Cytolytic proteins and peptide toxins are classical virulence factors of several bacterial pathogens which disrupt epithelial barrier function, damage cells and activate or modulate host immune ...responses. Such toxins have not been identified previously in human pathogenic fungi. Here we identify the first, to our knowledge, fungal cytolytic peptide toxin in the opportunistic pathogen Candida albicans. This secreted toxin directly damages epithelial membranes, triggers a danger response signalling pathway and activates epithelial immunity. Membrane permeabilization is enhanced by a positive charge at the carboxy terminus of the peptide, which triggers an inward current concomitant with calcium influx. C. albicans strains lacking this toxin do not activate or damage epithelial cells and are avirulent in animal models of mucosal infection. We propose the name 'Candidalysin' for this cytolytic peptide toxin; a newly identified, critical molecular determinant of epithelial damage and host recognition of the clinically important fungus, C. albicans.
Relapse remains the most common cause of treatment failure for patients with acute myeloid leukemia (AML) who undergo allogeneic stem cell transplantation (alloSCT), and carries a grave prognosis. ...Multiple studies have identified the presence of measurable residual disease (MRD) assessed by flow cytometry before alloSCT as a strong predictor of relapse, but it is not clear how these findings apply to patients who test positive in molecular MRD assays, which have far greater sensitivity. We analyzed pretransplant blood and bone marrow samples by reverse-transcription polymerase chain reaction in 107 patients with NPM1-mutant AML enrolled in the UK National Cancer Research Institute AML17 study. After a median follow-up of 4.9 years, patients with negative, low (<200 copies per 105ABL in the peripheral blood and <1000 copies in the bone marrow aspirate), and high levels of MRD had an estimated 2-year overall survival (2y-OS) of 83%, 63%, and 13%, respectively (P < .0001). Focusing on patients with low-level MRD before alloSCT, those with FLT3 internal tandem duplications(ITDs) had significantly poorer outcome (hazard ratio HR, 6.14; P = .01). Combining these variables was highly prognostic, dividing patients into 2 groups with 2y-OS of 17% and 82% (HR, 13.2; P < .0001). T-depletion was associated with significantly reduced survival both in the entire cohort (2y-OS, 56% vs 96%; HR, 3.24; P = .0005) and in MRD-positive patients (2y-OS, 34% vs 100%; HR, 3.78; P = .003), but there was no significant effect of either conditioning regimen or donor source on outcome. Registered at ISRCTN (http://www.isrctn.com/ISRCTN55675535).
•Pretransplant MRD level is predictive of outcome; thresholds of 200 copies per 105ABL in blood and 1000 copies in marrow are discriminatory.•Relapse in patients with pretransplant MRD positivity below these levels is largely restricted to those with FLT3-ITD.
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The fungus C. albicans uses adhesins to interact with human epithelial surfaces in the processes of colonization and pathogenesis. The C. albicans ALS (agglutinin-like sequence) gene family encodes ...eight large cell-surface glycoproteins (Als1-Als7 and Als9) that have adhesive function. This study utilized C. albicans Δals mutant strains to investigate the role of the Als family in oral epithelial cell adhesion and damage, cytokine induction and activation of a MAPK-based (MKP1/c-Fos) signaling pathway that discriminates between yeast and hyphae. Of the eight Δals mutants tested, only the Δals3 strain showed significant reductions in oral epithelial cell adhesion and damage, and cytokine production. High fungal:epithelial cell multiplicities of infection were able to rescue the cell damage and cytokine production phenotypes, demonstrating the importance of fungal burden in mucosal infections. Despite its adhesion, damage and cytokine induction phenotypes, the Δals3 strain induced MKP1 phosphorylation and c-Fos production to a similar extent as control cells. Our data demonstrate that Als3 is involved directly in epithelial adhesion but indirectly in cell damage and cytokine induction, and is not the factor targeted by oral epithelial cells to discriminate between the yeast and hyphal form of C. albicans.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We previously reported that a bi-phasic innate immune MAPK response, constituting activation of the mitogen-activated protein kinase (MAPK) phosphatase MKP1 and c-Fos transcription factor, ...discriminates between the yeast and hyphal forms of Candida albicans in oral epithelial cells (ECs). Since the vast majority of mucosal Candida infections are vaginal, we sought to determine whether a similar bi-phasic MAPK-based immune response was activated by C. albicans in vaginal ECs. Here, we demonstrate that vaginal ECs orchestrate an innate response to C. albicans via NF-κB and MAPK signaling pathways. However, unlike in oral ECs, the first MAPK response, defined by c-Jun transcription factor activation, is delayed until 2 h in vaginal ECs but is still independent of hypha formation. The 'second' or 'late' MAPK response, constituting MKP1 and c-Fos transcription factor activation, is identical to oral ECs and is dependent upon both hypha formation and fungal burdens. NF-κB activation is immediate but independent of morphology. Furthermore, the proinflammatory response in vaginal ECs is different to oral ECs, with an absence of G-CSF and CCL20 and low level IL-6 production. Therefore, differences exist in how C. albicans activates signaling mechanisms in oral and vaginal ECs; however, the activation of MAPK-based pathways that discriminate between yeast and hyphal forms is retained between these mucosal sites. We conclude that this MAPK-based signaling pathway is a common mechanism enabling different human epithelial tissues to orchestrate innate immune responses specifically against C. albicans hyphae.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Signal transducers and activators of transcription 5(STAT5) are cytokine induced signaling proteins, which regulate key immunological processes, such as tolerance induction, maintenance of ...homeostasis, and CD4 T-effector cell differentiation. In this study, transcriptional targets of STAT5 in CD4 T cells were studied by Chromatin Immunoprecipitation (ChIP). Genomic mapping of the sites cloned and identified in this study revealed the striking observation that the majority of STAT5-binding sites mapped to intergenic (>50 kb upstream) or intronic, rather than promoter proximal regions. Of the 105 STAT5 responsive binding sites identified, 94% contained the canonical (IFN-γ activation site) GAS motifs. A number of putative target genes identified here are associated with tumor biology. Here, we identified Fos-related antigen 2 (FRA2) as a transcriptional target of IL-2 regulated STAT5. FRA2 is a basic -leucine zipper (bZIP) motif 'Fos' family transcription factor that is part of the AP-1 transcription factor complex and is also known to play a critical role in the progression of human tumours and more recently as a determinant of T cell plasticity. The binding site mapped to an internal intron within the FRA2 gene. The epigenetic architecture of FRA2, characterizes a transcriptionally active promoter as indicated by enrichment for histone methylation marks H3K4me1, H3K4me2, H3K4me3, and transcription/elongation associated marks H2BK5me1 and H4K20me1. FRA2 is regulated by IL-2 in activated CD4 T cells. Consistently, STAT5 bound to GAS sequence in the internal intron of FRA2 and reporter gene assays confirmed IL-2 induced STAT5 binding and transcriptional activation. Furthermore, addition of JAK3 inhibitor (R333) or Daclizumab inhibited the induction in TCR stimulated cells. Taken together, our data suggest that FRA2 is a novel STAT5 target gene, regulated by IL-2 in activated CD4 T cells.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Kidney transplant recipients (KTRs) with “operational tolerance” (OT) maintain a functioning graft without immunosuppressive (IS) drugs, thus avoiding treatment complications. Nevertheless, IS drugs ...can influence gene-expression signatures aiming to identify OT among treated KTRs.
We compared five published signatures of OT in peripheral blood samples from 18 tolerant, 183 stable, and 34 chronic rejector KTRs, using gene-expression levels with and without adjustment for IS drugs and regularised logistic regression.
IS drugs explained up to 50% of the variability in gene-expression and 20–30% of the variability in the probability of OT predicted by signatures without drug adjustment. We present a parsimonious consensus gene-set to identify OT, derived from joint analysis of IS-drug-adjusted expression of five published signature gene-sets. This signature, including CD40, CTLA4, HSD11B1, IGKV4–1, MZB1, NR3C2, and RAB40C genes, showed an area under the curve 0⋅92 (95% confidence interval 0⋅88–0⋅94) in cross-validation and 0⋅97 (0⋅93–1⋅00) in six months follow-up samples.
We advocate including adjustment for IS drug therapy in the development stage of gene-expression signatures of OT to reduce the risk of capturing features of treatment, which could be lost following IS drug minimisation or withdrawal. Our signature, however, would require further validation in an independent dataset and a biomarker-led trial.
FP7-HEALTH-2012-INNOVATION-1 305147:BIO-DrIM (SC,IR-M,PM,DSt); MRC G0801537/ID:88245 (MPH-F); MRC MR/J006742/1 (IR-M); Guy's&StThomas’ Charity R080530&R090782; CONICYT-Bicentennial-Becas-Chile (EN-L); EU:FP7/2007–2013 HEALTH-F5–2010–260687: The ONE Study (MPH-F); Czech Ministry of Health NV19–06–00031 (OV); NIHR-BRC Guy's&StThomas' NHS Foundation Trust and KCL (SC); UK Clinical Research Networks portfolio:7521.
Parasite-derived glycosylphosphatidylinositol (GPI) is believed to be a major inducer of the pathways leading to pathology and morbidity during Plasmodium falciparum infection and has been termed a ...malaria "toxin." The generation of neutralizing anti-GPI ("antitoxic") antibodies has therefore been hypothesized to be an important step in the acquisition of antidisease immunity to malaria; however, to date the GPI-neutralizing capacity of antibodies induced during natural Plasmodium falciparum infection has not been evaluated. Here we describe the development of an in vitro macrophage-based assay to assess the neutralizing capacity of malarial GPI-specific IgG. We demonstrate that IgG from Plasmodium falciparum-exposed individuals can significantly inhibit the GPI-induced activation of macrophages in vitro, as shown by reduced levels of tumor necrosis factor production and attenuation of CD40 expression. The GPI-neutralizing capacity of individual IgG samples was directly correlated with the anti-GPI antibody titer. IgG from malaria-exposed individuals also neutralized the macrophage-activating effects of P. falciparum schizont extract (PfSE), but there was only a poor correlation between PfSE-neutralizing activity and the anti-GPI antibody titer, suggesting that PfSE contains other macrophage-activating moieties, in addition to GPI. In conclusion, we have established an in vitro assay to test the toxin-neutralizing activities of antimalarial antibodies and have shown that anti-GPI antibodies from malaria-immune individuals are able to neutralize GPI-induced macrophage activation; however, the clinical relevance of anti-GPI antibodies remains to be proven, given that malarial schizonts contain other proinflammatory moieties, in addition to GPI.
Discriminating between commensal and pathogenic states of opportunistic pathogens is critical for host mucosal defense and homeostasis. The opportunistic human fungal pathogen Candida albicans is ...also a constituent of the normal oral flora and grows either as yeasts or hyphae. We demonstrate that oral epithelial cells orchestrate an innate response to C. albicans via NF-κB and a biphasic MAPK response. Activation of NF-κB and the first MAPK phase, constituting c-Jun activation, is independent of morphology and due to fungal cell wall recognition. Activation of the second MAPK phase, constituting MKP1 and c-Fos activation, is dependent upon hypha formation and fungal burdens and correlates with proinflammatory responses. Such biphasic response may allow epithelial tissues to remain quiescent under low fungal burdens while responding specifically and strongly to damage-inducing hyphae when burdens increase. MAPK/MKP1/c-Fos activation may represent a "danger response" pathway that is critical for identifying and responding to the pathogenic switch of commensal microbes.
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