Isolation and Transplantation of Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.     

Scientific Basis for Use of Pyrus pashia Buch.-Ham. ex D. Don. Fruit in Gastrointestinal,Respiratory and Cardiovascular Ailments

Background

Pyrus pashia Buch.-Ham. ex D. Don. has been used conventionally by many communities in the Himalayan region for the management of gastrointestinal, respiratory, and vascular complications. Set against this background, this study was carried out to justify the scientific basis to validate folkloric uses of fruits of Pyrus pashia Buch.-Ham. ex D. Don. (Pp.Cr) in traditional systems of medicine.

Methods

The crude ethanol extract of fruits of Pyrus pashia Buch.-Ham. ex D. Don. (Pp.Cr) was tested in vitro on isolated rabbit jejunum, tracheal, and aorta preparations. The responses of tissues were recorded using isotonic transducers coupled with a PowerLab data acquisition system.

Results

The Pp.Cr on application (0.01–5.0 mg/ml) to isolated rabbit jejunum preparation exhibited relaxation through decrease in magnitude and frequency of spontaneous contractions. The Pp.Cr also exerted a relaxant (0.01–5.0 mg/ml) effect on K+(80 mM) induced contractions in isolated rabbit jejunum preparations and caused shifting of the Ca2+ curves (1.0–3.0 mg/ml) toward right in a manner similar to that of verapamil (3μM), possibly suggesting presence of Ca2+ channel blocking activity. Subsequently, Pp.Cr in a concentration-dependent fashion (0.01–10.0 mg/ml) caused relaxation of CCh (1μM) and K+ (80 mM) induced contractions in isolated rabbit tracheal preparations in a manner comparable to that of dicyclomine, suggesting that the observed relaxant effect is likely to be mediated through antimuscarinic and/or Ca2+ channel blocking activities. Moreover, when evaluated against isolated rabbit aortic preparations, the Pp.Cr in concentrations up to 10 mg/ml exhibited a contractile response that was found to be abolished subsequent to pretreatment of isolated tissue preparation with cyproheptadine (1μM), phentolamine (1μM), and losartan (1μM), suggesting that Pp.Cr may have some α-adrenergic, muscarinic, serotonergic, and angiotensin II activities.

Conclusions

The aqueous ethanolic extract of Pyrus pashia (Pp.Cr) exhibited spasmolytic, bronchodilator, and vaso-constrictive activities possibly through different mechanisms. The spasmolytic and bronchodilator activities are likely to be mediated through blockade of Ca2+ channels, while vasoconstrictive activity may be due to presence of a α-adrenergic, muscarinic, serotonergic, and angiotensin II agonistic component.     

Na+/H+ Exchanger Isoform 1-Induced Osteopontin Expression Facilitates Cardiomyocyte Hypertrophy

Enhanced expression and activity of the Na⁺/H⁺ exchanger isoform 1 (NHE1) has been implicated in cardiomyocyte hypertrophy in various experimental models. The upregulation of NHE1 was correlated with an increase in osteopontin (OPN) expression in models of cardiac hypertrophy (CH), and the mechanism for this remains to be delineated. To determine whether the expression of active NHE1-induces OPN and contributes to the hypertrophic response in vitro, cardiomyocytes were infected with the active form of the NHE1 adenovirus or transfected with OPN silencing RNA (siRNA-OPN) and characterized for cardiomyocyte hypertrophy. Expression of NHE1 in cardiomyocytes resulted in a significant increase in cardiomyocyte hypertrophy markers: cell surface area, protein content, ANP mRNA and expression of phosphorylated-GATA4. NHE1 activity was also significantly increased in cardiomyocytes expressing active NHE1. Interestingly, transfection of cardiomyocytes with siRNA-OPN significantly abolished the NHE1-induced cardiomyocyte hypertrophy. siRNA-OPN also significantly reduced the activity of NHE1 in cardiomyocytes expressing NHE1 (68.5±0.24%; P<0.05), confirming the role of OPN in the NHE1-induced hypertrophic response. The hypertrophic response facilitated by NHE1-induced OPN occurred independent of the extracellular-signal-regulated kinases and Akt, but required p90-ribosomal S6 kinase (RSK). The ability of OPN to facilitate the NHE1-induced hypertrophic response identifies OPN as a potential therapeutic target to reverse the hypertrophic effect induced by the expression of active NHE1.     

Identification of miRNAs Involved in Reprogramming Acinar Cells into Insulin Producing Cells

Reprogramming acinar cells into insulin producing cells using adenoviral (Ad)-mediated delivery of Pdx1, Ngn3 and MafA (PNM) is an innovative approach for the treatment of diabetes. Here, we aimed to investigate the molecular mechanisms involved in this process and in particular, the role of microRNAs. To this end, we performed a comparative study of acinar-to-β cell reprogramming efficiency in the rat acinar cell line AR42J and its subclone B13 after transduction with Ad-PNM. B13 cells were more efficiently reprogrammed than AR42J cells, which was demonstrated by a strong activation of β cell markers (Ins1, Ins2, IAPP, NeuroD1 and Pax4). miRNome panels were used to analyze differentially expressed miRNAs in acinar cells under four experimental conditions (i) non-transduced AR42J cells, (ii) non-transduced B13 cells, (iii) B13 cells transduced with Ad-GFP vectors and (iv) B13 cells transduced with Ad-PNM vectors. A total of 59 miRNAs were found to be differentially expressed between non-transduced AR42J and B13 cells. Specifically, the miR-200 family was completely repressed in B13 cells, suggesting that these cells exist in a less differentiated state than AR42J cells and as a consequence they present a greater plasticity. Adenoviral transduction per se induced dedifferentiation of acinar cells and 11 miRNAs were putatively involved in this process, whereas 8 miRNAs were found to be associated with PNM expression. Of note, Ad-PNM reprogrammed B13 cells presented the same levels of miR-137-3p, miR-135a-5p, miR-204-5p and miR-210-3p of those detected in islets, highlighting their role in the process. In conclusion, this study led to the identification of miRNAs that might be of compelling importance to improve acinar-to-β cell conversion for the future treatment of diabetes.     

Myosin heavy chain composition of muscle spindles in human biceps brachii.

Data on the myosin heavy chain (MyHC) composition of human muscle spindles are scarce in spite of the well-known correlation between MyHC composition and functional properties of skeletal muscle fibers. The MyHC composition of intrafusal fibers from 36 spindles of human biceps brachii muscle was studied in detail by immunocytochemistry with a large battery of antibodies. The MyHC content of isolated muscle spindles was assessed with SDS-PAGE and immunoblots. Four major MyHC isoforms (MyHCI, IIa, embryonic, and intrafusal) were detected with SDS-PAGE. Immunocytochemistry revealed very complex staining patterns for each intrafusal fiber type. The bag(1) fibers contained slow tonic MyHC along their entire fiber length and MyHCI, alpha-cardiac, embryonic, and fetal isoforms along a variable part of their length. The bag(2) fibers contained MyHC slow tonic, I, alpha-cardiac, embryonic, and fetal isoforms with regional variations. Chain fibers contained MyHCIIa, embryonic, and fetal isoforms throughout the fiber, and MyHCIIx at least in the juxtaequatorial region. Virtually each muscle spindle had a different allotment of numbers of bag(1), bag(2) and chain fibers. Taken together, the complexity in intrafusal fiber content and MyHC composition observed indicate that each muscle spindle in the human biceps has a unique identity.     

Inhibition by eicosapentaenoic acid of IL-1β-induced PGHS-2 expression in human microvascular endothelial cells: involvement of lipoxygenase-derived metabolites and p38 MAPK pathway

Prostaglandin H synthase 2 (PGHS-2), a highly inducible isoenzyme, is responsible for overproduction of the prostaglandins (PGs) in inflammatory sites.We established that among fish oil polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA), but not docosahexaenoic acid (DHA), greatly decreased interleukin-1β (IL-1β)-induced PGHS-2 expression in human pulmonary microvascular endothelial cells (HPMECs). Lipoxygenase products 12 (S)-hydroperoxyeicosapentaenoic acid ((S)-HpEPE), 15 (S)-HpEPE and leukotriene (LT) D5 reproduced similar inhibitory effect, suggesting that they may be the intermediate metabolites responsible for PGHS-2 down-regulation by EPA. Accordingly, the EPA effect is prevented by nordihydroguaiaretic acid (NDGA) and by REV 5901, nonspecific and specific 5-lipoxygenase inhibitors, respectively. Besides, inhibition of cyclooxygenase activity by ibuprofen, indomethacin or aspirin was not able to prevent this effect. Moreover, cyclooxygenase metabolites of EPA (PGs D3, E3 and I3) markedly potentiate IL-1β-induced PGHS-2 expression, probably by increasing intracellular cAMP levels. Peroxisome proliferator-activated receptors (PPARs) are known to be activated by fatty acids (FAs) such as EPA. We found here that HPMECs express only weak amounts of PPARα and PPARγ whose activation by synthetic agonists, Wy-14,643 and ciglitazone, does not cause any inhibition of IL-1β-induced PGHS-2 expression. This finding ruled out the involvement of PPARs in the EPA inhibitory effect. In addition, we established that EPA, which failed to inhibit nuclear factor-κB (NF-κB) activation, suppressed p38 mitogen-activated protein kinase (MAPK) phosphorylation in stimulated HPMECs.Our data demonstrate that EPA, unlike DHA, down-regulates PGHS-2 expression in HPMECs probably through its 5-lipoxygenase-dependent metabolites and advocates a beneficial role for this FA in limiting inflammatory response.     

The MUC1 mucin regulates the tumorigenic properties of human esophageal adenocarcinomatous cells

MUC1 is a membrane-bound mucin known to participate in tumor proliferation. It has been shown that MUC1 pattern of expression is modified during esophageal carcinogenesis, with a progressive increase from metaplasia to adenocarcinoma. The principal cause of development of esophageal adenocarcinoma is gastro-esophageal reflux and MUC1 was previously shown to be up-regulated by several bile acids present in reflux. In this report, our aim was thus to determine whether MUC1 plays a role in biological properties of human esophageal cancer cells. For that, a stable MUC1-deficient esophageal cancer cell line was established using a shRNA approach. In vitro (proliferation, migration and invasion) and in vivo (tumor growth following subcutaneous xenografts in SCID mice) biological properties of MUC1-deficient cells were analyzed. Our results show that esophageal cancer cells lacking MUC1 were less proliferative and had decreased migration and invasion properties. These alterations were accompanied by a decreased activity of NFKB p65, Akt and MAPK (p44/42, JNK and p38) pathways. MCM6 and TSG101 tumor-associated markers were also decreased. Subcutaneous xenografts showed a significant decrease in tumor size when cells did not express MUC1. Altogether, the data indicate that MUC1 plays a key role in proliferative, migrating and invasive properties of esophageal cancer cells as well as in tumor growth promotion. MUC1 mucin appears thus as a good therapeutic target to slow down esophageal tumor progression.     

Stabilization of the Dimeric Birch Pollen Allergen Bet v 1 Impacts Its Immunological Properties

Many allergens share several biophysical characteristics, including the capability to undergo oligomerization. The dimerization mechanism in Bet v 1 and its allergenic properties are so far poorly understood. Here, we report crystal structures of dimeric Bet v 1, revealing a noncanonical incorporation of cysteine at position 5 instead of genetically encoded tyrosine. Cysteine polysulfide bridging stabilized different dimeric assemblies, depending on the polysulfide linker length. These dimers represent quaternary arrangements that are frequently observed in related proteins, reflecting their prevalence in unmodified Bet v 1. These conclusions were corroborated by characteristic immunologic properties of monomeric and dimeric allergen variants. Hereby, residue 5 could be identified as an allergenic hot spot in Bet v 1. The presented results refine fundamental principles in protein chemistry and emphasize the importance of protein modifications in understanding the molecular basis of allergenicity.     

Reduced glucosylceramide in the mouse model of Fabry disease: Correction by successful enzyme replacement therapy

Fabry disease is an X-linked lysosomal storage disease (LSD) caused by deficient activity of α-Galactosidase A (α-Gal A). As a result, glycosphingolipids, mainly globotriaosylceramide (Gb3), progressively accumulate in body fluids and tissues. Studies aiming at the identification of secondary lipid alterations in Fabry disease may be potentially useful for the monitorization of the response to enzyme replacement therapy (ERT) and development of future therapies. The focus of this study was to evaluate if α-Gal A deficiency has an effect on two key groups of molecules of sphingolipids metabolism: glucosylceramides (GlucCers) and ceramides (Cers). Studies performed in a mouse model of Fabry disease showed reduced level of GlucCer and normal level of Cer in plasma, liver, spleen, kidney and heart. Moreover, analysis of GlucCer isoforms in Fabry knockout mice showed that GlucCer isoforms are unequally reduced in different tissues of these animals. ERT had a specific effect on the liver's GlucCer levels of Fabry knockout mice, increasing hepatic GlucCer to the levels observed in wild type mice. In contrast to Fabry knockout mice, plasma of Fabry patients had normal GlucCer and Cer but an increased GlucCer/Cer ratio. This alteration showed a positive correlation with plasma globotriaosylsphingosine (lyso-Gb3) concentration. In conclusion, this work reveals novel secondary lipid imbalances caused by α-Gal A deficiency.