CD40–CD40L interaction

is important for B-cell development, antibody production by B cells, germinal centre formation, interleukin-12 (IL-12) production, CD8+ T cell effector function and optimal T cell-dependent antibody responses [6]. We hypothesized that CD40L might undergo epigenetic deregulation in pSS via putative X-inactivation escape. We enrolled 26 women (age 56 ± 15.4 years) fulfilling the American European Consensus Group for pSS [7] and 22 healthy control women (age 41 ± 14.6 years) who did not have KU-57788 molecular weight autoimmune or infectious diseases. Sixteen of 26 pSS women were gone through menopause versus 3 of 22 female controls. Among the 26 patients with pSS, 76% tested positive for anti-SSA antibodies and/or anti-SSB antibodies; 9 patients showed extra-glandular involvement: active synovitis or inflammatory arthralgia (n = 5), renal involvement (n = 1), autoimmune cytopenia

and myositis (n = 1), purpura (n = 1), or lung involvement (n = 1). One patient had mucosa-associated lymphoid-tissue (MALT) gastric lymphoma. Ten patients received hydroxychloroquine, 4 methotrexate and none more potent immunosuppressive drugs. All patients underwent the same clinical, biological and immunological screening. To classify patients with active and non-active disease, we used 2 arbitrary Cediranib (AZD2171) cut-offs (5 or 7) of the EULAR Sjogren’s Syndrome Disease see more Activity Index (ESSDAI) [8]. The study was approved by the local ethics committee, and informed consent was obtained from all subjects. Peripheral blood mononuclear cells (PBMCs) from blood samples for all subjects

were isolated by density-gradient centrifugation. CD4+ T cells were isolated by positive selection (Miltenyi Biotec, Paris). The purity of CD4+ T cells was >96% in all experiments. After CD4+ T cell isolation, cells were analysed ex vivo at 4 h after polyclonal activation with 5 ng/ml phorbolmyristate acetate (PMA) (Sigma-Aldrich, Saint Quentin Fallavier, France) and 500 ng/ml ionomycin (Sigma-Aldrich) and after 4 days of culture and activation with phytohemagglutinin A (PHA, 5 μg/l) (Sigma-Aldrich) and interleukin2 (IL-2, 20 UI/ml) (Roche Diagnostics, Meylan, France), respectively. After 4 days of culture, cells were again stimulated with PMA and ionomycin to induce CD40L expression. A freshly prepared demethylating agent [5-azacytidineC (5-AzaC), 1 μm; Sigma-Aldrich] was added at day 1 of culture and then every day until day 3. The cell medium consisted of RPMI 1640 glutamax Gibco supplemented with 10% SVF, penicillin (100 U/ml), streptomycin (100 μg/ml), buffer HEPES 10 mm, pyruvate of sodium 1 mm and amino acids (Invitrogen, Saint-Aubin, France).

5 suggest that mCRAMP is negatively regulating the antibody response to a TD antigen, TNP-OVA/Alum. Since our in vitro data suggest a differential regulation of B and T cells, we sought to determine the mechanism by which more TNP-specific IgG1 is made by Camp−/− mice compared with WT mice. ELISpot analysis of the spleens at 4 days after the

second immunization with TNP-OVA/Alum shows that Camp−/− mice have more TNP-specific IgG1+ ASCs than WT (Fig. 6A). Since our in vitro data in Fig. 4 suggested that mCRAMP had no effect on isotype switching to IgG1, one potential explanation could be that Opaganib concentration the production of IL-4 was increased, similar to our findings in Fig. 2 with purified T cells in vitro. RT-PCR was performed to determine the level of total IL-4

mRNA in total spleen. Figure 6B shows that Camp−/− spleens contain more IL-4 mRNA than WT spleens. In addition, intracellular staining for IL-4 showed that the numbers of CD4+IL-4+ T cells were significantly increased in the Camp−/− mice (Fig. 6C). Overall, these results suggest that mCRAMP negatively regulates TD antibody responses by regulation of T-cell IL-4 production. Analysis of AMPs has shown that their cellular expression is widespread and their functions are diverse. Camp−/− mouse are more susceptible to, and fail to clear, numerous infections [1], supporting a role for AMPs in host defense and immune regulation. Our data showing that mouse B and T cells are capable of expressing and responding to mCRAMP further add to this complexity.

Importantly, while the use of Camp−/− mice has aided in the study of AMP biology, selleck products it is not definitive in differentiating the direct antimicrobial activity from the immune regulation. In addition, our data show that mCRAMP has the ability to regulate B and T cells in vivo, although there is still no clarity as to the exact source of mCRAMP and the mechanism by which it regulates B- and T-cell function. Using the Camp−/− mouse 24, we investigated the role of mCRAMP in regulating adaptive immune responses. Our data show that Camp−/− mice immunized with TNP-OVA/Alum produced more TNP-specific IgG1 antibody Aldehyde dehydrogenase when compared with WT mice. In contrast, Kurosaka et al. showed that mCRAMP acted as an immune adjuvant and enhanced TD antibody production in WT mice 3. The most obvious difference in the experiment design, which may contribute to the opposing findings, is that we studied effects of endogenously produced mCRAMP by comparing antibody responses in WT versus Camp−/− mice, while Kurosaka et al. 3 added additional exogenous mCRAMP to WT mice. The administration of exogenous mCRAMP to a WT mouse that is also making mCRAMP in response to the immunization may or may not accurately model the role of mCRAMP during an antibody response. In support of this possibility, previous studies have demonstrated that exogenous and endogenous mCRAMP function differently in macrophage activation 15.

Several clinical trials have demonstrated that allergen-SIT induces functional Treg with the capacity to modify the course of allergic diseases 4, 8, 74. Recently, it has been shown that the increased number of FOXP3+CD25+ Treg in nasal mucosa after grass pollen immunotherapy correlated with clinical efficacy and suppression of seasonal allergic inflammation, thus supporting the role of Treg in the induction of allergen-specific tolerance in human subjects 4. Several mechanisms involving Treg in tolerance induction after allergen-specific

SIT has been documented. Such mechanisms include increased capacity of Treg to suppress Th1 and Th2 cells 75, 76, induction of IL-10 and TGF-β 75, 77, decreased allergen-stimulated T-cell proliferation 77 or suppression of effector cells Fulvestrant in vitro 78. Although, in some cases, immunological changes have not been detected 79, similar findings have been also

reported in sublingual-specific immunotherapy, in which a sublingual application of the allergen extracts is employed. Classical events associated with the downregulation of allergic responses such as induction of IL-10 in T cells, suppression of Th2 cells, decreased eosinophil infiltration to nasal mucosa or increased serum allergen-specific IgG4 levels have also been reported in sublingual-specific immunotherapy 9. Another alternative that has been successfully employed for the induction Aprepitant of peripheral tolerance to allergens is peptide BGB324 immunotherapy. Mixtures of short peptides derived from the major cat allergen Fel d 1 and the bee venom allergen phospholipase A2 induced downregulation of systemic Th1 and Th2 cell responses to allergens 80 together with concomitant induction of IL-10 production 81, 82. Our understanding of the mechanisms underlying allergic diseases as well as those operating in healthy immune responses to allergens and allergen-SIT has significantly increased over the past decade. Peripheral T-cell tolerance to allergens represents an essential mechanism not only in healthy immune response to allergens

but also in successful allergen-SIT. Both CD4+CD25+FOXP3+ Treg and IL-10 and/or TGF-β–secreting TR1 cells play an essential role in the establishment of a healthy well-balanced immune response to allergens. Recent advances in the field of Treg biology have partially delineated the mechanisms involved in the in vivo generation of functional Treg. The identification of new molecules implicated in these processes is emerging. These aspects, together with a better understanding of the role that specific DC subsets play in the generation of functional Treg, will contribute to the design of more efficient and safer immunotherapy against allergic diseases in the near future. The M. Akdis and C.A.

There are limited data from which to address this issue. The often quoted follow-up studies of living donors are limited by several significant methodological flaws, studies are retrospective, predominantly Caucasian and the rate of loss to follow up is high. Baseline BMI has not been reported in many of the older studies and obese patients are almost certainly under-represented in the long term follow-up statistics used

to educate prospective donors regarding the risks of nephrectomy. Studies Selleck Lenvatinib reporting baseline characteristics of obese donors suggest that they are at higher risk of future kidney disease.59,63,74,75 A study from a centre59 with a high use of obese donors, in which 31% of donors had a BMI > 30 kg/m2 gives a detailed analysis of the baseline characteristics of obese donors. Obese donors had a significantly higher pre-nephrectomy BP (137/79 vs 126/73 mmHg), increased history

of donor hypertension (14% vs 4%), more adverse lipid profiles, higher fasting glucose levels (although within the normal range) and had a family history of diabetes (47% vs 33%), when compared with donors with a BMI  < 25 kg/m2. Data are available at 1 year for approximately 60% of donors in this study, and demonstrates that BP and fasting glucose remained higher, albeit in the acceptable range, and did not incrementally increase post nephrectomy. The post-nephrectomy GFR and rates of microalbuminuria were not different in the obese, within this short timeframe. Donors who are overweight or obese are more likely to gain weight post donation than those of normal weight.76 There is this website a probable relationship between BMI and subsequent hypertension.74,76–78 Obese patients are more likely

to have higher BP at the time of donation and it is unknown if nephrectomy alters G protein-coupled receptor kinase the age of onset or severity of hypertension. A German study of 152 donors, with 93% followed for a mean of 11 years and with pre-nephrectomy BMI of 26 ± 4 kg/m2, demonstrated that baseline BMI was correlated with mean arterial pressure but not change in BP post donation.78 There is no evidence of association between the baseline BMI and development of proteinuria or decline in GFR post donation in predominantly Caucasian populations.78,79 However, the number of donors who were obese at baseline is too small to be able to determine this with any certainty. The study from the Mayo Clinic79 had long-term follow up on 73% of donors with a median follow up of 12 years. Only data on weight are available and is not differentiated for gender. Median weight at donation was 70 kg and weight gain at follow up was 7.5 kg. Baseline weight, change in weight and relative weight (measured/ideal weight) was not a significant predictor of current serum creatinine or change in creatinine. The flaws are use of creatinine rather than GFR and the number of patients who were obese at baseline is unknown.

Generally, IgG is infused via the intravenous (IVIG) or subcutaneous (SCIG) route. For IVIG infusion, published data demonstrate that higher IgG doses and trough levels

provide patients with improved protection from infection. The same conclusions are not yet accepted for SCIG; data from two recent Phase III studies and a recent post-hoc analysis, however, suggest the same correlation between higher SCIG dose and serum IgG concentration and decreased incidence of infection seen with IVIG. Other measures Dactolisib research buy of clinical efficacy have not been considered similarly. Thus, combined analyses of these and other published SCIG studies were performed; a full comparison of the 13 studies was, however, limited by non-standardized definitions https://www.selleckchem.com/products/crenolanib-cp-868596.html and reporting. Despite these limitations, our analyses

indicate that certain clinical outcomes improve at higher SCIG doses and associated higher serum IgG concentrations, and suggest that there might be opportunity to improve patient outcomes via SCIG dose adjustment. “
“Klebsiella pneumoniae (Kp) is one of the most common pathogens in nosocomial infections and is becoming increasingly multidrug resistant. However, the underlying molecular pathogenesis of this bacterium remains elusive, limiting the therapeutic options. Understanding the mechanism of its pathogenesis may facilitate the development of anti-bacterial therapeutics. Here, we show that Lyn, a pleiotropic Src tyrosine kinase, is involved in host defense against Kp by regulating phagocytosis process and simultaneously Tau-protein kinase downregulating inflammatory responses. Using acute infection mouse models, we observed that lyn−/− mice were more susceptible to Kp with increased mortality and severe lung injury compared with WT mice. Kp infected-lyn−/− mice exhibited elevated inflammatory cytokines (IL-6 and TNF-α), and increased superoxide in the lung and other organs. In addition, the phosphorylation of p38 and NF-κB p65 subunit increased markedly in response to Kp infection in lyn−/− mice. We also demonstrated

that the translocation of p65 from cytoplasm to nuclei increased in cultured murine lung epithelial cells by Lyn siRNA knockdown. Furthermore, lipid rafts clustered with activated Lyn and accumulated in the site of Kp invasion. Taken together, these findings revealed that Lyn may participate in host defense against Kp infection through the negative modulation of inflammatory cytokines. “
“Accumulating evidence suggests that Th17 cells and Tregs may exhibit development plasticity and that CD4+ Tregs can differentiate into IL-17-producing T cells; however, whether Th17 cells can reciprocally convert into Tregs has not been described. In this study, we generated Th17 clones from tumor-infiltrating T lymphocytes (TILs).

Two patients with Mod-PTB had unilateral pleural disease, while one had lymph node involvement. Overall, 32 of 36 patients with PTB were AFB smear/culture and/or radiology positive. Four patients were diagnosed based on radiology, clinical diagnosis and response selleck chemicals llc to treatment. Tuberculous lymphadenitis (LNTB) was diagnosed by histopathological

staining of fine needle aspirate or excision biopsy with AFB smear and culture. Pleural TB was diagnosed on the basis of pleural fluid biochemical findings, AFB culture, histopathological findings on pleural biopsy, supportive radiological evidence on X-rays and/or contrast-enhanced CT scan and response to antituberculous treatment. Diagnosis of meningeal TB was based on CSF biochemical findings, supported by AFB culture and findings on contrast-enhanced CT and/or MRI [25]. Patients with L-ETB comprised those with LNTB (n = 19) and unilateral pleural disease (n = 12). see more Patients with D-ETB comprised those with tuberculous meningitis (n = 1), bilateral pleurisy (n = 1),

abdominal (n = 2), spinal (n = 4) and miliary TB (n = 2). Bacille Calmette-Guerin (BCG)-vaccinated asymptomatic healthy volunteers who were staff of AKU were used as endemic controls (ECs). Tuberculin skin testing (TST) was assessed by administering five tuberculin units on the volar surface of the right arm subcutaneously and read by a RVX-208 single reader at 48 h. An induration of ≥10 mm was used as a cut-off for positive responses (TST+), which are considered to be indicative latent infection. TST+ (n = 21) and TST− (n = 21) ECs were included in the study. Reagents. Mycobacterium tuberculosis H37Rv whole cell sonicate (MTBs) and recombinant antigens ESAT6 and CFP10 were provided through the NIH Tuberculosis vaccine testing and reagent material contract (NO1-A1-40091) awarded to Colorado State University, USA. Whole blood assay.  Heparinized venous blood was diluted 1 : 10 in RPMI-1640 medium each and set up in 96-well tissue

culture plates as per protocol [26]. Cells were stimulated with MTBs (10 μg/ml) and ESAT-6 and CFP10 (5 μg/ml each) and cultured for up to 5 days. Supernatants were collected for cytokine measurements at 2 and 5 days post-stimulation, spun to collect cellular debris and stored at −70 °C until tested. ELISA for IFNγ, CXCL9, CXCL10, CCL2 and IL10.  IFNγ standards and monoclonal antibody pairs for capture and detection were obtained from Pharmingen, San Diego, CA, USA. Reagents for CCL2, CXCL9 and CXCL10 were obtained from R&D Systems, Minneapolis, MN, USA. All measurements were carried out according to the manufacturer’s recommendations and as described previously [16]. The limit of detection for each read-out was following: IFNγ, 15.6 pg/ml; IL10, 7.8 pg/ml; CXCL9, 69 pg/ml; CXCL10, 23 pg/ml and CCL2, 23 pg/ml, respectively. Statistical analysis.

PBMCs were harvested and

washed with phosphate-buffered saline (PBS) plus 0·5% bovine serum albumin (BSA). Four-colour immunophenotyping was carried out in PBS 0·5% BSA for 15 min at 4°C using the following FK506 mouse fluorochrome conjugated antibodies: anti-CD45 peridinin chlorophyll (PerCP) (clone TU116), anti-IgD phycoerythrin (PE) (IA6-2; all from BD Biosciences, San Jose, CA, USA), anti-CD19 allophycocyanin (APC) (clone SJ25-C1), anti-CD24 fluorescein isothiocyanate (FITC) (clone SN3), anti-CD38 PE (clone HIT2), anti-CD27 FITC (M-T27; all from Invitrogen/Caltag, Karlsruhe, Germany) and anti-CD21 FITC (clone 1F8, Dako, Glostrup, Denmark). Flow cytometric analysis was performed on a FACSCalibur

instrument (BD Biosciences) and the data were analysed using CellQuest software version 3·1 (BD Biosciences). The following antibody combinations were used: (1) anti-CD27 FITC, anti-IgD PE, anti-CD45 PerCP, anti-CD19 APC; (2) anti-CD24-FITC, anti-CD38 PE, anti-CD45 PerCP, anti-CD19 APC; and (3) anti-CD21 FITC, anti-CD38 PE, anti-CD45 PerCP, anti-CD19 APC. Additionally, immunofluorescent staining using the whole blood method was performed in 21 individuals and compared to the approach described above. Whole blood was washed twice with PBS. After the final washing step cells were resuspended in PBS 0·5% BSA and immunofluorescent staining was performed as described above. At the end of the staining step erythrocytes BYL719 in vitro were lysed using the FACSLysing Solution (BD Biosciences), according to the manufacturer’s instructions. The gating strategies are explained in Fig. 1. Absolute numbers of cells were calculated by multiplying the relative proportion of a particular B cell population

with the absolute number of lymphocytes obtained by an automatically analysed differential white blood count obtained on the same day. The data were analysed using GraphPad Prism®, SAS/STAT® and Microsoft Office Excel® software. Age-dependent changes of B cell populations were analysed using a generalized additive model. A smoothing spline was estimated Inositol oxygenase via non-parametric regression. Reference values were established for seven age groups. Medians and interquartile ranges (25th–75th percentiles) were calculated for each age group. Statistical dependence between two variables was tested using Spearman’s rank correlation coefficient. P-values < 0·05 were regarded as statistically significant. Age-dependent changes in frequencies and absolute counts of total B cells as well as distinct B cell subsets are shown in Figs 2 and 3. The frequency of total CD19+ B cells within the lymphocytes decreased with age. The composition of the B cell subsets showed age-dependent changes.

© 2014 Wiley Periodicals, Inc. Microsurgery, 2014. “
“Extension of the elbow is required to oppose gravity; however, activation of the triceps brachii is

frequently underestimated during the surgical planning for brachial plexus injuries. This report aims to describe a novel technique of distal nerve transfer designed Decitabine for elbow extension reconstruction in patients sustaining a C5–C7 nerve root injury. We report a patient sustaining a brachial plexus injury with triceps palsy and preserved finger extension motion; after careful intraneural dissection of the radial nerve, a fascicle innervating the extensor digitorum communis muscle was sectioned, derouted and connected to a motor branch to the lateral head of the triceps. Eleven months after surgery, elbow extension strength scored MRC M4. No deficits on finger extension were observed. © 2011 Wiley Periodicals, Inc. Microsurgery, 2012. “
“Lipoprostaglandin E1 (lipo-PGE1) AZD6244 in vitro has been found accumulating in injured vascular regions. This study examined the localization of

lipo-PGE1 in the anastomotic region. The study was divided into three parts. First, we performed anastomosis of the rat femoral artery and vein (n = 17). Lipo-PGE1 labeled with 1,1′-dioctadecyl-1,3,3′,3′-tetramethyl-indocarbocyanine was infused intravenously. Hematoxylin-Eosin staining and fluorescence microscopic findings showed that lipo-PGE1 markedly accumulated at the anastomotic site when compared to the contralateral non anastomotic region. Then, we measured laser Doppler flow (LDF) of a lower leg before and after infusion of lipo-PGE1 (n = 7) and saline (n = 7). Increase of blood flow was maintained 1 hour after the infusion of lipo-PGE1 (144% ± 25.0%) when compared to saline infusion. Finally, we performed immunohistochemical and electron microscopic examinations

and found that Lipo-PGE1 was incorporated in vascular smooth muscle cells of the anastomotic region. These findings suggest selective accumulation of the lipo-PGE1 in the vascular STK38 anastomosis site and affect on the blood flow of repaired vessels. © 2011 Wiley-Liss, Inc. Microsurgery, 2011. “
“Distal fingertip replantation is associated with good functional and aesthetic results. Venous anastomosis is the most challenging procedure. For replantation with an artery anastomosis-only procedure (no venous anastomosis), some protocols have been designed to relieve venous congestion involve anticoagulation and the creation of wounds for persistent bleeding. This report presents the authors’ experience of fingertip survival after artery anastomosis-only replantation with no persistent external bleeding. Twelve Tamai zone I fingertip total amputation patients who underwent artery anastomosis-only replantations were recruited from February 2009 to June 2012. Nerve repair was performed if identified. The patients were not subjected to conventional external bleeding methods.

8a). Furthermore, because it is possible that endogenous TCR α-chains are necessary for DN T-cell selection or function and because we cannot monitor the frequency Selleckchem Temsirolimus of the TCR-Tg Vα5 chain by FACS (mAb against Vα5 is not commercially available), we

also bred 7/16-5 × HBeAg dbl-Tg mice on to a TCR α-chain KO background. The absence of endogenous TCR α-chains did not affect the presence of DN T cells in the periphery (Fig. 8b). This demonstrates that TCR-Tg Vα5 is sufficient to confer the DN T-cell phenotype. To examine the APC requirement for DN T-cell expansion, DN progenitor T cells from 7/16-5 × HBeAg dbl-Tg mice were fractionated, and cultured with different APC populations in the presence of HBeAg peptide p120–140. As shown in Fig. 9(a),

fractionated B cells do not support the proliferation or survival of DN T cells even with a relatively high concentration of antigen. This is DAPT in vivo surprising because B cells are the primary APCs for HBcAg and present p120–140 and HBc/HBeAgs efficiently to HBc/HBeAg-specific CD4+ T cells.40,41 To examine non-B-cell APC function, an APC fraction from B-cell KO (μMT) mice was used for co-culture with DN T-cell progenitors. Non-B cells supported the proliferation of DN T cells efficiently even at low concentrations (0·2 μg/ml) of p120–140 peptide (Fig. 9a). It was also interesting that APCs from μMT mice support the survival of DN T cells even in the absence of antigen. It appeared that in the induction phase of DN T-cell expansion, specific soluble factors or surface co-stimulatory molecules from DC or MΦ, but not from B cells specifically support the survival and proliferation of DN T many cells. Although IL-2 is not necessary for the proliferation of DN T cells, there are several other soluble factors involved in the proliferation of T cells. Notably, IL-7 and IL-15 are prominent candidates for the induction of IL-2-independent proliferation.

Interleukin-7 is known as a regulator of proliferation of T cells, in IL-2-dependent and IL-2-independent circumstances.42 Both IL-15 and IL-7 are also known to mediate homeostatic proliferation of naive T cells.42,43 Additionally, IL-15 is produced by DCs and can have IL-2-like function. To test the effect of IL-7 and IL-15 on the proliferation of DN T cells, we cultured purified DN progenitor cells with different APCs in the presence of antigen and cytokines. As observed in the previous experiment, DC/MΦ supported the proliferation of DN T-cell progenitor cells, whereas B cells did not, even at the higher concentration of antigen. However, when exogenous IL-15 was added to the B-cell APC culture, it rescued the proliferation of DN T-cell progenitor cells in the presence of p120–140 (Fig. 9b). This result suggests that IL-15 produced by DC/MΦ may play an important role in the proliferation of DN T cells. The DN T cells express high levels of IL-15R on their surface, whereas DN gated splenocytes from control mice do not express IL-15R (Fig. 10).

The two PSs used are hypericin (HYP) and 1,9-dimethyl methylene blue (DMMB). Quizartinib HYP is a natural naphthodianthrone endowed with fungicidal activity on yeast, especially on C. albicans.[9] DMMB is a hydrophobic derivative of the well-known

phenothiazinium PS methylene blue (MB).[16] The activity of different phenothiazinium salts against C. albicans has been studied only very recently.[11] Considering that phenothiazines are the most widely used PSs in clinical aPDT, especially in oral infections where C. albicans is an important pathogen, we studied whether HYP could provide any advantages over phenothiazinium salts for clinical candidiasis. DMMB was chosen as it is substantially more hydrophobic than MB or toluidine blue, the phenothiazinium salts currently in use, and it has not been studied for C. albicans. In addition, we investigate selleck chemicals llc the reactive oxygen species (ROS) involved in the phototoxic effect to ascertain mechanistic differences between both PS. Culture Media: Sabouraud Dextrose

Agar CM0041 (Oxoid Ltd., Hampshire, England). Chloramphenicol (Sigma-Aldrich®, St Louis, MO, USA). ROS quenchers: catalase (CAT) and superoxide dismutase (SOD), both from Sigma-Aldrich®. Sodium azide (SA) and mannitol (MAN) were purchased from Panreac® (Barcelona, Spain). Solvents and chemicals: ethanol (Alcohocel®; Barcelona, Spain), PBS buffer (Bio-Rad® Laboratories, Redmond, WA, USA), distiled water and physiological serum (Fresenius Kabi España®, Barcelona, Spain) and dimethyl sulphoxide (DMSO) (Panreac®). Hypericin was purchased from Sigma-Aldrich® and HWI-Analytik® Gmbh (Ruelzheim, Germany). A stock solution was prepared in DMSO and diluted immediately prior to use with distiled water or PBS to the desired concentration. 1,9-dimethyl methylene blue was purchased from Sigma-Aldrich® (Gillingham, UK). A stock solution was prepared in distiled water. Working solutions were prepared in distiled water or

PBS immediately before using with the desired concentrations. Yeast were irradiated 4��8C using light-emitting diode (LED)-based lamps. For DMMB, the lamp emitted at 639.8 ± 10 nm with and irradiance 19.0 mW cm−2, whereas for HYP the wavelength was 602 ± 10 nm and the irradiance 10.3 mW cm−2. Two fluences were used namely 18 and 37 J cm−2. Azole-resistant C. albicans strains namely AZN9635, 456325H and AMO7/0267, were obtained from Canisius Wilhemina Hospital (Nijmegen, The Netherlands). The susceptible C. albicans ATCC 10231 strain was acquired from the American Type Culture Collection (ATCC, Rockville, MD, USA) and C. albicans CETC 1001 from the Spanish Type Culture Collection (CECT, Valencia, Spain). The yeast were grown aerobically overnight in Sabouraud dextrose agar added with chloramphenicol (0.5 mg l−1) plates (SB) at 35 °C.