Multiple sclerosis is the most common chronic inflammatory disease of the central nervous system in young adults. Despite the fact that numerous lines of evidence link both the risk of disease development and the disease course to the serum level of 25-hydroxyvitamin D it still remains elusive whether multiple sclerosis patients benefit from boosting the serum level of 25-hydroxyvitamin D, mainly because interventional clinical trials that directly address the therapeutic effects of vitamin D in multiple sclerosis are sparse. We here present the protocol of an interventional clinical phase II study to test the hypothesis, that high-dose vitamin D supplementation of multiple sclerosis patients is safe and superior to low-dose supplementation with respect to beneficial therapeutic effects.The EVIDIMS trial is a German multi-center, stratified, randomized, controlled and double-blind clinical phase II pilot study. Eighty patients with the diagnosis of definite multiple sclerosis or clinically isolated syndrome who are on a stable immunomodulatory treatment with interferon-β1b will be randomized to additionally receive either high-dose (average daily dose 10.200 IU) or low-dose (average daily dose 200 IU) cholecalciferol for a total period of 18 months. The primary outcome measure is the number of new lesions detected on T2-weighted cranial MRI at 3 tesla. Secondary endpoints include additional magnetic resonance imaging and optical coherence tomography parameters for neuroinflammation and -degeneration, clinical parameters for disease activity, as well as cognition, fatigue, depression, and quality of life. Safety and tolerability of high-dose vitamin D supplementation are further outcome parameters.In light of the discrepancy between existing epidemiological and preclinical data on the one hand and available clinical data on the other the EVIDIMS trial will substantially contribute to the evaluation of the efficacy of high-dose vitamin D supplementation in MS patients. The study design presented here fulfills the criteria of a high-quality clinical phase II trial in MS.NCT01440062ClinicalTrials.gov Identifier:  Multiple sclerosis (MS) is the most common chronic inflammatory disease of the central nervous system (CNS) in western countries . Based oNumerous lines of evidence link both the risk of disease development and the disease course to the serum level of 25-hydroxyvitamin D which is the biologically inactive storage form of vitamin D (extensively reviewed in ): it is Against this background, we here present the study protocol for a randomized controlled clinical phase II pilot study on the effect of vitamin D supplementation on MRI in patients with relapsing-remitting MS or clinically isolated syndrome (CIS). We hypothesize, that high-dose supplementation is superior to low-dose supplementation with respect to imaging parameters including MRI and optical coherence tomography (OCT) as well as clinical parameters.EVIDIMS is a national, multi-center, stratified, randomized, controlled, and double-blind clinical phase II trial conducted at approximately ten sites located in or close to Berlin, Germany. Recruitment will start in 12/2011. Eighty patients with relapsing-remitting MS or CIS who are on a stable immunomodulatory treatment with interferon-β1b will be randomized to either high-dose (20.400 IU) or low-dose (400 IU) 25-hydroxyvitamin D supplementation to be taken every other day for 18 months add-on to interferon-β1b. The detailed study design is provided below according to the revised CONSORT statement ,28. The Inclusion criteria for participation in the EVIDIMS trial comprise the diagnosis of either definite relapsing-remitting MS according to the revised 2005 McDonald criteria  or CIS, http://www.dge.de. In both arms, vitamin D treatment will be add-on to a stable immunomodulatory treatment regimen with interferon-β1b. Regular study visits are carried out after 1, 3, 6, 9, 12, and 18 months. Investigations to be performed on the respective study visits are displayed in table Eligibility of patients will be determined at the screening visit. At baseline visit, patients who qualified for participation in the study will be stratified according to gender, disease course, and 25-hydroxyvitamin D serum level at screening and then randomized 1:1 into two intervention groups. Group A will receive 20.400 IU cholecalciferol orally every other day; group B will receive 400 IU cholecalciferol orally every other day, which corresponds to the daily dose recommended by the German Nutrition Society The primary endpoint is the cumulative number of new hyperintense lesions on cranial T2-weighted MRI during the treatment period of 18 months. Additional secondary MRI endpoints comprise number and volume of T1-hypointense and T2-hyperintense lesions, proportion of patients without any new T1-hypointense and T2-hyperintense lesions, number of contrast enhancing lesions, brain atrophy as determined by normalized brain volumetry, and changes in brain metabolism as determined by magnetic resonance spectroscopy (MRS). All MRI/MRS investigations will be performed on a 3 tesla MRI scanner . All MRI parameters will be assessed by an experienced evaluator who is blinded to both clinical data and treatment allocation. Secondary clinical endpoints include the annualized relapse rate and the proportion of relapse-free patients. A relapse is defined as de novo development or aggravation or re-occurrence of a preexisting neurological abnormality compatible with MS which lasts a minimum of 24 hours, is separated by at least 30 days from a preceding clinical event and does not occur in the context of fever. Additional secondary endpoints comprise the disease progression as determined by EDSS , Multiple Sclerosis Functional Composite (MSFC), cognition as determined by the Faces Symbol Test (FST) and the Symbol Digit Modalities Test (SDMT), fatigue and depression as well as quantitative assessment of motor function ,32. Addia priori determined sample size of 40 patients per intervention arm, in total 80 patients. Based on this sample size, a mean reduction of 1.5 new T2-hyperintense lesions in the high-dose treatment compared to the low-dose regimen will be detected with a power of 0.83.Since available data on the effect of vitamin D on the development of new T2-hyperintense lesions on cranial MRI are not sufficient for an exact statistical sample size calculation the study is designed as pilot study with an Both, patients and the entire study staff will remain blinded throughout the complete treatment period of 18 months. Treatment allocations will only be disclosed after the final database lock. Substantial efforts are made to maintain blinding: Since high-dose formulation (oil) does not match the low-dose formulation (tablet), patients in the high-dose arm will take 400 IU tablets in addition to 20.000 IU oil, whereas patients in the low-dose arm will receive an identical volume of placebo-oil in addition to a 400 IU tablet. The patients are primarily handled by a treating physician who is in charge of the evaluation of inclusion or exclusion criteria, adverse events, relapses, side effects, etc. All neurological examinations will be done by an independent evaluating physician. Accordingly, evaluation of all other paraclinical parameters including MRI and OCT will be done by independent examiners. Any patient-related crosstalk between treating and evaluating physicians or examiners is prohibited unless required because of safety concerns. An easy unblinding procedure allows for rapid unblinding of a patient in case of a medical necessity. Unblinding inevitably results in the exclusion of the respective patient.Evaluation of endpoints will be carried out by both intention-to-treat and per-protocol analyses. Statistical tests and presentation will be appropriate to the category and distribution of the respective variables. Analysis of OCT data will be performed by statistical models which account for intra-patient inter-eye relations. The test level for statistical significance of differences between both treatment arms is defined as p = 0.05 for all tests.The EVIDIMS trial is funded in part by a limited grant of Bayer Vital GmbH, Germany and supported by the German Research Foundation (DFG Exc 257).In light of the discrepancy between existing epidemiological and preclinical data on the one hand and available clinical data on the other the study design presented here has the potential to substantially contribute to the evaluation of the efficacy of vitamin D supplementation in MS and CIS patients. The randomized, controlled, double-blinded study design and the implementation of independent evaluation of outcome parameters fulfill the criteria for a high-quality clinical phase II trial in MS . Some asIn conclusion, vitamin D has the potential for a safe, orally available and cheap treatment option in MS and the EVIDIMS trial may help to close the existing gap between available promising preclinical and the lacking clinical data on the immunomodulatory efficacy of vitamin D in MS.The EVIDIMS trial is currently recruiting patients.CIS: Clinically isolated syndrome; CNS: Central nervous system; ECG: Electrocardiogram; EDSS: Expanded disability status scale; EVIDIMS: Efficacy of Vitamin D Supplementation in Multiple Sclerosis; FST: Faces symbol test; ICH-GCP: International Conference on Harmonization of Good Clinical Practice; IU: International units; MRI: Magnetic resonance imaging; MRS: Magnetic resonance spectroscopy; MS: Multiple sclerosis; MSFC: Multiple sclerosis functional composite; OCT: Optical coherence tomography; QoL: Quality of life; SDMT: Symbol digit modalities test.FP received travel support and speaker honoraria by Bayer Vital GmbH. JD received speaker honoraria by Bayer Vital GmbH. The authors declare that they have no competing interests. The partial funder of the study  is neither involved in the study design nor the interpretation of data or publication of results.JD designed the study, drafted the study protocol and drafted the manuscript. SO was in charge of all regulatory affairs and critically revised both, the study protocol and manuscript. HS is the biometrician of the study and critically revised both, the study protocol and manuscript. FP designed the study and also critically revised both, the study protocol and manuscript. All authors have given final approval of the version to be published.


We provide an overview of computational systems biology approaches as applied to the study of chemical- and drug-induced toxicity. The concept of “toxicity pathways” is described in the context of the 2007 US National Academies of Science report, “Toxicity testing in the 21st Century: A Vision and A Strategy.” Pathway mapping and modeling based on network biology concepts are a key component of the vision laid out in this report for a more biologically based analysis of dose-response behavior and the safety of chemicals and drugs. We focus on toxicity of the liver (hepatotoxicity) – a complex phenotypic response with contributions from a number of different cell types and biological processes. We describe three case studies of complementary multi-scale computational modeling approaches to understand perturbation of toxicity pathways in the human liver as a result of exposure to environmental contaminants and specific drugs. One approach involves development of a spatial, multicellular “virtual tissue” model of the liver lobule that combines molecular circuits in individual hepatocytes with cell–cell interactions and blood-mediated transport of toxicants through hepatic sinusoids, to enable quantitative, mechanistic prediction of hepatic dose-response for activation of the aryl hydrocarbon receptor toxicity pathway. Simultaneously, methods are being developing to extract quantitative maps of intracellular signaling and transcriptional regulatory networks perturbed by environmental contaminants, using a combination of gene expression and genome-wide protein-DNA interaction data. A predictive physiological model (DILIsym™) to understand drug-induced liver injury (DILI), the most common adverse event leading to termination of clinical development programs and regulatory actions on drugs, is also described. The model initially focuses on reactive metabolite-induced DILI in response to administration of acetaminophen, and spans multiple biological scales. The basis of this risk assessment paradigm would be perturbation of cellular responses using a carefully selected suite of in vitro assays. Central to this vision is the idea of “toxicity pathways” – innate cellular signaling pathways that are perturbed by chemicals and pharmaceuticals, and the determination of chemical concentration ranges where those perturbations are likely to be excessive, thereby leading to adverse health effects if present for a prolonged duration in an organism. A key element of the proposed approach is the use of computational systems biology models as a tool to generate hypotheses about cellular level dose-response based on existing data sets, and to identify data and knowledge gaps that can help guide the design of in vitro assays, focused animal studies, and improved in vitro – in vivo extrapolation (IVIVE) methods.The 2007 report by the National Research Council (NRC) of the U.S. National Academies of Science, titled “Toxicity testing in the 21st Century: A Vision and A Strategy” -α nuclear receptor (NR) pathway in primary human hepatocytes is used as an example for computational reconstruction of a toxicity pathway network from genomic data. We then use the example of aryl hydrocarbon receptor (AhR) activation in the liver to outline the process of developing a multi-scale spatial model of the liver lobule and interactions among multiple hepatic cell types consequent to exposure to toxic agents. Finally, we outline a predictive physiological model (DILIsym™) to understand drug-induced liver injury (DILI) in response to administration of acetaminophen, which spans multiple scales from the organ/tissue-level to the molecular and cellular levels. These varied modeling approaches, applied across different pathways and tissues, will be pivotal in creating twenty-first century toxicity pathways. Changes in the topology and dynamic behavior of these pathways subsequent to recognition of the external ligand account for the particular shape of the dose-response curve for specific phenotypic end points. A finite number of core “stress response pathways” mediate the response of cells to various chemical stimuli to maintain homeostasis, or to make specific cell-fate decisions such as proliferation, differentiation, or apoptosis  in individual cells are mediated by cell-membrane or cytosolic “receptor” molecules and downstream signaling and transcriptional networks, which together comprise intracellular regulatory network motifs that underlie cellular homeostasis and fate decisions including phenotypic transitions negative feedback, which enables homeostasis and acceleration of response time in gene circuits positive feedback, which generates switching behavior between multiple phenotypic states coherent feed-forward loop, which can introduce a time delay in activation as well as detect persistence in the activating signal incoherent feed-forward loop, which can function as a pulse generator and response accelerator  models can play a key role in this process, allowing mechanistic prediction of the dose-response based on pathway dynamics  are a common approach to modeling dynamical systems like intracellular signaling circuits. An assumption with deterministic ODE models is that the molecular components of the network of interest exist in a well-mixed volume such as the cytosol or nucleus, and that the amounts or concentrations of all molecular species in the network can be approximated by continuous variables. A typical deterministic model consists of a set of coupled ODEs, each describing the rate of change in the concentration or abundance of a molecular component, and incorporating terms that account for the known biochemical interactions among the various molecular species  is assumed to take either of two values, 0 (off) and 1 (on), is often a good approximation of the gene expression patterns in these processes. The state of each gene is updated according to its current state and that of other regulatory genes it is connected to in the network, as governed by a preset Boolean logical rule table. This binary-state assumption reduces the dependence on various kinetic parameters in the model, instead making full use of the large available database of qualitative protein–protein and protein–gene interactions. As with deterministic and stochastic models, a simulation of a Boolean network model should converge to an attractor state representing the binary gene expression pattern of a particular phenotypic state , also referred to as “individual-based modeling”  use an integrated visualization/programing interface , glucocorticoid receptor (GR), and progesterone receptor (PR), as well as the PPARs, liver X receptors (LXRs), retinoic acid receptors (RARs), and retinoid X receptors (RXRs). NRs act on chromatin combinatorially and dynamically throughout the genome to regulate transcriptional responses to physiological and environmental stimuli phosphorylation of PPARα in the cytosol;(ii)translocation of PPARα to the nucleus;(iii)heterodimerization with its binding partner retinoid X receptor alpha (RXRα);(iv)binding of the heterodimer at DNA-response-elements (PPREs) in the promoters of target genes; and(v)alterations in binding of co-activators and co-repressors.In the canonical picture, activation of PPARα in liver parenchymal cells causes downstream alterations in gene expression through a series of coordinated steps:In recent years, genome-wide profiling of TF-binding activity has provided an unprecedented global picture of gene regulation. Chromatin immunoprecipitation (ChIP) in combination with microarray hybridization (ChIP-chip) or high-throughput sequencing (ChIP-seq) has been widely used for genome-wide location analysis and characterization of TF-chromatin interactions both for NRs and other TFs genes with the NR directly bound to their promoters;(ii)genes with the NR indirectly bound to their promoters by tethering to other directly bound TFs(iii)non-NR-bound genes regulated by other TFs.Accordingly, we are using a combination of: (i) microarray-based gene expression data; (ii) published ChIP-on-chip analyses of genome-wide NR binding; and (iii) curated lists of directly bound NR target genes to develop a comprehensive picture of NR-mediated transcriptional regulation. Differentially expressed genes responding to specific NR ligands are classified into three groups:The non-NR TFs that regulate genes in groups (ii) and (iii) are identified from the TRANSFAC  database of TF-DNA interactions. These transcriptional interactions may be summarized in a “latent regulatory network”  belongs to a class of toxicants known as halogenated aromatic hydrocarbons (HAHs). The toxic effects of HAHs in mammals are mediated through binding to the AhR Accumulation of TCDD in the target tissue.(ii)Formation of a complex with AhR.(iii)Activation of growth-regulatory genes by the AhR-TCDD complex.(iv)Cellular responses to the altered expression of growth-regulatory gene products.(v)The effect of these cellular events on tumor promotion and progression.The hepatic dose-response of TCDD culminating in liver cancer consists of the following key steps  through (v) are not well understood.The carcinogenic effects of TCDD are believed to be mediated by tumor promotion rather than initiation  and non-parenchymal cells (hepatic stellate cells and Kupffer cells), along with oval cell proliferation, can serve as an ontological tool to assemble diverse (i)cellular heterogeneity across the lobule;(ii)multiple cell types in the liver lobule that participate in liver injury.A realistic spatial model of the liver lobule and drug/chemical-induced toxic effects needs to account for:The virtual tissue formalism can be used to develop a spatial agent-based model of the human liver lobule (the “lobule ABM”), with individual hepatocytes represented by single agents. There have been some initial efforts toward development of agent-based representations of the liver Output from a standard TCDD physiologically based pharmacokinetic (PBPK) model This TCDD input dose acts upon the individual agents (hepatocytes) in the model, which occupy heterogeneous states by virtue of differential gene expression: e.g., graded expression of the Ah receptor along the lobule The agents (hepatocytes) are implemented as intracellular signaling cascades: i.e., key molecular events associated with the AhR toxicity pathway Figures A and 5C.Subsequent steps incorporate additional events into the signaling cascade shown in Figure Drug-induced liver injury is the most common adverse drug event leading to termination of clinical development programs and regulatory actions on drugs, as well as the most common cause of acute liver failure in the United States  and methapyrilene (MP).N-acetyl-p-benzoquinone imine (NAPQI). As GSH concentration is depleted, NAPQI forms protein adducts and induces mitochondrial oxidant stress, leading to cell death. The DILIsym™ model includes the basic biochemistry to describe these processes, and model simulations are consistent with molecular data, e.g., changes in liver GSH following APAP administration  yields the toxic reactive metabolite, n Figure , and then Figure . Formalin Figure . Howevern Figure ,b, suggen Figure ,b. In adn Figure . Togethen Figure . Using tn Figure A, and inn Figure B. This eN-acetyl-cysteine (NAC) is the standard therapy for APAP overdose  and in species-specificity . The model for MP was constructed using in vitro data, including the log P, pKa, metabolic partitioning in rat and mouse hepatocytes, and covalent binding in mouse, rat, and human microsomes. Multiple doses of MP were evaluated in simulated mice, rats, and humans. The DILIsym™ model predicted hepatotoxicity in rats between 150 and 200 mg/kg, consistent with the available literature  and related drugs . Further model development is ongoing and focuses on expanding model capabilities to address other mechanisms of hepatotoxicity.in vitro toxicology testing strategies and determination of pathway targets, as well as improved estimation of dose-response characteristics from a network biology perspective.We have presented some concepts relevant to implementation of a new vision for toxicity testing in the twenty-first century for chemical and pharmaceutical molecules NAS/NRC, , centereThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The competent immune system controls disease effectively due to induction, function, and regulation of effector lymphocytes. Immunosurveillance is exerted mostly by cytotoxic T-lymphocytes (CTLs) while specific immune suppression is associated with tumor malignancy and progression. In squamous cell carcinoma of the head and neck, the presence, activity, but also suppression of tumor-specific CTL have been demonstrated. Functional CTL may exert a selection pressure on the tumor cells that consecutively escape by a combination of molecular and cellular evasion mechanisms. Certain of these mechanisms target antitumor effector cells directly or indirectly by affecting cells that regulate CTL function. This results in the dysfunction or apoptosis of lymphocytes and dysregulated lymphocyte homeostasis. Another important tumor-escape mechanism is to avoid recognition by dysregulation of antigen processing and presentation. Thus, both induction of functional CTL and susceptibility of the tumor and its microenvironment to become T cell targets should be considered in CTL-based immunotherapy. Squamous cell carcinomas of the head and neck (SCCHN) are the sixth most frequent type of malignancy worldwide. SCCHN accounts for approximately 6% of all cancer cases and for about 650,000 new cases and 350,000 deaths worldwide each year –3. WhileMortality from SCCHN remains high because of the development of distant metastases and the emergence of therapy-resistant local and regional recurrences. It is therefore essential to develop a deeper understanding of the biology of this disease for more effective alternative therapies such as immunotherapy. As basis for immunotherapeutic approaches, interactions between tumors and the host immune system have been a subject of many studies. It has been shown that a naturally induced T-cell response recognizing SCCHN exists that could potentially target and possibly kill the tumor cells. Most cases in which this may have happened will remain obscure because they never become visible. Those cases that become clinically apparent show a different constellation. On one hand, antitumor immune effects can be observed; on the other, a deleterious effect on immune cells is exerted by the tumor. Tumor progression itself is therefore invariably linked to selective and pervasive impairment of immune cells. The identification and characterization of a variety of human tumor antigens with possible use for immunotherapy and immunomonitoring  and expeFor the development of strategies to prevent or reverse tumor-induced effects and to protect immune cells in the tumor microenvironment, studies of both (a) naturally occurring immune cells that could be recruited for immunotherapeutic strategies targeting specifically the tumor cells and (b) direct and indirect mechanisms responsible for dysfunction and death of immune cells in SCCHN are essential , 12. This review focuses on the mechanisms of tumor-mediated interference with the host immune system and CTL in particular concerning SCCHN. We describe tumor-escape mechanisms from the immune system at the tumor site and in the periphery and describe strategies to redirect the immune system to a more effective antitumor response.Two distinct causes of SCCHN are known. SCCHN is either caused by the spontaneous accumulation of multiple genetic alterations modulated by genetic predisposition and chronic inflammation, enhanced by environmental influences such as tobacco and alcohol abuse or by infection with oncogenic human papillomavirus (HPV). Carcinogens are regarded the most important factors. Thus, two main etiologies can be defined: tumors induced by toxic substances or by the activity of the viral oncogenes of HPV. Both etiologies involve a multistep process and result in alterations affecting two large groups of genes: oncogenes and tumor suppressor genes. However, it has been demonstrated that another set of genes is also altered. These are related to immune response mechanisms like the MHC complex. This observation is important because it shows that immune recognition is relevant and probably a force driving selection in the tumor towards immune-resistant variants (see below).HPV-associated SCCHN defines a distinct subgroup that could specifically be targeted by novel preventive or therapeutic measures. More than 100 human papillomavirus (HPV) subtypes are known to date. Of these, 15 have been shown to be oncogenic in humans. HPV type 16 and 18 seem to play the major role in the etiology of HPV an associated SCCHN, particularly those arising in the oropharynx , 14. HPVThe lower rate of carcinogenic-risk factors and p53 mutations and a younger patient population suggests that factors, currently unknown, are associated with viral entry, propagation/transformation, and immune evasion in HPV-associated SCCHN patients , 17. FaiTherefore, with these two different etiologies, different treatment options according to the genesis of their malignancy may be developed for future patients. Immunogenic tumors in patients mounting specific immune responses may be treated by induction or enhancement of specific CTL responses. Such responses may have clinical impact for primary therapy or in adjuvant settings when tumor burden has been reduced.Effective antitumor responses in individuals with SCCHN depend on the presence and function of immune cells that are able to recognize and eliminate tumor cells. These tumor antigen-specific T cells are known to be present in the peripheral circulation and tissues of patients with cancer. They can be monitored by multicolor flow-cytometry directly using tetrameric peptide-MHC class I and II complexes, so called tetramers. These molecules bind to the cognate T cell receptor. These stainings are combined with T cell markers  and if desired with makers for the differentiation  and the functional  or dysfunctional  status of these cells. A number of studies have investigated the frequency of tumor-specific T cells in the peripheral circulation of patients and in the tumor , 18–24. Wild-type (wt) sequence p53 peptides like other tumor epitopes are processed and presented to the host immune cells either directly by the tumor cells or by professional antigen presenting cells (APCs) such as dendritic cells (DC). This results in an increased number of wt p53 peptide-specific T cells and, in some instances, p53-specific antibodies , 26, 27.+ CD8+ T cells specific for the HLA-A2.1-restricted wt p53264-272 peptide is significantly higher in the peripheral circulation of HLA-A2.1+ patients with head and neck cancer than that in normal donors [+CD8+ effector cells alone has no effect on tumor progression. In this study, two patients with sufficient numbers of TIL were available to test in vitro responsiveness after polyclonal stimulation with anti-CD3 mAb. Only a low IFN-γ expression of the CD3+CD8+ T cells could be measured indicating a poor responsiveness to this stimulus. At the same time, a significantly increased number of regulatory T cells (Treg) were found at the tumor site compared to the periphery [+ tumor-specific T lymphocytes [+ microvesicles in the circulation of patients with head and neck and other cancers also suggests a potential mechanism for systemic elimination of CD95+ activated CD8+ T cells. However, neither the tumor origin of microvesicles present in patients' sera nor their in vivo participation in T cell apoptosis has been confirmed. It could be demonstrated that the frequency of tetramerl donors . Intereseriphery . It has eriphery . Treg areriphery . This imphocytes . To datephocytes , or tumo+ T lymphocytes directed against HPV E7 epitopes documenting a natural immune response [γ treatment that enhanced antigen processing and presentation by the tumor cells. Further phenotypic characterization of the HPV-specific T cells revealed an increase in terminally differentiated/lytic T cells (CD8+CD45RA+CCR7−). This population was also characterized by a high frequency of staining for the degranulation marker CD107a in E7 tetramer+ T cells, compared with bulk CD8+ T cells, consistent with their terminally differentiated lytic, degranulated status. These cells may account for the unsuccessful antiviral immune response [A subgroup of head and neck cancer is associated with human papilloma virus. Virus-derived antigens are considered superior targets for T cells than tumor-associated self-antigens because they have higher affinity to MHC and are more immunogenic. As opposed to tumor-associated self-antigens, thymic negative selection of T cells recognizing these virus derived antigens has not reduced the pool from which tumor reactive T cells can be recruited. Therefore, HPV-encoded oncogenic proteins, such as E6 and E7, are promising tumor-specific antigens in addition to the fact that they are considered obligatory for tumor growth. Surprisingly, few studies have characterized endogenous T cell immunity to HPV-encoded oncogenes in SCCHN as a prerequisite for immunotherapeutic targeting of these antigens. Two studies show an increased frequency of CD8response , 22. Theresponse  to these+ T-helper lymphocytes may be required for optimal antitumor effects by generating and maintaining antitumor immune responses through interactions with CTL and other cells [+ T-helper cells [110–124 CD4+ T cells to enhance the generation and antitumor function of CD8 effector cells was demonstrated. The results emphasize the crucial role of T helper-defined epitopes in shaping the immune response to multiepitope cancer vaccines targeting p53. This suggests that future vaccination strategies targeting tumor cells should incorporate helper and cytotoxic T cell-defined epitopes [Several current studies suggest that cancer vaccines have increased efficacy if they incorporate tumor-specific cytotoxic as well as helper epitopes. Although CTL are considered to play the primary role in tumor eradication, it is also hypothesized that the participation of tumor antigen-specific CD4er cells , 33. As er cells , 34. By Homeostasis of lymphocytes, that circulates through the tissues and the blood, is maintained by refreshing the pool via the thymic output of naive lymphocytes and expansion of antigen-specific lymphocytes upon adequate stimulation and contraction of the lymphocyte pool by death of lymphocytes in the periphery that have completed their functions –37. +, Fas+ T cells in the peripheral circulation were in the process of apoptosis. This indicates that the Fas/FasL pathway is involved in spontaneous apoptosis of circulating CD95 (Fas+) T lymphocytes. Interestingly, these cells showed decreased expression of CD3-zeta-chain. Expression of T cell receptor- or Fc-gamma receptor III-associated signal-transducing zeta chain is important for the functional integrity of immune cells [+ T cells in patients with SCCHN that were altered relative to normal controls. This may reflect increased apoptosis of expanded or tumor-contracted CD8+ T cells, which define the TCR Vbeta profile of antigen-responsive T-cell populations in patients with cancer [+ T effector cells via Fas/FasL [+ effector cells, adverting the cancer community that traditional cancer therapy might contribute to tumor progression by collaborating with the peripheral tolerance process. In addition, Treg in patients with SCCHN kill CD4+ T effector cells via granzyme B in the presence of IL-2 [One of several mechanisms by which tumors escape from the host immune system is induction of apoptosis in effector T cells . It was ne cells . Fas/Fasne cells . This mane cells , 40. Thene cells  that alsne cells . Preferene cells . This obh cancer . Reportsh cancer , 43 suppFas/FasL . FasL isFas/FasL . These F of IL-2 . Signali of IL-2 . Patient of IL-2 . A high  of IL-2 .Taken together it appears that apoptosis of lymphocytes in the periphery as well as at the tumor site leads to rapid and selective tumor-specific lymphocyte turnover followed by a loss of effector cells and thus failure to control tumor growth in cancer patients.+ regulatory T cells (Treg), Treg have been well characterized as a distinct lineage of T cells [+ Treg are depleted in an adult individual, fatal multiorgan autoimmunity finally results [+ Treg de novo in the peripheral immune compartment (denominated inducible Treg or iTreg) or to selectively expand Treg in peripheral blood and lymphoid organs are less clear. Essential roles of IL-2, TGF-β, and TCR signaling in iTreg generation, expansion, function, and survival have been established [Because of the identification of the forkhead box transcription factor Foxp3 as an essential transcription factor in CD4 T cells . Their t T cells , 50. Whe results  and the  results , 53. Howablished –56. Howeablished . In addi+CD25+ Tregs are depleted [It has become increasingly clear that malignant transformation and cancer progression are immunologically recognizable events and the immunologic status of the host as well as the inflammatory conditions of the tumor microenvironment influences significantly the outcome of the patient . In earldepleted , 66. Remdepleted .Thus, one therapeutic possibility for restoration of anti-tumor immunity in patients with cancer is to eliminate tumor antigen (TA)-specific Treg and to boost simultaneously TA-specific T helper and CTL responses. The fact that Treg and activated T effector cells share receptors and common metabolites in their differentiation, function, survival, and expansion  suggests that regulation of the effector and suppressor compartment is dichotomic. Thus, one new challenge in modern immunotherapy is to understand the signaling pathways that command the interplay of effector and suppressor responses in physiologic conditions and in inflammation. A detailed knowledge of these pathways might enable us to design immunotherapeutic strategies that selectively promote expansion, survival, and function of effector or Treg responses in pathologies where one of the two compartments is in disadvantage resulting in autoreactive killer responses in the absence of Treg or in immunosuppression in the case of an excessive Treg response. Revert immunosuppression in cancer to anti-tumor immunity is essential to increase the quality and success of traditional cancer therapy as well as the response to tumor vaccines.in vitro and in vivo [Clinical trials for tumor vaccines using TA antigens and antigen presenting cells (APCs) or DC are now under way for the treatment of different types of cancer. To date, more than 1000 tumor vaccines have been reported . The col in vivo . The rep in vivo . Thus, aβ1, and the TCR are determinant in regulating homeostasis of suppressor cell and effector cell populations [First studies investigating the dynamics of Treg and effector responses indicate that molecular signaling pathways in response to IL-2, TGF-ulations –56, 79. in vivo, in the maintenance of immune system homeostasis and self-tolerance. The latter function is emphasized by the finding that mice deficient in IL-2 or components of the IL-2 receptor (IL-2Rα or IL-2Rβ) succumb to lymphoproliferative autoimmune syndrome, with the effect of IL-2Rβ lack being more severe [β1 is a pluripotent cytokine that has pronounced effects on T cell-mediated immune suppression as well as on the control of autoimmunity. The role of TGF-β is in influencing the constitutive expression of Foxp3. IL-2, the transcription factor NFκB, might be essential in regulating Treg and T effector cell homeostasis in conditions of inflammation. Elucidating the role of TGFβ and NF-κB in Treg and T effector cell dynamics is promising because both are up-regulated during cancer inflammation and are common regulators of Treg and Th17 cell differentiation. The design of studies that elucidate these signaling pathways in patients with cancer constitutes one step towards immunotherapeutic strategies that enforce immunogenicity of tumor vaccines.IL-2 plays a dominant role e severe , 81. TGFTaken together, these complex immunoregulatory mechanisms lead to an immunoediting of the T cell and in particular the CTL response by the tumor in order to avoid elimination by the immune system. However, also the tumor cell immunogenicity is edited by the immunoresponse.Tumor-T cell interaction leads to a negative selection pressure on tumor cells that are being recognized by the immune system. T cell recognition is therefore reduced in tumor cells by downregulation of molecules important for antigen processing and presentation or costimulation . Several observations demonstrate an immune selection of SCCHN tumor cells as discussed below. This selection process and the resulting immune escape variants in the tumor indicate that an effective CTL response must have taken place during the development of the malignancy. The CTL-mediated cytolysis of immunogenic tumor cells is the driving force of the selection process towards non-CTL-susceptible tumor cell variants. The immune-evaded tumor cells have several features making them resistant to further natural CTL attack. Reduced expression of costimulating molecules on the cell surface leads to inadequate T-cell activation, even if the tumor is recognized and in turn to tolerance induction . Expression of MHC class I is altered in up to 50% of SCCHN –86. BothExpression of components of the antigen processing machinery (APM), namely, LMP2, LMP7 and TAP1 is frequently downregulated or completely lost in tumor lesions as compared to surrounding tissue as well as in cell lines obtained from SCCHN cancer patients. As a consequence of the downregulated APM components, fewer antigen is processed and loaded onto MHC-complexes that are by themselves decreased in number or dysfunctional. In combination, these three mechanisms compromise recognition of the progressed tumor by tumor-specific T cells. In cell lines, the expression of APM-components could be restored by incubation of with IFN-g , 88. FurTumors can also interfere with the immune system by producing and releasing numerous factors that modulate functions of immune cells or directly induce apoptosis. These factors take action in the tumor microenvironment and beyond.For effective vaccination, a successful stimulation of the immune system and an effective modulation of suppressive effects exerted by the tumor cells are necessary .Downregulation of dysfunction of APM components by the tumor may disturb both the induction of tumor-specific T cells in the initial phase of the immune response and subsequently during the effector-phase the proper recognition of the tumor. This effect is augmented by absent or reduced presence of MHC class-I molecules on the cellular surface. These cells are considered to have a more aggressive phenotype  which maγ was able to restore T cell recognition and killing [In experimental systems, incubation of SCCHN cell lines with IFN- killing , 88. The+ T cells in the peripheral circulation and at the tumor site, has been observed [+ T cells from apoptosis [Tumor-induced increased apoptosis of lymphocytes, including NK-cells and tumor-specific CD8observed –43, 92. poptosis .+CD25+ compartment, including CD4+CD25highFoxp3+ [+CD25+ T and CD4+CD25highFoxp3+ cells increased in the patients after receiving oncologic therapy, but suppressor function and survival of these cells were significantly elevated. The observed expansion of Treg expressing Foxp3 correlated with increased suppression mediated by these cells in patients with non evident disease [de novo induced Treg and T responder cells by inflammatory cytokines derived from the strong inflammatory response that usually accompanies radio/chemotherapy. The immune system controls the level and the activation state of each cellular compartment through homeostatic regulation, a process that is triggered during development and after the induction of a lymphopenic state by external stimuli [The observation that Treg are increased in peripheral blood and at the tumor site in patients with cancer suggests that the development of treatment paradigms that seek to not only increase the frequency of tumor-specific T cells but to do so in conjunction with strategies that selectively inactivate or remove suppressor T cells is a must. However, the monitoring of Treg frequency and function in patients with cancer is not only required to improve the success of cancer vaccines and traditional cancer therapy but might also provide a broader basis for the development of more reliable prognostic factors, improving a tumor-free outcome after therapy. On the other hand, selective depletion of Treg in patients that have received traditional cancer therapy might be essential to avoid or decrease recurrent disease. We have shown that hematologic recovery in response to oncologic therapy results in expansion of the CD4ghFoxp3+ . Importa disease . The eff stimuli . Radio/c stimuli . Thus, T stimuli , 97. In p53 may serve as a model antigen for the development of broadly applicable antitumor vaccines in SCCHN. A number of p53-derived epitopes that can be used for the design of vaccines have been identified , 99. SinIn vitro stimulation of CD8+ T cells with wt p53 peptide-pulsed autologous dendritic cells can be used to induce either HLA-A2-restricted, wt p53149-157 and/or wt p53264-272 peptide-specific responses from epitope-specific precursors. Interestingly, using these single epitopes, wt p53 peptide-specific CD8+ T cells were generated in only a third of healthy donors or subjects with cancer [h cancer , 102. ThSince it may prove difficult to determine in an individual case the responsiveness prevaccination, a vaccine consisting of more than one epitope may be the more promising approach. Oncogenic HPV genotypes, particularly the HPV types 16 and 18, are found in a subset of SCCHN mostly lacking the risk factors of alcohol and tobacco abuse. It therefore seems to be a suitable malignancy for vaccination against HPV-associated targets. Thus, these HPV-associated SCCHN may be preventing by (a) the existing prophylactic HPV-vaccines or (b) treated by vaccines designed to induce an appropriate antitumor immune response against HPV-specific tumor antigens . Lately,Neutralizing antibodies specific for the viral capsid proteins may prevent infection by HPV. Prophylactic HPV vaccines have been developed and approved. As antigen, they contain capsid protein L1 of the most prevalent HPV types. They were introduced in 2006 and have the estimated potential to reduce the burden of cervical cancer, the tumor entity they were originally developed for, by approximately 70% , 106 andHowever, they may be of benefit in posttherapeutic situations where infected lesions have been removed surgically to prevent formation of new lesions due to reinfection . These sThe rationale for vaccines targeting HPV-associated SCCHN is that virus-related oncogenes are obligatory to tumor growth. Vaccines with therapeutic potential must target the two HPV oncogenic proteins, E6 and E7 as antigens that are important in the induction and maintenance of cellular transformation and are coexpressed in the majority of HPV-associated carcinomas. Two studies have investigated if an endogenous T-cell immunity to HPV-encoded oncogenes E6 and E7 in SCCHN patients exists , 22. ThiTumors consist of heterogeneous populations of cells. According to the cancer stem cells (CSCs) hypothesis, CSCs are a subpopulation of the tumor more capable than other cells to self-propagate, initiate new tumors differentiate into bulk tumor, and therefore sustain tumor growth. Because of these properties, CSCs have been moved into the focus of targeted therapies. The current knowledge of the existence of CSCs begins to lead to studies of their specific elimination . It is bThe development of CSC targeted therapy has to overcome three major hindrances, that relatively to the bulk tumor population are increased (i) chemoresistance, (ii) resistance to radiotherapy, and (iii) immunescape mechanisms.Since radio- and chemotherapies have already been optimized towards the limits of clinical benefit and yet tolerable side effects, a very attractive alternative approach of specifically targeting CSC is to develop antitumor T cell vaccines. One of the possible reasons that these therapies lacked efficacy in past clinical trials could be attributed to the fact that rather bulk tumor than CSC have been targeted. This may change with the identification of tumor-specific epitopes derived from CSC markers. One such as CSC model-target for head and neck cancer and others is the recently described CD8 defined T-cell epitope of aldehyde dehydrogenase-1 (ALDH1) . ExampleIn several studies, the efficacy of potential therapies directed against stem cell-associated signaling pathways are tested. For example, T cell immunity against embryonic stem cells antigen SOX2 and SOX6 has been explored in glioma stem cells , 78. SinSuccess of these potential therapies will depend on how well immunological responses to CSC can be modulated for example by vaccine adjuvants upregulating antigen processing and presentation. Recently, a reduced activity of the 26S proteasome in breast cancer cells and in gliomas was observed as a feature of CSC . ProteasThe classification of conclusive CSC markers followed by the identification of defined T cell-recognized CSC epitopes in the future may lead to the clinical application of anti-CSC vaccination strategies. Several approaches are currently being evaluated . WhetherImmunotherapy is considered an attractive treatment modality because it specifically targets the cancer avoiding or minimizing side effects. Ideally, its therapeutic effects will also reach distant metastasis and will be sustainable lasting beyond the presence of the cancer due to immunologic memory.Therapeutic strategies should consider that SCCHN has at least two distinct etiologies. One is chronic alcohol and tobacco abuse and the other is related to oncogenic human papillomavirus infection and transformation. Both etiologies will differ significantly in the antigenic make up of the tumor cells based on presence of self or viral antigens, respectively. In both cases, however, immunotherapeutic approaches should aim at induction of adequate antigen processing and presentation by the tumor cells to become visible for the immune system as target. Furthermore, tumor induced immune dysregulation should be redirected in favor of tumor rejection and finally an adequate stimulation of effector T cells capable of in vivo expansion and survival in the tumor-microenvironment is thought critical for improving clinical results.


To assess the likelihood of biased trials contained in 12 recently published meta-analyses.A review of component randomised controlled trials of systematic reviews with 12 meta analyses.12 recently published systematic reviews with 503 component randomised trials, which were published in the BMJ, Lancet, JAMA and The Annals of Internal Medicine up until May 2012.Systematic reviews were eligible for inclusion if they included only randomised controlled trials (RCTs). We obtained the full text for the component RCTs of the 12 systematic reviews. We excluded RCTs which were not available in English from our review and analysis.2 >0.30) in age differences, when there should have been none, with two having significant or substantial heterogeneity (I2 > 0.50). In three meta-analyses we found that the age of intervention group was statistically significantly higher than in the control group. Two meta-analysis had a distribution of p-values that were inconsistent with chance. Meta-regression explained some of the observed heterogeneity in two meta-analyses as a consequence of poor allocation concealment.Five of the 12 meta-analyses exhibited heterogeneity (IThe majority of a sample of recent meta-analyses showed that there were signs of imbalance and or heterogeneity in ages between treatment groups, when there should have been none. Systematic reviewers might consider using the techniques described here to assess the validity of their findings.


Arabidopsis thaliana has been studied intensively by a range of proteomics approaches and is now one of the most well characterized eukaryotic ribosomal proteomes. Plant cytosolic ribosomes are distinguished from other eukaryotic ribosomes by unique proteins, unique post-translational modifications and an abundance of ribosomal proteins for which multiple divergent paralogs are expressed and incorporated. Study of the A. thaliana ribosome has now progressed well beyond a simple cataloging of protein parts and is focused strongly on elucidating the functions of specific ribosomal proteins, their paralogous isoforms and covalent modifications. This review summarises current knowledge concerning the Arabidopsis cytosolic ribosomal proteome and highlights potentially fruitful areas of future research in this fast moving and important area.The cytosolic ribosomal proteome of  Ribosomes – the ribonucleoprotein complexes responsible for catalyzing translation – the mRNA-guided synthesis of proteins from aminoacyl-tRNA, GTP and ATP substrates – have fascinated biologists since their Nobel Prize-winning discovery by George E. Palade in 1955  design bioactive agents to alter their function , phosphorous (P), and energy while translation itself represents a further major demand on N and energy reserves Warner, . UnderstThe gross structure of ribosomes is essentially the same between prokaryotes and eukaryotes in that they are both comprised of ribosomal RNAs (rRNAs) and proteins (r-proteins) in large and small subunits. However, the ribosomes of eukaryotes exhibit greater structural complexity, reflecting the greater complexity of molecular mechanics observed in eukaryotic translation  to translate with a greater priority toward fidelity and control over speed of ribosome biogenesis; (2) to efficiently and accurately translate mRNAs having (and encoding proteins having) a wider range of primary and secondary structures; (3) to have greater control over the relative rates of translation of specific mRNAs; (4) to have a greater capacity for spatiotemporal ribosome heterogeneity in order to tailor the translation process for different subcellular locations, cell types, and developmental stages , rat (Rattus norvegicus), human (Homo sapiens), and Arabidopsis  have been the most extensively characterized. Primarily through the extensive r-protein sequencing and gene cloning efforts of Wool et al.  exhibit no sequence divergence within them while others  include some members predicted to generate proteotypic peptides and others that would not -based Peptide Mass Fingerprinting (PMF) to identify protein spots corresponding to 87 distinct r-protein gene products representing 60 of the 80 r-protein families. The authors highlighted low molecular weights, high pIs and low numbers of tryptic cleavage sites as possible reasons for the non-detection of the other 20 predicted families of r-proteins in their study. A key finding of the study was that at least 21 of the 60 detected r-protein families were represented by two or more distinct r-proteins (distinct AGIs) and that >45% of the distinct r-proteins detected were represented by 2–13 separate spots. Indeed, this confirmed earlier predictions by Barakat et al. . Protein assignments based on ∼850 peptide identifications (mostly based on MALDI-TOF-MS with 172 based on MS/MS) provided evidence for the ribosomal incorporation of 14 previously undetected r-protein families, bringing the total number of detected families to 74 and leaving only six undetected. This study also presented new evidence to support the identification of particular r-protein family members by reporting the masses of ions assigned to tryptic peptides predicted to belong to only one specific gene product. On this basis, Chang et al.  and a custom data analysis pipeline to provide deep proteome coverage and high-confidence paralog-specific identifications. This analysis, based on 1446 high-quality MS/MS spectra matching to 795 peptide sequences, provided high-confidence evidence for the presence of 79 of the 80 predicted r-protein families in the ribosomes of Arabidopsis, including five previously undetected r-protein families: S29, S30, L29, L36a, and L39.Prompted by the above observations, Carroll et al.  undertooArabidopsis ribosomes is the extremely basic (predicted pI of 13.4) and small (3.5 kDa) L41. The four paralogous L41 genes in the A. thaliana genome  encode identical proteins with the amino acid sequence MRAKWKKKRMRRLKRKRRKMRQRSK. The strong conservation between eukaryotes of genes encoding this putative r-protein suggests that it is most likely a component of Arabidopsis ribosomes. X-ray crystallography has shown that the yeast ortholog of Arabidopsis L41 forms a bridge between the 40S and 60S subunits .Each of the major efforts to qualitatively define the A. thaliana ribosomes, orthologs of the mammalian Receptor of Activated C Kinase (RACK1) are the most consistently detected. The RACK1A protein encoded by At1g18080 has been reported in all major proteomic surveys of A. thaliana ribosomes to date  and At2g39820 (eIF6B). While both proteins have been demonstrated to interact physically with RACK1  representing some 30% of newly synthesized proteins, it would seem efficient to have proteasome complexes localized at the point of protein synthesis to prevent the escape of potentially toxic defective proteins into the cytoplasm. Another possible explanation may lie in the major role played by the proteasome in ribosome biogenesis  in eukaryotic cells  of A. thaliana leaf cytosolic ribosomes responded significantly to sucrose feeding – a treatment that elicits dramatic changes in gene expression. Similar experiments involving different treatments seem likely to reveal even broader ribosome dynamics involving a wider range of r-proteins.With an abundance of qualitative proteomics data suggesting the extreme heterogeneity of cytosolic ribosome populations from whole plant tissues, ribosomal proteomics is now heavily focused on understanding the spatiotemporal distribution and physiological function of this heterogeneity. Ribosome heterogeneity could and probably does occur at many different spatiotemporal scales – from slow developmental changes or constitutive differences in the ribosome populations of distinct organs to rapid changes in minor subcellular populations of ribosomes within single cells. Hence, a major task that will be important for reverse-engineering the physiological function of ribosome heterogeneity will be the use of quantitative proteomic approaches to correlate variations in the relative abundances and modification states of different r-protein paralogs across tissues, cell types, subcellular-fractions, developmental stages, and environmental and genetic perturbations with ribosome properties and processes upstream and downstream of ribosomes. With this goal in mind, Hummel et al.  and collA. thaliana r-protein mutants have already emerged . Quantitative proteomic comparisons of these ribosomes with those pulled down using control mRNAs might help reveal regulatory elements within test mRNAs that promote their recruitment by polysomes while also revealing the types of ribosomes they attract.Another targeted approach might be to genetically perturb the expression of particular r-proteins and then monitor and cross-correlate changes in ribosome composition with changes in the translatome. However, given the high potential for pleiotropic effects when disrupting translation machinery, perhaps an appropriate approach would be to employ inducible, possibly cell type-specific, silencing, or over expression of r-proteins so that time-course profiling of the ribosomal proteome and translatome can be used to distinguish primary (early) and secondary (later) effects of specific r-protein perturbations.Another potentially powerful approach to understand how changes in ribosome composition are related to changes in translation may be to apply next-generation ribosome footprinting whereby the exact locations of ribosomes on transcripts is determined by deep sequencing the regions of transcripts that are protected by ribosomes (Ingolia et al., Clearly, as far as the structures and functions of ribosomes are concerned, there are still many more questions than answers. Despite being discovered so long ago, ribosomes remain one of the most interesting and crucially important targets for basic and applied biological research. However, being the complex natural nano-machines that they are, they do not give up their secrets easily and will no doubt remain the focus of many research careers well into the foreseeable future.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.http://www.frontiersin.org/Plant_Proteomics/10.3389/fpls.2013.00032/abstractThe Supplementary Material for this article can be found online at


Squamous metaplasia presenting in noninvasive mucinous cystic neoplasm (MCN) of the pancreas is extremely rare. We described a case of 39-year-old Chinese female with a 5-year history of a slow growing mass in the left upper abdomen and an 18-month history of surgical incision exudation. The patient underwent cystojejunostomy, laparotomy and distal pancreatectomy consecutively because of the initial diagnosis of “pancreatic cyst”. The histological section showed columnar mucin-producing epithelium formed small papillary projections and extensively visible squamous metaplasia. Therefore the diagnosis of “noninvasive MCN with intermediate-grade dysplasia of the pancreas and extensive squamous metaplasia” was made finally. The squamous component of the pancreas may be derived from pluripotent stem cells, and may be in association with cystojejunostomy.http://www.diagnosticpathology.diagnomx.eu/vs/1322364365718540The virtual slide(s) for this article can be found here  Mucinous cystic neoplasm (MCN) is a rare exocrine pancreatic tumor that can be classified into four histopathological types : noninvaA 39-year-old Chinese female was referred to our hospital with a 5-year history of a slow growing mass in the left upper abdomen and an 18-month history of surgical incision exudation. The painless mass was found incidentally with an initial diagnosis of pancreatic cyst 5 years previously, and the palliative bypass procedure of cystojejunostomy was performed. However, due to the increasing size of the mass, the patient underwent a laparotomy 2 years later. This revealed a pancreatic tumor which was inoperable because of the major adhesion surrounding the neoplasm, stomach, greater omentum, mesentery and abdominal wall. Some of the anastomotic stoma tissue was excised for pathological examination, and a diagnosis of noninvasive MCN with intermediate-grade dysplasia was made. After 18 months, the surgical incision began to produce exudate. On admission to hospital, physical examination revealed a single, deep-seated, painless mass and two incisional sinuses with exudation in the left upper abdomen, without tenderness or muscular tension. Laboratory investigations were unremarkable, and serum levels of carbohydrate antigen 19–9 and carcinoembryonic antigen were within normal ranges. Abdominal ultrasonography and computer tomography scan revealed a 7.8 cm × 7.3 cm, heterogeneous hypoechoic or low-density mass with poorly defined margins in the tail region of the pancreas, compressing other adjacent organs. The mass was composed of several large loculi with an irregular thickening of the cyst wall and papillary excrescences projecting into the cystic cavity , the epithelial component with squamous differentiation was strongly immunoreactive with high molecular weight cytokeratin HCK, 34βE12) and CK5/6  theThe preoperative diagnosis of MCN is very important, since other types of pancreatic neoplasm may be treated differently and the grade of neoplasm does accurately predict the outcome ,5. The mAll MCNs should be resected to prevent malignant transformation but can be monitored for an appropriate time if the lesion is small without the presence of mural nodules -15. Our We have described a case of 39-year-old Chinese female with a 5-year history of a slow growing mass in the left upper abdomen and an 18-month history of surgical incision exudation. The patient underwent cystojejunostomy, laparotomy and distal pancreatectomy consecutively because of the initial diagnosis of “pancreatic cyst”. The histological section showed columnar mucin-producing epithelium formed small papillary projections and extensively visible squamous metaplasia. Therefore the diagnosis of “Noninvasive MCN with intermediate-grade dysplasia of the pancreas and extensive squamous metaplasia” was made finally. The squamous component of the pancreas may be derived from pluripotent stem cells, and may be in association with cystojejunostomy and the inflammation. More studies of series of patients with MCN and longer follow-up times are needed to explore whether extensive squamous metaplasia represents neoplasm progression and/or predicts a poor prognosis.HCK: High molecular weight cytokeratin; IHC: Immunohistochemistry; LCK: Low molecular weight cytokeratin; MCN: Mucinous cystic neoplasm; MEBMMSO: Mixed-epithelial papillary cystadenomas of borderline malignancy of mullerian type with squamous overgrowth; PR: Progesterone receptor.The authors declare that they have no competing interests.The research for this article was supported by grants from National Natural Science Foundation Of China (Nos. 30771120 and 81072103).PFL, YMW and ZW drafted the manuscript. QQZ was responsible for collecting the patient material. YXL and YL carried out the tissue staining, immunoassays, and electron microscopic examination. PFL and ZW conducted the analysis of the histological features and clinicopathological relations. All authors read and approved the final manuscript.Written informed consent was obtained from the patient for publication of this Case Report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


The prevalence, and associated healthcare burden, of diabetes mellitus is increasing worldwide. Mortality and morbidity are associated with diabetic complications in multiple organs and tissues, including the eye, kidney and cardiovascular system, and new therapeutics to treat these complications are required urgently. Triethylenetetramine (TETA) is one such experimental therapeutic that acts to chelate excess copper (II) in diabetic tissues and reduce oxidative stress and cellular damage.Here we have performed two independent metabolomic studies of serum to assess the suitability of the streptozotocin (STZ)-induced rat model for studying diabetes and to define metabolite-related changes associated with TETA treatment. Ultraperformance liquid chromatography-mass spectrometry studies of serum from non-diabetic/untreated, non-diabetic/TETA-treated, STZ-induced diabetic/untreated and STZ-induced diabetic/TETA-treated rats were performed followed by univariate and multivariate analysis of data.Multiple metabolic changes related to STZ-induced diabetes, some of which have been reported previously in other animal and human studies, were observed, including changes in amino acid, fatty acid, glycerophospholipid and bile acid metabolism. Correlation analysis suggested that treatment with TETA led to a reversal of diabetes-associated changes in bile acid, fatty acid, steroid, sphingolipid and glycerophospholipid metabolism and proteolysis.Metabolomic studies have shown that the STZ-induced rat model of diabetes is an appropriate model system to undertake research into diabetes and potential therapies as several metabolic changes observed in humans and other animal models were also observed in this study. Metabolomics has also identified several biological processes and metabolic pathways implicated in diabetic complications and reversed following treatment with the experimental therapeutic TETA. Diabetes mellitus (DM) is a chronic debilitating condition that is rapidly increasing in prevalence worldwide, as a consequence of increases in obesity, changing patterns of diet and physical activity, and ageing populations. The World Health Organization estimated that 154 million people in the world had DM at the beginning of the 21st century [DM is a metabolic disorder characterized by hyperglycemia. The hyperglycemia is caused as a consequence of a deficiency in insulin in type 1 diabetes (T1D), and is a feature of late type 2 diabetes (T2D) along with insulin resistance. T2D is significantly more prevalent than T1D. Molecular pathophysiological mechanisms that precede hyperglycemia, or are observed with the clinical symptoms of DM, include, among others, alterations in lipid and amino acid metabolism -5, changDM is a disease associated with multiple metabolic abnormalities so the application of metabolomic techniques to study it and potential interventions is appropriate . A recenA number of animal models have been used to study DM  and hereOne potential treatment under investigation to treat diabetic complications (though not to reverse hyperglycemia) is the copper (II)-selective chelator triethylenetatramine (TETA) . Recent The two primary objectives of the research described were to (i) assess the metabolic changes in the STZ-induced rat model of DM and compare these changes to metabolic changes observed in published research in relation to other animal models of DM and (ii) to investigate the metabolic response to TETA treatment in the STZ-induced rat model of DM. Serum was chosen as an appropriate biofluid to integrate the diabetes-induced changes that occur in multiple tissues. The investigation of the serum metabolome was chosen as DM is defined as a metabolic disorder and changes in metabolism are expected. The application of metabolomics to study the effect of TETA treatment on the reversal of diabetic complications is also appropriate; the mode of action of TETA is to chelate copper and reduce oxidative stress in cells and tissues. Markers of oxidative stress (including oxidized lipids) are expected to be observed in the serum metabolome. This study had the potential to identify metabolic biomarkers to apply in other studies - for example, the monitoring of drug safety and efficacy in clinical trials. Two independent but similar biological studies were performed at different times, each assessing metabolic differences in four groups: (i) non-diabetic/untreated rats, (ii) non-diabetic/TETA-treated rats, (iii) STZ-induced diabetic/untreated rats and (iv) STZ-induced diabetic/TETA-treated rats. Ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) and univariate/multivariate data analysis methods were applied to determine differences in the serum metabolomes associated with STZ-induced DM and TETA treatment.Treatment of the rats with intraperitoneal STZ resulted in elevated plasma glucose levels consistent with diabetes (>30 mmol/L), whereas non-treated rats without STZ injection had normal glucose levels (<7 mmol/L) in each of the two studies. Time-independent replicate studies were performed with metabolomic analyses undertaken in November 2009 (study 1) and May 2010 (study 2). Half the rats in the non-diabetic and diabetic groups were treated with TETA, that is, there were four groups in total. In studies 1 and 2 the numbers of rats, which were present in each of the four classes at time of sacrifice, were as follows: non-diabetic/untreated , nondiabetic/TETA-treated , STZ-induced diabetic/untreated  and STZ-induced diabetic/TETA-treated . Following quality assurance procedures, 4,826 and 7,425 metabolic features were detected in positive ion mode in studies 1 and 2, respectively, and 1,369 and 3,469 metabolic features in negative ion mode in studies 1 and 2, respectively. These metabolic features were taken forward for further data analysis. Of these metabolic features, 51.9% and 40.3% of metabolic features were putatively identified in the two studies, respectively, following the process of metabolite identification. This highlights that not all metabolites are identified in untargeted metabolic studies.Principal components analysis (PCA) was performed applying all detected metabolic features to assess the variability in the data and determine whether outliers were present. Figure P < 0.05) between different pairwise combinations of the four study groups. A single metabolite can be detected as different metabolic features, each with the same retention time but a different m/z value caused by the detection of different ionic species of the same metabolite . Also, a single metabolic feature can relate to multiple stereoisomers. Therefore, multiple metabolic features can correspond to a single metabolite or stereoisomer. The number of statistically significant (P < 0.05) metabolic features for each comparison and animal study and the number of metabolic features observed as statistically significant in both studies are shown in Table Univariate statistical analyses were performed to identify metabolic features whose relative concentrations were statistically different (P < 0.05) (i) when comparing STZ-induced diabetic/untreated versus non-diabetic/untreated rats, (ii) in both independent studies and (iii) with the same direction of relative change (up-regulation or down-regulation) in both independent studies. This provides further confidence that the metabolites defined as 'biologically interesting' are relevant and deserve further investigation. Specific metabolite classes that are over-represented in the results include amino acids and related metabolites (10 metabolites), bile acids (5 metabolites), dipeptides (2 metabolites), long- and short chain fatty acids and related metabolites (23 metabolites), glycerophospholipids (41 metabolites), nucleosides, nucleotides and purine metabolites (6 metabolites), sphingolipids (4 metabolites) and vitamin D metabolites (3 metabolites).Table The statistical analysis showed a limited number of metabolic features whose change in concentration was statistically significant when comparing STZ-induced diabetic/TETA-treated versus STZ-induced diabetic/untreated rats. These are shown in Table Diabetes is a multi-factorial metabolic disease. To study metabolic alterations in an experimental, STZ-induced animal model of DM, we applied UPLC-MS based metabolic profiling. Investigation of serum from animals 12 weeks after induction of the diabetes-like insult with comparison to non-diabetic controls, both in the presence and absence of oral treatment with TETA, were performed. Identification of changes in relative metabolite concentrations revealed changes of specific metabolic pathways or areas of metabolism in response to DM and treatment with TETA.In addition to the expected hyperglycemia, changes in the serum concentrations of amino acids and related metabolites, bile acids, dipeptides, short and long-chain fatty acids and related metabolites, glycerophospholipids, nucleosides/nucleotides/purine metabolites, organic acids, sphingolipids and vitamin D metabolites were observed. These metabolic changes could be a result of mechanisms related to DM and associated tissue-specific complications but also unexpected secondary actions of treatment with STZ. However, the findings of our study are compatible with known altered mechanisms in DM, making it reasonable to believe that these changes are related to the diabetic state. A selection of biologically important alterations related to potential tissue-specific changes and observed in human and other animal models of DM will be discussed below. These previously observed changes highlight the applicability of the STZ-induced rat model to the study of metabolic perturbations in DM.Changes in branched-chain amino acid metabolism related to altered catabolism have been reported previously in the pre-diabetic state in humans ,5 and inArginine, proline and oxoproline, which all decreased in concentration in the diabetic rats, are metabolically closely connected and are downstream products of the urea cycle. Creatinine is also present at lower concentrations in this study. Alterations to urea cycle intermediates in humans  and animOther amino acid-related changes included an elevation of 5-methoxytryptophan and a decrease in tryptophan concentrations in diabetic animals; these changes have been found previously in the retina . So the Creatine is decreased in this study and this has been observed previously in animal models of DM, but in urine and cardiac tissue. The decrease in cardiac tissue may be related to increased demands of creatine/phosphocreatine for energy production through rapid re-synthesis of ATP from ADP in muscle and tissue with high energy demands .Bile acids have an important role to play in regulation of lipid, glucose and energy metabolism through the farnesoid × receptor and the facilitation of postprandial nutrient absorption by the gut . Our stuChanges in other lipid species were also observed. Multiple fatty acids were observed to change, with seven long-chain fatty acids present at higher concentrations in diabetic rats. These changes indicate either an impairment of adipose tissue storage of circulating fatty acids and inhibition of hepatic fatty acid esterification or an alteration to the uptake and utilization of fatty acids via fatty acid beta-oxidation in mitochondria. Both arise from insulin insensitivity causing increased concentrations of non-esterified fatty acids in plasma and ultimately increased beta-oxidation of fatty acids in liver and skeletal muscle mitochondria producing short chain fatty acids and ketone bodies. Increased ketone bodies were observed in this study consistent with insulin deficiency in diabetic animals. Free fatty acids have been shown to cause insulin resistance in many tissues, including cardiac and skeletal muscle .Glycerophospholipids showed both increases and decreases in their concentrations in diabetic rats in this study, implicating changes in cellular membranes and lipoproteins in blood. Here, many lysoglycerophosphocholines (lysoPCs) were decreased in diabetic rats, which indicate a perturbation in the equilibrium between lysoPC production  and lysoPC acetylation . Although increases in lysoPC concentrations, which are pro-inflammatory mediators, accompany inflammatory responses in DM , here wSphingolipids were also observed to change and may be related to signaling and plasma membrane changes. Dysfunctional sphingolipid metabolism has been suggested to contribute to metabolic stress in DM and to the pathogenesis of diabetic retinopathy ,41. In mThe second objective of this research was to identify metabolites, classes of metabolites and/or metabolic pathways that are perturbed in DM and return to a pre-diabetes state following treatment with TETA. Some of the already known mechanisms of action and effects of TETA  includeMultivariate PCA analysis showed no clear indication of metabolic differences between STZ-induced diabetic/TETA-treated and STZ-induced diabetic/untreated rats in study 1 or study 2. Univariate analysis showed one metabolic feature whose relative concentration change was shown to be statistically significant in both study 1 and study 2, putatively identified as hydroxybutanoate and/or methyl-hydroxybutanoic acid. This change most likely relates to the formation of ketone bodies, which is well known in DM.To further investigate any potential changes in the serum metabolic profile of STZ-induced diabetic rats in the presence or absence of treatment with TETA, pairwise correlation analysis was performed. Correlation analysis was chosen to investigate the complex interaction and regulatory mechanisms of biochemicals  in mammalian systems. Metabolites are directly or indirectly correlated to other metabolites in metabolic networks and other direct or indirect correlations between metabolites and other biochemicals are central to the regulation of biological systems. Differences between pairwise correlations were detected without statistically significant changes in the concentrations of correlated metabolites. Therefore, the investigation of these complex correlation networks can provide further inferences about the effect of TETA treatment.Study 2 was chosen as the sample sizes for all four groups were higher than for study 1 where two groups had only six rats at the end of the study because of death of rats during the study. There were 6,514 245 and 1,965 153 pairwise comparisons in positive and negative ion modes relating to 3,610 and 1,983 metabolic features, respectively. Metabolic features were filtered to provide only those features that show: (a) a high positive or negative pairwise correlation for non-diabetic/untreated rats; (b) a loss of this correlation or a switch from a high positive correlation to/from a high negative correlation for STZ-induced diabetic/untreated rats ; and (c) a return to a high positive or negative correlation in the STZ-induced diabetic/TETA-treated rats. We found that 30,784 and 4,040 metabolic features showed this pattern in positive and negative ion modes, respectively. To filter the data further, only metabolic features exhibiting this correlation pattern with ten or more different metabolites were investigated further. This provided 857 and 77 metabolic features in positive and negative ion modes, respectively, and related to 506 uniquely identified metabolites. These metabolites are listed in Additional file + and Cu2+). Further investigations of these metabolic features showed that the mass difference between isotopic peaks does not relate to expected mass differences that would be observed for charged non-covalent associations composed of metabolite and Cu (II) with a charge state of +2. The mass differences observed did relate to expected mass isotopic differences between 12C and 13C for doubly and triply charged organic species. These are most probably peptides in view of the hundreds of different features detected (it would not be expected that there would be hundreds of TETA metabolites present at high concentrations), though the absence of TETA metabolites cannot be proven without further targeted studies. It should be noted that 28% of all detected metabolic features in positive ion mode were allocated a multiply charged assignment that is significantly greater than observed in previous animal- or human-based studies in Manchester. This is observed across all four groups and therefore is not a direct result of disease or TETA treatment. However, greater than 20 multiply-charged species were statistically significant between non-diabetic/untreated and STZ-induced diabetic/untreated rats. These results highlight that the methods applied in this discovery study have the potential to identify non-metabolic changes and strengthen the applicability of the method. The changes in the correlation network of peptides are most likely related to changes in proteolysis (or protein catabolism) activity in the diabetic state and then following TETA treatment. Increased proteolysis in insulin resistance and DM has been reported, most likely from the removal of the anti-catabolic effect of insulin [Specific classes of metabolites were observed as potentially important in defining metabolic changes related to TETA treatment of STZ-induced diabetic rats. Of the unique metabolic features, 337 of 506 (66.7%) were definitively assigned as multiple charged species (predominantly doubly and triply charged species) by the isotopic pattern observed for these metabolic features. Of these 337, 336 species were detected in positive ion mode. These species could be peptides, doubly charged metabolites of TETA (as TETA is a polyamine) or charged non-covalent associations composed of metabolite and Cu (II) with a charge state of +2 (defined as adduct ions). The charge state of some metabolites detected in our study may be influenced by the presence of metal ions  Act (1986), and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Wistar adult male rats were included in the study and were fed with standard laboratory chow and kept in a 12:12 h light:dark cycle. Two independent replicate studies were performed to reduce the probability of reporting false positive observations. The replicate animal and metabolomic studies were separated in time. Animal study 1 was performed from July to October 2009 and study 2 from February to April 2010. For each study, the STZ-induced diabetic group (n = 20) consisted of age-matched animals that received an intraperitoneal injection of STZ  and showed blood glucose levels of ≥30 mmol/L on two consecutive measurements 3 and 6 days after the injection. Assessment of the glycemic state of the animals was carried out by measuring blood glucose concentrations. This method can be easily applied by collecting a small amount of venous blood and is known to correlate well with serum levels of fructosamine and glycosylated haemoglobin. Given that only a small blood sample is required, it alleviates the stress associated with the serial blood sampling required for a glucose tolerance test. Non-diabetic animals (n = 20) were age matched and received an intraperitoneal injection of Na citrate buffer. Ten animals from each group (STZ-induced diabetic and non-diabetic) were treated with oral TETA  by gavage from the day after STZ injection until the day before they were sacrificed. Animals were housed in collective cages (maximum of four per cage) and had free access to water and food. Twelve weeks after STZ administration, blood samples were collected from the tail vein in non-fasting animals into 2 ml tubes , placed on ice and subsequently centrifuged at 2,400g). The supernatant was transferred to a separate 2 ml Eppendorf tube and was dried . Samples were stored at 4°C until analysis. A pooled QC sample was prepared by the pooling of 30 μl aliquots from each sample and vortex mixing (60 seconds). 80 μl aliquots of the pooled QC sample were deproteinized and lyophilized as described above. Samples collected in animal study 1 and animal study 2 were prepared and analyzed in November 2009 and May 2010, respectively.Samples were randomized before sample preparation. Serum samples were thawed on ice, deproteinized and the sample extract lyophilized in a similar approach as previously described . This prRat serum extracts and QC samples were analyzed applying an Acquity UPLC system  coupled to an electrospray hybrid LTQ-Orbitrap XL mass spectrometer . All samples were analyzed separately in positive and negative ion modes. The UPLC and MS methods applied have been described previously . QC sampRaw data files (.RAW) were converted to the NetCDF format using the File converter program in XCalibur (ThermoFisher Scientific). Deconvolution of the NetCDF format files were performed using the XCMS software, an open-source deconvolution program available for LC-MS data as described previously  and each® scripting language [All univariate and multivariate analyses were carried out using the Matlablanguage  and explUnivariate analysis was performed using the Mann-Whitney U test, a non-parametric method for assessing whether two independent samples of observations come from the same distribution. No assumption is made of a normal distribution and the test is identical to a one-way analysis of variance (ANOVA) with the data replaced by their ranks. For multivariate analysis all missing values were annotated as 0 and in univariate analysis annotated as 'NaN'. All analyses were performed on data from both ion modes separately but the results have been combined post-data analysis to allow biological interpretation.Pairwise metabolite correlations were calculated for data acquired in study 2 separately for the four different groups  using the non-parametric Spearman rank correlation method and by applying the bootstrapping method (n = 100). Final results only included comparisons where there were a minimum of 6 versus 6 pair-wise peak correlations. All missing values were annotated 'NaN'. Changes in the correlation network were then calculated by comparison of data from (a) non-diabetic/untreated, (b) STZ-induced diabetic/untreated and (c) STZ-induced diabetic/TETA-treated groups. Metabolic features with a high correlation (greater than +0.5 or less than -0.5) in non-diabetic/untreated and STZ-induced diabetic/TETA-treated rats but with a loss of the correlation coefficient (correlation coefficient change >0.5) in STZ-induced diabetic/untreated rats were retained as biologically important in relation to TETA-treatment of STZ-induced DM. Only metabolic features exhibiting this correlation pattern with ten or more different metabolites were investigated further.Metabolic features, characterized by a unique accurate mass and retention time, were putatively annotated according to level 2 the Metabolomics Standards Initiative guidelines  applyingDM: diabetes mellitus; FAAH: fatty acid amide hydrolase; IL: interleukin; lysoPC: lysoglycerophosphocholine; mTOR: mammalian target of rapamycin; NF: nuclear factor; PCA: principal components analysis; QC: quality control; S1P: sphingosine-1-phosphate; STZ: streptozotocin; T1D: type 1 diabetes; T2D: type 2 diabetes; TETA: triethylenetetramine; UPLC-MS: ultra performance liquid chromatography-mass spectrometry.GJC declares a potential conflict of interest, a non-financial relationship with PhiERA Limited, Auckland, New Zealand. All other authors have no competing interests.MU and GC jointly conceived the study, jointly performed the experimental design, performed the animal study, jointly analyzed and interpreted the data and jointly prepared the manuscript. MB jointly performed the experimental design and jointly analyzed and interpreted the data. KAH jointly analyzed and interpreted the data. PB jointly conceived the study, jointly performed the experimental design, jointly analyzed and interpreted the data and jointly prepared the manuscript. WBD jointly performed the experimental design, performed the metabolomic study, analyzed and interpreted the data and prepared the manuscript. All authors have read and approved the manuscript for publication.File describing all uniquely identified metabolites showing changes in pairwise correlation coefficients between non-diabetic/untreated and STZ-induced diabetic/untreated rats and changes in pairwise correlation coefficients between STZ-induced diabetic/TETA-treated and STZ-induced diabetic/untreated rats.Click here for file