2) of 6–10 weeks of age were used as the source of BM for in vitro cultures.

GMKO mice [43], GM-CSF receptor βcKO mice [44] on C56BL/6 background, and GM-CSF transgenic mice on SJL × C57BL/6 mixed background [45] were generated, and maintained in the RG7204 purchase animal facility of The Walter & Eliza Hall Institute (WEHI) Animal Facility. All mouse procedures were approved by the WEHI animal ethics committee. Cultures were setup as previously described [4, 12, 46]. Briefly, BM cells were extracted, and erythrocytes were removed by exposure to 0.168 M NH4Cl. Cells were cultured at a density of 1.5 × 106–3.0 × 106 cells per mL in RPMI 1640 medium with 10% (v/v) fetal bovine serum containing either recombinant mouse Flt3L (made in-house), recombinant GM-CSF (R&D systems), or both at 37°C in 10% CO2. DCs induced by Flt3L, GM-CSF, or both are termed FL-DCs, GM-DCs,

or GMFL-DCs, respectively. OT-I T cells (H-2Kb-restricted anti-OVA257–264) and OT-II T cells (I-Ab-restricted anti-OVA323–339) were purified from pooled lymph nodes (inguinal, axillary, brachial, cervical, and mesenteric) by Ab depletion of non-T cells (non-CD8 T cells for purification of OT-I T cells and non-CD4 T cells for purification of OT-II T cells). T cells were then dye labeled by incubating them for 10 min at 37°C in FCS free PBS containing 0.1% BSA and 2.5 mM CFSE. The T-cell preparations were routinely >80% pure, as determined by flow cytometry. The capacity of the FL-DCs, GM-DCs, or GMFL-DCs to generate Histone demethylase an antigen-specific T-cell AZD1152-HQPA supplier stimulatory response was evaluated using isolated OT-1 and OT-II T cells. FL-DCs, GM-DCs, or GMFL-DCs were plated at 104 cells per well in U-bottom 96-well plates and pulsed for 45 min at 37°C at the indicated concentration of OVA. Cells were washed and resuspended with 5 × 104 CFSE-labeled OT-I/OT-II cells. Proliferation of the T cells was determined after 60 h of culture as described

above. To quantify proliferation, the T cells were stained with anti-CD4 or -CD8 (for OT-II and OT-I, respectively) and anti-TCRVα2 antibodies, and resuspended in 100 μL of balanced-salt solution and 2% FCS-containing 2.5 × 104 blank calibration particles (BD Biosciences Pharmingen). Samples were analyzed by flow cytometry on a FACScallibur (Beckton Dickinson) and the total number of live dividing lymphocytes (propidium iodide-negative, CFSElo) was calculated from the number of dividing cells per 5 × 103 beads. Each determination was done in duplicate. Samples were then analyzed using Flowjo Software (Tree Star Inc). As previously described [22], BM cells were suspended in nycodenz medium (1.086 g/cm3) and cells of lighter density were isolated by centrifugation. The cells of lighter density were then coated with biotinylated monoclonal antibodies to the following lineage markers: CD3 (KT3–1.1), CD19 (ID3), CD45R (B220, RA36B2), CD11b (M1/70), CD11c (N418), Ly6G (IA8), Ly6C.2 (5075–3.6), NK1.1 (PK136), CD127 (IL-7R; A7R34–2.2), and Ter119.

Methods:  This is a retrospective study on patients with proliferative lupus nephritis who had received PSI treatment. Results:  Seven patients were included. Two patients had concomitant membranous lupus nephropathy. The indications for PSI included mycophenolate mofetil intolerance (n = 4), history of malignancy (n = 2) and leucopoenia (n = 1). Five patients were given PSI when disease was active. Two had treatment discontinued because of acute cholecystitis and leucopoenia, respectively. In the other three patients combined steroid and PSI treatment as induction therapy led to improvements in serology, Small molecule library renal function and

proteinuria. In two patients treated with PSI and low-dose steroid as maintenance immunosuppression, both maintained stable lupus serology, renal function and proteinuria over 18 months. Side-effects included dyslipidemia and oral ulcers. Conclusion:  Proliferation signal inhibitors warrants Sirolimus research buy further investigation as an alternative immunosuppressive treatment in lupus nephritis. “
“Aim:  The aims of the study were to translate the Kidney Disease Quality of Life – Short

Form version 1.3 (KDQOL-SF ver. 1.3) questionnaire into Iranian (Farsi), and to then assess it in terms of validity and reliability on Iranian patients. Methods:  The questionnaire was first translated into Farsi by two independent translators, and then subsequently translated back into English. After translation disparities had been reconciled, the final Iranian questionnaire was tested. An initial test–retest reliability evaluation was performed over a 10 day period on a sample of 20 patients recruited from a larger group (212 patients with end-stage renal disease on haemodialysis). Afterwards, reliability was estimated by internal consistency, and validity was assessed

using PTK6 known group comparisons and constructs for the patient group as a whole. Finally, the factor structure of the questionnaire was extracted by performing exploratory factor analysis. Results:  All of the scales in the questionnaire showed good test–retest reliability (i.e. intraclass correlations between test and retest scores were >0.7). All of the scales met the minimal criteria (0.7) for internal consistency and Cronbach’s-α ranged 0.71–0.93. Furthermore, results from a discriminate validity evaluation showed that the questionnaire could be used to discriminate between subgroups of the patients. Finally, a principal component analysis of the disease-specific scales indicated that this part of the questionnaire could be summarized into an 11 factor structure that jointly accounted for 79.81% of the variance. Conclusion:  The Iranian version of the KDQOL-SF questionnaire is both highly reliable and valid for use with Iranian patients on haemodialysis. “
“Aim:  Alport syndrome (AS) is a progressive renal disease characterized by hematuria and progressive renal failure.

Compared to the full-length CCL3, CCL3(5–70) shows enhanced binding affinity to CCR1 and CCR5 (Table 1) [74]. CCL4 and CCL4L1 mature proteins differ Epacadostat mouse only in one amino acid: a conservative S to G change at amino acid 47

of the mature protein (Fig. 2) [48,78]. Few studies have been compared the functions of CCL4 and CCL4L1. Modi et al. reported a functional redundancy of the human CCL4 and CCL4L1 chemokines: their competitive binding assays, cell motility and anti-HIV-1 replication experiments revealed similar activities of the CCL4 and CCL4L1 proteins [67]. However, structural analysis of the CCL4 and CCL4L1 proteins revealed the importance of amino acid 47 of the mature protein: this amino acid (S) in CCL4 protein forms a hydrogen bond with amino acid Thr44, thus conferring structural stability to the loop defined by the β-turn between the second and third strands of the β-sheet

[79]. However, the glycine (G) at that position in the CCL4L1 protein cannot form this hydrogen bond. This loop is believed to be essential for the binding of CCL4 to the glycosaminoglycans (GAGs) [80]. It has been suggested that the immobilization of chemokines on GAGs forms stable, solid-phase chemokine Z-VAD-FMK datasheet foci and gradients crucial for directing leucocyte trafficking in vivo. Their higher effective local concentration increases their binding to cell surface receptors and influences chemokine T1/2in vivo[81–84]. Hence, the destabilization of this loop could reduce the stability of CCL4L1 binding to GAGs and therefore modify their functional features in vivo. It is important to note that the available data about functional studies of CCL4 and CCL4L1 were obtained by in vitro experiments, Thiamine-diphosphate kinase where the binding of these chemokines to GAGs is neglected. The apparent functional redundancy of CCL4 and CCL4L1 in vitro warrants further in vivo studies examining their GAG binding capabilities. Additionally, regulation of CCL4 and CCL4L1 expression appears different. Lu et al. reported an independent expression

of the CCL4 and CCL4L1 genes in monocytes and B lymphocytes [85]. This observation suggests that differential expression of these proteins in different cells provides an advantage to the host and that these proteins might have different functions in vivo. Both CCL4 and CCL4L1 genes produce alternatively spliced mRNAs that lack the second exon, which give rise to the CCL4Δ2 and CCL4L1Δ2 variants (Figs 1c and 2) [48,78]. The predicted CCL4Δ2 and CCL4L1Δ2 proteins of only 29 aa would only maintain the first two amino acids from the CCL4 and CCL4L1 proteins, lacking three of the four cysteine residues critical for intramolecular disulphide bonding. Therefore, CCL4Δ2 and CCL4L1Δ2 may not be structurally considered chemokines. Despite the difficulty in predicting protein folding, these variants do not seem to be able to bind to CCR5 and thus may have no CCL4/CCL4L1 activity [48].

“
“Dengue is a mosquito-borne viral disease selleck screening library of humans,

and animal models that recapitulate human immune responses or dengue pathogenesis are needed to understand the pathogenesis of the disease. We recently described an animal model for dengue virus (DENV) infection using humanized NOD-scid IL2rγnull mice (NSG) engrafted with cord blood haematopoietic stem cells. We sought to further improve this model by co-transplantation of human fetal thymus and liver tissues into NSG (BLT-NSG) mice. Enhanced DENV-specific antibody titres were found in the sera of BLT-NSG mice compared with human cord blood haematopoietic stem cell-engrafted NSG mice. Furthermore, B cells generated during the acute phase and in memory from splenocytes of immunized BLT-NSG mice secreted DENV-specific IgM antibodies with neutralizing activity. Human T cells in engrafted BLT-NSG mice secreted

interferon-γ in response to overlapping DENV peptide pools and HLA-A2 restricted peptides. The BLT-NSG mice will allow assessment of human immune responses to DENV vaccines and the effects of previous immunity on subsequent DENV infections. Dengue virus (DENV) is a mosquito-borne member of click here the Flavivirus genus and includes four serotypes (DENV-1, DENV-2, DENV-3 and DENV-4). The virus infects approximately 50 million individuals each year, leading to over 500 000 hospitalizations. Infection results in a range of symptoms from mild fever to acute febrile illness (dengue fever). In a small percentage of cases, however, individuals develop a severe capillary leakage syndrome, dengue haemorrhagic fever and dengue shock syndrome, which can be life-threatening.1,2 Studies in humans suggest that dengue haemorrhagic fever and dengue shock syndrome are more likely to occur in individuals experiencing diglyceride their second DENV infections and in infants born to DENV-immune mothers. Experimental manipulation of in vivo immune responses to DENV is a critical step in exploration of the role of previous immunity in subsequent DENV infection

and testing of candidate vaccines and therapeutics. Progress in understanding the pathogenesis of dengue haemorrhagic fever has come largely from controlled well-designed clinical studies of patients with mild and severe forms of dengue disease in endemic areas.3–10 Most patients who present to hospital live in endemic areas and are experiencing a secondary infection; however, the serotype of the previous DENV infection is difficult to determine. Furthermore, controlled virus challenge studies are not feasible in humans, and it is difficult to assess the contribution of antibodies or T cells to DENV pathogenesis. Immunodeficient mice bearing components of a human immune system (humanized mice) present a novel approach for studying human immune responses to DENV.

These include the ability of TcdA to induce the release of the pro-inflammatory mediators IL-1β,[62] TNF-α,[63] IFN-γ,[64] CXCL1,[48] CXCL2[49] and CCL3,[65] as well as the fact that both IFN-γ−/−[64] and CCR1−/−[65] mice have a milder form of enteritis in response to TcdA injection. Despite the useful insights provided by the ileal loop model into the actions of C. difficile toxins, it should be noted that the model has some important shortcomings. First, it is a surgery-based model, which entails the injection

of C. difficile toxin preparations into the animal and not infection with the bacterium itself; second, it targets the wrong organ for disease, i.e. ileum instead of the colon; and third, it does not reflect any interaction of C. difficile with the host’s microbiota. The current Protease Inhibitor Library supplier work is the first to assess the induction of the CHIR-99021 in vitro UPR during acute C. difficile infection. A number of recent studies have implicated the UPR in the response to different forms of intestinal inflammation. These include the protective role(s) of XBP1,[17] ATF6[18] and eIF2α phosphorylation[19] against dextran sodium

sulphate-induced colitis. Despite the phosphorylation of eIF2α and the slight up-regulation of the phospho-eIF2α targets Wars and Gadd34 in the caeca and colons of C. difficile-infected mice (which serve as an early indication of phospho-eIF2α exerting its downstream effect), the lack of Xbp1 splicing and the absence of ER chaperone up-regulation in these tissues cast serious doubt on the activation of the UPR in this model of infection. Although numerous laboratories have shown that the UPR output can be modulated in a context-specific manner,[66, 67] a more likely explanation for the current set of findings is the phosphorylation of eIF2α by a kinase other than PERK. Of the four kinases that can phosphorylate Selleck Erlotinib eIF2α, Protein Kinase RNA-activated (PKR) is the most plausible candidate. The phosphorylation of AKT and STAT3, as well as eIF2α,

in the C. difficile-infected mice gives further credence to this hypothesis because, in addition to phosphorylating eIF2α, PKR is an upstream inducer of both AKT and STAT3 phosphorylation.[68] AKT plays an important role in promoting intestinal epithelial homeostasis and wound repair during intestinal inflammation.[69] Furthermore, the protective effect of lysophosphatidic acid against C. difficile toxin-induced cell death in vitro is in part due to its induction of AKT phosphorylation.[70] Therefore, the phosphorylation of AKT in the C. difficile-infected mice may be a pro-survival signal that aims to counteract and contain the inflicted epithelial damage. The phosphorylation of STAT3 in the C. difficile-infected mice should be viewed from a broader perspective. First, the use of STAT3IEC-KO mice has shown that activation of intestinal epithelial STAT3 regulates immune homeostasis in the gut by promoting IL-22-dependent mucosal wound healing.

On average, 25% of macrophage-associated conidia are phagocytosed after 1 h, 16% of these cell-associated conidia are killed after

4 h and conidial germination was inhibited in more than 95% of the conidia of A. fumigatus.[53] Comparable studies on zygomycetes would gain insights into the clearance of the fungal burden and forecast the potential of zygomycetes as causative agents of emerging fungal diseases. Moreover, transcriptomic analysis will help to understand the virulence and survival mechanism of the fungi when it confronts macrophages, e.g. the role of chromatin Opaganib cell line remodelling as a central regulator of survival strategies which facilitates a reprogramming of cellular energy metabolism in macrophage-internalised fungal cells and provide protection against DNA damage as shown for Candida glabrata.[54] Many studies have established that virulence factors can be differentially expressed as a function of experimental conditions,

but clinical isolates of Cryptococcus neoformans behave differently under the same experimental conditions, a diversity that could participate in the variable outcome of infection in humans.[55] It has been hypothesised that the survival strategies for soil-borne, potentially human pathogenic fungi (e.g. C. neoformans) after ingestion by macrophages and amoebae were similar.[56] Consequently, selleck chemicals llc it will be worthwhile to compare the mechanisms of phagocytosis with amoebae which served as interacting partners in the environment resulting in a training of the fungal pathogen to successfully cope with the amoeboid immune cells of the human immune system towards adaption to the human host during evolution. Just recently, the whole genome of Acanthamoeba castellanii (Ac) was determined, the first representative from a solitary free-living amoebozoan.[57] Ac utilises a diverse repertoire of predicted

pattern recognition receptors, many with predicted orthologous functions in the innate immune systems of higher organisms.[57] The exploration of the Liothyronine Sodium connection between both, receptors of amoebae and immune cells will shed light into the evolutionary origin of the interplay between fungal pathogen and innate immune cells. This work was financially supported by the Deutsche Forschungsgemeinschaft (DFG) Jena School for Microbial Communication (JMRC project #66) and within CRC/TR 124 FungiNet: project Z1 to KV. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We express our gratitude to Paul M. Kirk (Royal Botanic Gardens Kew, UK) for providing the numbers of species for the subphyla and orders of the zygomycetes. The authors declare that no conflict of interest exists. “
“This multicentre observational study evaluated the feasibility, efficacy and toxicity of antifungal combination therapy (combo) as treatment of proven or probable invasive fungal diseases (IFDs) in patients with haematological malignancies.

We coincubated the APC and the ADC with poly

(I:C) and observed, as shown in Fig. 3D, a small increase in the cross-presentation of both epitopes (396 and 276) that correlated with increasing concentrations of poly (I:C). Thus, it is possible that the small reduction in cross-presentation of NP396 after the RNAase FK506 datasheet treatment (Fig. 3C, iv) could be due to reduced APC activation as a result of poorer TLR engagement. We could not detect any significant increases in cross-presentation of NP396 or GP276, when poly (I:C) was added to untreated ADC, probably because the system reached its maximum capacity (Fig. 3E). To investigate whether cross-presentation of LCMV antigens was processed differently by DC and Mø, we tested different inhibitors that target different components of the intracellular processing pathways. Employing the proteasome inhibitor lactacystin and the ER protein transport inhibitor brefeldin A (BFA), we were able to interfere with cross-presentation in a dose-independent manner (Fig. 4A), indicating the involvement of the cytosolic pathway. To assess the relative contribution of the proteasome-independent vacuolar processing pathway, we applied leupeptin

to inhibit serine and cysteine proteases (cathepsins B, L, and S), and pepstatin A click here to inhibit aspartic proteases (cathepsin D). Figure 4A shows limited interference with leupeptin that was more pronounced with DC2.4 than with BMA, whereas pepstatin A was ineffective with either cell types. These results would suggest that low/modest antigen processing (cathepsin D independent) took place in the endosomal/phagosomal compartments. To interfere with phagosomal acidification, we pretreated the APC with different doses of chloroquine and observed 50% inhibition at the highest dose Epothilone B (EPO906, Patupilone) with DC2.4, whereas with BMA a complete inhibition was evident (Fig. 4A).

Another regulator of phagosomal acidification, NADPH oxidase (NOX2), which mediates the transfer of electrons across endocytic and plasma membranes, was required by the APC. This was observed because cross-presentation of NP396 was reduced following treatment with diphenyleneiodonium chloride (NOX2 inhibitor) that increases phagosomal acidification. As controls, we employed peptide-labeled APC with high- (Fig. 4B) or low-peptide concentrations (Fig. 4C), with all the inhibitors tested and did not find any significant effects on antigen presentation when compared with untreated controls. To examine if the cross-presentation results we obtained translate in vivo, we investigated the cross-priming of LCMV-infected ADC. Generally, after LCMV infection of B6 mice, one can detect two immunodominant epitopes, GP33, and NP396, and two subdominant ones, GP276 and NP205. We confirmed these data with tetramer staining after introducing 200 pfu of LCMV i.v. (Fig. 5A) and testing 8 days p.i.

Myeloid DCs are central in the

orchestration of innate and acquired immune responses and in the maintenance of self-tolerance [1]. DC development involves three functionally and phenotypically distinct stages for which the terms “precursors,” “immature,” and “mature” are commonly used [2-5]. DCs precursors originate in the bone marrow, circulate via the bloodstream to reach target tissues, and take up residence at sites of potential pathogen entry, where they differentiate into immature DCs (iDCs) specialized for antigen capture [2, 4, 6]. Peripheral blood monocytes recruited from the circulation to inflammatory sites can also serve as iDC precursors [7, 8]. iDC redistribution in the tissues is determined by the local microenvironment through the production of chemotactic mediators, activation selleck products of inflammatory chemokine receptors, and regulation of adhesion molecules [7, 8]. Tissue

injury, inflammation, and transformation cause dramatic changes of the microenvironment, modulating iDC phenotype and function and promoting maturation into (m)DCs [7-14]. A common denominator of injured and inflamed tissues is the presence of low partial oxygen pressure (pO2), which creates a unique microenvironment affecting cell phenotype, gene expression profile, and functional behavior Kinase Inhibitor Library [10, 11, 15, 16]. Response to hypoxia is primarily under the molecular control of a family of hypoxia-inducible transcription factors, composed of the constitutive HIF-1β subunit and an O2-sensitive α subunit (HIF-1α/-2α), which binds and transactivates the hypoxia responsive element (HRE) present in the promoter of many hypoxia-inducible genes [11, 15-17]. DC development

from monocytic precursors recruited at pathological sites occurs under the setting of reduced pO2. Recent studies have reported that HIF-1α accumulates in hypoxic Sodium butyrate DCs and that O2 levels similar to those present in diseased tissues can impact on DC differentiation, maturation, and activation [10, 11, 18-24]. Hypoxia promotes the onset of a migratory phenotype in iDCs through the upregulation of inflammatory chemokine receptors and motility-related genes with consequent increased responsiveness to specific chemoattractants [18-20] and a proinflammatory state in mDCs by increasing the expression of genes coding for proinflammatory and Th1-priming chemokines/cytokines [24]. DCs integrate stimulatory and inhibitory signals present in the microenvironment through a defined repertoire of cell surface receptors, and deregulated expression of these molecules may result in aberrant responses characterized by amplification of inflammation and loss of tolerance [5, 7-9, 25-27].

Candida albicans is a common pathogenic yeast that normally exists in the human microflora, but that can also cause infections. It is an opportunistic pathogen that usually lives as a commensal in the healthy human host. Alterations in the balance between the commensal and the host, such as those that occur in the immunocompromised patients may trigger infection of the mucosal epithelia, followed by dissemination via the bloodstream and buy Obeticholic Acid colonization of internal organs [6, 7]. Deltamethrin,

a synthetic pyrethroid type II, is highly effective against a broad spectrum of insects. The main sources of general population exposure to this pesticide are contaminated food and water, and it has been reported that deltamethrin is readily absorbed by the oral route [8]. Several studies have shown Caspase inhibitor clinical trial that pyrethroid insecticide exposure caused alterations in biochemical and haematological profile and reproduction in the exposed animals [9]. While, studies describing the oxidative stress mechanisms in pyrethroid-induced toxicity are limited,

deltamethrin was observed to suppress the immune functions. It is also reported to alter blood parameters and antioxidant defense of mice in previous studies [10, 11]. An investigation therefore was undertaken to assess impact of deltamethrin-induced alteration of host resistance to infection (C. albicans challenge). Animals.  The study was conducted in Swiss albino male mice (30–32 g). Female guinea pigs (250 g) were used for the preparation of complement most for plaque forming cell (PFC) assay. The Central Animal

House Facility of the University provided the animals. The study was approved by the Institutional Animal Ethics Committee. The animals were given mild anesthesia using di-ethylether. The animals were bred and maintained under standard conditions: temperature 25 ± 2 °C and photoperiod of 12 h. Commercial pellet diet and water were given ad libitum. Animals were divided in five different groups.  Group I:  Control animals, treated with corn oil orally, and normal saline intraperitoneally (i.p.) for 10 days. After taking the blood from orbital plexus of mice for haemagglutination titre (HT) assay, animals were sacrificed by cervical dislocation under mild anesthesia and their liver and spleen were aseptically removed. The spleens of few animals (n = 5–6) were used for PFC assay, whereas spleen from rest of the animals (n = 5–6) were homogenized with a tissue homogenizer (Potter-Elvehjem homogenizer) using 5 ml of saline and used for infection investigation. Colony forming unit (CFU) was counted in liver and spleen by the method of Srivastava et al., [12]. Chemicals.  Antibiotic antimycotic solution (100X), fetal bovine serum (FBS), yeast extract, peptone, dextrose, agar, Hank’s balanced salt solution (HBSS), Histopaque-1077, phosphate buffer saline (PBS) and RPMI-1640 medium were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). Deltamethrin.

17 In general, duplex PCR amplification of BT2 yielded clear MK-2206 price Scedosporium-specific bands. Although the closely related species P. desertorum was also amplified, it gave a signal exclusively with the group-specific probe PS_P on the blot. This assay was found positive in five of six

clinically relevant Scedosporium species. Non-specific signals were found for S. dehoogii strains when probes of P. apiosperma, P. boydii, and P. minutispora were applied. No other cross-reactions with non-target Scedosporium species or other clinically relevant fungi were observed. The detection limit of the PCR-RLB method was found to be 50 cells μl−1 or 0.2 pg genomic DNA. Fifty-nine sputum samples, comprising five culture-positive samples and 54 culture-negative samples, were analysed by PCR-RLB hybridisation assay (Table 1). Twenty-two of the samples proved to be negative by PCR-RLB. The PCR-RLB hybridisation assay permitted the detection of members of the P. apiosperma/P. boydii complex in 32 of 52 patients (61.5%). Pseudallescheria

apiosperma was detected in 20 samples, while P. boydii and S. aurantiacum were detected in 17 and eight samples, respectively. Only two samples were found positive for S. prolificans and P. minutispora, respectively. RG7204 concentration Eight samples contained two distinct species or three species simultaneously. Figure 1 shows a typical result of PCR-RLB for some sputum samples and for a number of Scedosporium reference strains. Four of the five Scedosporium culture-positive samples proved also to be positive with PCR-RLB hybridisation assay. All isolates of the P. boydii/P. apiosperma complex were identified morphologically,

except one strain Forskolin in vitro recovered from sample 10 which was identified as S. aurantiacum and confirmed by sequencing the ITS1-ITS2 (99% identity with NCBI sequence AJ889599 from S. aurantiacum strain IHEM 144-458) and BT2 region (100% identity with the NCBI sequence AJ888441 from S. aurantiacum strain IHEM 15-458); this last sample gave a positive signal by PCR-RLB hybridisation exclusively with the S. aurantiacum-specific probe. Considering all analysed samples, PCR-RLB yielded more positive results than culturing (47 vs. 5, respectively). Among the 54 Pseudallescheria/Scedosporium culture-negative samples analysed, 21 were also found negative by PCR-RLB. Twenty-six DNA extracts gave a positive signal with one species-specific probe, while six samples gave a positive reaction with two distinct species-specific probes and one sample with three probes. Antifungal treatment (mostly with the azoles itraconazole or voriconazole) during the months preceding the sampling took place in seven of the patients. However, for the remaining Pseudallescheria/Scedosporium culture-negative samples producing discrepant results (26 samples), the patients did not receive any antifungal treatment preceding the sampling date and Scedosporium species were never detected by culture in previous or later sputum samples.