Platelet‐Rich Plasma Increases Growth and Motility of Adipose Tissue‐Derived Mesenchymal Stem Cells and Controls Adipocyte Secretory Function

Adipose tissue‐derived mesenchymal stem cells (Ad‐MSC) and platelet derivatives have been used alone or in combination to achieve regeneration of injured tissues. We have tested the effect of platelet‐rich plasma (PRP) on Ad‐MSC and adipocyte function. PRP increased Ad‐MSC viability, proliferation rate and G1‐S cell cycle progression, by at least 7‐, 2‐, and 2.2‐fold, respectively, and reduced caspase 3 cleavage. Higher PRP concentrations or PRPs derived from individuals with higher platelet counts were more effective in increasing Ad‐MSC growth. PRP also accelerated cell migration by at least 1.5‐fold. However, PRP did not significantly affect mature adipocyte viability, differentiation and expression levels of PPAR‐γ and AP‐2 mRNAs, while it increased leptin production by 3.5‐fold. Interestingly, PRP treatment of mature adipocytes also enhanced the release of Interleukin (IL)‐6, IL‐8, IL‐10, Interferon‐γ, and Vascular Endothelial Growth Factor. Thus, data are consistent with a stimulatory effect of platelet derivatives on Ad‐MSC growth and motility. Moreover, PRP did not reduce mature adipocyte survival and increased the release of pro‐angiogenic factors, which may facilitate tissue regeneration processes. © 2015 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc. J. Cell. Biochem. 116: 2408–2418, 2015. © 2015 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.     

Marked inhibition of retinal neovascularization in rats following soluble-flt-1 gene transfer

BACKGROUND: In mouse models of retinopathy of prematurity (ROP) inhibitors of vascular endothelial growth factor (VEGF) functions administered systemically completely block retinal neovascularization. In contrast, selective ocular VEGF depletion has achieved an approx. 50% inhibition of retinal neovascular growth. It is unclear whether a more complete inhibition of new blood vessel development can be obtained with an anti-VEGF therapy localized to the eye. Therefore, the objective of the present study was to determine the effect of local anti-VEGF therapy in a different animal model which closely mimics human ROP. METHODS: Rats were exposed to alternating cycles of high and low levels of oxygen for 14 days immediately after birth; thereafter, they were intravitreally injected with an adenoviral vector expressing a secreted form of the VEGF receptor flt-1 (Ad.sflt), which acts by sequestering VEGF. Contralateral eyes were injected with the control vector carrying the reporter gene expressing beta-galactosidase (Ad.betaGal). RESULTS: At the peak of retinal neovascular growth, i.e. post-natal day 21 (P21), we observed up to 97.5% decrease in retinal neovascularization in animals injected with Ad.sflt. At the end of observation (P28), no significant difference in retinal vessel number was detected in both oxygen-injured and normoxic Ad.sflt-treated retinas compared with untreated or Ad.betaGal-treated retinas. CONCLUSION: Adenoviral-mediated sflt-1 gene transfer induces a near-complete inhibition of ischemia-induced retinal neovascularization in rats without affecting pre-existing retinal vessels.     

Expression of HECA-452 antigen correlates with disease relapse in mycosis fungoides

OBJECTIVE: To determine the expression of HECA-452 epitope in mycosis fungoides (MF), assess whether its expression increases in relapsed MF compared with nonrelapsed MF and determine the potential prognostic relevance of HECA-452 expression. STUDY DESIGN: HECA-452 expression was evaluated by immunohistochemistry in a consecutive series of 20 MF. In all patients we evaluated the disease-free survival rate according to HECA-452 expression in a univariate analysis. RESULTS: We found a low expression in 5 MF (25%), a moderate expression in 8 MF (40%) and a high expression in 7 MF (35%) in the intraepidermal area. All patients were disease-free after appropriate therapy. Four of 20 patients (20%) relapsed within 2 years. HECA-452 expression significantly correlated with disease relapse in these patients. In fact, among the 7 patients whose lesions had a high expression, 4 had a disease recurrence (57%), whereas 0 of 13 (0%) with a low or moderate expression relapsed (p < 0.05). CONCLUSION: HECA-452 expression correlates with disease relapse in MF. Correlation with disease progression suggests that HECA-452 could be of prognostic relevance in the early stage of mycosis fungoides.     

A Wide Range of 3243A>G/tRNALeu(UUR) (MELAS) Mutation Loads May Segregate in Offspring through the Female Germline Bottleneck

Segregation of mutant mtDNA in human tissues and through the germline is debated, with no consensus about the nature and size of the bottleneck hypothesized to explain rapid generational shifts in mutant loads. We investigated two maternal lineages with an apparently different inheritance pattern of the same pathogenic mtDNA 3243A>G/tRNA^Leu(UUR) (MELAS) mutation. We collected blood cells, muscle biopsies, urinary epithelium and hair follicles from 20 individuals, as well as oocytes and an ovarian biopsy from one female mutation carrier, all belonging to the two maternal lineages to assess mutant mtDNA load, and calculated the theoretical germline bottleneck size (number of segregating units). We also evaluated “mother-to-offspring” segregations from the literature, for which heteroplasmy assessment was available in at least three siblings besides the proband. Our results showed that mutation load was prevalent in skeletal muscle and urinary epithelium, whereas in blood cells there was an inverse correlation with age, as previously reported. The histoenzymatic staining of the ovarian biopsy failed to show any cytochrome-c-oxidase defective oocyte. Analysis of four oocytes and one offspring from the same unaffected mother of the first family showed intermediate heteroplasmic mutant loads (10% to 75%), whereas very skewed loads of mutant mtDNA (0% or 81%) were detected in five offspring of another unaffected mother from the second family. Bottleneck size was 89 segregating units for the first mother and 84 for the second. This was remarkably close to 88, the number of “segregating units” in the “mother-to-offspring” segregations retrieved from literature. In conclusion, a wide range of mutant loads may be found in offspring tissues and oocytes, resulting from a similar theoretical bottleneck size.     

Bisphenol-A Impairs Insulin Action and Up-Regulates Inflammatory Pathways in Human Subcutaneous Adipocytes and 3T3-L1 Cells

Current evidence indicates that chemical pollutants may interfere with the homeostatic control of nutrient metabolism, thereby contributing to the increased prevalence of metabolic disorders. Bisphenol-A (BPA) is a lipophilic compound contained in plastic which is considered a candidate for impairing energy and glucose metabolism. We have investigated the impact of low doses of BPA on adipocyte metabolic functions. Human adipocytes derived from subcutaneous adipose tissue and differentiated 3T3-L1 cells were incubated with BPA, in order to evaluate the effect on glucose utilization, insulin sensitivity and cytokine secretion. Treatment with 1nM BPA significantly inhibited insulin-stimulated glucose utilization, without grossly interfering with adipocyte differentiation. Accordingly, mRNA levels of the adipogenic markers PPARγ and GLUT4 were unchanged upon BPA exposure. BPA treatment also impaired insulin-activated receptor phosphorylation and signaling. Moreover, adipocyte incubation with BPA was accompanied by increased release of IL-6 and IFN-γ, as assessed by multiplex ELISA assays, and by activation of JNK, STAT3 and NFkB pathways. Treatment of the cells with the JNK inhibitor SP600125 almost fully reverted BPA effect on insulin signaling and glucose utilization. In conclusion, low doses of BPA interfere with inflammatory/insulin signaling pathways, leading to impairment of adipose cell function.     

<Emphasis Type="SmallCaps">d</Emphasis>-Aspartate affects secretory activity in rat Harderian gland: molecular mechanism and functional significance

In this paper, the role of d-aspartate in the rat Harderian gland (HG) was investigated by histochemical, ultrastructural, and biochemical analyses. In this gland, substantial amounts of endogenous d-Asp were detected, along with aspartate racemases that convert d-Asp to l-Asp and vice versa. We found that the gland was capable of uptaking and accumulating exogenously administered d-Asp. d-Asp acute treatment markedly increased lipid and porphyrin secretion and induced a powerful hyperaemia in inter-acinar interstitial tissue. Since d-Asp is known to be recognized by NMDA receptors, the expression of such receptors in rat HG led us to the hypothesis that d-Asp acute treatment induced the activation of the extracellular signal-regulated protein kinase (ERK) and nitric oxide synthase (NOS) pathways mediated by NMDA. Interestingly, as a result of enhanced oxidative stress due to increased porphyrin secretion, the revealed activation of the stress-activated protein kinase/c-jun N-terminal kinase (SAPK/JNK) pro-apoptotic pathway was probably triggered by the gland itself to preserve its cellular integrity.     

CIN85 regulates the ligand-dependent endocytosis of the IgE receptor: a new molecular mechanism to dampen mast cell function

Ligation of the high-affinity receptor for IgE (Fc epsilonRI), constitutively expressed on mast cells and basophils, promotes cell activation and immediate release of allergic mediators. Furthermore, Fc epsilonRI up-regulation on APC from atopic donors is involved in the pathophysiology of allergic diseases. In consideration of the clinical relevance of the IgE receptor, the down-modulation of Fc epsilonRI expression in mast cells may represent a potential target for handling atopic diseases. In an effort to identify new molecular mechanisms involved in attenuating Fc epsilonRI expression and signaling, we focused our attention on CIN85, a scaffold molecule that regulates, in concert with the ubiquitin ligase Cbl, the clathrin-mediated endocytosis of several receptor tyrosine kinases. In the present study, we show that endogenous CIN85 is recruited in Cbl-containing complexes after engagement of the Fc epsilonRI on a mast cell line and drives ligand-induced receptor internalization. By confocal microscopic analysis, we provide evidence that CIN85 directs a more rapid receptor sorting in early endosomes and delivery to a lysosomal compartment. Furthermore, biochemical studies indicate that CIN85 plays a role in reducing the expression of receptor complex. Finally, we demonstrate that CIN85-overexpressing mast cells are dramatically impaired in their ability to degranulate following Ag stimulation, suggesting that the accelerated internalization of activated receptors by perturbing the propagation of Fc epsilonRI signaling may contribute to dampen the functional response. This role of CIN85 could be extended to include other multimeric immune receptors, such as the T and B cell receptors, providing a more general molecular mechanism for attenuating immune responses.     

Coupling Demographic and Genetic Variability from Archived Collections of European Anchovy (Engraulis encrasicolus)

It is well known that temporal fluctuations in small populations deeply influence evolutionary potential. Less well known is whether fluctuations can influence the evolutionary potentials of species with large census sizes. Here, we estimated genetic population parameters from as survey of polymorphic microsatellite DNA loci in archived otoliths from Adriatic European anchovy (Engraulis encrasicolus), a fish with large census sizes that supports numerous local fisheries. Stocks have fluctuated greatly over the past few decades, and the Adriatic fishery collapsed in 1987. Our results show a significant reduction of mean genetic parameters as a consequence of the population collapse. In addition, estimates of effective population size (N_e) are much smaller than those expected in a fishes with large population census sizes (N_c). Estimates of N_e indicate low effective population sizes, even before the population collapse. The ratio N_e/N_e ranged between 10⁻⁶ and 10⁻⁸, indicating a large discrepancy between the anchovy gene pool and population census size. Therefore, anchovy populations may be more vulnerable to fishery effort and environmental change than previously thought.