Astrocyte-targeted gene delivery of interleukin 2 specifically increases brain-resident regulatory T cell numbers and protects against pathological neuroinflammation.

The ability of immune-modulating biologics to prevent and reverse pathology has transformed recent clinical practice. Full utility in the neuroinflammation space, however, requires identification of both effective targets for local immune modulation and a delivery system capable of crossing the blood-brain barrier. The recent identification and characterization of a small population of regulatory T (Treg) cells resident in the brain presents one such potential therapeutic target. Here, we identified brain interleukin 2 (IL-2) levels as a limiting factor for brain-resident Treg cells. We developed a gene-delivery approach for astrocytes, with a small-molecule on-switch to allow temporal control, and enhanced production in reactive astrocytes to spatially direct delivery to inflammatory sites. Mice with brain-specific IL-2 delivery were protected in traumatic brain injury, stroke and multiple sclerosis models, without impacting the peripheral immune system. These results validate brain-specific IL-2 gene delivery as effective protection against neuroinflammation, and provide a versatile platform for delivery of diverse biologics to neuroinflammatory patients.

P17 induces chemotaxis and differentiation of monocytes via MRGPRX2-mediated mast cell-line activation.

BACKGROUND: P17, a peptide isolated from Tetramorium bicarinatum ant venom, is known to induce an alternative phenotype of human monocyte-derived macrophages via activation of an unknown G protein-coupled receptor (GPCR). OBJECTIVE: We sought to investigate the mechanism of action and the immunomodulatory effects of P17 mediated through MRGPRX2 (Mas-related G protein-coupled receptor X2). METHODS: To identify the GPCR for P17, we screened 314 GPCRs. Upon identification of MRGPRX2, a battery of in silico, in vitro, ex vivo, and in vivo assays along with the receptor mutation studies were performed. In particular, to investigate the immunomodulatory actions, we used ß-hexosaminidase release assay, cytokine releases, quantification of mRNA expression, cell migration and differentiation assays, immunohistochemical labeling, hematoxylin and eosin, and immunofluorescence staining. RESULTS: P17 activated MRGPRX2 in a dose-dependent manner in ß-arrestin recruitment assay. In LAD2 cells, P17 induced calcium and ß-hexosaminidase release. Quercetin- and short hairpin RNA-mediated knockdown of MRGPRX2 reduced P17-evoked ß-hexosaminidase release. In silico and in vitro mutagenesis studies showed that residue Lys8 of P17 formed a cation-π interaction with the Phe172 of MRGPRX2 and [Ala8]P17 lost its activity partially. P17 activated LAD2 cells to recruit THP-1 and human monocytes in Transwell migration assay, whereas MRGPRX2-impaired LAD2 cells cannot. In addition, P17-treated LAD2 cells stimulated differentiation of THP-1 and human monocytes, as indicated by the enhanced expression of macrophage markers cluster of differentiation 11b and TNF-α by quantitative RT-PCR. Immunohistochemical and immunofluorescent staining suggested monocyte recruitment in mice ears injected with P17. CONCLUSIONS: Our data provide novel structural information regarding the interaction of P17 with MRGPRX2 and intracellular pathways for its immunomodulatory action.

Simultaneous removal of heavy metals and dye from wastewater: modelling and experimental study.

In this work, heavy metals were removed simultaneously using wheat bran as an adsorbent. For batch experiments, the Box-Behnken design of response surface methodology was used and the effect of dye on metal removal was analysed. It has been observed that the presence of dye has reduced the removal of each metal in the range of 100-20% with no appreciable reduction in dye adsorption. The optimum pH, temperature, and adsorbent dose were found to be 7.59, 33.23 °C, and 2.90 g/L, respectively, for 79.70% chromium, 99.9% cadmium and 87.27% copper removal. It was found that Langmuir isotherm fits well with the experimental data (RMSE value up to 0.033). The maximum adsorption capacity obtained for copper, chromium, cadmium and dye were 2.17 mg/g, 1.76 mg/g, 1.52 mg/g and 3.215 mg/g, respectively. The continuous study was performed for parameters, i.e. bed height (0.15-0.45 m), flow rate (5-15 mL/min) and initial metal concentration (100-500 mg/L). In continuous study, dye acted as an interfering species and as a result breakthrough and exhaustion time decreased. The modelling and simulation of continuous adsorption process were performed. A dynamic mathematical model was developed for continuous fixed bed adsorption column to compare the breakthrough curve with experimental results.

Interleukin-35 Prevents the Elevation of the M1/M2 Ratio of Macrophages in Experimental Type 1 Diabetes.

Macrophages play an important role in the early development of type 1 diabetes (T1D). Based on the phenotype, macrophages can be classified into pro-inflammatory (M1) and anti-inflammatory (M2) macrophages. Despite intensive research in the field of macrophages and T1D, the kinetic response of M1/M2 ratio has not been studied in T1D. Thus, herein, we studied the M1 and M2 macrophages in the early development of T1D using the multiple low dose streptozotocin (MLDSTZ) mouse model. We determined the proportions of M1 and M2 macrophages in thymic glands, pancreatic lymph nodes and spleens on days 3, 7 and 10 after the first injection of STZ. In addition, we investigated the effect of IL-35 in vivo on the M1/M2 ratio and IL-35+ plasmacytoid dendritic cells in diabetic mice and in vitro on the sorted macrophages. Our results revealed that the M1/M2 ratio is higher in STZ-treated mice but this was lowered upon the treatment with IL-35. Furthermore, IL-35 treated mice had lower blood glucose levels and a higher proportion of IL-35+ cells among pDCs. Macrophages treated with IL-35 in vitro also had a higher proportion of M2 macrophages. Together, our data indicate that, under diabetic conditions, pro-inflammatory macrophages increased, but IL-35 treatment decreased the pro-inflammatory macrophages and increased anti-inflammatory macrophages, further suggesting that IL-35 prevents hyperglycemia by maintaining the anti-inflammatory phenotype of macrophages and other immune cells. Thus, IL-35 should be further investigated for the treatment of T1D and other autoimmune disorders.

GIP receptor suppresses PAC1receptor-mediated neuronal differentiation via formation of a receptor heterocomplex.

Pituitary adenylate cyclase-activating peptide (PACAP) receptor (PAC1R) is a class B Gprotein-coupled receptor (GPCR) that is widely expressed in the human body and is involved in neuronal differentiation. As class B GPCRs are known to form heterocomplexes with family members, we hypothesized that PAC1R mediates neuronal differentiation through interaction with a class B GPCR. We used the BRET assay to identify potential interactions between PAC1R and 11 class B GPCRs. Gastric inhibitory polypeptide receptor (GIPR) and secretin receptor were identified as putative binding partners of PAC1R. The effect of heterocomplex formation by PAC1R on receptor activation was evaluated with the cyclic (c)AMP, luciferase reporter, and calcium signaling assays; and the effects on receptor internalization and subcellular localization were examined by confocal microscopy. The results suggested he PAC1R/GIPR heterocomplex suppressed signaling events downstream of PAC1R, including cAMP production, serum response element and calcium signaling, and ß-arrestin recruitment. Protein-protein interaction was analyzed in silico, and induction of neuronal differentiation by the PAC1R heterocomplex was assessed in SH-SY5Y neuronal cells by measure the morphological changes and marker genes expression by real-time quantitative PCR and western blot. Over-expression of GIPR suppressed PACAP/PAC1R-mediated neuronal differentiation and the differentiation markers expression in SH-SY5Y cells. GIPR regulates neuronal differentiation through heterocomplex formation with PAC1R.

Purification and identification of a surfactin biosurfactant and engine oil degradation by Bacillus velezensis KLP2016.

Engine oil used in automobiles is a threat to soil and water due to the recalcitrant properties of its hydrocarbons. It pollutes surrounding environment which affects both flora and fauna. Microbes can degrade hydrocarbons containing engine oil and utilize it as a substrate for their growth. Our results demonstrated that cell-free broth of Bacillus velezensis KLP2016 (Gram + ve, endospore forming; Accession number KY214239) recorded an emulsification index (E24%) from 52.3% to 65.7% against different organic solvents, such as benzene, pentane, cyclohexane, xylene, n-hexane, toluene and engine oil. The surface tension of the cell-free broth of B. velezensis grown in Luria-Bertani broth at 35 °C decreased from 55 to 40 mN m-1at critical micelle concentration 17.2 µg/mL. The active biosurfactant molecule of cell-free broth of Bacillus velezensis KLP2016 was purified by Dietheylaminoethyl-cellulose and size exclusion chromatography, followed by HPLC (RT = 1.130), UV-vis spectrophotometry (210 nm) and thin layer chromatography (Rf = 0.90). The molecular weight of purified biosurfactant was found to be ~ 1.0 kDa, based on Electron Spray Ionization-MS. A concentration of 1980 × 10-2 parts per million of CO2 was trapped in a KOH solution after 15 days of incubation in Luria-Bertani broth containing 1% engine oil. Our results suggest that bacterium Bacillus velezensis KLP2016 may promise a new dimension to solving the engine oil pollution problem in near future.

Interleukin-35 Prevents Development of Autoimmune Diabetes Possibly by Maintaining the Phenotype of Regulatory B Cells.

The anti-inflammatory role of regulatory B cells (Breg cells) has been associated with IL-35 based on studies of experimental autoimmune uveitis and encephalitis. The role of Breg cells and IL-35+ Breg cells for type 1 diabetes (T1D) remains to be investigated. We studied PBMCs from T1D subjects and healthy controls (HC) and found lowered proportions of Breg cells and IL-35+ Breg cells in T1D. To elucidate the role of Breg cells, the lymphoid organs of two mouse models of T1D were examined. Lower proportions of Breg cells and IL-35+ Breg cells were found in the animal models of T1D compared with control mice. In addition, the systemic administration of recombinant mouse IL-35 prevented hyperglycemia after multiple low dose streptozotocin (MLDSTZ) injections and increased the proportions of Breg cells and IL-35+ Breg cells. A higher proportion of IFN-γ+ cells among Breg cells were found in the PBMCs of the T1D subjects. In the MLDSTZ mice, IL-35 administration decreased the proportions of IFN-γ+ cells among the Breg cells. Our data illustrate that Breg cells may play an important role in the development of T1D and that IL-35 treatment prevents the development of hyperglycemia by maintaining the phenotype of the Breg cells under an experimental T1D condition.

Protective Effect of Genistein against Compound 48/80 Induced Anaphylactoid Shock via Inhibiting MAS Related G Protein-Coupled Receptor X2 (MRGPRX2).

Anaphylactoid shock is a fatal hypersensitivity response caused by non-IgE mediated mast cell activation. These reactions are mediated by a family of G protein-coupled receptors (GPCRs) known as Mas related GPCRX2 (MRGPRX2). Several US FDA approved drugs which are used in day to day life have been reported to cause anaphylactoid shock. Surprisingly, no therapeutic drugs are available which can directly target MRGPRX2 for treatment of anaphylactoid shock. Genistein is a non-steroidal polyphenol known for its diverse physiological and pharmacological activities. In recent studies, Genistein has been reported for its anti-inflammatory activity on mast cells. However, the effects and mechanistic pathways of Genistein on anaphylactoid reaction remain unknown. In the present study, we designed a battery of in-vitro, in-silico and in-vivo experiments to evaluate the anti-anaphylactoid activity of Genistein in order to understand the possible molecular mechanisms of its action. The in-vitro results demonstrated the inhibitory activity of Genistein on MRGPRX2 activation. Further, a mouse model of anaphylactoid shock was used to evaluate the inhibitory activity of Genistein on blood vessel leakage and hind paw edema. Taken together, our findings have demonstrated a therapeutic potential of Genistein as a lead compound in the treatment of anaphylactoid shock via MRGPRX2.

Reactive adsorption of Safranin O: surface - pore diffusion modeling and degradation study.

Granular activated carbon was doped with iron (Fe-AC) and was used to study the removal of Safranin O (SO) using the Fe-AC/H2O2 system for reactive adsorption and Fe-AC for adsorption. Fe-AC and H2O2 doses were optimized to obtain maximum removal of SO. Maximum removal was found to be 96.1% after 5 h using 1.0 g/L Fe-AC and 5.0 mM hydrogen peroxide doses for 10 mg/L initial SO concentration. Kinetic study suggested the suitability of the pseudo-first-order model for reactive adsorption. The Langmuir isotherm explained well the sorption of SO onto Fe-AC. Parallel-pore-reactive adsorption model was applied and validated. By fitting the experimental data to the model, it is observed that the surface reaction rate coefficient, kr, was found to be five times that of the apparent rate constant, kapp. Parameters such as the external liquid film mass transfer coefficient, macro-pore and micro-pore diffusivities were estimated by regression analysis. Pore diffusion and surface reaction were found to be rate controlling for adsorption and reactive adsorption, respectively. An oxidative degradation of SO took place via hydroxylation and ring cleavage processes.

PAN substitutions A37S, A37S/I61T and A37S/V63I attenuate the replication of H7N7 influenza A virus by impairing the polymerase and endonuclease activities.

Substitutions in the PA N-terminus (PAN) of influenza A viruses are associated with viral pathogenicity. During our previous study, which identified PAN-V63I and -A37S/I61T/V63I/V100A substitutions as virulence determinants, we observed a severe decrease in virus growth and transcription/replication capacity posed by PAN-A37S/V100A substitution. To further delineate the significance of substitutions at these positions, we generated mutant H7N7 viruses bearing the substitutions PAN-A37S, -A37S/I61T, -A37S/V63I, -V100A, -I61T/V100A and -V63I/V100A by reverse genetics. Our results showed that all mutant viruses except PAN-V100A showed a significantly reduced growth capability in infected cells. At the same time, the PAN-A37S, -A37S/I61T and -A37S/V63I mutant viruses displayed decreased viral transcription and replication by diminishing virus RNA synthesis activity. Biochemical assays indicated that the substitutions PAN-A37S, -A37S/I61T and -A37S/V63I suppressed the polymerase and endonuclease activities when compared with those of the wild-type. Together, our results demonstrated that the PAN-A37S, -A37S/I61T and -A37S/V63I substitutions contributed to a decreased pathogenicity of avian H7N7 influenza A virus.

Heparan Sulfate Proteoglycans Are Important for Islet Amyloid Formation and Islet Amyloid Polypeptide-induced Apoptosis.

Deposition of ß cell toxic islet amyloid is a cardinal finding in type 2 diabetes. In addition to the main amyloid component islet amyloid polypeptide (IAPP), heparan sulfate proteoglycan is constantly present in the amyloid deposit. Heparan sulfate (HS) side chains bind to IAPP, inducing conformational changes of the IAPP structure and an acceleration of fibril formation. We generated a double-transgenic mouse strain (hpa-hIAPP) that overexpresses human heparanase and human IAPP but is deficient of endogenous mouse IAPP. Culture of hpa-hIAPP islets in 20 mm glucose resulted in less amyloid formation compared with the amyloid load developed in cultured islets isolated from littermates expressing human IAPP only. A similar reduction of amyloid was achieved when human islets were cultured in the presence of heparin fragments. Furthermore, we used CHO cells and the mutant CHO pgsD-677 cell line (deficient in HS synthesis) to explore the effect of cellular HS on IAPP-induced cytotoxicity. Seeding of IAPP aggregation on CHO cells resulted in caspase-3 activation and apoptosis that could be prevented by inhibition of caspase-8. No IAPP-induced apoptosis was seen in HS-deficient CHO pgsD-677 cells. These results suggest that ß cell death caused by extracellular IAPP requires membrane-bound HS. The interaction between HS and IAPP or the subsequent effects represent a possible therapeutic target whose blockage can lead to a prolonged survival of ß cells.

A novel small-molecule compound disrupts influenza A virus PB2 cap-binding and inhibits viral replication.

OBJECTIVES: The conserved residues 318-483 in the PB2 subunit of influenza A polymerase is an independently folded cap-binding domain (PB2cap) that exhibits a distinct binding mode from other host cap-binding proteins, which suggests that PB2cap might be an ideal drug target. This study aimed to identify a new class of anti-influenza inhibitors that specifically disrupts the interaction between PB2cap and host cap structures. METHODS: An innovative fluorescence polarization assay was established for primary screening, followed by cap-binding inhibitory activity, antiviral efficacy and cytotoxicity evaluations of the selected compounds. The best compound was characterized by multi-cycle virus growth assay, cross-protection test, synergism evaluation, mini-replicon assay, binding affinity analysis, docking simulation and mouse study. RESULTS: Several PB2 cap-binding inhibitors were discovered. The compound 7-(4-hydroxy-2-oxo-2H-chromen-3-yl)-6H,7H,8H-chromeno[3',4':5,6]pyrano[3,2-c]chromene-6,8-dione, designated PB2-39, was identified as a potent inhibitor of replication of multiple subtypes of influenza A virus, including H1N1, H3N2, H5N1, H7N7, H7N9 and H9N2 in vitro and H1N1, H5N1 and H7N9 in vivo. Combinational treatment with the influenza virus release inhibitor zanamivir and PB2-39 exerted a synergistic anti-influenza effect. Mechanistic experiments supported that PB2-39 suppressed viral polymerase activity. Docking and binding affinity analyses demonstrated that PB2-39 interacted with the PB2 cap-binding pocket, suggesting its role as a cap-binding competitor. CONCLUSIONS: Our study provides new insights for the strategic development of novel cap-binding inhibitors of influenza A viruses.

Glycyrrhizic Acid Reduces Heart Rate and Blood Pressure by a Dual Mechanism.

Beta adrenergic receptors are crucial for their role in rhythmic contraction of heart along with their role in the pathological conditions such as tachycardia and high risk of heart failure. Studies report that the levels of beta-1 adrenergic receptor tend to decrease by 50%, whereas, the levels of beta-2 adrenergic receptor remains constant during the risk of heart failure. Beta blockers-the antagonistic molecules for beta-adrenergic receptors, function by slowing the heart rate, which thereby allows the left ventricle to fill completely during tachycardia incidents and hence helps in blood pumping capacity of heart and reducing the risk of heart failure. In the present study, we investigate the potential of glycyrrhizic acid (GA) as a possible principal drug molecule for cardiac arrhythmias owing to its ability to induce reduction in the heart rate and blood pressure. We use in vitro and in silico approach to study GA's effect on beta adrenergic receptor along with an in vivo study to examine its effect on heart rate and blood pressure. Additionally, we explore GA's proficiency in eliciting an increase in the plasma levels of vasoactive intestinal peptide, which by dilating the blood vessel consequently, can be a crucial aid during the occurrence of a potential heart attack. Therefore, we propose GA as a potential principal drug molecule via its potential in modulating heart rate and blood pressure.

Cross-protection of influenza A virus infection by a DNA aptamer targeting the PA endonuclease domain.

Amino acid residues in the N-terminal of the PA subunit (PAN) of the influenza A virus polymerase play critical roles in endonuclease activity, protein stability, and viral RNA (vRNA) promoter binding. In addition, PAN is highly conserved among different subtypes of influenza virus, which suggests PAN to be a desired target in the development of anti-influenza agents. We selected DNA aptamers targeting the intact PA protein or the PAN domain of an H5N1 virus strain using systematic evolution of ligands by exponential enrichment (SELEX). The binding affinities of selected aptamers were measured, followed by an evaluation of in vitro endonuclease inhibitory activity. Next, the antiviral effects of enriched aptamers against influenza A virus infections were examined. A total of three aptamers targeting PA and six aptamers targeting PAN were selected. Our data demonstrated that all three PA-selected aptamers neither inhibited endonuclease activity nor exhibited antiviral efficacy, whereas four of the six PAN-selected aptamers inhibited both endonuclease activity and H5N1 virus infection. Among the four effective aptamers, one exhibited cross-protection against infections of H1N1, H5N1, H7N7, and H7N9 influenza viruses, with a 50% inhibitory concentration (IC50) of around 10 nM. Notably, this aptamer was identified at the 5th round but disappeared after the 10th round of selection, suggesting that the identification and evaluation of aptamers at early rounds of selection may be highly helpful for screening effective aptamers. Overall, our study provides novel insights for screening and developing effective aptamers for use as anti-influenza drugs.

Tensile nanostructured hierarchically porous non-precious transition metal-based electrocatalyst for durable anion exchange membrane-based water electrolysis.

Electrochemical water electrolysis is a promising method for sustainable hydrogen production while transiting towards hydrogen economy. Among many, the Anion Exchange Membrane (AEM) based water electrolyzer is an emerging yet potentially affordable technology on maturity for producing large-scale hydrogen accommodating the usage of Non-Platinum Group Metal (non-PGM) based inexpensive electrocatalysts. Herein, we demonstrate the excellent performance of a bifunctional Nickel Copper Phosphide-Nickel sulphide (NCP-NS) electrocatalyst with a unique tensile nanostructure obtained via a facile, controlled ambient galvanic displacement route. An AEM electrolyzer with a larger active area of 10 cm2 stacked with the symmetric NCP-NS electrodes and a membrane demonstrates scalability with a requirement of a mere 1.66 V to reach a current density of 10 mA cm-2. The nickel-copper phosphide boosts the kinetics of charge transfer between the electrode and electrolyte interface, while a unique combination of a few nickel sulphide phases present in the catalyst provides sufficiently appropriate active sites for the overall water electrolysis. For the first time, we report a room temperature performance of âˆ¼ 230 mA cm-2 at 2 V for a non-PGM-based bifunctional electrocatalyst with exceptional durability for over 300 h of operation in an AEM water electrolyser with a retention rate of 95 %-97 % at a current density range of 80-800 mA cm-2.

Investigating the effects of radiation, T cell depletion, and bone marrow transplantation on murine gut microbiota.

Microbiome research has gained much attention in recent years as the importance of gut microbiota in regulating host health becomes increasingly evident. However, the impact of radiation on the microbiota in the murine bone marrow transplantation model is still poorly understood. In this paper, we present key findings from our study on how radiation, followed by bone marrow transplantation with or without T cell depletion, impacts the microbiota in the ileum and caecum. Our findings show that radiation has different effects on the microbiota of the two intestinal regions, with the caecum showing increased interindividual variation, suggesting an impaired ability of the host to regulate microbial symbionts, consistent with the Anna Karenina principle. Additionally, we observed changes in the ileum composition, including an increase in bacterial taxa that are important modulators of host health, such as Akkermansia and Faecalibaculum. In contrast, radiation in the caecum was associated with an increased abundance of several common commensal taxa in the gut, including Lachnospiraceae and Bacteroides. Finally, we found that high doses of radiation had more substantial effects on the caecal microbiota of the T-cell-depleted group than that of the non-T-cell-depleted group. Overall, our results contribute to a better understanding of the complex relationship between radiation and the gut microbiota in the context of bone marrow transplantation and highlight the importance of considering different intestinal regions when studying microbiome responses to environmental stressors.

Assessing the effectiveness of Interleukin-2 therapy in experimental type 1 diabetes.

AIM: Much focus of immunotherapy for type 1 diabetes (T1D) has been devoted on selectively boosting regulatory T (Treg) cells using low dose IL-2 due to their constitutive expression of IL-2Rα, CD25. However, several clinical trials using a low dose of IL-2 only showed a limited improvement of metabolic control. It can therefore be hypothesized that further decreasing IL-2 dosage may increase the selective responsiveness of Treg cells. METHODS: We induced experimental T1D using multiple low dose streptozotocin (STZ) injections and treated the mice with an ultra-low dose IL-2 (uIL-2, approximately 7-fold lower than low dose). Immune response was studied using multicolor flow cytometry. RESULTS: We found that uIL-2 did not protect STZ mice from developing hyperglycemia. It did neither increase Treg cell proportions, nor did it correct the phenotypic shift of Treg cells seen in T1D. It only partially decreased the proportion of IFN-γ+ T cells. Likewise, uIL-2 also did not protect the dysfunction of regulatory B (Breg) cells. Strikingly, when administered in combination with an anti-inflammatory cytokine IL-35, uIL-2 abrogated IL-35's protective effect. Low dose IL-2, on the other hand, protected half of the STZ mice from developing hyperglycemia. No difference was found in the Treg and Breg response, and it only tended to decrease CD80 expression in macrophages and dendritic cells. CONCLUSION: In conclusion, further decreasing IL-2 dosage may not be a suitable approach for T1D therapy, and the limited success suggests that an alternative low dose IL-2 therapy strategy or other immunotherapies should be considered.

Diabetic complications and prospective immunotherapy.

The incidence of Diabetes Mellitus is increasing globally. Individuals who have been burdened with diabetes for many years often develop complications as a result of hyperglycemia. More and more research is being conducted highlighting inflammation as an important factor in disease progression. In all kinds of diabetes, hyperglycemia leads to activation of alternative glucose metabolic pathways, resulting in problematic by-products including reactive oxygen species and advanced glycation end products. This review takes a look into the pathogenesis of three specific diabetic complications; retinopathy, nephropathy and neuropathy as well as their current treatment options. By considering recent research papers investigating the effects of immunotherapy on relevant conditions in animal models, multiple strategies are suggested for future treatment and prevention of diabetic complications with an emphasis on molecular targets associated with the inflammation.

Discovery of small-molecule modulators of the secretin receptor: Purmorphamine as novel anti-hypertensive agent.

The Secretin/Secretin receptor (SCTR) axis is well-known for its important role in water/salt homeostasis and blood pressure control. Recent studies revealed that absence of Secretin could lead to hypertension in animals and the administration of external Secretin leads to a sharp drop in blood pressure. Therefore, Secretin receptor has emerged as a crucial drug target of interest. In this report, using structure based drug design strategy, we have identified a small compound-based Secretin receptor modulator (i.e. purmorphamine or KSD179019). The virtual docking of KSD179019 with SCTR crystal structure and homology models revealed similar binding interactions. Based on active pharmacophores of KSD179019, several derivatives were designed and sythesized. SAR studies revealed that KSD179019 is the most effective SCTR modulator and chosen for further biological evaluation, including drug like properties and anti-hypertensive effect. KSD179019 not only has a similar blood pressure lowering effect as SCT peptide, but more importantly, it has a much longer half-life (∼8 h) and can be taken orally. Preliminary preclinical studies revealed extended bioavailability and low toxicity of this compound.

Gene Delivery of Manf to Beta-Cells of the Pancreatic Islets Protects NOD Mice from Type 1 Diabetes Development.

In type 1 diabetes, dysfunctional glucose regulation occurs due to the death of insulin-producing beta-cells in the pancreatic islets. Initiation of this process is caused by the inheritance of an adaptive immune system that is predisposed to responding to beta-cell antigens, most notably to insulin itself, coupled with unknown environmental insults priming the autoimmune reaction. While autoimmunity is a primary driver in beta-cell death, there is growing evidence that cellular stress participates in the loss of beta-cells. In the beta-cell fragility model, partial loss of islet mass requires compensatory upregulation of insulin production in the remaining islets, driving a cellular stress capable of triggering apoptosis in the remaining cells. The Glis3-Manf axis has been identified as being pivotal to the relative fragility or robustness of stressed islets, potentially operating in both type 1 and type 2 diabetes. Here, we have used an AAV-based gene delivery system to enhance the expression of the anti-apoptotic protein Manf in the beta-cells of NOD mice. Gene delivery substantially lowered the rate of diabetes development in treated mice. Manf-treated mice demonstrated minimal insulitis and superior preservation of insulin production. Our results demonstrating the therapeutic potential of Manf delivery to enhance beta-cell robustness and avert clinical diabetes.