Non-stimulatory Conditions Research Articles

Differential dysregulation of granule subsets in WASH-deficient neutrophil leukocytes resulting in inflammation

Dysregulated secretion in neutrophil leukocytes associates with human inflammatory disease. The exocytosis response to triggering stimuli is sequential; gelatinase granules modulate the initiation of the innate immune response, followed by the release of pro-inflammatory azurophilic granules, requiring stronger stimulation. Exocytosis requires actin depolymerization which is actively counteracted under non-stimulatory conditions. Here we show that the actin nucleator, WASH, is necessary to maintain azurophilic granules in their refractory state by granule actin entrapment and interference with the Rab27a-JFC1 exocytic machinery. On the contrary, gelatinase granules of WASH-deficient neutrophil leukocytes are characterized by decreased Rac1, shortened granule-associated actin comets and impaired exocytosis. Rac1 activation restores exocytosis of these granules. In vivo, WASH deficiency induces exacerbated azurophilic granule exocytosis, inflammation, and decreased survival. WASH deficiency thus differentially impacts neutrophil granule subtypes, impairing exocytosis of granules that mediate the initiation of the neutrophil innate response while exacerbating pro-inflammatory granule secretion.

Microfluidics in male reproduction: is ex vivo culture of primate testis tissue a future strategy for ART or toxicology research?

The significant rise in male infertility disorders over the years has led to extensive research efforts to recapitulate the process of male gametogenesis in vitro and to identify essential mechanisms involved in spermatogenesis, notably for clinical applications. A promising technology to bridge this research gap is organ-on-chip (OoC) technology, which has gradually transformed the research landscape in ART and offers new opportunities to develop advanced in vitro culture systems. With exquisite control on a cell or tissue microenvironment, customized organ-specific structures can be fabricated in in vitro OoC platforms, which can also simulate the effect of in vivo vascularization. Dynamic cultures using microfluidic devices enable us to create stimulatory effect and non-stimulatory culture conditions. Noteworthy is that recent studies demonstrated the potential of continuous perfusion in OoC systems using ex vivo mouse testis tissues. Here we review the existing literature and potential applications of such OoC systems for male reproduction in combination with novel bio-engineering and analytical tools. We first introduce OoC technology and highlight the opportunities offered in reproductive biology in general. In the subsequent section, we discuss the complex structural and functional organization of the testis and the role of the vasculature-associated testicular niche and fluid dynamics in modulating testis function. Next, we review significant technological breakthroughs in achieving in vitro spermatogenesis in various species and discuss the evidence from microfluidics-based testes culture studies in mouse. Lastly, we discuss a roadmap for the potential applications of the proposed testis-on-chip culture system in the field of primate male infertility, ART and reproductive toxicology.

Utility Of Salivary CA-125 And LDH As Tumor Markers In Oral Malignancy

Background: The mortality and morbidity associated with oral squamous cell carcinoma(OSCC) can be greatly reduced if tumor markers which can detect OSCC at an early stage are available. This study attempts to use saliva as the diagnostic medium to determine the correlation of salivary CA 125 and LDH in tobacco users with and without potentially malignant disorders. Changes in CA 125 and LDH level can be used as a marker in patients with and without premalignant disease and can be used as a deterrent in continuation of the habit. Aims: To assess the role of CA 125 & LDH as a tumour marker in Oral cancer patients. Materials and methods: Cross sectional observational study .Time period: December 2015 to August 2017. Study area: OPD of tertiary care hospital in pune. Total participants: 150 persons. Under non-stimulatory conditions whole saliva sample was collected from each individual and was centrifuged. The resulting supernatant was used for estimation of CA 125 and LDH levels. Results: In present study, mean Salivary LDH level was found to be lowest among cases of healthy individuals while it was highest among individuals who were diagnosed and underwent treatment for malignancy. However there was insignificant difference seen between levels of diagnosed case of oral malignancy and the ones who underwent treatment for oral malignancy. Also mean Salivary CA-125 was lowest in healthy individuals and highest in individuals diagnosed with oral malignancy and the levels were seen to be reduced significantly in the one who underwent treatment for oral malignancies than those who didn't. Conclusion: Salivary CA 125 and LDH has a role in early detection of oral malignancies and can be used as a diagnostic marker. However LDH levels are persistently raised even after treatment for oral malignancy.

Characterization and potential roles of calretinin in rodent spermatozoa

Calretinin has been detected in various excitable cells but the presence and putative roles of such a calcium-binding protein has never been characterized in sperm.Epididymal spermatozoa were collected from C57Bl6 (wild-type, WT) or calretinin knockout (CR−/−) mice and Wistar rats. A specific staining for calretinin was detected by immunofluorescence in the principal piece of the flagellum, both in WT mouse and rat spermatozoa. Western blots confirmed the expression of calretinin in rat and WT spermatozoa as well as its absence in CR−/− mice.No significant difference was observed in the spontaneous acrosome reaction between WT and CR−/− sperm. The addition of the calcium-ionophore A-23187, Thapsigargin or Progesterone to WT or CR−/− incubated spermatozoa induced increases in the acrosome reaction but the stimulatory effects were identical in both genotypes. Motility measurements assessed by computer-assisted sperm analysis indicated that, under basal non-stimulatory conditions, CR-/- sperm exhibited a lower curvilinear velocity and a smaller lateral head movement amplitude, although no difference was observed for the beat cross frequency. After incubation with 25 mM NH4Cl, the curvilinear velocity, the amplitude of the lateral head movement and the hyperactivation were increased, while the beat cross frequency was decreased, in both genotypes.Evaluation of the in vivo fertility potential indicated that the CR−/− litter sizes were clearly reduced compared to the WT litter sizes.Our study describes, for the first time, the expression of calretinin in sperm. These data extend the potential implication of calcium-binding proteins in the sperm calcium-signaling cascade and bring new insights into the understanding of sperm physiology.

Testosterone and FSH modulate Sertoli cell extracellular secretion: Proteomic analysis

Spermatogenesis is a highly complicated biological process that occurs in the epithelium of the seminiferous tubules. It is regulated by a complex network of endocrine and paracrine factors and by juxtacrine testicular cross-talk. Sertoli cells (SC) play a key role in spermatogenesis due to their production of trophic, differentiation and immune-modulating factors, but many of the molecular pathways of SC action remain controversial and unclear. Over the last two decades, research has focused on extracellular vesicles as an important mechanism of intercellular communication. The aim of this study was to investigate the presence of extracellular vesicles (EVs) in SC and the modulation of their content in SC after FSH and testosterone stimulation. Highly purified porcine pre-pubertal Sertoli cells were isolated according to previously established methods. After 48h of culture with FSH or FSH+testosterone stimulation, we identified sertolian EVs containing specific mRNAs. Proteomic analysis of EVs content identified 29 proteins under non-stimulatory conditions, most of which were related to receptor binding activity. FSH stimulation induced increases in inhibin-alpha, inhibin-beta, plakoglobin, haptoglobin, D-3-phosphoglycerate dehydrogenase and sodium/potassium-transporting ATPase in sertolian EVs. Testosterone stimulation enhanced the abundance of inhibin-alpha, inhibin-beta, tissue-type plasminogen activator, epidermal growth factor-like protein 8, elongating factor 1-gamma and D-3-phosphoglycerate dehydrogenase. These results are likely to help determine the unknown molecular secretion of Sertoli cells.

Heterogeneous Niche Activity of Ex-Vivo Expanded MSCs as Factor for Variable Outcomes in Hematopoietic Recovery.

Ex-vivo expanded mesenchymal stromal cells (MSCs) are increasingly used for paracrine support of hematopoietic stem cell (HSC) regeneration, but inconsistent outcomes have hindered ongoing clinical trials. Here, we show that significant heterogeneity in the niche activity of MSCs is created during their culture in various serum-supplemented media. The MSCs cultured under stimulatory or non-stimulatory culture conditions exhibited differences in colony forming unit-fibroblast contents, expression levels of cross-talk molecules (Jagged-1 and CXCL-12) and their support for HSC self-renewal. Accordingly, the enhancing effects of MSCs on hematopoietic engraftment were only visible when HSCs were co-transplanted with MSCs under stimulatory conditions. Of note, these differences in MSCs and their effects on HSCs were readily reversed by switching the cultures, indicating that the difference in niche activity can be caused by distinct functional state, rather than by clonal heterogeneity. Supporting the findings, transcriptomic analysis showed distinct upstream signaling pathways such as inhibition of P53 and activation of ER-stress response gene ATF4 for MSCs under stimulatory conditions. Taken together, our study shows that the niche activity of MSCs can vary rapidly by the extrinsic cues during culture causing variable outcomes in hematopoietic recoveries, and point to the possibility that MSCs can be pre-screened for more predictable efficacy in various cell therapy trials.

Stimulatory Effects of Serum for Ex-Vivo Culture of Mesenchymal Stromal Cells Can be a Parameter for Heterogeneity in Hematopoietic Supporting Activity

Although hematopoietic supporting activity of mesenchymal stromal cells (MSCs) have been widely used in clinical trials to facilitate the hematopoietic recovery, variations have been observedin supporting effects. In the present study, we show that the niche activity of MSCs is regulated by stimulatory effects of fetal bovine serum during ex-vivo culture of MSCs.First, we observed extensive variations in the numbers of colonies (CFU-F)(up to 40-folds) differences in numbers when human bone marrow (BM)-derived MSCs were cultured under different batches of fetal bovine serum (FBS),and two batches with high (SS) and low (NSS) stimulating effects were selected. 5 independent BM-MSCs cultured with stimulating serum exhibitedsignificantly lower doubling times, higher numbers of CFU-F and higher % of CD146+ cells. When umbilical cord blood-derived CD34+ cells were co-cultured on each MSC group, significantly higher expansion of CD34+CD90+ cells and higher numbers of colonies were observed than those cultured with non-stimulatory serum. Similarly, numbers of primitive hematopoietic cells analyzed by long-term cultureinitiating cells (LTC-IC) were significantly higher in stimulating serum conditions than in non-stimulating condition. Furthermore, MSCs under stimulatory serum exhibited higher level expression of niche cross-talk molecules (CXCL-12 and Jagged-1) than MSCs under non-stimulatory condition, indicating higher support of HSC self-renewal. Whenexamined for murine model, the stimulatory serum caused similar difference in colonogenic activity (2.5-folds higher CFU-F) and phenotypic marker for mesenchymal progenitors (PDGFR-alpha+ cells). Moreover, when each group MSCs were co-transplanted with HSCs into congenic recipient, significantly higher reconstitution of donor-derived cells were observed without changes in the lympho-myeloid lineage distribution in the MSCs with stimulatory serum group than in MSCs with non-stimulatory group (51.2% vs 8.2%, respectively, p<0.005). In contrast, priming of HSCs with 2hr pre-incubation with each group MSCs before transplantation or co-transplantation of MSCs cultured under hypoxic conditions did not exhibit additional increase in the reconstitution level by donor-derived cells.Of note, the enhancing effects of stimulatory serum on MSCs or on the co-cultured HSCs were reversed when the MSC culture conditions were switched to non-stimulatory serum, indicating that the enhancing effects were caused by extrinsic stimulating effects rather than clonal difference of MSC subpopulation. To explore the differential signals between MSCs grown under stimulatory and non-stimulatory condition, microarray-based analysis of gene expression were analyzed. The gene ontology (GO) analysis showed that extensive signals related to cell growth and survival was enriched in the MSCs grown in stimulatory conditions compared to those in non-stimulatory conditions. Furthermore, upstream signal analysis showed that signals for P53, TRIB3, TGF-beta1, RABL6 or ATF4 were significantly up-regulated in the MSCs under stimulatory serum condition. Taken together, our study shows that MSCs can exhibit significant heterogeneity in the hematopoietic niche activity depending on the stimulatory effects of the serum during ex-vivo culture to cause difference in hematological recovery, and suggest that optimization of CFU-F-based screening of MSCs can produce more predictable outcomes in clinical trials. DisclosuresNo relevant conflicts of interest to declare.

Human amniotic mesenchymal stromal cells (hAMSCs) as potential vehicles for drug delivery in cancer therapy: an in vitro study.

IntroductionIn the context of drug delivery, mesenchymal stromal cells (MSCs) from bone marrow and adipose tissue have emerged as interesting candidates due to their homing abilities and capacity to carry toxic loads, while at the same time being highly resistant to the toxic effects. Amongst the many sources of MSCs which have been identified, the human term placenta has attracted particular interest due to its unique, tissue-related characteristics, including its high cell yield and virtually absent expression of human leukocyte antigens and co-stimulatory molecules. Under basal, non-stimulatory conditions, placental MSCs also possess basic characteristics common to MSCs from other sources. These include the ability to secrete factors which promote cell growth and tissue repair, as well as immunomodulatory properties. The aim of this study was to investigate MSCs isolated from the amniotic membrane of human term placenta (hAMSCs) as candidates for drug delivery in vitro.MethodsWe primed hAMSCs from seven different donors with paclitaxel (PTX) and investigated their ability to resist the cytotoxic effects of PTX, to upload the drug, and to release it over time. We then analyzed whether the uptake and release of PTX was sufficient to inhibit proliferation of CFPAC-1, a pancreatic tumor cell line sensitive to PTX.ResultsFor the first time, our study shows that hAMSCs are highly resistant to PTX and are not only able to uptake the drug, but also release it over time. Moreover, we show that PTX is released from hAMSCs in a sufficient amount to inhibit tumor cell proliferation, whilst some of the PTX is also retained within the cells.ConclusionTaken together, for the first time our results show that placental stem cells can be used as vehicles for the delivery of cytotoxic agents.

Connective tissue growth factor modulates adult β-cell maturity and proliferation to promote β-cell regeneration in mice.

Stimulation of endogenous β-cell expansion could facilitate regeneration in patients with diabetes. In mice, connective tissue growth factor (CTGF) is expressed in embryonic β-cells and in adult β-cells during periods of expansion. We discovered that in embryos CTGF is necessary for β-cell proliferation, and increased CTGF in β-cells promotes proliferation of immature (MafA−) insulin-positive cells. CTGF overexpression, under nonstimulatory conditions, does not increase adult β-cell proliferation. In this study, we tested the ability of CTGF to promote β-cell proliferation and regeneration after partial β-cell destruction. β-Cell mass reaches 50% recovery after 4 weeks of CTGF treatment, primarily via increased β-cell proliferation, which is enhanced as early as 2 days of treatment. CTGF treatment increases the number of immature β-cells but promotes proliferation of both mature and immature β-cells. A shortened β-cell replication refractory period is also observed. CTGF treatment upregulates positive cell-cycle regulators and factors involved in β-cell proliferation, including hepatocyte growth factor, serotonin synthesis, and integrin β1. Ex vivo treatment of whole islets with recombinant human CTGF induces β-cell replication and gene expression changes consistent with those observed in vivo, demonstrating that CTGF acts directly on islets to promote β-cell replication. Thus, CTGF can induce replication of adult mouse β-cells given a permissive microenvironment.

Fibulin-1 Predicts Disease Progression in Patients With Idiopathic Pulmonary Fibrosis

The underlying mechanisms of idiopathic pulmonary fibrosis (IPF) are unknown. This progressive disease has high mortality rates, and current models for prediction of mortality have limited value in identifying which patients will progress. We previously showed that the glycoprotein fibulin-1 is involved in enhanced proliferation and wound repair by mesenchymal cells and, thus, may contribute to lung fibrosis in IPF. Serum, lung tissue, and lung function values were obtained from four independent locations (Sydney, NSW, and Perth, WA, Australia; San Francisco, CA; and Modena, Italy). Patients with IPF were followed for a minimum of 1 year and progression was defined as a significant decline in lung function or death. Primary parenchymal lung fibroblasts of 15 patients with and without IPF were cultured under nonstimulatory conditions. Fibulin-1 levels in serum, and secreted or deposited by fibroblasts, were measured by western blot and in lung tissue by immunohistochemistry. Serum fibulin-1 levels were increased in patients with IPF compared with subjects without lung disease (P = .006). Furthermore, tissue fibulin-1 levels were increased in patients with IPF (P = .02) and correlated negatively with lung function (r = -0.9, P < .05). Primary parenchymal fibroblasts from patients with IPF produced more fibulin-1 than those from subjects without IPF (P < .05). Finally, serum fibulin-1 levels at first blood draw predicted disease progression in IPF within 1 year (area under the curve , 0.71; 95% CI, 0.57-0.86; P = .012). Fibulin-1 is a novel potential biomarker for disease progression in IPF and raises the possibility that it could be used as a target for the development of new treatments.

Aging Enhances the Basal Production of IL-6 and CCL2 in Vascular Smooth Muscle Cells

Increased circulating cytokine levels are a prominent feature of aging that may contribute to atherosclerosis. However, the role vascular cells play in chronic inflammation induced by aging is not clear. Here, we examined the role of aging on inflammatory responses of vascular cells. In an ex vivo culture system, we examined the inflammatory response of aortas from young (2-4 months) and aged (16-18 months) mice under nonstimulatory conditions. We found that basal levels of interleukin-6 were increased in aged aortas. Aged aortic vascular smooth muscle cells (VSMC) exhibited a higher basal secretion of interleukin-6 than young VSMC. Gene and protein expression analysis revealed that aged VSMC exhibited upregulation of chemokines (eg, CCL2), adhesion molecules (eg, intracellular adhesion molecule 1), and innate immune receptors (eg, Toll-like receptor [TLR] 4), which all contribute to atherosclerosis. Using VSMC from aged TL4(-/-) and Myd88(-/-) mice, we demonstrate that signaling via TLR4 and its signal adaptor, MyD88, are in part responsible for the age-elevated basal interleukin-6 response. Aging induces a proinflammatory phenotype in VSMC due in part to increased signaling of TLR4 and MyD88. Our results provide a potential explanation as to why aging leads to chronic inflammation and enhanced atherosclerosis.

MiR-155, a Modulator of FOXO3a Protein Expression, Is Underexpressed and Cannot Be Upregulated by Stimulation of HOZOT, a Line of Multifunctional Treg

MicroRNAs (miRNAs) play important roles in regulating post-transcriptional gene repression in a variety of immunological processes. In particular, much attention has been focused on their roles in regulatory T (Treg) cells which are crucial for maintaining peripheral tolerance and controlling T cell responses. Recently, we established a novel type of human Treg cell line, termed HOZOT, multifunctional cells exhibiting a CD4+CD8+ phenotype. In this study, we performed miRNA profiling to identify signature miRNAs of HOZOT, and therein identified miR-155. Although miR-155 has also been characterized as a signature miRNA for FOXP3+ natural Treg (nTreg) cells, it was expressed quite differently in HOZOT cells. Under both stimulatory and non-stimulatory conditions, miR-155 expression remained at low levels in HOZOT, while its expression in nTreg and conventional T cells remarkably increased after stimulation. We next searched candidate target genes of miR-155 through bioinformatics, and identified FOXO3a, a negative regulator of Akt signaling, as a miR-155 target gene. Further studies by gain- and loss-of-function experiments supported a role for miR-155 in the regulation of FOXO3a protein expression in conventional T and HOZOT cells.

Activation and repression of glucose-stimulated ChREBP requires the concerted action of multiple domains within the MondoA conserved region

Carbohydrate response element-binding protein (ChREBP) is a glucose-dependent transcription factor that stimulates the expression of glycolytic and lipogenic genes in mammals. Glucose regulation of ChREBP has been mapped to its conserved NH(2)-terminal region of 300 amino acids, designated the MondoA conserved region (MCR). Within the MCR, five domains (MCR1-5) have a particularly high level of conservation and are likely to be important for glucose regulation. We carried out a large-scale deletion and substitution mutational analysis of the MCR domain of ChREBP. This analysis revealed that MCRs 1-4 function in a concerted fashion to repress ChREBP activity in basal (nonstimulatory) conditions. Deletion of the entire MCR1-4 segment or the combination of four specific point mutations located across this region leads to a highly active, glucose-independent form of ChREBP. However, deletion of any individual MCR domain and the majority of point mutations throughout MCR1-4 rendered ChREBP inactive. These observations suggest that the MCR1-4 region interacts with an additional coregulatory factor required for activation. This possibility is supported by the observation that the MCR1-4 region can compete for activity with wild-type ChREBP in stimulatory conditions. In contrast, mutations in the MCR5 domain result in increased activity, suggesting that this domain may be the target of intramolecular repression in basal conditions. Thus, the MCR domains act in a complex and coordinated manner to regulate ChREBP activity in response to glucose.

A Specialized Polarized Membrane Domain Found On Normal but Not Leukemic Hematopoietic Progenitor Cells Is Required for Homing to the Bone Marrow Microenvironment.

Abstract 82Hematopoietic stem/progenitor cells (HSPCs) reside in a bone marrow niche, where adhesive interactions with osteoblasts provide essential cues for their proliferation and survival. In co-cultures of osteoblasts with primary human normal CD34+ cells, CD34+CD38- cells, or the KG1a progenitor cell line, we previously showed, using live cell imaging approaches, that HSPCs made prolonged contact with the osteoblast surface via a specialized membrane domain enriched in prominin 1 (CD133), the very late antigen-4 (VLA-4), the phosphatidylethanolamine (PE) analogue rhodamine-PE, and the tetraspanins CD63 and CD81. At the contact site, portions of the specialized domain of the CD34+ cells containing these molecules were taken up by osteoblasts and internalized into signaling endosomes within the osteoblasts. This caused the osteoblasts to downregulate Smad signaling and to increase their production of stromal-derived factor-1 (SDF-1), a chemokine responsible for HSPC homing to bone marrow (Gillette J. et al, Nat. Cell Biol. 11(3): 303–311, 2009). We have now evaluated the functional significance of these specialized membrane domains for in vivo homing of normal HSPCs to the bone marrow microenvironment. G-CSF mobilized human peripheral blood CD34+ cells from two normal donors were treated for 30 minutes with the cholesterol sequestration agent methyl-β-cyclodextrin (MβCD). This treatment resulted in disruption of the CD34+ cell membrane domains but had no effect on cell viability, proliferation or colony forming capacity in vitro. However, in two independent experiments, we observed a three-fold decrease in homing of MβCD-treated CD34+ cells to the bone marrow of NOD/SCID IL2rψcnull mice 16 hours after transplantation as compared to mock-treated CD34+ cells (p=0.0002). In contrast to homing studies, long-term human cell engraftment determined by CD45 cell surface expression 2 months after transplantation in two independent experiments was not significantly different in mice transplanted with MβCD-treated CD34+ cells compared to mock-treated CD34+ cells (p=0.13). Rapid repolarization of the membrane domain after transplantation may have resulted in engraftment of MβCD-treated CD34+ cells at levels similar to those observed with mock-treated CD34+ cells. Given the known homing/engraftment defect of actively cycling HSPC, we compared membrane domains on quiescent and cycling CD34+ cells from two normal donors. At baseline, 55–68% of CD34+ cells were in the G0 phase of the cell cycle as measured by Hoechst/Pyronin Y staining and specialized membrane domains were detected on a similar percentage of CD34+ cells for both donors. After culture for 4 days in the presence of stimulatory cytokines (SCF, TPO and Flt3), less than 10% of the CD34+ cells remained in G0 and, similarly, less than 10% of the cells analyzed by microscopy had a specialized membrane domain. After 4 days, cells were continued in culture for 2 days under non-stimulatory conditions (SCF alone). Under these conditions, for one donor, the percentage of cells in G0 increased from 9% to 18% and the percentage of cells with membrane domains increased similarly from 6% to 11%. For the other donor, the percentage of cells in G0 and with membrane domains both remained unchanged after 2 days in non-stimulatory culture. Surprisingly, the polarized membrane domains detected on normal CD34+ cells were not found on peripheral blood blast cells from patients diagnosed with relapsed AML (n=2 patients) or CML (n=1 patient). Accordingly, in a preliminary experiment, no difference in homing was observed 16 hours after transplantation of MβCD- or mock-treated peripheral blood cells from an AML patient in NOD/SCID IL2rψcnull mice. Additional homing and engraftment studies using cells from leukemic patients are ongoing. In combination, these findings indicate that specialized membrane domains are found on normal but not leukemic HSPCs and that these domains are required for homing to the bone marrow microenvironment. Disruption of these domains in actively cycling progenitor cells may provide an explanation for the previously demonstrated homing/engraftment defect of cycling cells compared to quiescent cells in the G0 phase of the cell cycle. Disclosures:No relevant conflicts of interest to declare.

Proteins altered by elevated levels of palmitate or glucose implicated in impaired glucose-stimulated insulin secretion

BackgroundDevelopment of type 2 diabetes mellitus (T2DM) is characterized by aberrant insulin secretory patterns, where elevated insulin levels at non-stimulatory basal conditions and reduced hormonal levels at stimulatory conditions are major components. To delineate mechanisms responsible for these alterations we cultured INS-1E cells for 48 hours at 20 mM glucose in absence or presence of 0.5 mM palmitate, when stimulatory secretion of insulin was reduced or basal secretion was elevated, respectively.ResultsAfter culture, cells were protein profiled by SELDI-TOF-MS and 2D-PAGE. Differentially expressed proteins were discovered and identified by peptide mass fingerprinting. Complimentary protein profiles were obtained by the two approaches with SELDI-TOF-MS being more efficient in separating proteins in the low molecular range and 2D-PAGE in the high molecular range. Identified proteins included alpha glucosidase, calmodulin, gars, glucose-6-phosphate dehydrogenase, heterogenous nuclear ribonucleoprotein A3, lon peptidase, nicotineamide adenine dinucleotide hydrogen (NADH) dehydrogenase, phosphoglycerate kinase, proteasome p45, rab2, pyruvate kinase and t-complex protein. The observed glucose-induced differential protein expression pattern indicates enhanced glucose metabolism, defense against reactive oxygen species, enhanced protein translation, folding and degradation and decreased insulin granular formation and trafficking. Palmitate-induced changes could be related to altered exocytosis.ConclusionThe identified altered proteins indicate mechanism important for altered β-cell function in T2DM.

A Novel ABCC8 (SUR1)-dependent Mechanism of Metabolism-Excitation Uncoupling

ATP/ADP-sensing (sulfonylurea receptor (SUR)/K(IR)6)(4) K(ATP) channels regulate the excitability of our insulin secreting and other vital cells via the differential MgATP/ADP-dependent stimulatory actions of their tissue-specific ATP-binding cassette regulatory subunits (sulfonylurea receptors), which counterbalance the nearly constant inhibitory action of ATP on the K(+) inwardly rectifying pore. Mutations in SUR1 that abolish its stimulation have been found in infants persistently releasing insulin. Activating mutations in SUR1 have been shown to cause neonatal diabetes. Here, analyses of K(IR)6.2-based channels with diabetogenic receptors reveal that MgATP-dependent hyper-stimulation of mutant SUR can compromise the ability of K(ATP) channels to function as metabolic sensors. I demonstrate that the channel hyperactivity rises exponentially with the number of hyperstimulating subunits, so small subpopulations of channels with more than two mutant SUR can dominate hyperpolarizing currents in heterozygous patients. I uncovered an attenuated tolbutamide inhibition of the hyperstimulated mutant, which is normally sensitive to the drug under non-stimulatory conditions. These findings show the key role of SUR in sensing the metabolic index in humans and urge others to (re)test mutant SUR/K(IR)6 channels from probands in physiologic MgATP.

Loss of Connexin36 Channels Alters β-Cell Coupling, Islet Synchronization of Glucose-Induced Ca2+ and Insulin Oscillations, and Basal Insulin Release

Normal insulin secretion requires the coordinated functioning of beta-cells within pancreatic islets. This coordination depends on a communications network that involves the interaction of beta-cells with extracellular signals and neighboring cells. In particular, adjacent beta-cells are coupled via channels made of connexin36 (Cx36). To assess the function of this protein, we investigated islets of transgenic mice in which the Cx36 gene was disrupted by homologous recombination. We observed that compared with wild-type and heterozygous littermates that expressed Cx36 and behaved as nontransgenic controls, mice homozygous for the Cx36 deletion (Cx36(-/-)) featured beta-cells devoid of gap junctions and failing to exchange microinjected Lucifer yellow. During glucose stimulation, islets of Cx36(-/-) mice did not display the regular oscillations of intracellular calcium concentrations ([Ca(2+)](i)) seen in controls due to the loss of cell-to-cell synchronization of [Ca(2+)](i) changes. The same islets did not release insulin in a pulsatile fashion, even though the overall output of the hormone in response to glucose stimulation was normal. However, under nonstimulatory conditions, islets lacking Cx36 showed increased basal release of insulin. These data show that Cx36-dependent signaling is essential for the proper functioning of beta-cells, particularly for the pulsatility of [Ca(2+)](i) and insulin secretion during glucose stimulation.

Avoidance of stimulation improves engraftment of cultured and retrovirally transduced hematopoietic cells in primates.

Recent reports suggest that cells in active cell cycle have an engraftment defect compared with quiescent cells. We used nonhuman primates to investigate this finding, which has direct implications for clinical transplantation and gene therapy applications. Transfer of rhesus CD34(+) cells to culture in stem cell factor (SCF) on the CH-296 fibronectin fragment (FN) after 4 days of culture in stimulatory cytokines maintained cell viability but decreased cycling. Using retroviral marking with two different gene transfer vectors, we compared the engraftment potential of cytokine-stimulated cells versus those transferred to nonstimulatory conditions (SCF on FN alone) before reinfusion. In vivo competitive repopulation studies showed that the level of marking originating from the cells continued in culture for 2 days with SCF on FN following a 4-day stimulatory transduction was significantly higher than the level of marking coming from cells transduced for 4 days and reinfused without the 2-day culture under nonstimulatory conditions. We observed stable in vivo overall gene marking levels of up to 29%. This approach may allow more efficient engraftment of transduced or ex vivo expanded cells by avoiding active cell cycling at the time of reinfusion.

Mechanism of Phosphorylation of Protein Kinase B/Akt by a Constitutively Active 3-Phosphoinositide-dependent Protein Kinase-1

Phosphorylation of Thr(308) in the activation loop and Ser(473) at the carboxyl terminus is essential for protein kinase B (PKB/Akt) activation. However, the biochemical mechanism of the phosphorylation remains to be characterized. Here we show that expression of a constitutively active mutant of mouse 3-phosphoinositide-dependent protein kinase-1 (PDK1(A280V)) in Chinese hamster ovary cells overexpressing the insulin receptor was sufficient to induce PKB phosphorylation at Thr(308) to approximately the same extent as insulin stimulation. Phosphorylation of PKB by PDK1(A280V) was not affected by treatment of cells with inhibitors of phosphatidylinositol 3-kinase or by deletion of the pleckstrin homology (PH) domain of PKB. C(2)-ceramide, a cell-permeable, indirect inhibitor of PKB phosphorylation, did not inhibit PDK1(A280V)-catalyzed PKB phosphorylation in cells and had no effect on PDK1 activity in vitro. On the other hand, co-expression of full-length protein kinase C-related kinase-1 (PRK1/PKN) or 2 (PRK2) inhibited PDK1(A280V)-mediated PKB phosphorylation. Replacing alanine at position 280 with valine or deletion of the PH domain enhanced PDK1 autophosphorylation in vitro. However, deletion of the PH domain of PDK1(A280V) significantly reduced PDK1(A280V)-mediated phosphorylation of PKB in cells. In resting cells, PDK1(A280V) localized in the cytosol and at the plasma membrane. However, PDK1(A280V) lacking the PH domain localized predominantly in the cytosol. Taken together, our findings suggest that the wild-type PDK1 may not be constitutively active in cells. In addition, activation of PDK1 is sufficient to phosphorylate PKB at Thr(308) in the cytosol. Furthermore, the PH domain of PDK1 may play both positive and negative roles in regulating the in vivo function of the enzyme. Finally, unlike the carboxyl-terminal fragment of PRK2, which has been shown to bind PDK1 and allow the enzyme to phosphorylate PKB at both Thr(308) and Ser(473), full-length PRK2 and its related kinase PRK1/PKN may both play negative roles in PKB-mediated downstream biological events.

Prostacyclin and prostaglandin E2inhibit proinflammatory cytokine-induced macrophage colony-stimulating factor production in cultured human glomerular mesangial cells

Summary: Monocyte/macrophages within the mesangium plays some important roles in the progression of renal glomerular injury in which prostanoids exert a broad range of actions. We have examined the production of macrophage colony-stimulating factor (M-CSF), a monocyte-specific cytokine, by human glomerular mesangial cells (MC) and its regulation by prostacyclin and prostaglandin E2 (PGE2). the MCSF production by MC under non-stimulatory conditions was below detectable levels by ELISA, and was also at a trace level in the steady-state M-CSF mRNA expression. Proinflammatory cytokines, interleukin-1β (IL-1β) or tumour necrosis factor-α (TNF-α) induced the M-CSF production in the protein and mRNA levels. Both beraprost, a stable analogue of prostacyclin, and PGE2 attenuated the IL-1β- or TNF-α-driven M-CSF production. Indomethacin, a non-selective cyclooxygenase inhibitor, enhanced the IL-1β- or TNF at-induced M-CSF production. Beraprost and PGE2 showed similar inhibitory effects in the presence of indomethacin. Forskolin, a direct activator of adenylate cyclase, and dibutyryl cAMP decreased the M-CSF production. These results indicate that: (i) human MC have capacity to produce M-CSF; (ii) exogenous prostacyclin and PGE2 downregulate the IL-1β- or TNF-α-driven M-CSF production possibly by an increase of intracellular cAMP; and (iii) endogenous prostanoids can exert on the M-CSF production.