Human mesenchymal stem cell proliferation is regulated by PGE2 through differential activation of cAMP-dependent protein kinase isoforms

The conditions used for in vitro differentiation of hMSCs contain substances that affect the activity and expression of cyclooxygenase enzymes (COX1/COX2) and thereby the synthesis of prostanoids. hMSC constitutively produce PGE2 when cultivated in vitro. In this study we have investigated effects of PGE2 on proliferation of hMSC. We here demonstrate that one of the main control molecules in the Wnt pathway, GSK-3β, is phosphorylated at the negative regulatory site ser-9 after treating the cells with PGE2. This phosphorylation is mediated by elevation of cAMP and subsequent activation of PKA. Furthermore, PGE2 treatment leads to enhanced nuclear translocation of β-catenin, thus influencing cell proliferation. The presence of two PKA isoforms, types I and II, prompted us to investigate their individual contribution in PGE2-mediated regulation of proliferation. Specific activation of PKA type II with synthetic cAMP analogues, resulted in enhancement of proliferation. On the other side, we found that treatment of hMSC with high concentrations of PGE2 inhibited cell proliferation by arresting the cells in G₀/G₁ phase, an effect we found to be mediated by PKA I. Hence, the two different PKA isoforms seem to have opposing functions in the regulation of proliferation and differentiation in these cells.     

Endothelial microparticles released by activated protein C protect beta cells through EPCR/PAR1 and annexin A1/FPR2 pathways in islets

Islet transplantation is associated with early ischaemia/reperfusion, localized coagulation and redox‐sensitive endothelial dysfunction. In animal models, islet cytoprotection by activated protein C (aPC) restores islet vascularization and protects graft function, suggesting that aPC triggers various lineages. aPC also prompts the release of endothelial MP that bear EPCR, its specific receptor. Microparticles (MP) are plasma membrane procoagulant vesicles, surrogate markers of stress and cellular effectors. We measured the cytoprotective effects of aPC on endothelial and insulin‐secreting Rin‐m5f β‐cells and its role in autocrine and paracrine MP‐mediated cell crosstalk under conditions of oxidative stress. MP from aPC‐treated primary endothelial (EC) or β‐cells were applied to H₂O₂‐treated Rin‐m5f. aPC activity was measured by enzymatic assay and ROS species by dihydroethidium. The capture of PKH26‐stained MP and the expression of EPCR were probed by fluorescence microscopy and apoptosis by flow cytometry. aPC treatment enhanced both annexin A1 (ANXA1) and PAR‐1 expression in EC and to a lesser extent in β‐cells. MP from aPC‐treated EC (eM_aPC) exhibited high EPCR and annexin A1 content, protected β‐cells, restored insulin secretion and were captured by 80% of β cells in a phosphatidylserine and ANXA1‐dependent mechanism. eMP activated EPCR/PAR‐1 and ANXA1/FPR2‐dependent pathways and up‐regulated the expression of EPCR, and of FPR2/ALX, the ANXA1 receptor. Cytoprotection was confirmed in H₂O₂‐treated rat islets with increased viability (62% versus 48% H₂O₂), reduced apoptosis and preserved insulin secretion in response to glucose elevation (16 versus 5 ng/ml insulin per 10 islets). MP may prove a promising therapeutic tool in the protection of transplanted islets.     

Effects of endocrine disruptors on genes associated with 17beta-estradiol metabolism and excretion

In order to provide a global analysis of the effects of endocrine disruptors on the hormone cellular bioavailability, we combined 17beta-estradiol (E2) cellular flow studies with real-time PCR and Western blot expression measurements of genes involved in the hormone metabolism and excretion. Three endocrine disruptors commonly found in food were chosen for this study, which was conducted in the estrogen receptor (ER) negative hepatoblastoma HepG2 cell line: bisphenol A (BPA), genistein (GEN) and resveratrol (RES). We showed that 24h after a single dose treatment with genistein, resveratrol or bisphenol A, the expression of ATP-binding cassette transporters (the multidrug resistance or MDR, and the multidrug resistance associated proteins or MRP) uridine diphosphate-glucuronosyltransferases (UGT) and/or sulfotransferases (ST) involved in 17beta-estradiol elimination process were significantly modulated and that 17beta-estradiol cellular flow was modified. Resveratrol induced MDR1 and MRP3 expressions, bisphenol A induced MRP2 and MRP3 expressions, and both enhanced 17beta-estradiol efflux. Genistein, on the other hand, inhibited ST1E1 and UGT1A1 expressions, and led to 17beta-estradiol cellular retention. Thus, we demonstrate that bisphenol A, genistein and resveratrol modulate 17beta-estradiol cellular bioavailability in HepG2 and that these modulations most probably involve regulations of 17beta-estradiol phase II and III metabolism proteins. Up to now, the estrogenicity of environmental estrogenic pollutants has been based on the property of these compounds to bind to ERs. Our results obtained with ER negative cells provide strong evidence for the existence of ER-independent pathways leading to endocrine disruption.     

Gene transfer of Ig-fusion proteins into B cells prevents and treats autoimmune diseases

Based on the tolerogenic properties of IgG carriers and B cell Ag presentation, we developed a retrovirally mediated gene expression approach for treatment of autoimmune conditions. In this study, we show that the IgG-Ag retroviral constructs, expressing myelin basic protein (MBP) or glutamic acid decarboxylase in B cells, can be used for the treatment of murine models for multiple sclerosis and diabetes. Transduction of syngeneic B cells with MBP-IgG leads to the amelioration of ongoing experimental allergic encephalomyelitis induced by the transfer of primed cells from PLxSJL F(1) mice with ongoing disease and could be effective even after symptoms appeared. This effect is specific and does not involve bystander suppression because treatment with MBP-IgG does not affect disease induced after immunization with proteolipid protein immunodominant peptide plus MBP. Interestingly, if donor B cells are derived from gld mice (Fas ligand-negative), then tolerance is not induced with a model Ag although there was no evidence for Fas ligand-mediated deletion of target T cells. In spontaneous diabetes in nonobese diabetic mice, we were able to stop the ongoing autoimmune process by treatment at 7-10 wk with glutamic acid decarboxylase-IgG retrovirally transduced B cells, or attenuate it with B cells transduced with an insulin B chain (B9-23) epitope IgG fusion protein. Furthermore, IgG fusion protein gene therapy can also protect primed recipients from Ag-induced anaphylactic shock, and thus does not cause immune deviation. These results demonstrate proof of principle for future efforts to develop this approach in a clinical setting.     

Common loci underlie field resistance to soybean sudden death syndrome in Forrest,Pyramid, Essex,and Douglas

Soybean [Glycine max (L.) Merr.] sudden death syndrome (SDS) caused by Fusarium solani f. sp. glycines results in severe yield losses. Resistant cultivars offer the most-effective protection against yield losses but resistant cultivars such as ’Forrest’ and ’Pyramid’ vary in the nature of their response to SDS. Loci underlying SDS resistance in ’Essex’ × Forrest are well defined. Our objectives were to identify and characterize loci and alleles that underlie field resistance to SDS in Pyramid×’Douglas’. SDS disease incidence and disease severity were determined in replicated field trials in six environments over 4 years. One hundred and twelve polymorphic DNA markers were compared with SDS disease response among 90 recombinant inbred lines from the cross Pyramid×Douglas. Two quantitative trait loci (QTLs) for resistance to SDS derived their beneficial alleles from Pyramid, identified on linkage group G by BARC-Satt163 (261-bp allele, P=0.0005, R²=16.0%) and linkage group N by BARC-Satt080 (230-bp allele, P=0.0009, R²=15.6%). Beneficial alleles of both QTLs were previously identified in Forrest. A QTL for re- sistance to SDS on linkage group C2 identified by BARC-Satt307 (292-bp allele, P=0.0008, R²=13.6%) derived the beneficial allele from Douglas. A beneficial allele of this QTL was previously identified in Essex. Recombinant inbred lines that carry the beneficial alleles for all three QTLs for resistance to SDS were significantly (P≤0.05) more resistant than other recombinant inbred lines . Among these recombinant inbred lines resistance to SDS was environmentally stable. Therefore, gene pyramiding will be an effective method for developing cultivars with stable resistance to SDS. Received: 20 October 1999 / Accepted: 22 May 2001     

Impact of Routine Cerebral CT Angiography on Treatment Decisions in Infective Endocarditis

Background

Infective endocarditis (IE) is commonly complicated by cerebral embolization and hemorrhage secondary to intracranial mycotic aneurysms (ICMAs). These complications are associated with poor outcome and may require diagnostic and therapeutic plans to be modified. However, routine screening by brain CT and CT angiography (CTA) is not standard practice. We aimed to study the impact of routine cerebral CTA on treatment decisions for patients with IE.

Methods

From July 2007 to December 2012, we prospectively recruited 81 consecutive patients with definite left-sided IE according to modified Duke’s criteria. All patients had routine brain CTA conducted within one week of admission. All patients with ICMA underwent four-vessel conventional angiography. Invasive treatment was performed for ruptured aneurysms, aneurysms ≥5 mm, and persistent aneurysms despite appropriate therapy. Surgical clipping was performed for leaking aneurysms if not amenable to intervention. Results: The mean age was 30.43±8.8 years and 60.5% were males. Staph aureus was the most common organism (32.3%). Among the patients, 37% had underlying rheumatic heart disease, 26% had prosthetic valves, 23.5% developed IE on top of a structurally normal heart and 8.6% had underlying congenital heart disease. Brain CT/CTA revealed that 51 patients had evidence of cerebral embolization, of them 17 were clinically silent. Twenty-six patients (32%) had ICMA, of whom 15 were clinically silent. Among the patients with ICMAs, 11 underwent endovascular treatment and 2 underwent neurovascular surgery. The brain CTA findings prompted different treatment choices in 21 patients (25.6%). The choices were aneurysm treatment before cardiac surgery rather than at follow-up, valve replacement by biological valve instead of mechanical valve, and withholding anticoagulation in patients with prosthetic valve endocarditis for fear of aneurysm rupture.

Conclusions

Routine brain CT/CTA resulted in changes in the treatment plan in a significant proportion of patients with IE, even those without clinically evident neurological disease. Routine brain CT/CTA may be indicated in all hospitalized patients with IE.     

The sodium proton exchanger NHE9 regulates phagosome maturation and bactericidal activity in macrophages

Acidification of phagosomes is essential for the bactericidal activity of macrophages. Targeting machinery that regulates pH within the phagosomes is a prominent strategy employed by various pathogens that have emerged as major threats to public health. Nascent phagosomes acquire the machinery for pH regulation through a graded maturation process involving fusion with endolysosomes. Meticulous coordination between proton pumping and leakage mechanisms is crucial for maintaining optimal pH within the phagosome. However, relative to mechanisms involved in acidifying the phagosome lumen, little is known about proton leakage pathways in this organelle. Sodium proton transporter NHE9 is a known proton leakage pathway located on the endosomes. As phagosomes acquire proteins through fusions with endosomes during maturation, NHE9 seemed a promising candidate for regulating proton fluxes on the phagosome. Here, using genetic and biophysical approaches, we show NHE9 is an important proton leakage pathway associated with the maturing phagosome. NHE9 is highly expressed in immune cells, specifically macrophages; however, NHE9 expression is strongly downregulated upon bacterial infection. We show that compensatory ectopic NHE9 expression hinders the directed motion of phagosomes along microtubules and promotes early detachment from the microtubule tracks. As a result, these phagosomes have shorter run lengths and are not successful in reaching the lysosome. In accordance with this observation, we demonstrate that NHE9 expression levels negatively correlate with bacterial survival. Together, our findings show that NHE9 regulates lumenal pH to affect phagosome maturation, and consequently, microbicidal activity in macrophages.     

High‐fat diet‐induced obesity augments the deleterious effects of estrogen deficiency on bone: Evidence from ovariectomized mice

Several epidemiological studies have suggested that obesity complicated with insulin resistance and type 2 diabetes exerts deleterious effects on the skeleton. While obesity coexists with estrogen deficiency in postmenopausal women, their combined effects on the skeleton are poorly studied. Thus, we investigated the impact of high‐fat diet (HFD) on bone and metabolism of ovariectomized (OVX) female mice (C57BL/6J). OVX or sham operated mice were fed either HFD (60%fat) or normal diet (10%fat) for 12 weeks. HFD‐OVX group exhibited pronounced increase in body weight (~86% in HFD and ~122% in HFD‐OVX, p < 0.0005) and impaired glucose tolerance. Bone microCT‐scanning revealed a pronounced decrease in trabecular bone volume/total volume (BV/TV) (−15.6 ± 0.48% in HFD and −37.5 ± 0.235% in HFD‐OVX, p < 0.005) and expansion of bone marrow adipose tissue (BMAT; +60.7 ± 9.9% in HFD vs. +79.5 ± 5.86% in HFD‐OVX, p < 0.005). Mechanistically, HFD‐OVX treatment led to upregulation of genes markers of senescence, bone resorption, adipogenesis, inflammation, downregulation of gene markers of bone formation and bone development. Similarly, HFD‐OVX treatment resulted in significant changes in bone tissue levels of purine/pyrimidine and Glutamate metabolisms, known to play a regulatory role in bone metabolism. Obesity and estrogen deficiency exert combined deleterious effects on bone resulting in accelerated cellular senescence, expansion of BMAT and impaired bone formation leading to decreased bone mass. Our results suggest that obesity may increase bone fragility in postmenopausal women.