Skin pharmacokinetics of miltefosine in the treatment of post-kala-azar dermal leishmaniasis in South Asia.

INTRODUCTION: Post-kala-azar dermal leishmaniasis (PKDL) arises as a dermal complication following a visceral leishmaniasis (VL) infection. Current treatment options for PKDL are unsatisfactory, and there is a knowledge gap regarding the distribution of antileishmanial compounds within human skin. The present study investigated the skin distribution of miltefosine in PKDL patients, with the aim to improve the understanding of the pharmacokinetics at the skin target site in PKDL. METHODS: Fifty-two PKDL patients underwent treatment with liposomal amphotericin B (20 mg/kg) plus miltefosine (allometric dosing) for 21 days. Plasma concentrations of miltefosine were measured on study days 8, 15, 22 and 30, while a punch skin biopsy was taken on day 22. A physiologically based pharmacokinetic (PBPK) model was developed to evaluate the distribution of miltefosine into the skin. RESULTS: Following the allometric weight-based dosing regimen, median miltefosine concentrations on day 22 were 43.73 µg/g (IQR: 21.94-60.65 µg/g) in skin and 33.29 µg/mL (IQR: 25.9-42.58 µg/mL) in plasma. The median individual concentration ratio of skin to plasma was 1.19 (IQR: 0.79-1.9). In 87% (45/52) of patients, skin exposure was above the suggested EC90 PK target of 10.6 mg/L associated with in vitro susceptibility. Simulations indicated that the residence time of miltefosine in the skin would be more than 2-fold longer than in plasma, estimated by a mean residence time of 604 versus 266 hours, respectively. CONCLUSION: This study provides the first accurate measurements of miltefosine penetration into the skin, demonstrating substantial exposure and prolonged retention of miltefosine within the skin. These findings support the use of miltefosine in cutaneous manifestations of leishmaniasis. In combination with parasitological and clinical data, these results are critical for the future optimization of combination therapies with miltefosine in the treatment of PKDL.

LDHB contributes to the regulation of lactate levels and basal insulin secretion in human pancreatic ß cells.

Using 13C6 glucose labeling coupled to gas chromatography-mass spectrometry and 2D 1H-13C heteronuclear single quantum coherence NMR spectroscopy, we have obtained a comparative high-resolution map of glucose fate underpinning ß cell function. In both mouse and human islets, the contribution of glucose to the tricarboxylic acid (TCA) cycle is similar. Pyruvate fueling of the TCA cycle is primarily mediated by the activity of pyruvate dehydrogenase, with lower flux through pyruvate carboxylase. While the conversion of pyruvate to lactate by lactate dehydrogenase (LDH) can be detected in islets of both species, lactate accumulation is 6-fold higher in human islets. Human islets express LDH, with low-moderate LDHA expression and ß cell-specific LDHB expression. LDHB inhibition amplifies LDHA-dependent lactate generation in mouse and human ß cells and increases basal insulin release. Lastly, cis-instrument Mendelian randomization shows that low LDHB expression levels correlate with elevated fasting insulin in humans. Thus, LDHB limits lactate generation in ß cells to maintain appropriate insulin release.

Molecular determinants and intracellular targets of taurine signalling in pancreatic islet ß-cells.

AIM: Despite its abundance in pancreatic islets of Langerhans and proven antihyperglycemic effects, the impact of the essential amino acid, taurine, on islet ß-cell biology has not yet received due consideration, which prompted the current studies exploring the molecular selectivity of taurine import into ß-cells and its acute and chronic intracellular interactions. METHODS: The molecular aspects of taurine transport were probed by exposing the clonal pancreatic BRIN BD11 ß-cells and primary mouse and human islets to a range of the homologs of the amino acid (assayed at 2-20 mM), using the hormone release and imaging of intracellular signals as surrogate read-outs. Known secretagogues were employed to profile the interaction of taurine with acute and chronic intracellular signals. RESULTS: Taurine transporter TauT was expressed in the islet ß-cells, with the transport of taurine and homologs having a weak sulfonate specificity but significant sensitivity to the molecular weight of the transporter. Taurine, hypotaurine, homotaurine, and ß-alanine enhanced insulin secretion in a glucose-dependent manner, an action potentiated by cytosolic Ca2+ and cAMP. Acute and chronic ß-cell insulinotropic effects of taurine were highly sensitive to co-agonism with GLP-1, forskolin, tolbutamide, and membrane depolarization, with an unanticipated indifference to the activation of PKC and CCK8 receptors. Pre-culturing with GLP-1 or KATP channel inhibitors sensitized or, respectively, desensitized ß-cells to the acute taurine stimulus. CONCLUSION: Together, these data demonstrate the pathways whereby taurine exhibits a range of beneficial effects on insulin secretion and ß-cell function, consistent with the antidiabetic potential of its dietary low-dose supplementation.

GLP-1 metabolite GLP-1(9-36) is a systemic inhibitor of mouse and human pancreatic islet glucagon secretion.

AIMS/HYPOTHESIS: Diabetes mellitus is associated with impaired insulin secretion, often aggravated by oversecretion of glucagon. Therapeutic interventions should ideally correct both defects. Glucagon-like peptide 1 (GLP-1) has this capability but exactly how it exerts its glucagonostatic effect remains obscure. Following its release GLP-1 is rapidly degraded from GLP-1(7-36) to GLP-1(9-36). We hypothesised that the metabolite GLP-1(9-36) (previously believed to be biologically inactive) exerts a direct inhibitory effect on glucagon secretion and that this mechanism becomes impaired in diabetes. METHODS: We used a combination of glucagon secretion measurements in mouse and human islets (including islets from donors with type 2 diabetes), total internal reflection fluorescence microscopy imaging of secretory granule dynamics, recordings of cytoplasmic Ca2+ and measurements of protein kinase A activity, immunocytochemistry, in vivo physiology and GTP-binding protein dissociation studies to explore how GLP-1 exerts its inhibitory effect on glucagon secretion and the role of the metabolite GLP-1(9-36). RESULTS: GLP-1(7-36) inhibited glucagon secretion in isolated islets with an IC50 of 2.5 pmol/l. The effect was particularly strong at low glucose concentrations. The degradation product GLP-1(9-36) shared this capacity. GLP-1(9-36) retained its glucagonostatic effects after genetic/pharmacological inactivation of the GLP-1 receptor. GLP-1(9-36) also potently inhibited glucagon secretion evoked by ß-adrenergic stimulation, amino acids and membrane depolarisation. In islet alpha cells, GLP-1(9-36) led to inhibition of Ca2+ entry via voltage-gated Ca2+ channels sensitive to ω-agatoxin, with consequential pertussis-toxin-sensitive depletion of the docked pool of secretory granules, effects that were prevented by the glucagon receptor antagonists REMD2.59 and L-168049. The capacity of GLP-1(9-36) to inhibit glucagon secretion and reduce the number of docked granules was lost in alpha cells from human donors with type 2 diabetes. In vivo, high exogenous concentrations of GLP-1(9-36) (>100 pmol/l) resulted in a small (30%) lowering of circulating glucagon during insulin-induced hypoglycaemia. This effect was abolished by REMD2.59, which promptly increased circulating glucagon by >225% (adjusted for the change in plasma glucose) without affecting pancreatic glucagon content. CONCLUSIONS/INTERPRETATION: We conclude that the GLP-1 metabolite GLP-1(9-36) is a systemic inhibitor of glucagon secretion. We propose that the increase in circulating glucagon observed following genetic/pharmacological inactivation of glucagon signalling in mice and in people with type 2 diabetes reflects the removal of GLP-1(9-36)'s glucagonostatic action.

Beta-cell death and dysfunction drives hyperglycaemia in organ donors.

BACKGROUND: Donor hyperglycaemia following brain death has been attributed to reversible insulin resistance. However, our islet and pancreas transplant data suggest that other mechanisms may be predominant. We aimed to determine the relationships between donor insulin use and markers of beta-cell death and beta-cell function in pancreas donors after brain death. METHODS: In pancreas donors after brain death, we compared clinical and biochemical data in 'insulin-treated' and 'not insulin-treated donors' (IT vs. not-IT). We measured plasma glucose, C-peptide and levels of circulating unmethylated insulin gene promoter cell-free DNA (INS-cfDNA) and microRNA-375 (miR-375), as measures of beta-cell death. Relationships between markers of beta-cell death and islet isolation outcomes and post-transplant function were also evaluated. RESULTS: Of 92 pancreas donors, 40 (43%) required insulin. Glycaemic control and beta-cell function were significantly poorer in IT donors versus not-IT donors [median (IQR) peak glucose: 8 (7-11) vs. 6 (6-8) mmol/L, p = .016; C-peptide: 3280 (3159-3386) vs. 3195 (2868-3386) pmol/L, p = .046]. IT donors had significantly higher levels of INS-cfDNA [35 (18-52) vs. 30 (8-51) copies/ml, p = .035] and miR-375 [1.050 (0.19-1.95) vs. 0.73 (0.32-1.10) copies/nl, p = .05]. Circulating donor miR-375 was highly predictive of recipient islet graft failure at 3 months [adjusted receiver operator curve (SE) = 0.813 (0.149)]. CONCLUSIONS: In pancreas donors, hyperglycaemia requiring IT is strongly associated with beta-cell death. This provides an explanation for the relationship of donor IT with post-transplant beta-cell dysfunction in transplant recipients.

European Society for Organ Transplantation (ESOT) Consensus Statement on the Role of Pancreas Machine Perfusion to Increase the Donor Pool for Beta Cell Replacement Therapy.

The advent of Machine Perfusion (MP) as a superior form of preservation and assessment for cold storage of both high-risk kidney's and the liver presents opportunities in the field of beta-cell replacement. It is yet unknown whether such techniques, when applied to the pancreas, can increase the pool of suitable donor organs as well as ameliorating the effects of ischemia incurred during the retrieval process. Recent experimental models of pancreatic MP appear promising. Applications of MP to the pancreas, needs refinement regarding perfusion protocols and organ viability assessment criteria. To address the "Role of pancreas machine perfusion to increase the donor pool for beta cell replacement," the European Society for Organ Transplantation (ESOT) assembled a dedicated working group comprising of experts to review literature pertaining to the role of MP as a method of improving donor pancreas quality as well as quantity available for transplant, and to develop guidelines founded on evidence-based reviews in experimental and clinical settings. These were subsequently refined during the Consensus Conference when this took place in Prague.

NKCC transport mediates the insulinotropic effects of taurine and other small neutral amino acids.

AIMS: Despite its high concentration in pancreatic islets of Langerhans and broad range of antihyperglycemic effects, the route facilitating the import of dietary taurine into pancreatic ß-cell and mechanisms underlying its insulinotropic activity are unclear. We therefore studied the impact of taurine on beta-cell function, alongside that of other small neutral amino acids, L-alanine and L-proline. MAIN METHODS: Pharmacological profiling of insulin secretion was conducted using clonal BRIN BD11 ß-cells, the impact of taurine on the metabolic fate of glucose carbons was assessed using NMR and the findings were verified by real-time imaging of Ca2+ dynamics in the cytosol of primary mouse and human islet beta-cells. KEY FINDINGS: In our hands, taurine, alanine and proline induced secretory responses that were dependent on the plasma membrane depolarisation, import of Ca2+, homeostasis of K+ and Na+ as well as on cell glycolytic and oxidative metabolism. Taurine shifted the balance between the oxidation and anaplerosis towards the latter, in BRIN BD11 beta-cells. Furthermore, the amino acid signalling was significantly attenuated by inhibition of Na+-K+-Cl- symporter (NKCC). SIGNIFICANCE: These data suggest that taurine, like L-alanine and L-proline, acutely induces glucose-dependent insulin-secretory responses by modulating electrogenic Na+ transport, with potential role of intracellular K+ and Cl- in the signal transduction. The acute action delineated would be consistent with antidiabetic potential of dietary taurine supplementation.

The ischaemic preconditioning paradox and its implications for islet isolation from heart-beating and non heart-beating donors.

The impact of ischaemia can severely damage procured donor organs for transplantation. The pancreas, and pancreatic islets in particular, is one of the most sensitive tissues towards hypoxia. The present study was aimed to assess the effect of hypoxic preconditioning (HP) performed ex-vivo in islets isolated from heart-beating donor (HBD) and non heart-beating donor (NHBD) rats. After HP purified islets were cultured for 24 h in hypoxia followed by islet characterisation. Post-culture islet yields were significantly lower in sham-treated NHBD than in HBD. This difference was reduced when NHBD islets were preconditioned. Similar results were observed regarding viability, apoptosis and in vitro function. Reactive oxygen species generation after hypoxic culture was significantly enhanced in sham-treated NHBD than in HBD islets. Again, this difference could be diminished through HP. qRT-PCR revealed that HP decreases pro-apoptotic genes but increases HIF-1 and VEGF. However, the extent of reduction and augmentation was always substantially higher in preconditioned NHBD than in HBD islets. Our findings indicate a lower benefit of HBD islets from HP than NHBD islets. The ischaemic preconditioning paradox suggests that HP should be primarily applied to islets from marginal donors. This observation needs evaluation in human islets.

Acceptance and Perception of Artificial Intelligence Usability in Eye Care (APPRAISE) for Ophthalmologists: A Multinational Perspective.

Background: Many artificial intelligence (AI) studies have focused on development of AI models, novel techniques, and reporting guidelines. However, little is understood about clinicians' perspectives of AI applications in medical fields including ophthalmology, particularly in light of recent regulatory guidelines. The aim for this study was to evaluate the perspectives of ophthalmologists regarding AI in 4 major eye conditions: diabetic retinopathy (DR), glaucoma, age-related macular degeneration (AMD) and cataract. Methods: This was a multi-national survey of ophthalmologists between March 1st, 2020 to February 29th, 2021 disseminated via the major global ophthalmology societies. The survey was designed based on microsystem, mesosystem and macrosystem questions, and the software as a medical device (SaMD) regulatory framework chaired by the Food and Drug Administration (FDA). Factors associated with AI adoption for ophthalmology analyzed with multivariable logistic regression random forest machine learning. Results: One thousand one hundred seventy-six ophthalmologists from 70 countries participated with a response rate ranging from 78.8 to 85.8% per question. Ophthalmologists were more willing to use AI as clinical assistive tools (88.1%, n = 890/1,010) especially those with over 20 years' experience (OR 3.70, 95% CI: 1.10-12.5, p = 0.035), as compared to clinical decision support tools (78.8%, n = 796/1,010) or diagnostic tools (64.5%, n = 651). A majority of Ophthalmologists felt that AI is most relevant to DR (78.2%), followed by glaucoma (70.7%), AMD (66.8%), and cataract (51.4%) detection. Many participants were confident their roles will not be replaced (68.2%, n = 632/927), and felt COVID-19 catalyzed willingness to adopt AI (80.9%, n = 750/927). Common barriers to implementation include medical liability from errors (72.5%, n = 672/927) whereas enablers include improving access (94.5%, n = 876/927). Machine learning modeling predicted acceptance from participant demographics with moderate to high accuracy, and area under the receiver operating curves of 0.63-0.83. Conclusion: Ophthalmologists are receptive to adopting AI as assistive tools for DR, glaucoma, and AMD. Furthermore, ML is a useful method that can be applied to evaluate predictive factors on clinical qualitative questionnaires. This study outlines actionable insights for future research and facilitation interventions to drive adoption and operationalization of AI tools for Ophthalmology.

Acute pancreatitis in children - morbidity and outcomes at 1 year.

OBJECTIVE: To establish short-term and medium-term complications 1-year postdiagnosis, of acute pancreatitis (AP) in children aged 0-14 years. DESIGN: One-year follow-up of a prospective monthly surveillance of new cases of AP in children under 15 years through the British Paediatric Surveillance Unit (BPSU) from April 2013 to April 2014. SETTING: A monthly surveillance of >3700 consultant paediatricians and paediatric surgeons in the UK and Ireland using the BPSU. PATIENTS: Children aged 0-14 years with a new diagnosis of AP. MAIN OUTCOME MEASURES: The outcomes following AP, including the incidence of complications and comorbidity at diagnosis and at 1 year. RESULTS: Of the 94 new confirmed cases of AP identified in the UK during the study period, 90 cases (96%) were included in the 1-year follow-up. 30 patients (32%) developed further episode(s) of AP. Over one-fifth of patients developed one or more major complication. At initial admission, the most common of these was pancreatic necrosis (n=8, 9%), followed by respiratory failure (n=7, 7%). Reported complications by 1 year were pseudocyst formation (n=9, 10%), diabetes requiring insulin therapy (n=4, 4%) and maldigestion (n=1, 1%). At 1-year postdiagnosis, only 59% of children made a full recovery with no acute or chronic complications or recurrent episodes of AP. Two patients died, indicating a case fatality of ~2.0%. CONCLUSIONS: AP in childhood is associated with significant short-term and medium-term complications and comorbidities including risk of recurrence in approximately a third of cases.

A Worldwide Survey of Activities and Practices in Clinical Islet of Langerhans Transplantation.

A global online survey was administered to 69 islet transplantation programs, covering 84 centers and 5 networks. The survey addressed questions on program organization and activity in the 2000-2020 period, including impact on activity of national health care coverage policies. We obtained full data from 55 institutions or networks worldwide and basic activity data from 6 centers. Additional data were obtained from alternative sources. A total of 94 institutions and 5 networks was identified as having performed islet allotransplantation. 4,365 islet allotransplants (2,608 in Europe, 1,475 in North America, 135 in Asia, 119 in Oceania, 28 in South America) were reported in 2,170 patients in the survey period. From 15 centers active at the start of the study period, the number of simultaneously active islet centers peaked at 54, to progressively decrease to 26 having performed islet allotransplants in 2020. Notably, only 16 centers/networks have done >100 islet allotransplants in the survey period. Types of transplants performed differed notably between North America and the rest of the world, in particular with respect to the near-absence of simultaneous islet-kidney transplantation. Absence of heath care coverage has significantly hampered transplant activity in the past years and the COVID-19 pandemic in 2020.

Instructor-Blinded Study of Pharmacy Student Learning When a Flipped Online Classroom Was Implemented during the COVID-19 Pandemic.

A multi-cohort instructor-blinded research study was completed at the School of Pharmacy, University of Waterloo, to test the impact on study learning endpoints when an online flipped classroom teaching style was implemented during the COVID-19 pandemic. The learning endpoints were gain in factual knowledge and gain in self-confidence in clinical skills (assessing a patient, developing a care plan for a minor ailment, and implementing the care plan by counselling patients on the condition). Gain in factual knowledge was assessed with an instructor-blinded multiple-choice test administered before and after the course. Gain in self-confidence in clinical skills was assessed with a survey asking students to report their self-confidence in completing 10 clinical tasks on a 5-item Likert scale. Students being taught in an online flipped classroom cohort during the COVID-19 pandemic trended toward having a higher gain in self-confidence throughout the course but a lower gain in factual knowledge when compared with a traditional classroom cohort in the previous year.

Model-Based Assessment of the Contribution of Monocytes and Macrophages to the Pharmacokinetics of Monoclonal Antibodies.

PURPOSE: We have hypothesized that a high concentration of circulating monocytes and macrophages may contribute to the fast weight-based clearance of monoclonal antibodies (mAbs) in young children. Exploring this hypothesis, this work uses modeling to clarify the role of monocytes and macrophages in the elimination of mAbs. METHODS: Leveraging pre-clinical data from mice, a minimal physiologically-based pharmacokinetic model was developed to characterize mAb uptake and FcRn-mediated recycling in circulating monocytes, macrophages, and endothelial cells. The model characterized IgG disposition in complex scenarios of site-specific FcRn deletion and variable endogenous IgG levels. Evaluation was performed for predicting IgG disposition with co-administration of high dose IVIG. A one-at-a-time sensitivity analysis quantified the role of relevant cellular parameters on IgG elimination in various scenarios. RESULTS: The plasma AUC of mAbs was highly sensitive to endothelial cell parameters, but had near-nil sensitivity to monocyte and macrophage parameters, even in scenarios with 90% loss of FcRn expression/activity. In mice with normal FcRn expression, simulations suggest that less than 2% of an IV dose is eliminated in macrophages, while endothelial cells are predicted to dominate mAb elimination. CONCLUSIONS: The model suggests that the role of monocytes and macrophages in IgG homeostasis includes extensive uptake and highly efficient FcRn-mediated protection, but not appreciable degradation when FcRn is present. Therefore, it is very unlikely that a high concentration of circulating monocytes can contribute to explaining the fast weight-based clearance of mAbs in very young children, even if FcRn expression/activity was 90% lower in children than in adults.

Basement membrane proteins improve human islet survival in hypoxia: Implications for islet inflammation.

Enzymatic digestion of the pancreas during islet isolation is associated with disintegration of the islet basement membrane (IBM) that can cause reduction of functional and morphological islet integrity. Attempts to re-establish IBM by coating the surface of culture vessels with various IBM proteins (IBMP) have resulted in loss of islet phenotype and function. This study investigated the capability of Collagen-IV, Laminin-521 and Nidogen-1, utilised as single or combined media supplements, to protect human islets cultured in hypoxia. When individually supplemented to media, all IBMP significantly improved islet survival and in-vitro function, finally resulting in as much as a two-fold increase of islet overall survival. In contrast, combining IBMP enhanced the production of chemokines and reactive oxygen species diminishing all positive effects of individually added IBMP. This impact was concentration-dependent and concerned nearly all parameters of islet integrity. Predictive extrapolation of these findings to data from 116 processed human pancreases suggests that more than 90% of suboptimal pancreases could be rescued for clinical islet transplantation increasing the number of transplantable preparations from actual 25 to 40 when adding Nidogen-1 to pretransplant culture. This study suggests that media supplementation with essential IBMP protects human islets from hypoxia. Amongst those, certain IBMP may be incompatible when combined or applied at higher concentrations. STATEMENT OF SIGNIFICANCE: Pancreatic islet transplantation is a minimally-invasive treatment that can reverse type 1 diabetes in certain patients. It involves infusing of insulin-producing cell-clusters (islets) from donor pancreases. Unfortunately, islet extraction is associated with damage of the islet basement membrane (IBM) causing reduced islet function and cell death. Attempts to re-establish the IBM by coating the surface of culture vessels with IBM proteins (IBMP) have been unsuccessful. Instead, we dissolved the most relevant IBM components Collagen-IV, Laminin-521 and Nidogen-1 in media routinely used for clinical islet culture and transplantation. We found human islet survival and function was substantially improved by IBMP, particularly Nidogen-1, when exposed to a hypoxic environment as found in vivo. We also investigated IBMP combinations. Our present findings have important clinical implications.

Pediatric Dose Selection for Therapeutic Proteins.

In selecting optimal dosing regimens in support of the clinical use of monoclonal antibodies and other therapeutic proteins in pediatric indications, the unique pharmacokinetic properties of this class of biologics, as well as the underlying physiologic and pathophysiologic processes and their modulation by childhood growth and development, needs to be appreciated. During drug development, first-in-pediatric dose selection is a capstone event in the pediatric investigation plan that relies heavily on extrapolation of pharmacokinetic and pharmacodynamic data from adult to pediatric populations. It is facilitated by combinations of pharmacometric approaches, including allometry, physiologically based pharmacokinetic modeling, and population pharmacokinetic analyses, although data on reliability and qualification of some of these tools in the context of therapeutic proteins are still limited but emerging. Presented data suggest nonlinear relationships between body weight and both clearance and volume of distribution for therapeutic proteins in pediatric populations, with allometric exponents of 0.75 and 0.8, respectively. For newborns and infants (<1 year), even higher nonlinearity seems to occur. Translation of the quantitative characterization of the pediatric pharmacokinetics of therapeutic proteins into dosing regimens for the drug label requires compromising between precision dosing and clinical practicability, with tiered dosing algorithms based on size or age strata being the currently most frequently applied methodology.

High Concentrations of Etanercept Reduce Human Islet Function and Integrity.

BACKGROUND: Most islet transplant groups worldwide routinely use the TNFα inhibitor Etanercept in their peri-transplant protocols. Surprisingly, there have been no published dose-response studies on the effects of Etanercept on human islets. Our study aimed to address this by treating cultured human islets with increasing concentrations of Etanercept. MATERIALS AND METHODS: Isolated human islets were cultured for 3-4 days in normoxic (21% oxygen) or in hypoxic (2% oxygen) atmosphere using Etanercept dissolved in a range of 2.5-40 µg/mL prior to islet characterisation. RESULTS: In normoxic atmosphere, it was found that 5 µg/mL is the most efficient dose to preserve islet morphological and functional integrity during culture. Increasing the dose to 10 µg/mL or more resulted in detrimental effects with respect to viability and glucose-stimulated insulin release. When human islets were cultured for 3 to 4 days in clinically relevant hypoxia and treated with 5 µg/mL Etanercept, post-culture islet survival (P < 0.001) and in vitro function (P < 0.01) were significantly improved. This correlated with a substantially reduced cytokine production (P < 0.05), improved mitochondrial function (P < 0.01), and reduced production of reactive oxygen species (P < 0.001) in hypoxia-exposed islets. CONCLUSION: These findings suggest that the therapeutic window of Etanercept is very narrow and that this should be considered when optimising the dosage and route of Etanercept administration in islet-transplant recipients or when designing novel drug-delivering islet scaffolds.

'Paediatric surgical research in the UK - Challenges and opportunities'.

Academic pediatric surgery faces challenges and opportunity. The author provides a brief overview of the landscape of academic surgery from a UK perspective and based on his considerable experience, makes suggestions for present and future directions.

Spatiotemporal analysis of human intestinal development at single-cell resolution.

Development of the human intestine is not well understood. Here, we link single-cell RNA sequencing and spatial transcriptomics to characterize intestinal morphogenesis through time. We identify 101 cell states including epithelial and mesenchymal progenitor populations and programs linked to key morphogenetic milestones. We describe principles of crypt-villus axis formation; neural, vascular, mesenchymal morphogenesis, and immune population of the developing gut. We identify the differentiation hierarchies of developing fibroblast and myofibroblast subtypes and describe diverse functions for these including as vascular niche cells. We pinpoint the origins of Peyer's patches and gut-associated lymphoid tissue (GALT) and describe location-specific immune programs. We use our resource to present an unbiased analysis of morphogen gradients that direct sequential waves of cellular differentiation and define cells and locations linked to rare developmental intestinal disorders. We compile a publicly available online resource, spatio-temporal analysis resource of fetal intestinal development (STAR-FINDer), to facilitate further work.

A Physiological Approach to Pharmacokinetics in Chronic Kidney Disease.

The conventional approach to approximating the pharmacokinetics of drugs in patients with chronic kidney disease (CKD) only accounts for changes in the estimated glomerular filtration rate. However, CKD is a systemic and multifaceted disease that alters many body systems. Therefore, the objective of this exercise was to develop and evaluate a whole-body mechanistic approach to predicting pharmacokinetics in patients with CKD. Physiologically based pharmacokinetic models were developed in PK-Sim v8.0 (www.open-systems-pharmacology.org) to mechanistically represent the disposition of 7 compounds in healthy human adults. The 7 compounds selected were eliminated by glomerular filtration and active tubular secretion by the organic cation transport system to varying degrees. After a literature search, the healthy adult models were adapted to patients with CKD by numerically accounting for changes in glomerular filtration rate, kidney volume, renal perfusion, hematocrit, plasma protein concentrations, and gastrointestinal transit. Literature-informed interindividual variability was applied to the physiological parameters to facilitate a population approach. Model performance in CKD was evaluated against pharmacokinetic data from 8 clinical trials in the literature. Overall, integration of the CKD parameterization enabled exposure predictions that were within 1.5-fold error across all compounds and patients with varying stages of renal impairment. Notable improvement was observed over the conventional approach to scaling exposure, which failed in all but 1 scenario in patients with advanced CKD. Further research is required to qualify its use for first-in-CKD dose selection and clinical trial planning for a wider selection of renally eliminated compounds, including those subject to anion transport.

Proteomic Profiling Reveals the Ambivalent Character of the Mesenchymal Stem Cell Secretome: Assessing the Effect of Preconditioned Media on Isolated Human Islets.

Previous studies in rodents have indicated that function and survival of transplanted islets can be substantially improved by mesenchymal stem cells (MSC). The few human islet studies to date have confirmed these findings but have not determined whether physical contact between MSC and islets is required or whether the benefit to islets results from MSC-secreted proteins. This study aimed to investigate the protective capacity of MSC-preconditioned media for human islets. MSC were cultured for 2 or 5 days in normoxia or hypoxia before harvesting the cell-depleted media for human islet culture in normoxia or hypoxia for 6-8 or 3-4 days, respectively. To characterize MSC-preconditioned media, proteomic secretome profiling was performed to identify angiogenesis- and inflammation-related proteins. A protective effect of MSC-preconditioned media on survival and in vitro function of hypoxic human islets was observed irrespective of the atmosphere used for MSC preconditioning. Islet morphology changed markedly when media from hypoxic MSC were used for culture. However, PDX-1 and insulin gene expression did not confirm a change in the genetic phenotype of these islets. Proteomic profiling of preconditioned media revealed the heterogenicity of the secretome comprising angiogenic and antiapoptotic as well as angiostatic or proinflammatory mediators released at an identical pattern regardless whether MSC had been cultured in normoxic or hypoxic atmosphere. These findings do not allow a clear discrimination between normoxia and hypoxia as stimulus for protective MSC capabilities but indicate an ambivalent character of the MSC angiogenesis- and inflammation-related secretome. Nevertheless, culture of human islets in acellular MSC-preconditioned media resulted in improved morphological and functional islet integrity suggesting a disbalance in favor of protective factors. Further approaches should aim to eliminate potentially detrimental factors to enable the production of advanced clinical grade islet culture media with higher protective qualities.