Congenital diaphragmatic hernia (CDH) is a malformation leading to pulmonary hypoplasia, which can be treated in utero by fetal tracheal occlusion (TO). However, the changes of gene expression induced by TO remain largely unknown but could be used to further improve the clinically used prenatal treatment of this devastating malformation. Therefore, we aimed to investigate the pulmonary transcriptome changes caused by surgical induction of diaphragmatic hernia (DH) and additional TO in the fetal rabbit model. Induction of DH was associated with 378 upregulated genes compared to controls when allowing a false-discovery rate (FDR) of 0.1 and a fold change (FC) of 2. Those genes were again downregulated by consecutive TO. But DH+TO was associated with an upregulation of 157 genes compared to DH and controls. When being compared to control lungs, 106 genes were downregulated in the DH group and were not changed by TO. Therefore, the overall pattern of gene expression in DH+TO is more similar to the control group than to the DH group. In this study, we further provide a database of gene expression changes induced by surgical creation of DH and consecutive TO in the rabbit model. Future treatment strategies could be developed using this dataset. We also discuss the most relevant genes that are involved in CDH.

Prenatal irradiation is known to perturb brain development. Epidemiological studies revealed that radiation exposure during weeks 8-15 of pregnancy was associated with an increased occurrence of mental disability and microcephaly. Such neurological deficits were reproduced in animal models, in which rodent behavioral testing is an often used tool to evaluate radiation-induced defective brain functionality. However, up to now, animal studies suggested a threshold dose of around 0.30 Gray (Gy) below which no behavioral alterations can be observed, while human studies hinted at late defects after exposure to doses as low as 0.10 Gy. Here, we acutely irradiated pregnant mice at embryonic day 11 with doses ranging from 0.10 to 1.00 Gy. A thorough investigation of the dose-response relationship of altered brain function and architecture following in utero irradiation was achieved using a behavioral test battery and volumetric 3D T2-weighted magnetic resonance imaging (MRI). We found dose-dependent changes in cage activity, social behavior, anxiety-related exploration, and spatio-cognitive performance. Although behavioral alterations in low-dose exposed animals were mild, we did unveil that both emotionality and higher cognitive abilities were affected in mice exposed to ≥0.10 Gy. Microcephaly was apparent from 0.33 Gy onwards and accompanied by deviations in regional brain volumes as compared to controls. Of note, total brain volume and the relative volume of the ventricles, frontal and posterior cerebral cortex, cerebellum, and striatum were most strongly correlated to altered behavioral parameters. Taken together, we present conclusive evidence for persistent low-dose effects after prenatal irradiation in mice and provide a better understanding of the correlation between their brain size and performance in behavioral tests.

IKAROS (encoded by IKZF1) is an important hematopoietic transcription factor critical for early B cell differentiation, with major defects known to lead to low B cell numbers and hypogammaglobulinemia. More perplexing is the link between IKZF1 variants and autoimmunity, including polymorphisms associated with susceptibility to SLE, and recently, rare variants driving monogenic autoimmunity. We identified a novel p.L188V mutation in IKZF1 in the index patient and her father and found this mutation to lead to loss of DNA binding. Peripheral B cells lacking a full complement of IKAROS function show upregulation of molecules accentuating B cell activation, while CD22, a key negative feedback circuit, is suppressed. The resulting hyperresponsiveness of peripheral B cells, in combination with elevated follicular helper T cell (Tfh) numbers, provides a putative mechanistic explanation for the association of IKZF1 variants with the emergence of autoimmune manifestations in this kindred.

Mesenchymal stem cells (MSCs) are a promising clinical therapy for ischemic stroke. However, critical parameters, such as the most effective administration route, remain unclear. Intravenous (i.v.) and intraarterial (i.a.) delivery routes have yielded varied outcomes across studies, potentially due to the unknown MSCs distribution. We investigated whether MSCs reached the brain following i.a. or i.v. administration after transient cerebral ischemia in rats, and evaluated the therapeutic effects of both routes. MSCs were labeled with dextran-coated superparamagnetic nanoparticles for magnetic resonance imaging (MRI) cell tracking, transmission electron microscopy and immunohistological analysis. MSCs were found in the brain following i.a. but not i.v. administration. However, the i.a. route increased the risk of cerebral lesions and did not improve functional recovery. The i.v. delivery is safe but MCS do not reach the brain tissue, implying that treatment benefits observed for this route are not attributable to brain MCS engrafting after stroke.

The present animal experiments were conducted to evaluate radioiodinated Hypericin (Hyp) for its regional distribution as well as theranostic potentials.

Several lines of evidence imply early alterations in endocannabinoid and phosphodiesterase 10A (PDE10A) signaling in Huntington disease (HD). Using [(18)F]MK-9470 and [(18)F]JNJ42259152 small-animal positron emission tomography (PET), we investigated for the first time cerebral changes in type 1 cannabinoid (CB1) receptor binding and PDE10A levels in vivo in presymptomatic, early symptomatic, and late symptomatic HD (R6/2) mice, in relation to glucose metabolism ([(18)F]FDG PET), brain morphology (magnetic resonance imaging) and motor function. Ten R6/2 and 16 wild-type (WT) mice were investigated at 3 different time points between the age of 4 and 13 weeks. Parametric CB1 receptor and PDE10A images were anatomically standardized to Paxinos space and analyzed voxelwise. Volumetric microMRI imaging was performed to assess HD pathology. In R6/2 mice, CB1 receptor binding was decreased in comparison with WT in a cluster comprising the bilateral caudate-putamen, globus pallidus, and thalamic nucleus at week 5 (-8.1% ± 2.6%, p = 1.7 × 10(-5)). Longitudinal follow-up showed further progressive decline compared with controls in a cluster comprising the bilateral hippocampus, caudate-putamen, globus pallidus, superior colliculus, thalamic nucleus, and cerebellum (late vs. presymptomatic age: -13.7% ± 3.1% for R6/2 and +1.5% ± 4.0% for WT, p = 1.9 × 10(-5)). In R6/2 mice, PDE10A binding potential also decreased over time to reach significance at early and late symptomatic HD (late vs. presymptomatic age: -79.1% ± 1.9% for R6/2 and +2.1% ± 2.7% for WT, p = 1.5 × 10(-4)). The observed changes in CB1 receptor and PDE10A binding were correlated to anomalies exhibited by R6/2 animals in motor function, whereas no correlation was found with magnetic resonance imaging-based striatal volume. Our findings point to early regional dysfunctions in endocannabinoid and PDE10A signaling, involving the caudate-putamen and lateral globus pallidus, which may play a role in the progression of the disease in R6/2 animals. PET quantification of in vivo CB1 and/or PDE10A binding may thus be useful early biomarkers for HD. Our results also provide evidence of subtle motor deficits at earlier stages than previously described.

The use of stably integrated reporter gene imaging provides a manner to monitor the in vivo fate of engrafted cells over time in a non-invasive manner. Here, we optimized multimodal imaging (small-animal PET, Cerenkov luminescence imaging (CLI) and bioluminescence imaging (BLI)) of mesenchymal stem cells (MSCs), by means of the human sodium iodide symporter (hNIS) and firefly luciferase (Fluc) as reporters.

The intronic microRNA (miR)-342 has been proposed as a potent tumor-suppressor gene. miR-342 is found to be downregulated or epigenetically silenced in multiple different tumor sites, and this loss of expression permits the upregulation of several key oncogenic pathways. In several different cell lines, lower miR-342 expression results in enhanced proliferation and metastasis potential, both in vitro and in xenogenic transplant conditions. Here, we sought to determine the function of miR-342 in an in vivo spontaneous cancer model, using the Ela1-TAg transgenic model of pancreatic acinar carcinoma. Through longitudinal magnetic resonance imaging monitoring of Ela1-TAg transgenic mice, either wild-type or knockout for miR-342, we found no role for miR-342 in the development, growth rate, or pathogenicity of pancreatic acinar carcinoma. These results indicate the importance of assessing miR function in the complex physiology of in vivo model systems and indicate that further functional testing of miR-342 is required before concluding it is a bona fide tumor-suppressor-miR.

Perfluorocarbon (PFC) particles are currently on the rise as cell labeling agents for ¹⁹F-MRI tracking of dendritic cell (DC)-based vaccines. In this work, we design theranostic PFC particles for single-step loading of DCs with both antigenic protein and with a liquid PFC for ¹⁹F-MRI detection of the antigen-loaded cells. Upon addition to DCs in vitro, the antigen-loaded PFC particles are efficiently internalized, resulting in intracellular presence of up to 40 pmol ¹⁹F atoms per cell. At the same time, the DCs become loaded with antigenic proteins, that can be efficiently processed, without important effects on cell viability or altering the DC's phenotype and the cell's capacity to respond to danger signals. In addition, antigen-loaded PFC particle containing DCs are capable of inducing extensive proliferation of antigen-specific CD8⁺ T cells in vitro. Importantly, the antigen-coated PFC particles allow in vitro ¹⁹F-MRI-based detection of the antigen-containing DCs with detection limits as low as 10³ cells μl⁻¹. The dual-modality characteristics of the designed particles could assure that only those DCs that have taken up the antigen, and hence are responsible for an immune response, are traceable via ¹⁹F-MRI. Taken together, these novel dual-modality particles represent an interesting strategy in the development of a traceable DC vaccine.

Transportin-SR2 (TRN-SR2, TNPO3, transportin 3) was previously identified as an interaction partner of human immunodeficiency virus type 1 (HIV-1) integrase and functions as a nuclear import factor of HIV-1. A possible role of capsid in transportin-SR2-mediated nuclear import was recently suggested by the findings that a chimeric HIV virus, carrying the murine leukemia virus (MLV) capsid and matrix proteins, displayed a transportin-SR2 independent phenotype, and that the HIV-1 N74D capsid mutant proved insensitive to transportin-SR2 knockdown.

The unambiguous detection of specific neuronal subtypes is up to now only possible with invasive techniques or optical imaging after genetic modification. High field magnetic resonance imaging (MRI) has the ability to visualize the brain structure and anatomy noninvasively, with high resolution--but missing the cell specific and functional information. Here we present a new tool for neuroimaging with MRI, enabling the selective detection of GABAergic neurons under in vivo conditions. The specific imaging contrast is achieved by a novel paramagnetic contrast agent, which responds to the activity of the enzyme glutamic acid decarboxylase--expressed solely by inhibitory neurons. The relaxivity of the complex is increased upon decarboxylation of two glutamic acid moieties, thus allowing increased water access to the inner and outer coordination spheres of the paramagnetic ion. The mechanism and specificity of activation were proven with tissue lysates and further applied to a differentiation protocol for murine embryonic stem cells. The relaxation enhancement was studied quantitatively and revealed decreased longitudinal relaxation times in the inhibitory neuron samples compared to the naïve stem cells in vitro and in vivo. Furthermore, this approach offers not only the discrimination of inhibitory, GABAergic neurons in the brain but also may expand the usefulness of MRI for functional imaging on a cellular level.

LEDGINs are novel allosteric HIV integrase (IN) inhibitors that target the lens epithelium-derived growth factor (LEDGF)/p75 binding pocket of IN. They block HIV-1 integration by abrogating the interaction between LEDGF/p75 and IN as well as by allosterically inhibiting the catalytic activity of IN.

Lens epithelium-derived growth factor/p75 (LEDGF/p75) is a transcriptional coactivator involved in stress response, autoimmune disease, cancer and HIV replication. A fusion between the nuclear pore protein NUP98 and LEDGF/p75 has been found in human acute and chronic myeloid leukemia and association of LEDGF/p75 with mixed-lineage leukemia (MLL)/menin is critical for leukemic transformation. During lentiviral replication, LEDGF/p75 tethers the pre-integration complex to the host chromatin resulting in a bias of integration into active transcription units (TUs). The consensus function of LEDGF/p75 is tethering of cargos to chromatin. In this regard, we determined the LEDGF/p75 chromatin binding profile. To this purpose, we used DamID technology and focused on the highly annotated ENCODE (Encyclopedia of DNA Elements) regions. LEDGF/p75 primarily binds downstream of the transcription start site of active TUs in agreement with the enrichment of HIV-1 integration sites at these locations. We show that LEDGF/p75 binding is not restricted to stress response elements in the genome, and correlation analysis with more than 200 genomic features revealed an association with active chromatin markers, such as H3 and H4 acetylation, H3K4 monomethylation and RNA polymerase II binding. Interestingly, some associations did not correlate with HIV-1 integration indicating that not all LEDGF/p75 complexes on the chromosome are amenable to HIV-1 integration.

The capacity to integrate transgenes into the host cell genome makes retroviral vectors an interesting tool for gene therapy. Although stable insertion resulted in successful correction of several monogenic disorders, it also accounts for insertional mutagenesis, a major setback in otherwise successful clinical gene therapy trials due to leukemia development in a subset of treated patients. Despite improvements in vector design, their use is still not risk-free. Lentiviral vector (LV) integration is directed into active transcription units by LEDGF/p75, a host-cell protein co-opted by the viral integrase. We engineered LEDGF/p75-based hybrid tethers in an effort to elicit a more random integration pattern to increase biosafety, and potentially reduce proto-oncogene activation. We therefore truncated LEDGF/p75 by deleting the N-terminal chromatin-reading PWWP-domain, and replaced this domain with alternative pan-chromatin binding peptides. Expression of these LEDGF-hybrids in LEDGF-depleted cells efficiently rescued LV transduction and resulted in LV integrations that distributed more randomly throughout the host-cell genome. In addition, when considering safe harbor criteria, LV integration sites for these LEDGF-hybrids distributed more safely compared to LEDGF/p75-mediated integration in wild-type cells. This approach should be broadly applicable to introduce therapeutic or suicide genes for cell therapy, such as patient-specific iPS cells.

The development of cancers involves the complex dysregulation of multiple cellular processes. With key functions in simultaneous regulation of multiple pathways, microRNA (miR) are thought to have important roles in the oncogenic formation process. miR-29a is among the most abundantly expressed miR in the pancreas. Together with altered expression in pancreatic cancer cell lines and biopsies, and known oncogenic functions in leukemia, this expression data has identified miR-29a as a key candidate for miR involvement in pancreatic cancer biology. Here we used miR-29a-deficient mice and the TAg model of pancreatic acinar carcinoma to functionally test the role of miR-29a in vivo. We found no impact of miR-29a loss on the development or growth of pancreatic tumours, nor on the survival of tumour-bearing mice. These results suggest that, despite differential expression, miR-29a is oncogenically neutral in the pancreatic acinar carcinoma context. If these results are extended to other models of pancreatic cancer, they would reduce the attractiveness of miR-29a as a potential therapeutic target in pancreatic cancer.

In humans, in utero exposure to ionising radiation results in an increased prevalence of neurological aberrations, such as small head size, mental retardation and decreased IQ levels. Yet, the association between early damaging events and long-term neuronal anomalies remains largely elusive.

The goal of this study was to measure functional and structural aspects of local neuroinflammation induced by intracerebral injection of lipopolysaccharide (LPS) in rats using TSPO microPET imaging with [(18)F]DPA-714, magnetic resonance imaging (MRI), in vitro autoradiography and immunohistochemistry (IHC) in order to characterize a small animal model for screening of new PET tracers targeting neuroinflammation.

Fibrosis and fat replacement in skeletal muscle are major complications that lead to a loss of mobility in chronic muscle disorders, such as muscular dystrophy. However, the in vivo properties of adipogenic stem and precursor cells remain unclear, mainly due to the high cell heterogeneity in skeletal muscles. Here, we use single-cell RNA sequencing to decomplexify interstitial cell populations in healthy and dystrophic skeletal muscles. We identify an interstitial CD142-positive cell population in mice and humans that is responsible for the inhibition of adipogenesis through GDF10 secretion. Furthermore, we show that the interstitial cell composition is completely altered in muscular dystrophy, with a near absence of CD142-positive cells. The identification of these adipo-regulatory cells in the skeletal muscle aids our understanding of the aberrant fat deposition in muscular dystrophy, paving the way for treatments that could counteract degeneration in patients with muscular dystrophy.

Reproducible and unbiased methods to quantify alveolar structure are important for research on many lung diseases. However, manually estimating alveolar structure through stereology is time consuming and inter-observer variability is high. The objective of this work was to develop and validate a fast, reproducible and accurate (semi-)automatic alternative. A FIJI-macro was designed that automatically segments lung images to binary masks, and counts the number of test points falling on tissue and the number of intersections of the air-tissue interface with a set of test lines. Manual selection remains necessary for the recognition of non-parenchymal tissue and alveolar exudates. Volume density of alveolar septa ([Formula: see text]) and mean linear intercept of the airspaces (Lm) as measured by the macro were compared to theoretical values for 11 artificial test images and to manually counted values for 17 lungs slides using linear regression and Bland-Altman plots. Inter-observer agreement between 3 observers, measuring 8 lungs both manually and automatically, was assessed using intraclass correlation coefficients (ICC). [Formula: see text] and Lm measured by the macro closely approached theoretical values for artificial test images (R2 of 0.9750 and 0.9573 and bias of 0.34% and 8.7%). The macro data in lungs were slightly higher for [Formula: see text] and slightly lower for Lm in comparison to manually counted values (R2 of 0.8262 and 0.8288 and bias of -6.0% and 12.1%). Visually, semi-automatic segmentation was accurate. Most importantly, manually counted [Formula: see text] and Lm had only moderate to good inter-observer agreement (ICC 0.859 and 0.643), but agreements were excellent for semi-automatically counted values (ICC 0.956 and 0.900). This semi-automatic method provides accurate and highly reproducible alveolar morphometry results. Future efforts should focus on refining methods for automatic detection of non-parenchymal tissue or exudates, and for assessment of lung structure on 3D reconstructions of lungs scanned with microCT.

The bone morphogenetic protein receptor II (BMPRII) signaling pathway is impaired in pulmonary arterial hypertension and mutations in the BMPR2 gene have been observed in both heritable and idiopathic pulmonary arterial hypertension. However, all BMPR2 mutation carriers do not develop pulmonary arterial hypertension, and inflammation could trigger the development of the disease in BMPR2 mutation carriers. Circulating levels and/or lung tissue expression of cytokines such as tumor necrosis factor-α or interleukin-18 are elevated in patients with pulmonary arterial hypertension and could be involved in the pathogenesis of pulmonary arterial hypertension. We consequently hypothesized that cytokines could trigger endothelial dysfunction in addition to impaired BMPRII signaling. Our aim was to determine whether impairment of BMPRII signaling might affect endothelium barrier function and adhesiveness to monocytes, in response to cytokines. BMPR2 was silenced in human lung microvascular endothelial cells (HLMVECs) using lentiviral vectors encoding microRNA-based hairpins. Effects of tumor necrosis factor-α and interleukin-18 on HLMVEC adhesiveness to the human monocyte cell line THP-1, adhesion molecule expression, endothelial barrier function and activation of P38MAPK were investigated in vitro. Stable BMPR2 silencing in HLMVECs resulted in impaired endothelial barrier function and constitutive activation of P38MAPK. Adhesiveness of BMPR2-silenced HLMVECs to THP-1 cells was enhanced by tumor necrosis factor-α and interleukin-18 through ICAM-1 adhesion molecule. Interestingly, tumor necrosis factor-α induced activation of P38MAPK and disrupted endothelial barrier function in BMPR2-silenced HLMVECs. Altogether, our findings showed that stable BMPR2 silencing resulted in impaired endothelial barrier function and activation of P38MAPK in HLMVECs. In BMPR2-silenced HLMVECs, cytokines enhanced adhesiveness capacities, activation of P38MAPK and impaired endothelial barrier function suggesting that cytokines could trigger the development of pulmonary arterial hypertension in a context of impaired BMPRII signaling pathway.