X-ray reflectivity measurements of increasingly more complex interfaces involving silicon (001) substrates reveal the existence of a thin low density layer intruding between the single-crystalline silicon and the amorphous native SiO$_2$ terminating it. The importance of accounting for this layer in modeling silicon/liquid interfaces and silicon-supported monolayers is demonstrated by comparing fits of the measured reflectivity curves by models including and excluding this layer. The inclusion of this layer, with 6-8 missing electrons per silicon unit cell area, is found to be particularly important for an accurate and high-resolution determination of the surface normal density profile from reflectivities spanning extended momentum transfer ranges, now measurable at modern 3rd generation synchrotron sources.
Therapeutic natural killer (NK) cell-mediated alloreactivity towards acute myeloid leukemia (AML) has largely been attributed to mismatches between killer immunoglobulin-like receptors (KIRs) on NK cells and their ligands, HLA class I molecules, on target cells. While adult acute B cell precursor leukemia (BCP-ALL) appears to be resistant to NK cell-mediated lysis, recent data indicate that pediatric BCP-ALL might yet be a target of NK cells. We here demonstrate in a donor-patient-specific NOD.Cg-Prkdc(scid) IL2rg(tmWjl)/Sz (NSG) xenotransplantation model that NK cells mediate considerable alloreactivity towards pediatric BCP-ALL in vivo. Notably, not only adoptively transferred mature, KIR(+) NK cells but also immature, KIR- NK cells arising early post transplantation in humanized NSG mice (huNSG) exerted substantial anti-leukemic activity. Low-dose and long-term treatment of huNSG mice with the DNA-demethylating agent 5-Aza-cytidine distinctly enhanced the anti-tumor response, interestingly without inducing common inhibitory KIR expression but rather by promoting the differentiation of various NK cell precursor subsets. Collectively, these data indicate that the future design of innovative therapy protocols should consider further exploitation of NK cell-mediated immune-responses for poor prognosis pediatric BCP-ALL patients.
Graft-versus-host disease (GvHD) is a major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation. However, the pathophysiology of GvHD remains poorly understood. In this study, we analyzed the induction of Th17 cells by monocytes of patients with GvHD in vitro, demonstrating that monocytes isolated from patients with acute skin and intestinal GvHD stage I-IV and chronic GvHD induce significantly increased levels of Th17 cells compared with patients without GvHD. S100 proteins are known to act as innate amplifier of inflammation. We therefore investigated the presence of S100 proteins in the stool, serum, and bowel tissue of patients with GvHD and the influence of S100 proteins on the induction of Th17 cells. Elevated levels of S100 proteins could be detected in patients with acute GvHD, demonstrating the release of these phagocyte-specific proteins during GvHD. Furthermore, stimulation of monocytes with S100 proteins was found to promote Th17 development, emphasizing the role of S100 proteins in Th17-triggered inflammation. Altogether, our results indicate that induction of Th17 cells by activated monocytes and the stimulatory effects of proinflammatory S100 proteins might play a relevant role in the pathogenesis of acute GvHD. Regarding our data, S100 proteins might be novel markers for the diagnosis and follow-up of GvHD.
We analyzed the influence of donor killer-cell immunoglobulin-like receptor (KIR) gene haplotypes on the risk for relapse and the probability of event-free survival (EFS) in children with acute lymphoblastic leukemia who received human leukocyte antigen-haploidentical transplantation of ex vivo T-cell-depleted peripheral blood stem cells. The KIR gene haplotype was evaluated in 85 donors, and the KIR B content score was determined in the 63 KIR haplotype B donors. Patients transplanted from a KIR haplotype B donor had a significantly better EFS than those transplanted from a KIR haplotype A donor (50.6% vs 29.5%, respectively; P = .033). Moreover, a high donor KIR B-content score was associated with a significantly reduced risk for relapse (Log-rank test for trend, P = .026). These data indicate that KIR genotyping should be included in the donor selection algorithm for haploidentical transplantation in children with acute lymphoblastic leukemia with the aim of choosing, whenever possible, a KIR haplotype B donor with a high KIR B-content score.
Natural killer (NK)-cell alloreactivity after allogeneic hematopoietic cell transplantation (HCT) is influenced by the interaction of killer-cell immunoglobulin-like receptors (KIRs) on donor NK cells and human leukocyte antigen (HLA) class I ligands on recipient cells. We investigated the influence of donor KIR haplotype and KIR-ligand mismatch (MM) on relapse in 57 patients with hematologic malignancies receiving haploidentical HCT after reduced intensity conditioning and graft CD3/CD19 depletion. Of the 57 donors, 17 had KIR haplotype A (29.8 %) and 40 had KIR haplotype B (70.2 %). A KIR-ligand MM was found in 34 of 57 patients (59.6 %). There was no difference between donor KIR haplotypes in non-relapse mortality (NRM, p?=?0.200) but had a significantly reduced incidence of relapse for patients with a haplotype B donor (p?=?0.001). In particular, patients in partial remission (PR) benefited more from a haplotype B graft (p?=?0.008) than patients in complete remission (CR, p?=?0.297). Evaluating KIR-ligand MM cumulative incidences of relapse (p?=?0.680) or NRM (p?=?0.579), we found no significant difference. In conclusion, in the setting of reduced intensity conditioning (RIC) and CD3/CD19-depleted haploidentical HCT, we could not confirm the positive data with KIR-ligand MM but observed a significant lower risk of relapse with a KIR haplotype B donor.
Single nucleotide polymorphisms (SNPs) have been associated with an increased incidence of invasive aspergillosis (IA) after allogeneic stem cell transplantation (allo-SCT). We analyzed 41 patients with proven/probable IA after allo-SCT for an association of SNPs, within the TLR2, TLR4, TLR5, TLR9, and NOD2/CARD15 genes, with susceptibility to IA. The control group consisted of 130 patients who had allo-SCT but did not develop IA. While no association was found for donor SNPs and the recipients risk of IA, analysis of recipient SNPs showed a significant association between the presence of recipient TLR5-Stop SNP (1174C> T) and the incidence of IA (P = 0.004). Multivariate analysis demonstrated that the recipient TLR5-Stop SNP appeared as an independent risk factor for IA after allo-SCT. Our study suggests that TLR5 is involved in host defense against Aspergillus fumigatus, and that the recipient TLR5-Stop SNP represents a risk factor for the development of IA after allo-SCT.
The molecular-scale structure of the ionic liquid [C18mim](+)[FAP](-) near its free surface was studied by complementary methods. X-ray absorption spectroscopy and resonant soft X-ray reflectivity revealed a depth-decaying near-surface layering. Element-specific interfacial profiles were extracted with submolecular resolution from energy-dependent soft X-ray reflectivity data. Temperature-dependent hard X-ray reflectivity, small- and wide-angle X-ray scattering, and infrared spectroscopy uncovered an intriguing melting mechanism for the layered region, where alkyl chain melting drove a negative thermal expansion of the surface layer spacing.
Chemically modified mRNA is capable of inducing therapeutic levels of protein expression while circumventing the threat of genomic integration often associated with viral vectors. We utilized this novel therapeutic tool to express the regulatory T cell transcription factor, FOXP3, in a time- and site-specific fashion in murine lung, in order to prevent allergic asthma in vivo. We show that modified Foxp3 mRNA rebalanced pulmonary T helper cell responses and protected from allergen-induced tissue inflammation, airway hyperresponsiveness, and goblet cell metaplasia in 2 asthma models. This protection was conferred following delivery of modified mRNA either before or after the onset of allergen challenge, demonstrating its potential as both a preventive and a therapeutic agent. Mechanistically, FOXP3 induction controlled Th2 and Th17 inflammation by regulating innate immune cell recruitment through an IL-10-dependent pathway. The protective effects of FOXP3 could be reversed by depletion of IL-10 or administration of recombinant IL-17A or IL-23. Delivery of Foxp3 mRNA to sites of inflammation may offer a novel, safe therapeutic tool for the treatment of allergic asthma and other diseases driven by an imbalance in helper T cell responses.
Epidermolysis bullosa (EB) is a heterogeneous group of inherited diseases characterized by the formation of blisters in the skin and mucosa. There is no cure or effective treatment for these potentially severe and fatal diseases. Over the past few years, several reports have proposed different molecular strategies as new therapeutic options for the management of EB. From classical vector-based gene therapy to cell-based strategies such as systemic application of bone marrow stem cells or local application of fibroblasts, a broad range of molecular approaches have been explored. This array also includes novel methods, such as protein replacement therapy, gene silencing and the use of induced pluripotent stem cells (iPCs). In this review, we summarize current concepts of how inherited blistering diseases might be treated in the future and discuss the opportunities, promises, concerns and risks of these innovative approaches.
The two-dimensional diffusion of isolated molecular tracers at the water-n-alkane interface was studied with fluorescence correlation spectroscopy. The interfacial diffusion coefficients of larger tracers with a hydrodynamic radius of 4.0 nm agreed well with the values calculated from the macroscopic viscosities of the two bulk phases. However, for small molecule tracers with hydrodynamic radii of only 1.0 and 0.6 nm, notable deviations were observed, indicating the existence of an interfacial region with reduced effective viscosity and increased mobility.
40-O -[2-Hydroxyethyl]rapamycin (RAD), a novel derivative of the immunosuppressive drug rapamycin, was analyzed for its immunomodulatory influence during the interaction of human monocyte-derived dendritic cells (moDC) with Aspergillus fumigatus. RAD is clinically used to prevent graft-versus -host disease as well as solid organ and bone marrow transplant rejection. However, it may constitute a risk factor for the development of opportunistic infections, such as invasive aspergillosis which is mainly caused by the most common airborne fungal pathogen A. fumigatus. moDC were generated in the presence or absence of RAD. In this setting, RAD had various modulating effects on the immune function of DC. A decrease of pro- and anti-inflammatory cytokines (IL-12, TNF-?, CCL20, IL-10) was observed. Furthermore, RAD reduced the expression of innate immunity receptors (TLR2, TLR4, dectin-1), impaired the maturation capacity of moDC observed through the reduction of costimulatory factors (CD40, CD80, CD83, CD86), and impaired their capacity to phagocytose and damage A. fumigatus. These data demonstrate that RAD influences the differentiation of DC. RAD modulates the cytokine response of DC to A. fumigatus and reduces their ability to kill germ tubes. Thus, RAD treatment might affect the risk of invasive aspergillosis independently of its capacity of blocking T cell activation.
Dendritic cells (DC) are the most important antigen presenting cells and play a pivotal role in host immunity to infectious agents by acting as a bridge between the innate and adaptive immune systems. Monocyte-derived immature DCs (iDC) were infected with viable resting conidia of Aspergillus fumigatus (Af293) for 12 hours at an MOI of 5; cells were sampled every three hours. RNA was extracted from both organisms at each time point and hybridised to microarrays. iDC cell death increased at 6 h in the presence of A. fumigatus which coincided with fungal germ tube emergence; >80% of conidia were associated with iDC. Over the time course A. fumigatus differentially regulated 210 genes, FunCat analysis indicated significant up-regulation of genes involved in fermentation, drug transport, pathogenesis and response to oxidative stress. Genes related to cytotoxicity were differentially regulated but the gliotoxin biosynthesis genes were down regulated over the time course, while Aspf1 was up-regulated at 9 h and 12 h. There was an up-regulation of genes in the subtelomeric regions of the genome as the interaction progressed. The genes up-regulated by iDC in the presence of A. fumigatus indicated that they were producing a pro-inflammatory response which was consistent with previous transcriptome studies of iDC interacting with A. fumigatus germ tubes. This study shows that A. fumigatus adapts to phagocytosis by iDCs by utilising genes that allow it to survive the interaction rather than just up-regulation of specific virulence genes.
The density deficit of water at hydrophobic interfaces, frequently called the hydrophobic gap, has been the subject of numerous experimental and theoretical studies in the past decade. Recent experiments give values for the interfacial depletion that consistently correspond to less than a monolayer of water. The main question which remained so far unanswered is its origin and the mechanisms affected by the chemistry and molecular geometry of a particular hydrophobic coating. In this work, we present a combined high-energy X-ray reflectivity and molecular dynamics simulation study of the water depletion at a perfluorinated hydrophobic interface with a spatial resolution on the molecular scale. A comparison of our experimental and computational results elucidates the underlying mechanisms that affect the extent of the interfacial depletion. The complex interplay between surface chemistry and topography precludes the existence of a direct and universal relation between the macroscopic contact angle and the nanoscopic water depletion.
Chemokines represent central players of the innate and adaptive immunity and are involved in the regulation of inflammatory events occurring during infectious complications or during graft vs. host disease (GvHD). Patients after allogeneic stem cell transplantation (alloSCT) are at a high risk for the development of acute GvHD or to suffer from fungal infections. Susceptibility to fungal infections and the course of GvHD can be genetically influenced by single nucleotide polymorphisms (SNPs), which regulate expression or biological activity of chemokines, and therefore have an impact on the outcome of invasive aspergillosis and GvHD. High lightened studies of abetting factors for GvHD revealed SNPs in TNFA, IL-6, IL-10, INF-?, CCL2, CCL5 (RANTES), IL-1Ra, IL-23R, IL-7Ralpha, IL-10RB, and CCR9 genes as prevalent considerable. Furthermore, additional SNPs were described to be significantly associated with fungal infections (Aspergillus fumigatus, Candida albicans), including markers in CCL3, CCL4, CCL20, CXCL2, CXCL8, CXCL10, CCR1, and CCR2. This review summarizes the current knowledge about the growing number of genetic markers in chemokine genes and their relevance for patients after alloSCT.
Infections with the opportunistic mold Aspergillus fumigatus show high morbidity and mortality. Risk factors for the development of invasive aspergillosis are neutropenia, T-cell depletion, CD34-selected stem cell products, corticosteroid therapy, and cytomegalovirus infections. Recently, a growing number of defined single nucleotide polymorphisms have been described that genetically determine susceptibility to A. fumigatus. This includes genes encoding for cytokines or chemokines and their receptors, toll-like receptor genes, and other genes involved in innate immunity. This review summarizes the current knowledge about the growing number of genetic markers and their relevance for the course and outcome of infections with A. fumigatus.
Dysregulation of the Th1/Th2 cytokine balance and a switch to a Th2 immune response contribute to the development of and the unfavorable outcome from invasive aspergillosis (IA). We explore in this paper the role of glycogen synthase kinase 3 (GSK-3) in human immature dendritic cells (iDCs) relative to infection caused by A. fumigatus by the use of GSK-3 inhibitors (LiCl, SB415286) and RNA interference technology. In iDCs exposed to A. fumigatus germ tubes, inhibition of GSK-3 with LiCl or SB415286, as well as transfection with small interfering RNA, led to markedly elevated expression of the anti-inflammatory cytokine IL-10. In contrast, pro-inflammatory cytokine response was only partially regulated by GSK-3. Screening of patients after allogeneic stem cell transplantation (with or without IA) for the presence of genetic markers (rs334558, rs6438552) in the GSK-3 gene revealed no significant association with an increased risk for IA. In conclusion, GSK-3 might be involved in the regulation of the anti-inflammatory response of iDCs in the context of infections due to A. fumigatus, albeit the exact mechanisms have to be clarified in future experiments.
Toll-like receptors (TLRs) recognize an increasingly broad range of pathogens, thus demonstrating the importance of these pattern-recognition receptors (PRRs) in host defense. Here, the role of TLR3 in the interaction of monocyte-derived dendritic cells (moDCs) with human cytomegalovirus (HCMV) was investigated by using the TB40E strain, which actively replicates in moDCs. Microarray analysis and quantitative real-time PCR revealed that TB40E infection of moDCs led to changes in the gene expression pattern. A variety of proinflammatory cytokines and chemokines (CXCL10, CXCL11, and CCL5), TLR3, and genes whose products function downstream of the TLR3 signaling pathway (e.g., IFN-alpha and IFN-beta) were significantly upregulated. By silencing TLR3 expression with short interfering RNA (siRNA), and subsequent stimulation with TLR3 ligand poly I:C, expression of IFN-beta was markedly reduced compared to cells transfected with a non-silencing control siRNA. However, expression of IFN-beta induced by HCMV was not diminished when TLR3 was silenced first. Thus the early HCMV-triggered immune response of human moDCs appears to be independent of TLR3 signaling.
The structure of two model room temperature ionic liquids, [BMIM](+)[PF(6)](-) and [BMIM](+)[BF(4)](-), near the solid/liquid interface with charged Al(2)O(3)(0001) (sapphire) was determined with subnanometer resolution by high energy (72.5 keV) x-ray reflectivity. [BMIM](+)[PF(6)](-) exhibits alternately charged, exponentially decaying, near-surface layering. By contrast, the smaller-anion compound, [BMIM](+)[BF(4)](-), shows only a single layer of enhanced electron density at the interface. The different layering behaviors, and their characteristic length scales, correspond well to the different bulk diffraction patterns, also measured in this study. Complementary measurements of the surface and interface energies showed no significant different between the two RTILs. The combined bulk-interface results support the conclusion that the interfacial ordering is dominated by the same electrostatic ion-ion interactions dominating the bulk correlations, with hydrogen bonding and dispersion interactions playing only a minor role.
Invasive aspergillosis is a significant cause of morbidity and mortality in patients after stem cell transplantation, in solid organ transplant recipients, and in patients with hematological malignancies. The interactions between human immature dendritic cells (iDCs) and Aspergillus fumigatus antigens are widely uncharacterized. We analyzed the immune response of iDCs to different recombinant A. fumigatus antigens (Aspf1 and Crf1). One of these antigens, the 18-kDa RNase Aspf1, triggered the increased level of expression of genes encoding proinflammatory cytokines and chemokines, and augmented the activation of NFkappaB and the apoptosis of iDCs. Furthermore, by fluorescence microscopy, we could demonstrate that in the first 3 h a major portion of Aspf1 accumulates on the cell surface. Finally, we could show an increased segregation of cytokines and chemokines after the stimulation of iDCs by an Aspf1 deletion mutant strain of A. fumigatus.
We present a dedicated experimental spin-echo resolved grazing incidence scattering (SERGIS) setup for the investigation of surfaces and thin films exhibiting large lateral length scales. This technique uses the neutron spin to encode one in-plane component of the wave-vector transfer in a grazing angle scattering experiment. Instead of the scattering angle, the depolarization of the scattered beam is measured. This allows one to achieve a very high in-plane momentum resolution without collimation of the incident neutron beam in the corresponding direction. SERGIS can therefore offer an alternative or complementary method to conventional grazing incidence neutron scattering experiments. We describe the experimental setup installed at the neutron sources ILL (Grenoble) and FRM II (Garching) and present data obtained with this setup on various samples exhibiting characteristic mesoscopic length scales in the range of several hundred nanometers. We also derive general formulas and error margins for the analysis and interpretation of SERGIS data and apply them to the cases of a one-dimensional structure and of an island morphology.
Monocytes play a major role in the cellular defence against Aspergillus fumigatus in immunocompromised patients. To obtain a better understanding of the mechanisms involved in this interaction, phagocytosis and gene expression profiling of human monocytes was carried out after incubation with A. fumigatus resting, swollen and germinating conidia and hyphae (for 3, 6 and 9 h). The majority of monocytes phagocytosed up to three conidia during the first 3 h of incubation. Microarray analysis showed an increased expression level of immune-relevant genes, which was dependent on the germination state of the fungus and the incubation period. Among these genes, those encoding interleukin-8, macrophage inflammatory protein 3-alpha (CCL20) and monocyte chemotactic protein-1 (CCL2) were found to be potential key regulators involved in the A. fumigatus-induced immune response. In addition, A. fumigatus was found to be an inducer of the genes encoding urokinase type plasminogen activator (uPA), urokinase type plasminogen activator receptor (uPAR),plasminogen activator inhibitor (PAI), pentraxin-3 (PTX3) and intercellular adhesion molecule-1 (ICAM-1), which, in combination, may contribute to thrombosis and local lung tissue injury.
Systemic fungal infections are a major cause of infection-related mortality in patients with hematologic malignancies. This report addresses the case of an adolescent patient with acute lymphoblastic leukemia who underwent three allogeneic hematopoietic stem cell transplantations and developed pulmonary aspergillosis. Combination therapy with liposomal amphotericin B (L-AmB, 3?mg/kg bw/day) and caspofungin (CAS, 50 mg/day) during the first allogeneic hematopoietic stem cell transplantation (HSCT) improved the pulmonary situation. After shifting the antifungal combination therapy to oral voriconazole (2 × 200?mg/day) and CAS, a new pulmonal lesion occurred alongside the improvements in the existing pulmonary aspergillosis. An antifungal combination during a second HSCT with L-AmB (3?mg/kg bw/day) and CAS showed an improvement in the pulmonary aspergillosis. A combination therapy with CAS and L-AmB (1?mg/kg bw/day) during the third HSCT led once again to progress the pulmonary aspergillosis, after increasing the L-AMB to 3?mg/kg bw/day for recovery. The presented case provides an example of how, despite severe immunosuppression, a combination of antifungal drugs administered intravenously at therapeutic dosages may be more efficient than either intravenous monotherapy or combinations of intravenous and oral antifungals in selecting pediatric and adolescent patients with proven fungal infections.
Gecko-inspired arrays of micropillars made of a liquid crystalline elastomer display thermoswitchable adhesive behavior as a consequence of elongation changes caused by reorientation of the mesogens at the nematic-isotropic (N-I) phase transition.
In this issue, Paek et al. describe two phenomena. First, they show that intermediate concentrations of a "transgenic" autoantigen may cause a lichen planus-like autoimmune disease. Second, and more importantly, they show that high doses of peptide antigen suppress the expression of the T-cell receptor and coreceptors, particularly CD8, and that this suppression improves this T-cell-mediated, destructive inflammatory skin disease that is similar to erosive lichen planus.
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