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.

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.

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.

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.

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.

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.

Vaccination against measles, mumps, and rubella (MMR) and yellow fever (YF) with live attenuated viruses can rarely cause life-threatening disease. Severe illness by MMR vaccines can be caused by inborn errors of type I and/or III interferon (IFN) immunity (mutations in IFNAR2, STAT1, or STAT2). Adverse reactions to the YF vaccine have remained unexplained. We report two otherwise healthy patients, a 9-yr-old boy in Iran with severe measles vaccine disease at 1 yr and a 14-yr-old girl in Brazil with viscerotropic disease caused by the YF vaccine at 12 yr. The Iranian patient is homozygous and the Brazilian patient compound heterozygous for loss-of-function IFNAR1 variations. Patient-derived fibroblasts are susceptible to viruses, including the YF and measles virus vaccine strains, in the absence or presence of exogenous type I IFN. The patients' fibroblast phenotypes are rescued with WT IFNAR1 Autosomal recessive, complete IFNAR1 deficiency can result in life-threatening complications of vaccination with live attenuated measles and YF viruses in previously healthy individuals.

STAT1 gain-of-function (GOF) is a primary immunodeficiency typically characterized by chronic mucocutaneous candidiasis (CMC), recurrent respiratory infections, and autoimmunity. Less commonly, also immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX)-like syndromes with CMC, and combined immunodeficiency without CMC have been described. Recently, our group and others have shown that different mutation-specific mechanisms underlie STAT1 GOF in vitro, including faster nuclear accumulation (R274W), and reduced mobility (R321, N574I) to near immobility in the nucleus (T419R) upon IFNγ stimulation. In this work, we evaluated the transcriptomic fingerprint of the aforementioned STAT1 GOF mutants (R274W, R321S, T419R, and N574I) relative to STAT1 wild-type upon IFNγ stimulation in an otherwise isogenic cell model. The majority of genes up-regulated in wild-type STAT1 cells were significantly more up-regulated in cells expressing GOF mutants, except for T419R. In addition to the common interferon regulated genes (IRG), STAT1 GOF mutants up-regulated an additional set of genes, that were in part shared with other GOF mutants or mutation-specific. Overall, R274W and R321S transcriptomes clustered with STAT1 WT, while T419R and N574I had a more distinct fingerprint. We observed reduced frequency of canonical IFNγ activation site (GAS) sequences in promoters of genes up-regulated by all the STAT1 GOF mutants, suggesting loss of DNA binding specificity for the canonical GAS consensus. Interestingly, the T419R mutation, expected to directly increase the affinity for DNA, showed the most pronounced effects on the transcriptome. T419R STAT1 dysregulated more non-IRG than the other GOF mutants and fewer GAS or degenerate GAS promotor sequences could be found in the promoter regions of these genes. In conclusion, our work confirms hyperactivation of common sets of IFNγ-induced genes in STAT1 GOF with additional dysregulation of mutation-specific genes, in line with the earlier observed mutation-specific mechanisms. Binding to more degenerate GAS sequences is proposed as a mechanism toward transcriptional dysregulation in R274W, R321S, and N574I. For T419R, an increased interaction with the DNA is suggested to result in a broader and less GAS-specific response. Our work indicates that multiple routes leading to STAT1 GOF are associated with common and private transcriptomic fingerprints, which may contribute to the phenotypic variation observed in vivo.

Non-typhoidal Salmonella (NTS) are a frequent cause of invasive infections in sub-Saharan Africa. They are frequently multidrug resistant (co-resistant to ampicillin, trimethoprim-sulfamethoxazole, and chloramphenicol), and resistance to third-generation cephalosporin and fluoroquinolone non-susceptibility have been reported. Third-generation cephalosporins and fluoroquinolones are often used to treat invasive NTS infections, but azithromycin might be an alternative. However, data on antibiotic treatment efficacy in invasive NTS infections are lacking. In this study, we aimed to assess the spatiotemporal distribution of antimicrobial resistance in invasive NTS infections in sub-Saharan Africa and to describe the available evidence and recommendations on antimicrobial treatment.

To develop safer retroviral murine leukemia virus (MLV)-based vectors, we previously mutated and re-engineered the MLV integrase: the W390A mutation abolished the interaction with its cellular tethering factors, BET proteins, and a retargeting peptide (the chromodomain of the CBX1 protein) was fused C-terminally. The resulting BET-independent MLVW390A-CBX was shown to integrate efficiently and more randomly, away from typical retroviral markers. In this study, we assessed the functionality and stability of expression of the redistributed MLVW390A-CBX vector in more depth, and evaluated safety using a clinically more relevant vector design encompassing a self-inactivated (SIN) LTR and a weak internal elongation factor 1α short (EFS) promoter. MLVW390A-CBX-EFS produced like MLVWT and efficiently transduced laboratory cells and primary human CD34+ hematopoetic stem cells (HSC) without transgene silencing over time, while displaying a more preferred, redistributed, and safer integration pattern. In a human mesoangioblast (MAB) stem cell model, the myogenic fusion capacity was hindered following MLVWT transduction, while this remained unaffected when applying MLVW390A-CBX. Likewise, smooth muscle cell differentiation of MABs was unaltered by MLVW390A-CBX-EFS. Taken together, our results underscore the potential of MLVW390A-CBX-EFS as a clinically relevant viral vector for ex-vivo gene therapy, combining efficient production with a preferable integration site distribution profile and stable expression over time.

Vaginal birth causes pelvic floor injury which may lead to urinary incontinence. Cell therapy has been proposed to assist in functional recovery. We aim to assess if intra-arterial injection of rat mesoangioblasts (MABs) and stable Vascular Endothelial Growth Factor (VEGF)-expressing MABs, improve recovery of urethral and vaginal function following simulated vaginal delivery (SVD). Female rats (n = 86) were assigned to either injection of saline (control), allogeneic-MABs (MABsallo), autologous-MABs (MABsauto) or allogeneic-MABs transduced to stably expressed VEGF (MABsallo-VEGF). One hour after SVD, 0.5 × 106 MABs or saline were injected into the aorta. Primary outcome was urethral (7d and 14d) and vaginal (14d) function; others were bioluminescent imaging for cell tracking (1, 3 and 7d), morphometry (7, 14 and 60d) and mRNAseq (3 and 7d). All MABs injected rats had external urethral sphincter and vaginal function recovery within 14d, as compared to only half of saline controls. Functional recovery was paralleled by improved muscle regeneration and microvascularization. Recovery rate was not different between MABsallo and MABsauto. MABsallo-VEGF accelerated functional recovery and increased GAP-43 expression at 7d. At 3d we detected major transcriptional changes in the urethra of both MABsallo and MABsallo-VEGF-injected animals, with upregulation of Rho/GTPase activity, epigenetic factors and dendrite development. MABSallo also upregulated transcripts that encode proteins involved in myogenesis and downregulated pro-inflammatory processes. MABsallo-VEGF also upregulated transcripts that encode proteins involved in neuron development and downregulated genes involved in hypoxia and oxidative stress. At 7d, urethras of MABsallo-VEGF-injected rats showed downregulation of oxidative and inflammatory response compared to MABSallo. Intra-arterial injection of MABsallo-VEGF enhances neuromuscular regeneration induced by untransduced MABs and accelerates the functional urethral and vaginal recovery after SVD.

Pulmonary hypoplasia, characterized by incomplete alveolar development, remains a major cause of mortality and morbidity in congenital diaphragmatic hernia. Recently demonstrated to differentiate from a common bipotent progenitor during development, the two cell types that line the alveoli type 1 and type 2 alveolar cells have shown to alter their relative ratio in congenital diaphragmatic hernia lungs.

Fetal gene therapy is one of the possible new therapeutic strategies for congenital or perinatal diseases with high mortality or morbidity. We developed a novel delivery strategy to inject directly into the fetal mouse trachea. Intratracheal (i.t.) injection at embryonic day 18 (E18) was more efficient in targeting the fetal lung than conventional intra-amniotic (i.a.) delivery. Viral vectors derived from adeno-associated virus serotype 6.2, with tropism for the airway epithelium and not earlier tested in the fetal mouse lung, were injected into the fetal trachea. Bioluminescence (BL) imaging (BLI) was combined with magnetic resonance (MR) imaging (MRI) for noninvasive and accurate localization of transgene expression in vivo. Histological analysis for β-galactosidase (β-gal) revealed 17.5% of epithelial cells transduced in the conducting airways and 1.5% in the alveolar cells. Stable gene expression was observed up to 1 month after injection. This study demonstrates that direct injection of rAAV2/6.2 in the fetal mouse trachea is superior to i.a. delivery for transducing the lung. Second, as stable gene transfer was detected up to 1 postnatal month, this approach may be useful to evaluate fetal gene therapy for pulmonary diseases such as cystic fibrosis, requiring both substantial numbers of transduced cells as well as prolonged gene expression to obtain a stable phenotypic effect.

The neonatal management of preterm born infants often results in damage to the developing lung and subsequent morbidity, referred to as bronchopulmonary dysplasia (BPD). Animal models may help in understanding the molecular processes involved in this condition and define therapeutic targets. Our goal was to identify molecular pathways using the earlier described preterm rabbit model of hyperoxia induced lung-injury. Transcriptome analysis by mRNA-sequencing was performed on lungs from preterm rabbit pups born at day 28 of gestation (term: 31 days) and kept in hyperoxia (95% O2) for 7 days. Controls were preterm pups kept in normoxia. Transcriptomic data were analyzed using Array Studio and Ingenuity Pathway Analysis (IPA), in order to identify the central molecules responsible for the observed transcriptional changes. We detected 2217 significantly dysregulated transcripts following hyperoxia, of which 90% could be identified. Major pathophysiological dysregulations were found in inflammation, lung development, vascular development and reactive oxygen species (ROS) metabolism. To conclude, amongst the many dysregulated transcripts, major changes were found in the inflammatory, oxidative stress and lung developmental pathways. This information may be used for the generation of new treatment hypotheses for hyperoxia-induced lung injury and BPD.

Testing of new therapeutic strategies for Parkinson's disease (PD) is currently hampered by the lack of relevant and reproducible animal models. Here, we developed a robust rat model for PD by injection of adeno-associated viral vectors (rAAV2/7) encoding α-synuclein into the substantia nigra, resulting in reproducible nigrostriatal pathology and behavioral deficits in a 4-week time period. Progressive dopaminergic dysfunction was corroborated by histopathologic and biochemical analysis, motor behavior testing and in vivo microdialysis. L-DOPA treatment was found to reverse the behavioral phenotype. Non-invasive positron emission tomography imaging and magnetic resonance spectroscopy allowed longitudinal monitoring of neurodegeneration. In addition, insoluble α-synuclein aggregates were formed in this model. This α-synuclein rat model shows improved face and predictive validity, and therefore offers the possibility to reliably test novel therapeutics. Furthermore, it will be of great value for further research into the molecular pathogenesis of PD and the importance of α-synuclein aggregation in the disease process.

Lens epithelium-derived growth factor (LEDGF/p75) is a cellular cofactor of HIV-1 integrase (IN) that interacts with IN through its IN binding domain (IBD) and tethers the viral pre-integration complex to the host cell chromatin. Here we report the generation of a human somatic LEDGF/p75 knockout cell line that allows the study of spreading HIV-1 infection in the absence of LEDGF/p75. By homologous recombination the exons encoding the LEDGF/p75 IBD (exons 11 to 14) were knocked out. In the absence of LEDGF/p75 replication of laboratory HIV-1 strains was severely delayed while clinical HIV-1 isolates were replication-defective. The residual replication was predominantly mediated by the Hepatoma-derived growth factor related protein 2 (HRP-2), the only cellular protein besides LEDGF/p75 that contains an IBD. Importantly, the recently described IN-LEDGF/p75 inhibitors (LEDGINs) remained active even in the absence of LEDGF/p75 by blocking the interaction with the IBD of HRP-2. These results further support the potential of LEDGINs as allosteric integrase inhibitors.