Islet Isolation from the porcine pancreas, using a continuous digestion method

Abstract: This paper describes a method for pig islet isolation with the following original features: first, we set up a reproducible, rapid, sterile, and simple method for pancreas procurement and preparation with double duct incannulation. Second, we demonstrated that UW solution can be used for pancreas digestion; third, we designed an original method for pancreas digestion, derived from the method originally described by Ricordi, except that a double digestion circuit made it possible to digest continuously, within one step, the pancreas, which makes the method operator-independent; finally we designed a simple method for islet purification based on a two-layer Histopaque/ UW density gradient and the use of the COBE 2991 cell processor.     

Packaging of prions into exosomes is associated with a novel pathway of PrP processing

Prion diseases are fatal, transmissible neurodegenerative disorders associated with conversion of the host-encoded prion protein (PrP(C)) into an abnormal pathogenic isoform (PrP(Sc)). Following exposure to the infectious agent (PrP(Sc)) in acquired disease, infection is propagated in lymphoid tissues prior to neuroinvasion and spread within the central nervous system. The mechanism of prion dissemination is perplexing due to the lack of plausible PrP(Sc)-containing mobile cells that could account for prion spread between infected and uninfected tissues. Evidence exists to demonstrate that the culture media of prion-infected neuronal cells contain PrP(Sc) and infectivity but the nature of the infectivity remains unknown. In this study we have identified PrP(C) and PrP(Sc) in association with endogenously expressing PrP neuronal cell-derived exosomes. The exosomes from our prion-infected neuronal cell line were efficient initiators of prion propagation in uninfected recipient cells and to non-neuronal cells. Moreover, our neuronal cell line was susceptible to infection by non-neuronal cell-derived exosome PrP(Sc). Importantly, these exosomes produced prion disease when inoculated into mice. Exosome-associated PrP is packaged via a novel processing pathway that involves the N-terminal modification of PrP and selection of distinct PrP glycoforms for incorporation into these vesicles. These data extend our understanding of the relationship between PrP and exosomes by showing that exosomes can establish infection in both neighbouring and distant cell types and highlight the potential contribution of differentially processed forms of PrP in disease distribution. These data suggest that exosomes represent a potent pool of prion infectivity and provide a mechanism for studying prion spread and PrP processing in cells endogenously expressing PrP.     

Evaluation of antibody response to BNT162b2 mRNA COVID-19 vaccine in patients affected by immune-mediated inflammatory diseases up to 5 months after vaccination

Clinical and Experimental Medicine - SARS-CoV-2 vaccination with mRNA product BNT162b2 elicited high immunogenicity in healthy subjects in trials. This study aims to better understand the factors...     

Increased prevalence of proliferative retinopathy in patients with type 1 diabetes who are deficient in glucose-6-phosphate dehydrogenase

Aims/hypothesis  

Impaired activity of the pentose phosphate pathway of glucose metabolism caused by hereditary deficiency of its key regulatory enzyme glucose-6-phosphate dehydrogenase (G6PD) has consequences that may worsen or attenuate the course of diabetic complications. Decreased availability of NADPH can predispose to oxidative stress and endothelial dysfunction, but can also limit the activity of the polyol pathway and cholesterol synthesis. Reduced availability of pentose phosphates for nucleic acid synthesis could impair cell proliferation. We sought to learn in which direction G6PD deficiency affects diabetic retinopathy.     

Stereospecific interactions are necessary for Alzheimer disease amyloid-β toxicity

Previous studies suggest membrane binding is a key determinant of amyloid β (Aβ) neurotoxicity. However, it is unclear whether this interaction is receptor driven. To address this issue, a D-handed enantiomer of Aβ42 (D-Aβ42) was synthesized and its biophysical and neurotoxic properties were compared to the wild-type Aβ42 (L-Aβ42). The results showed D- and L-Aβ42 are chemically equivalent with respect to copper binding, generation of reactive oxygen species and aggregation profiles. Cell binding studies show both peptides bound to cultured cortical neurons. However, only L-Aβ42 was neurotoxic and inhibited long term potentiation indicating L-Aβ42 requires a stereospecific target to mediate toxicity. We identified the lipid phosphatidylserine, as a potential target. Annexin V, which has very high affinity for externalized phosphatidylserine, significantly inhibited L-Aβ42 but not D-Aβ42 binding to the cultured cortical neurons and significantly rescued L-Aβ42 neurotoxicity. This suggests that Aβ mediated toxicity in Alzheimer disease is dependent upon Aβ binding to phosphatidylserine on neuronal cells.     

Evaluation of the effects of treatment with sAPPα on functional and histological outcome following controlled cortical impact injury in mice

Neural stem cells (NSCs) are tissue-specific, multipotent stem cells that can differentiate into three cell lineages in the central nervous system: neurons, astrocytes and oligodendrocytes. The therapeutic potential of NSCs has fueled attempts to characterize the expression of genes that regulate their fate. In this study, NSCs from embryonic day 15 (E15) mouse embryos were differentiated for 1 (DF-1) or 2 (DF-2) days, and the gene expression patterns in the NSCs and in the DF-1 and DF-2 cells were measured by microarray and real-time RT-PCR. Among the analyzed genes, 1898 genes were up-regulated in the DF-1 and DF-2 cells relative to the NSCs, whereas 1642 genes were down-regulated. The up-regulated genes included Gfap, Smad6, Fst, Tgfb2 and Cdkn2. The down-regulated genes included Ccnb1, Ccnd1 and Ccnd2. We identified gene networks that were associated with BMP and TGF-beta2 signaling pathways using Ingenuity Pathway Analysis. Our results suggest that the differentiation of E15 NSCs into astrocytes is based on a combinatorial network of various signaling pathways, including cell cycle, BMP and TGF-beta2 signaling.     

Amyloid precursor protein and amyloid precursor-like protein 2 have distinct roles in modulating myelination,demyelination, and remyelination of axons

The identification of factors that regulate myelination provides important insight into the molecular mechanisms that coordinate nervous system development and myelin regeneration after injury. In this study, we investigated the role of amyloid precursor protein (APP) and its paralogue amyloid precursor-like protein 2 (APLP2) in myelination using APP and APLP2 knockout (KO) mice. Given that BACE1 regulates myelination and myelin sheath thickness in both the peripheral and central nervous systems, we sought to determine if APP and APLP2, as alternate BACE1 substrates, also modulate myelination, and therefore provide a better understanding of the events regulating axonal myelination. In the peripheral nervous system, we identified that adult, but not juvenile KO mice, have lower densities of myelinated axons in their sciatic nerves while in the central nervous system, axons within both the optic nerves and corpus callosum of both KO mice were significantly hypomyelinated compared to wild-type (WT) controls. Biochemical analysis demonstrated significant increases in BACE1 and myelin oligodendrocyte glycoprotein and decreased NRG1 and proteolipid protein levels in both KO brain tissue. The acute cuprizone model of demyelination/remyelination revealed that whereas axons in the corpus callosum of WT and APLP2-KO mice underwent similar degrees of demyelination and subsequent remyelination, the myelinated callosal axons in APP-KO mice were less susceptible to cuprizone-induced demyelination and showed a failure in remyelination after cuprizone withdrawal. These data identified APP and APLP2 as modulators of normal myelination and demyelination/remyelination conditions. Deletion of APP and APLP2 identifies novel interplays between the BACE1 substrates in the regulation of myelination.     

Western Bluetongue virus serotype 3 in Sardinia,diagnosis and characterization

Over the last 20 years, Italy has experienced multiple incursions of different serotypes of Bluetongue virus (BTV), a Culicoides‐borne arbovirus, the causative agent of bluetongue (BT), a major disease of ruminants. The majority of these incursions originated from Northern Africa, likely because of wind‐blown dissemination of infected midges. Here, we report the first identification of BTV‐3 in Sardinia, Italy. BTV‐3 circulation was evidenced in sentinel animals located in the province of Sud Sardegna on September 19, 2018. Prototype strain BTV‐3 SAR2018 was isolated on cell culture. BTV‐3 SAR2018 sequence and partial sequences obtained by next‐generation sequencing from nucleic acids purified from the isolate and blood samples, respectively, were demonstrated to be almost identical (99–100% of nucleotide identity) to BTV‐3 TUN2016 identified in Tunisia in 2016 and 2017, a scenario already observed in past incursions of other BTV serotypes originating from Northern Africa.