Spectral flow cytometry identifies distinct nonneoplastic plasma extracellular vesicle phenotype in glioblastoma patients.

Background: Glioblastoma (GBM) is the most common malignant brain tumor and has a poor prognosis. Imaging findings at diagnosis and in response to treatment are nonspecific. Developing noninvasive assays to augment imaging would be helpful. Plasma extracellular vesicles (EVs) are a promising biomarker source for this. Here, we develop spectral flow cytometry techniques that demonstrate differences in bulk plasma EV phenotype between GBM patients and normal donors that could serve as the basis of a liquid biopsy. Methods: Plasma EVs were stained for EV-associated tetraspanins (CD9/CD63/CD81), markers indicating cell of origin (CD11b/CD31/CD41a/CD45), and actin/phalloidin (to exclude cell debris). EVs were analyzed using spectral flow cytometry. Multiparametric analysis using t-distributed stochastic neighbor embedding (t-SNE) and self-organizing maps on flow cytometry data (FlowSOM) was performed comparing GBM and normal donor (ND) plasma EVs. Results: Size exclusion chromatography plus spectral-based flow cytometer threshold settings enriched plasma EVs while minimizing background noise. GBM patients had increased CD9+, CD63+, CD81+, and myeloid-derived (CD11b+) EVs. Multiparametric analysis demonstrated distinct surface marker expression profiles in GBM plasma EVs compared to ND EVs. Fifteen plasma EV sub-populations differing in size and surface marker expression were identified, six enriched in GBM patients and two in normal donors. Conclusions: Multiparametric analysis demonstrates that GBM patients have a distinct nonneoplastic plasma EV phenotype compared to ND. This simple rapid analysis can be performed without purifying tumor EVs and may serve as the basis of a liquid biopsy.

Use of heparin to rescue immunosuppressive monocyte reprogramming by glioblastoma-derived extracellular vesicles.

OBJECTIVE: The profound immunosuppression found in glioblastoma (GBM) patients is a critical barrier to effective immunotherapy. Multiple mechanisms of tumor-mediated immune suppression exist, and the induction of immunosuppressive monocytes such as myeloid-derived suppressor cells (MDSCs) is increasingly appreciated as a key part of this pathology. GBM-derived extracellular vesicles (EVs) can induce the formation of MDSCs. The authors sought to identify the molecular consequences of these interactions in myeloid cells in order to identify potential targets that could pharmacologically disrupt GBM EV-monocyte interaction as a means to ameliorate tumor-mediated immune suppression. Heparin-sulfate proteoglycans (HSPGs) are a general mechanism by which EVs come into association with their target cells, and soluble heparin has been shown to interfere with EV-HSPG interactions. The authors sought to assess the efficacy of heparin treatment for mitigating the effects of GBM EVs on the formation of MDSCs. METHODS: GBM EVs were collected from patient-derived cell line cultures via staged ultracentrifugation and cocultured with monocytes collected from apheresis cones from healthy blood donors. RNA was isolated from EV-conditioned and unconditioned monocytes after 72 hours of coculture, and RNA-sequencing analysis performed. For the heparin treatment studies, soluble heparin was added at the time of EV-monocyte coculture and flow cytometry analysis was performed 72 hours later. After the initial EV-monocyte coculture period, donor-matched T-cell coculture studies were performed by adding fluorescently labeled and stimulated T cells for 5 days of coculture. RESULTS: Transcriptomic analysis of GBM EV-treated monocytes demonstrated downregulation of several important immunological and metabolic pathways, with upregulation of the pathways associated with synthesis of cholesterol and HSPG. Heparin treatment inhibited association between GBM EVs and monocytes in a dose-dependent fashion, which resulted in a concomitant reduction in MDSC formation (p < 0.01). The authors further demonstrated that reduced MDSC formation resulted in a partial rescue of immune suppression, as measured by effects on activated donor-matched T cells (p < 0.05). CONCLUSIONS: The authors demonstrated that GBM EVs induce broad but reproducible reprogramming in monocytes, with enrichment of pathways that may portend an immunosuppressive phenotype. The authors further demonstrated that GBM EV-monocyte interactions are potentially druggable targets for overcoming tumor-mediated immune suppression, with heparin inhibition of EV-monocyte interactions demonstrating proof of principle.

Glioblastoma may evade immune surveillance through primary cilia-dependent signaling in an IL-6 dependent manner.

Glioblastoma is the most common, malignant primary brain tumor in adults and remains universally fatal. While immunotherapy has vastly improved the treatment of several solid cancers, efficacy in glioblastoma is limited. These challenges are due in part to the propensity of glioblastoma to recruit tumor-suppressive immune cells, which act in conjunction with tumor cells to create a pro-tumor immune microenvironment through secretion of several soluble factors. Glioblastoma-derived EVs induce myeloid-derived suppressor cells (MDSCs) and non-classical monocytes (NCMs) from myeloid precursors leading to systemic and local immunosuppression. This process is mediated by IL-6 which contributes to the recruitment of tumor-associated macrophages of the M2 immunosuppressive subtype, which in turn, upregulates anti-inflammatory cytokines including IL-10 and TGF-ß. Primary cilia are highly conserved organelles involved in signal transduction and play critical roles in glioblastoma proliferation, invasion, angiogenesis, and chemoradiation resistance. In this perspectives article, we provide preliminary evidence that primary cilia regulate intracellular release of IL-6. This ties primary cilia mechanistically to tumor-mediated immunosuppression in glioblastomas and potentially, in additional neoplasms which have a shared mechanism for cancer-mediated immunosuppression. We propose potentially testable hypotheses of the cellular mechanisms behind this finding.

Superinduction of immunosuppressive glioblastoma extracellular vesicles by IFN-γ through PD-L1 and IDO1.

Background: Glioblastoma (GBM), the most common primary brain tumor, has a median survival of 15-16 months. Immunotherapy is promising but GBM-mediated immunosuppression remains a barrier. GBMs express the interferon-gamma (IFN-γ)-responsive immunosuppressive molecules programmed cell death ligand 1 (PD-L1) and indoleamine 2,3-dioxygenase 1 (IDO1). Extracellular vesicles (EVs) have also been implicated in GBM-mediated immunosuppression, in part through PD-L1. We therefore sought to determine if GBM IFN-γ exposure increased GBM EV-mediated immunosuppression and mechanisms underlying this. Methods: Human GBM-derived cells were cultured in the presence/absence of IFN-γ. EVs were harvested. PD-L1, IDO1, and EV-associated protein expression was assessed. GBM EVs (+/-IFN-γ) were cultured with healthy donor monocytes. Immunosuppressive myeloid-derived suppressor cell (MDSC) and nonclassical monocyte (NCM) frequency was determined. Impact of GBM (+/-IFN-γ) EV-treated monocytes on CD3/CD28-mediated T cell proliferation was assessed. The impact of PD-L1 and IDO1 knockdown in GBM EVs in this system was evaluated. Results: IFN-γ exposure increased PD-L1 and IDO1 expression in GBM cells and EVs without altering EV size or frequency. IFN-γ-exposed GBM EVs induced more MDSC and NCM differentiation in monocytes and these monocytes caused more T cell inhibition than IFN-γ-naive GBM EVs. PD-L1 and/or IDO1 knockdown in GBM cells abrogated the immunosuppressive effects of IFN-γ-exposed GBM EVs on monocytes. Conclusions: IFN-γ exposure such as might occur during an antitumor immune response results in superinduction of GBM EVs' baseline immunosuppressive effects on monocytes. These effects are mediated by increased PD-L1 and IDO1 expression in GBM EVs. These data highlight mechanisms of GBM EV-mediated immunosuppression and identify therapeutic targets (PD-L1, IDO1) to reverse these effects.

A comparison of barbed continuous suture versus conventional interrupted suture for fascial closure in total hip arthroplasty.

A barbed suture is a self-anchoring knotless suture hypothesized to shorten suture time and reduce the tension point of the wound. The purpose of this study was to compare the barbed suture and the interrupted suture for fascial closure in total hip arthroplasty. We retrospectively reviewed patients who underwent total hip arthroplasty from March 2014 to June 2020. We evaluated 324 cases among 274 patients consisting of 188 males and 86 females. We collected the following data: demographics, time for wound closure, the number of threads used, hemoglobin level, surgical site pain, and wound complications. Variables were analyzed for their association with closure time using multiple regression analyses between the barbed suture (the SFX group) and the interrupted suture (the Vicryl group). Mean closure time was 5.8 min lower and the mean number of sutures used was 2.2 lower in the SFX group versus the Vicryl group (P < 0.01 and < 0.01, respectively). There were no statistical intergroup differences in the mean largest hemoglobin drop, the incidence of transfusion, surgical site pain, and the incidence of wound complications. The use of barbed sutures for fascial closure in total hip arthroplasty effectively reduces the surgical time without increasing wound complications.

Plasma extracellular vesicles as a source of biomarkers in traumatic brain injury.

OBJECTIVE: The objective of this study was to isolate extracellular vesicles (EVs) from plasma in a cohort of patients with traumatic brain injury (TBI) and analyze their contents for novel biomarkers that could prove useful for rapid diagnosis and classification of brain injury during initial evaluation. METHODS: Plasma EVs were isolated by serial ultracentrifugation from patients with TBI (n = 15) and healthy controls (n = 5). Samples were obtained from the TRACK-TBI biorepository (2010-present). Size and concentration were determined by nanoparticle tracking. Glial fibrillary acidic protein (GFAP) concentration was determined in EV protein. EV RNA was isolated and deep sequencing of short noncoding RNA was performed. RESULTS: Plasma EVs are physically similar but contained approximately 10 times more GFAP in TBI patients with altered consciousness than patients and controls with normal consciousness. Eleven highly differentially expressed microRNAs (miRNAs) were identified between these groups. Genes targeted by these miRNAs are highly associated with biologically relevant cellular pathways, including organismal injury, cellular development, and organismal development. Multiple additional coding and noncoding RNA species with potential biomarker utility were identified. CONCLUSIONS: Isolating plasma EVs in patients with TBI is feasible. Increased GFAP concentration-a validated plasma TBI marker-in EVs from TBI patients with altered consciousness, along with differential expression of multiple miRNAs targeting TBI-relevant pathways, suggests that EVs may be a useful source of TBI biomarkers. Additional evaluation in larger patient cohorts is indicated.

The role of extracellular vesicles and PD-L1 in glioblastoma-mediated immunosuppressive monocyte induction.

BACKGROUND: Immunosuppression in glioblastoma (GBM) is an obstacle to effective immunotherapy. GBM-derived immunosuppressive monocytes are central to this. Programmed cell death ligand 1 (PD-L1) is an immune checkpoint molecule, expressed by GBM cells and GBM extracellular vesicles (EVs). We sought to determine the role of EV-associated PD-L1 in the formation of immunosuppressive monocytes. METHODS: Monocytes collected from healthy donors were conditioned with GBM-derived EVs to induce the formation of immunosuppressive monocytes, which were quantified via flow cytometry. Donor-matched T cells were subsequently co-cultured with EV-conditioned monocytes in order to assess effects on T-cell proliferation. PD-L1 constitutive overexpression or short hairpin RNA-mediated knockdown was used to determined the role of altered PD-L1 expression. RESULTS: GBM EVs interact with both T cells and monocytes but do not directly inhibit T-cell activation. However, GBM EVs induce immunosuppressive monocytes, including myeloid-derived suppressor cells (MDSCs) and nonclassical monocytes (NCMs). MDSCs and NCMs inhibit T-cell proliferation in vitro and are found within GBM in situ. EV PD-L1 expression induces NCMs but not MDSCs, and does not affect EV-conditioned monocytes T-cell inhibition. CONCLUSION: These findings indicate that GBM EV-mediated immunosuppression occurs through induction of immunosuppressive monocytes rather than direct T-cell inhibition and that, while PD-L1 expression is important for the induction of specific immunosuppressive monocyte populations, immunosuppressive signaling mechanisms through EVs are complex and not limited to PD-L1.

Transforming growth factor beta induces fibroblasts to express and release the immunomodulatory protein PD-L1 into extracellular vesicles.

Transforming growth factor-beta (TGFß) is an enigmatic protein with various roles in healthy tissue homeostasis/development as well as the development or progression of cancer, wound healing, fibrotic disorders, and immune modulation, to name a few. As TGFß is causal to various fibroproliferative disorders featuring localized or systemic tissue/organ fibrosis as well as the activated stroma observed in various malignancies, characterizing the pathways and players mediating its action is fundamental. In the current study, we found that TGFß induces the expression of the immunoinhibitory molecule Programed death-ligand 1 (PD-L1) in human and murine fibroblasts in a Smad2/3- and YAP/TAZ-dependent manner. Furthermore, PD-L1 knockdown decreased the TGFß-dependent induction of extracellular matrix proteins, including collagen Iα1 (colIα1) and alpha-smooth muscle actin (α-SMA), and cell migration/wound healing. In addition to an endogenous role for PD-L1 in profibrotic TGFß signaling, TGFß stimulated-human lung fibroblast-derived PD-L1 into extracellular vesicles (EVs) capable of inhibiting T cell proliferation in response to T cell receptor stimulation and mediating fibroblast cell migration. These findings provide new insights and potential targets for a variety of fibrotic and malignant diseases.

Hexokinase 2 couples glycolysis with the profibrotic actions of TGF-ß.

Metabolic dysregulation in fibroblasts is implicated in the profibrotic actions of transforming growth factor-ß (TGF-ß). Here, we present evidence that hexokinase 2 (HK2) is important for mediating the fibroproliferative activity of TGF-ß both in vitro and in vivo. Both Smad-dependent and Smad-independent TGF-ß signaling induced HK2 accumulation in murine and human lung fibroblasts through induction of the transcription factor c-Myc. Knockdown of HK2 or pharmacological inhibition of HK2 activity with Lonidamine decreased TGF-ß-stimulated fibrogenic processes, including profibrotic gene expression, cell migration, colony formation, and activation of the transcription factors YAP and TAZ, with no apparent effect on cellular viability. Fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) exhibited an increased abundance of HK2. In a mouse model of bleomycin-induced lung fibrosis, Lonidamine reduced the expression of genes encoding profibrotic markers (collagenΙα1, EDA-fibronectin, α smooth muscle actin, and connective tissue growth factor) and stabilized or improved lung function as assessed by measurement of peripheral blood oxygenation. These findings provide evidence of how metabolic dysregulation through HK2 can be integrated within the context of profibrotic TGF-ß signaling.

B7-1 drives TGF-ß stimulated pancreatic carcinoma cell migration and expression of EMT target genes.

B7-1 proteins are routinely expressed on the surface of antigen presenting cells (APC) and within the innate immune system. They function to establish a biologically optimal and dynamic balance between immune activation and inhibition or self-tolerance. Interactions between B7-1 and its receptors, which include CD28, CTLA4 and PD-L1, contribute to both stimulatory as well as inhibitory or homeostatic regulation. In the current study, we investigated whether the tumor-promoting actions of transforming growth factor beta (TGF-ß) disrupted this equilibrium in pancreatic cancer to promote malignant progression and an enhanced means to evade immune detection. The data show that B7-1 is (i) upregulated following treatment of pancreatic carcinoma cells with TGF-ß; (ii) induced by TGF-ß via both Smad2/3-dependent and independent pathways; (iii) required for pancreatic tumor cell in vitro migration/invasion; and (iv) necessary for TGF-ß regulated epithelial-mesenchymal transition (EMT) through induction of Snail family members. Results from the proposed studies provide valuable insights into mechanisms whereby TGF-ß regulates both the innate immune response and intrinsic properties of pancreatic tumor growth.

Analysis and Identification of Active Compounds from Gami-Soyosan Toxic to MCF-7 Human Breast Adenocarcinoma Cells.

Gami-soyosan is a medicinal herbal formulation prescribed for the treatment of menopausal symptoms, including hot flashes and osteoporosis. Gami-soyosan is also used to treat similar symptoms experienced by patients with breast cancer. The incidence of breast cancer in women receiving hormone replacement therapy is a big burden. However, little is known about the components and their mechanism of action that exhibit these beneficial effects of Gami-soyosan. The aim of this study was to simultaneously analyze compounds of Gami-soyosan, and determine their cytotoxic effects on estrogen receptor (ER)-positive MCF-7 human breast adenocarcinoma cells. We established a simultaneous analysis method of 18 compounds contained in Gami-soyosan and found that, among the various compounds in Gami-soyosan, gallic acid (1), decursin (17), and decursinol angelate (18) suppressed the viability of MCF-7 cells. Gallic acid (1), decursin (17), and decursinol angelate (18) induced apoptotic cell death and significantly increased poly (ADP-ribose) polymerase (PARP) cleavage and the Bcl-2-associated X protein/ B-cell lymphoma 2 (Bax/Bcl-2) ratio. Decursin (17) increased the expression of cleaved caspases-8, -9, -7, and -3. Decursinol angelate (18) increased the expression of cleaved caspase-8 and -7. These three components altered the different apoptosis signal pathways. Collectively, gallic acid (1), decursin (17), and decursinol angelate (18) may be used to inhibit cell proliferation synergistically in patients with ER-positive breast cancer.

In Vivo Estimation of Oncolytic Virus Populations within Tumors.

The use of replication-competent viruses as oncolytic agents is rapidly expanding, with several oncolytic viruses approved for cancer therapy. As responses to therapy are highly variable, understanding the dynamics of therapy is critical for optimal application of virotherapy in practice. Although mathematical models have been developed to understand the dynamics of tumor virotherapy, a scarcity of in vivo data has made difficult parametrization of these models. To tackle this problem, we studied the in vitro and in vivo spread of two oncolytic measles viruses that induce expression of the sodium iodide symporter (NIS) in cells. NIS expression enabled infected cells to concentrate radioactive isotopes that could be reproducibly and quantitatively imaged using SPECT/CT. We observed a strong linear relationship in vitro between infectious virus particles, viral N and NIS gene expression, and radioactive isotope uptake. In vivo radioisotope uptake was highly correlated with viral N and NIS gene expression. Similar expression patterns between viral N and NIS gene expression in vitro and in vivo implied that the oncolytic virus behaved similarly in both scenarios. Significant titers of viable virus were consistently isolated from tumors explanted from mice that had been injected with oncolytic measle viruses. We observed a weaker but positive in vivo relationship between radioisotope uptake and the viable virus titer recovered from tumors; this was likely due to anisotropies in the viral distribution in vivo These data suggest that methods that enable quantitation of in vivo anisotropies are required for continuing development of oncolytic virotherapy.Significance: These findings address a fundamental gap in our knowledge of oncolytic virotherapy by presenting technology that gives insight into the behavior of oncolytic viruses in vivo Cancer Res; 78(20); 5992-6000. ©2018 AACR.

Fatty acid synthase is required for profibrotic TGF-ß signaling.

Evidence is provided that the fibroproliferative actions of TGF-ß are dependent on a metabolic adaptation that sustains pathologic growth. Specifically, profibrotic TGF-ß signaling is shown to require fatty acid synthase (FASN), an essential anabolic enzyme responsible for the de novo synthesis of fatty acids. With the use of pharmacologic and genetic approaches, we show that TGF-ß-stimulated FASN expression is independent of Smad2/3 and is mediated via mammalian target of rapamycin complex 1. In the absence of FASN activity or protein, TGF-ß-driven fibrogenic processes are reduced with no apparent toxicity. Furthermore, as increased FASN expression was also observed to correlate with the degree of lung fibrosis in bleomycin-treated mice, inhibition of FASN was examined in a murine-treatment model of pulmonary fibrosis. Remarkably, inhibition of FASN not only decreased expression of profibrotic targets, but lung function was also stabilized/improved, as assessed by peripheral blood oxygenation.-Jung, M.-Y., Kang, J.-H., Hernandez, D. M., Yin, X., Andrianifahanana, M., Wang, Y., Gonzalez-Guerrico, A., Limper, A. H., Lupu, R., Leof, E. B. Fatty acid synthase is required for profibrotic TGF-ß signaling.

A distinct biomolecular profile identifies monoclonal mast cell disorders in patients with idiopathic anaphylaxis.

BACKGROUND: Clonal mast cell disorders are known to occur in a subset of patients with systemic reactions to Hymenoptera stings. This observation has prompted the question of whether clonal mast cell disorders also occur in patients with idiopathic anaphylaxis (IA). OBJECTIVE: We sought to determine the prevalence of clonal mast cell disorders among patients with IA, criteria to identify those patients who require a bone marrow biopsy, and whether the pathogenesis of IA involves a hyperresponsive mast cell compartment. METHODS: We prospectively enrolled patients with IA (≥3 episodes/y) who then underwent a medical evaluation that included a serum tryptase determination, allele-specific quantitative PCR (ASqPCR) for the KIT D816V mutation, and a bone marrow examination. Mast cells were cultured from peripheral blood CD34+ cells and examined for releasability after FcεRI aggregation. RESULTS: Clonal mast cell disease was diagnosed in 14% of patients referred with IA. ASqPCR for the KIT D816V mutation was a useful adjunct in helping identify those with systemic mastocytosis but not monoclonal mast cell activation syndrome. A modified overall clonal prediction model was developed by using clinical findings, a serum tryptase determination, and ASqPCR. There was no evidence of a hyperresponsive mast cell phenotype in patients with IA. CONCLUSION: Patients with clonal mast cell disease can present as having IA. Distinct clinical and laboratory features can be used to select those patients more likely to have an underlying clonal mast cell disorder (monoclonal mast cell activation syndrome or systemic mastocytosis) and thus candidates for a bone marrow biopsy.

Basolateral delivery of the type I transforming growth factor beta receptor is mediated by a dominant-acting cytoplasmic motif.

Delivery of biomolecules to the correct subcellular locales is critical for proper physiological function. To that end, we have previously determined that type I and II transforming growth factor beta (TGF-ß) receptors (TßRI and TßRII, respectively) localize to the basolateral domain in polarized epithelia. While TßRII targeting was shown to be regulated by sequences between amino acids 529 and 538, the analogous region(s) within TßRI is unknown. To address that question, sequential cytoplasmic TßRI truncations and point mutations identified a targeting motif between residues 158 and 163 (VxxEED) required for basolateral TßRI expression. Further studies documented that receptor internalization, down-regulation, direct recycling, or Smad signaling were unaffected by motif mutations that caused TßRI mislocalization. However, inclusion of amino acids 148-217 containing the targeting motif was able to direct basolateral expression of the apically sorted nerve growth factor receptor (NGFR, p75; extracellular and transmembrane regions) in a dominant manner. Finally, coexpression of apically targeted type I and type II TGF-ß receptors mediated Smad3 signaling from the apical membrane of polarized epithelial cells. These findings demonstrate that the absence of apical TGF-ß signaling in normal epithelia is primarily a reflection of domain-specific receptor expression and not an inability to couple with the signaling machinery.

Cell-penetrating peptides selectively targeting SMAD3 inhibit profibrotic TGF-ß signaling.

TGF-ß is considered a master switch in the pathogenesis of organ fibrosis. The primary mediators of this activity are the SMAD proteins, particularly SMAD3. In the current study, we have developed a cell-penetrating peptide (CPP) conjugate of the HIV TAT protein that is fused to an aminoterminal sequence of sorting nexin 9 (SNX9), which was previously shown to bind phosphorylated SMAD3 (pSMAD3). We determined that specifically preventing the nuclear import of pSMAD3 using the TAT-SNX9 peptide inhibited profibrotic TGF-ß activity in murine cells and human lung fibroblasts as well as in vivo with no demonstrable toxicity. TGF-ß signaling mediated by pSMAD2, bone morphogenetic protein 4 (BMP4), EGF, or PDGF was unaffected by the TAT-SNX9 peptide. Furthermore, while the TAT-SNX9 peptide prevented TGF-ß's profibrotic activity in vitro as well as in 2 murine treatment models of pulmonary fibrosis, a 3-amino acid point mutant that was unable to bind pSMAD3 proved ineffective. These findings indicate that specifically targeting pSMAD3 can ameliorate both the direct and indirect fibroproliferative actions of TGF-ß.

Profibrotic up-regulation of glucose transporter 1 by TGF-ß involves activation of MEK and mammalian target of rapamycin complex 2 pathways.

TGF-ß plays a central role in the pathogenesis of fibroproliferative disorders. Defining the exact underlying molecular basis is therefore critical for the development of viable therapeutic strategies. Here, we show that expression of the facilitative glucose transporter 1 (GLUT1) is induced by TGF-ß in fibroblast lines and primary cells and is required for the profibrotic effects of TGF-ß. In addition, enhanced GLUT1 expression is observed in fibrotic areas of lungs of both patients with idiopathic pulmonary fibrosis and mice that are subjected to a fibrosis-inducing bleomycin treatment. By using pharmacologic and genetic approaches, we demonstrate that up-regulation of GLUT1 occurs via the canonical Smad2/3 pathway and requires autocrine activation of the receptor tyrosine kinases, platelet-derived and epidermal growth factor receptors. Engagement of the common downstream effector PI3K subsequently triggers activation of the MEK and mammalian target of rapamycin complex 2, which cooperate in regulating GLUT1 expression. Of note, inhibition of GLUT1 activity and/or expression is shown to impair TGF-ß-driven fibrogenic processes, including cell proliferation and production of profibrotic mediators. These findings provide new perspectives on the interrelation of metabolism and profibrotic TGF-ß signaling and present opportunities for potential therapeutic intervention.-Andrianifahanana, M., Hernandez, D. M., Yin, X., Kang, J.-H., Jung, M.-Y., Wang, Y., Yi, E. S., Roden, A. C., Limper, A. H., Leof, E. B. Profibrotic up-regulation of glucose transporter 1 by TGF-ß involves activation of MEK and mammalian target of rapamycin complex 2 pathways.

IL-6 promotes an increase in human mast cell numbers and reactivity through suppression of suppressor of cytokine signaling 3.

BACKGROUND: IL-6, levels of which are reported to be increased in association with mastocytosis, asthma, and urticaria, is used in conjunction with stem cell factor to generate CD34(+) cell-derived primary human mast cell (HuMC) cultures. Despite these associations, the effects on and mechanisms by which prolonged exposure to IL-6 alters HuMC numbers and function are not well understood. OBJECTIVES: We sought to study the effect of IL-6 on HuMC function, the mechanisms by which IL-6 exerts its effects, and the relationship of these findings to mastocytosis. METHODS: HuMCs were cultured in stem cell factor with or without IL-6. Responses to FcεRI aggregation and expression of proteases and receptors, including the soluble IL-6 receptor (sIL-6R), were then quantitated. Epigenetic changes in suppressor of cytokine signaling 3 (SOCS3) were determined by using methylation-specific PCR. Serum samples from healthy control subjects and patients with mastocytosis were assayed for IL-6, tryptase, and sIL-6R. RESULTS: IL-6 enhanced mast cell (MC) proliferation, maturation, and reactivity after FcεRI aggregation. IL-6 reduced expression of SOCS3, which correlated with methylation of the SOCS3 promoter and increased expression and activation of signal transducer and activator of transcription 3. IL-6 also suppressed constitutive production of sIL-6R, and serum levels of sIL-6R were similarly reduced in patients with mastocytosis. CONCLUSION: IL-6 increases MC proliferation and formation of a more reactive phenotype enabled by suppressing proteolytic cleavage of sIL-6R from IL-6R and downregulation of the SOCS3 autoinhibitory pathway. We suggest IL-6 blockade might ameliorate MC-related symptoms and pathology in patients with MC-related diseases associated with increased IL-6 levels, including mastocytosis.

Predicting postoperative total calcium requirements after parathyroidectomy in secondary hyperparathyroidism.

BACKGROUND/AIMS: To prevent hypocalcemia after parathyroidectomy (PTX), parenteral calcium is required in addition to oral calcitriol and calcium. After switching to oral calcium, patients can be discharged from the hospital. The aim of this study was to analyze the clinical characteristics and outcomes of PTX performed at a single Korean center and to investigate the associated laboratory factors used to analyze the total amount of postoperative calcium required. METHODS: We enrolled 91 hemodialysis patients undergoing PTX from November 2003 to December 2011. We collected clinical and laboratory data preoperatively, 12 and 48 hours postoperatively, at discharge, and 3 and 6 months postoperatively. RESULTS: In total, 59 patients underwent PTX with autotransplantation (AT), 6 underwent total PTX without AT, 11 underwent subtotal PTX, and 15 underwent limited PTX. Total PTX without AT showed the lowest recurrence rate. At all postoperative time points, the mean levels of serum calcium, phosphorus, and intact parathyroid hormone (iPTH) decreased significantly, compared with preoperative levels; however, alkaline phosphatase (ALP) increased significantly from 48 hours postoperatively to discharge (p < 0.001). On multiple linear regression analysis, the total amount of injected calcium during hospitalization showed a significant correlation with preoperative ALP (p < 0.001), preoperative iPTH (p = 0.037), and Δphosphorus at 48 hours (p < 0.001). We developed an equation for estimating the total calcium requirement after PTX. CONCLUSIONS: Preoperative ALP, preoperative iPTH, and Δphosphorus at 48 hours may be significant factors in estimating the postoperative calcium requirement. The formula for postoperative calcium requirement after PTX may help to predict the duration of postoperative hospitalization.

Optimal hemoglobin level for anemia treatment in a cohort of hemodialysis patients.

BACKGROUND: Anemia is a major risk factor that contributes to mortality in patients with chronic kidney disease. There is controversy over the optimal hemoglobin (Hb) target in these patients. This study investigated the association between Hb level and mortality in a cohort of hemodialysis (HD) patients in Korea. METHODS: This study was a multicenter prospective observational study of maintenance HD patients that was performed for 5 years in western Seoul, Korea. Three hundred and sixty-two participants were enrolled. Laboratory values and mortality were accessed every 6 months. Repeated measures of laboratory values in each interval were averaged to obtain one semiannual mean value. The Hb values were divided into six groups: (1) Hb<9 g/dL; (2) 9 g/dL≤Hb<10 g/dL; (3) 10 g/dL≤Hb<11 g/dL; (4) 11 g/dL≤Hb<12 g/dL; (5) 12 g/dL≤Hb<13 g/dL; and (6) Hb≥13 g/dL. We analyzed the odds ratio for all-cause mortality, based on the Hb group, and adjusted for demographics and various laboratory values. Statistics were performed with SAS, version 9.1 software (SAS Institute Inc., Cary, NC, USA). RESULTS: Mortality odds ratios relative to the reference group (10-11 g/dL) in the fully adjusted model were 3.61 for<9 g/dL; 3.17 for 9-10 g/dL(⁎); 4.65 for 11-12 g/dL(⁎); 5.50 for 12-13 g/dL(⁎); and 2.05 for≥13 g/dL ((⁎) indicates P<0.05). CONCLUSION: In this study, a Hb level of 10-11 g/dL was associated with the lowest mortality among the groups with Hb level<13 g/dL. Larger interventional trials are warranted to determine the optimal Hb target for Korean HD patients.