Sustained Secretion Research Articles

Exendin-4 induces a novel extended effect of ischemic tolerance via crosstalk with IGF-1R

Glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) agonist exendin-4 (Ex-4), a drug that has been used in the clinical treatment of type 2 diabetes mellitus, also confers a neuroprotective effect against stroke. Although GLP-1 analogs were reported to induce sustained insulin secretion and glucose tolerance improved after cessation of treatment, no study has revealed whether Ex-4 exerts sustained neuroprotection against stroke and the underlying mechanism after treatment cessation. In this study, mice were pretreated with Ex-4 for 7 days, and middle cerebral artery occlusion (MCAO) was performed on different days after cessation of Ex-4 treatment. Ex-4 ameliorated neurological dysfunction and reduced the infarct volume induced by MCAO. These protective effects lasted for 6 days after the cessation of Ex-4 treatment and were associated with sustained upregulation of PI3K, AKT, mTOR, and HIF-1α levels, as well as HIF-1α downstream genes. Knockdown of GLP-1R or HIF-1α in the brain by short hairpin RNA abolished Ex-4 treatment-mediated neuroprotection. In normal mice, Ex-4 treatment led to instant upregulation of p-PI3K, p-AKT, p-mTOR, and HIF-1α expression levels, which quickly returned to normal after cessation of Ex-4 treatment, while the expression levels of insulin growth factor-1 receptor (IGF-1R) remained high for 6 days after Ex-4 cessation. Additionally, Ex-4 did not directly induce IGF-1 production, which was only induced by MCAO. Ex-4 induces extended cerebral ischemic tolerance. This neuroprotective effect is associated with activation of GLP-1R and upregulation of IGF-1R in the brain, and the latter then activates the PI3K/AKT/mTOR/HIF-1 signaling pathway via binding to IGF-1 secreted from the ischemic brain.

Development and characterization of a human Th17-driven ex vivo skin inflammation model.

Skin models mimicking features of psoriasis‐related inflammation are needed to support the development of new drugs in dermatology. Reconstructed skin models lack tissue complexity, including a fully competent skin barrier, and presence and/or diversity of immune cells. Here, we describe InflammaSkin®, a novel human Th17‐driven ex vivo skin inflammation model. In this model, skin‐resident T cells are in situ activated by intradermal injection of anti‐CD3 and anti‐CD28 antibodies and Th17 cell polarization is sustained by culture in a chemically defined medium supplemented with IL‐1β, IL‐23 and TGF‐β for seven days. The acquired Th17 signature is demonstrated by the sustained secretion of IL‐17A, IL‐17AF, IL‐17F, IL‐22, IFN‐γ, and to some degree IL‐15 and TNF‐α observed in the activated ex vivo skin inflammation model compared with the non‐activated skin model control. Furthermore, expression of S100A7 and Keratin‐16 by keratinocytes and loss of epidermal structure integrity occur subsequently to in situ Th17cell activation, demonstrating cellular crosstalk between Th17 cells and keratinocytes. Finally, we demonstrate the use of this model to investigate the modulation of the IL‐23/IL‐17 immune axis by topically applied anti‐inflammatory compounds. Taken together, we show that by in situ activation of skin‐resident Th17 cells, the InflammaSkin® model reproduces aspects of inflammatory responses observed in psoriatic lesions and could be used as a translational tool to assess efficacy of test compounds.

Fatty acid transport protein-2 regulates glycemic control and diabetic kidney disease progression.

Kidney disease is one of the most devastating complications of diabetes, and tubular atrophy predicts diabetic kidney disease (DKD) progression to end-stage renal disease. We have proposed that fatty acids bound to albumin contribute to tubular atrophy by inducing lipotoxicity, after filtration across damaged glomeruli, and subsequent proximal tubule reabsorption by a fatty acid transport protein-2-dependent (FATP2-dependent) mechanism. To address this possibility, genetic (Leprdb/db eNOS-/-) and induced (high-fat diet plus low-dose streptozotocin) mouse models of obesity and DKD were bred with global FATP2 gene-deleted mice (Slc27a2) and then phenotyped. DKD-prone mice with the Slc27a2-/- genotype demonstrated normalization of glomerular filtration rate, reduced albuminuria, improved kidney histopathology, and longer life span compared with diabetic Slc27a2+/+ mice. Genetic and induced DKD-prone Slc27a2-/- mice also exhibited markedly reduced fasting plasma glucose, with mean values approaching euglycemia, despite increased obesity and decreased physical activity. Glucose lowering in DKD-prone Slc27a2-/- mice was accompanied by β cell hyperplasia and sustained insulin secretion. Together, our data indicate that FATP2 regulates DKD pathogenesis by a combined lipotoxicity and glucotoxicity (glucolipotoxicity) mechanism.

B-cell depletion with rituximab in the COVID-19 pandemic: where do we stand?

B-cell depletion with rituximab in the COVID-19 pandemic: where do we stand?

Signalling, trafficking and glucoregulatory properties of glucagon-like peptide-1 receptor agonists exendin-4 and lixisenatide.

Background and PurposeAmino acid substitutions at the N‐termini of glucagon‐like peptide‐1 (GLP‐1) receptor agonist peptides result in distinct patterns of intracellular signalling, sub‐cellular trafficking and efficacy in vivo. Here, we to determine whether sequence differences at the ligand C‐termini of clinically approved GLP‐1 receptor agonists exendin‐4 and lixisenatide lead to similar phenomena.Experimental ApproachExendin‐4, lixisenatide and N‐terminally substituted analogues with biased signalling characteristics were compared across a range of in vitro trafficking and signalling assays in different cell types. Fluorescent ligands and new time‐resolved FRET approaches were developed to study agonist behaviours at the cellular and sub‐cellular level. Anti‐hyperglycaemic and anorectic effects of each parent ligand and their biased derivatives were assessed in mice.Key ResultsLixisenatide and exendin‐4 showed equal binding affinity, but lixisenatide was fivefold less potent for cAMP signalling. Both peptides induced extensive GLP‐1 receptor clustering in the plasma membrane and were rapidly endocytosed, but the GLP‐1 receptor recycled more slowly to the cell surface after lixisenatide treatment. These combined deficits resulted in reduced maximal sustained insulin secretion and reduced anti‐hyperglycaemic and anorectic effects in mice with lixisenatide. N‐terminal substitution of His1 by Phe1 to both ligands had favourable effects on their pharmacology, resulting in improved insulin release and lowering of blood glucose.Conclusion and ImplicationsChanges to the C‐terminus of exendin‐4 affect signalling potency and GLP‐1 receptor trafficking via mechanisms unrelated to GLP‐1 receptor occupancy. These differences were associated with changes in their ability to control blood glucose and therefore may be therapeutically relevant.

278-OR: Efficacy and Safety of Anti-interleukin (IL)-21 in Combination with Liraglutide in Adults Recently Diagnosed with Type 1 Diabetes

Objective: To evaluate the effect of anti-IL-21 and liraglutide, alone and in combination, compared to placebo, on preservation of β-cell function after 54 weeks of treatment in adults with recently diagnosed type 1 diabetes. Methods: This was a multicenter, double-dummy, double-blind, efficacy, safety and pharmacokinetic randomized control trial (NCT02443155) in adults recently diagnosed with type 1 diabetes and non-fasting C-peptide peak ≥0.2 nmol/L, comprising a 54-week treatment period followed by a 26-week observation period. Primary endpoint: area under the curve (AUC)0-4h for meal-stimulated C-peptide at week 54 relative to baseline. Results: At week 54, combination treatment was associated with a significant improvement of 48% in meal-stimulated C-peptide secretion versus placebo (Table). The insulin requirement was significantly lowered by 0.13 U/kg. Trends for better glycemic control and lower risk of hypoglycemic episodes at 54 weeks compared to placebo were also observed. No safety concerns were identified. Treatment benefits were not sustained at 26 weeks after end of treatment. Conclusion: Treatment with anti-IL-21 and liraglutide for 54 weeks was safe and resulted in sustained insulin secretion and lower insulin dose. Trends for improved glucose control and fewer hypoglycemic episodes compared to placebo were observed. Disclosure M.G. von Herrath: Employee; Self; Novo Nordisk A/S. S.C. Bain: Advisory Panel; Self; Boehringer Ingelheim International GmbH, Lilly Diabetes, Merck Sharp & Dohme Corp., Napp Pharmaceuticals, Novo Nordisk A/S, Sanofi. Research Support; Self; Novo Nordisk A/S, Sanofi. Speaker’s Bureau; Self; AstraZeneca. B.W. Bode: Advisory Panel; Self; InPen, Medtronic. Consultant; Self; ADOCIA, Eli Lilly and Company, Lexicon Pharmaceuticals, Inc., Novo Nordisk A/S, Pfizer Inc. Research Support; Self; Dexcom, Inc., Diasome Pharmaceuticals, Inc., Eli Lilly and Company, Gan & Lee Pharmaceuticals, Medtronic, Novo Nordisk A/S, Provention Bio, Inc., Senseonics, Xeris Pharmaceuticals, Inc. Speaker’s Bureau; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., InPen, Janssen Pharmaceuticals, Inc., MannKind Corporation, Medtronic, Novo Nordisk A/S, Sanofi, Sanofi, Senseonics, Xeris Pharmaceuticals, Inc. Stock/Shareholder; Self; Glytec. J. Clausen: Other Relationship; Self; Novo Nordisk A/S. K. Coppieters: Other Relationship; Self; Novo Nordisk A/S. L. Gaysina: None. J. Gumprecht: Consultant; Self; Astra, Bioton, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Merck & Co., Inc., Merck Sharp & Dohme Corp., Mundipharma International, Novo Nordisk A/S, Polfa Tarchomin S.A., Sanofi, Servier. Speaker’s Bureau; Self; Astra, Bioton, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Merck & Co., Inc., Merck Sharp & Dohme Corp., Mundipharma International, Novo Nordisk A/S, Polfa Tarchomin S.A., Sanofi, Servier. T.K. Hansen: None. C. Mathieu: Advisory Panel; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company, Intrexon, MannKind Corporation, Medtronic, Merck Sharp & Dohme Corp., Novartis AG, Novo Nordisk Inc., Pfizer Inc., Roche Diagnostic USA, Sanofi. Research Support; Self; Intrexon, Novo Nordisk Inc., Sanofi. Speaker’s Bureau; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company, Merck Sharp & Dohme Corp., Novartis AG, Novo Nordisk Inc., Sanofi. C. Morales Portillo: Board Member; Self; Novo Nordisk, Lilly, MSD, Astra, Sanofi, Abbot. Research Support; Self; Novo Nordisk, Sanofi, AstraZeneca, Pzifer, Lilly, Merck, Lexicon, FPS, Hanmi, Janssen, Boehringer, Takeda, Roche, Theracos. Speaker’s Bureau; Self; Sanofi, Novo Nordisk, AstraZeneca, Roche, Lilly, Boehringer, MSD, Ferrer, Janssen, Abbot. O. Mosenzon: Advisory Panel; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Merck Sharp & Dohme Corp., Novo Nordisk A/S, Sanofi. Research Support; Self; AstraZeneca, Novo Nordisk A/S. Speaker’s Bureau; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Merck Sharp & Dohme Corp., Novo Nordisk A/S, Sanofi, Teva Pharmaceutical Industries Ltd. S. Segel: Employee; Self; Novo Nordisk A/S. Employee; Spouse/Partner; Novo Nordisk A/S. Stock/Shareholder; Self; Novo Nordisk A/S. Stock/Shareholder; Spouse/Partner; Novo Nordisk A/S. G.M. Tsoukas: Advisory Panel; Self; Amgen, Novo Nordisk Inc. Speaker’s Bureau; Self; Amgen Canada. T. Pieber: Advisory Panel; Self; ADOCIA, Arecor, AstraZeneca, Eli Lilly and Company, Novo Nordisk A/S, Sanofi. Research Support; Self; AstraZeneca, Novo Nordisk A/S. Speaker’s Bureau; Self; Novo Nordisk A/S, Roche Diagnostics K.K. Funding Novo Nordisk A/S

Reduced Heterochromatin Formation on the pFAR4 Miniplasmid Allows Sustained Transgene Expression in the Mouse Liver

Non-viral gene delivery into the liver generally mediates a transient transgene expression. A comparative analysis was performed using two gene vectors, pFAR4 and pKAR4, which differ by the absence or presence of an antibiotic resistance marker, respectively. Both plasmids carried the same eukaryotic expression cassette composed of a sulfamidase (Sgsh) cDNA expressed from the human alpha antitrypsin liver-specific promoter. Hydrodynamic injection of the pFAR4 construct resulted in prolonged sulfamidase secretion from the liver, whereas delivery of the pKAR4 construct led to a sharp decrease in circulating enzyme. After induction of hepatocyte division, a rapid decline of sulfamidase expression occurred, indicating that the pFAR4 derivative was mostly episomal. Quantification analyses revealed that both plasmids were present at similar copy numbers, whereas Sgsh transcript levels remained high only in mice infused with the pFAR4 construct. Using a chromatin immunoprecipitation assay, it was established that the 5′ end of the expression cassette carried by pKAR4 exhibited a 7.9-fold higher heterochromatin-to-euchromatin ratio than the pFAR4 construct, whereas a bisulfite treatment did not highlight any obvious differences in the methylation status of the two plasmids. Thus, by preventing transgene expression silencing, the pFAR4 gene vector allows a sustained transgene product secretion from the liver.

Inflammasome expression is higher in ovarian tumors than in normal ovary.

Chronic inflammation fundamentally influences cancer risk and development. A mechanism of chronic inflammation is the formation of inflammasome complexes which results in the sustained secretion of the pro-inflammatory cytokines IL1β and IL18. Inflammasome expression and actions vary among cancers. There is no information on inflammasome expression in ovarian cancer (OvCa). To determine if ovarian tumors express inflammasome components, mRNA and protein expression of NLRP3 (nucleotide-binding domain, leucine-rich repeat family, pyrin domain containing 3), caspase-1, IL1β, and IL18 expression in hen and human OvCa was assessed. Chicken (hen) OvCa a valid model of spontaneous human OvCa. Hens were selected into study groups with or without tumors using ultrasonography; tumors were confirmed by histology, increased cellular proliferation, and expression of immune cell marker mRNA. mRNA expression was higher for hallmarks of inflammasome activity (caspase-1, 5.9x increase, p = 0.04; IL1β, 4x increase, p = 0.04; and IL18, 7.8x increase, p = 0.0003) in hen OvCa compared to normal ovary. NLRP3, caspase-8 and caspase-11 mRNA did not differ significantly between tumor and non-tumor containing ovaries. Similar results occurred for human OvCa. Protein expression by immunohistochemistry paralleled mRNA expression and was qualitatively higher in tumors. Increased protein expression of caspase-1, IL1β, and IL18 occurred in surface epithelium, tumor cells, and immune cells. The aryl hydrocarbon receptor (AHR), a potential tumor suppressor and NLRP3 regulator, was higher in hen (2.4x increase, p = 0.002) and human tumors (1.8x increase, p = 0.038), suggesting a role in OvCa. Collectively, the results indicate that inflammasome expression is associated with hen and human OvCa, although the NLR sensor type remains to be determined.

Engineering Secretory Amyloids for Remote and Highly Selective Destruction of Metastatic Foci.

Functional amyloids produced in bacteria as nanoscale inclusion bodies are intriguing but poorly explored protein materials with wide therapeutic potential. Since they release functional polypeptides under physiological conditions, these materials can be potentially tailored as mimetic of secretory granules for slow systemic delivery of smart protein drugs. To explore this possibility, bacterial inclusion bodies formed by a self-assembled, tumor-targeted Pseudomonas exotoxin (PE24) are administered subcutaneously in mouse models of human metastatic colorectal cancer, for sustained secretion of tumor-targeted therapeutic nanoparticles. These proteins are functionalized with a peptidic ligand of CXCR4, a chemokine receptor overexpressed in metastatic cancer stem cells that confers high selective cytotoxicity in vitro and in vivo. In the mouse models of human colorectal cancer, time-deferred anticancer activity is detected after the subcutaneous deposition of 500 µg of PE24-based amyloids, which promotes a dramatic arrest of tumor growth in the absence of side toxicity. In addition, long-term prevention of lymphatic, hematogenous, and peritoneal metastases is achieved. These results reveal the biomedical potential and versatility of bacterial inclusion bodies as novel tunable secretory materials usable in delivery, and they also instruct how therapeutic proteins, even with high functional and structural complexity, can be packaged in this convenient format.

Mesenchymal stem cells ascarrier of the therapeutic agent in the gene therapy of blood disorders.

Nonhematopoietic stem cells as a delivery platform of therapeutic useful genes have attracted widespread attention in recent years, owing to gained a long lifespan, easy separation, high proliferation, and high transfection capacity. Mesenchymal stem/stromal cells (MSCs) are the choice of the cells for gene and cell therapy due to high self-renewal capacity, high migration rate to the site of the tumor, and with immune suppressive and anti-inflammatory properties. Hence, it has a high potential of safety genetic modification of MSCs for antitumor gene expression and has paved the way for the clinical application of these cells to target the therapy of cancers and other diseases. The aim of gene therapy is targeted treatment of cancers and diseases through recovery, change, or enhancement cell performance to the sustained secretion of useful therapeutic proteins and induction expression of the functional gene in intended tissue. Recent developments in the vectors designing leading to the increase and durability of expression and improvement of the safety of the vectors that overcome a lot of problems, such as durability of expression and the host immune response. Nowadays, gene therapy approach is used by MSCs as a delivery vehicle in the preclinical and the clinical trials for the secretion of erythropoietin, recombinant antibodies, coagulation factors, cytokines, as well as angiogenic inhibitors in many blood disorders like anemia, hemophilia, and malignancies. In this study, we critically discuss the status of gene therapy by MSCs as a delivery vehicle for the treatment of blood disorders. Finally, the results of clinical trial studies are assessed, highlighting promising advantages of this emerging technology in the clinical setting.

Sustained Secretion of the Antimicrobial Peptide S100A7 Is Dependent on the Downregulation of Caspase-8

Antimicrobial peptides (AMPs) are the body's natural innate immune defense against a spectrum of pathogens and can also modulate cell proliferation, chemotaxis, angiogenesis, wound healing, and immune cell activity. Harnessing these diverse functions for prophylactic use is contingent upon understanding the regulatory mechanisms governing their unconventional secretion from cells. Analysis of the secretion of S100A7 (Psoriasin), an abundant AMP stored in differentiated keratinocytes of the skin, has revealed an unexpected biphasic secretory response to bacterial exposure. The core components regulating S100A7 secretion are NFκB/p38MAPK, caspase-1, and interleukin (IL)-1α. The initial activation of this core machinery is mediated by Toll-like receptor signaling, whereas the chronic response is mediated by Caspase-8 downregulation. Interestingly, there is a concomitant downregulation of Caspase-8 in inflammatory skin diseases wherein S100A7 is constitutively released. These results highlight the potential of targeting these components to control the release of AMPs from the skin in both homeostatic and disease conditions.

Sustained Neurotrophin Release from Protein Nanoparticles Mediated by Matrix Metalloproteinases Induces the Alignment and Differentiation of Nerve Cells.

The spatial and temporal availability of cytokines, and the microenvironments this creates, is critical to tissue development and homeostasis. Creating concentration gradients in vitro using soluble proteins is challenging as they do not provide a self-sustainable source. To mimic the sustained cytokine secretion seen in vivo from the extracellular matrix (ECM), we encapsulated a cargo protein into insect virus-derived proteins to form nanoparticle co-crystals and studied the release of this cargo protein mediated by matrix metalloproteinase-2 (MMP-2) and MMP-8. Specifically, when nerve growth factor (NGF), a neurotrophin, was encapsulated into nanoparticles, its release was promoted by MMPs secreted by a PC12 neuronal cell line. When these NGF nanoparticles were spotted onto a cover slip to create a uniform circular field, movement and alignment of PC12 cells via their extended axons along the periphery of the NGF nanoparticle field was observed. Neural cell differentiation was confirmed by the expression of specific markers of tau, neurofilament, and GAP-43. Connections between the extended axons and the growth cones were also observed, and expression of connexin 43 was consistent with the formation of gap junctions. Extensions and connection of very fine filopodia occurred between growth cones. Our studies indicate that crystalline protein nanoparticles can be utilized to generate a highly stable cytokine gradient microenvironment that regulates the alignment and differentiation of nerve cells. This technique greatly simplifies the creation of protein concentration gradients and may lead to therapies for neuronal injuries and disease.

Long-term facilitation of catecholamine secretion from adrenal chromaffin cells of neonatal rats by chronic intermittent hypoxia.

In neonates, catecholamine (CA) secretion from adrenal medullary chromaffin cells (AMC) is an important mechanism for maintaining homeostasis during hypoxia. Nearly 90% of premature infants experience chronic intermittent hypoxia (IH) because of high incidence of apnea of prematurity, which is characterized by periodic stoppage of breathing. The present study examined the effects of repetitive hypoxia, designed to mimic apnea of prematurity, on CA release from AMC of neonatal rats. Neonatal rats were exposed to either control conditions or chronic intermittent hypoxia (IH) from ages postnatal days 0-5 (P0-P5), and CA release from adrenal medullary slices was measured after challenge with repetitive hypoxia (5 episodes of 30-s hypoxia, Po2 ~35 mmHg). In response to repetitive hypoxia, chronic IH-treated AMC exhibited sustained CA release, and this phenotype was not seen in control AMC. The sustained CA release was associated with long-lasting elevation of intracellular Ca2+ concentration ([Ca2+]i), which was due to store-operated Ca2+ entry (SOCE). 2-Aminoethoxydiphenyl borate, an inhibitor of SOCE, prevented the long-lasting [Ca2+]i elevation and CA release. Repetitive hypoxia increased H2O2 abundance, and polyethylene glycol (PEG)-catalase, a scavenger of H2O2 blocked this effect. PEG-catalase also prevented repetitive hypoxia-induced SOCE activation, sustained [Ca2+]i elevation, and CA release. These results demonstrate that repetitive hypoxia induces long-term facilitation of CA release in chronic IH-treated neonatal rat AMC through sustained Ca2+ influx mediated by SOCE.NEW & NOTEWORTHY Apnea of prematurity and the resulting chronic intermittent hypoxia are major clinical problems in neonates born preterm. Catecholamine release from adrenal medullary chromaffin cells maintains homeostasis during hypoxia in neonates. Our results demonstrate that chronic intermittent hypoxia induces a hitherto uncharacterized long-term facilitation of catecholamine secretion from neonatal rat chromaffin cells in response to repetitive hypoxia, simulating hypoxic episodes encountered during apnea of prematurity. The sustained catecholamine secretion might contribute to cardiovascular morbidities in infants with apnea of prematurity.

Sensitivity Study for the Key Parameters in Heterospheroid Preparation with Insulin-Secreting β-Cells and Mesenchymal Stem Cells.

The outcome of islet transplantation in clinics has been determined by the success of tissue engraftment. The strong immune attack that occurs upon transplantation of β-cells plays a central role as this attack results in the failure of transplanted tissue. To improve tissue engraftment, deleterious effects of immune reactions should be minimized for β-cell function and survival. Here, we report a systematic analysis of the effect of insulin-secreting β-cell (MIN6) and mesenchymal stem cell (MSC) number and size on the function of β-cells and present immune protection potential of heterospheroid structures through MSCs and synthetic scaffolds. We prepared 3D heterospheroids with MSCs and MIN6 cells through a hanging-drop approach. To precisely estimate the influence of critical parameters on heterospheroid size and insulin secretion function of β-cells, we prepared heterospheroids using two independent input variables: (i) initial cell number in each droplet and (ii) MIN6:MSC ratio. We studied the influence of initial cell numbers of 200 and 500, and six different MIN6:MSC ratios (1:0, 0:1, 1:1, 2:1, 5:1, and 10:1) for the preparation of heterospheroids through the hanging drop. Next, we used PEG hydrogels as a semipermeable physical barrier to improve immune protection from cytokines. Through encapsulation of our heterospheroids within PEG hydrogel, we were able to observe sustained β-cell survival and insulin secretion despite exposure of heterospheroids with proinflammatory cytokines. Insulin secretion was further promoted with glucagon like peptide-1 (GLP-1) incorporation within PEG hydrogel structure. This study is significant to demonstrate the synergistic effects of MIN6-MSC and scaffold-MIN6 interactions and to improve therapeutic efficacy of islet transplantation. Overall, this study comprehensively presents the optimum conditions for the preparation of MIN6-MSC spheroids, utilizes MSCs and GLP-1 functional PEG hydrogels as a scaffold to retain insulin secretion function and further demonstrates protection of heterospheroids exposed to proinflammatory cytokines.

SAT-472 Increased Thyroid Cancer Risk in Acromegaly: A Report of 3 Cases

Introduction: There is a high incidence of thyroid cancer in patients with acromegaly. We present three patients who were diagnosed with acromegaly and thyroid cancer within a short time interval. Case 1: A 34-year-old female who had uvulopalatopharngoplasty for sleep apnea presented for evaluation of elevated IGF-1 1226 ng/mL (88-246). She reported increased shoe size, growth of facial hair, and increased hand size causing her to resize her wedding band multiple times. GH did not suppress on oral glucose tolerance testing (OGTT) and MRI revealed a 2.2 cm pituitary macroadenoma. On initial exam, she was noted to have to have thyromegaly and ultrasound showed a 1.3 cm solid nodule with microcalcifications. She underwent transsphenoidal hypophysectomy (TSSH) and a month later was sent for FNA biopsy of the nodule which was consistent with papillary carcinoma (PTC). She underwent total thyroidectomy two months later with 1 cm PTC on final pathology. Case 2: A 60-year-old female presented for evaluation of thyroid nodules. An ultrasound revealed a right solid 2.2 cm isoechoic nodule and a left 1.2cm solid nodule with calcifications. At the initial encounter, physical exam was notable for prominence of the forehead and jawline and sausage digits. History was significant for carpal tunnel syndrome, osteoarthritis, DM2, excessive perspiration and snoring. Screening IGF-1 was elevated 771 ng/mL (81-225) and GH failed to suppress on OGTT. FNA biopsy of the left nodule showed PTC. Pituitary MRI revealed an 8mm microadenoma. She underwent TSSH and then a total thyroidectomy three months later with a 1cm PTC, follicular variant on final pathology. Case 3: A 30-year-old female presented with amenorrhea, increased hand/foot size and changes in the jaw. IGF-1 was 729 ng/mL(63-373) and GH failed to suppress on OGTT. MRI showed a 2.5cm pituitary adenoma with cavernous sinus invasion. She underwent TSSH and was on medical treatment for acromegaly for about two years when she was noted to have a thyroid nodule on palpation. Ultrasound showed a 10 mm isthmus solid isoechoic nodule and a 15 mm left midpole solid hypoechoic nodule. FNA of the isthmus nodule was consistent with PTC. Total thyroidectomy pathology showed a 0.7cm PTC. Conclusion: Carcinomas are the third most frequent cause of mortality in acromegalic patients, after vascular and respiratory diseases. Sustained secretion of GH and IGF-1 is known to induce proliferation and inhibit apoptosis. Thyroid nodular disease is more frequent in acromegalic patients, and thyroid cancer is one of the most commonly detected cancers in acromegaly. It remains controversial whether acromegaly increases the risk of malignant transformation of thyroid nodules. In all of our patients with acromegaly, stage I PTC was likely present at the time of diagnosis. Periodic thyroid ultrasound exam should be considered an important part of monitoring patients with acromegaly.

Ultrasound-responsive nanobubble-mediated gene transfection in the cerebroventricular region by intracerebroventricular administration in mice

AimIntracerebroventricular (ICV) administration of ultrasound-responsive bubbles and cranial ultrasound irradiation is reported as a transfection system for the cerebroventricular region. This study aimed to characterize the transfection system with respect to transfection efficiency, spatial distribution of transgene expression, and safety. MethodsPlasmid DNA was transfected to mouse brain by ICV injection of ultrasound-responsive nanobubbles, followed by ultrasound irradiation to brain. Spatial distribution of transgene expression in the cerebroventricular region was investigated using multicolor deep imaging. ResultThis transfection system efficiently transferred the transgene to the choroid plexus with no morphological change or cerebral hemorrhage. Moreover, sustained secretion of transgenic protein was achieved by transferring the transgene encoding the secretable protein. ConclusionWe successfully developed an ultrasound-responsive nanobubbles-mediated method for gene transfection into the cerebroventricular region via ICV administration in mice.

Sustained Secretion of the Antimicrobial Peptide S100A7 Is Mediated by the Wound Healing Machinery

Antimicrobial peptides (AMPs) are the body’s natural innate immune defense against a spectrum of pathogenic insults and provide an attractive strategy towards combating the growing problem of antibiotic resistant microorganisms. The prophylactic use of AMPs is contingent upon understanding the regulatory mechanisms governing the release of AMPs from intracellular stores, which occurs via the non-conventional secretory pathway. Analysis of S100A7 (Psoriasin), an abundant AMP in the skin, from its endogenous stores within differentiated keratinocytes has revealed an unexpected biphasic secretory response to bacterial exposure. The core components regulating S100A7 secretion are NFκB/p38MAPK, caspase-1 and IL-1α. The early activation of this core machinery is mediated by toll-like receptor signaling, whereas the chronic response is regulated by the wound-healing machinery mediated by Caspase-8 downregulation. Interestingly, in inflammatory skin diseases in which S100A7 is excessively released, there is a concomitant downregulation of caspase-8, indicating that these pathological scenarios are commandeering the normal wound healing response. These results highlight the potential benefits of targeting intracellular wound healing pathways as a means of exerting control over the release of AMPs from the skin in both homeostatic and disease conditions.

Ex vivo conditioning with IL-12 protects tumor-infiltrating CD8+ T cells from negative regulation by local IFN-γ.

Optimal ex vivo expansion protocols for adoptive cell therapy (ACT) must yield T cells able to effectively home to tumors and survive the inhospitable conditions of the tumor microenvironment (TME), while simultaneously exerting persistent anti-tumor effector functions. Our previous work has shown that ex vivo activation in the presence of IL-12 can induce optimal expansion of murine CD8+ T cells, thus resulting in significant tumor regression after ACT mostly via sustained secretion of IFN-γ. In this report, we further elucidate the mechanism of this potency, showing that IL-12 additionally counteracts the negative regulatory effects of autocrine IFN-γ. IL-12 not only downregulates PD-1 expression by T cells, thus minimizing the effects of IFN-γ-induced PD-L1 upregulation by tumor stromal cells, but also inhibits IFNγR2 expression, thereby protecting T cells from IFN-γ-induced cell death. Thus, the enhanced anti-tumor activity of CD8+ T cells expanded ex vivo in the presence of IL-12 is due not only to the ability of IL-12-stimulated cells to secrete sustained levels of IFN-γ, but also to the additional capacity of IL-12 to counter the negative regulatory effects of autocrine IFN-γ.

Exosome-delivered microRNAs promote IFN-α secretion by human plasmacytoid DCs via TLR7.

The excessive production of type I IFNs is a hallmark and a main pathogenic mechanism of many autoimmune diseases, including systemic lupus erythematosus (SLE). In these pathologies, the sustained secretion of type I IFNs is dependent on the improper activation of plasmacytoid DCs (pDCs) by self-nucleic acids. However, the nature and origin of pDC-activating self-nucleic acids is still incompletely characterized. Here, we report that exosomes isolated from the plasma of SLE patients can activate the secretion of IFN-α by human blood pDCs in vitro. This activation requires endosomal acidification and is recapitulated by microRNAs isolated from exosomes, suggesting that exosome-delivered microRNAs act as self-ligands of innate single-stranded endosomal RNA sensors. By using synthetic microRNAs, we identified an IFN induction motif that is responsible for the TLR7-dependent activation, maturation, and survival of human pDCs. These findings identify exosome-delivered microRNAs as potentially novel TLR7 endogenous ligands able to induce pDC activation in SLE patients. Therefore, microRNAs may represent novel pathogenic mediators in the onset of autoimmune reactions and potential therapeutic targets in the treatment of type I IFN-mediated diseases.

Sustained secretion of immunoglobulin by long-lived plasma cells from human nasal biopsies

Abstract Background During secondary immune responses, plasma cell survival and antibody production is mediated by extrinsic factors provided by the local environment. It is unknown whether there are long-lived plasma cells (LLPCs) in the human nasal mucosa. Objective To identify LLPCs and characterize functionally specific survival niche in the human nasal mucosa. Methods Nasal polyp (NP) biopsies were used for ex vivo air-liquid interface culture. Protein levels of immunoglobulins in tissue homogenates were detected by Bio-Plex. The expression of LLPCs related markers including NGF, TrkA, P75, IL-6, APRIL, CD138, CD20, CD4, CD8, ECP, Bcl2, Ki-67, CD3 were detected by means of quantitative RT-PCR, immunohistochemistry (IHC) and ELISA. Results Using an ex vivo biopsies culture, we found sustained IgG, IgA, IgE, and IgM secretion >4 wk of culture. IL-6, APRIL NGF, TrkA, and P-75 were up-regulated in CRSwNP compared with controls by ELISA or IHC. We assessed TrkA+ cells, ECP+ eosinophils, and tryptase+ mast cells represent the major interstitial cell sources of NGF, while tryptase+ mast cells and CD138+ plasma cells represent the major sources for TrkA in CRSwNP by immunohistochemistry. We performed histological analysis of fresh nasal polyp tissues, and of tissue fragments harvested 10, 20, 32 days after cultured respectively and found that there’s a small portion of plasma cell in CRSwNP expressed Bcl2 pre- and post-culture. However, post-cultured plasma cells do not express Ki67. Conclusion This study demonstrates that the human nasal mucosa harbors a population of nonproliferating plasma cells that are instructed by the microenvironment for prolonged survival and Ab secretion.