External antigen uptake by Langerhans cells with reorganization of epidermal tight junction barriers

Outermost barriers are critical for terrestrial animals to avoid desiccation and to protect their bodies from foreign insults. Mammalian skin consists of two sets of barriers: stratum corneum (SC) and tight junctions (TJs). How acquisition of external antigens (Ags) by epidermal Langerhans cells (LCs) occur despite these barriers has remained unknown. We show that activation-induced LCs elongate their dendrites to penetrate keratinocyte (KC) TJs and survey the extra-TJ environment located outside of the TJ barrier, just beneath the SC. Penetrated dendrites uptake Ags from the tip where Ags colocalize with langerin/Birbeck granules. TJs at KC–KC contacts allow penetration of LC dendrites by dynamically forming new claudin-dependent bicellular- and tricellulin-dependent tricellular TJs at LC–KC contacts, thereby maintaining TJ integrity during Ag uptake. Thus, covertly under keratinized SC barriers, LCs and KCs demonstrate remarkable cooperation that enables LCs to gain access to external Ags that have violated the SC barrier while concomitantly retaining TJ barriers to protect intra-TJ environment

The Lantern, 2015-2016

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Effects of Intrauterine Global Restriction on Reward Processing of Offspring

Obesity in recent years has become the leading cause of death and is associated with over 60 high risk diseases. Evidence indicates that the in utero environment significantly impacts fetal development via the process of gestational programming. In particular, maternal nutrition appears to play a significant part in the development of offspring addictive tendencies. Tyrosine hydroxylase (TH), the rate limiting enzyme for the production of the reward neurotransmitter, dopamine, acts in the ventral tegmental area (VTA) to reinforce rewarding behaviors. In this study, we used global caloric maternal restriction during gestation to produce intrauterine growth restricted (IGUR) offspring in rats, to understand how expression of TH expression is affected in the reward pathway of IUGR offspring. We hypothesize that alterations in VTA TH expression will explain observed increases in binge-like consumption of rewarding substances, such as sucrose observed in IUGR offspring

GMP-compatible and xeno-free cultivation of mesenchymal progenitors derived from human-induced pluripotent stem cells

Abstract Background Human mesenchymal stem cells are a strong candidate for cell therapies owing to their regenerative potential, paracrine regulatory effects, and immunomodulatory activity. Yet, their scarcity, limited expansion potential, and age-associated functional decline restrict the ability to consistently manufacture large numbers of safe and therapeutically effective mesenchymal stem cells for routine clinical applications. To overcome these limitations and advance stem cell treatments using mesenchymal stem cells, researchers have recently derived mesenchymal progenitors from human-induced pluripotent stem cells. Human-induced pluripotent stem cell-derived progenitors resemble adult mesenchymal stem cells in morphology, global gene expression, surface antigen profile, and multi-differentiation potential, but unlike adult mesenchymal stem cells, it can be produced in large numbers for every patient. For therapeutic applications, however, human-induced pluripotent stem cell-derived progenitors must be produced without animal-derived components (xeno-free) and in accordance with Good Manufacturing Practice guidelines. Methods In the present study we investigate the effects of expanding mesodermal progenitor cells derived from two human-induced pluripotent stem cell lines in xeno-free medium supplemented with human platelet lysates and in a commercial high-performance Good Manufacturing Practice-compatible medium (Unison Medium). Results The results show that long-term culture in xeno-free and Good Manufacturing Practice-compatible media somewhat affects the morphology, expansion potential, gene expression, and cytokine profile of human-induced pluripotent stem cell-derived progenitors but supports cell viability and maintenance of a mesenchymal phenotype equally well as medium supplemented with fetal bovine serum. Conclusions The findings support the potential to manufacture large numbers of clinical-grade human-induced pluripotent stem cell-derived mesenchymal progenitors for applications in personalized regenerative medicine. Graphical abstrac

Systemic immune system alterations in early stages of Alzheimer's disease

Immune activation and inflammation play significant roles in the pathogenesis of Alzheimer's disease (AD). To test whether AD patients showed systemic manifestations of inflammation, blood from 41 patients with early stages of AD and 31 aged-match elderly controls were evaluated. Cellular markers for monocyte/macrophage (MO) activation and CD8 T lymphocyte were increased in early AD patients. Expression of monocyte CCR2, the receptor for monocyte chemoattractant protein-1 (MCP-1), was decreased; however, plasma MCP-1 levels were significantly increased and were related to the degree of MO activation in AD. These findings suggest that AD pathogenesis may be influenced by systemic immunologic dysfunction and provides potential immunologic targets for therapeutic intervention

<i>B</i>. <i>burgdorferi</i> RecA binds preferentially to ssDNA.

<p>6% PAGE 0.5X TBE gel analysis of RecA binding to 95 nt ssDNA and 95 bp dsDNA. The indicated concentrations of RecA were assayed for binding with 10 nM (molecules) of the indicated 5-endlabeled DNAs in a buffer containing 25 mM HEPES (pH 7.6), 2 mM MgCl₂, 1 mM DTT, 50 mM NaCl and, where indicated, 2 mM ATP, by incubation at 30°C for 5 min. This was followed by protein crosslinking with glutaraldehyde (final concentration 0.033%) at 20°C for 5 min prior to application of the binding reactions to native a PAGE gel. The gel migration position of RecA-DNA complexes is indicated by asterisks and the position of the wells is noted. See the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187382#sec002" target="_blank">Materials and methods</a> section for details.</p

<i>B</i>. <i>burgdorferi</i> RecA-promoted strand exchange with oligonucleotide substrates is strongly stimulated by SSB.

<p>A) Schematic representation of the oligonucleotide substrates used. The ssDNA donor oligonucleotide is 70 nt in length while the 5’-endlabeled duplex target DNA is 35 bp. * denote the ³²P-endlabels, red/shaded lines represent endlabeled strands.B) 8% PAGE 1X TAE/0.1% SDS gel analysis of strand exchange reactions with RecA, -/+ SSB. Strand exchange was assayed in a buffer containing 25 mM HEPES (pH 7.6), 2 mM MgCl₂, 1 mM DTT, 100 μg/ml BSA, 50 mM NaCl and 2 mM ATP, AMPPNP or ATP[γS] in 60 μL reaction volumes. The assay was conducted as a staged reaction with pre-incubation of RecA with the donor ssDNA (30°C, 5 min) followed by addition of 5’-endlabeled duplex target DNA (S) and continued incubation at 37°C. RecA was present at 2 μM, donor ssDNA at 1.4 μM (nt) and 5’-endlabeled target duplex (S) was present at 1.4 μM (nt). When added, SSB was added after pre-incubation of RecA with the single stranded donor and was present at 300 nM. The proteins and nucleotide co-factor present are indicated in the loading key above the gels. C) 8% PAGE 1X TAE/0.1% SDS gel analysis of strand exchange reactions with RecA and a titration of SSB added before RecA (1st) or after RecA incubation with single-stranded donor DNA (2nd). Reactions were performed with ATP[γS] as the nucleotide co-factor.</p

<i>B</i>. <i>burgdorferi</i> RecA-promoted strand exchange with long substrates.

<p>A) Schematic of the strand exchange assay using ϕX174 virion DNA (closed circular ssDNA; css) and XhoI-linearized ϕX174 duplex DNA (linear dsDNA; lds). As strand exchange initiates, joint molecule (JM) intermediates form. As strand exchange progresses, the final strand exchange products of nicked circular DNA (NC) and linear ssDNA (lss) are formed. Partial, intermolecular strand exchange between multiple donor and target molecules can also give rise to large aggregates (aggr.). B) 0.8% agarose 1X TAE gel analysis of strand exchange reactions performed with RecA and SSB. Strand exchange was assayed in a buffer containing 25 mM HEPES (pH 7.6), 1 mM DTT, 100 μg/ml BSA, 50 mM NaCl, 2 mM ATP and the indicated concentration of MgCl₂ in 120 μL reaction volumes. The assay was conducted as a staged reaction with pre-incubation of RecA with the donor ssDNA (css; 30°C, 5 min) followed by addition of SSB and linear duplex DNA (lds) and continued incubation at 37°C. RecA was present at 2 μM, ϕX174 virion (css) at 5.1 μM (nt) and XhoI-linearized ϕX174 duplex DNA (lds) at 5.1 μM (nt). SSB was added after pre-incubation of RecA with the single stranded donor and was present at 0.9 μM. The concentration of added MgCl₂ is noted in the loading key above the gel. The migration position of the substrate DNA is noted to the left of the gel and of the products to the right. Under our gel conditions css and lss have identical gel mobilities. M denotes mock incubation of the substrate DNAs without added RecA or SSB. C) 0.8% agarose 1X TAE gel analysis of SSB order-of-addition effects and ATP hydrolysis requirements of strand exchange reactions performed with long substrates. Strand exchange reactions were performed as noted in B) using buffer conditions with the optimal 5 mM MgCl₂ concentration and 0.9 μM of added SSB. The order of SSB addition and nucleotide cofactor present is indicated in the loading key above the gel. When added first, SSB was incubated with ϕX174 virion at 30°C for 5 min prior to the addition of RecA and linear ϕX174 duplex DNA followed by continued incubation at 37°C for the times indicated above the gel. When SSB was added second, RecA was incubated with ϕX174 virion at 30°C for 5 min prior to the addition of SSB and linear duplex DNA, followed by continued incubation at 37°C for the times indicated above the gel. M denotes mock incubation of the substrate DNAs without added RecA or SSB.</p