Point Mutations in the 14-α Sterol Demethylase Cyp51A or Cyp51C Could Contribute to Azole Resistance in Aspergillus flavus.

Infections caused by Aspergillus species are being increasingly reported. Aspergillus flavus is the second most common species within this genus causing invasive infections in humans, and isolates showing azole resistance have been recently described. A. flavus has three cyp51-related genes (cyp51A, cyp51B, and cyp51C) encoding 14-α sterol demethylase-like enzymes which are the target of azole drugs. In order to study triazole drug resistance in A. flavus, three strains showing reduced azole susceptibility and 17 azole susceptible isolates were compared. The three cyp51-related genes were amplified and sequenced. A comparison of the deduced Cyp51A, Cyp51B, and Cyp51C protein sequences with other protein sequences from orthologous genes in different filamentous fungi led to a protein identity that ranged from 50% to 80%. Cyp51A and Cyp51C presented several synonymous and non-synonymous point mutations among both susceptible and non-susceptible strains. However, two amino acid mutations were present only in two resistant isolates: one strain harbored a P214L substitution in Cyp51A, and another a H349R in Cyp51C that also showed an increase of cyp51A and cyp51C gene expression compared to the susceptible strain ATCC2004304. Isolates that showed reduced in vitro susceptibility to clinical azoles exhibited a different susceptibility profile to demethylation inhibitors (DMIs). Although P214L substitution might contribute to azole resistance, the role of H349R substitution together with changes in gene expression remains unclear.This research was funded by Fondo de Investigacion Sanitaria (FIS PI18CIII/00045) and also by Plan Nacional de I+D+i 2013–2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD16/CIII/0004/0003), co-financed by European Development Regional Fund ERDF “A way to achieve Europe”, Operative program Intelligent Growth 2014-2020. J.L. holds a predoctoral fellowship from the Fondo de Investigación Sanitaria (F17CIII/00037).S

CCR8 leads to eosinophil migration and regulates neutrophil migration in murine allergic enteritis

Allergic enteritis (AE) is a gastrointestinal form of food allergy. This study aimed to elucidate cellular and molecular mechanisms of AE using a murine model. To induce AE, BALB/c wild type (WT) mice received intraperitoneal sensitization with ovalbumin (an egg white allergen) plus ALUM and feeding an egg white (EW) diet. Microarray analysis showed enhanced gene expression of CC chemokine receptor (CCR) 8 and its ligand, chemokine CC motif ligand (CCL) 1 in the inflamed jejunum. Histological and FACS analysis showed that CCR8 knock out (KO) mice exhibited slightly less inflammatory features, reduced eosinophil accumulation but accelerated neutrophil accumulation in the jejunums, when compared to WT mice. The concentrations of an eosinophil chemoattractant CCL11 (eotaxin-1), but not of IL-5, were reduced in intestinal homogenates of CCR8KO mice, suggesting an indirect involvement of CCR8 in eosinophil accumulation in AE sites by inducing CCL11 expression. The potential of CCR8 antagonists to treat allergic asthma has been discussed. However, our results suggest that CCR8 blockade may promote neutrophil accumulation in the inflamed intestinal tissues, and not be a suitable therapeutic target for AE, despite the potential to reduce eosinophil accumulation. This study advances our knowledge to establish effective anti-inflammatory strategies in AE treatment.Fil: Blanco-Pérez, Frank. Paul-ehrlich-institut;Fil: Kato, Yoichiro. Tokyo Women's Medical University;Fil: Gonzalez-Menendez, Irene. Universitätsklinikum Tübingen Medizinische Fakultät;Fil: Laiño, Jonathan Emiliano. Paul-ehrlich-institut;Fil: Ohbayashi, Masaharu. Toyohashi Sozo University;Fil: Burggraf, Manja. Paul-ehrlich-institut;Fil: Krause, Maren. Paul-ehrlich-institut;Fil: Kirberg, Jörg. Paul-ehrlich-institut;Fil: Iwakura, Yoichiro. Tokyo University Of Science;Fil: Martella, Manuela. Universitätsklinikum Tübingen Medizinische Fakultät;Fil: Quintanilla-Martinez, Leticia. Universitätsklinikum Tübingen Medizinische Fakultät;Fil: Shibata, Noriyuki. Tokyo Women's Medical University;Fil: Vieths, Stefan. Paul-ehrlich-institut;Fil: Scheurer, Stephan. Paul-ehrlich-institut;Fil: Toda, Masako. Paul-ehrlich-institut; . Tohoku University

The inflammation in cutaneous lichen planus is dominated by IFN‐ϒ and IL‐21—A basis for therapeutic JAK1 inhibition

Cutaneous lichen planus (CLP) and psoriasis (PSO) are both common chronic inflammatory skin diseases for which development of new treatments requires the identification of key targets. While PSO is a typical Th17/IL-17-disorder, there is some evidence that Th1/IFN-ɣ dominate the inflammatory process in CLP. Nonetheless, the immunopathogenesis of CLP is not fully explained and key immunological factors still have to be recognized. In this study, we compared the immune signature of CLP lesions with the well-characterized inflammation present in PSO skin. First, we analysed the histological and immunohistological characteristics of CLP and PSO. Second, we assessed the cytokine expression (IL1A, IL1B, IL4, IL6, IL8, IL10, IL17A, IL19, IL21, IL22, IL23A, IL13, IFNG, TNF, IL12A, IL12B and IL36G) of lesional skin of CLP with PSO by qPCR. Histology revealed a similar epidermal thickness in CLP and PSO. Immunohistochemically, both diseases presented with an inflammatory infiltrate mainly composed by CD3+CD4+ T cells rather than CD3+CD8+. Importantly, mRNA analysis showed a distinct cytokine signature: while levels of IL12B, IL1A, IL6 and IL23 were similar between the two groups, the characteristic PSO-associated cytokines IL8, IL17A, IL22, IL19 and IL36G were expressed at very low levels in CLP. In contrast, CLP lesional skin was dominated by the expression of IFNG, IL21, IL4, IL12A and TNF. Immunohistochemistry confirmed the dominance of IL-21, IFN-ɣ and also pSTAT1 in the dermal infiltrate of CLP, while IL-17A was more present in PSO. Collectively, this study improves our understanding of the immunological factors dominating CLP. The dominating cytokines and signalling proteins identified suggest that anti-cytokine therapeutics like JAK inhibitors may be beneficial in CLP

Mast cells partly contribute to allergic enteritis development: Findings in two different mast cell-deficient mice

Allergic enteritis (AE) is a gastrointestinal form of food allergy. The presence of mast cells and granulocytes has been detected in the inflamed tissues in AE. In this study, we aimed to elucidate the role of mast cells in AE development using two mast cell-deficient mouse strains: KIT(W-sh/W-sh) bearing the W-sash (W(sh)) inversion mutation and Cpa3Cre/+, which lack mast cells due to Cre-mediated mast cell eradication, were used in an AE experimental model. The development of clinical symptoms (e.g. drop in body temperature and weight loss) were abolished in both strains, whereas inflammatory levels of AE (e.g. villous atrophy, edema, and granulocyte accumulation) were reduced mainly in KITW-sh/W-sh mice. FACS analysis of the KITW-sh/W-sh intestinal lamina propria, showed a reduction in the eosinophil (CD45+CD11b+SiglecF+cells) and neutrophil (CD45+CD11b+SiglecF−Ly6G+ cells) accumulation. Cpa3Cre/+ mice showed reduced eosinophil (CD45+CD11b+SiglecF+cells) accumulation, but neutrophil (CD45+CD11b+SiglecF−Ly6G+ cells) accumulation was retained at AE sites. The concentrations of CC chemokine ligand 1 (CCL1), a known CC chemokine receptor 8 ligand leading to eosinophil recruitment, was reduced in intestinal homogenates of both mast cell-deficient mouse strains. These results suggest that mast cells play a role in AE development in part by expressing CCL1 and contributing to eosinophil accumulation at AE. This study offers implications for establishing AE treatments that target infiltrating leucocytes in AE tissues.Fil: Blanco Pérez, Frank. No especifíca;Fil: Gonzalez Menendez, Irene. No especifíca;Fil: Stassen, Michael. Johannes Gutenberg Universitat Mainz; AlemaniaFil: Kato, Yoichiro. Tokyo Women's Medical University; JapónFil: Laiño, Jonathan Emiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Kirberg, Jörg. No especifíca;Fil: Krause, Maren. No especifíca;Fil: Martella, Manuela. No especifíca;Fil: Shibata, Noriyuki. Tokyo Women's Medical University; JapónFil: Quintanilla-Martinez, Leticia. No especifíca;Fil: Feyerabend, Thorsten B.. No especifíca;Fil: Rodewald, Hans Reimer. No especifíca;Fil: Galli, Stephen J.. University of Stanford; Estados UnidosFil: Vieths, Stefan. No especifíca;Fil: Scheurer, Stephan. No especifíca;Fil: Toda, Masako. No especifíca

Cancer immunotherapy is accompanied by distinct metabolic patterns in primary and secondary lymphoid organs observed by non-invasive in vivo18F-FDG-PET

Purpose: Cancer immunotherapy depends on a systemic immune response, but the basic underlying mechanisms are still largely unknown. Despite the very successful and widespread use of checkpoint inhibitors in the clinic, the majority of cancer patients do not benefit from this type of treatment. In this translational study, we investigated whether noninvasive in vivo positron emission tomography (PET) imaging using 2-[18F]fluoro-2-deoxy-D-glucose (18F-FDG) is capable of detecting immunotherapy-associated metabolic changes in the primary and secondary lymphoid organs and whether this detection enables the prediction of a successful anti-cancer immune response. Methods: RIP1-Tag2 mice with progressed endogenous insular cell carcinomas underwent a combined cancer immunotherapy consisting of CD4+ T cells plus monoclonal antibodies (mAbs) against programmed death ligand-1 (PD-L1) and lymphocyte activation gene-3 (LAG-3) or a sham treatment after radiation-mediated immune cell depletion. A second cohort of RIP1-Tag2 mice underwent exclusive checkpoint inhibitor therapy (CIT) using anti-PD-L1/LAG-3 mAbs or sham treatment without initial immune cell depletion to mimic the clinical situation. All mice were monitored by 18F-FDG-PET combined with anatomical magnetic resonance imaging (MRI). In addition, we retrospectively analyzed PET / computed tomography (CT) scans (PET/CT) regarding 18F-FDG uptake of CIT-treated metastatic melanoma patients in the spleen (n=23) and bone marrow (BM; n=20) as well as blood parameters (n=17-21). Results: RIP1-Tag2 mice with advanced insular cell carcinomas treated with combination immunotherapy exhibited significantly increased 18F-FDG uptake in the spleen compared to sham-treated mice. Histopathology of the spleens from treated mice revealed atrophy of the white pulp with fewer germinal centers and an expanded red pulp with hyperplasia of neutrophils than those of sham-treated mice. Immunohistochemistry and flow cytometry analyses of the spleens revealed a lower number of T cells and a higher number of neutrophils compared to those in the spleens of sham-treated mice. Flow cytometry of the BM showed enhanced activation of T cells following the treatment schemes that included checkpoint inhibitors. The ratio of 18F-FDG uptake at baseline to the uptake at follow-up in the spleens of exclusively CIT-treated RIP1-Tag2 mice was significantly enhanced, but the ratio was not enhanced in the spleens of the sham-treated littermates. Flow cytometry analysis confirmed a reduced number of T cells in the spleens of exclusively CIT-treated mice compared to that of sham-treated mice. A retrospective analysis of clinical 18F-FDG-PET/CT scans revealed enhanced 18F-FDG uptake in the spleens of some successfully CIT-treated patients with metastatic melanoma, but there were no significant differences between responders and non-responders. The analysis of the BM in clinical 18F-FDG-PET/CT scans with a computational segmentation tool revealed significantly higher baseline 18F-FDG uptake in patients who responded to CIT than in non-responders, and this relationship was independent of bone metastasis, even in the baseline scan. Conclusions: Thus, we are presenting the first translational study of solid tumors focusing on the metabolic patterns of primary and secondary lymphoid organs induced by the systemic immune response after CIT. We demonstrate that the widely available 18F-FDG-PET modality is an applicable translational tool that has high potential to stratify patients at an early time point

CK2b regulates thrombopoiesis and Ca21-Triggered platelet activation in arterial thrombosis

© 2017 by The American Society of Hematology. Platelets, anucleated megakaryocyte (MK)-derived cells, play a major role in hemostasis and arterial thrombosis. Although protein kinase casein kinase 2 (CK2) is readily detected in MKs and platelets, the impact of CK2-dependent signaling on MK/platelet (patho-) physiology has remained elusive. The present study explored the impact of the CK2 regulatory b-subunit on platelet biogenesis and activation. MK/platelet-specific genetic deletion of CK2β (ck2β-/-) in mice resulted in a significant macrothrombocytopenia and an increased extramedullar megakaryopoiesis with an enhanced proportion of premature platelets. Although platelet life span was only mildly affected, ck2β-/- MK displayed an abnormal microtubule structure with a drastically increased fragmentation within bone marrow and a significantly reduced proplatelet formation in vivo. In ck2β-/- platelets, tubulin polymerization was disrupted, resulting in an impaired thrombopoiesis and an abrogated inositol 1,4,5-Triphosphate receptor-dependent intracellular calcium (Ca21) release. Presumably due to a blunted increase in the concentration of cytosolic Ca21, activation-dependent increases of a and dense-granule secretion and integrin aIIbb3 activation, and aggregation were abrogated in ck2β-/- platelets. Accordingly, thrombus formation and stabilization under high arterial shear rates were significantly diminished, and thrombotic vascular occlusion in vivo wassignificantly blunted in ck2β-/- mice, accompanied by a slight prolongation of bleeding time. Following transient middle cerebral artery occlusion, ck2b-/- mice displayed significantly reduced cerebral infarct volumes, developed significantly less neurological deficits, and showed significantly better outcomes after ischemic stroke than ck2βfl/fl mice. The present observations reveal CK2b as a novel powerful regulator of thrombopoiesis, Ca2+-dependent platelet activation, and arterial thrombosis in vivo

Differential Expression of Melanopsin Isoforms Opn4L and Opn4S during Postnatal Development of the Mouse Retina

Photosensitive retinal ganglion cells (pRGCs) respond to light from birth and represent the earliest known light detection system to develop in the mouse retina. A number of morphologically and functionally distinct subtypes of pRGCs have been described in the adult retina, and have been linked to different physiological roles. We have previously identified two distinct isoforms of mouse melanopsin, Opn4L and Opn4S, which are generated by alternate splicing of the Opn4 locus. These isoforms are differentially expressed in pRGC subtypes of the adult mouse retina, with both Opn4L and Opn4S detected in M1 type pRGCs, and only Opn4L detected in M2 type pRGCs. Here we investigate the developmental expression of Opn4L and Opn4S and show a differential profile of expression during postnatal development. Opn4S mRNA is detected at relatively constant levels throughout postnatal development, with levels of Opn4S protein showing a marked increase between P0 and P3, and then increasing progressively over time until adult levels are reached by P10. By contrast, levels of Opn4L mRNA and protein are low at birth and show a marked increase at P14 and P30 compared to earlier time points. We suggest that these differing profiles of expression are associated with the functional maturation of M1 and M2 subtypes of pRGCs. Based upon our data, Opn4S expressing M1 type pRGCs mature first and are the dominant pRGC subtype in the neonate retina, whereas increased expression of Opn4L and the maturation of M2 type pRGCs occurs later, between P10 and P14, at a similar time to the maturation of rod and cone photoreceptors. We suggest that the distinct functions associated with these cell types will develop at different times during postnatal development

Existence of reprogrammed lymphoma stem cells in a murine ALCL-like model.

Funder: DJCLS (R14/22) MSCA-ITN-2015-ETN AlkatrasFunder: DFG (CRC850)Funder: DFG (CRC850) BMBF (DeCaRe, FKZ 01ZX1409B)Funder: DJCLS (R14/22) MSCA-ITN-2015-ETN Alkatras DFG (FOR 2033 B1)Funder: DJCLS (R14/22) MSCA-ITN-2015-ETN Alkatras Grant from the Government of Baden-Württemberg (BSL)While cancer stem cells are well established in certain hematologic and solid malignancies, their existence in T cell lymphoma is unclear and the origin of disease is not fully understood. To examine the existence of lymphoma stem cells, we utilized a mouse model of anaplastic large cell lymphoma. Established NPM-ALK+ lymphomas contained heterogeneous cell populations ranging from mature T cells to undifferentiated hematopoietic stem cells. Interestingly, CD4-/CD8- double negative (DN) lymphoma cells aberrantly expressed the T cell receptor α/β chain. Serial transplantation of sorted CD4/CD8 and DN lymphoma subpopulations identified lymphoma stem cells within the DN3/DN4 T cell population, whereas all other subpopulations failed to establish serial lymphomas. Moreover, transplanted lymphoma DN3/DN4 T cells were able to differentiate and gave rise to mature lymphoma T cells. Gene expression analyses unmasked stem-cell-like transcriptional regulation of the identified lymphoma stem cell population. Furthermore, these lymphoma stem cells are characterized by low CD30 expression levels, which might contribute to limited long-term therapeutic success in patients treated with anti-CD30-targeted therapies. In summary, our results highlight the existence of a lymphoma stem cell population in a NPM-ALK-driven CD30+ mouse model, thereby giving the opportunity to test innovative treatment strategies developed to eradicate the origin of disease

PiggyBac transposon tools for recessive screening identify B-cell lymphoma drivers in mice.

B-cell lymphoma (BCL) is the most common hematologic malignancy. While sequencing studies gave insights into BCL genetics, identification of non-mutated cancer genes remains challenging. Here, we describe PiggyBac transposon tools and mouse models for recessive screening and show their application to study clonal B-cell lymphomagenesis. In a genome-wide screen, we discover BCL genes related to diverse molecular processes, including signaling, transcriptional regulation, chromatin regulation, or RNA metabolism. Cross-species analyses show the efficiency of the screen to pinpoint human cancer drivers altered by non-genetic mechanisms, including clinically relevant genes dysregulated epigenetically, transcriptionally, or post-transcriptionally in human BCL. We also describe a CRISPR/Cas9-based in vivo platform for BCL functional genomics, and validate discovered genes, such as Rfx7, a transcription factor, and Phip, a chromatin regulator, which suppress lymphomagenesis in mice. Our study gives comprehensive insights into the molecular landscapes of BCL and underlines the power of genome-scale screening to inform biology

Clustering COVID-19 ARDS patients through the first days of ICU admission. An analysis of the CIBERESUCICOVID Cohort

Background Acute respiratory distress syndrome (ARDS) can be classified into sub-phenotypes according to different inflammatory/clinical status. Prognostic enrichment was achieved by grouping patients into hypoinflammatory or hyperinflammatory sub-phenotypes, even though the time of analysis may change the classification according to treatment response or disease evolution. We aimed to evaluate when patients can be clustered in more than 1 group, and how they may change the clustering of patients using data of baseline or day 3, and the prognosis of patients according to their evolution by changing or not the cluster.Methods Multicenter, observational prospective, and retrospective study of patients admitted due to ARDS related to COVID-19 infection in Spain. Patients were grouped according to a clustering mixed-type data algorithm (k-prototypes) using continuous and categorical readily available variables at baseline and day 3.Results Of 6205 patients, 3743 (60%) were included in the study. According to silhouette analysis, patients were grouped in two clusters. At baseline, 1402 (37%) patients were included in cluster 1 and 2341(63%) in cluster 2. On day 3, 1557(42%) patients were included in cluster 1 and 2086 (57%) in cluster 2. The patients included in cluster 2 were older and more frequently hypertensive and had a higher prevalence of shock, organ dysfunction, inflammatory biomarkers, and worst respiratory indexes at both time points. The 90-day mortality was higher in cluster 2 at both clustering processes (43.8% [n = 1025] versus 27.3% [n = 383] at baseline, and 49% [n = 1023] versus 20.6% [n = 321] on day 3). Four hundred and fifty-eight (33%) patients clustered in the first group were clustered in the second group on day 3. In contrast, 638 (27%) patients clustered in the second group were clustered in the first group on day 3.Conclusions During the first days, patients can be clustered into two groups and the process of clustering patients may change as they continue to evolve. This means that despite a vast majority of patients remaining in the same cluster, a minority reaching 33% of patients analyzed may be re-categorized into different clusters based on their progress. Such changes can significantly impact their prognosis