The engraftment of bone marrow-derived cells has been described not only during diseases of the central nervous system (CNS) but also under healthy conditions. However, previous studies pointing to ...an ample bone marrow cell engraftment used irradiation-induced bone marrow chimeras that evoked severe alterations of the CNS micromilieu including disturbances of the blood brain barrier (BBB), damage of endothelial cells and local induction of proinflammatory cytokines. On the other hand, parabiosis experiments using temporarily joined circulatory systems generally yielded low levels of myeloid cell chimerism thereby potentially underestimating bone marrow cell turnover with the CNS. To avoid these drawbacks we established a protocol using the alkylating agent busulfan prior to allogenic bone marrow transplantation from CX3CR1(GFP/+) donors. This regimen resulted in a stable and high peripheral myeloid chimerism, significantly reduced cytokine induction and preserved BBB integrity. Importantly, bone marrow cell recruitment to the CNS was strongly diminished under these conditions and only weakly enhanced during local neurodegeneration induced by facial nerve axotomy. These results underscore the requirement of local CNS conditioning for efficient recruitment of bone marrow cells, establish busulfan as an alternative treatment for studying bone marrow chimeras and suggest a critical re-evaluation of earlier chimeric studies involving irradiation or parabiosis regimens.
Common understanding suggests that the normal function of a "healthy" immune system safe-guards and protects against the development of malignancies, whereas a genetically impaired one might increase ...the likelihood of their manifestation. This view is primarily based on and apparently supported by an increased incidence of such diseases in patients with specific forms of immunodeficiencies that are caused by high penetrant gene defects. As I will review and discuss herein, such constellations merely represent the tip of an iceberg. The overall situation is by far more varied and complex, especially if one takes into account the growing difficulties to define what actually constitutes an immunodeficiency and what defines a cancer predisposition. The enormous advances in genome sequencing, in bioinformatic analyses and in the functional
and
assessment of novel findings together with the availability of large databases provide us with a wealth of information that steadily increases the number of sequence variants that concur with clinically more or less recognizable immunological problems and their consequences. Since many of the newly identified hard-core defects are exceedingly rare, their tumor predisposing effect is difficult to ascertain. The analyses of large data sets, on the other hand, continuously supply us with low penetrant variants that, at least in statistical terms, are clearly tumor predisposing, although their specific relevance for the respective carriers still needs to be carefully assessed on an individual basis. Finally, defects and variants that affect the same gene families and pathways in both a constitutional and somatic setting underscore the fact that immunodeficiencies and cancer predisposition can be viewed as two closely related errors of development. Depending on the particular genetic and/or environmental context as well as the respective stage of development, the same changes can have either a neutral, predisposing and, in some instances, even a protective effect. To understand the interaction between the immune system, be it "normal" or "deficient" and tumor predisposition and development on a systemic level, one therefore needs to focus on the structure and dynamic functional organization of the entire immune system rather than on its isolated individual components alone.
Mammalian genomes are organized into megabase-scale topologically associated domains (TADs). We demonstrate that disruption of TADs can rewire long-range regulatory architecture and result in ...pathogenic phenotypes. We show that distinct human limb malformations are caused by deletions, inversions, or duplications altering the structure of the TAD-spanning WNT6/IHH/EPHA4/PAX3 locus. Using CRISPR/Cas genome editing, we generated mice with corresponding rearrangements. Both in mouse limb tissue and patient-derived fibroblasts, disease-relevant structural changes cause ectopic interactions between promoters and non-coding DNA, and a cluster of limb enhancers normally associated with Epha4 is misplaced relative to TAD boundaries and drives ectopic limb expression of another gene in the locus. This rewiring occurred only if the variant disrupted a CTCF-associated boundary domain. Our results demonstrate the functional importance of TADs for orchestrating gene expression via genome architecture and indicate criteria for predicting the pathogenicity of human structural variants, particularly in non-coding regions of the human genome.
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•Disruptions of TADs lead to de novo enhancer-promoter interactions and misexpression•Misexpression occurs when CTCF-associated TAD boundary elements are disrupted•Structural variations disrupting TAD structures can cause malformation syndromes•Different phenotypes can result from one enhancer acting on different target genes
Disease-associated structural variants, when affecting CTCF-associated boundary elements, cause pathogenicity by disrupting the structure of topologically associated chromatin domains leading to ectopic promoter interactions and altered gene expression.
The development of a convex probe endobronchial ultrasound (CP-EBUS) videobronchoscope to allow real-time transbronchial needle aspiration (TBNA) has been a significant advance in minimally invasive ...lung cancer staging and diagnosis. Several recent studies have demonstrated CP-EBUS-TBNA to have recovery rivalling that of the current gold standard, cervical mediastinoscopy. These same studies have indicated that the safety of this procedure has no reported complications. The present case study presents two infectious complications from full extension endobronchial ultrasound transbronchial needle aspiration and discusses possible aetiologies of these infections, as well as implications for future application of this technology.
Staphylococcal superantigen‐like (SSL) proteins, one of the major virulence factor families produced by Staphylococcus aureus, were previously demonstrated to be immune evasion molecules that ...interfere with a variety of innate immune defences. However, in contrast to characterised SSLs, which inhibit immune functions, we show that SSL13 is a strong activator of neutrophils via the formyl peptide receptor 2 (FPR2). Moreover, our data show that SSL13 acts as a chemoattractant and induces degranulation and oxidative burst in neutrophils. As with many other staphylococcal immune evasion proteins, SSL13 shows a high degree of human specificity. SSL13 is not able to efficiently activate mouse neutrophils, hampering in vivo experiments. In conclusion, SSL13 is a neutrophil chemoattractant and activator that acts via FPR2. Therefore, SSL13 is a unique SSL member that does not belong to the immune evasion class but is a pathogen alarming molecule. Our study provides a new concept of SSLs; SSLs not only inhibit host immune processes but also recruit human neutrophils to the site of infection. This new insight allows us to better understand complex interactions between host and S. aureus pathological processes.
Background. Azole resistance is an emerging problem in Aspergillus fumigatus and complicates the management of patients with Aspergillus-related diseases. Selection of azole resistance may occur ...through exposure to azole fungicides in the environment. In the Netherlands a surveillance network was used to investigate the epidemiology of resistance selection in A. fumigatus. Methods. Clinical A. fumigatus isolates were screened for azole resistance in 8 university hospitals using azole agar dilution plates. Patient information was collected using an online questionnaire and azole-resistant A. fumigatus isolates were analyzed using gene sequencing, susceptibility testing, and genotyping. Air sampling was performed to investigate the presence of resistant isolates in hospitals and domiciles. Results. Between December 2009 and January 2011, 1315 A. fumigatus isolates from 921 patients were screened. A new cyp51A-mediated resistance mechanism (TR 46 /Y121F/T289A) was observed in 21 azole-resistant isolates from 15 patients in 6 hospitals. TR 46 /Y121F/T289A isolates were highly resistant to voriconazole (minimum inhibitory concentration ≥16 mg/L). Eight patients presented with invasive aspergillosis due to TR 46 /Y121F/T289A, and treatment failed in all 5 patients receiving primary therapy with voriconazole. TR 46 /Y121F/T289A Aspergillus fumigatus was recovered from 6 of 10 sampled environmental sites. Conclusions. We describe the emergence and geographical migration of a voriconazole highly resistant A. fumigatus that was associated with voriconazole treatment failure in patients with invasice aspergillosis. Recovery of TR 46 /Y121F/T289A from the environment suggests an environmental route of resistance selection. Exposure of A. fumigatus to azole fungicides may facilitate the emergence of new resistance mechanisms over time, thereby compromising the use of azoles in the management of Aspergillus-related diseases.
This study investigates whether childhood health acts as a mechanism through which socioeconomic status is transferred across generations. The study uses data from the Panel Study of Income Dynamics ...to track siblings and to estimate fixed-effects models that account for unobserved heterogeneity at the family level. The results demonstrate that disadvantaged social background is associated with poor childhood health. Subsequently, poor health in childhood has significant, direct, and large adverse effects on educational attainment and wealth accumulation. In addition, childhood health appears to have indirect effects on occupational standing, earnings, and wealth via educational attainment and adult health status. The results further show that socioeconomic health gradients are best understood as being embedded within larger processes of social stratification.
Integrins are used as prognostic indicators in breast cancer. Following engagement with extracellular matrix proteins, their signaling influences numerous cellular processes including migration, ...proliferation, and death. Integrin signaling varies between cell types through differential expression of integrin subunits, and changes within a given cell upon exposure to a cell agonist or through changes in its surroundings. These variations in signaling can profoundly affect the phenotypic, tumorogenecity and metastatic properties of cancer cells. In the present study, we investigated if there were differences in the expression of integrins, integrin structures, and integrin co-receptors within three breast cancer cells and if these differences effected integrin signaling.
Expression of integrins, urokinase receptor and vascular endothelial cell growth factor receptor (VEGFR) in metastatic MDA-MB-435 and MDA-MB-231, non-metastatic MCF7 and non-breast cancer Hek-293 cells was measured by flow cytometry. Cell adhesion was assessed using collagen, fibrinogen, fibronectin and vitronectin coated plates. Changes in kinase levels following PMA stimulation, and cell adhesion-induced activation of kinases were determined by western blot analysis. Distribution of actin stress fibers and focal adhesions was assessed by immunocytochemistry.
All cells expressed αv integrins, while high β5 and αvβ5 expression was restricted to the cancer cells and high β3 and αvβ3 expression was restricted to MDA-MB-435 cells. The two metastatic cells were the least adhesive, but all cells adhered well to most proteins in the absence of PMA. All proliferating cells expressed activated pSrc, but only proliferating metastatic cells expressed high pMEK levels. PMA treatment resulted in time-dependent changes in activated kinase levels, and only MDA-MB-231 cells constitutively expressed high levels of activated pMEK. MDA-MB-435 cells formed more stress fibers and focal adhesions and only exhibited adhesion-induced activation of pMEK and pFAK. All cells expressed the urokinase receptor, but MCF7 cells had markedly higher VEGFR expression. Adhesion induced differential expression of pFAK, pMEK and pERK.
This study demonstrates that breast cancers vary in their expression of integrins, their capacity to form focal adhesion and to signal through integrins. These differences likely contribute to phenotypic variations between cancer lines and account for some of the heterogeneity of breast cancer.
Zika virus (ZIKV) has recently emerged as a global health concern owing to its widespread diffusion and its association with severe neurological symptoms and microcephaly in newborns
. However, the ...molecular mechanisms that are responsible for the pathogenicity of ZIKV remain largely unknown. Here we use human neural progenitor cells and the neuronal cell line SK-N-BE2 in an integrated proteomics approach to characterize the cellular responses to viral infection at the proteome and phosphoproteome level, and use affinity proteomics to identify cellular targets of ZIKV proteins. Using this approach, we identify 386 ZIKV-interacting proteins, ZIKV-specific and pan-flaviviral activities as well as host factors with known functions in neuronal development, retinal defects and infertility. Moreover, our analysis identified 1,216 phosphorylation sites that are specifically up- or downregulated after ZIKV infection, indicating profound modulation of fundamental signalling pathways such as AKT, MAPK-ERK and ATM-ATR and thereby providing mechanistic insights into the proliferation arrest elicited by ZIKV infection. Functionally, our integrative study identifies ZIKV host-dependency factors and provides a comprehensive framework for a system-level understanding of ZIKV-induced perturbations at the levels of proteins and cellular pathways.
Reactive oxygen species (ROS) are generated by virus-infected cells; however, the physiological importance of ROS generated under these conditions is unclear. Here we found that the inflammation and ...cell death induced by exposure of mice or cells to sources of ROS were not altered in the absence of canonical ROS-sensing pathways or known cell-death pathways. ROS-induced cell-death signaling involved interactions among the cellular ROS sensor and antioxidant factor KEAP1, the phosphatase PGAM5 and the proapoptotic factor AIFM1. Pgam5
mice showed exacerbated lung inflammation and proinflammatory cytokines in an ozone-exposure model. Similarly, challenge with influenza A virus led to increased infiltration of the virus, lymphocytic bronchiolitis and reduced survival of Pgam5
mice. This pathway, which we have called 'oxeiptosis', was a ROS-sensitive, caspase independent, non-inflammatory cell-death pathway and was important for protection against inflammation induced by ROS or ROS-generating agents such as viral pathogens.
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