Mesenchymal cells from human amniotic fluid survive and migrate after transplantation into adult rat brain

Amniotic fluid has been recently suggested as an alternative source of mesenchymal stem cells. However, the fate of amniotic fluid-derived mesenchymal stem cells (AF-MSCs) after in vivo transplantation has yet to be determined. In the present study we explored whether human AF-MSCs could survive and migrate following transplantation into the striatum of normal and ischemic rat. We found that the grafted cells could survive and migrate towards multiple brain regions in the normal animals, while they moved towards the injured region in the ischemic rat. Double-immunostaining analyses showed that the implanted human AF-MSCs express markers for immature neurons (Doublecortin) at 10 days, and for astrocytes (GFAP) at 10, 30 and 90 after transplantation. This study provides the first evidence that human amniotic fluid contains cells having the potential to survive and integrate into adult rat brain tissue and, therefore, to function as effective stem cells for therapeutic strategies.     

Impaired Endothelium-Mesenchymal Stem Cells Cross-talk in Systemic Sclerosis: a Link Between Vascular and Fibrotic Features

Introduction

To assess if an impaired cross-talk between endothelial cells (ECs) and perivascular/multipotent mesenchymal stem cells (MSCs) might induce a perturbation of vascular repair and leading to a phenotypic switch of MSC toward myofibroblast in Systemic Sclerosis (SSc).

Methods

We investigated different angiogenic and profibrotic molecules in a tridimentional matrigel assay, performing co-cultures with endothelial cells (ECs) and bone marrow derived MSCs from patients and healthy controls (HC). After 48 hours of co-culture, cells were sorted and analyzed for mRNA and protein expression.

Results

ECs-SSc showed a decreased tube formation ability which is not improved by co-cultures with different MSCs. After sorting, we showed: i. an increased production of vascular endothelial growth factor A (VEGF-A) in SSc-MSCs when co-cultured with SSc-ECs; ii. an increased level of transforming growth factor beta (TGF-β) and platelet growth factor BB (PDGF-BB) in SSc-ECs when co-cultured with both HC- and SSc-MSCs; iii. an increase of TGF-β, PDGF-R, alpha smooth muscle actin (α-SMA) and collagen 1 (Col1) in both HC- and SSc-MSCs when co-cultured with SSc-ECs.

Conclusion

We showed that during SSc, the ECs-MSCs crosstalk resulted in an altered expression of different molecules involved in the angiogenic processes, and mainly SSc-ECs seem to modulate the phenotypic switch of perivascular MSCs toward a myofibroblast population, thus supporting the fibrotic process.     

Modulation of Intestinal Microbiota by the Probiotic VSL#3 Resets Brain Gene Expression and Ameliorates the Age-Related Deficit in LTP

The intestinal microbiota is increasingly recognized as a complex signaling network that impacts on many systems beyond the enteric system modulating, among others, cognitive functions including learning, memory and decision-making processes. This has led to the concept of a microbiota-driven gut–brain axis, reflecting a bidirectional interaction between the central nervous system and the intestine. A deficit in synaptic plasticity is one of the many changes that occurs with age. Specifically, the archetypal model of plasticity, long-term potentiation (LTP), is reduced in hippocampus of middle-aged and aged rats. Because the intestinal microbiota might change with age, we have investigated whether the age-related deficit in LTP might be attenuated by changing the composition of intestinal microbiota with VSL#3, a probiotic mixture comprising 8 Gram-positive bacterial strains. Here, we report that treatment of aged rats with VSL#3 induced a robust change in the composition of intestinal microbiota with an increase in the abundance of Actinobacteria and Bacterioidetes, which was reduced in control-treated aged rats. VSL#3 administration modulated the expression of a large group of genes in brain tissue as assessed by whole gene expression, with evidence of a change in genes that impact on inflammatory and neuronal plasticity processes. The age-related deficit in LTP was attenuated in VSL#3-treated aged rats and this was accompanied by a modest decrease in markers of microglial activation and an increase in expression of BDNF and synapsin. The data support the notion that intestinal microbiota can be manipulated to positively impact on neuronal function.     

The HIV Matrix Protein p17 Promotes the Activation of Human Hepatic Stellate Cells through Interactions with CXCR2 and Syndecan-2

Background

The human immunodeficiency virus type 1 (HIV-1) p17 is a matrix protein involved in virus life''s cycle. CXCR2 and Syndecan-2, the two major coreceptors for the p17 protein, are expressed in hepatic stellate cells (HSCs), a key cell type involved in matrix deposition in liver fibrotic disorders.

Aim

In this report we have investigated the in vitro impact of p17 on HSCs transdifferentiation and function and underlying signaling pathways involved in these processes.

Methods

LX-2 cells, a human HSC line, and primary HSC were challenged with p17 and expressions of fibrogenic markers and of p17 receptors were assessed by qRT-PCR and Western blot. Downstream intracellular signaling pathways were evaluated with qRT-PCR and Western blot as well as after pre-treatment with specific pathway inhibitors.

Results

Exposure of LX2 cells to p17 increases their contractile force, reshapes the cytoskeleton fibers and upregulates the expression of transdifferentiation markers including αSMA, COL1α1 and endothelin-1 through the activation of Jak/STAT and Rho signaling pathways. These effects are lost in HSCs pre-incubated with a serum from HIV positive person who underwent a vaccination with a p17 peptide. Confocal laser microscopy studies demonstrates that CXCR2 and syndecan-2 co-associate at the plasma membrane after exposure to p17. Immunostaining of HIV/HCV liver biopsies from co-infected patients reveals that the progression of liver fibrosis correlates with a reduced expression of CXCR2.

Conclusions

The HIV matrix protein p17 is pro-fibrogenic through its interactions both with CXCR2 and syndecan-2 on activated HSCs.     

N-glycosylation of the mammalian dipeptidyl aminopeptidase-like protein 10 (DPP10) regulates trafficking and interaction with Kv4 channels

The dipeptidyl aminopeptidase-like protein 10 (DPP10) is a type II transmembrane protein homologue to the serine protease DPPIV/CD26 but enzymatically inactive. In the mammalian brain, DPP10 forms a complex with voltage-gated potassium channels of the Kv4 family, regulating their cell surface expression and biophysical properties. DPP10 is a glycoprotein containing eight predicted N-glycosylation sites in the extracellular domain. In this study we investigated the role of N-glycosylation on DPP10 trafficking and functional activity. Using site-directed mutagenesis (N to Q) we showed that N-glycosylation occured at six positions. Glycosylation at these specific residues was necessary for DPP10 trafficking to the plasma membrane as observed by flow cytometry. The surface expression levels of the substitutions N90Q, N119Q, N257Q and N342Q were reduced by more than 60%. Hence the interaction with the Kv4.3/KChIP2a channel complex was disrupted preventing the hastening effect of wild type DPP10 on current kinetics. Interestingly, N257 was crucial for this function and its substitution to glutamine completely blocked DPP10 sorting to the cell surface and prevented DPP10 dimerization. In summary, we demonstrated that glycosylation was necessary for both DPP10 trafficking to the cell surface and functional interaction with Kv4 channels.     

Chemical and immunological modifications of an arabinogalactan present in tea preparations of Phyllanthus niruri after treatment with gastric fluid

An arabinogalactan (AG) obtained from tea preparations of Phyllanthus niruri was previously investigated and presented immunological properties when tested with peritoneal mice macrophages. AG was now submitted to acidic and neutral gastric conditions using human gastric fluids and aq. HCl solution. Since the acidic procedures gave rise to the same free monosaccharidic composition, the acid hydrolyzate of AG at pH 2.00 was treated with ethanol to form insoluble (AG-P) and soluble fractions (AG-S). These were analyzed using (13)C NMR, HPSEC, and GC-MS for monosaccharide composition and methylation analyses. The results showed an intense partial degradation, including cleavages of the main chain. AG-S presented the monosaccharides released from the native polymer and some oligosaccharides as shown by methylation data. AG-P contained larger molecular fragments comprising the internal units from AG, which were not attacked by the hydrolysis condition. Both fractions were tested in peritoneal mice macrophages and remained active, promoting an increase of superoxide anion production of 2.0 and 2.3-fold, at 250 microg/mL, for AG-S and AG-P, respectively. When compared to AG, a slight diminished response was observed, revealing a structure-activity relation. The significance of the results is that most plant extracts are orally ingested and will reach the gastrointestinal tract before performing a biological function, so checking these changes is crucial to propose future clinical therapies based on the rational use of phytomedicine.     

Three-dimensional reconstruction of the oral cavity using the free anterolateral thigh flap.

Oral cavity reconstruction after removal of locally advanced tumors is particularly difficult because anatomical restoration must accurately reproduce the original structure and enable effective and fast rehabilitation of mastication, swallowing, and phonation. The authors report their 2-year experience with 17 patients surgically treated for oral cavity cancer with reconstruction performed with the free anterolateral thigh flap. Thanks to its thinness and pliability, this flap has proven to be perfectly adaptable to the structural peculiarities of the resected areas and has enabled the authors to considerably reduce the cosmetic and functional complications in the donor area observed with other flaps (such as the radial forearm flap). Flap grafting has always been complete and regular, and no intraoperative and postoperative complications have been observed. Swallowing recovery has always been satisfactory. On the basis of the authors' results, their current approach to oral cavity reconstruction is based on the use of flaps that enable anatomical restoration of the resected areas and reduce morbidity of the donor site. They believe that the anterolateral thigh flap can offer all of these opportunities, and the surgery can be simultaneously performed by two surgical teams.