Chronic Persistent Infection Research Articles

Making antigen invisible: a coinhibitory molecule regulates the interaction between T cells and dendritic cells

Evaluation of: Fife BT, Pauken KE, Eagar TN et al. Interactions between PD-1 and PD-L1 promote tolerance by blocking the TCR-induced stop signal. Nat. Immunol. 10(11), 1185–1191 (2009).Antigen-specific downregulation of T-cell effector function is critical for maintaining self-tolerance but it can promote pathogen persistence in chronic infections; consequently, the restoration of T-cell effector functions is a major goal of therapeutic vaccines against chronic viral infections and malignancies. Recently, a number of T-cell inhibitory receptors, most prominently programmed death-1 (PD-1) and cytotoxic T-lymphocyte antigen-4, have been described that are associated with T-cell exhaustion and tolerance. Blocking these receptors can restore T-cell function and, depending on the model, lead to autoimmune disease or successful viral elimination. Antibodies to PD-1 and cytotoxic T-lymphocyte antigen-4 are currently being tested in clinical trials in several malignant diseases and chronic hepatitis C as they are promising candidates for combination with both prophylactic and therapeutic vaccines. Given the central role of T-cell inhibitory receptors in the regulation of immune responses, understanding their molecular mode of action is of major importance. In the report from Fife and colleagues, two-photon laser scanning microscopy of mouse lymphoid and peripheral tissue has been employed to study the interaction of tolerized PD-1-expressing T cells with antigen-bearing dendritic cells in vivo. While tolerized T cells moved freely and did not make prolonged contacts with dendritic cells, addition of an antibody that blocked the interaction between PD-1 and its ligand PD-L1 lowered T-cell motility, enhanced T-cell–dendritic cell contacts and caused autoimmune disease in the nonobese diabetic mouse model of autoimmune diabetes. The authors conclude that PD-1–PD-L1 interactions mediate peripheral tolerance by inhibiting T-cell receptor-induced stop signals.

Impact of Chronic Pulmonary Infection with Pseudomonas aeruginosa on Transfection Mediated by Viral and Nonviral Vectors

Pseudomonas aeruginosa plays a crucial role in the lung pathology of cystic fibrosis (CF). We showed that acute infection with P. aeruginosa has a substantial impact on gene transfer into lung epithelial cells mediated by polyplexes. As an extension of those studies we report here on the effect of chronic pulmonary infection with P. aeruginosa on transfection of lung epithelial cells by viral and nonviral vectors. As an in vivo model of the persistent chronic infection in patients with CF we used C57BL/6 mice intratracheally infected with P. aeruginosa encapsulated in agar beads. Two weeks after infection the presence of viable bacteria in the lungs was confirmed, mostly in the bronchial lumen. In lung tissue sections stained with hematoxylin and eosin, extensive inflammatory infiltrations were found. At that time point the mice received an intratracheal dose of luciferase gene complexed with either Lipofectamine (Lf), a GL67 lipid mixture (GL67), or polyethylenimine (PEI) or with lentivirus (LV) as a carrier system. Luciferase activity was determined by a luminescence assay in supernatants of lung homogenates. The transfection level induced by PEI/DNA polyplexes complexed with serum albumin was decreased in infected mice. Lf-mediated transfection was almost completely blocked in infected mice. Transfection levels in mice treated with LV or plain PEI/DNA polyplexes were unchanged in infected animals as compared with control mice. The only carrier that displayed a clearly increased transfection level in infected mice was the GL67 lipid mixture, which is tentatively ascribed to the presence of polyethylene glycol in this carrier.

Crystal structure and oligomeric state of the RetS signaling kinase sensory domain

The opportunistic pathogen Pseudomonas aeruginosa may cause both acute and chronic-persistent infections in predisposed individuals. Acute infections require the presence of a functional type III secretion system (T3SS), whereas chronic P. aeruginosa infections are characterized by the formation of drug-resistant biofilms. The T3SS and biofilm formation are reciprocally regulated by the signaling kinases LadS, RetS, and GacS. RetS downregulates biofilm formation and upregulates expression of the T3SS through a unique mechanism. RetS forms a heterodimeric complex with GacS and thus prevents GacS autophosphorylation and downstream signaling. The signals that regulate RetS are not known but RetS possesses a distinctive periplasmic sensor domain that is believed to serve as receptor for the regulatory ligand. We have determined the crystal structure of the RetS sensory domain at 2.0 A resolution. The structure closely resembles those of carbohydrate binding modules of other proteins, suggesting that the elusive ligands are likely carbohydrate moieties. In addition to the conserved beta-sandwich structure, the sensory domain features two alpha helices which create a unique surface topology. Protein-protein crosslinking and fluorescence energy transfer experiments also revealed that the sensory domain dimerizes with a dissociation constant of K(d) = 580 +/- 50 nM, a result with interesting implications for our understanding of the underlying signaling mechanism.

T-Cell Dysfunction and Inhibitory Receptors in Hepatitis C Virus Infection

Dysfunction of the virus-specific T cells is a cardinal feature in chronic persistent viral infections such as one caused by hepatitis C virus (HCV). In chronic HCV infection, virus-specific dysfunctional CD8 T cells often overexpress various inhibitory receptors. Programmed cell death 1 (PD-1) was the first among these inhibitory receptors that were identified to be overexpressed in functionally impaired T cells. The roles of other inhibitory receptors such as cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and T cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3) have also been demonstrated in T-cell dysfunctions that occur in chronic HCV patients. Blocking these inhibitory receptors in vitro restores the functions of HCV-specific CD8 T cells and allows enhanced proliferation, cytolytic activity and cytokine production. Therefore, the blockade of the inhibitory receptors is considered as a novel strategy for the treatment of chronic HCV infection.

Chlamydophila pneumoniaeInfection and Its Role in Neurological Disorders

Chlamydophila pneumoniae is an intracellular pathogen responsible for a number of different acute and chronic infections. The recent deepening of knowledge on the biology and the use of increasingly more sensitive and specific molecular techniques has allowed demonstration of C. pneumoniae in a large number of persons suffering from different diseases including cardiovascular (atherosclerosis and stroke) and central nervous system (CNS) disorders. Despite this, many important issues remain unanswered with regard to the role that C. pneumoniae may play in initiating atheroma or in the progression of the disease. A growing body of evidence concerns the involvement of this pathogen in chronic neurological disorders and particularly in Alzheimer's disease (AD) and Multiple Sclerosis (MS). Monocytes may traffic C. pneumoniae across the blood-brain-barrier, shed the organism in the CNS and induce neuroinflammation. The demonstration of C. pneumoniae by histopathological, molecular and culture techniques in the late-onset AD dementia has suggested a relationship between CNS infection with C. pneumoniae and the AD neuropathogenesis. In particular subsets of MS patients, C. pneumoniae could induce a chronic persistent brain infection acting as a cofactor in the development of the disease. The role of Chlamydia in the pathogenesis of mental or neurobehavioral disorders including schizophrenia and autism is uncertain and fragmentary and will require further confirmation.

Dissemination of Mycobacteria to the Thymus Renders Newly Generated T Cells Tolerant to the Invading Pathogen

The ability of the thymus to generate a population of T cells that is, for the most part, self-restricted and self-tolerant depends to a great extent on the Ags encountered during differentiation. We recently showed that mycobacteria disseminate to the thymus, which raised the questions of how mycobacteria within the thymus influence T cell differentiation and whether such an effect impacts host-pathogen interactions. Athymic nude mice were reconstituted with thymic grafts from Mycobacterium avium-infected or control noninfected donors. T cells generated from thymi of infected donors seemed generally normal, because they retained the ability to reconstitute the periphery and to respond to unspecific stimuli in vitro as well as to antigenic stimulation with third-party Ags, such as OVA, upon in vivo immunization. However, these cells were unable to mount a protective immune response against a challenge with M. avium. The observation that thymic infection interferes with T cell differentiation, generating T cells that are tolerant to pathogen-specific Ags, is of relevance to understand the immune response during chronic persistent infections. In addition, it has potential implications for the repertoire of T cells generated in patients with a mycobacterial infection recovering from severe lymphopenia, such as patients coinfected with HIV and receiving antiretroviral therapy.

Genome‐wide DNA methylation profiles in precancerous conditions and cancers

Alterations of DNA methylation, which result in chromosomal instability and silencing of tumor-related genes, are among the most consistent epigenetic changes observed in human cancers. Analysis of tissue specimens has revealed that DNA methylation alterations participate in multistage carcinogenesis, even from the early and precancerous stages, especially in association with chronic inflammation and/or persistent viral infection, such as chronic hepatitis or liver cirrhosis resulting from infection with hepatitis B or C virus. DNA methylation alterations can account for the histological heterogeneity and clinicopathological diversity of human cancers. Overexpression of DNA methyltransferase 1 is not a secondary result of increased cell proliferative activity, but is significantly correlated with accumulation of DNA hypermethylation in CpG islands of tumor-related genes. Alteration of DNA methyltransferase 3b splicing may result in chromosomal instability through DNA hypomethylation in pericentromeric satellite regions. Genome-wide analysis of DNA methylation status has revealed that the DNA methylation profile at the precancerous stage is basically inherited by the corresponding cancers developing in individual patients. DNA methylation status is not simply altered at the precancerous stage; rather, DNA methylation alterations at the precancerous stage may confer vulnerability to further genetic and epigenetic alterations, generate more malignant cancers, and thus determine patient outcome. Therefore, genome-wide DNA methylation profiling may provide optimal indicators for carcinogenetic risk estimation and prognostication, and thus provide an avenue for cancer prevention and therapy on an individual basis.

Prevalence of Chlamydophila pneumoniae is higher in aorta and coronary artery than in carotid artery of coronary artery disease patients

Coronary artery disease (CAD) is a public health problem accounting for an estimated one-third of deaths overall. A potential link between infectious agents and atherosclerosis has been suggested. Data obtained from several seroepidemiological studies have suggested that infection with Chlamydophila pneumoniae, Helicobacter pylori, cytomegalovirus and herpes simplex virus-1 can initiate or maintain the atherosclerotic process. However, there is no single study in which multiple infectious agents have been detected together in different vascular locations in the same population. This would help in determining if there is any leading pathogen in atheromatous plaques of CAD patients. Hence, we screened for C. pneumoniae, H. pylori, CMV and HSV-1 in different vascular locations of CAD patients using quantitative real-time (RT) PCR. We performed multiplex RT-PCR for detecting pathogens, viz. C. pneumoniae, H. pylori, CMV and HSV-1 in different vascular locations of CAD patients. Percent positivity scores for C. pneumoniae, H. pylori, CMV and HSV-1 in different vascular locations were as follows: aorta (64.7, 35.3, 11.7 and 11.7 respectively); carotid (27.2, 27.2, 9 and 0 respectively); coronary artery (58.3, 33.3, 16.6 and 8.3 respectively). Combined positivity for C. pneumoniae (C. pneumoniae IgA and RT-PCR for C. pneumoniae) was the highest compared with all other groups. Aorta and coronary artery were more susceptible to these pathogens as compared with carotid artery. Moreover, CAD patients' characteristics were associated with C. pneumoniae positivity (C. pneumoniae IgA and RT-PCR), suggesting thereby that C. pneumoniae may have caused chronic persistent infection in CAD.

Feline programmed death and its ligand: Characterization and changes with feline immunodeficiency virus infection

Programmed death (PD)-1 and its ligand, PD-L1, are co-stimulatory molecules expressed on T cells and antigen-presenting cells, respectively, that modulate T cell receptor signals. Altered PD expression or signalling contributes to pathogen persistence in chronic infections. The sequence of the feline PD genes was derived from gene amplification with primers conserved across human and canine homologs, and by sequence extension through rapid amplification of cDNA ends. Feline PD-1 was similar to that of other mammalian species and consisted of extracellular, transmembrane and cytoplasmic regions. Functional motif analysis of the translated amino acid sequence predicted immunoreceptor tyrosine-based inhibitory and switch motifs, and a SH3-binding region, in the cytoplasmic tail. PD-1 and PD-L1 were expressed in resting lymphocytes and dendritic cells, and up-regulated on mitogen-activated or irradiated lymphocytes of both CD4 and CD8-positive subsets. In vitro infection with the feline immunodeficiency virus (FIV) significantly decreased PD-1, but not PD-L1, gene expression in lymphocytes at 24 h, and decreased expression of both genes at 168 h. No significant changes in gene or protein expression from FIV infection were noted in dendritic cells. Blood lymphocytes from cats chronically FIV-infected expressed significantly higher PD protein than lymphocytes from FIV-negative cats. These findings indicate that both feline PD-1 and PD-L1 are expressed by resting lymphocytes and dendritic cells. Apoptosis and cell activation increased protein expression on lymphocytes, while in vitro acute FIV infection decreased PD-1 gene expression. Increased PD levels in lymphocytes from chronically FIV-infected cats suggests that alterations in T cell co-signalling may contribute to immune dysfunction in lentiviral infection.

Correction: Mycobacterium tuberculosis Universal Stress Protein Rv2623 Regulates Bacillary Growth by ATP-Binding: Requirement for Establishing Chronic Persistent Infection

Correction: Mycobacterium tuberculosis Universal Stress Protein Rv2623 Regulates Bacillary Growth by ATP-Binding: Requirement for Establishing Chronic Persistent Infection

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells <em>In situ</em>

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in determining outcomes that range from clearance to chronic persistent infections. We describe here a method for assessing the spatial and quantitative relationships between immune effector (E) virus-specific CD8+ T cells and infected targets (T) that combines in situ tetramer (IST) staining to detect virus-specific CD8+ T cells and in situ hybridization (ISH) to detect viral-RNA+ cells in the tissues where the battle between immune defenses and infection takes place. The combination of IST staining and ISH, abbreviated ISTH, enables visualization and mapping of the locations of immune effector cells and targets, and facile determination of E:T ratios. These parameters in turn can then be used to determine the relationships between spatial proximity, and the timing and magnitude of the immune response that predict outcomes in early infection.

A technique to simultaneously visualize virus-specific CD8+ T cells and virus-infected cells in situ.

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in determining outcomes that range from clearance to chronic persistent infections. We describe here a method for assessing the spatial and quantitative relationships between immune effector (E) virus-specific CD8+ T cells and infected targets (T) that combines in situ tetramer (IST) staining to detect virus-specific CD8+ T cells and in situ hybridization (ISH) to detect viral-RNA+ cells in the tissues where the battle between immune defenses and infection takes place. The combination of IST staining and ISH, abbreviated ISTH, enables visualization and mapping of the locations of immune effector cells and targets, and facile determination of E:T ratios. These parameters in turn can then be used to determine the relationships between spatial proximity, and the timing and magnitude of the immune response that predict outcomes in early infection.

Virus‐specific CD8 T cells: activation, differentiation and memory formation

CD8 T cells are pivotal for the control of many intracellular pathogens, and besides their role in immediate control of infections, CD8 T cells have the capacity to differentiate into long-lived antigen-independent memory CD8 T cells, at least in situations of acute and resolved infections. The population of memory cells is heterogeneous with respect to their phenotype, their anatomical localization and their functional capacities in order to afford optimal protection against secondary infections. In the past years, it has become clear that multiple in vivo parameters are involved in shaping the composition of the memory CD8 T cell population, including antigen load, duration and strength of CD8 T cell stimulation, the level of inflammation, availability of CD4 T cell help and CD8 T cell precursor frequencies. With respect to the timing when CD8 T cells are committed to become memory cells, several models have been proposed. In contrast to acute, resolved infection, the continued in vivo exposure to high levels of antigen during persistent chronic viral infection precludes the development of long-lived antigen-independent memory CD8 T cells and might even result in severe dysfunction of virus-specific CD8 T cells.

Immunotherapeutics effects of IL-7 during a chronic viral infection (44.13)

Abstract Viral persistence in chronic infections is associated with ineffective T cell responses. Here, we evaluated the therapeutic use of IL-7 to enhance T cell responses during a chronic infection with lymphocytic choriomeningitis virus (LCMV). Mice infected with Clone 13 strain of LCMV were treated with IL-7 at various intervals spanning the first 30 days after infection. Our studies show that the ability of IL-7 to enhance T cell responses to a chronic viral infection is constrained by high viral load. Early in the infection (days 0-15 PI), when the viral load is very high, IL-7 administration induced minimal enhancement of T cell responses to LCMV. However, with reducing viral load (after day 15 PI), IL-7 augmented virus-specific T cell responses, albeit temporarily. An extended duration of IL-7 treatment after the initial phase of T cell expansion (days 8-30 PI), markedly enhanced the number of virus-specific CD8 T cells. Moreover, extended IL-7 therapy altered the cell surface phenotype, and augmented the cytokine-producing ability of LCMV-specific T cells. Surprisingly, despite the augmentation of virus-specific T cell responses, IL-7 therapy had a modest effect on circulating viral load in chronically infected mice. In summary, these findings suggested that IL-7 administration could be used as an adjunct to anti-viral immunotherapy to bolster T cell responses to chronic viral infections. This work was supported by PHS grant AI68841 to M. Suresh.

A role for CMV infection in expansion of dysfunctional IL-7 receptor alpha low memory CD8+ T cell with aging. (83.11)

Abstract We recently reported an age-associated expansion of human memory CD8+ T cells expressing low levels of IL-7 receptor alpha chain (IL-7Rα) that is crucial for cell survival. These cells have limited T cell receptor (TCR) repertoire and impaired TCR-mediated proliferation. Chronic persistent viral infection is suggested as a factor that expands memory CD8+ T cells with aging and suppresses their function. Thus, we explored the role for cytomegalovirus (CMV) infection in expanding IL-7Rαlow memory CD8+ T cells and suppressing their TCR function via leukocyte Ig-like receptor-1 (LIR-1) with inhibitory function. CMV-infected donors have a higher frequency of IL-7Rαlow memory CD8+ T cells than CMV-uninfected donors in the elderly but not in the young. Most expanded IL-7Rαlow cells express LIR-1 and such expression correlates with replicative senescent marker CD57. The frequency of IL-7Rαlow memory CD8+ T cells is also higher in CMV-uninfected elderly donors than in CMV-infected and -uninfected young donors, suggesting that a factor(s) additional to CMV infection exists for expanding these cells. The expression of IL-2/15 receptor beta chain (IL-2/15Rβ) on IL-7Rαlow memory CD8+ T cells is higher in the elderly than in the young, suggesting IL-15-mediated expansion of them. Our data suggest that age-associated expansion of IL-7Rαlow memory CD8+ T cells is driven by both CMV infection and IL-15.

Lazy, Dynamic or Minimally Recrudescent? On the Elusive Nature and Location of the Mycobacterium Responsible for Latent Tuberculosis

In the absence of symptoms characteristic of tuberculosis (TB), a condition termed clinical latency, diagnosis is currently impossible by detection of the microorganism itself and resorts to the demonstration of an immunological memory response to antigens of Mycobacterium tuberculosis (Mtb). Whether latency is synonymous to chronic persistent infection with viable Mtb in all instances has been difficult to establish. The physical and physiological state of Mtb during latency is much disputed: are organisms mostly dormant, in a nonreplicating state of persistence, and characterized by lipid inclusions and metabolic adaptation to hypoxia, or do they continue to replicate and sometimes even escape from the fringes of granulomatous lesions or alveolar epithelial cells into adjacent airways, thereby inducing recurring immune responses? The physical nature of Mtb during latency is important as it determines which antimicrobial agents may be used to kill it, which immunomodulating strategies (including post-exposure vaccines) may be appropriate to contain it, and which diagnostic measures may be most useful to discriminate latent from reactivating infection. Two major viewpoints exist: one argues that Mtb persists mostly in a lazy state within granulomatous lesions, but periodically recrudesces, and that there is considerable heterogeneity for different sites within the lesion and within the infected lung. Throughout latency, there is a dynamic immunological interplay between Mtb and the host, necessitating continuous recruitment of cells into the granuloma, and reactivation occurs when this dynamic cellular exchange becomes dysregulated. Another view holds that dormant Mtb reside within alveolar epithelial cells in the lung apices and in adipocytes, with reactivation being associated with the upregulation of resuscitation promoting factors within Mtb and the escape of newly dividing microorganisms into alveoli and bronchi in the form of lipid pneumonia. These views need not be mutually exclusive. However, if minimal intermittent recrudescence were to take place within the alveolar space, this would contradict the very definition of latency, which implies that no access of Mtb to the airways exists during latency.

Formation of Biofilm in Patients with Chronic Otitis Media and Cholesteatoma

Background and Objectives:In many chronic infections, bacteria are present as a form of biofilms. Recently, bacterial bio- film has been implicated in persistent or recurrent chronic otolaryngologic infections, because they are highly resistant to treatment with antimicrobial agents and capable of shedding individual bacteria to the surrounding tissues. In this study, we aimed to evaluate biofilm in chronic middle ear infections. Subjects and Method:A total of 85 cases with chronic middle ear infections were included in this study. Histomorphologic studies for middle ear granulations and cholesteatomas were performed using light and electron microscopy. Results:In middle ear granulations, about 33% of the cases showed the evidence of bacterial biofilm. In comparison, about 64% of the cases in cholesteatoma tissues have the evidences of bacterial biofilm. Conclusion:Biofilms are associated with chronic middle ear infection and they may play a role in the pathogenesis of refractory or persistent chronic middle ear infections. (Korean J Otorhinolaryngol-Head Neck Surg 2009;52:124-8)

Human CD4 and CD8 regulatory T cells in infectious diseases and vaccination

Regulatory T cells (Tregs) are crucial players in balancing inflammation and antigen specific immune responses. In chronic infectious diseases, Tregs dampen inflammation to limit tissue damage, but they can also inhibit ensuing effector immunity, thereby impairing pathogen clearance. Chronic persistent infections by human pathogens such as parasites, viruses, and (myco)bacteria can all result in the induction of both CD4 + and CD8 + Tregs. However, among the many different subsets of Tregs that are induced, mostly CD4 + Tregs have been studied. A remarkably increased frequency has been observed at the site of infection, supporting a role in pathogen containment. Indeed, antigen specificity has been demonstrated for several pathogen derived antigens. Here we review different classes of human Tregs in infectious diseases, including CD4 and CD8 Treg subsets. A better understanding of the induction and activity of Tregs is relevant for the design of better vaccines that optimally induce effector immunity without co-induction of excessive Treg activity.

Molecular detection of Chlamydophila abortus in post-abortion sheep at oestrus and subsequent lambing

Enzootic abortion of ewes (EAE), resulting from infection with the bacterium Chlamydophila abortus ( C. abortus), is a major cause of lamb loss in Europe. The purpose of this study was to assess the potential impact of the shedding of organisms in post-abortion ewes at oestrus and subsequent lambing on the epidemiology of EAE. Using a newly developed C. abortus specific real-time PCR assay, few chlamydial genomes could be detected in vaginal swabs taken from post-abortion ewes at oestrus. At subsequent parturition, all ewes lambed normally with no macroscopic or microbiological evidence of infection. Real-time PCR analysis of placental samples identified very few or no chlamydial genomes, which contrasted significantly with samples taken at the time of abortion, where an average of 2.7 × 10 7 chlamydial genomes per microgram of total tissue DNA was detected. Few genomes could also be detected from vaginal and cervical tissue samples and lymph nodes taken post-mortem. The results, although not discounting the possibility of a chronic low level persistent infection in post-abortion ewes, suggest that the low levels of chlamydial DNA detected during the periovulation period and at lambing do not significantly impact on the epidemiology of EAE. In terms of flock management, the products of abortion should be considered the major and principal source of infection for transmission to naïve ewes.

Alterations of DNA methylation and clinicopathological diversity of human cancers

Alterations of DNA methylation can account for the histological heterogeneity, reflected in the stepwise progression and complex biological characteristics of human cancers, that genetic alterations alone cannot explain. Analysis of DNA methylation status in tissue samples can be an aid to understanding the molecular mechanisms of multistage carcinogenesis. Human cancer cells show a drastic change in DNA methylation status, that is, overall DNA hypomethylation and regional DNA hypermethylation, which results in chromosomal instability and silencing of tumor-suppressor genes. Overexpression of DNA methyltransferase (DNMT) 1 is not a secondary result of increased cell proliferative activity but may underline the CpG island methylator phenotype of cancers. Splicing alteration of DNMT3B may result in chromosomal instability through DNA hypomethylation of pericentromeric satellite regions. Alterations of DNA methylation are observed even in the precancerous stage frequently associated with chronic inflammation and/or persistent viral infection or with cigarette smoking. Precancerous conditions showing alterations of DNA methylation may generate more malignant cancers. Aberrant DNA methylation is significantly associated with aggressiveness of cancers and poorer outcome of cancer patients. Genome-wide analysis of DNA methylation status based on array-based technology may identify DNA methylation profiles that can be used as appropriate indicators for carcinogenetic risk estimation and prognostication.