Drug and Disease Model Resources: A Consortium to Create Standards and Tools to Enhance Model-Based Drug Development

Model-based drug development (MBDD) is accepted as a vital approach in understanding patients' drug-related benefit and risk by integrating quantitative information integration from diverse sources collected throughout drug development.1 This perspective introduces the activities of the Drug and Disease Model Resources (DDMoRe) consortium, founded in 2011 through the Innovative Medicines Initiative Joint Undertaking (IMI-JU)2 as a European public–private partnership to address a lack of common tools, languages, and standards for modeling and simulation (M&S) to improve model-based knowledge integration.

ESTABLISHING A DRUG AND DISEASE MODEL LIBRARY
A drug and disease model library is being created to store and promote the reusability of models. DDMoRe has identified a number of high interest disease areas to exemplify the implementation of its language standards within the open source interoperability framework. A collection of diabetes and oncology models, both existing and requiring development, were selected for their value in drug development. Thirty published pharmacokinetic, pharmacodynamic, pharmacokinetic-pharmacodynamic, physiologically based pharmacokinetic, disease and systems pharmacology/biology models covering differing phases of the drug-development process will initially be stored in the library and new models in key therapeutic areas added over time. In each therapeutic area, relevant models were identified from the literature, but gaps were also highlighted in model coverage which will direct new model development over the course of the DDMoRe project.

DRAFTING A NEW MODEL DESCRIPTION LANGUAGE
The model coding language (MCL) has been conceived to provide a single, unified language that can capture the definition of models together with their parameters and data attributes, irrespective of the target software for performing the M&S task.
The MCL will bring together features of various languages to code models and is intended to be flexible, extensible and easy to code, understand and use. This will reduce rework and recoding in passing models between software platforms thereby helping to promote consistency and reusability of models and propagate knowledge throughout the drugdevelopment process.
Forming a model description language, the MCL is combined with a complementary task execution language (TEL), which describes how the input from the MCL will be used to generate a desired output. The TEL will allow connecting the users' modeling aims, the immediate task of execution of a modeling-related task, and the overarching analysis workflow within the interoperability framework. Specifications for both aspects of the model description language (MCL and TEL) have been drafted, reviewed, and will be made public shortly, but a depiction of the key components constituting the model description language is given in Figure 2.

DEVELOPING AN OPEN SOURCE INTEROPERABILITY FRAMEWORK
For the DDMoRe project to be successful, a platform must be developed that enables the integration of existing software, tools, and desired modeling workflows, as well as improving the integration efficiency for new methodologies.
There will be mechanisms to translate the new modeling language into the specific languages of different softwares such as NONMEM, Monolix, R, and WinBUGS. Once basic commands are supported, an engine will be incorporated that orchestrates the execution of complex modeling workflows, involving simulation of trial designs, parameter estimation, derivation of inferences, and analysis reporting. The framework will be extended to allow interactions with the modeling Ontology KnowledgeBase server that will provide assistance with the annotation of models to facilitate sharing and reuse.
The infrastructure of the framework will also comprise a distributed tool execution framework, connectors within this framework, and rule sets that define constraints on how the tools will be executed (exemplified illustration of process flow given in Supplementary Figure S1 online).

CREATING A MODELING FRAMEWORK PROTOTYPE
To demonstrate and facilitate the development of the languages, examples were defined addressing various aspects of the modeling workflow. Although simpler models and modeling tasks will be illustrated using warfarin pharmacokineticpharmacodynamic models, more complex models involving integrated physiological systems, disease progression, categorical and time-to-event data, and multilevel random effects models will be illustrated in the oncology, diabetes, and Alzheimer's disease areas. All examples will exercise the main integration points of the framework, namely running an analysis workflow that will allow execution and manipulation of modeling tasks. The prototypes will also be used to address other relevant tasks, such as meta-analyses, simulation, and optimal design problems.
An initial prototype has been developed that provides a client for modeling with MCL objects, and a server for the execution of corresponding workflow tasks.

CREATING STANDARDS IN MARKUP LANGUAGES, METADATA FRAMEWORKS, AND ONTOLOGIES
To facilitate interoperability of data, models, and related metadata in storage and system-to-system interchange, there is a need for a common set of comprehensive standards. The needs of MBDD were explored in terms of prevailing standards, with respect to markup languages (ML) that allow annotation of documents, controlled vocabularies (ontologies), and model components. A relevant set of MLs was identified (SBML, SED-ML, NuML, CellML, and UncerML) and evaluated to determine whether they could form part of a set for DDMoRe. Beyond providing the modeling community with a complete set of interoperable MLs, with the first schema to be made publically available in March 2013, the eventual goal is to create a coherent ontology-based solution in support of classification and reasoning over data and variable types in databases and model libraries.
To achieve external acceptance and compatibility of the developed ontology and meta standards, collaborations have been established with the Clinical Data Interchange Standards Consortium, the Ricordo 4 project to scope the integration of semantic relationships within the framework, the Pistoia Alliance to achieve endorsement, and the IMI consortia Open PHACTS (http://www.openphacts.org) and EHR4CR (http:// www.ehr4cr.eu) to ensure compatibility of standards.

INTEGRATING EXISTING MODELING SOFTWARE INTO THE OPEN SOURCE INTEROPERABILITY FRAMEWORK
DDMoRe will integrate some existing softwares such as NONMEM, Monolix, and WinBUGS, and various R and To describe models, data, metadata, relations, tasks, and designs A single, unified language to capture the definition of models and modeling tasks A modular platform for integrating tools and reusing models to remove barriers and promote consistency A collection of specific disease area models to make quantitive knowledge accessible and sharable

Figure 2
Modeling description language (MDL). Although the MCL will allow the user to define new models, the TEL will provide the functionality to update the objects and execution settings and handles the modeling tasks. MCL, model coding language; TEL, task execution language. Matlab-based tools into the interoperability framework. It will also provide means to integrate other existing softwares by adopting standards for the design, analysis, and interpretation phases of the M&S workflow and embedding the application programming interface and modeling language developed.

DEVELOPING NEW MODELING APPROACHES AND SOFTWARE
The participation of numerous partners provides DDMoRe with the unique opportunity to integrate existing modeling tools and address gaps in the current array of modeling software tools and related methodological aspects to facilitate MBDD.
Clinical trial simulation is at the center of quantitative decision making to derive an optimal drug development path for future trials or optimal portfolio management in the pharmaceutical industry. DDMoRe is developing a clinical trial simulation engine to address most realistic trial scenarios, for which a prototype has already been released, and has explored demand among industry partners for adaptive optimal design methodologies to increase trial informativeness. New methodologies for parameter estimation in complex models, new strategies for covariate and random effects modeling, and new diagnostics for time-to-event data and random effects components have already been developed and are among the 16 DDMoRe scientific contributions to the 2012 PAGE conference. 5

TRAINING AND EDUCATION
An initiative such as DDMoRe can only show its potential if it is accompanied early by a comprehensive training program to influence the drug-development environment in a persistent way. Training will cover methodological and technological aspects of the project, and model-based training exercises will cover a number of drug-development scenarios across a variety of therapeutic areas. To this end, a survey on the technical and conceptual requirements in drug/disease M&S was conducted to identify opportunities for training and education purposes and will be published shortly.

OUTLOOK
This is an abbreviated summary of the activities of DDMoRe for the first of the 5 years of this partnership. It is by no means a complete detailed account of all deliverables and tangible outcomes, which will be provided through subsequent publications.
It was a considerable effort to bring more than two dozen organizations into a common enterprise, with many more colleagues being actively involved in the various developments, but it is also important to notice that this initiative can only succeed on its set goals if the community not directly involved is kept informed, allowed to comment, and interact. In this regard, we conducted an Advisory Board meeting with external stakeholders to gain feedback on plans and progress. Because external acceptance and contribution is vital for the long-term success of this initiative, we will continue to engage with stakeholders and the community by making outcomes public at http://www.ddmore.eu and through communications at conferences and workshops.


extfieldsofstudy: Biology Medicine

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Genes involved in pancreatic islet cell rejuvenation

Pancreas plays an important role in maintaining the glucose homeostasis. The deterioration of β-cells in the pancreas is a crucial factor in the progression of diabetes mellitus; therefore, the restoration of β-cell mass and its function is of vital importance for effective therapeutic strategies. The precise mechanism for increase in functional β-cell mass is still unknown. This review focuses on the importance of certain genes which are involved in the rejuvenation of pancreas. These genes are divided according to their functions into three categories: participate either in proliferation (mitotic division of differentiated β-cells), neogenesis/transdifferentiation (development from precursor cells) or inhibition of β-cell apoptosis (programmed cell death). The rate of β-cell rejuvenation is the balance among the rates of β-cell proliferation, neogenesis and apoptosis. Understanding these genes and their pathways may lead to the discovery of new drugs, target based gene delivery and development of safer antidiabetic drugs.

Introduction
Diabetes is a major cause of health concern in the world and is growing in epidemic proportions. It is assumed that in the next ten years it will become number one disease of the world 1 . Type-1 diabetes mellitus (T1DM) is an autoimmune disease while type 2 is mostly a lifestyle disease. Majority of people suffer mainly due to type-2 diabetes and is responsible for the current diabetes explosion. The detection early markers for the disease and its prevention is an active area of research to develop target based novel drugs.

Dysfunctional pancreas in diabetes
Insulin, a key polypeptide hormone secreted by the pancreas, targets several tissues for the utilization of glucose and thus maintains the glucose homeostasis.
Type 2 diabetes mellitus (T2DM) develops from a combination of genetic and acquired factors (such as changes in metabolic homeostasis) that impair β-cell function on one side, and tissue insulin sensitivity on the other 2,3 . Normally, β-cell mass can adapt to changes in metabolic homeostasis. Recurrence of these changes in metabolism creates a stress on pancreas often predating the on-set of T2DM by many years. This pancreatic stress causes β-cell mass expansion, through enhanced proliferation and neogenesis. The progression from this stress condition to a state of diabetes is inevitably associated with a decrease in the β-cell mass 2-4 . This β-cell loss arises due to an increase in β-cell apoptosis, which clearly outweighs replication and neogenesis.
The war against diabetes through the development of new drugs is an ongoing continuous process 5 . With Table I. Genes involved in beta cell proliferation

Genes proteins Functions References
Reg gene family (RegI, II, IIIα, IIIβ, IIIγ) Increase islets cell size and density, Regeneration of pancreas 16,22,24,25,27 Sox9 Stimulates proliferation and survival of pluripotent progenitors. 28 Hnf - Figure) also contributes for regeneration of pancreas in which gene/gene products play in conjunction either with (1) or (2). All these mechanism may contribute synergistically for the regeneration of pancreas. See Tables 1, 2 and 3 for gene/gene products involved in the above processes for pancreatic rejuvenation. Reproduced with permission from Nature Publishing Group, London, UK 6 .
the technological advancement, efforts are being made to rejuvenate the pancreatic cells or create artificial pancreas. Pancreatic rejuvenation can happen either due to proliferation of existing β-cells or differentiation of progenitor cells to β-cells 6 (figure), or due to decrease in β-cell apoptosis.

β-Cell proliferation
Islets regeneration refers to an increase in β-cell mass by proliferation and replication of existing islet cells. Several mouse studies 7-9 here shown that β-cells do not proliferate, however, lineage tracing studies [10][11][12][13][14] have confirmed that human β-cells proliferate and give rise to a population of progenitor/stem cell. Various genes and transcription factors are involved in this process viz. Reg (Regenerating islets derived proteins), Sox9, Hnf-6, NeuroD1, Neurogenin-3 and Netrin-1 (Table I). Besides these genes, certain peptides or their analogues such as glucagon like peptide-1/exendin-4 are also involved in islet regeneration. These observations are confirmed by using dipeptidyl peptidase (DPP) IV inhibitor sitagliptin in mice 15 .
So far, five REG proteins have been reported in humans that belong to Reg gene family. Some of the members of this family have been implicated in β-cell replication and/or neogenesis as shown in in vivo studies using transgenic and knockout mice 16 . These also preserve the β-cell mass in autoimmune type 1 diabetes 17 . This Reg family of genes are expressed in both young and old mice that were subjected to partial pancreatectomy 18 . In isolated rat islets, Reg1 mRNA levels were significantly increased by glucose, amino acids, foetal serum or specific growth factors such as insulin, growth hormone and platelet-derived growth factors (PDGF) 19 . PDGF receptor signalling controls age-dependent β-cell proliferation in mouse and human pancreatic islet cells 20 . Disruption of RegI gene resulted in a significantly decreased rate of DNA synthesis and diminished β-cell hyperplasia in response to obesity, confirming the role of endogenous RegI in islets cell growth 21 . A study conducted by Huszarik et al 22 showed upregulation of RegII during diabetogenic process and also after adjuvant therapy in NOD mice. While all Reg family mRNAs can be detected from total pancreas, RegII and RegIIIα genes have been detected in pancreatic islet cells as confirmed by immunofluorescence 23 and RegIIIα expression was remarkably increased during pregnancy in rats 24 . Mice overexpressing RegIII β was resistant to streptozotocin induced diabetes mellitus 25 . RegIIIγ, another member of Reg family of genes is also found to be involved in regeneration of pancreas. REG III protein was found to be expressed only in regenerating islets and not in normal rat pancreas 26 and its gene expression level induced 10-100 folds on day 3 of pancreatectomy 27 . These data suggest that there is a strong link between Reg gene family and rejuvenation of pancreatic islets.

Transcription factors in β-cell proliferation
Certain transcription factors (Sox9, Hnf-6, Ngn-3 and NeuroD1) are also found to be involved in the proliferation of β-cells. SOX9 is the first specific marker and maintenance factor of multi-potential progenitors during pancreatic organogenesis. SOX9, in the embryonic pancreas stimulates proliferation and prevents apoptosis of pluripotent progenitor cells. It controls pancreatic progenitor cell maintenance by modulating Notch signal transduction. The phenotypic alterations in the Sox9-deficient pancreas shows a striking resemblance to the pancreatic defects associated with mutations in components of the Notch signalling pathway, thus establishing a possible link between Sox9 and the notch signal transduction pathway for stem cell maintenance 28 . The hepatocyte nuclear factor 6 (Hnf-6), homeodomain-containing transcription factor, is an important regulator of endocrine development. HNF6 is expressed in early pancreatogenesis in all endodermally derived cells, but is not detected in differentiated endocrine cells at late-gestation 29 . Hnf-6 null mice embryos showed impaired endocrine differentiation and perturbed duct morphogenesis during embryogenesis 30 . In addition to defects in endocrine development, Hnf-6 null embryos showed defects in duct development 31 . Loss of Hnf-6 from Ngn-3 expressing cells did not affect β-cell function or glucose homeostasis suggesting that Hnf-6 is dispensable for later events of endocrine differentiation. These data confirm that HNF6 has both early and late functions in the developing pancreas and is essential for maintenance of Ngn-3 expression and proper pancreatic duct morphology 32 . NeuroD1, a downstream target of Ngn-3, carries on the endocrine differentiation programme initiated by Ngn3 and participates in the maintenance of the differentiated phenotype of the mature islet cells 33 . During pancreatic endocrine development, Ngn-3 acts early to determine endocrine cell fate, while NeuroD1 directs endocrine cell differentiation 34 . At early stage of life, mice lacking a functional NeuroD1 (also called as BETA2) gene exhibit a striking reduction in the number of insulinproducing β-cells and failed to develop mature islets with a marked hyperglycaemia. Attempts to rescue the diabetic phenotype by administration of insulin were unsuccessful, suggesting that the mutant animals were unable to respond to insulin, have become insulin resistant, or perhaps contained additional defects 35 . Thus BETA-2 is required for the expansion of the pancreatic β-cells population, as well as other islet cell types which are involved in the development of endocrine cells into islets of Langerhans 34 .
Netrins are laminin-like diffusible chemotactic proteins involved in pancreatic morphogenesis and play a role in the regulation of duct-cell and foetal islet cell migration. In adult rat pancreas, Netrin-1 mRNA was practically undetectable. After duct ligation, its expression was very low in the head part of the pancreas whereas it was strongly upregulated in the tail part at 3 rd , 5 th and 7 th day of post-ligation with the maximum expression on day 5 36 . Netrin-1 mRNA was found to be expressed by islet cells and exocrine cells with ductal characteristics. These observations suggest that Netrin-1 plays a role in pancreatic morphogenesis, both prenatally and in the regenerating adult rat pancreas.

Transdifferentiation of pancreas
Islet neogenesis specifically refers to an increase in β-cell mass via transdifferentiation of adult pancreatic stem cells, putatively found in the ductal epithelium or acinar tissue. Trans-differentiation involves in the conversion of alpha or delta cells of the pancreas into insulin producing β-cells. Various genes/proteins contribute to this process. These include INGAP, Gastrin,MafA,Foxa2,Nkx2.2,Nkx6.1,Pax4,etc. (Table II).
INGAP (islets neogenesis associated protein) is a member of the C-lectin protein family that serves as the initiator of a cascade of events that culminates in islet neogenesis and can reverse diabetes in streptozotocininduced diabetic C57BL/6J mice 37 . These studies were further confirmed in beagle dogs 38 . There was also a significant increase in insulin gene expression in the INGAP treated animals. INGAP is also found in human pancreas during pathological states involving islet neogenesis which further suggests that INGAP is of primary importance in the process of islet neogenesis 39 . Gastrin, a classical gut hormone secreted by G cells in the stomach lining is found to stimulate pancreatic β-cell neogenesis. Intravenous infusion of gastrin into the ligated duct cells, resulted in a doubling of the β-cell mass in rats 40 , due to high expression of gastrin/ cholecystokinin (CCK) B receptors in duct ligated cells 41 . These observations were confirmed using EGF plus gastrin combinatorial therapy, which showed an increase in insulin positive cells in human islet 42 . In another study using GLP-1 and gastrin in NOD mice, there was a significant reduction in blood glucose due to increase in pancreatic β-cell mass and insulin content 43 . MafA a member of Maf subfamily of leucine zippers is capable of strongly activating the insulin promoter. Maf factors play important roles in cellular differentiation of exocrine cells to β-cells 44 . MafA is restricted to β-cells and known to be important in the embryonic development of pancreas 45 . It has been observed that MafA expression is decreased during the diabetic condition 46 .

Transcription factors in pancreatic neogenesis
There are several transcription factors involved both in neogenesis and replication. However, it is not clear whether these work alone or in combination with other transcription factors in a coordinated manner. The list of the transcription factors and their role in transdifferentiation is summarized in Table II. Pancreatic duodenal homeobox-1 (Pdx-1), a homeobox transcription factor, besides being involved as a regulator of pancreatic development (the differentiation and gene expression in the β-cell) 47 , Pdx1 also turns out to be a major player in the maintenance of an adequate pool of healthy β-cells in adults 48,49 . It maintains the homeostasis between β-cell neogenesis and apoptosis. In mice with a 50 per cent reduction in Pdx1, the isolated islets showed more susceptibility to apoptosis at basal glucose concentrations along with impaired ability to maintain β-cell mass with age. Its expression is shown to be down-regulated during hyperglycaemic condition 50 . The survival functions of Pdx1 may be mediated by insulin/IGF signaling acting through the forkhead transcription factor Foxo1 (forkhead/winged helix transcription factor). Foxa2 (formerly known as Hnf-3) is a key regulator of foregut development that plays an essential role in the cell type-specific transcription of the Pdx-1 gene in the pancreas 51 . On deletion of Foxa2 in mice, there is a significant downregulation of Pdx-1 mRNA 52 . This shows that  Gastrin Induces islet β-cell neogenesis from pancreatic exocrine duct cells. 40

Nkx6.1
Maintaining & expanding population of β-cell precursors as these progress from precursors to differentiated β-cell.

Pax4
Expressed in endocrine cell progenitors and directs formation of β and delta cells.
is an essential upstream factor that regulates Pdx-1 mRNA levels in β-cells.
Nkx2.2 is another essential pancreatic transcription factor that affects the expression of ghrelin during pancreatic development 53 . Nkx2.2-null mice lose all β-cells and the majority of the α-cells; and the islet is predominantly populated by gherlin expressing cells. The discovery that ghrelin cells often replace the other endocrine populations in the pancreas 54 suggests a lineage relationship between the ghrelin producing epsilon cells and other hormone-producing populations 55 . In another study, disruption of the Nkx2.2 gene has shown to lead to the accumulation of incomplete differentiated β-cells that express some β-cell markers, but not insulin 56 . This illustrates the role of Nkx2.2 in pancreatic endocrine cell differentiation. The phenotypic effects of Nkx2.2 mutant mice may in part result from the loss of other homeodomain transcription factor Nkx6.1. In pancreas, Nkx6.1 also follows an expression pattern similar to that of Nkx2.2; but restricted to the β-cells alone 57 . Deletion of the Nkx6.1 gene in mice caused a marked reduction of β-cells. In the same study, with the double knockout animal model (Nkx2.2/Nkx6.1) showed the same phenotype as of Nkx2.2 single mutant 58 . This shows that Nkx6.1 functions downstream of Nkx2.2 in pancreatic development. The paired homeobox transcription factor Pax4 appears to be a strong candidate for specifying β-cell lineages. Mice deficient in Pax4 fail to develop beta and delta cells within the pancreas suggesting that Pax4 expression commits selected endocrine precursors towards the beta and delta cell lineage 59 . The Pax4 genes are expressed during the embryonic pancreatic development, but later on these are restricted to β-cells alone. Inactivation of Pax4 in mice results in the improper maturation of alpha-and β-cells 60 . This indicates the role of PAX4 in the maintenance of progenitors and in maturation of β-cells.

Inhibition of β-cell apoptosis
Under normal circumstances, apoptosis is highly regulated to maintain normal physiological function of the cells. In diabetes, during excess stress, the pancreatic cells not only undergo apoptosis but also become necrotic and are unable to secrete insulin. A study conducted by Butler et al 61 indicated that increased apoptosis rather than decreased neogenesis/ proliferation might be the main mechanism leading to reduced β-cell mass in T2DM. Thus, decrease in the rate of apoptosis itself, may increase the β-cell mass via proliferation. Several genes/proteins are involved in pancreatic apoptosis and their functions are summarized in Table III. Perforin (pore forming protein) is a cytolytic protein, initiating apoptosis by inducing minimal cell membrane damage while effectively releasing Table III. Genes involved in inhibition of beta cell apoptosis

Pdx-1
Granzymes Possesses anti-apoptotic activity that helps facilitate the maintenance of β-cell mass.
A serine protease, that activates Bid (a pro-apoptotic factor) essential for death-receptor induced apoptosis of islets. Thioredoxin-interacting protein (TXNIP) It is a pro-apoptotic factor that plays an essential role in glucose toxicity-induced β-cell apoptosis.

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granzymes from the endosomal compartment into the cytosol 62 . Of the granzyme family, granzyme A and B are the most common in human and mouse 63 . Granzyme A induces single-strand DNA breaks, while granzyme B cleaves specific substrates including caspases and the pro-apoptotic molecule Bid 64 . Activated Bid is targeted to the mitochondria where it sequesters antiapoptotic members of the Bcl-2 family, allowing the oligomerization of Bax and/or Bak which mediates loss of mitochondrial outer membrane potential, release of cytochrome C and causes irreversible apoptosis 65 . During hyperglycaemic condition its expression is elevated in β-cells, thus increasing the rate of apoptosis 51 . Deficiency in Bid prevents β-cells from undergoing mitochondrial apoptotic pathway 66,67 . Caspase-3 has been extensively studied in various tissues due to its role as the principal executioner of apoptosis 68 . As such, Caspase-3 is an attractive target to inhibit apoptosis in diseased conditions including diabetes 69 . Caspase-3 null (Casp3 _ / _ ) mice were found to be protected from developing diabetes in a multiplelow-dose streptozotocin autoimmune diabetic model 70 . This illustrates the importance of Caspase-3 in β-cell death and its activity is found to be increased during the diabetic conditions. An attractive regulator of β-cell replication and survival after birth is Survivin. This protein blocks the functions of Caspases in the mitochondria-dependent cell death pathway, protecting cells from apoptosis 71 . Deletion of Survivin within the mice endocrine pancreas results in diabetes manifested by hyperglycaemia and polyuria. Exogenous expression of Survivin in streptozotocin -induced diabetic model protects the β-cells from apoptosis 72 . Thus, it may play a role in the replication and/or survival of matured β-cells 73 . Surprisingly it is modestly upregulated in diabetic patients 74 which may help to assuage diabetes.
Beside survivin, there are other proteins present in pancreas which prevent apoptosis. BCL-xL, an anti-apoptotic protein coded by the "survival gene", is involved in the inhibition of apoptosis. Marked overexpression of Bcl-xL, resulted in a severe defect in insulin secretion and hyperglycaemia in transgenic mice 75 . Under conditions of stress, β-cells require BCL-xL to maintain their survival in vivo 48 . Myc is a potent inducer of both β-cell proliferation and apoptosis in vitro 76 . Myc sensitizes cells to a variety of apoptotic triggers rather than directly inducing apoptosis by itself 77 . Sustained Myc activation leads to initial hyperplasia and increased apoptosis later on, suggesting that apoptosis ultimately predominates over proliferation 78 . In chronic hyperglycemia, an increase in the expression of Myc was observed in pancreas 79 . A thioredoxin-interacting protein (TXNIP), a regulatory protein, is involved in the inhibition of thioredoxin and thereby modulates the cellular redox state and promotes oxidative stress 80 . Its overexpression in β-cells is found to induce apoptosis 81 , and the process involves the activation of intrinsic mitochondrial pathway, while the Endoplasmic reticulum (ER)mediated cell death remains unaffected. It was demonstrated that their mRNA expression levels are elevated during the diabetic conditions 82 . In a recent study, huntingtin-interacting protein  is found to posses anti-apoptotic property of β-cells. This also plays an important role in glucose-stimulated insulin secretion 83 . Bace2 (Beta site amyloid precursor protein cleaving enzyme 2), a proteolytic enzyme acts as a negative regulator of β-cell mass via inhibiting Tmem27 expression. Overexpression of Tmem27 or ablation of Bace2 leads to an increased β-cell mass by inhibiting apoptosis 84 .

Future prespective
Several lifestyle diseases such as T2DM, obesity are increasing significantly. It has been predicted that in near future diabetes will overtake all the current infectious and non-infectious diseases. In spite of rapid progress in our understanding regarding the pathophysiology of diabetes, challenges remain high due to increased complexity of the disease contributed by genetic and environmental factors. In future, there need to be more emphasis on the prognosis and better treatment for diabetes associated diseases, and on the discovery of reliable biomarker(s) for its early detection. Development of new technologies i.e. rejuvenation of the pancreas by small molecules or gene targeted drug delivery, pancreatic transplantation, stem cell therapy and creating artificial pancreas, etc. will be the mainstay of the diabetes research. Thus, it becomes essential to have better understanding of the various pathways at the molecular level that are involved in pancreatic islet cell rejuvenation.


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Epithelial-Mesenchymal Transition Associates with Maintenance of Stemness in Spheroid-Derived Stem-Like Colon Cancer Cells

Despite earlier studies demonstrating characteristics of colon cancer stem cells (CCSCs) and the role of epithelial-mesenchymal transition (EMT) in tumor development, it remains controversial as to the relationship between CCSCs and EMT. In this study, in order to present an insight into this relationship in colon cancer, we developed HCT116 and HT29 sphere models, which are known to be the cells enriching cancer stem cells. Compared to their parental counterparts, spheroid cells displayed lower homotypic/heterotypic adhesion but higher in vitro migratory/invasive capacity, as well as higher tumorigenic and metastatic potential in vivo. The spheroid cells also demonstrated down-regulated E-cadherin and up-regulated α-SMA and Vimentin expression, which is the typical phenotype of EMT. In order to explore whether this phenomenon is associated to activation of Wnt/β-catenin pathway, we detected several key signaling molecules. Compared with their parental cells, HCT116 and HT29 spheroid cells demonstrated down-regulated expression of GSK3β, but up-regulated expression of Slug and Snail. And also, the up-regulation of nucleus β-catenin in spheroid cells indicated that the free β-catenin transferred from cytoplasm to cell nucleus. Our findings indicate that spheroid cells have the characteristics of colon cancer stem cells, and EMT may account for their stemness and malignancy. And persistent activation of Wnt/β-catenin pathway may play an important role in the EMT of CCSCs.

Introduction
Colorectal cancer is the third leading cause of cancer deaths worldwide, and the 5-year relative survival rate is only 53.8-65.2% despite diagnostic and therapeutic advances [1,2]. Tumor recurrence and metastasis are two critical survival-influencing factors of colorectal cancer (CRC). There is a growing understanding that epithelial-mesenchymal transition (EMT) contributes to tumor invasion and metastasis [3,4]. As we all know, EMT is a highly conserved cellular program that allows polarized, welldifferentiated epithelial cells to convert to unpolarized, motile mesenchymal cells. This process is considered to promote colorectal cancer cell to invade the basement membrane and the surrounding microenvironment, such as the lymph and blood vascular systems, ultimately contribute to intra or extravasation [5].
Recently, increasing evidence suggests that tumor initiation and metastases are dependent on a small sub-population of tumor cells termed cancer stem cells (CSCs) bearing infinite self-renewal potential and the capacity to differentiate into diverse populations comprising a tumor [6,7,8,9]. According to this model, cancer stem cells sustain carcinogenesis, angiogenesis, metastasis, and recurrence process of colorectal cancer [10]. In other words, cancer stem cells are in charge of the malignancy of colorectal cancer. But how do the cancer stem cells maintain their stemness, such as ability of migration, invasion, and metastasis? Many researchers observed that some cancer cells (such as breast cancer, colon cancer, etc.) can obtain the characteristics like cancer stem cells through epithelial-mesenchymal transition [11,12,13,14]. That is to say, EMT may assign cancer cell the stem-like bionomics, which indicates that EMT may participate in the maintenance of stemness of CCSC. But the detailed relationship between cancer stem cells and EMT has not been reported. Meanwhile, prevalent activation of Wnt/b-catenin signaling pathway in sporadic CRC, is relevant to EMT [15]. The current study aimed to demonstrate if the bionomics of colon cancer stem cells and its maintenance of stemness are related with EMT or not, and illustrate the role of Wnt/b-catenin signaling pathway in this process.

Ethics Statement
All experiments involving human participants (including the collection of human colon cancer samples) have been approved by the Medical Research Ethics Committee of Sun Yat-sen University, and conducted according to the principles expressed in the Declaration of Helsinki. All participants involved in the study signed the informed consent forms. And all animal experiments were conducted according to relevant national and international guidelines. And this project was approved by the Medical Research Animal Ethics Committee of Sun Yat-sen University.
Cell culture HCT116 (ATCC, CCL-247) and HT29 (ATCC, HTB-38) colon cancer cell lines were maintained in DMEM/F12 supplemented with 10% FBS, 200 U/ml penicillin and 200 mg/ml streptomycin. Tumorsphere media (also called as serum free medium, SFM) was composed of DMEM/F12 media supplemented with 16B27 (Invitrogen), EGF (20 ng/ml, Peprotech), bFGF (10 ng/ml, Peprotech), routine insulin (5 mg/ml, Invitrogen), 200 U/ml penicillin and 200 mg/ml streptomycin. For 3D floating culture, HCT116 and HT29 cells grown in two dimensional monolayer were digested with trypsin, resuspended, and then seeded at a density of 2610 6 cells in SFM in 100 mm ultra-low attachment dishes (Corning) at 37uC in a humidified 5% CO2/95% air atmosphere. The cells derived from monolayer cultures and suspension spheres on day 7 after primary culture were detected for the expression of CD133. The cells were washed twice in cold PBS, and subsequently cell suspensions were incubated at 4uC with 1:10 FITC-conjugated mouse monoclonal antihuman CD133 antibody (Ab, Miltenyi biotec) for 45 minutes in the dark. After incubation, the cells were washed twice in cold PBS with 1% BSA and resuspended in 400 ml cold PBS with 1% BSA for flow cytometry analysis within 1 hour (h).

Immunofluorescence staining
The anti-Lgr5 Ab (Abcam), anti-CK20 Ab, anti-E-cadherin Ab, anti-b-catenin Ab, anti-a-SMA Ab and anti-Vimentin Ab (Santa Cruz Biotechnologies) were prepared according to the Manufacturer's protocols. Cells were fixed with 4% of paraformaldehyde and permeabilized with 0.05% of Triton X-100 in PBS at room temperature for 20 min. Samples were blocked with 1% of bovine serum albumin (Sigma) and incubated with appropriate primary antibody at 37uC for 1 h. After washing extensively, they were incubated with Alexa Fluor-488 goat anti-mouse IgG (Invitrogen) or Fluor-Cy3 goat anti-rabbit IgG (Jackson Immunoresearch) at 37uC for 1 h. Counterstaining of nuclei with DAPI (Invitrogen) was also performed. Cells were then washed and mounted for observation under a scanning confocal microscope (LSM-710, Zeiss). Here, throughout this manuscript, data from at least three independent experiments have been analyzed to verify reproducibility of results.

Homogeneity and heterogeneity adhesion assay
The adhesion ability of cells to ECM was tested using fibronectin (FN)-coated 96-well plates (Corning). Single-cell suspensions of spheroid and adherent cells were plated (10 5 /well), and incubated at 37uC for 2 h, then washed with PBS to remove the non-adhesive cells. Delt OD of 570 nm wavelength was determined to reflect the FN-adherent cells using Cell Counting Kit-8 (Dojindo, Japan). The adhesion ability of cells to homogeneous cells was tested using monolayer cells-paved 96-well plates. Spheroid and adherent cells were plated at 10 5 per well, and incubated at 37uC for 60 min, 90 min, and 120 min. Then the non-adhesive cells were counted and homotypic adhesive activity was calculated using the formula: homotypic adhesion (%) = (total cell number -non-adhesive cells)/total cells6100%.

Migration and invasion assays
Migration and invasion assays were performed in 24-well transwell chambers with 8 mm-pore polycarbonate filter inserts (Corning). A total of 5610 4 or 10 5 dissociated spheroid or adherent cells were seeded on uncoated or Matrigel-coated inserts in 100 ml of serum-free medium inserts for migration or invasion assays respectively. The lower chambers were filled with 0.5 ml of 10% FBS-supplemented DMEM/F12 medium. After 24 h and/or 48 h, cells on the upper side of the filter were removed and the cells on the lower surface of the insert were fixed and stained with crystal violet. The number of stained cells was counted under a light microscope. Assays were performed in triplicates.

Western blot analysis
GSK3b, b-catenin, Slug, and Snail expression were analyzed in total cell extracts or in nucleus extracts separated by 10% SDS- polyacrylamide gel electrophoresis and transferred to Polyvinylidene Fluoride Membrane (Millipore). The membranes were washed in Tris-buffered saline with Tween (TBST, composed of 10 mM Tris-HCl, pH 8, 150 mM NaCl, and 0.05% Tween 20), blocked 1 h at room temperature with 5% nonfat milk in TBST, then probed 1 h at room temperature with anti-E-cadherin Ab, anti-a-SMA Ab, anti-Vimentin Ab, anti-GSK3b Ab, anti-bcatenin Ab, anti-Snail Ab (Santa Cruz Biotechnologies), anti-Slug Ab (BD Pharmingen), anti-a-Tublin Ab and anti-GAPDH Ab (Peprotech). After incubation with horseradish peroxide-conjugated secondary antibody, immunoreactive proteins were detected by ECL detection system (Millipore). Quantitative analyses of immunoblotting signals were obtained via densitometry analysis using LAS4000 Image Software (Fuji Film). Protein concentrations were determined using the BCA protein assay kit (ThermoScientific Biosciences).

In vivo tumorigenesis
To determine whether spheroid cells are more tumorigenic than their adherent counterparts in vivo, we carried out tumor development and liver metastasis experiments. Single cells were resuspended in PBS. A 100-ml suspension containing 2610 6 adherent cells or spheroid cells were injected subcutaneously (s.c.) into the flanks of 4-to 6-week-old male BALB/C-nu mouse, obtained from the Jackson Laboratory. Subcutaneous tumor diameters were measured with a digital caliper every two or three days, and tumor volume in mm 3 was calculated using the formula: Volume = width 2 6length60.52. Then 3 or 4 weeks later, the mice were killed and frozen tissue sections (6 mm) of subcutaneous tumors were made for pathological detection.

In vivo liver metastasis
Liver metastatic model was used to determine the metastatic potential of spheroid cells. Liver metastases were established by intrasplenic injection of 4610 6 HCT116 spheroid or adherent cells and splenectomy was performed 10 min after intrasplenic injection to avoid spleen metastasis. The overall health condition was recorded in detail once a day. Liver metastasis foci were examined and measured. And tissue section was made for hematoxylin-eosin staining to confirm the pathological source.

Statistical analysis
Statistical significance was determined by one-way ANOVA followed by Bonferroni post-hoc test for multiple comparisons or Student's t-test. For all tests, P,0.05 or ,0.01 was considered statistically significant or very significant.

Morphological and biological characterization of colonospheres
When cultured in serum free medium supplemented with 16B27, 20 ng/ml EGF and 10 ng/ml bFGF, both HCT116 and HT29 colon cancer cells grow in large round, unattached floating spheroid colonies (termed colonospheres) (Figure 1). Morphologically, the normal polarity of adherent cells vanished in colonspheres. Then we determined the frequency of sphere-forming cells by performing an extreme limiting dilution analysis (ELDA) for parental and spheroid-derived HCT-116 cells. The frequency to form colonsphere was found to be 5.5 fold higher in spheroid cell than parental cell line, indicating that the spheroid cell had higher self-renew capacity.
The spheroid cells showed enhanced expression of colon CSC markers-CD133 and Lgr5, but attenuated expression of differen-tiated epithelial cell marker CK20 compared to their corresponding parental cells. The proportion of the CD133 positive cells was determined by flow cytometry in parental and spheroid cells, they were found to be more than 80% in colonospheres, as opposed to #1% observed in parental cell lines (Figure 1). When the colonospheres were subjected to immunofluorescence staining for Lgr5, we observed bright Lgr5 staining on the surface of each spheroid cell indicating the presence of CSCs in colonosphere (Figure 1). The results suggest that CSCs were efficiently enriched in colonospheres. For multiple-differentiation assay, the spheroid cells were transferred serum-containing medium. Then the phenotype were detected after 4 weeks' culture. And we observed that the spheroid cells re-attached to the plastic and changed into adherent cells morphologically, along with down-regulated CD133/Lgr5 expression and up-regulated CK20 (not shown). The results suggest that spheroid cells have the capacity of multidifferentiation [10].

Spheroid cells display lower adhesion but higher in vitro migratory/invasive capacity
To corroborate malignant profile of the obtained colon cancer spheroid cells, we conducted in vitro assays to evaluate the adhesion and migratory/invasive capacity of spheroid cells in comparison with their adherent counterparts. The results of cell adhesion assay demonstrated that spheroid cells have lower capacities of both adhesion to FN (one of the key ECM components) and adhesion to their homogeneous neighbors, as compared with their parental cells (Figure 2A and 2B).
We found that spheroid cells display a significant increase in cell motility using transwell migration chambers. HCT116/HT29 spheroid cells showed a 3.8-fold (p,0.01) and 4-fold (p,0.001) increase in chemotactic potential at 24 h respectively, compared with their adherent counterparts ( Figure 2C). And also, significantly more HCT116/HT29 spheroid cells were able to invade Matrigel-coated inserts in Transwell migration chambers than their parental counterparts (p,0.001 and p,0.05, respectively) ( Figure 2D). These results indicate that colon cancer spheroid cells, compared to their adherent counterparts are endowed with lower adhesion, and higher migratory/invasive capacity, a functional phenotype associated with tumor aggressiveness.

Spheroid cells possess higher tumorigenic and metastatic potential in vivo
To determine whether spheroid cells are more tumorigenic and metastatic than their adherent counterparts in vivo, we carried out tumor development and liver metastasis experiments. When 2610 6 cells were inoculated subcutaneously into nude mice, all mice developed tumors. Transplanted tumors were confirmed as colon cancers with hematoxylin-eosin staining. And the volumes of subcutaneous tumors in spheroid cells groups were significantly higher than that of adherent cells groups ( Figure 3A, 3B and 3C).
As the liver metastasis was concerned, four mice (4/5) developed liver metastasis in HCT116 spheroid cells group, but only one mouse (1/5) did in adherent group ( Figure 3D). Metastatic tumors were also confirmed as colon cancers with hematoxylin-eosin staining ( Figure 3E). The result of liver metastatic foci counting shows the similar results ( Figure 3F). The overall conditions of the mice in spheroid cells group are worse than those in adherent cells group, and some of them developed ascites and severe emaciation. The results of in vivo experiments illustrated that the spheroid-derived cells possess higher tumorigenic and metastatic capacities than their parental counterparts. EMT-phenotype of spheroid cells associated with Wnt/bcatenin signaling pathway To demonstrate whether there were connections between the malignant profiles of spheroid cells and EMT, we detected their EMT-phenotype. The results of IF and Western Blotting showed that down-regulation of E-cadherin expression and up-regulation of a-SMA and Vimentin expression in HCT116 and HT29 colonospheres, compared with their parental cells (Figure 4 and 5). That is to say, the spheroid cells have the characteristic of mesenchymal cells. The activation of Wnt/b-catenin pathway is reported to induce EMT [16,17]. Then the key molecules in Wnt/ b-catenin pathway were detected using Western Blotting. Compared with their parental cells, HCT116 and HT29 spheroid cells demonstrated down-regulation of GSK3b expression but upregulation of Slug and Snail expression. And also, the upregulation of nucleus b-catenin expression in spheroid cells indicated that the free b-catenin transfer from cytoplasm to cell nucleus ( Figure 5).

Discussion
The concept of tumor heterogeneity, which presumed there were different phenotypes in the tumor tissue, was first proposed by Fidler IJ in the 70 s of the last century [18]. The cancer stem cell theory was shaped based on this concept. Cancer stem cells have been defined as ''cells within a tumor that possess the capacity for self-renewal and that can cause the heterogeneous lineages of cancer cells that constitute the tumor'' at a recent American Association of Cancer Research (AACR) workshop [19]. Up to now, the existence of cancer stem cells was confirmed in several solid tumors. And the recent identification of colon cancer tumor-initiating cells adds further support to the cancer stem cell hypothesis [7]. According to this theory, the small subpopulation of cancer stem cell bears the whole characteristic of malignancy, including invasion and metastasis.
There were several methods to obtain cancer stem cells. The extensively used method to isolate or enrich colon cancer stem cells is cell sorting based on cell surface markers, such as CD133, CD44, Musasai-1, etc [20]. Then, the dye-effluxing side population cells expressing ABCG2, an ATP-binding cassette halftransporter, were isolated as cancer stem cells [21,22,23]. In the current study, we obtained tumor spheres from HCT116 and HT29 colon cancer cell lines by culturing in serum-free medium [24,25]. And the following analysis indicated that CD133 and Lgr5 expression of these spheroid cells was higher than their parental counterparts, while CK20 expression was lower which represents the differentiated endothelial cell. And when were cultured in serum containing medium, the spheroid cells reattached to the plastic and displayed down-regulated CD133/Lgr5 and up-regulated CK20 expression (not shown). The results suggest that spheroid cells have the capacity of multi-differentiation. Moreover, the spheroid cells can form the same spheres, indicating that they have the self-renew capacity. From these results, we can draw the conclusion that cancer stem cells may be enriched in these spheroid cells.
We performed a series of functional experiments, in order to take a further insight into the biological behavior of these spheroid cells. Firstly, the results of adhesion assay showed that spheroid cells demonstrate lower capacities of both adhesions to FN (one of the key ECM components) and adhesion to their homogeneous neighbors. That was to say, this kind of cells are easier to detach from the ECM and to release from the bulk tumor. And also, spheroid cells display higher capacity of in vitro migration and invasion, as compared to their adherent counterparts. As we all know, capacity of migration and solubilization of basement membrane are mandatory for the metastasis of cancer cells. So spheroid cells can migrate to the chemotactic factor more easily. And they may have more facility of enzymes secretion to solubilize the basement membrane, and easier to invade the microvessel then migrate to other organs along with blood flow. To determine whether spheroid cells are more tumorigenic and metastatic in vivo, tumor development and liver metastasis experiments were carried out. And the results showed that the volumes of subcutaneous tumor in spheroid cells groups were significantly higher than that of adherent cells. And also, liver metastatic foci counting showed the similar results. The findings of in vivo experiments illustrated that the spheroid-derived cells possess higher tumorigenic and metastatic capacities than their parental counterparts.
The cells at the invasive fronts exhibit alterations in epithelial structure and function, leading to a dissolution of adherens junctions, loss of apical-basal polarity, loss of epithelial markers, reorganization of the actin cytoskeleton, induction of a mesenchymal gene-expression program and then enhanced motility and invasion: a process referred to as epithelial-mesenchymal transition (EMT) [26,27,28]. Generally, E-cadherin, b-catenin and a-catenin bind to form a complex. And the connection of E-cadherin-bcatenin-a-catenin complex with Actin cytoskeleton is very important for the maintenance of cell polarity and cell adhesion. So, the proper localization of E-cadherin to adherens junctions is very important for the establishment and stabilization of these intercellular junctions amongst HCT116/HT29 cells. This is an important function, especially in the context of tumorigenesis and metastasis, because a loss of E-cadherin on the cell surface has been shown to play a role in tumor progression and metastasis [16,29]. In our study, spheroid cells demonstrated down-regulation of E-cadherin, and up-regulation of a-SMA and Vimentin, and the latter two molecules are markers of mesenchymal cells. So, the spheroid cells display the phenotype of EMT according to our results. And the biological behaviors of spheroid cells consistent with the mesenchymal cells, such as nonpolar morphological character, lower adhesion but higher in vitro migratory/invasive capacity.
Recent studies have reported the pivotal role of Wnt/b-catenin signaling pathway in the self-renewal of epithelial stem cells [30,31]. In contrast, dysregulation of Wnt/b-catenin signaling pathway has been implicated in colon carcinogenesis [32,33]. It is reported that highly conserved Wnt/b-catenin signaling pathway closely associated with EMT [34]. Wnt/b-catenin signaling pathway contain many components, including Wnt proteins, Frizzled (Fz), Dishevelled (Dsh), APC/GSK3b/Axin complex, bcatenin, TCF/LEF transcription factor, and some target genes (cmyc, Cycline D, Survivin, Snail, Slug, etc.). Generally, in the absence of Wnt, the APC/GSK3b/Axin complex (destruction complex) resides in the cytoplasm, where it binds and phosphorylates b-catenin. The latter then leaves the complex to be ubiquitinated by b-TrCP (which binds to the phosphorylated 'degron' motif in b-catenin), is then degraded by the proteasome. In the condition of activation of Wnt/b-catenin signaling pathway, Wnt induces the association of Axin with phosphorylated LRP. The APC/GSK3b/Axin complex falls apart, and b-catenin is stabilized. The accumulated b-catenin in cytoplasm translocates to nucleus, and binds to TCF/LEF, enhances the expression of target genes, such as Slug and Snail, which induce EMT and metastasis of colon cancer [35]. The activation of Wnt signal, inhibition of APC/GSK3b/Axin complex, nuclear translocation of b-catenin, and up-regulation of Slug and Snail are key points of EMT regulation in colon cancer [36]. In our study, down-regulation of GSK3b and up-regulation of Slug and Snail expression were detected. And also, the up-regulation of nucleus b-catenin expression in spheroid cells indicates that the free b-catenin transfer from cytoplasm to cell nucleus.
In summary, our study provides evidence of the existence of cancer stem cells. The spheroid cells derived from HCT116 and HT29 cell lines enrich stem-like cells. Compared to their adherent counterparts, floating-sphere-derived cells displayed lower homotypic/heterotypic adhesion but higher in vitro migratory/invasive capacity, as well as higher tumorigenic and metastatic potential in vivo. And they demonstrate typical phenotype of EMT (downregulated E-cadherin and up-regulated Vimentin), which may account for their malignancy. And activation of Wnt/b-catenin pathway (down-regulation of GSK3b and up-regulation of Slug, Snail and nucleus b-catenin) may play an important role in the EMT of CCSCs.


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Targeting the “Cytokine Storm” for Therapeutic Benefit

ABSTRACT Inflammation is the body's first line of defense against infection or injury, responding to challenges by activating innate and adaptive responses. Microbes have evolved a diverse range of strategies to avoid triggering inflammatory responses. However, some pathogens, such as the influenza virus and the Gram-negative bacterium Francisella tularensis, do trigger life-threatening “cytokine storms” in the host which can result in significant pathology and ultimately death. For these diseases, it has been proposed that downregulating inflammatory immune responses may improve outcome. We review some of the current candidates for treatment of cytokine storms which may prove useful in the clinic in the future and compare them to more traditional therapeutic candidates that target the pathogen rather than the host response.

I n the event of tissue damage, whether caused by injury or infection, inflammation is the body's first coordinated line of defense. It is responsible for activating both innate and adaptive immune responses so that the damage can be resolved and homeostasis restored. The characteristic signs of inflammation include heat, redness, swelling, and pain and are easily recognizable (1). There are four stages to a classical self-limiting inflammatory response: (i) recognition of the problem, (ii) recruitment of leukocytes and other immune system components, (iii) elimination of the threat, and (iv) resolution of the inflammatory state (i.e., a return to homeostasis).

RECOGNITION
In the case of infection, inflammation begins when the cells of the innate immune system recognize a pathogen-associated molecular pattern (PAMP) possessed by the invading organism. PAMPs are often an essential feature of the microbe and therefore are highly conserved, increasing recognition (2). The receptors on host phagocytic cells that recognize PAMPs are known as pattern recognition receptors (PRRs), of which there are several different categories. Soluble PRRs such as mannose binding lectin act as opsonins, preparing the microbe for phagocytosis (3). Intracellular PRRs, notably the Nod-like receptors (NLRs), are found in the cytosol for the detection of intracellular pathogens (4). Retinoic acid-inducible gene (RIG)-like receptors (RLRs) share a caspase recruitment domain (CARD) with NLRs and are mainly responsible for viral detection (5,6). Transmembrane PRRs include the Toll-like receptors (TLRs) and C-type lectin receptors. Activation of a subset of NLRs, NLRP1, NLRP3, and NLRC4, induces the formation of a multiprotein complex called the inflammasome. Upon assembly, caspase proteins are cleaved from their proforms to an active state leading to the processing of interleukin-1␤ (IL-1␤) and IL-18 (7).
Once the PRR is activated and ligand binding occurs, a signaling cascade is triggered, which results in expression of specific proinflammatory cytokines. Cytokines play a vital role throughout the four stages of inflammation. During the early phase of infection, these protein messenger molecules act as signals to the immune system, regulating the duration and gravity of the immune response to damage or infection. Depending upon the specific cytokine that has been secreted, their role can be to activate (proinflammatory) or dampen (anti-inflammatory) the host response. For example, stimulated TLRs induce proinflammatory cytokines, while the production of the anti-inflammatory cytokine IL-10 is important during the later stages of infection in controlling disease-induced tissue pathology (8). In the case of sterile inflammation caused by tissue damage, trauma, and ischemia, PRRs recognize certain host-specific molecules that are only released during cell injury or necrotic death, termed damage-associated molecular patterns (DAMPs). These molecules include heat shock proteins and high-mobility group box 1 (HMGB1) and are recognized in much the same way as PAMPs (9).

RECRUITMENT
Once recognition has occurred and inflammation has been initiated, certain host cells begin to secrete chemokines. Chemokines are relatively small proteins with a molecular weight of less than 10 kDa which activate and mediate the migration of leukocytes to the site of infection or inflammation (10). Many different types of cells are able to secrete these chemotactic cytokines, including phagocytic cells such as macrophages and neutrophils, though endothelial cells are responsible for over half of all produced. Chemokines activate integrins and bind to intercellular adhesion molecules (ICAMs) (11). Subsequently, cells roll along the endothelium, up a chemokine gradient to the site of inflammation where they transmigrate through cell junctions into the damaged or infected tissue (10,12).

RESOLUTION
Following recruitment of immune cells to the site of inflammation, resolution of the damage begins. The cytokines induced by PAMPs and produced by leukocytes are proinflammatory cytokines and include tumor necrosis factor alpha (TNF-␣), IL-6, and members of the IL-1 family, all of which have different proinflammatory roles. TNF-␣ and IL-1␤ induce vasodilation and permeability, allowing immune cells to reach the site of damage, while IL-␤ and IL-6 induce complement and opsonization (2). As well as mediating the inflammatory response, proinflammatory cyto-kines can affect the brain, inducing behavioral and physiological symptoms such as fever, nausea, and anorexia (13).

RETURN TO HOMEOSTASIS
Throughout its activation, the inflammatory response must be regulated to prevent a damaging systemic inflammation, also known as a "cytokine storm." A number of cytokines with antiinflammatory properties are responsible for this, such as IL-10 and transforming growth factor ␤ (TGF-␤) (14). Each cytokine acts on a different part of the inflammatory response. For example, products of the T h 2 immune response suppress the T h 1 immune response and vice versa (15). Without the ability to resolve the inflammation, the collateral damage to surrounding cells has the potential to be catastrophic, resulting in sepsis and even death. However, if it is controlled correctly, inflammation can be resolved effectively, with little or no long-term damage to the host (16).

PATHOGENS
Pathogens attempt to skew the response of the finely balanced immune system in order to evade immune responses and have evolved a diverse range of strategies to favor their own growth, survival, and replication. At one extreme, some pathogens have strategies to appear invisible to the immune system and thus fail to induce an effective immune response, while at the other extreme, other pathogenic organisms are capable of hyperstimulating the immune system, commonly known as a cytokine storm. This can prevent the clearance of infection and induce tissue damage (i.e., necrosis, a potentially fatal condition). Many reviews have focused on immune evasion as a means to establish infection (17), but the importance of cytokine storms in disease is only just becoming apparent.
Diverse pathogenic viruses (e.g., influenza A) and bacteria (e.g., Francisella tularensis) have been found to induce cytokine storms or hypercytokinemia ( Fig. 1) (18)(19)(20). These pathogens disrupt the delicate balance of a suitable inflammatory response, tipping it from being beneficial to destructive by causing large amounts of positive feedback in immune cells and upregulation of proinflammatory markers, in particular cytokines TNF-␣, IL-1␤, IL-8, and IL-6. This soon results in symptoms such as hypotension, fever, and edema and can eventually cause organ dysfunction and death (21).
One of the most studied examples of an organism that can cause cytokine storms is influenza A virus, in particular the pandemic subtypes. For example, the H1N1 strain that caused the 1918 pandemic has been shown to induce higher levels of proinflammatory immune cells and cytokines in the lungs than seasonal influenza viruses. This contributes to its high virulence and may account for the unusually high mortality rate seen in otherwise healthy young adults during the outbreak (22). Severe influenza infections caused by highly virulent subtypes such as H1N1 and H5N1 are characterized by overinduction of proinflammatory cytokines TNF-␣, IL-1␤, IL-6, IL-8, and monocyte chemotactic protein-1 (MCP-1) (23,24), which eventually results in multiple organ dysfunction and failure and increased vascular hyperpermeability (23).
Infection by the inhalation route of the zoonotic bacterium F. tularensis can also result in a systemic inflammatory response. Less than 10 F. tularensis type A strain bacteria are required to initiate disease (25). F. tularensis is an intracellular pathogen and, upon infection, rapidly invades macrophages, where it can multiply in the cytoplasm to high levels (reviewed in reference 26). Interestingly, it has been shown in animal models that when they are infected via the inhalational route, there is a delay of several days between the initial infection and induction of cytokines and chemokines, allowing bacterial replication and dissemination uncontrolled by the immune system (27,28). Once activated, however, proinflammatory cytokines such as IL-6 are quickly upregulated by up to 1,000 times their resting level. As with influenza, the unchecked hypercytokinemia and subsequent secondary cascades such as coagulation eventually result in widespread necrosis, organ and system failure, and death (25).

THERAPEUTIC STRATEGIES FOR TREATING INFECTIOUS DISEASE
The last century saw enormous leaps forward in the advancement of medicine, resulting in the development of more and more strategies to protect against infectious diseases, many of which have been very successful. Some of these, such as antibiotics, target the pathogen, but increasingly, approaches to elicit a beneficial immune response are being developed as our understanding of the human immune response and host-pathogen interactions develops.

TARGETING THE PATHOGEN
Antibiotics are the best known and most widely used weapon to combat bacterial infections. When antibiotics were discovered in the first half of the 20th century (29), they were heralded as wonder drugs, the beginning of the end for infectious diseases. However, the strong selective pressure exerted by antibiotics, combined with inappropriate use, resulted in the rapid emergence of resistance. Some species of bacteria, such as Mycobacterium tuberculosis, have become multidrug resistant (MDR) or even extensively drug resistant (XDR). XDR M. tuberculosis has now been reported in over 45 countries (30). Indeed, there are now worrying reports of totally drug-resistant M. tuberculosis in India (31). As resistance renders many antibiotics ineffective, there is a pressing need for new compounds for use in the clinic. However, very few new classes of antibiotic have been discovered in the last three decades (32,33), most new antibiotics appearing on the market being derivatives of beta-lactams and quinolones. The situation with antivirals is even more desperate: there are far fewer licensed antiviral treatments available than there are antibiotics, and those that are available suffer from being highly specific and thus only target a narrow proportion of viruses. One of the underlying issues is that viruses exploit host cell machinery; thus, identifying effective compounds that inhibit the viral life cycle without affecting the host is challenging. For example, the nucleoside analogue ribavirin targets viral nucleic acid replication. The compound is activated by viral, but not human, enzymes, thus preventing replication (34,35). Primarily used to treat hepatitis C virus (HCV) as part of combination therapy, it has also been shown to be effective against other viruses, such as measles virus, influenza virus, and arenaviruses, in particular, the virus causing Lassa hemorrhagic fever (36). However, it has a high prevalence of side effects and is thought to be teratogenic in humans (35). Similarly to antibiotics, resistance is also an issue with anti-viral drugs, especially for those viruses which have high rates of mutation. Herpes simplex virus, for example, has developed resistance to the antiviral acyclovir. Resistance in patients on longterm treatment regimens for recurrent herpes outbreaks began to emerge within a decade of the drug's original release in the 1980s (37).
Due to the lack of promising antibiotics and antiviral compounds in development, alternative approaches have been considered. For example, two historically evaluated approaches, phage therapy and passive protection, have experienced an increase in interest. While they were largely disregarded after the discovery of antibiotics, they are now being considered again, as levels of antibiotic resistance continue to rise (38). While bacteriophages are easier to produce than antibiotics and have been shown to have very few, if any, side effects, they must be used as a cocktail of several different phages in order to prevent resistance from rapidly emerging. They are also highly specific, so an exact diagnosis, possibly even to the strain or serotype level, must be made before the correct bacteriophage can be administered (38).
Similarly, the idea of using antibodies to directly and immediately boost the immune system during infection has a long history of use but is rarely used today. Sera from immune individuals or animals have been used to treat disease such as Corynebacterium diphtheriae as early as the end of the 19th century (39). However, problems with side effects such as serum sickness and narrow specificity caused this approach to fall out of favor for treatment of most diseases. The exception was for prophylaxis of rabies: part of the postexposure rabies treatment consists of rabies immune globulin, which provides short-term, immediate protection with minimal side effects. More recently, developments in monoclonal antibody technology and antibody humanization have made passive therapy a more attractive option by decreasing the risk of adverse side effects.

TARGETING THE HOST
Both chronic infections and acute infections result in the induction of cytokine storms. It is therefore becoming apparent that a combination therapy approach involving an antimicrobial compound along with an immunotherapy may produce a more favorable outcome, and this is an area of intense investigation.
For an immunomodulatory therapeutic to be considered for treatment of infection, it must not deleteriously affect "helpful" elements of the immune response. It also needs to be specific for highly conserved networks that are essential to the host in either maintaining immune homeostasis and/or combating infection. In this review, we focus on promising new therapeutic approaches in this area (Fig. 2) and discuss the advantages and disadvantages which will influence whether they gain acceptance for the clinic.

PROINFLAMMATORY CYTOKINES
The therapeutic use of cytokines as nonspecific immunomodulators that boost the host defenses has traditionally been used to treat long-term or chronic diseases, such as hepatitis, and several are already licensed for human use, including IL-2 and interferons (IFNs) (40). One of the most widely used therapeutic cytokines is IFN-␣, a type 1 interferon which inhibits viral replication. It is used in combination with ribavirin for the treatment of chronic HCV, resulting in greater viral RNA clearance together than when administered alone (41,42).
Having a strong proinflammatory response at the time of infection often results in survival of the host following infection with what would normally be a lethal dose of a microbial pathogen. If treatment is initiated either just before or the same time as infection, then the induction of proinflammatory cytokines by stimulatory molecules such as CpG oligonucleotides has beneficial effects on host survival. Prophylactic use of CpG has been demonstrated to be effective in murine models of F. tularensis subsp. holarctica live vaccine strain (LVS) (43), Burkholderia mallei (44), and Burkholderia pseudomallei (45) infection. However, it should be noted that such treatments are not universally successful. For example, recent studies have demonstrated that a preexposure CpG treatment strategy failed to protect mice that were subsequently infected with the highly virulent F. tularensis strain SchuS4 (46). Furthermore, there are no reported data on the efficacy of CpG treatment given postexposure against highly virulent pathogens. Indeed, unpublished observations from our laboratory suggest that CpG treatment following infection/onset of symptoms may indeed be detrimental to the host. This therefore implies that causing a rapid increase in proinflammatory cytokine production once infection has occurred may not be suitable.
An alternative approach to induction of proinflammatory host responses could be the use of proinflammatory cytokines themselves. However, the use of common proinflammatory cytokines like IL-1␤, IL-6, and TNF-␣ induces the pathophysiological effects associated with severe infection (47,48). Indeed, intravenous administration of IL-1␤ has been shown to cause generalized fatigue, headache, nausea, vomiting, myalgias, and arthralgias (49).
The timing of administration of proinflammatory cytokine treatments requires careful management, since there is a danger that their use may exacerbate the symptoms of disease in infected individuals. It is therefore likely that the window for treatment has probably closed once symptoms present. This uncertainty, coupled with a paucity of effective triggers for the use of proinflammatory treatments, suggests that such an approach is unlikely to find widespread application.

TARGETING THE OVERACTIVE IMMUNE RESPONSE
Once an infection has progressed to a late stage and an individual begins to suffer symptoms of disease (e.g., fever, pyrexia), the immune response generated at this point can be detrimental to the host if cascades are not appropriately controlled. Therefore, balancing the inflammatory network may represent a more effective means of treatment for postsymptomatic infections than stimulating a broad response with proinflammatory cytokines. One cause of death in infectious disease is the collateral damage caused by the immune response as it attempts to clear the pathogen rather than the effect of virulence factors produced by the organism. By controlling the proinflammatory response (e.g., leukocyte recruitment to the site of infection), an immunomodulatory treatment has the potential to reduce this tissue damage by preventing immune "overcrowding." While such an approach may not clear the infection, it can support survival until a successful adaptive immune response is mounted, allowing antimicrobial therapy to be effective. It is clear that for infections with pathogens such as influenza A virus and F. tularensis, where a dysregulated immune response can cause significant damage, one therapeutic strategy would be to bring the inflammatory response back under control.

FIG 2
When a cytokine storm has arisen, conventional therapeutics may not be sufficient. Strategies to combat this cytokine storm have included compounds that target fundamental immune pathways, such as the chemokine network and the cholinergic anti-inflammatory pathway, and more specific strategies have included the use of HMGB1 antibodies and COX-2 inhibitors. All these lead to a downregulation of the cytokine storm, reducing the risk of tissue damage and allowing time for conventional therapies to target the pathogen directly.

STIMULATING THE CHOLINERGIC ANTI-INFLAMMATORY PATHWAY
The cholinergic anti-inflammatory pathway uses the neurotransmitter acetylcholine (Ach) to interact specifically with ␣7 subunit of nicotinic acetylcholine (␣7nAch) receptors on innate immune cells such as macrophages. These receptors are able to respond to Ach from a number of sources, including other immune cells and the vagus nerve, and their activation results in the suppression of proinflammatory cytokines. NF-B, the main transcription factor for proinflammatory cytokines, is activated by PAMPs such as lipopolysaccharide (LPS) and triggers a pathway which results in the translocation of NF-B and the transcription of proinflammatory genes. Stimulation of the vagus nerve can inhibit this pathway, downregulating the immune response and even reversing the symptoms of sepsis (50,51).
It has been shown that direct electrical stimulation of the vagus nerve can substantially reduce the levels of LPS-induced TNF-␣ in both the liver and serum of rats (52), as well as inhibiting secondary sepsis cascades such as systemic coagulation (53), increasing the rates of survival in both cases. However, electrical stimulation of the nervous system in humans would be too invasive and risky to be considered a feasible treatment for sepsis and hypercytokinemia, and so pharmaceutical methods of activating the ␣7nAch receptor are currently being investigated.
Nicotine is a nonselective agonist of the ␣7Ach receptor and is able to suppress the production of proinflammatory cytokines by mimicking the binding of acetylcholine. It has been demonstrated that nicotine can selectively reduce the inflammatory response in a number of infection scenarios, including Legionella pneumophila (54) and Chlamydia pneumoniae (55) infection; however, it is highly unlikely that nicotine will ever be used clinically due to its toxicity, addictive nature, and lack of specificity. GTS-21, also known as DMXB-A, is another selective ␣7Ach receptor agonist already undergoing clinical trials for schizophrenia and Alzheimer's disease (56)(57)(58). It produces the same inflammatory modulation as nicotine but is nontoxic, does not result in an addiction, and has no known side effects (59). GTS-21 significantly reduces TNF-␣ and the late mediator of sepsis, HMGB1, downregulates IFN-␥ pathways, and prevents the LPS-induced suppression of IL-10 and STAT 3 mechanisms (60), all of which contribute to a significant increase in survival in murine sepsis (59). In 2011, GTS-21 underwent in vivo human trials for the treatment of sepsis. The effects of orally administered GTS-21 at the highest known safe dose were examined in response to endotoxin-induced sepsis. The effects, while clearly dose-dependent, were highly variable between subjects and the mean plasma concentration of GTS-21 was low, resulting in a lack of statistically significant results. Within each individual, however, low levels of IL-6 and TNF-␣ were observed, proportional to the GTS-21 plasma concentration, indicating that high doses or different methods of administration may produce a more significant effect (61). There may also be potential for other ␣7Ach receptor agonists such as CNI-1495, which has already been shown to increase survival in murine sepsis models (62,63).

PROSTAGLANDINS AND CYCLOOXYGENASE INHIBITORS
Prostaglandins are a large, varied family of fatty acids, a number of which, such as prostaglandin E 2 , are early markers of inflammation. These prostaglandins are produced by activated macrophages during infection and increase symptoms of sepsis and sys-temic inflammation, such as vascular permeability and edema, as well as stimulating other immune cells (64). Prostaglandins are synthesized by cyclooxygenase (COX) enzymes, COX-1 and COX-2, which can be inhibited by pharmaceuticals in order to prevent the production of inflammatory prostaglandins. There are many COX inhibitors already widely and cheaply available, many of which are classed as nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin and ibuprofen. However, many NSAIDs are not suitable for long-term use, as the lack of specificity causes side effects in the gastrointestinal tract (65).
Selective inhibition of COX-1 has been shown to increase mortality and disease symptoms in experimental sepsis and systemic inflammation (66). In comparison, selective COX-2 inhibitors have been shown to significantly reduce levels of inflammation, without the damaging gastrointestinal side effects. One such drug is celecoxib, a safe, inexpensive COX-2 inhibitor which has shown promising results in decreasing symptoms of severe systemic inflammation caused by influenza infection in mice. Infection with the virus results in the production of very high levels of COX-2, especially in alveolar epithelial cells. In particular, lethal strains such as H5N1 induce COX-2 at higher levels than nonlethal, seasonal strains (67). The downregulation of COX-2, through prostaglandin inhibition, results in a decrease in proinflammatory cytokine levels and leukocyte activation without causing immune suppression. This event effects viral clearance and disrupts the formation of protective immunity (68,69). A combination therapy of celecoxib along with an antiviral such as zanamivir results in a significant increase in survival (68). Overall, the data currently available in the literature suggest that the use of COX-2 inhibitors as therapeutics may represent a promising approach for the treatment of viral and bacterial infectious diseases.

PLATELET-ACTIVATING FACTOR INHIBITORS
The phospholipid platelet-activating factor (PAF) plays an important and varied role in mediating the inflammatory response. It is produced by a number of different cells and acts on the PAF receptor, which is primarily found on the plasma membrane of cells such as leukocytes, platelets, and endothelial cells (70). The effects induced by PAF binding are dependent on the type of cell the PAF receptor is located on. Binding to the PAF receptor on platelets activates platelet aggregation and coagulation cascades, while binding to receptors on endothelial surfaces encourages neutrophil adhesion and permeability (71). PAF binding also increases the production of proinflammatory cytokines such as TNF-␣, IL-8, and IL-1␤, as well as contributing to the formation of pulmonary edema and organ dysfunction when overexpressed during severe sepsis and systemic inflammation (70,71).
PAF plays a key role in inflammation and its concentration is controlled by the enzyme PAF acetylhydrolase (PAF-AH) (72). However, during systemic inflammation, the levels of PAF-AH are suppressed, preventing the PAF-linked immune response from being controlled (73). As a natural, highly efficient process, it has been hypothesized that artificially increasing the concentration of PAF-AH could control the effects of PAF by inhibiting the PAF cascade before it can bind to the receptor. Recombinant PAF-AH has been expressed in Escherichia coli and shown promising early results in both rodents and human clinical trials, especially when combined with antibiotic therapy (73), resulting in decreased mortality and levels of inflammation, especially when administered early in infection, without demonstrating serious side effects (74). However, a subsequent phase III clinical trial showed no significant reduction in mortality or sepsis and eventually was discontinued (75). A number of pharmaceutical PAF receptor antagonists have also been tested and successfully shown to reduce symptoms and mortality in diseases where systemic inflammation and PAF play key roles, such as the antagonist UK-74,505 in dengue virus infection (76).

CHEMOKINE MANIPULATION
Chemokines and their receptors are a large and diverse family of proteins. It has become clear that these molecules contribute to a large number of biological functions. Among these, however, the recruitment of leukocytes to specific tissues is the most extensively studied and likely the most important (77). Over the last decade, a key role for chemokines and their receptors has been demonstrated in many inflammatory diseases, including atherosclerosis, rheumatoid arthritis, and gastrointestinal diseases (78). This, in turn, has led to the idea that modulating the chemokine response may be a useful target for generating novel therapeutics (79).
It has been reported that pretreatment of mice with monocyte chemotactic protein-1 (MCP-1) completely protected against lethal systemic infection by Pseudomonas aeruginosa or Salmonella enterica serovar Typhimurium (80). Further therapeutic agents have been developed, including an IL-8 (CXCL8) inhibitor to block inflammatory states and angiogenesis (81), GRO␣ E6A to treat malaria, and I-309 (a human monocyte chemoattractant) to treat tumors (82). This approach is made more attractive by the unusually attractive pharmacokinetics of injected chemokine proteins; unlike most biological therapeutics, where the protein is rapidly cleared from the bloodstream, a single injection of chemokine protein results in a sustained increase in activity as a result of the rapid equilibrium with the abundant, promiscuous chemokine receptor DARC (Duffy antigen/receptor for chemokines), which is present on red blood cells. Red blood cells, therefore, act as a storage depot for the injected chemokine which is then gradually released over the following hours into the plasma in order to facilitate leukocyte trafficking.
Research looking to identify treatments that block specific chemokines/chemokine receptors has proved challenging. This is predominantly due to the chemokine network having a significant level of redundancy within the system (i.e., there are more chemokines than chemokine receptors), with some chemokine receptors binding multiple chemokine ligands (83,84). Therefore, the blockage of one chemokine-receptor interaction does not fully stop its function and, as a result, research involving inhibitors or antagonists for a specific target rarely repeat the promising results found using knockout mice, e.g., studies of experimental autoimmune encephalomyelitis (85,86). Nevertheless, promising early results have demonstrated that the administration of PF-04178903, an antagonist of the chemokine receptor CCR2, prior to challenge with the influenza H1N1A/Puerto Rico/8/34 strain reduced the mortality and the morbidity in a mouse model of infection while also reducing pulmonary immune pathology (87). In addition, the development of broad-spectrum chemokine inhibitors (BSCIs) that can affect multiple chemokine signaling pathways simultaneously while leaving other cytokine signals unaffected offer the potential to target the chemokine network specifically in order to dampen the overactive immune response. A compound called NR58-3.14.3 was shown to be effective against bacterial endotoxin-induced inflammation in the skin (88), and extensive data suggest that this compound is a useful therapeutic for asthma, atherosclerosis, stroke, and bronchiolitis obliterans syndrome.

MANIPULATION OF REGULATORY T CELLS
Regulatory T (Treg) cells are the host's natural anti-inflammatory cell and are important in maintaining homeostasis and controlling tissue damage that occurs during infection. Two main types of Treg cell have been described: those that are called induced and others that are called natural. Induced Treg cells (Tr1 cells) are dependent on IL-10 for their differentiation and regulation (89,90). Natural Treg cells have been studied and characterized in much more detail. Typically, these CD4 ϩ T cells express IL-2R alpha (CD25), glucocorticoid-induced TNF receptor family-related gene (GITR), and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) (91,92). Highlighting their role as Treg cell mediators, depletion of each of these proteins individually can lead to autoimmunity. However, the expression of these markers alone is not sufficient to determine whether a Th cell is of the regulatory subset because activated nonregulatory T cells have the potential to express all these markers. The discovery of Forkhead box P3 (Foxp3) as a more specific marker of natural Treg cells has been essential to their experimental study. Foxp3 is a transcription factor that has now been shown to be the key regulator in the development of natural Treg cells (93,94). The importance of this transcription factor is exemplified by the human disease immunodysregulation polyendocrinopathy enteropathy X-linked syndrome, in which a mutation in the Foxp3 gene leads to severe autoimmunity (95)(96)(97).
It has been suggested that one Treg cell can influence numerous surrounding cells (98). With such potency, it is no surprise that these cells have been investigated as possible therapies to many diseases. Manipulation of Treg cell numbers by the addition of cytokines or targeting cell surface proteins has been shown to be effective in controlling many aspects of inflammation. Removal of Tregs with cocktails of antibodies has been shown to affect survival of pathogens within several mouse models of infection. The literature in the field of Treg manipulation for treatment of diseases is extensive (reviewed in references [99][100][101]. Targeting these cells directly may be difficult in the human context but their effector molecules may act as useful targets to combat the overactive immune response observed in several diseases.

NOVEL THERAPIES PROMOTING IMMUNE RESOLUTION FOLLOWING INFECTION
Resolution of tissue damage is no longer seen as a passive process but more accurately as an active process that involves a range of biomolecules. Resolvins, lipoxins, and protectins are proresolution molecules involved in restoring tissue homeostasis. They elicit their effects via a range of mechanisms and are able to reduce neutrophil infiltration (102), increase the uptake of apoptotic neutrophils (103), and increase cellular exit via the lymphatic system. Collectively, these molecules work in concert in order to return the immune system to a resting state.
Resolvins are a set of newly identified lipid-based mediators that are derived from omega-3 polyunsaturated fatty acid (EPA) and docosahexaenoic acid (DHA). What separates these molecules from the traditional anti-inflammatory compounds is their ability to promote resolution without necessarily dampening down the inflammatory response (104). Resolvins can be further divided into two groups: the E series and the D series, derived from EPA and DHA, respectively. Both types of resolvins have been shown to have an effect in vivo in several mouse models of diseases and stop neutrophil recruitment in peritonitis (105,106). Resolvin E1 has been shown to increase host survival in models of colitis (107), and resolvin D2 has been shown to protect from ischemiareperfusion kidney damage (108). This family of lipid mediators offer a novel and exciting avenue to treat inflammatory diseases. The ability to control tissue damage without disrupting the beneficial inflammatory response means these molecules represent a promising therapeutic strategy for treating infection that is worthy of further investigation.

SUMMARY
Inflammation is an essential part of an effective immune response, without which successful resolution of cellular damage and infection would not be possible. The inflammatory response is responsible for initial recognition of an invader or trauma, recruitment of the correct cells to allow resolution of the problem, and, eventually, a return to homeostasis. Pathogens are constantly adapting to be one step ahead of the immune system by evading or suppressing certain aspects. The immune system is unable to adapt at the same rate as microbes, and so pharmaceuticals have been developed to support the body's defenses, such as antibiotics and antivirals. However, many pathogens have developed resistance to such drugs. Therefore, attention has turned to immunomodulation as a therapeutic approach to enhance the efficacy of antimicrobials. Unfortunately, generically enhancing the broad immune response can sometimes worsen the outcome of disease. By modulating rather than upregulating the immune response via mechanisms such as the cholinergic anti-inflammatory pathway, COX-2 pathways, and PAF, the damaging positive feedback loops of sepsis and cytokine storms can be prevented. This may allow a longer window for diagnosis and treatment. Methods such as direct stimulation of the vagus nerve, treatment with nicotine, and PAF-AH have proven to be effective in murine studies but are unsuitable for clinical use in humans. However, more appropriate compounds are currently undergoing trials, including the nicotinic AchR agonist GTS-21. Though still in its infancy as an immune modulation drug, it shows promise in vitro and requires more investigation of its effects in vivo. Within the next decade, it is very possible that immune modulators such as GTS-21 and PAF-AH may be widely available and at the forefront of disease therapy.