Quantification of human papillomavirus cell-free DNA from low-volume blood plasma samples by digital PCR.

The incidence rate of human papillomavirus-driven oropharyngeal cancer (HPV-OPC) is increasing in countries with high human development index. HPV cell-free DNA (cfDNA) isolated from 3 to 4 mL blood plasma has been successfully used for therapy surveillance. A highly discussed application of HPV-cfDNA is early detection of HPV-OPC. This requires sensitive and specific cfDNA detection as cfDNA levels can be very low. To study the predictive power of pre-diagnostic HPV-cfDNA, archived samples from epidemiological cohorts with limited plasma volume are an important source. To establish a cfDNA detection workflow for low plasma volumes, we compared cfDNA purification methods [MagNA Pure 96 (MP96) and QIAamp ccfDNA/RNA] and digital PCR systems (Biorad QX200 and QIAGEN QIAcuity One). Final assay validation included 65 low-volume plasma samples from oropharyngeal cancer (OPC) patients with defined HPV status stored for 2-9 years. MP96 yielded a 28% higher cfDNA isolation efficiency in comparison to QIAamp. Both digital PCR systems showed comparable analytical sensitivity (6-17 copies for HPV16 and HPV33), but QIAcuity detected both types in the same assay. In the validation set, the assay had 80% sensitivity (n = 28/35) for HPV16 and HPV33 and a specificity of 97% (n = 29/30). In samples with ≥750 µL plasma, the sensitivity was 85% (n = 17/20), while in samples with <750 µL plasma, it was 73% (n = 11/15). Despite the expected drop in sensitivity with decreased plasma volume, the assay is sensitive and highly specific even in low-volume samples and thus suited for studies exploring HPV-cfDNA as an early HPV-OPC detection marker in low-volume archival material.IMPORTANCEHPV-OPC has a favorable prognosis compared to HPV-negative OPC. However, the majority of tumors is diagnosed after regional spread, thus making intensive treatment necessary. This can cause lasting morbidity with a large impact on quality of life. One potential method to decrease treatment-related morbidity is early detection of the cancer. HPV cfDNA has been successfully used for therapy surveillance and has also been detected in pre-diagnostic samples of HPV-OPC patients. These pre-diagnostic samples are only commonly available from biobanks, which usually only have small volumes of blood plasma available. Hence, we have developed a workflow optimized for small-volume archival samples. With this method, a high sensitivity can be achieved despite sample limitations, making it suitable to conduct further large-scale biobank studies of HPV-cfDNA as a prognostic biomarker for HPV-OPC.

Iptacopan in Idiopathic Immune Complex-Mediated Membranoproliferative Glomerulonephritis: Protocol of the APPARENT Multicenter, Randomized Phase 3 Study.

Introduction: Immune complex-mediated membranoproliferative glomerulonephritis (IC-MPGN) is an ultra-rare, fast-progressing kidney disease that may be idiopathic (primary) or secondary to chronic infection, autoimmune disorders, or monoclonal gammopathies. Dysregulation of the alternative complement pathway is implicated in the pathophysiology of IC-MPGN; and currently, there are no approved targeted treatments. Iptacopan is an oral, highly potent proximal complement inhibitor that specifically binds to factor B and inhibits the alternative pathway (AP). Methods: This randomized, double-blind, placebo-controlled phase 3 study (APPARENT; NCT05755386) will evaluate the efficacy and safety of iptacopan in patients with idiopathic (primary) IC-MPGN, enrolling up to 68 patients (minimum of 10 adolescents) aged 12 to 60 years with biopsy-confirmed IC-MPGN, proteinuria ≥1 g/g, and estimated glomerular filtration rate (eGFR) ≥30 ml/min per 1.73 m2. All patients will receive maximally tolerated angiotensin-converting enzyme inhibitor/angiotensin receptor blocker and vaccination against encapsulated bacteria. Patients with any organ transplant, progressive crescentic glomerulonephritis, or kidney biopsy with >50% interstitial fibrosis/tubular atrophy, will be excluded. Patients will be randomized 1:1 to receive either iptacopan 200 mg twice daily (bid) or placebo for 6 months, followed by open-label treatment with iptacopan 200 mg bid for all patients for 6 months. The primary objective of the study is to evaluate the efficacy of iptacopan versus placebo in proteinuria reduction measured as urine protein-to-creatinine ratio (UPCR) (24-h urine) at 6 months. Key secondary end points will assess kidney function measured by eGFR, patients who achieve a proteinuria-eGFR composite end point, and patient-reported fatigue. Conclusion: This study will provide evidence toward the efficacy and safety of iptacopan in idiopathic (primary) IC-MPGN.

Microcavity well-plate for automated parallel bioelectronic analysis of 3D cell cultures.

Three-dimensional (3D) in vitro cell culture models serve as valuable tools for accurately replicating cellular microenvironments found in vivo. While cell culture technologies are rapidly advancing, the availability of non-invasive, real-time, and label-free analysis methods for 3D cultures remains limited. To meet the demand for higher-throughput drug screening, there is a demanding need for analytical methods that can operate in parallel. Microelectrode systems in combination with microcavity arrays (MCAs), offer the capability of spatially resolved electrochemical impedance analysis and field potential monitoring of 3D cultures. However, the fabrication and handling of small-scale MCAs have been labour-intensive, limiting their broader application. To overcome this challenge, we have established a process for creating MCAs in a standard 96-well plate format using high-precision selective laser etching. In addition, to automate and ensure the accurate placement of 3D cultures on the MCA, we have designed and characterized a plug-in tool using SLA-3D-printing. To characterize our new 96-well plate MCA-based platform, we conducted parallel analyses of human melanoma 3D cultures and monitored the effect of cisplatin in real-time by impedance spectroscopy. In the following we demonstrate the capabilities of the MCA approach by analysing contraction rates of human pluripotent stem cell-derived cardiomyocyte aggregates in response to cardioactive compounds. In summary, our MCA system significantly expands the possibilities for label-free analysis of 3D cell and tissue cultures, offering an order of magnitude higher parallelization capacity than previous devices. This advancement greatly enhances its applicability in real-world settings, such as drug development or clinical diagnostics.

Results of a randomized double-blind placebo-controlled Phase 2 study propose iptacopan as an alternative complement pathway inhibitor for IgA nephropathy.

Targeting the alternative complement pathway is an attractive therapeutic strategy given its role in the pathogenesis of immunoglobulin A nephropathy (IgAN). Iptacopan (LNP023) is an oral, proximal alternative complement inhibitor that specifically binds to Factor B. Our randomized, double-blind, parallel-group adaptive Phase 2 study (NCT03373461) enrolled patients with biopsy-confirmed IgAN (within previous three years) with estimated glomerular filtration rates of 30 mL/min/1.73 m2 and over and urine protein 0.75 g/24 hours and over on stable doses of renin angiotensin system inhibitors. Patients were randomized to four iptacopan doses (10, 50, 100, or 200 mg bid) or placebo for either a three-month (Part 1; 46 patients) or a six-month (Part 2; 66 patients) treatment period. The primary analysis evaluated the dose-response relationship of iptacopan versus placebo on 24-hour urine protein-to-creatinine ratio (UPCR) at three months. Other efficacy, safety and biomarker parameters were assessed. Baseline characteristics were generally well-balanced across treatment arms. There was a statistically significant dose-response effect, with 23% reduction in UPCR achieved with iptacopan 200 mg bid (80% confidence interval 8-34%) at three months. UPCR decreased further through six months in iptacopan 100 and 200 mg arms (from a mean of 1.3 g/g at baseline to 0.8 g/g at six months in the 200 mg arm). A sustained reduction in complement biomarker levels including plasma Bb, serum Wieslab, and urinary C5b-9 was observed. Iptacopan was well-tolerated, with no reports of deaths, treatment-related serious adverse events or bacterial infections, and led to strong inhibition of alternative complement pathway activity and persistent proteinuria reduction in patients with IgAN. Thus, our findings support further evaluation of iptacopan in the ongoing Phase 3 trial (APPLAUSE-IgAN; NCT04578834).

Single-cell characterization of neovascularization using hiPSC-derived endothelial cells in a 3D microenvironment.

The formation of vascular structures is fundamental for in vitro tissue engineering. Vascularization can enable the nutrient supply within larger structures and increase transplantation efficiency. We differentiated human induced pluripotent stem cells toward endothelial cells in 3D suspension culture. To investigate in vitro neovascularization and various 3D microenvironmental approaches, we designed a comprehensive single-cell transcriptomic study. Time-resolved single-cell transcriptomics of the endothelial and co-evolving mural cells gave insights into cell type development, stability, and plasticity. Transfer to a 3D hydrogel microenvironment induced neovascularization and facilitated tracing of migrating, coalescing, and tubulogenic endothelial cell states. During maturation, we monitored two pericyte subtypes evolving mural cells. Profiling cell-cell interactions between pericytes and endothelial cells revealed angiogenic signals during tubulogenesis. In silico discovered ligands were tested for their capability to attract endothelial cells. Our data, analyses, and results provide an in vitro roadmap to guide vascularization in future tissue engineering.

Impact of the South Atlantic Anomaly on radiation exposure at flight altitudes during solar minimum.

The South Atlantic Anomaly (SAA) is a geographical region over the South Atlantic Ocean where the inner Van Allen radiation belt extends down particularly close to Earth. This leads to highly increased levels of ionizing radiation and related impacts on spacecraft in Low Earth Orbits, e.g., correspondingly increased radiation exposure of astronauts and electronic components on the International Space Station. According to an urban legend, the SAA is also supposed to affect the radiation field in the atmosphere even down to the altitudes of civil aviation. In order to identify and quantify any additional contributions to the omnipresent radiation exposure due to the Galactic Cosmic Radiation at flight altitudes, comprehensive measurements were performed crossing the geographical region of the SAA at an altitude of 13 km in a unique flight mission-Atlantic Kiss. No indication of increased radiation exposure was found.

Targeting the Alternative Complement Pathway With Iptacopan to Treat IgA Nephropathy: Design and Rationale of the APPLAUSE-IgAN Study.

Introduction: Targeting the alternative complement pathway (AP) is an attractive therapeutic strategy because of its role in immunoglobulin A nephropathy (IgAN) pathophysiology. Iptacopan (LNP023), a proximal complement inhibitor that specifically binds to factor B and inhibits the AP, reduced proteinuria and attenuated AP activation in a Phase 2 study of patients with IgAN, thereby supporting the rationale for its evaluation in a Phase 3 study. Methods: APPLAUSE-IgAN (NCT04578834) is a multicenter, randomized, double-blind, placebo-controlled, parallel-group, Phase 3 study enrolling approximately 450 adult patients (aged ≥18 years) with biopsy-confirmed primary IgAN at high risk of progression to kidney failure despite optimal supportive treatment. Eligible patients receiving stable and maximally tolerated doses of angiotensin-converting enzyme inhibitors (ACEis) or angiotensin receptor blockers (ARBs) will be randomized 1:1 to either iptacopan 200 mg or placebo twice daily for a 24-month treatment period. A prespecified interim analysis (IA) will be performed when approximately 250 patients from the main study population complete the 9-month visit. The primary objective is to demonstrate superiority of iptacopan over placebo in reducing 24-hour urine protein-to-creatinine ratio (UPCR) at the IA and demonstrate the superiority of iptacopan over placebo in slowing the rate of estimated glomerular filtration rate (eGFR) decline (total eGFR slope) estimated over 24 months at study completion. The effect of iptacopan on patient-reported outcomes, safety, and tolerability will be evaluated as secondary outcomes. Conclusions: APPLAUSE-IgAN will evaluate the benefits and safety of iptacopan, a novel targeted therapy for IgAN, in reducing complement-mediated kidney damage and thus slowing or preventing disease progression.

Single-cell profiling of GP2-enriched pancreatic progenitors to simultaneously create acinar, ductal, and endocrine organoids.

Rationale: Pancreatic lineage specification follows the formation of tripotent pancreatic progenitors (PPs). Current protocols rebuilding PPs in vitro have an endocrine lineage bias and are mostly based on PDX1/NKX6-1 coexpression neglecting other markers decisive for PP heterogeneity and lineage potential. However, true tripotent PPs are of utmost interest to study also exocrine disorders such as pancreatic cancer and to simultaneously generate all three pancreatic lineages from the same ancestor. Methods: Here, we performed a comprehensive compound testing to advance the generation of multipotent progenitors, which were further characterized for their trilineage potential in vitro and in vivo. The heterogeneity and cell-cell communication across the PP subpopulations were analyzed via single-cell transcriptomics. Results: We introduce a novel PP differentiation platform based on a comprehensive compound screening with an advanced design of experiments computing tool to reduce impurities and to increase Glycoprotein-2 expression and subsequent trilineage potential. Superior PP tripotency was proven in vitro by the generation of acinar, endocrine, and ductal cells as well as in vivo upon orthotopic transplantation revealing all three lineages at fetal maturation level. GP2 expression levels at PP stage ascribed varying pancreatic lineage potential. Intermediate and high GP2 levels were superior in generating endocrine and duct-like organoids (PDLO). FACS-based purification of the GP2high PPs allowed the generation of pancreatic acinar-like organoids (PALO) with proper morphology and expression of digestive enzymes. scRNA-seq confirmed multipotent identity, positioned the GP2/PDX1/NKX6-1high population next to human fetal tip and trunk progenitors and identified novel ligand-receptor (LR) interactions in distinct PP subpopulations. LR validation experiments licensed midkine and VEGF signaling to increase markers labelling the single cell clusters with high GP2 expression. Conclusion: In this study, we guide human pluripotent stem cells into multipotent pancreatic progenitors. This common precursor population, which has the ability to mature into acinar, ductal and functional ß-cells, serves as a basis for studying developmental processes and deciphering early cancer formation in a cell type-specific context. Using single-cell RNA sequencing and subsequent validation studies, we were able to dissect PP heterogeneity and specific cell-cell communication signals.

Long-Term Effects of Everolimus-Facilitated Tacrolimus Reduction in Living-Donor Liver Transplant Recipients with Hepatocellular Carcinoma.

BACKGROUND The study objective was to evaluate the effect of everolimus (EVR) in combination with reduced tacrolimus (rTAC) compared with a standard TAC (sTAC) regimen on hepatocellular carcinoma (HCC) recurrence in de novo living-donor liver transplantation recipients (LDLTRs) with primary HCC at liver transplantation through 5 years after transplantation. MATERIAL AND METHODS In this multicenter, non-interventional study, LDLTRs with primary HCC, who were previously randomized to either everolimus plus reduced tacrolimus (EVR+rTAC) or standard tacrolimus (sTAC), and who completed the 2-year core H2307 study, were followed up. Data were collected retrospectively (end of core to the start of follow-up study), and prospectively (during the 3-year follow-up study). RESULTS Of 117 LDLTRs with HCC at LT in the core H2307 study (EVR+rTAC, N=56; sTAC, N=61), 86 patients (EVR+rTAC, N=41; sTAC, N=45) entered the follow-up study. Overall HCC recurrence was lower but statistically non-significant in the EVR+rTAC group (3.6% vs 11.5% in sTAC; P=0.136) at 5 years after LT. There was no graft loss or chronic rejection. Acute rejection and death were comparable between treatment groups. Higher mean estimated glomerular filtration rate in the EVR+rTAC group (76.8 vs 65.8 mL/min/1.73 m² in sTAC) was maintained up to 5 years. Reported adverse events were numerically lower in the EVR+rTAC group (41.0% vs 53.5% sTAC) but not statistically significant. CONCLUSIONS Although statistically not significant, early EVR initiation reduced HCC recurrence, with comparable efficacy and safety, and better long-term renal function, than that of sTAC treatment.

Alternative Complement Pathway Inhibition With Iptacopan for the Treatment of C3 Glomerulopathy-Study Design of the APPEAR-C3G Trial.

Introduction: Complement 3 glomerulopathy (C3G) is a rare kidney disease characterized by dysregulation of the alternative pathway (AP) of the complement system. About 50% of patients with C3G progress to kidney failure within 10 years of diagnosis. Currently, there are no approved therapeutic agents for C3G. Iptacopan is an oral, first-in-class, potent, and selective inhibitor of factor B, a key component of the AP. In a Phase II study, treatment with iptacopan was associated with a reduction in proteinuria and C3 deposit scores in C3G patients with native and transplanted kidneys, respectively. Methods: APPEAR-C3G (NCT04817618) is a randomized, double-blind, and placebo-controlled Phase III study to evaluate the efficacy and safety of iptacopan in C3G patients, enrolling 68 adults with biopsy-confirmed C3G, reduced C3 (<77 mg/dl), proteinuria ≥1.0 g/g, and estimated glomerular filtration rate (eGFR) ≥30 ml/min per 1.73 m2. All patients will receive maximally tolerated angiotensin-converting enzyme inhibitor/angiotensin receptor blocker and vaccination against encapsulated bacteria. Patients with any organ transplantation, progressive crescentic glomerulonephritis (GN), monoclonal gammopathy of undetermined significance, or kidney biopsy with >50% interstitial fibrosis/tubular atrophy, will be excluded. Patients will be randomized 1:1 to receive either iptacopan 200 mg twice daily or placebo for 6 months, followed by open-label treatment with iptacopan 200 mg twice daily for all patients for 6 months. The primary objective is to evaluate the efficacy of iptacopan versus placebo on proteinuria reduction urine protein:creatinine ratio (UPCR) (24 h urine). Key secondary endpoints will assess kidney function measured by eGFR, histological disease total activity score, and fatigue. Conclusion: This study aims to demonstrate the clinical benefits of AP inhibition with iptacopan in C3G.

Predictive Value of Early Postoperative Course of Serum Cortisol After Transsphenoidal Surgery for Cushing's Disease.

OBJECTIVE: To identify early available predictors for the long-term outcome of patients after transsphenoidal surgery (TSS) in the management of Cushing's disease. METHODS: This single-center, retrospective study included 93 consecutive patients with Cushing's disease (follow-up 12-129 months, mean 48, median 38) who underwent TSS (21 had previous operations elsewhere). Six cases had early re-operation, and the resulting data were evaluated instead of the respective first operation. During the postoperative course, serum cortisol levels were assessed every four hours at least until the next morning. An association of parameters with long-term outcomes was tested using binary logistic regression. Receiver operating characteristic curves were used to determine sensitivity, specificity, positive predictive value, and negative predictive value of different cut-off values of serum cortisol in the postoperative course in the event of recurrence after remission. RESULTS: Eighty out of 93 patients (86%) showed postoperative remission (after primary treatment, 60 out of 72 patients, 90.3%). Of these, 8 patients (10%) developed recurrence of hypercortisolism. Compared to patients with persisting long-term remission, those with recurrence differed in cortisol levels starting from 4 pm on the day of surgery plus an event of increasing cortisol during the early postoperative course ("peak"). Binary logistic regression showed the association between a peak of serum cortisol in the early postoperative course with an increased probability of recurrence. CONCLUSIONS: Patients with a peak of serum cortisol in the early postoperative course show an increased recurrence rate. A cut-off value of serum cortisol for clear identification of patients with later recurrence could not be determined.

New operational dose quantity ambient doseH* in the context of galactic cosmic radiation in aviation.

The International Commission on Radiation Units and Measurements recently proposed new operational quantities for external radiation exposure. Among those, the ambient dose is intended to replace the ambient dose equivalent as estimator for the effective dose. Following its definition, the measurement of the ambient dose requires a much more detailed knowledge about the radiation field than the ambient dose equivalent. The implications for radiation protection in aviation concerning galactic cosmic radiation that would follow the adoption of the ambient dose as operational quantity at flight altitudes were investigated in this work using model calculations. It was found that the ambient dose is about 10% higher than the ambient dose equivalent for conditions relevant in commercial aviation and overestimates the effective dose by about 30%.

Differentiation of human pluripotent stem cells into pancreatic duct-like organoids.

The recapitulation of human developmental processes and pathological manifestations requires access to specific cell types and precursor stages during embryogenesis and disease. Here, we describe a scalable in vitro differentiation protocol to guide human pluripotent stem cells stepwise into pancreatic duct-like organoids. The protocol mimics pancreatic duct development and was successfully used to model the onset and progression of pancreatic ductal adenocarcinoma; the approach is suitable for multiple downstream applications. However, the protocol is cost- and time-intensive. For complete details on the use and execution of this protocol, please refer to Breunig et al. (2021).

CDKN2A-Mutated Pancreatic Ductal Organoids from Induced Pluripotent Stem Cells to Model a Cancer Predisposition Syndrome.

Patient-derived induced pluripotent stem cells (iPSCs) provide a unique platform to study hereditary disorders and predisposition syndromes by resembling germline mutations of affected individuals and by their potential to differentiate into nearly every cell type of the human body. We employed plucked human hair from two siblings with a family history of cancer carrying a pathogenic CDKN2A variant, P16-p.G101W/P14-p.R115L, to generate patient-specific iPSCs in a cancer-prone ancestry for downstream analytics. The differentiation capacity to pancreatic progenitors and to pancreatic duct-like organoids (PDLOs) according to a recently developed protocol remained unaffected. Upon inducible expression of KRASG12Dusing a piggyBac transposon system in CDKN2A-mutated PDLOs, we revealed structural and molecular changes in vitro, including disturbed polarity and epithelial-to-mesenchymal (EMT) transition. CDKN2A-mutated KRASG12DPDLO xenotransplants formed either a high-grade precancer lesion or a partially dedifferentiated PDAC-like tumor. Intriguingly, P14/P53/P21 and P16/RB cell-cycle checkpoint controls have been only partly overcome in these grafts, thereby still restricting the tumorous growth. Hereby, we provide a model for hereditary human pancreatic cancer that enables dissection of tumor initiation and early development starting from patient-specific CDKN2A-mutated pluripotent stem cells.

Engineering islets from stem cells for advanced therapies of diabetes.

Diabetes mellitus is a metabolic disorder that affects more than 460 million people worldwide. Type 1 diabetes (T1D) is caused by autoimmune destruction of ß-cells, whereas type 2 diabetes (T2D) is caused by a hostile metabolic environment that leads to ß-cell exhaustion and dysfunction. Currently, first-line medications treat the symptomatic insulin resistance and hyperglycaemia, but do not prevent the progressive decline of ß-cell mass and function. Thus, advanced therapies need to be developed that either protect or regenerate endogenous ß-cell mass early in disease progression or replace lost ß-cells with stem cell-derived ß-like cells or engineered islet-like clusters. In this Review, we discuss the state of the art of stem cell differentiation and islet engineering, reflect on current and future challenges in the area and highlight the potential for cell replacement therapies, disease modelling and drug development using these cells. These efforts in stem cell and regenerative medicine will lay the foundations for future biomedical breakthroughs and potentially curative treatments for diabetes.

Single-cell-resolved differentiation of human induced pluripotent stem cells into pancreatic duct-like organoids on a microwell chip.

Creating in vitro models of diseases of the pancreatic ductal compartment requires a comprehensive understanding of the developmental trajectories of pancreas-specific cell types. Here we report the single-cell characterization of the differentiation of pancreatic duct-like organoids (PDLOs) from human induced pluripotent stem cells (hiPSCs) on a microwell chip that facilitates the uniform aggregation and chemical induction of hiPSC-derived pancreatic progenitors. Using time-resolved single-cell transcriptional profiling and immunofluorescence imaging of the forming PDLOs, we identified differentiation routes from pancreatic progenitors through ductal intermediates to two types of mature duct-like cells and a few non-ductal cell types. PDLO subpopulations expressed either mucins or the cystic fibrosis transmembrane conductance regulator, and resembled human adult duct cells. We also used the chip to uncover ductal markers relevant to pancreatic carcinogenesis, and to establish PDLO co-cultures with stellate cells, which allowed for the study of epithelial-mesenchymal signalling. The PDLO microsystem could be used to establish patient-specific pancreatic duct models.

Modeling plasticity and dysplasia of pancreatic ductal organoids derived from human pluripotent stem cells.

Personalized in vitro models for dysplasia and carcinogenesis in the pancreas have been constrained by insufficient differentiation of human pluripotent stem cells (hPSCs) into the exocrine pancreatic lineage. Here, we differentiate hPSCs into pancreatic duct-like organoids (PDLOs) with morphological, transcriptional, proteomic, and functional characteristics of human pancreatic ducts, further maturing upon transplantation into mice. PDLOs are generated from hPSCs inducibly expressing oncogenic GNAS, KRAS, or KRAS with genetic covariance of lost CDKN2A and from induced hPSCs derived from a McCune-Albright patient. Each oncogene causes a specific growth, structural, and molecular phenotype in vitro. While transplanted PDLOs with oncogenic KRAS alone form heterogenous dysplastic lesions or cancer, KRAS with CDKN2A loss develop dedifferentiated pancreatic ductal adenocarcinomas. In contrast, transplanted PDLOs with mutant GNAS lead to intraductal papillary mucinous neoplasia-like structures. Conclusively, PDLOs enable in vitro and in vivo studies of pancreatic plasticity, dysplasia, and cancer formation from a genetically defined background.

Efficacy and Safety of Everolimus With Reduced Tacrolimus in Liver Transplant Recipients: 24-month Results From the Pooled Analysis of 2 Randomized Controlled Trials.

BACKGROUND AND METHODS: Data from 2 randomized liver transplant trials (N = 772; H2304 [deceased donor, n = 488], H2307 [living donor, n = 284]) were pooled to further evaluate the efficacy and safety of everolimus with reduced tacrolimus (EVR + rTAC) versus standard tacrolimus (sTAC) regimen at month 24. RESULTS: EVR + rTAC was comparable to sTAC for composite efficacy failure of treated biopsy-proven acute rejection, graft loss, or death (9.8% versus 10.8%; difference, -1.0%; 95% confidence interval, -5.4 to 3.4; P = 0.641) at month 24. EVR + rTAC was superior to sTAC for the mean change in estimated glomerular filtration rate (eGFR) from randomization to month 24 (-8.37 versus -13.40 mL/min/1.73 m2; P = 0.001). A subanalysis of renal function by chronic kidney disease (CKD) stage at randomization showed significantly lower decline in eGFR from randomization to month 24 for patients with CKD stage 1/2 (eGFR ≥ 60 mL/min/1.73 m2) in EVR + rTAC group versus sTAC (-12.82 versus -17.67 mL/min/1.73 m2, P = 0.009). In patients transplanted for hepatocellular carcinoma (HCC) beyond Milan criteria, HCC recurrence was numerically lower although not statistically significant with EVR + rTAC versus sTAC group (5.9% [1 of 17] versus 23.1% [6 of 26], P = 0.215), while comparable in patients within Milan criteria (2.9% [3 of 102] versus 2.1% [2 of 96], P = 1.000), irrespective of pretransplant alpha-fetoprotein levels. CONCLUSIONS: EVR + rTAC versus sTAC showed comparable efficacy and safety with significantly better renal function, particularly in patients with normal/mildly decreased renal function (CKD stage 1/2) at randomization and a trend toward lower HCC recurrence in patients transplanted with HCC beyond Milan at month 24. Further long-term data would be required to confirm these results.

Inceptor counteracts insulin signalling in ß-cells to control glycaemia.

Resistance to insulin and insulin-like growth factor 1 (IGF1) in pancreatic ß-cells causes overt diabetes in mice; thus, therapies that sensitize ß-cells to insulin may protect patients with diabetes against ß-cell failure1-3. Here we identify an inhibitor of insulin receptor (INSR) and IGF1 receptor (IGF1R) signalling in mouse ß-cells, which we name the insulin inhibitory receptor (inceptor; encoded by the gene Iir). Inceptor contains an extracellular cysteine-rich domain with similarities to INSR and IGF1R4, and a mannose 6-phosphate receptor domain that is also found in the IGF2 receptor (IGF2R)5. Knockout mice that lack inceptor (Iir-/-) exhibit signs of hyperinsulinaemia and hypoglycaemia, and die within a few hours of birth. Molecular and cellular analyses of embryonic and postnatal pancreases from Iir-/- mice showed an increase in the activation of INSR-IGF1R in Iir-/- pancreatic tissue, resulting in an increase in the proliferation and mass of ß-cells. Similarly, inducible ß-cell-specific Iir-/- knockout in adult mice and in ex vivo islets led to an increase in the activation of INSR-IGF1R and increased proliferation of ß-cells, resulting in improved glucose tolerance in vivo. Mechanistically, inceptor interacts with INSR-IGF1R to facilitate clathrin-mediated endocytosis for receptor desensitization. Blocking this physical interaction using monoclonal antibodies against the extracellular domain of inceptor resulted in the retention of inceptor and INSR at the plasma membrane to sustain the activation of INSR-IGF1R in ß-cells. Together, our findings show that inceptor shields insulin-producing ß-cells from constitutive pathway activation, and identify inceptor as a potential molecular target for INSR-IGF1R sensitization and diabetes therapy.

Three-year outcomes from the CRADLE study in de novo pediatric kidney transplant recipients receiving everolimus with reduced tacrolimus and early steroid withdrawal.

CRADLE was a 36-month multicenter study in pediatric (≥1 to <18 years) kidney transplant recipients randomized at 4 to 6 weeks posttransplant to receive everolimus + reduced-exposure tacrolimus (EVR + rTAC; n = 52) with corticosteroid withdrawal at 6-month posttransplant or continue mycophenolate mofetil + standard-exposure TAC (MMF + sTAC; n = 54) with corticosteroids. The incidence of composite efficacy failure (biopsy-proven acute rejection [BPAR], graft loss, or death) at month 36 was 9.8% vs 9.6% (difference: 0.2%; 80% confidence interval: -7.3 to 7.7) for EVR + rTAC and MMF + sTAC, respectively, which was driven by BPARs. Graft loss was low (2.1% vs 3.8%) with no deaths. Mean estimated glomerular filtration rate at month 36 was comparable between groups (68.1 vs 67.3 mL/min/1.73 m2 ). Mean changes (z-score) in height (0.72 vs 0.39; P = .158) and weight (0.61 vs 0.82; P = .453) from randomization to month 36 were comparable, whereas growth in prepubertal patients on EVR + rTAC was better (P = .050) vs MMF + sTAC. The overall incidence of adverse events (AEs) and serious AEs was comparable between groups. Rejection was the leading AE for study drug discontinuation in the EVR + rTAC group. In conclusion, though AE-related study drug discontinuation was higher, an EVR + rTAC regimen represents an alternative treatment option that enables withdrawal of steroids as well as reduction of CNIs for pediatric kidney transplant recipients. ClinicalTrials.gov: NCT01544491.