Protecting pancreatic b-cells against cytokines: novel function of islet neogenesis associated protein (INGAP) and combinatorial treatment with NF-kB inhibitor SCA-B1-NBD

One of the most challenging health epidemics facing the 21st century is Diabetes Mellitus, characterized by destruction of the insulin-producing β-cells. Inflammation, in particular pro-inflammatory cytokines, plays a key role in initiation and progression of β-cell death. The goal of our research is to develop a novel approach to preserve and expand the pancreatic β-cell mass. This PhD thesis investigates the β-cell protective capacity of two candidate proteins: (i) Islet Neogenesis Associated Protein (INGAP), co-discovered by Dr. Rosenberg as the first neogenic molecule to reverse diabetes by regenerating insulin-producing β-cells (a derived bioactive peptide, INGAP-P, is currently in phase 2 clinical trials); and (ii) a fusion protein, originally engineered at McGill by Dr. Schneider, made up of a β-cell specific single-chain antibody (SCA-B1) fused with an NF-κB inhibitor peptide (NBD) that has been shown to protect mice from streptozotocin-induced diabetes. Hypothesis: In addition to its neogenic properties, INGAP protects β-cells against cytokine-induced cell death by inhibiting cytokine-induced signaling; this effect can be further enhanced by combinatorial treatment with the β-specific NF-κB inhibitor, SCA-B1-NBD. Pre-treatment with recombinant full-length protein INGAP (rINGAP) and INGAP-P partially inhibited cytokine-mediated cytotoxicity, improved viability, reduced NO levels, as well as cytokine-induced apoptosis. While both molecules conferred survival to β-cells, rINGAP appeared to be a more potent and longer-lasting cytoprotective agent compared to INGAP-P. Analysis of signaling events revealed that INGAP partially blocks cytokine-induced apoptosis by inhibiting NF-κB signaling, decreasing phosphorylation of p65, IκBα and kinase complex IKK. INGAP-mediated inhibition of NF-κB signaling was followed by decreased expression of its downstream target, NOS2 gene expression and its gene product, iNOS protein. Our data suggest that rINGAP inhibited cytokine-induced p65 phosphorylation in an IKK-dependent and independent manner. rINGAP partially inhibited other signaling pathways implicated in regulation of iNOS – reduced activation of JAK2 and STAT3, JNK and p38. Finally, cytokine treatment induced early AKT phosphorylation which decreased over time. rINGAP pre-treatment partially inhibited cytokine-mediated effects on AKT.We successfully expressed and purified SCA-B1-NBD and validated its functionality as per: (i) binding β-cells specifically and (ii) partially inhibiting the NF-κB signaling pathway in RINm5F and INS-1 cell lines as well as in human islets. Pre-treatment with SCA-B1-NBD improved viability, reduced cytokine-induced NOS2 expression, iNOS protein and NO levels and partially inhibited apoptosis by decreasing activation of caspase-3 and PARP. Co-treatment of the cell lines with INGAP and SCA-B1-NBD exhibited minimal additive effects against cytokine-induced β-cell death. The limited augmented effects of this combinatorial treatment could be explained in part by the fact that both proteins affect the same signaling pathway, NF-κB. Additional studies, including experimentation with timing and dosage of in vitro administration of each drug as well as in vivo models of diabetes requiring both regenerative and anti-apoptotic interventions, are necessary in order to confidently conclude whether this combinatorial regimen is effective against diabetes.This is the first time INGAP has been shown to protect β-cells against cytokines and to inhibit NF-κB signaling. These studies have broadened our current understanding of INGAP-mediated effects as well as provided novel insight into its anti-apoptotic potential and the underlying intracellular signaling mechanisms. Additionally, successful production and validation of β-cell specific SCA-B1-NBD protective effects have laid the necessary foundation for future in vitro and in vivo studies on pancreatic β-cell mass preservation.Une des pires épidémies de santé du XXIe siècle est le Diabetes Mellitus, caractérisé par la destruction des cellules β productrices d'insuline. L'inflammation, en particulier les cytokines pro-inflammatoires, jouer un rôle clé dans l'initiation et la progression de la mort des cellules β. Le but de notre recherche est de développer une nouvelle approche pour préserver et accroître la masse des cellules β pancréatiques. Cette thèse étudie la capacité protectrice des cellules β de deux protéines candidates: (i) Islet Neogenesis Associated Protein (INGAP), co-découverte par le Dr Rosenberg comme la première molécule néogénique inversant le diabète dans les modèles animaux en régénérant les cellules β productrices d'insuline (son peptide bioactif dérivé, INGAP-P, est actuellement en phase 2 d'essais cliniques); et (ii) une protéine de fusion, initialement conçue à McGill par le Dr Schneider, composée d'un anticorps monocaténaire spécifique de cellules β (SCA-B1) fusionné avec un peptide inhibiteur de NF-κB (NBD) qui protège les souris contre le diabète induit par streptozotocin.Hypothèse: En plus de ses propriétés néogènes, l'INGAP protège les cellules β contre la mort de cellules induite par les cytokines en inhibant la signalisation induite par ces cytokines; cet effet peut être encore amélioré en combinant un traitement avec l'inhibiteur NF-κB, β-spécifique, SCA-B1-NBD.Le prétraitement avec la protéine INGAP recombinante entière (rINGAP) et INGAP-P a partiellement inhibé la cytotoxicité induite par les cytokines, amélioré la viabilité, réduit les niveaux de NO et a aussi réduit l'apoptose induite par les cytokines. Alors que les deux molécules permettent la survie des cellules β, le rINGAP semble être un agent cytoprotecteur plus puissant et plus durable que l'INGAP-P. L'analyse des événements de signalisation a révélé que l'INGAP bloque partiellement l'apoptose induite par les cytokines en inhibant la signalisation de NF-κB, en diminuant la phosphorylation de p65, IκBα et le complexe de kinase IKK. L'inhibition induite par INGAP de la signalisation de NF-κB a été suivie par une expression diminuée de sa cible aval, l'expression du gène NOS2 et de son produit génique, la protéine iNOS. Nos données suggèrent également que rINGAP a inhibé la phosphorylation de p65 induite par les cytokines de manière dépendante et indépendante de l'IKK. rINGAP a partiellement inhibé d'autres voies de signalisation impliquées dans la régulation de iNOS - activation réduite de JAK2 et STAT3, JNK et p38. Enfin, le traitement par les cytokines a induit une phosphorylation précoce de l'AKT qui a diminué avec le temps. Le prétraitement par rINGAP a partiellement inhibé les effets induits par les cytokines sur l'AKT.Nous avons généré et purifié avec succès le SCA-B1-NBD et validé sa fonctionnalité selon: (i) la liaison spécifique aux cellules β et (ii) l'inhibition partielle de la signalisation de NF-κB dans les lignées cellulaires RINm5F et INS-1 ainsi que dans les ilots humains. Le prétraitement avec SCA-B1-NBD a amélioré la viabilité, réduit l'expression de NOS2 induite par les cytokines, de la protéine iNOS et des niveaux de NO et inhibé partiellement l'apoptose en diminuant l'activation de la caspase-3 et de la PARP. Le co-traitement des lignées cellulaires avec INGAP et SCA-B1-NBD a montré des effets additifs minimaux contre la mort des cellules β induite par les cytokines. Les effets positifs limités de ce traitement combinatoire pourraient s'expliquer en partie par le fait que les deux protéines affectent la même voie de signalisation, NF-κB. Des études additionnelles, y compris l'expérimentation avec le temps de l'administration et la posologie in vitro de chaque médicament ainsi que des modèles in vivo de diabète nécessitant à la fois des interventions régénératrices et anti-apoptotiques, sont nécessaires pour conclure avec certitude si ce régime combinatoire est efficace contre le diabète

Islet neogenesis associated protein (INGAP) protects pancreatic β cells from IL-1β and IFNγ-induced apoptosis

AbstractThe goal of this study was to determine whether recombinant Islet NeoGenesis Associated Protein (rINGAP) and its active core, a pentadecapeptide INGAP104–118 (Ingap-p), protect β cells against cytokine-induced death. INGAP has been shown to induce islet neogenesis in diabetic animals, to stimulate β-cell proliferation and differentiation, and to improve islet survival and function. Importantly, Ingap-p has shown promising results in clinical trials for diabetes (phase I/II). However, the full potential of INGAP and its mechanisms of action remain poorly understood. Using rat insulinoma cells RINm5F and INS-1 treated with interleukin-1β (IL-1β) and interferon‐gamma (IFN‐γ), we demonstrate here that both rINGAP and Ingap-p inhibit apoptosis, Caspase-3 activation, inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production, and explore the related signaling pathways. As expected, IL-1β induced nuclear factor kappa B (NF-κB), p38, and JNK signaling, whereas interferon‐gamma (IFN‐γ) activated the JAK2/STAT1 pathway and potentiated the IL-1β effects. Both rINGAP and Ingap-p decreased phosphorylation of IKKα/β, IkBα, and p65, although p65 nuclear translocation was not inhibited. rINGAP, used for further analysis, also inhibited STAT3, p38, and JNK activation. Interestingly, all inhibitory effects of rINGAP were observed for the cytokine cocktail, not IL-1β alone, and were roughly equal to reversing the potentiating effects of INFγ. Furthermore, rINGAP had no effect on IL-1β/NF-κB-induced gene expression (e.g., Ccl2, Sod2) but downregulated several IFNγ-stimulated (Irf1, Socs1, Socs3) or IFNγ-potentiated (Nos2) genes. This, however, was observed again only for the cytokine cocktail, not IFNγ alone, and rINGAP did not inhibit the IFNγ-induced JAK2/STAT1 activation. Together, these intriguing results suggest that INGAP does not target either IL-1β or IFNγ individually but rather inhibits the signaling crosstalk between the two, the exact mechanism of which remains to be investigated. In summary, our study characterizes the anti-inflammatory effects of INGAP, both protein and peptide, and suggests a new therapeutic utility for INGAP in the treatment of diabetes.</jats:p

Characterization of MSCs expressing islet neogenesis associated protein (INGAP): INGAP secretion and cell survival in vitro and in vivo

Mesenchymal stem/stromal cells (MSCs) are emerging as a new therapy for diabetes. Here we investigate the properties of MSCs engineered to express Islet Neogenesis Associated Protein (INGAP) previously shown to reverse diabetes in animal models and evaluate their potential for anti-diabetic applications in mice. Mouse bone marrow-derived MSCs retrovirally transduced to co-express INGAP, Firefly Luciferase and EGFP (INGAP-MSCs), were characterized in vitro and implanted intraperitoneally (IP) into non-diabetic and diabetic C57BL/6 mice (Streptozotocin model) and tracked by live bioluminescence imaging (BLI). Distribution and survival of IP injected INGAP-MSCs differed between diabetic and non-diabetic mice, with a rapid clearance of cells in the latter, and a stronger retention (up to 4 weeks) in diabetic mice concurring with homing towards the pancreas. Interestingly, INGAP-MSCs inhibited the progression of hyperglycemia starting at day 3 and lasting for the entire 6 weeks of the study. Pursuing greater retention, we investigated the survival of INGAP-MSCs in hydrogel matrices. When mixed with Matrigel™ and injected subcutaneously into non-diabetic mice, INGAP-MSCs remained in the implant up to 16 weeks. In vitro tests in three matrices (Matrigel™, Type I Collagen and VitroGel®-MSC) demonstrated that INGAP-MSCs survive and secrete INGAP, with best results at the density of 1–2 x 106 cells/mL. However, all matrices induced spontaneous adipogenic differentiation of INGAP-MSCs in vitro and in vivo, which requires further investigation of its potential impact on MSC therapeutic properties. In summary, based on their ability to stop the rise in hyperglycemia in STZ-treated mice, INGAP-MSCs are a promising therapeutic tool against diabetes but require further research to improve cell delivery and survival

Convalescent plasma for hospitalized patients with COVID-19: an open-label, randomized controlled trial

AbstractThe efficacy of convalescent plasma for coronavirus disease 2019 (COVID-19) is unclear. Although most randomized controlled trials have shown negative results, uncontrolled studies have suggested that the antibody content could influence patient outcomes. We conducted an open-label, randomized controlled trial of convalescent plasma for adults with COVID-19 receiving oxygen within 12 d of respiratory symptom onset (NCT04348656). Patients were allocated 2:1 to 500 ml of convalescent plasma or standard of care. The composite primary outcome was intubation or death by 30 d. Exploratory analyses of the effect of convalescent plasma antibodies on the primary outcome was assessed by logistic regression. The trial was terminated at 78% of planned enrollment after meeting stopping criteria for futility. In total, 940 patients were randomized, and 921 patients were included in the intention-to-treat analysis. Intubation or death occurred in 199/614 (32.4%) patients in the convalescent plasma arm and 86/307 (28.0%) patients in the standard of care arm—relative risk (RR) = 1.16 (95% confidence interval (CI) 0.94–1.43,P = 0.18). Patients in the convalescent plasma arm had more serious adverse events (33.4% versus 26.4%; RR = 1.27, 95% CI 1.02–1.57,P = 0.034). The antibody content significantly modulated the therapeutic effect of convalescent plasma. In multivariate analysis, each standardized log increase in neutralization or antibody-dependent cellular cytotoxicity independently reduced the potential harmful effect of plasma (odds ratio (OR) = 0.74, 95% CI 0.57–0.95 and OR = 0.66, 95% CI 0.50–0.87, respectively), whereas IgG against the full transmembrane spike protein increased it (OR = 1.53, 95% CI 1.14–2.05). Convalescent plasma did not reduce the risk of intubation or death at 30 d in hospitalized patients with COVID-19. Transfusion of convalescent plasma with unfavorable antibody profiles could be associated with worse clinical outcomes compared to standard care.</jats:p