Genetic Engineering of the Kidney to Permanently Silence MHC Transcripts During ex vivo Organ Perfusion

Organ gene therapy represents a promising tool to correct diseases or improve graft survival after transplantation. Polymorphic variation of the major histocompatibility complex (MHC) antigens remains a major obstacle to long-term graft survival after transplantation. Previously, we demonstrated that MHC-silenced cells are protected against allogeneic immune responses. We also showed the feasibility to silence MHC in the lung. Here, we aimed at the genetic engineering of the kidney toward permanent silencing of MHC antigens in a rat model. We constructed a sub-normothermic ex vivo perfusion system to deliver lentiviral vectors encoding shRNAs targeting β2-microglobulin and the class II transactivator to the kidney. In addition, the vector contained the sequence for a secreted nanoluciferase. After kidney transplantation (ktx), we detected bioluminescence in the plasma and urine of recipients of an engineered kidney during the 6 weeks of post-transplant monitoring, indicating a stable transgene expression. Remarkably, transcript levels of β2-microglobulin and the class II transactivator were decreased by 70% in kidneys expressing specific shRNAs. Kidney genetic modification did not cause additional cell death compared to control kidneys after machine perfusion. Nevertheless, cytokine secretion signatures were altered during perfusion with lentiviral vectors as revealed by an increase in the secretion of IL-10, MIP-1α, MIP-2, IP-10, and EGF and a decrease in the levels of IL-12, IL-17, MCP-1, and IFN-γ. Biodistribution assays indicate that the localization of the vector was restricted to the graft. This study shows the potential to generate immunologically invisible kidneys showing great promise to support graft survival after transplantation and may contribute to reduce the burden of immunosuppression

PD-L1 status and Immune checkpoint inhibitors in kidney cancer: ignorance, lack of knowledge or both

<jats:title>Abstract</jats:title><jats:p>The East African margin between the Somali Basin in the north and the Natal Basin in the south formed as a result of the Jurassic/Cretaceous dispersal of Gondwana. While the initial movements between East and West Gondwana left (oblique) rifted margins behind, the subsequent southward drift of East Gondwana from 157 Ma onwards created a major shear zone, the Davie Fracture Zone (DFZ), along East Africa. To document the structural variability of the DFZ, several deep seismic lines were acquired off northern Mozambique. The profiles clearly indicate the structural changes along the shear zone from an elevated continental block in the south (14°–20°S) to non-elevated basement covered by up to 6-km-thick sediments in the north (9°–13°S). Here, we compile the geological/geophysical knowledge of five profiles along East Africa and interpret them in the context of one of the latest kinematic reconstructions. A pre-rift position of the detached continental sliver of the Davie Ridge between Tanzania/Kenya and southeastern Madagascar fits to this kinematic reconstruction without general changes of the rotation poles.</jats:p&gt

Cholemic Nephropathy Causes Acute Kidney Injury and Is Accompanied by Loss of Aquaporin 2 in Collecting Ducts

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Disease-Associated miRNA-mRNA Networks in Oral Lichen Planus

<div><p>Background</p><p>A large number of pathophysiological mechanisms are regulated by microRNAs (miRNAs), which represent a new class of posttranscriptional regulators of gene expression. To date, little is known about their role in oral lichen planus (OLP), a chronic inflammatory mucocutaneous disease of unknown etiology which is being discussed as a potentially premalignant condition of oropharyngeal cancer. The aim of the present investigation was to assess the pathophysiological impact of miRNAs and to determine regulatory miRNA networks which are directly linked to potentially disease-associated target transcripts in OLP.</p><p>Methods</p><p>Native tissue samples were collected from the oral mucosa of seven patients with OLP. The control group was composed of native tissue from elective oral surgery. The mRNA profiling was performed using the Affymetrix Human Gene 1.0 ST Array while miRNA profiling was performed using the microRNA Galaxy Array. Subsequent validation of initial results was carried out using TaqMan real time PCR.</p><p>Results</p><p>We identified 24 differentially regulated miRNA and 2,694 regulated transcripts. Linking the miRNAs to their potential targets we found 11 potential miRNA-mRNA pairs, of which several are functionally related to premalignant as well as to inflammatory events.</p><p>Conclusions</p><p>Our data shows miRNA associated with transcripts which are regulated when comparing OLP patients with healthy control individuals. This suggests that miRNAs may potentially regulate disease-relevant transcripts, proposing the concept of therapeutic interventions based on miRNAs.</p></div

Sorafenib tosylate inhibits directly necrosome complex formation and protects in mouse models of inflammation and tissue injury

Necroptosis contributes to the pathophysiology of several inflammatory, infectious and degenerative disorders. TNF-induced necroptosis involves activation of the receptor-interacting protein kinases 1 and 3 (RIPK1/3) in a necrosome complex, eventually leading to the phosphorylation and relocation of mixed lineage kinase domain like protein (MLKL). Using a high-content screening of small compounds and FDA-approved drug libraries, we identified the anti-cancer drug Sorafenib tosylate as a potent inhibitor of TNF-dependent necroptosis. Interestingly, Sorafenib has a dual activity spectrum depending on its concentration. In murine and human cell lines it induces cell death, while at lower concentrations it inhibits necroptosis, without affecting NF-.B activation. Pull down experiments with biotinylated Sorafenib show that it binds independently RIPK1, RIPK3 and MLKL. Moreover, it inhibits RIPK1 and RIPK3 kinase activity. In vivo Sorafenib protects against TNF-induced systemic inflammatory response syndrome (SIRS) and renal ischemia-reperfusion injury (IRI). Altogether, we show that Sorafenib can, next to the reported Braf/Mek/Erk and VEGFR pathways, also target the necroptotic pathway and that it can protect in an acute inflammatory RIPK1/3-mediated pathology

Bevacizumab-associated glomerular microangiopathy

Bevacizumab is a humanized monoclonal IgG1 antibody, which neutralizes vascular endothelial growth factor and is used for treating multiple cancer types. As a known and frequent adverse event, this therapy can lead to renal damage including proteinuria and nephrotic syndrome. In a retrospective approach, we analyzed 17 renal biopsies from patients receiving bevacizumab treatment. We observed a distinctive histopathological pseudothrombotic pattern different from the previously reported thrombotic microangiopathy. Since this pattern includes some features similar to acute and chronic thrombotic microangiopathy, focal segmental glomerulosclerosis and cryoglobulinemic membranoproliferative glomerulonephritis, biopsies with these diagnoses were included for comparison. Clinical, laboratory, light microscopic, immunohistochemical (including a proximity ligation assay), proteomic and electron microscopic features were assessed. Nephrotic syndrome was present in 15 of the 17 bevacizumab-treated patients. All 17 displayed a patchy pattern of variably PAS-positive hyaline pseudothrombi occluding markedly dilated glomerular capillaries in their biopsies. Mass spectrometry-based proteome analysis revealed a special protein pattern demonstrating some features of thrombotic microangiopathy and some of cryoglobulinemic glomerulonephritis, including a strong accumulation of IgG in the pseudothrombi. Proximity ligation assay did not show interaction of IgG with C1q, arguing for accumulation without classic pathway complement activation. In contrast to thrombi in thrombotic microangiopathy cases, the hyaline pseudothrombi did not contain clusters of CD61-positive platelets. Electron microscopy of bevacizumab cases did not show fibrin polymers or extensive loss of podocyte foot processes. Even though cases of bevacizumab-associated microangiopathy share some features with thrombotic microangiopathy, its overall histopathological pattern is quite different from acute or chronic thrombotic microangiopathy cases. We conclude that bevacizumab therapy can lead to a unique hyaline occlusive glomerular microangiopathy, likely arising from endothelial leakage followed by subendothelial accumulation of serum proteins. It can be diagnosed by light microscopy and is an important differential diagnosis in cancer patients with nephrotic syndrome

Natural Compound Library Screening Identifies New Molecules for the Treatment of Cardiac Fibrosis and Diastolic Dysfunction.

High-throughput natural compound library screening identified 15 substances with antiproliferative effects in human cardiac fibroblasts. Using multiple in vitro fibrosis assays and stringent selection algorithms, we identified the steroid bufalin (from Chinese toad venom) and the alkaloid lycorine (from Amaryllidaceae species) to be effective antifibrotic molecules both in vitro and in vivo, leading to improvement in diastolic function in 2 hypertension-dependent rodent models of cardiac fibrosis. Administration at effective doses did not change plasma damage markers or the morphology of kidney and liver, providing the first toxicological safety data. Using next-generation sequencing, we identified the conserved microRNA 671-5p and downstream the antifibrotic selenoprotein P1 as common effectors of the antifibrotic compounds

Natural Compound Library Screening Identifies New Molecules for the Treatment of Cardiac Fibrosis and Diastolic Dysfunction

BACKGROUND: Myocardial fibrosis is a hallmark of cardiac remodeling and functionally involved in heart failure development, a leading cause of deaths worldwide. Clinically, no therapeutic strategy is available that specifically attenuates maladaptive responses of cardiac fibroblasts, the effector cells of fibrosis in the heart. Therefore, our aim was to develop novel antifibrotic therapeutics based on naturally derived substance library screens for the treatment of cardiac fibrosis. METHODS: Antifibrotic drug candidates were identified by functional screening of 480 chemically diverse natural compounds in primary human cardiac fibroblasts, subsequent validation, and mechanistic in vitro and in vivo studies. Hits were analyzed for dose-dependent inhibition of proliferation of human cardiac fibroblasts, modulation of apoptosis, and extracellular matrix expression. In vitro findings were confirmed in vivo with an angiotensin II–mediated murine model of cardiac fibrosis in both preventive and therapeutic settings, as well as in the Dahl salt-sensitive rat model. To investigate the mechanism underlying the antifibrotic potential of the lead compounds, treatment-dependent changes in the noncoding RNAome in primary human cardiac fibroblasts were analyzed by RNA deep sequencing. RESULTS: High-throughput natural compound library screening identified 15 substances with antiproliferative effects in human cardiac fibroblasts. Using multiple in vitro fibrosis assays and stringent selection algorithms, we identified the steroid bufalin (from Chinese toad venom) and the alkaloid lycorine (from Amaryllidaceae species) to be effective antifibrotic molecules both in vitro and in vivo, leading to improvement in diastolic function in 2 hypertension-dependent rodent models of cardiac fibrosis. Administration at effective doses did not change plasma damage markers or the morphology of kidney and liver, providing the first toxicological safety data. Using next-generation sequencing, we identified the conserved microRNA 671-5p and downstream the antifibrotic selenoprotein P1 as common effectors of the antifibrotic compounds. CONCLUSIONS: We identified the molecules bufalin and lycorine as drug candidates for therapeutic applications in cardiac fibrosis and diastolic dysfunction

Natural Compound Library Screening Identifies New Molecules for the Treatment of Cardiac Fibrosis and Diastolic Dysfunction

BACKGROUND: Myocardial fibrosis is a hallmark of cardiac remodeling and functionally involved in heart failure development, a leading cause of deaths worldwide. Clinically, no therapeutic strategy is available that specifically attenuates maladaptive responses of cardiac fibroblasts, the effector cells of fibrosis in the heart. Therefore, our aim was to develop novel antifibrotic therapeutics based on naturally derived substance library screens for the treatment of cardiac fibrosis. METHODS: Antifibrotic drug candidates were identified by functional screening of 480 chemically diverse natural compounds in primary human cardiac fibroblasts, subsequent validation, and mechanistic in vitro and in vivo studies. Hits were analyzed for dose-dependent inhibition of proliferation of human cardiac fibroblasts, modulation of apoptosis, and extracellular matrix expression. In vitro findings were confirmed in vivo with an angiotensin II–mediated murine model of cardiac fibrosis in both preventive and therapeutic settings, as well as in the Dahl salt-sensitive rat model. To investigate the mechanism underlying the antifibrotic potential of the lead compounds, treatment-dependent changes in the noncoding RNAome in primary human cardiac fibroblasts were analyzed by RNA deep sequencing. RESULTS: High-throughput natural compound library screening identified 15 substances with antiproliferative effects in human cardiac fibroblasts. Using multiple in vitro fibrosis assays and stringent selection algorithms, we identified the steroid bufalin (from Chinese toad venom) and the alkaloid lycorine (from Amaryllidaceae species) to be effective antifibrotic molecules both in vitro and in vivo, leading to improvement in diastolic function in 2 hypertension-dependent rodent models of cardiac fibrosis. Administration at effective doses did not change plasma damage markers or the morphology of kidney and liver, providing the first toxicological safety data. Using next-generation sequencing, we identified the conserved microRNA 671-5p and downstream the antifibrotic selenoprotein P1 as common effectors of the antifibrotic compounds. CONCLUSIONS: We identified the molecules bufalin and lycorine as drug candidates for therapeutic applications in cardiac fibrosis and diastolic dysfunction