeric ratios determined in sediments indicated that these compounds were originated mainly from petrogenic sources. The PAHs profile reveals the dominance of 3 and 4 rings PAHs in sediments and dominance of 4 rings PAHs in bivalves.

Submicroscopic infection of placenta by Plasmodium produces Th1/Th2 cytokine imbalance, inflammation and hypoxia in women from north-west Colombia.

BACKGROUND: A large-scale study was set up in order to study the epidemiology, clinical aspects, and immunopathology of gestational and placental malaria in north-west Colombia. In this region, recent reports using a qPCR technique, confirmed frequencies of infection, by Plasmodium falciparum or Plasmodium vivax, up to 45%. Given the high rates of infection observed both in mother and placenta, a first exploratory study was proposed in order to characterize the effect on the inflammation status, tissue damage and hypoxia in Plasmodium spp. infected placentas. METHODS: A descriptive, prospective, cross-sectional design was applied to pregnant women with (PM+) and without (PM-) placental malaria. Messenger RNA expression of Fas, FasL; COX-1, COX-2, HIF, VEGF, and the cytokines IL-2, IL-4, IL-10, IFN-γ and TNF, were measured in peripheral and placental blood using a quantitative PCR. The percentage of apoptotic cells was determined with a TUNEL assay. RESULTS: In total 50 placentas were studied: 25 were positive for submicroscopic infection and 25 were negative for Plasmodium infection. Expression of IL-4 and IL-10 was observed high in placental tissue of PM+, while IL-2 was high in peripheral blood of the same group. Expression of TNF and IFNγ in peripheral blood of the PM + group was high. Similarly, the apoptotic index and Fas expression were significantly high in PM+. However, FasL expression was observed low in PM + compared to PM-. Inflammation markers (HIF, VEGF) and hypoxia markers (COX-1, COX-2) were high in the PM + group. CONCLUSION: During placental malaria expression of some pro-inflammatory cytokines is up-regulated and markers of hypoxia and tissue damage are increased in cases of submicroscopic infection.

Methodology for Anti-Gene Anti-IGF-I Therapy of Malignant Tumours.

The aim of this study was to establish the criteria for methodology of cellular "anti-IGF-I" therapy of malignant tumours and particularly for glioblastoma multiforme. The treatment of primary glioblastoma patients using surgery, radiotherapy, and chemotherapy was followed by subcutaneous injection of autologous cancer cells transfected by IGF-I antisense/triple helix expression vectors. The prepared cell "vaccines" should it be in the case of glioblastomas or other tumours, have shown a change of phenotype, the absence of IGF-I protein, and expression of MHC-I and B7. The peripheral blood lymphocytes, PBL cells, removed after each of two successive vaccinations, have demonstrated for all the types of tumour tested an increasing level of CD8(+) and CD8(+)28(+) molecules and a switch from CD8(+)11b(+) to CD8(+)11. All cancer patients were supervised for up to 19 months, the period corresponding to minimum survival of glioblastoma patients. The obtained results have permitted to specify the common criteria for "anti-IGF-I" strategy: characteristics sine qua non of injected "vaccines" (cloned cells IGF-I(-) and MHC-I(+)) and of PBL cells (CD8(+) increased level).

Gene expression analysis of Paracoccidioides brasiliensis transition from conidium to yeast cell.

Paracoccidioides brasiliensis infectious process relies on the initial expression of virulence factors that are assumed to be controlled by molecular mechanisms through which the conidia and/or mycelial fragments convert to yeast cells. In order to analyze the profile of the thermally-induced dimorphic gene expression, 48 h C-L transition cultures which had been incubated at 36 degrees C were studied. By this time approximately 50% of the conidial population had already reverted to yeast form cells. At this transition time, an EST-Orestes library was constructed and characterized. As a result, 79 sequences were obtained, of which 39 (49.4%) had not been described previously in other libraries of this fungus and which could represent novel exclusive C-Y transition genes. Two of these sequences are, among others, cholestanol delta-isomerase, and electron transfer flavoprotein-ubiquinoneoxidoreductase (ETF-QO). The other 40/79 (50.6%) sequences were shared with Mycelia (M), Yeast (Y) or Mycelia to yest transition (M-Y) libraries. An important component of this group of sequences is a putative response regulator receiver SKN7, a protein of high importance in stress adaptation and a regulator of virulence in some bacteria and fungi. This is the first report identifying genes expressed during the C-Y transition process, the initial step required to understand the natural history of P. brasiliensis conidia induced infection.

Identificación de algunos genes asociados al proceso de germinación de la conidia al micelio en Paracoccidioides brasiliensis / Identification of genes associated with germination of conidia to form mycelia in the fungus Paracoccidioides brasiliensis

Introducción. Paracoccidioides brasiliensis es un hongo dimórfico térmico, que a temperatura ambiente se presenta como un moho productor de conidias, mientras que en el huésped se comporta como una levadura de gemación múltiple. Los mecanismos moleculares que rigen la germinación de conidia a micelio aún se desconocen. Objetivo. Estudiar en P. brasiliensis la cinética del proceso de germinación de conidia a micelio y determinar los genes expresados durante este proceso mediante la construcción y el análisis de una librería EST (Expressed Sequence Tag). Materiales y métodos. Para el estudio de la cinética de germinación, se produjeron y aislaron conidias de P. brasiliensis. Estas fueron incubadas en cultivos líquidos a 18°C por 24, 48, 72 y 96 horas, y se examinaron por microscopía de luz. A partir de conidias cultivadas por 96 horas, se construyó y caracterizó una librería EST, la cual representaría los genes expresados durante el proceso de germinación. Resultados. Durante el proceso de germinación de conidia a micelio, se observó 11,7±1,2%, 30±0,6%, 43±1,3% y 66±2,4% de germinación a las 24, 48, 72 y 96 horas de incubación, respectivamente. Además, se obtuvo una librería del proceso de germinación consistente en 129 secuencias agrupadas en cuatro secuencias contiguas y siete secuencias únicas, para un total de 11 posibles genes. Ocho secuencias (72,7%) no habían sido descritas anteriormente en otras librerías informadas para este hongo y podrían representar genes específicos de la germinación de conidia a micelio. Conclusiones. Éste es el primer reporte en el que se identifican genes no descritos anteriormente, que son expresados durante la germinación de conidia a micelio, proceso de gran importancia en la biología de P. brasiliensis.

Introduction. Paracoccidioides brasiliensis is a thermo-dimorphic fungus. At room temperature it grows as a mold that produces conidia, whereas in the vertebrate host it grows as a multiple-budding yeast. The molecular mechanisms involved in the germination from the conidia to the mycelia process remain unknown. Objective. The kinetics of conidia to mycelia germination process were studied in the dimorphic fungus P. brasiliensis. Gene expression during this process was evaluated by construction and analysis of an EST library. Materials and methods. For the germination kinetics study, P. brasiliensis conidia were isolated as single cell units. Then, they were cultured at 18° C in BHI (brain-heart infusion) broth for 24, 48, 72 and 96 hr. After each perion, they were examined by light microscopy. From conidia harvested at 96 hr, an EST library was constructed; at this stage the gene expression was presumed to be maximal for the germination process. Results. During the conidia to the mycelia developmental process, the following germination rates were observed: at 24 hr, 11.7±1.2%; at 48 hr, 30±0.6%; at 72 hr, 43±1.3%; and at 96 hr, 66±2.4%. At the 96 hour stage, an EST library was constructed. It consisted of 129 sequences grouped in 4 contigs and 7 singlets for a total of 11 possible genes. Eight of the sequences had not been described previously in other EST libraries of this fungus. Conclusions. New genes were identified that were expressed during the conidia to the mycelia germination process and may represent genes specific to the germination process.

[Identification of genes associated with germination of conidia to form mycelia in the fungus Paracoccidioides brasiliensis]. / Identificación de algunos genes asociados al proceso de germinación de la conidia al micelio en Paracoccidioides brasiliensis.

INTRODUCTION: Paracoccidioides brasiliensis is a thermo-dimorphic fungus. At room temperature it grows as a mold that produces conidia, whereas in the vertebrate host it grows as a multiple-budding yeast. The molecular mechanisms involved in the germination from the conidia to the mycelia process remain unknown. OBJECTIVE: The kinetics of conidia to mycelia germination process were studied in the dimorphic fungus P. brasiliensis. Gene expression during this process was evaluated by construction and analysis of an EST library. MATERIALS AND METHODS: For the germination kinetics study, P. brasiliensis conidia were isolated as single cell units. Then, they were cultured at 18 degrees C in BHI (brain-heart infusion) broth for 24, 48, 72 and 96 hr. After each perion, they were examined by light microscopy. From conidia harvested at 96 hr, an EST library was constructed; at this stage the gene expression was presumed to be maximal for the germination process. RESULTS: During the conidia to the mycelia developmental process, the following germination rates were observed: at 24 hr, 11.7+/-1.2%; at 48 hr, 30+/-0.6%; at 72 hr, 43+/-1.3%; and at 96 hr, 66+/-2.4%. At the 96 hour stage, an EST library was constructed. It consisted of 129 sequences grouped in 4 contigs and 7 singlets for a total of 11 possible genes. Eight of the sequences had not been described previously in other EST libraries of this fungus. CONCLUSIONS: New genes were identified that were expressed during the conidia to the mycelia germination process and may represent genes specific to the germination process.

Lysozyme plays a dual role against the dimorphic fungus Paracoccidioides brasiliensis.

In order to determine the role of lysozyme, an antimicrobial peptide belonging to the innate immune system, against the dimorphic fungus Paracoccidioides brasiliensis, co-cultures of the MH-S murine alveolar macrophages cell line with P. brasiliensis conidia were done; assays to evaluate the effect of physiological and inflammatory concentrations of lysozyme directly on the fungus life cycle were also undertaken. We observed that TNF-alpha-activated macrophages significantly inhibited the conidia to yeast transition (p = 0.0043) and exerted an important fungicidal effect (p = 0.0044), killing 27% more fungal propagules in comparison with controls. Nonetheless, after adding a selective inhibitor of lysozyme, the fungicidal effect was reverted. When P. brasiliensis propagules were exposed directly to different concentrations of lysozyme, a dual effect was observed. Physiologic concentrations of the enzyme facilitated the conidia-to-yeast transition process (p < 0.05). On the contrary, inflammatory concentrations impaired the normal temperature-dependent fungal transition (p < 0.0001). When yeast cells were exposed to lysozyme, irrespective of concentration, the multiple-budding ability was badly impaired (p < 0.0001). In addition, ultra-structural changes such as subcellular degradation, fusion of lipid vacuoles, lamellar structures and interruption of the fibrillar layer were observed in lysozyme exposed conidia. These results suggest that lysozyme appears to exert a dual role as part of the anti-P. brasiliensis defense mechanisms.

Lysozyme plays a dual role against the dimorphic fungus Paracoccidioides brasiliensis / A lisozima desempenha um papel duplo contra o fungo dimórfico Paracoccidioides brasiliensis

In order to determine the role of lysozyme, an antimicrobial peptide belonging to the innate immune system, against the dimorphic fungus Paracoccidioides brasiliensis, co-cultures of the MH-S murine alveolar macrophages cell line with P. brasiliensis conidia were done; assays to evaluate the effect of physiological and inflammatory concentrations of lysozyme directly on the fungus life cycle were also undertaken. We observed that TNF-α-activated macrophages significantly inhibited the conidia to yeast transition (p = 0.0043) and exerted an important fungicidal effect (p = 0.0044), killing 27 percent more fungal propagules in comparison with controls. Nonetheless, after adding a selective inhibitor of lysozyme, the fungicidal effect was reverted. When P. brasiliensis propagules were exposed directly to different concentrations of lysozyme, a dual effect was observed. Physiologic concentrations of the enzyme facilitated the conidia-to-yeast transition process (p < 0.05). On the contrary, inflammatory concentrations impaired the normal temperature-dependant fungal transition (p < 0.0001). When yeast cells were exposed to lysozyme, irrespective of concentration, the multiple-budding ability was badly impaired (p < 0.0001). In addition, ultra-structural changes such as subcellular degradation, fusion of lipid vacuoles, lamellar structures and interruption of the fibrilar layer were observed in lysozyme exposed conidia. These results suggest that lysozyme appears to exert a dual role as part of the anti-P. brasiliensis defense mechanisms.

Com a finalidade de determinar o papel da lisozima, um peptídeo antimicrobiano que pertence ao sistema imune inato, contra o fungo dimórfico Paracoccidioides brasiliensis, foram feitas co-culturas de uma linha de macrófagos alveolares murinos (MH-S) com as conídias do fungo na presença ou não do TNF-α e/ou um inibidor da lisozima; também foram feitos ensaios que avaliaram o efeito das concentrações fisiológicas e inflamatórias de lisozima diretamente sobre o ciclo de vida do fungo. Observamos que os macrófagos ativados com a citoquina tiveram um efeito significativo na inibição da transição conídia/levedura (p = 0,0043) e exerceram um efeito fungicida importante (p = 0,0044), matando mais de 27 por cento das propágulas do fungo em comparação com os macrófagos não ativados. No entanto, após ser o inibidor seletivo da lisozima adicionado, o efeito fungicida foi revertido. Quando os propágulos do fungo foram expostos diretamente a diferentes concentrações da lisozima, um duplo efeito foi observado. Assim, as concentrações fisiológicas da enzima facilitaram o processo de transição conídia-levedura (p < 0,05). Contrariamente, as concentrações inflamatórias prejudicaram a transição fúngica (p < 0,0001). Quando as leveduras foram expostas a qualquer concentração de lisozima, sua capacidade de multi-brotação foi gravemente prejudicada (p < 0,0001). Além disso, mudanças ultra-estruturais, como a sub degradação, a fusão dos vacúolos dos lípidos, estruturas lamelares e interrupção da camada fibrilar foram observadas em conídios expostos à lisozima. Estes resultados sugerem que a lisozima poderia exercer um duplo papel no mecanismo antifúngico contra P. brasiliensis.

Involvement of extracellular matrix proteins in the course of experimental paracoccidioidomycosis.

We aimed at determining involvement of extracellular matrix proteins (ECMp) and an ECM-binding adhesin (32-kDa protein) from Paracoccidioides brasiliensis, in the course of experimental paracoccidioidomycosis. BALB/c mice were infected with P. brasiliensis conidia previously incubated with soluble laminin, fibronectin and fibrinogen or a mAb against the fungal adhesin. Inflammatory response, chitin levels and cytokine production at different postinfection periods were determined. Chitin was significantly decreased in lungs of mice infected with ECMp-treated conidia when compared with controls at week 8, especially with laminin and fibrinogen. Contrariwise, when animals were infected with mAb-treated conidia no differences in chitin content were found. The observed inflammatory reaction in lungs was equivalent in all cases. IFN-gamma increased significantly in lungs from mice infected with soluble ECMp - (at day 4 and week 12) or mAb-treated conidia (at week 12) when compared with animals infected with untreated conidia. Significant increased levels of tumour necrosis factor-alpha were observed at 8 weeks in animals infected with ECMp-treated conidia while no differences were observed during the remaining periods. These findings point toward an inhibitory effect of ECMp on P. brasiliensis conidia infectivity and suggest that these proteins may interfere with conidia initial adhesion to host tissues probably modulating the immune response in paracoccidioidomycosis.

Virus del papiloma humano, respuesta inmune y cáncer cervical: una relación compleja / Human papillomavirus, immune response and cervical cancer: a complex relationship

El cáncer de cuello uterino se considera como un grave problema de salud pública con una alta incidencia en los países en desarrollo. La infección, permanencia y replicación del virus de papiloma humano (HPV, por sus siglas en inglés) de alto riesgo a nivel cervical están relacionadas con el desarrollo del cáncer de cuello uterino. En condiciones normales, el sistema inmune es capaz de controlar y eliminar la infección por acción de la inmunidad innata, la activación de una respuesta tipo celular y la creación de anticuerpos dirigidos principalmente a las proteínas de la cápside del virión (L1 y L2). A pesar de toda la maquinaria de protección inmune del hospedero, el virus posee estrategias de evasión, conservando un número reducido de copias en las células basales proliferantes y aprovechando la corta vida natural del queratinocito. En esta revisión se tratarán los diferentes mecanismos inmunológicos del hospedero en la respuesta a la infección por el HPV.

Short report: Inhibition by tumor necrosis factor-alpha-activated macrophages of the transition of Paracoccidioides brasiliensis conidia to yeast cells through a mechanism independent of nitric oxide.

It is known that peritoneal murine macrophages activated with interferon-gamma exert a fungicidal effect against Paracoccidioides brasiliensis conidia by a nitric oxide (NO)-mediated mechanism. This NO-mediated effect can also be induced by other cytokines such as tumor necrosis factor-alpha (TNF-alpha). The aim of this study was to determine if TNF-alpha-activated peritoneal murine macrophages infected with P. brasiliensis were able to show fungistatic/fungicidal effects mediated by NO. The results indicated that although macrophage activation with TNF-alpha did not result in NO production, these cells played an important role in inhibiting the conidia from becoming yeast cells. In vivo, the NO-independent inhibitory effect would prove of importance for the establishment of P. brasiliensis in host tissues.

Fibrotic sequelae in pulmonary paracoccidioidomycosis: histopathological aspects in BALB/c mice infected with viable and non-viable Paracoccidioides brasiliensis propagules

Patients with paracoccidioidomycosis often present pulmonary fibrosis and exhibit important respiratory limitations. Based on an already established animal model, the contribution of viable and non-viable P. brasiliensis propagules to the development of fibrosis was investigated. BALB/c male mice, 4-6 weeks old were inoculated intranasally either with 4x10(6 )viable conidia (Group I), or 6.5x10(6) fragmented yeast cells (Group II). Control animals received PBS. Six mice per period were sacrificed at 24, 48, 72h (initial) and 1, 2, 4, 8, 12 and 16 weeks post-challenge (late). Paraffin embedded lungs were sectioned and stained with H&E, trichromic (Masson), reticulin and Grocott's. During the initial period PMNs influx was important in both groups and acute inflammation involving 34 per cent to 45 per cent of the lungs was noticed. Later on, mononuclear cells predominated. In group I, the inflammation progressed and granulomas were formed and by the 12th week they fussed and became loose. Thick collagen I fibers were observed in 66.6 per cent and 83.3 per cent of the animals at 8 and 12 weeks, respectively. Collagen III, thick fibers became apparent in some animals at 4weeks and by 12 weeks, 83 per cent of them exhibited alterations in the organization and thickness of these elements. In group II mice, this pattern was different with stepwise decrease in the number of inflammatory foci and lack of granulomas. Although initially most animals in this group had minor alterations in thin collagen I fibers, they disappeared by the 4th week. Results indicate that tissue response to fragmented yeast cells was transitory while viable conidia evoked a progressive inflammatory reaction leading to granuloma formation and to excess production and/or disarrangement of collagens I and III; the latter led to fibrosis.