From Alien Species to Alien Communities: Host- and Habitat-Associated Microbiomes in an Alien Amphibian.

Alien species can host diverse microbial communities. These associated microbiomes may be important in the invasion process and their analysis requires a holistic community-based approach. We analysed the skin and gut microbiome of Eleutherodactylus johnstonei from native range populations in St Lucia and exotic range populations in Guadeloupe, Colombia, and European greenhouses along with their respective environmental microbial reservoir through a 16S metabarcoding approach. We show that amphibian-associated and environmental microbial communities can be considered as meta-communities that interact in the assembly process. High proportions of bacteria can disperse between frogs and environment, while respective abundances are rather determined by niche effects driven by the microbial community source and spatial environmental properties. Environmental transmissions appeared to have higher relevance for skin than for gut microbiome composition and variation. We encourage further experimental studies to assess the implications of turnover in amphibian-associated microbial communities and potentially invasive microbiota in the context of invasion success and impacts. Within this novel framework of "nested invasions," (meta-)community ecology thinking can complement and widen the traditional perspective on biological invasions.

A comparative study of two separation methods to isolate monocytes.

Separation of specific blood cells is necessary for a deeper insight into their role in health and disease. To obtain such cells, efficient and robust isolation methods are needed. We compare here the Fab-based Traceless Affinity Cell Selection (TACS®) technology and the Magnetic Activated Cell Sorting (MACS®) technology to isolate human monocytes from whole blood and buffy coats as well as the differentiation of the isolated monocytes to dendritic cells (DCs). TACS® is a positive selection technology using immune affinity chromatography based on CD-specific low affinity Fab-fragments for the reversible capture and release of target cells. The positive selection by MACS® is based on magnetic beads coated with specific high affinity monoclonal antibodies to catch the target cells. The target cells separated by TACS® are "label-free" while cells positively isolated by MACS® will carry the cell specific label. Our data show that the separation methods described here are well suited to obtain functional monocytes of high quality and purity. A differentiation of the cells into DCs leads to comparable results with the exception that CD1a expression levels on immature and mature DCs are elevated when monocytes are isolated using the TACS® technology. Taken together, our results suggest that the TACS® method may be of advantage when preparing monocytes and monocyte-derived DCs for functional analyses, while the MACS® method seems to be capable of higher monocyte recoveries. © 2018 International Society for Advancement of Cytometry.

Efficient immunoaffinity chromatography of lymphocytes directly from whole blood.

We show that defined lymphocytes can be rapidly purified by immunoaffinity chromatography starting directly from whole blood. The method relies on low-affinity Fab-fragments attached to a column-matrix combined with the reversible Strep-tag technology. Compared to established cell enrichment protocols, the Strep-tag affinity chromatography of cells is independent of erythrocyte lysis or centrifugation steps, allowing for simple cell-enrichment with good yields, high purities, and excellent functionality of purified cells.

Mid-regional pro-adrenomedullin (MR-proADM) and mid-regional pro-atrial natriuretic peptide (MR-proANP) in severe aortic valve stenosis: association with outcome after transcatheter aortic valve implantation (TAVI).

BACKGROUND: This study aimed to assess the association of mid-regional (MR) pro-adrenomedullin (MR-proADM) and MR-pro-A-type natriuretic peptide (MR-proANP) in comparison to N-terminal pro-natriuretic peptide (NT-proBNP) with outcome in patients with aortic stenosis (AS) treated with transcatheter aortic valve implantation (TAVI). METHODS: One hundred consecutive TAVI patients were included in this prospective study. Association of preinterventional levels of MR-proADM, MR-proANP, NT-proBNP, C-reactive protein (CrP), and high-sensitive cardiac Troponin T (hsTN) with 30-day and 1-year outcome was analyzed. RESULTS: There was no association with 30-day outcome, but all markers were associated with 1-year cardiovascular events and all-cause mortality. The combined biomarker analysis further improved risk prediction. CONCLUSIONS: In TAVI patients MR-proADM, MR-proANP, and NT-proBNP are promising predictors of adverse events within 1 year. Integration of these biomarkers into decision pathways may help to identify patients at higher risk.

Computational Drug Repositioning by Target Hopping: A Use Case in Chagas Disease.

BACKGROUND: Drug repositioning aims to identify novel indications for existing drugs. One approach to repositioning exploits shared binding sites between the drug targets and other proteins. Here, we review the principle and algorithms of such target hopping and illustrate them in Chagas disease, an in Latin America widely spread, but neglected disease. CONCLUSION: We demonstrate how target hopping recovers known treatments for Chagas disease and predicts novel drugs, such as the antiviral foscarnet, which we predict to target Farnesyl Pyrophosphate Synthase in Trypanosoma cruzi, the causative agent of Chagas disease.

MicroRNA-155 Exerts Cell-Specific Antiangiogenic but Proarteriogenic Effects During Adaptive Neovascularization.

BACKGROUND: Adaptive neovascularization after arterial occlusion is an important compensatory mechanism in cardiovascular disease and includes both the remodeling of pre-existing vessels to collateral arteries (arteriogenesis) and angiogenic capillary growth. We now aimed to identify regulatory microRNAs involved in the modulation of neovascularization after femoral artery occlusion in mice. METHODS AND RESULTS: Using microRNA-transcriptome analysis, we identified miR-155 as a downregulated microRNA during hindlimb ischemia. Correspondingly, inhibition of miR-155 in endothelial cells had a stimulatory effect on proliferation and angiogenic tube formation via derepression of its direct target gene angiotensin II type 1 receptor. Surprisingly, miR-155-deficient mice showed an unexpected phenotype in vivo, with a strong reduction of blood flow recovery after femoral artery ligation (arteriogenesis) dependent on the attenuation of leukocyte-endothelial interaction and a reduction of proarteriogenic cytokine expression. Consistently, miR-155-deficient macrophages exhibit a specific alteration of the proarteriogenic cytokine expression profile, which is partly mediated by the direct miR-155 target gene SOCS-1. CONCLUSIONS: Our data demonstrate that miR-155 exerts an antiangiogenic but proarteriogenic function in the regulation of neovascularization via the suppression of divergent cell-specific target genes and that its expression in both endothelial and bone marrow-derived cells is essential for arteriogenesis in response to hindlimb ischemia in mice.

Therapeutic activity of multiple common γ-chain cytokine inhibition in acute and chronic GVHD.

The common γ chain (CD132) is a subunit of the interleukin (IL) receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Because levels of several of these cytokines were shown to be increased in the serum of patients developing acute and chronic graft-versus-host disease (GVHD), we reasoned that inhibition of CD132 could have a profound effect on GVHD. We observed that anti-CD132 monoclonal antibody (mAb) reduced acute GVHD potently with respect to survival, production of tumor necrosis factor, interferon-γ, and IL-6, and GVHD histopathology. Anti-CD132 mAb afforded protection from GVHD partly via inhibition of granzyme B production in CD8 T cells, whereas exposure of CD8 T cells to IL-2, IL-7, IL-15, and IL-21 increased granzyme B production. Also, T cells exposed to anti-CD132 mAb displayed a more naive phenotype in microarray-based analyses and showed reduced Janus kinase 3 (JAK3) phosphorylation upon activation. Consistent with a role of JAK3 in GVHD, Jak3(-/-) T cells caused less severe GVHD. Additionally, anti-CD132 mAb treatment of established chronic GVHD reversed liver and lung fibrosis, and pulmonary dysfunction characteristic of bronchiolitis obliterans. We conclude that acute GVHD and chronic GVHD, caused by T cells activated by common γ-chain cytokines, each represent therapeutic targets for anti-CD132 mAb immunomodulation.

Galectin-3 predicts short- and long-term outcome in patients undergoing transcatheter aortic valve implantation (TAVI).

Until now, no reliable biomarker has become available for short- or long-term outcome prediction in patients undergoing transcatheter aortic valve implantation (TAVI). Our goal was to investigate whether galectin-3 is also suited for risk assessment in TAVI patients. Galectin-3, a novel marker indicative for myocardial fibrosis, has prognostic value in heart failure. We included 101 patients undergoing TAVI in this prospective, single-center, observational study. Baseline galectin-3 levels were correlated to the VARC 30-day safety and one-year efficacy endpoint as well as to total mortality and cardiovascular events at one year. At baseline, mean galectin-3 level for the entire group was 18.1 (± 11.1) ng/ml. Of the 101 patients, 36 had a galectin-3 value above the cut off value of 17.8 ng/ml. These patients had significantly higher systolic pulmonary artery and capillary wedge pressures. The hazard ratio in patients with galectin-3 > 17.8 ng/ml was 3.36 (95% confidence interval, CI: 1.47-7.69; p=0.004) for the VARC 30-day safety endpoint, 5.12 (95% CI: 2.10-12.47; p<0.001) for one-year cardiovascular events, and 4.48 (95% CI: 1.56-12.91; p=0.005) for all-cause mortality. This prediction remained stable even after adjusting for possible confounders including age, sex, glomerular filtration rate, and NT-proBNP. Furthermore, the prediction was even more valuable when combining galectin-3 with NTproBNP. In summary elevated galectin-3 levels are associated with adverse outcome after TAVI. Combining galectin-3 with NT-proBNP provides additive predictive value of risk stratification.

Neutrophil granulocytes recruited upon translocation of intestinal bacteria enhance graft-versus-host disease via tissue damage.

Acute graft-versus-host disease (GVHD) considerably limits wider usage of allogeneic hematopoietic cell transplantation (allo-HCT). Antigen-presenting cells and T cells are populations customarily associated with GVHD pathogenesis. Of note, neutrophils are the largest human white blood cell population. The cells cleave chemokines and produce reactive oxygen species, thereby promoting T cell activation. Therefore, during an allogeneic immune response, neutrophils could amplify tissue damage caused by conditioning regimens. We analyzed neutrophil infiltration of the mouse ileum after allo-HCT by in vivo myeloperoxidase imaging and found that infiltration levels were dependent on the local microbial flora and were not detectable under germ-free conditions. Physical or genetic depletion of neutrophils reduced GVHD-related mortality. The contribution of neutrophils to GVHD severity required reactive oxygen species (ROS) because selective Cybb (encoding cytochrome b-245, beta polypeptide, also known as NOX2) deficiency in neutrophils impairing ROS production led to lower levels of tissue damage, GVHD-related mortality and effector phenotype T cells. Enhanced survival of Bcl-xL transgenic neutrophils increased GVHD severity. In contrast, when we transferred neutrophils lacking Toll-like receptor-2 (TLR2), TLR3, TLR4, TLR7 and TLR9, which are normally less strongly activated by translocating bacteria, into wild-type C57BL/6 mice, GVHD severity was reduced. In humans, severity of intestinal GVHD strongly correlated with levels of neutrophils present in GVHD lesions. This study describes a new potential role for neutrophils in the pathogenesis of GVHD in both mice and humans.

Inflammatory neovascularization during graft-versus-host disease is regulated by αv integrin and miR-100.

Acute graft-versus-host disease (GvHD) is a complex process involving endothelial damage and neovascularization. Better understanding of the pathophysiology of neovascularization during GvHD could help to target this process while leaving T-cell function intact. Under ischemic conditions, neovascularization is regulated by different micro RNAs (miRs), which potentially play a role in inflamed hypoxic GvHD target organs. We observed strong neovascularization in the murine inflamed intestinal tract (IT) during GvHD. Positron emission tomography imaging demonstrated abundant αvß3 integrin expression within intestinal neovascularization areas. To interfere with neovascularization, we targeted αv integrin-expressing endothelial cells, which blocked their accumulation in the IT and reduced GvHD severity independent of immune reconstitution and graft-versus-tumor effects. Additionally, enhanced neovascularization and αv integrin expression correlated with GvHD severity in humans. Expression analysis of miRs in the inflamed IT of mice developing GvHD identified miR-100 as significantly downregulated. Inactivation of miR-100 enhanced GvHD indicating a protective role for miR-100 via blocking inflammatory neovascularization. Our data from the mouse model and patients indicate that inflammatory neovascularization is a central event during intestinal GvHD that can be inhibited by targeting αv integrin. We identify negative regulation of GvHD-related neovascularization by miR-100, which indicates common pathomechanistic features of GvHD and ischemia.

Inhibition of protein geranylgeranylation and farnesylation protects against graft-versus-host disease via effects on CD4 effector T cells.

Despite advances in immunosuppressive regimens, acute graft-versus-host disease remains a frequent complication of allogeneic hematopoietic cell transplantation. Pathogenic donor T cells are dependent on correct attachment of small GTPases to the cell membrane, mediated by farnesyl- or geranylgeranyl residues, which, therefore, constitute potential targets for graft-versus-host disease prophylaxis. A mouse model was used to study the impact of a farnesyl-transferase inhibitor and a geranylgeranyl-transferase inhibitor on acute graft-versus-host disease, anti-cytomegalovirus T-cell responses and graft-versus-leukemia activity. Treatment of mice undergoing allogeneic hematopoietic cell transplantation with farnesyl-transferase inhibitor and geranylgeranyl-transferase inhibitor reduced the histological severity of graft-versus-host disease and prolonged survival significantly. Mechanistically, farnesyl-transferase inhibitor and geranylgeranyl-transferase inhibitor treatment resulted in reduced alloantigen-driven expansion of CD4 T cells. In vivo treatment led to increased thymic cellularity and polyclonality of the T-cell receptor repertoire by reducing thymic graft-versus-host disease. These effects were absent when squalene production was blocked. The farnesyl-transferase inhibitor and geranylgeranyl-transferase inhibitor did not compromise CD8 function against leukemia cells or reconstitution of T cells that were subsequently responsible for anti-murine cytomegalovirus responses. In summary, we observed an immunomodulatory effect of inhibitors of farnesyl-transferase and geranylgeranyl-transferase on graft-versus-host disease, with enhanced functional immune reconstitution. In the light of the modest toxicity of farnesyl-transferase inhibitors such as tipifarnib in patients and the potent reduction of graft-versus-host disease in mice, farnesyl-transferase and geranylgeranyl-transferase inhibitors could help to reduce graft-versus-host disease significantly without having a negative impact on immune reconstitution.

The small molecule inhibitor QLT0267 Radiosensitizes squamous cell carcinoma cells of the head and neck.

BACKGROUND: The constant increase of cancer cell resistance to radio- and chemotherapy hampers improvement of patient survival and requires novel targeting approaches. Integrin-Linked Kinase (ILK) has been postulated as potent druggable cancer target. On the basis of our previous findings clearly showing that ILK transduces antisurvival signals in cells exposed to ionizing radiation, this study evaluated the impact of the small molecule inhibitor QLT0267, reported as putative ILK inhibitor, on the cellular radiation survival response of human head and neck squamous cell carcinoma cells (hHNSCC). METHODOLOGY/PRINCIPAL FINDINGS: Parental FaDu cells and FaDu cells stably transfected with a constitutively active ILK mutant (FaDu-IH) or empty vectors, UTSCC45 cells, ILK(floxed/floxed(fl/fl)) and ILK(-/-) mouse fibroblasts were used. Cells grew either two-dimensionally (2D) on or three-dimensionally (3D) in laminin-rich extracellular matrix. Cells were treated with QLT0267 alone or in combination with irradiation (X-rays, 0-6 Gy single dose). ILK knockdown was achieved by small interfering RNA transfection. ILK kinase activity, clonogenic survival, number of residual DNA double strand breaks (rDSB; gammaH2AX/53BP1 foci assay), cell cycle distribution, protein expression and phosphorylation (e.g. Akt, p44/42 mitogen-activated protein kinase (MAPK)) were measured. Data on ILK kinase activity and phosphorylation of Akt and p44/42 MAPK revealed a broad inhibitory spectrum of QLT0267 without specificity for ILK. QLT0267 significantly reduced basal cell survival and enhanced the radiosensitivity of FaDu and UTSCC45 cells in a time- and concentration-dependent manner. QLT0267 exerted differential, cell culture model-dependent effects with regard to radiogenic rDSB and accumulation of cells in the G2 cell cycle phase. Relative to corresponding controls, FaDu-IH and ILK(fl/fl) fibroblasts showed enhanced radiosensitivity, which failed to be antagonized by QLT0267. A knockdown of ILK revealed no change in clonogenic survival of the tested cell lines as compared to controls. CONCLUSIONS/SIGNIFICANCE: Our data clearly show that the small molecule inhibitor QLT0267 has potent cytotoxic and radiosensitizing capability in hHNSCC cells. However, QLT0267 is not specific for ILK. Further in vitro and in vivo studies are necessary to clarify the potential of QLT0267 as a targeted therapeutic in the clinic.