Pdx1 Is Transcriptionally Regulated by EGR-1 during Nitric Oxide-Induced Endoderm Differentiation of Mouse Embryonic Stem Cells.

The transcription factor, early growth response-1 (EGR-1), is involved in the regulation of cell differentiation, proliferation, and apoptosis in response to different stimuli. EGR-1 is described to be involved in pancreatic endoderm differentiation, but the regulatory mechanisms controlling its action are not fully elucidated. Our previous investigation reported that exposure of mouse embryonic stem cells (mESCs) to the chemical nitric oxide (NO) donor diethylenetriamine nitric oxide adduct (DETA-NO) induces the expression of early differentiation genes such as pancreatic and duodenal homeobox 1 (Pdx1). We have also evidenced that Pdx1 expression is associated with the release of polycomb repressive complex 2 (PRC2) and P300 from the Pdx1 promoter; these events were accompanied by epigenetic changes to histones and site-specific changes in the DNA methylation. Here, we investigate the role of EGR-1 on Pdx1 regulation in mESCs. This study reveals that EGR-1 plays a negative role in Pdx1 expression and shows that the binding capacity of EGR-1 to the Pdx1 promoter depends on the methylation level of its DNA binding site and its acetylation state. These results suggest that targeting EGR-1 at early differentiation stages might be relevant for directing pluripotent cells into Pdx1-dependent cell lineages.

Efficacy and safety of intramuscular administration of allogeneic adipose tissue derived and expanded mesenchymal stromal cells in diabetic patients with critical limb ischemia with no possibility of revascularization: study protocol for a randomized controlled double-blind phase II clinical trial (The NOMA Trial).

BACKGROUND: Chronic lower limb ischemia develops earlier and more frequently in patients with type 2 diabetes mellitus. Diabetes remains the main cause of lower-extremity non-traumatic amputations. Current medical treatment, based on antiplatelet therapy and statins, has demonstrated deficient improvement of the disease. In recent years, research has shown that it is possible to improve tissue perfusion through therapeutic angiogenesis. Both in animal models and humans, it has been shown that cell therapy can induce therapeutic angiogenesis, making mesenchymal stromal cell-based therapy one of the most promising therapeutic alternatives. The aim of this study is to evaluate the feasibility, safety, and efficacy of cell therapy based on mesenchymal stromal cells derived from adipose tissue intramuscular administration to patients with type 2 diabetes mellitus with critical limb ischemia and without possibility of revascularization. METHODS: A multicenter, randomized double-blind, placebo-controlled trial has been designed. Ninety eligible patients will be randomly assigned at a ratio 1:1:1 to one of the following: control group (n = 30), low-cell dose treatment group (n = 30), and high-cell dose treatment group (n = 30). Treatment will be administered in a single-dose way and patients will be followed for 12 months. Primary outcome (safety) will be evaluated by measuring the rate of adverse events within the study period. Secondary outcomes (efficacy) will be measured by assessing clinical, analytical, and imaging-test parameters. Tertiary outcome (quality of life) will be evaluated with SF-12 and VascuQol-6 scales. DISCUSSION: Chronic lower limb ischemia has limited therapeutic options and constitutes a public health problem in both developed and underdeveloped countries. Given that the current treatment is not established in daily clinical practice, it is essential to provide evidence-based data that allow taking a step forward in its clinical development. Also, the multidisciplinary coordination exercise needed to develop this clinical trial protocol will undoubtfully be useful to conduct academic clinical trials in the field of cell therapy in the near future. TRIAL REGISTRATION: ClinicalTrials.gov NCT04466007 . Registered on January 07, 2020. All items from the World Health Organization Trial Registration Data Set are included within the body of the protocol.

Corrigendum: Cost-Effective, Safe, and Personalized Cell Therapy for Critical Limb Ischemia in Type 2 Diabetes Mellitus.

[This corrects the article DOI: 10.3389/fimmu.2019.01151.].

AICAR Stimulates the Pluripotency Transcriptional Complex in Embryonic Stem Cells Mediated by PI3K, GSK3ß, and ß-Catenin.

Pluripotent stem cells maintain the property of self-renewal and differentiate into all cell types under clear environments. Though the gene regulatory mechanism for pluripotency has been investigated in recent years, it is still not completely understood. Here, we show several signaling pathways involved in the maintenance of pluripotency. To investigate whether AMPK is involved in maintaining the pluripotency in mouse embryonic stem cells (mESCs) and elucidating the possible molecular mechanisms, implicated D3 and R1/E mESC lines were used in this study. Cells were cultured in the absence or presence of LIF and treated with 1 mM and 0.5 mM 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR), 2 mM metformin, compound C, and the PI3K inhibitor LY294002 for 24, 72, and 120 h. The levels of Nanog, Oct3/4, and REX1 and Brachyury, Notch2, and Gata4 mRNAs and Nanog or OCT3/4 protein levels were analyzed. Alkaline phosphatase and the cellular cycle were determined. The pGSK3ß, GSK3ß, p-ß-catenin, and ß-catenin protein levels were also investigated. We found that AMPK activators such as AICAR and metformin increase mRNA expression of pluripotency markers and decrease mRNA expression of differentiation markers in R1/E and D3 ES cells. AICAR increases phosphatase activity and arrests the cellular cycle in the G1 phase in these cells. We describe that AICAR effects were mediated by AMPK activation using a chemical inhibitor or by silencing this gene. AICAR effects were also mediated by PI3K, GSK3ß, and ß-catenin in R1/E ES cells. According to our findings, we provide a mechanism by which AICAR increases and maintains a pluripotency state through enhanced Nanog expression, involving AMPK/PI3K and p-GSK3ß Ser21/9 pathways backing up the AICAR function as a potential target for this drug controlling pluripotency. The highlights of this study are that AICAR (5-aminoimidazole-4-carboxamied-1-b-riboside), an AMP protein kinase (AMPK) activator, blocks the ESC differentiation and AMPK is a key enzyme for pluripotency and shows valuable data to clarify the molecular pluripotency mechanism.

Mesenchymal Stromal Cell-Based Therapies as Promising Treatments for Muscle Regeneration After Snakebite Envenoming.

Snakebite envenoming is a global neglected disease with an incidence of up to 2.7 million new cases every year. Although antivenoms are so-far the most effective treatment to reverse the acute systemic effects induced by snakebite envenoming, they have a limited therapeutic potential, being unable to completely neutralize the local venom effects. Local damage, such as dermonecrosis and myonecrosis, can lead to permanent sequelae with physical, social, and psychological implications. The strong inflammatory process induced by snake venoms is associated with poor tissue regeneration, in particular the lack of or reduced skeletal muscle regeneration. Mesenchymal stromal cells (MSCs)-based therapies have shown both anti-inflammatory and pro-regenerative properties. We postulate that using allogeneic MSCs or their cell-free products can induce skeletal muscle regeneration in snakebite victims, improving all the three steps of the skeletal muscle regeneration process, mainly by anti-inflammatory activity, paracrine effects, neovascularization induction, and inhibition of tissue damage, instrumental for microenvironment remodeling and regeneration. Since snakebite envenoming occurs mainly in areas with poor healthcare, we enlist the principles and potential of MSCs-based therapies and discuss regulatory issues, good manufacturing practices, transportation, storage, and related-procedures that could allow the administration of these therapies, looking forward to a safe and cost-effective treatment for a so far unsolved and neglected health problem.

Cost-Effective, Safe, and Personalized Cell Therapy for Critical Limb Ischemia in Type 2 Diabetes Mellitus.

Cell therapy is a progressively growing field that is rapidly moving from preclinical model development to clinical application. Outcomes obtained from clinical trials reveal the therapeutic potential of stem cell-based therapy to deal with unmet medical treatment needs for several disorders with no therapeutic options. Among adult stem cells, mesenchymal stem cells (MSCs) are the leading cell type used in advanced therapies for the treatment of autoimmune, inflammatory and vascular diseases. To date, the safety and feasibility of autologous MSC-based therapy has been established; however, their indiscriminate use has resulted in mixed outcomes in preclinical and clinical studies. While MSCs derived from diverse tissues share common properties depending on the type of clinical application, they markedly differ within clinical trials in terms of efficacy, resulting in many unanswered questions regarding the application of MSCs. Additionally, our experience in clinical trials related to critical limb ischemia pathology (CLI) shows that the therapeutic efficacy of these cells in different animal models has only been partially reproduced in humans through clinical trials. Therefore, it is crucial to develop new research to identify pitfalls, to optimize procedures and to clarify the repair mechanisms used by these cells, as well as to be able to offer a next generation of stem cell that can be routinely used in a cost-effective and safe manner in stem cell-based therapies targeting CLI.

Zn2+ chelation by serum albumin improves hexameric Zn2+-insulin dissociation into monomers after exocytosis.

ß-cells release hexameric Zn2+-insulin into the extracellular space, but monomeric Zn2+-free insulin appears to be the only biologically active form. The mechanisms implicated in dissociation of the hexamer remain unclear, but they seem to be Zn2+ concentration-dependent. In this study, we investigate the influence of albumin binding to Zn2+ on Zn2+-insulin dissociation into Zn2+-free insulin and its physiological, methodological and therapeutic relevance. Glucose and K+-induced insulin release were analyzed in isolated mouse islets by static incubation and perifusion experiments in the presence and absence of albumin and Zn2+ chelators. Insulin tolerance tests were performed in rats using different insulin solutions with and without Zn2+ and/or albumin. Albumin-free buffer does not alter quantification by RIA of Zn2+-free insulin but strongly affects RIA measurements of Zn2+-insulin. In contrast, accurate determination of Zn2+-insulin was obtained only when bovine serum albumin or Zn2+ chelators were present in the assay buffer solution. Albumin and Zn2+ chelators do not modify insulin release but do affect insulin determination. Preincubation with albumin or Zn2+ chelators promotes the conversion of "slow" Zn2+-insulin into "fast" insulin. Consequently, insulin diffusion from large islets is ameliorated in the presence of Zn2+ chelators. These observations support the notion that the Zn2+-binding properties of albumin improve the dissociation of Zn2+-insulin into subunits after exocytosis, which may be useful in insulin determination, insulin pharmacokinetic assays and islet transplantation.

Regulation of mitochondrial function and endoplasmic reticulum stress by nitric oxide in pluripotent stem cells.

Mitochondrial dysfunction and endoplasmic reticulum stress (ERS) are global processes that are interrelated and regulated by several stress factors. Nitric oxide (NO) is a multifunctional biomolecule with many varieties of physiological and pathological functions, such as the regulation of cytochrome c inhibition and activation of the immune response, ERS and DNA damage; these actions are dose-dependent. It has been reported that in embryonic stem cells, NO has a dual role, controlling differentiation, survival and pluripotency, but the molecular mechanisms by which it modulates these functions are not yet known. Low levels of NO maintain pluripotency and induce mitochondrial biogenesis. It is well established that NO disrupts the mitochondrial respiratory chain and causes changes in mitochondrial Ca2+ flux that induce ERS. Thus, at high concentrations, NO becomes a potential differentiation agent due to the relationship between ERS and the unfolded protein response in many differentiated cell lines. Nevertheless, many studies have demonstrated the need for physiological levels of NO for a proper ERS response. In this review, we stress the importance of the relationships between NO levels, ERS and mitochondrial dysfunction that control stem cell fate as a new approach to possible cell therapy strategies.

Nitric Oxide Prevents Mouse Embryonic Stem Cell Differentiation Through Regulation of Gene Expression, Cell Signaling, and Control of Cell Proliferation.

Nitric oxide (NO) delays mouse embryonic stem cell (mESC) differentiation by regulating genes linked to pluripotency and differentiation. Nevertheless, no profound study has been conducted on cell differentiation regulation by this molecule through signaling on essential biological functions. We sought to demonstrate that NO positively regulates the pluripotency transcriptional core, enforcing changes in the chromatin structure, in addition to regulating cell proliferation, and signaling pathways with key roles in stemness. Culturing mESCs with 2 µM of the NO donor diethylenetriamine/NO (DETA/NO) in the absence of leukemia inhibitory factor (LIF) induced significant changes in the expression of 16 genes of the pluripotency transcriptional core. Furthermore, treatment with DETA/NO resulted in a high occupancy of activating H3K4me3 at the Oct4 and Nanog promoters and repressive H3K9me3 and H3k27me3 at the Brachyury promoter. Additionally, the activation of signaling pathways involved in pluripotency, such as Gsk3-ß/ß-catenin, was observed, in addition to activation of PI3 K/Akt, which is consistent with the protection of mESCs from cell death. Finally, a decrease in cell proliferation coincides with cell cycle arrest in G2/M. Our results provide novel insights into NO-mediated gene regulation and cell proliferation and suggest that NO is necessary but not sufficient for the maintenance of pluripotency and the prevention of cell differentiation. J. Cell. Biochem. 117: 2078-2088, 2016. © 2016 Wiley Periodicals, Inc.

Using stem cells to produce insulin.

INTRODUCTION: Tremendous progress has been made in generating insulin-producing cells from pluripotent stem cells. The best outcome of the refined protocols became apparent in the first clinical trial announced by ViaCyte, based on the implantation of pancreatic progenitors that would further mature into functional insulin-producing cells inside the patient's body. AREAS COVERED: Several groups, including ours, have contributed to improve strategies to generate insulin-producing cells. Of note, the latest results have gained a substantial amount of interest as a method to create a potentially functional and limitless supply of ß-cell to revert diabetes mellitus. This review analyzes the accomplishments that have taken place over the last few decades, summarizes the state-of-art methods for ß-cell replacement therapies based on the differentiation of embryonic stem cells into glucose-responsive and insulin-producing cells in a dish and discusses alternative approaches to obtain new sources of insulin-producing cells. EXPERT OPINION: Undoubtedly, recent events preface the beginning of a new era in diabetes therapy. However, in our opinion, a number of significant hurdles still stand in the way of clinical application. We believe that the combination of the private and public sectors will accelerate the process of obtaining the desired safe and functional ß-cell surrogates.

Role of nitric oxide in the maintenance of pluripotency and regulation of the hypoxia response in stem cells.

Stem cell pluripotency and differentiation are global processes regulated by several pathways that have been studied intensively over recent years. Nitric oxide (NO) is an important molecule that affects gene expression at the level of transcription and translation and regulates cell survival and proliferation in diverse cell types. In embryonic stem cells NO has a dual role, controlling differentiation and survival, but the molecular mechanisms by which it modulates these functions are not completely defined. NO is a physiological regulator of cell respiration through the inhibition of cytochrome c oxidase. Many researchers have been examining the role that NO plays in other aspects of metabolism such as the cellular bioenergetics state, the hypoxia response and the relationship of these areas to stem cell stemness.

Transient Downregulation of Nanog and Oct4 Induced by DETA/NO Exposure in Mouse Embryonic Stem Cells Leads to Mesodermal/Endodermal Lineage Differentiation.

The function of pluripotency genes in differentiation is a matter of investigation. We report here that Nanog and Oct4 are reexpressed in two mouse embryonic stem cell (mESC) lines following exposure to the differentiating agent DETA/NO. Both cell lines express a battery of both endoderm and mesoderm markers following induction of differentiation with DETA/NO-based protocols. Confocal analysis of cells undergoing directed differentiation shows that the majority of cells expressing Nanog express also endoderm genes such as Gata4 and FoxA2 (75.4% and 96.2%, resp.). Simultaneously, mRNA of mesodermal markers Flk1 and Mef2c are also regulated by the treatment. Acetylated histone H3 occupancy at the promoter of Nanog is involved in the process of reexpression. Furthermore, Nanog binding to the promoter of Brachyury leads to repression of this gene, thus disrupting mesendoderm transition.

Impact of exposure to low concentrations of nitric oxide on protein profile in murine and human pancreatic islet cells.

Homeostatic levels of nitric oxide (NO) protect efficiently against apoptotic death in both human and rodent pancreatic ß cells, but the protein profile of this action remains to be determined. We have applied a 2 dimensional LC-MS-MALDI-TOF/TOF-based analysis to study the impact of protective NO in rat insulin-producing RINm5F cell line and in mouse and human pancreatic islets (HPI) exposed to serum deprivation condition. 24 proteins in RINm5F and 22 in HPI were identified to undergo changes in at least one experimental condition. These include stress response mitochondrial proteins (UQCRC2, VDAC1, ATP5C1, ATP5A1) in RINm5F cells and stress response endoplasmic reticulum proteins (HSPA5, PDIA6, VCP, GANAB) in HPI. In addition, metabolic and structural proteins, oxidoreductases and chaperones related with protein metabolism are also regulated by NO treatment. Network analysis of differentially expressed proteins shows their interaction in glucocorticoid receptor and NRF2-mediated oxidative stress response pathways and eNOS signaling. The results indicate that exposure to exogenous NO counteracts the impact of serum deprivation on pancreatic ß cell proteome. Species differences in the proteins involved are apparent.

Regulation of pancreatic ß-cell survival by nitric oxide: clinical relevance.

The reduction of pancreatic ß-cell mass is an important factor in the development of type 1 and type 2 diabetes. Understanding the mechanisms that regulate the maintenance of pancreatic ß-cell mass as well as ß-cell death is necessary for the establishment of therapeutic strategies. In this context, nitric oxide (NO) is a diatomic, gaseous, highly reactive molecule with biological activity that participates in the regulation of pancreatic ß-cell mass. Two types of cellular responses can be distinguished depending on the level of NO production. First, pancreatic ß-cells exposed to inflammatory cytokines, lipid stress or hyperglycaemia produce high concentrations of NO, mainly due to the activation of inducible NO synthase (iNOS), thus promoting cell death. Meanwhile, under homeostatic conditions, low concentrations of NO, constitutively produced by endothelial NO synthase (eNOS), promote cell survival. Here, we will discuss the current knowledge of the NO-dependent mechanisms activated during cellular responses, emphasizing those related to the regulation of cell survival.

Islet cell development.

Over the last years, there has been great success in driving stem cells toward insulin-expressing cells. However, the protocols developed to date have some limitations, such as low reliability and low insulin production. The most successful protocols used for generation of insulin-producing cells from stem cells mimic in vitro pancreatic organogenesis by directing the stem cells through stages that resemble several pancreatic developmental stages. Islet cell fate is coordinated by a complex network of inductive signals and regulatory transcription factors that, in a combinatorial way, determine pancreatic organ specification, differentiation, growth, and lineage. Together, these signals and factors direct the progression from multipotent progenitor cells to mature pancreatic cells. Later in development and adult life, several of these factors also contribute to maintain the differentiated phenotype of islet cells. A detailed understanding of the processes that operate in the pancreas during embryogenesis will help us to develop a suitable source of cells for diabetes therapy. In this chapter, we will discuss the main transcription factors involved in pancreas specification and beta-cell formation.

Taurine supplementation modulates glucose homeostasis and islet function.

Taurine is a conditionally essential amino acid for human that is involved in the control of glucose homeostasis; however, the mechanisms by which the amino acid affects blood glucose levels are unknown. Using an animal model, we have studied these mechanisms. Mice were supplemented with taurine for 30 d. Blood glucose homeostasis was assessed by intraperitoneal glucose tolerance tests (IPGTT). Islet cell function was determined by insulin secretion, cytosolic Ca2+ measurements and glucose metabolism from isolated islets. Islet cell gene expression and translocation was examined via immunohistochemistry and quantitative real-time polymerase chain reaction. Insulin signaling was studied by Western blot. Islets from taurine-supplemented mice had: (i) significantly higher insulin content, (ii) increased insulin secretion at stimulatory glucose concentrations, (iii) significantly displaced the dose-response curve for glucose-induced insulin release to the left, (iv) increased glucose metabolism at 5.6 and 11.1-mmol/L concentrations; (v) slowed cytosolic Ca2+ concentration ([Ca2+]i) oscillations in response to stimulatory glucose concentrations; (vi) increased insulin, sulfonylurea receptor-1, glucokinase, Glut-2, proconvertase and pancreas duodenum homeobox-1 (PDX-1) gene expression and (vii) increased PDX-1 expression in the nucleus. Moreover, taurine supplementation significantly increased both basal and insulin stimulated tyrosine phosphorylation of the insulin receptor in skeletal muscle and liver tissues. Finally, taurine supplemented mice showed an improved IPGTT. These results indicate that taurine controls glucose homeostasis by regulating the expression of genes required for glucose-stimulated insulin secretion. In addition, taurine enhances peripheral insulin sensitivity.

Nicotinamide induces differentiation of embryonic stem cells into insulin-secreting cells.

The poly(ADP-ribose) polymerase (PARP) inhibitor, nicotinamide, induces differentiation and maturation of fetal pancreatic cells. In addition, we have previously reported evidence that nicotinamide increases the insulin content of cells differentiated from embryonic stem (ES) cells, but the possibility of nicotinamide acting as a differentiating agent on its own has never been completely explored. Islet cell differentiation was studied by: (i) X-gal staining after neomycin selection; (ii) BrdU studies; (iii) single and double immunohistochemistry for insulin, C-peptide and Glut-2; (iv) insulin and C-peptide content and secretion assays; and (v) transplantation of differentiated cells, under the kidney capsule, into streptozotocin (STZ)-diabetic mice. Here we show that undifferentiated mouse ES cells treated with nicotinamide: (i) showed an 80% decrease in cell proliferation; (ii) co-expressed insulin, C-peptide and Glut-2; (iii) had values of insulin and C-peptide corresponding to 10% of normal mouse islets; (iv) released insulin and C-peptide in response to stimulatory glucose concentrations; and (v) after transplantation into diabetic mice, normalized blood glucose levels over 7 weeks. Our data indicate that nicotinamide decreases ES cell proliferation and induces differentiation into insulin-secreting cells. Both aspects are very important when thinking about cell therapy for the treatment of diabetes based on ES cells.

Nitric oxide mediates the survival action of IGF-1 and insulin in pancreatic beta cells.

Generation of low levels of nitric oxide (NO) contributes to beta cell survival in vitro. The purpose of this study was to explore the link between NO and the survival pathway triggered by insulin-like growth factor-1 (IGF-1) and insulin in insulin producing RINm5F cells and in pancreatic islets. Results show that exposure of cells to IGF-1/insulin protects against serum deprivation-induced apoptosis. This action is prevented with inhibitors of NO generation, PI3K and Akt. Moreover, transfection with the negative dominant form of the tyrosine kinase c-Src abrogates the effect of IGF-1 and insulin on DNA fragmentation. An increase in the expression level of NOS3 protein and in the enzyme activity is observed following exposure of serum-deprived RINm5F cells to IGF-1 and insulin. Phosphorylation of IRS-1, IRS-2 and to less extent IRS-3 takes place when serum-deprived RINm5F cells and rat pancreatic islets are exposed to either IGF-1, insulin, or diethylenetriamine nitric oxide adduct (DETA/NO). In human islets, IRS-1 and IRS-2 proteins are present and tyrosine phosphorylated upon exposure to IGF-1, insulin and DETA/NO. Both rat and human pancreatic islets undergo DNA fragmentation when cultured in serum-free medium and IGF-1, insulin and DETA/NO protect efficiently from this damage. We then conclude that generation of NO participates in the activation of survival pathways by IGF-1 and insulin in beta cells.

Gastrointestinal stem cells. I. Pancreatic stem cells.

The transplantation of islets isolated from donor pancreas has renewed the interest in cell therapy for the treatment of diabetes. In addition, the capacity that stem cells have to differentiate into a wide variety of cell types makes their use ideal to generate beta-cells for transplantation therapies. Several studies have reported the generation of insulin-secreting cells from embryonic and adult stem cells that normalized blood glucose values when transplanted into diabetic animal models. Finally, although much work remains to be done, there is sufficient evidence to warrant continued efforts on stem cell research to cure diabetes.

Stimulators of AMP-activated protein kinase inhibit the respiratory burst in human neutrophils.

In the present study, we have examined the potential ability of 5'-AMP-activated protein kinase (AMPK) to modulate NADPH oxidase activity in human neutrophils. AMPK activated with either 5'-aminoimidazole-4-carboxamide ribonucleoside (AICAR) or with 5'-AMP significantly attenuated both phorbol 12-myristate 13-acetate (PMA) and formyl methionyl leucyl phenylalanine-stimulated superoxide anion O2- release by human neutrophils, consistently with a reduced translocation to the cell membrane and phosphorylation of a cytosolic component of NADPH oxidase, namely p47phox. AMPK was found to be present in human neutrophils and to become phosphorylated in response to either AICAR or other stimulators of its enzyme activity. Furthermore, AICAR also strongly reduced PMA-dependent H2O2 release, and induced the phosphorylation of c-jun N-terminal kinase 1 (p46), p38 mitogen-activated protein kinase and extracellular signal-regulated kinase. Present data demonstrate for the first time that the activation of AMPK, in states of low cellular energy charge (such as under high levels of 5'-AMP) or other signals, could be a factor contributing to reduce the host defense mechanisms.