Chronic systemic immunosuppression in cell replacement therapy restricts its clinical application. This study sought to explore the potential of cell-based immune modulation as an alternative to immunosuppressive drug therapy in the context of pancreatic islet transplantation. Human amniotic epithelial cells (AEC) possess innate anti-inflammatory and immunosuppressive properties that were utilized to create localized immune privilege in an in vitro islet cell culture system. Cellular constructs composed of human islets and AEC (islet/AEC) were bioengineered under defined rotational cell culture conditions. Insulin secretory capacity was validated by glucose challenge and immunomodulatory potential characterized using a peripheral blood lymphocyte (PBL) proliferation assay. Results were compared to control constructs composed of islets or AEC cultured alone. Studies employing AEC-conditioned medium examined the role of soluble factors, and fluorescence immunocytochemistry was used to identify putative mediators of the immunosuppressive response in isolated AEC monocultures. Sustained, physiologically appropriate insulin secretion was observed in both islets and islet/AEC constructs. Activation of resting PBL proliferation occurred on exposure to human islets alone but this response was significantly (p < 0.05) attenuated by the presence of AEC and AEC-conditioned medium. Mitogen (phytohaemagglutinin, 5 µg/ml)-induced PBL proliferation was sustained on contact with isolated islets but abrogated by AEC, conditioned medium, and the islet/AEC constructs. Immunocytochemical analysis of AEC monocultures identified a subpopulation of cells that expressed the proapoptosis protein Fas ligand. This study demonstrates that human islet/AEC constructs exhibit localized immunosuppressive properties with no impairment of β-cell function. The data suggest that transplanted islets may benefit from the immune privilege status conferred on them as a consequence of their close proximity to human AEC. Such an approach may reduce the need for chronic systemic immunosuppression, thus making islet transplantation a more attractive treatment option for the management of insulindependent diabetes.Key words: Human islets; Human amniotic epithelial cells; Immune privilege; Rotational cell culture system; Peripheral blood lymphocytes; Immunosuppression; Insulin; Fas ligand INTRODUCTIONimmune isolation that circumvents the need for systemic immunosuppression has been the subject of extensive research. Macro-and microencapsulation devices have Islet transplantation offers a more physiological approach to the restoration of glucose homeostasis than been the preferred option, resulting in limited clinical application (6,39), but loss of capsule integrity and imexogenous insulin therapy (8,30), but its use is restricted to a discrete population of individuals with type 1 diabepaired gaseous and nutrient exchange undermine longterm β-cell function. Nanocapsule devices, formed from tes who experience frequent and unpredictable episodes of hypoglyce...

MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression and are implicated in the etiology of several neuropsychiatric disorders, including substance use disorders (SUDs). Using in silico genome-wide sequence analyses, we identified miR-495 as a miRNA whose predicted targets are significantly enriched in the Knowledgebase of Addiction-Related Genes (ARG) database (KARG; http://karg.cbi.pku.edu.cn). This small non-coding RNA is also highly expressed within the nucleus accumbens (NAc), a pivotal brain region underlying reward and motivation. Using luciferase reporter assays, we found that miR-495 directly targeted the 3’UTRs of Bdnf, Camk2a, and Arc. Furthermore, we measured miR-495 expression in response to acute cocaine in mice and found that it is downregulated rapidly and selectively in the NAc, along with concomitant increases in ARG expression. Lentiviral-mediated miR-495 overexpression in the NAc shell (NAcsh) not only reversed these cocaine-induced effects, but also downregulated multiple ARG mRNAs in specific SUD-related biological pathways, including those that regulate synaptic plasticity. miR-495 expression was also downregulated in the NAcsh of rats following cocaine self-administration. Most importantly, we found that NAcsh miR-495 overexpression suppressed the motivation to self-administer and seek cocaine across progressive ratio, extinction, and reinstatement testing, but had no effect on food reinforcement, suggesting that miR-495 selectively affects addiction-related behaviors. Overall, our in silico search for post-transcriptional regulators identified miR-495 as a novel regulator of multiple ARGs that play a role in modulating motivation for cocaine.

The neuronal RNA-binding protein HuD is involved in synaptic plasticity and learning and memory mechanisms. These effects are thought to be due to HuD-mediated stabilization and translation of target mRNAs associated with plasticity. To investigate the potential role of HuD in drug addiction, we first used bioinformatics prediction algorithms together with microarray analyses to search for specific genes and functional networks upregulated within the forebrain of HuD overexpressing mice (HuD ). When this set was further limited to genes in the knowledgebase of addiction-related genes database (KARG) that contains predicted HuD-binding sites in their 3' untranslated regions (3'UTRs), we found that HuD regulates networks that have been associated with addiction-like behavior. These genes included Bdnf and Camk2a, 2 previously validated HuD targets. Since addiction is hypothesized to be a disorder stemming from altered gene expression causing aberrant plasticity, we sought to test the role of HuD in cocaine conditioned placed preference (CPP), a model of addiction-related behaviors. HuD mRNA and protein were upregulated by CPP within the nucleus accumbens of wild-type C57BL/6J mice. These changes were associated with increased expression of Bdnf and Camk2a mRNA and protein. To test this further, we trained HuD and wild-type mice in CPP and found that HuD mice showed increased cocaine CPP compared with controls. This was also associated with elevated expression of HuD target mRNAs and proteins, CaMKIIα and BDNF. These findings suggest HuD involvement in addiction-related behaviors such as cocaine conditioning and seeking, through increased plasticity-related gene expression.

Patients who presented to their family doctors with previously uninvestigated dyspepsia of at least two weeks' duration were recruited into a placebo controlled trial of treatment with ranitidine (150 mg twice daily) for six weeks. All patients were examined by endoscopy before treatment, and for those with macroscopical abnormalities the examination was repeated after treatment. Of the 604 patients recruited, 559 had endoscopy, of whom 171 (30%) had no apparent abnormality. Of the 388 patients remaining, one third had two or more lesions. The high incidence of underlying disease was coupled with low accuracy in unaided clinical diagnosis. After endoscopy 496 patients with persistent symptoms (median duration six to eight weeks) were randomly allocated to treatment and then reviewed every two weeks. Complete remission of symptoms occurred in 76% of patients who were taking ranitidine and in 55% who were taking placebo (p less than 0.000004). Of those with non-ulcer dyspepsia, significantly more became symptom free taking ranitidine compared with placebo (p less than 0.002). Ranitidine healed most duodenal ulcers (80%) and gastric ulcers (90%) within four weeks. Tolerance to ranitidine was good, and the incidence of complaints was similar on placebo.

The RNA-binding protein (RBP) HuD is involved in neuronal differentiation, regeneration, synaptic plasticity and learning and memory. RBPs not only bind to mRNAs but also interact with several types of RNAs including circular RNAs (circRNAs), a class of non-coding RNAs generated by pre-mRNA back-splicing. This study explored whether HuD could regulate the expression of neuronal circRNAs. HuD controls target RNA’s fate by binding to Adenylate-Uridylate Rich Elements (AREs). Using bioinformatics analyses, we found HuD-binding ARE-motifs in about 26% of brain-expressed circRNAs. By RNA immunoprecipitation (RIP) from the mouse striatum followed by circRNA arrays, we identified over 600 circRNAs bound to HuD. Among these, 226 derived from genes where HuD also bound to their associated mRNAs including circHomer1a , which we previously characterized as a synaptic HuD target circRNA. Binding of HuD to two additional plasticity–associated circRNAs, circCreb1 , and circUfp2 , was validated by circRNA-specific qRT-PCR. Interestingly, we found that circUpf2 is also enriched in synaptosomes. Pathway analyses confirmed that the majority of HuD-bound circRNAs originate from genes regulating nervous system development and function. Using striatal tissues from HuD overexpressor (HuD-OE) and knock out (KO) mice for circRNA expression analyses we identified 86 HuD-regulated circRNAs. These derived from genes within the same biological pathways as the HuD RIP. Cross-correlation analyses of HuD-regulated and HuD-bound circRNAs identified 69 regulated in either HuD-OE or HuD-KO and 5 in both sets. These include circBrwd1 , circFoxp1 , and circMap1a , which derive from genes involved in neuronal development and regeneration, and circMagi1 and circLppr4 , originating from genes controlling synapse formation and linked to psychiatric disorders. These circRNAs form competing endogenous RNA (ceRNA) networks including microRNAs and mRNAs. Among the HuD target circRNAs, circBrwd1 and circFoxp1 are regulated in an opposite manner to their respective mRNAs. The expressions of other development- and plasticity-associated HuD target circRNAs such as circSatb2, cirHomer1a and circNtrk3 are also altered after the establishment of cocaine conditioned place preference (CPP). Collectively, these data suggest that HuD interactions with circRNAs regulate their expression and transport, and that the ensuing changes in HuD-regulated ceRNA networks could control neuronal differentiation and synaptic plasticity.