One of the key questions in the study of mammalian gene regulation is how epigenetic methylation patterns on histones and DNA are initiated and established. These stable, heritable, covalent modifications are largely associated with the repression or silencing of gene transcription, and when deregulated can be involved in the development of human diseases such as cancer. This article reviews examples of viruses and bacteria known or thought to induce epigenetic changes in host cells, and how this might contribute to disease. The heritable nature of these processes in gene regulation suggests that they could play important roles in chronic diseases associated with microbial persistence; they might also explain so-called ‘hit-and-run’ phenomena in infectious disease pathogenesis. However, not all histone modifications are truly epigenetic, as very few satisfy the heritable part of the definition. To establish and mediate epigenetic memory, such modifications must be transmitted during DNA replication An epigenetic trait was recently defined as ‘a stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence’ Epigenetic processes have been heavily implicated in the development of cancer, in which repression or silencing of tumour suppressor genes is remarkably common  [4–6]. AThe aim of this paper is to briefly review examples of epigenetic control of host genes by viral and bacterial pathogens, and highlight how this could be relevant in disease pathogenesis.As obligate intracellular parasites, viruses have developed numerous ways of hijacking cell processes to facilitate the completion of their life cycle and sometimes to evade the immune responses of their host. Viruses that cause persistent (often latent) infections are likely to benefit from heritable epigenetic changes in host transcription that produce an environment for their latent or persistent state without having to continuously express the initiating effectors in vitro, but LANA appears to interact preferentially with DNMT3A in vivoCDH13), a gene that is methylated in several types of cancer CDH13 regulatory locus was also substantially methylated in KSHV-positive BCBL-1 PEL-derived cells, so it is possible LANA recruits DNMT3A and initiates de novo DNA methylation in B cell lymphomagenesis CDH13 (or other repressed promoters), but its ability to interact with other chromatin modifying proteins might provide clues. The capacity of LANA to associate with the corepressors mSin3, SAP30 and CIR KSHV has a distinct tropism for B lymphocytes, but it also infects endothelial cells. Similar to EBV, KSHV establishes life-long latency in humans, but unlike EBV, the specific cell type in which it remains latent is unknown. As the name indicates, the virus is associated with Kaposi's sarcoma, but also with primary effusion lymphoma (PEL) and multicentric Castleman's disease), two rare forms of B cell lymphoma More recently it was reported that LANA also binds to the promoter of the gene encoding the transforming growth factor (TGF)-β type II receptor (TGF-βRII) leading to DNA methylation and downregulation of transcription CDH13 and TGF-βRII. Therefore, it is possible that the interactions of KSHV factors with host chromatin modifiers and the epigenetic reprogramming of infected cells is also associated with a requirement of KSHV to epigenetically regulate its own genome.Furthermore, an investigation into the roles of KSHV encoded miRNAs revealed that ectopic expression of miR-K12–4-5p1 could indirectly increase the levels of DNMT1, DNMT3A and DNMT3B mRNAs EBV is associated with several human tumours of B cell, T cell and epithelial origin, including EBV-associated Burkitt's lymphoma (BL), post-transplant lymphoproliferative disease, diffuse large B cell lymphoma, some forms of Hodgkin's lymphoma, and the epithelium-derived nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC) In vitro, EBV can very efficiently induce the activation and continuous proliferation of resting human B cells. The resulting lymphoblastoid cell lines (LCLs) carry the viral genome as extrachromosomal episomes, and express only nine latency-associated EBV proteins. These include six EB nuclear antigens  and three latent membrane proteins , and there are also several untranslated RNA species Data on the persistence of EBV in humans are consistent with the viral genome residing long-term in a resting memory B cell population. It is now considered probable that to establish persistence, EBV initially infects resting (naive) B cells and drives these to proliferate as activated B blasts. This expanding infected B blast population then migrates into germinal centres, where they differentiate to become centroblasts, centrocytes and finally resting memory B cells. Although the precise series of events that the EBV-positive B cells undergo to reach the memory compartment is unknown, it appears to involve regulated shutdown of latent protein expression from an initial state called latency III (as found in LCLs), via latency II  until latency 0, a state in quiescent memory B cells in which no EBV proteins can be detected. Occasionally memory cells divide and EBNA1 is transiently expressed to ensure maintenance of the viral episome (a state known as latency I) (BCL2L11) as a target that is repressed by latent EBV through a mechanism requiring the functional cooperation of the EBNAs 3A and 3C Because evidence indicated that expression of EBNA3 in BL cells dramatically enhanced their survival potential BIM transcription initially involves the epigenetic modification H3K27me3 (histone H3 trimethylated at lysine 27), probably through polycomb repressive complex (PRC)2, a known mediator of this modification in humans  INK4A, p14ARF, p73, E-cadherin and PTEN tumour suppressors, relative to EBV-negative GC. It has been suggested that these epigenetic changes could be caused by elevated levels of DNMT1, which in these cells might be induced by the expression of EBV LMP2  is associated with hepatocellular carcinoma (HCC), and several studies have indicated that the presence of HBV in HCC correlates with aberrant DNA methylation of the host genome INK4A, which is normally repressed in cycling cells by EZH2 via H3K27me3 and frequently undergoes DNA hypermethylation in cancer, is often overexpressed in HPV-positive carcinoma There are over 100 types of human papillomavirus (HPV) and several, such as HPV16 and 18, are associated with the development of malignancies that include cervical carcinoma. It has recently been reported that a keratinocyte cell line carrying HPV16 episomes has reduced levels of E-cadherin The presence of HPV in cervical carcinoma-derived cells also correlates with DNA hypermethylation of CpG islands at the 3.3 kb repeats in the subtelomeric regions of multiple chromosomes BLU and RASSF1) that are adjacent (head to tail) on the genome HPV in low-grade squamous intraepithelial lesions is associated with increased DNA methylation of the promoters of genes encoding two putative tumour suppressors  sequences significantly correlates with high levels of DNA methylation RASSF1 methylation in small cell lung cancer, where this promoter is also sometimes hypermethylated Simian vacuolating virus (SV)40 is a polyomavirus that infects monkeys and humans, and is another oncogenic virus suspected of inducing epigenetic changes in host cells. SV40, similar to HPV, is associated with INK4A, p15INK4B and p73 Nevertheless, more specific sites of aberrant DNA methylation might await discovery in SV40 infected cells, possibly because expression of large T antigen also upregulates DNMT1, with concomitant increases in DNA methyltransferase activity and genomic methylation Although adenoviruses generally exhibit a lytic rather than latent life cycle, the viral oncoprotein small e1a has been reported to globally restrict histone 3 lysine 18 acetylation (H3K18Ac) and repress transcription of multiple genes INK4A and p73, become hypermethylated More than a decade ago it was reported that infection of CD4+ T cells by either HIV-1 or human T cell leukaemia virus (HTLV)-1 results in an increase in cellular DNA methyltransferase activity GNE, which encodes a key enzyme in sialic acid biosynthesis, and represses its transcription HIV infection of T lymphocytes in culture also causes promoter methylation of the gene Paramecium bursaria chlorella virus (PBCV)-1 infects Chlorella green algae and would probably not be considered a typical pathogen, it is worthy of consideration because it carries a gene encoding a viral SET (vSET) domain protein (Although Helicobacter pylori infection of human gastric mucosae (see below). Moreover, many microbes have evolved ways of eluding the immune response and, again, epigenetic changes in host cells have been implicated in these processes.Some chronic bacterial infections are also associated with malignancy, the most well-known and widely studied being C. pneumoniae, similar to PBCV-1, encodes a SET domain protein. The chlamydial SET domain protein directly interacts with chlamydial histone H1-like proteins Hc1 and Hc2, and has a histone methyltransferase activity capable of methylating murine histone H3 in vitroAn obligate intracellular pathogen that causes acute respiratory diseases, C. rectus revealed bacteria translocated to the placenta from the original site of infection in 46.7% of the cases. Concurrently, downregulation of the Igf2 gene in the placenta was found to be associated with higher levels of DNA methylation at its promoter Igf2 is an imprinted gene that is implicated in placental development and fetal growth rectus can lead to increased risk of preterm births Experiments in which mice were orally infected with A. phagocytophilum, 19 were downregulated Listeria monocytogenes, Clostridium perfringens, Streptococcus pneumoniae and Mycobacteria spp., can alter host histone acetylation and/or HDAC activity This Rickettsiales intracellular pathogen replicates in human neutrophils and their bone marrow progenitors. In these cells it has to survive despite the presence of the multiple antimicrobial mechanisms employed by these primary defence cells, mechanisms mediated by a number of so-called ‘defence genes’. In a recent report that assessed the expression of 23 defence genes after infection of a monocytic cell line with H. pylori infection is closely associated with intestinal metaplasia and the development of gastric cancer H. pylori infection H. pylori and cancer progression was not entirely consistent Infection with this Gram-negative bacterium can result in chronic gastritis and peptic ulcers, and chronic H. pylori induced DNA methylation of CDH1, the E-cadherin gene H. pyloriH. pylori itself, that is responsible for the induction of aberrant DNA methylation in cells of the gastric mucosa Co-cultivation of human gastric cell lines with C. pneumoniae, C. rectus, and H. pylori) are also linked to chronic disease.It might be significant that all the bacteria that are most clearly associated with stable epigenetic modifications of host genes (e.g. H. pylori) contributing to disease is conceptually satisfying. What is less obvious is the significance of such heritable changes in host cell behaviour during acute pathogen-mediated disease. Perhaps a unifying theme is the modulation of the immune response, inflammation and intracellular host defences. This requires further exploration.It is easy to understand from the examples of persistent viral infections (e.g. KSHV and EBV) how the ability to epigenetically influence host gene expression could have evolved and how it might play a major role in the pathogenesis of chronic disease. Similarly, although the mechanisms are less well understood, the idea of epigenetic changes in host cells associated with chronic bacterial infections regulate patterns of repressive histone modifications that could then precipitate aberrant DNA methylation and the reprogramming of infected cells and their progeny. This can involve the repression of tumour suppressor genes, for which there will be strong selection in the development in cancer. Examples include KSHV and EBV.2.Infection by diverse viruses appears to induce expression of DNMTs and/or polycomb group (PcG) proteins such as EZH2. Examples include KSHV, EBV, HBV, HIV-1, HTLV-1, HPV, SV40 and adenoviruses. The important caveat with these data is that the expression of DNMTs and EZH2 correlates with cell proliferation rate; therefore a high level of expression might be a consequence of the proportion of cells proliferating in tumours and/or chronically inflamed tissue.3.C. pneumoniae,At least one virus, PBCV-1, and perhaps many bacteria, including 4.Stimulation of host HDAC activity might be a common feature of microbial infection. This could contribute to the epigenetic repression of ‘defence genes’ in host cells. The possibility that this occurs in non-infected bystander cells responding to the uptake of soluble factors is also worth exploring.5.Inflammation, whether or not it is induced by infection, can be a potent initiator of aberrant methylation of PcG target genes. Therefore chronic inflammation might predispose cells to malignant transformation.6.The stable heritable nature of epigenetic change implies that the initial inducers of transcriptional repression and chromatin modification need only be expressed transiently. Although in most cases, effectors are continuously present, this provides a rational explanation for potential ‘hit and run’ mechanisms in infectious disease pathogenesis.In summary, from data available currently, several themes are emerging.For many pathogens, it is very difficult to make even educated guesses about the extent or the mechanics of epigenetic change they might induce. Currently available data largely provide snapshots of what is happening to ‘usual suspect’ host genes after infection. More comprehensive global studies are now necessary and possible.We are now in the era of epigenetics et al. , showed that the E4-ORF3 protein of oncogenic adenoviruses induces widespread epigenetic silencing of tumour suppressor p53-target genes.While this article was in the final stages of preparation, Soria


TLR9 polymorphisms between clinical severity variables in MM survivors.Genetic variation in immune response genes is associated with susceptibility and severity of infectious diseases. Toll-like receptor (TLR) 9 polymorphisms are associated with susceptibility to develop meningococcal meningitis (MM). The aim of this study is to compare genotype distributions of two TLR9 -1237 and TLR9 +2848 polymorphisms and compared these between thirteen clinical variables associated with prognostic factors predicting adverse outcome of bacterial meningitis in children.We used DNA samples of a cohort of 390 children who survived MM. Next, we determined the genotype frequencies of TLR9 -1237 TC and CC genotypes were associated with a decreased incidence of a positive blood culture for Neisseria (N.) meningitidis  0.5. 95% confidence interval (CI) 0.3 – 0.9). The TLR9 +2848 AA mutant was associated with a decreased incidence of a positive blood culture for N. meningitidis . Cerebrospinal fluid (CSF) leukocytes per μL were higher in patients carrying the TLR9 -1237 TC or CC genotypes compared to carriers of the TT wild type (WT)  4987 versus 955, IQR 3938). CSF blood/glucose ratios were lower in TLR9 -1237 TC or CC carriers than in carriers of the TT WT . CSF leukocytes/μL were higher in patients carrying the TLR9 +2848 AA mutant compared to carriers of GG or GA .The We identified TLR9 genotypes associated with protection against meningococcemia and enhanced local inflammatory responses inside the central nervous system, important steps in MM pathogenesis and defense. Neisseria (N.) meningitidis is a common causing pathogen of BM, both in the Western world as in developing countries. The clinical course of meningococcal meningitis (MM) is highly diverse and depends both on pathogen characteristics as on the individual immune response of the affected patient. Host-bacteria interactions are crucial in defense against MM [N. meningitidis may lead to bacterial colonization in one patient and to fatal MM or meningococcal septic shock in the other. Survivors of BM have a high risk to develop neurological sequelae, ranging from subtle learning and behavioral disorders to deafness, paresis, and severe encephalopathy [TLR9 +2848 SNP is associated with a decreased susceptibility to MM [TLR9 -1237 C variant allele creates a potential nuclear factor kappa B (NFκB) binding site that increases the transcriptional activity of TLR9 and enhances cellular production of pro-inflammatory cytokines [The susceptibility, severity and prognosis of infectious diseases depend on the ability of the host immune system to respond to pathogens. Genetic variation of immune response genes is associated with susceptibility to and severity of infectious diseases . Bacteriainst MM . Acquisialopathy -7. Innatalopathy . TLR9 isalopathy . TLR9 isalopathy . In a prty to MM . Recent ytokines .TLR9 -1237 and TLR9 +2848 single nucleotide polymorphisms (SNPs) between thirteen clinical severity variables in order to identify patients at risk for severe disease and sequelae.The purpose of this study is to compare the genotype distributions of N. meningitidis or meningococcal antigens in the CSF. A total of 560 children were asked to participate in the study and to return a sterile swab after collecting their buccal DNA, of whom 390 patients (70%) returned a buccal swab and informed consent form. Patients were diagnosed between January 1990 and December 1995 and this cohort was previously described in detail by Koomen et al.[The study population consists of 390 Dutch Caucasian children who survived MM. These patients were identified by the Dutch Reference Laboratory for Bacterial Meningitis. The diagnosis of MM was based on a positive cerebrospinal fluid (CSF) culture with patients 0% returnen et al.. Data foChildren with ‘complex onset’ of meningitis , relapsing meningitis, or meningitis in the neonatal period were excluded.The Medical Ethical Committee of the VU University Medical Center, Amsterdam, The Netherlands approved this study.TLR9 -1237 (rs5743836) and TLR9 +2848 (rs352140) SNPs were analyzed in buccal DNA by TaqMan analysis using the standard TaqMan protocol. The AbiPrism® 7000 Sequence Detection System  was used to obtain data. Primers and probes we used have been described previously [TLR9 SNPs were chosen based on a study by Lazarus et al. In three ethnic groups they found 20 TLR9 SNPs. A set of four frequent TLR9 SNPs  accounted for more than 75% of all chromosomes in all three populations. Genotyping of both TLR9 -1237 T > C and TLR9 +2848 G > A allows all four locus haplotypes to be distinguished [nguished .We performed a literature search to identify severity variables. We used clinical variables: duration of clinical illness before admission, rectal temperature, convulsions, level of consciousness at admission, ICU admission, main clinical diagnosis at discharge (meningitis or meningitis with sepsis), and post meningitis hearing loss. Convulsions were defined as convulsions reported before or at admission or during hospitalization. Post meningitis hearing loss was defined as > 25 dB perceptive hearing loss that was not present before meningitis occurred. Laboratory variables at admission included: blood culture results, CSF leukocyte numbers, CSF/blood glucose ratios, CSF protein concentrations, blood leukocyte numbers, and C-reactive protein (CRP) concentrations in serum. The selected variables were in accordance with a recent systematic review summarizing the evidence regarding prognostic factors predicting death or sequelae after BM in children . We distTLR9 -1237 and +2848 genotypes. For statistical analysis, SPSS for Windows 17.0 and Graphpad Prism 5 were used. Genotype distributions were checked for deviations of the Hardy-Weinberg equilibrium (HWE). Recessive and dominant models were used to model the relations between genotype distributions and clinical variables. Histograms were used to assess normality of the clinical variables. T-tests, Mann–Whitney U tests, and χ2 test or Fisher’s exact tests were used where appropriate. Outliers (< 4%) were excluded by the Grubbs’ test (p < 0.01) before continuous testing. P values < 0.05 were considered statistically significant.Within selected severity groups, we compared the distribution of Table TLR9 -1237 and TLR9 +2848 respectively. Figure TLR9 -1237 TC or CC genotypes compared to carriers of the TT WT genotype  4987 versus median 955, IQR 3938).Continuous variables i.e. duration of clinical illness, rectal temperature, CSF leukocyte numbers, CSF/blood glucose ratios, CSF protein concentrations, blood leukocytes, and serum CRP concentrations were compared between carriers of wild type (WT) alleles and mutant alleles in MM patients for TLR9 -1237 TC or CC carriers than in carriers of the TT WT   .CSF/blood glucose ratios were significantly lower in No significant differences in genotype distributions of −1237 and +2848 SNPs were found for the other continuous variables (data not shown).TLR9 -1237 TC or CC mutant carriers were compared to the TT WT carriers. Significant associations are shown in Table TLR9 -1237 TC and CC genotypes were associated with a decreased incidence of a positive blood culture for N. meningitidis  0.5, 95% confidence interval (CI) 0.3 – 0.9). These genotypes were also associated with CSF leukocyte levels > 1000 per μL , and with a CSF/blood glucose ratio ≤ 0.4 .Dichotomous variables, i.e. blood culture, convulsions in patient history, level of consciousness at admission, ICU admission, sepsis and hearing loss and the dichotomized continuous variables (as described above) were compared between WT carriers and mutant carriers. TLR9 -1237 genotype distribution were observed between the groups classified by the other severity variables (data not shown).No significant differences in TLR9 +2848 mutant carriers were compared with WT carriers. Significant associations are shown in Table TLR9 +2848 AA was associated with a decreased incidence of a positive blood culture . The TLR9 +2848 AA mutant was also significantly more present in children with > 600 and > 1000 leukocytes per μL .TLR9 +2848 genotype distribution were observed between the groups classified by the other severity variables (data not shown).No significant differences for TLR9 haplotype I was very significantly associated with blood cultures positive for N. meningitidis . Haplotype I was also significantly associated with decreased CSF leukocytes . No significant differences in haplotype distribution were observed for other severity variables (data not shown).Table TLR9 -1237 and +2848 SNPs are associated with severity variables in a cohort of MM survivors. In order to assess the biological consequence of this statistical association we focus on the essential steps in BM pathogenesis and the recognition of N. meningitidis by TLR9. Meningococci colonize the nasopharynx and may penetrate the mucosal barrier of the upper respiratory tract by transcellular passage of epithelial cells [N. meningitidis leads to activation of phagocytosing cells via TLR9 [We demonstrate that the al cells . During al cells . After pal cells . Survivavia TLR9 . Upon suvia TLR9 ,25.TLR9 SNPs with protection against meningococcemia, a prerequisite for meningeal invasion, and with elevated CSF leukocyte levels during MM. A decreased incidence of positive blood cultures in children carrying TLR9 -1237 C allele and TLR9 +2848 AA genotypes may represent a reduction in the occurrence of secondary bacteremia due to more pronounced host immune response in the CSF. A possible mechanistic explanation and biological consequence is increased NFκB binding to the TLR9 promotor region, leading to increased transcriptional regulation of TLR9[in silico regulatory SNP detection method as described by McIntyre et al.[TLR9 -1237 “C” variant was associated with significantly increased binding of NFκB, avian reticuloendotheliosis viral oncogene homolog A (RelA) and signal transducer and activator of transcription (STAT3) to the TLR9 -1237 C variant  harbouring the TC genotype show higher expression of both TLR9 and IL-6 and increased B-cell proliferation in response to CpG [TLR9-1237-C allele with cerebral malaria, indicating that enhanced TLR9 mediated immune responses are also relevant inside the CNS [We associated n of TLR9. We confre et al.: The TLRe to CpG . AnotherTLR9 KO mice displayed reduced survival and elevated levels of bacteremia compared to WT mice [TLR9 polymorphisms with prevention of bacteremia and higher levels of leukocytes in the CSF. The link between systemic inflammation and pleocytosis was previously studied. Intravenous injection of LPS prior to intracisternal LPS injection in rabbits led to impaired pleocytosis, reduced levels of TNF-α, and impaired leukocyte influx into the CNS reflecting an impaired inflammatory response in the CNS [In a mouse meningococcal bacteremia model the role of TLR9 in preventing bacteremia was also confirmed.  WT mice . We asso the CNS .TLR9 SNPs with bacteremia, no association was found with clinical sepsis. We propose that TLR9 -1237 and +2848 polymorphisms have a beneficial effect on preventing bacteremia and increase the leukocyte influx in the CNS, reflecting an enhanced immune response inside the CNS.Clinical studies show that the most severely affected patients with MM or septic shock with a rapidly evolving septic shock associated with high mortality have significant lower levels of pleocytosis . Althoug. We concluded that TLR9 polymorphisms have a small but possibly important contribution to warrant balance between beneficiary and injurious effects of inflammation in the CNS. For exact consequences of these SNPs, future studies focussing on TLR9 SNPs and long term consequences of BM should be performed.Although CNS inflammation is necessary to guarantee sterility of the CNS, its injurious properties are also evident. An adequate but balanced inflammatory response inside the CNS is essential in limiting adverse outcome of diseaseThis study does have certain aspects that limit the interpretations. Survivors of MM were retrospectively included in our study. DNA from children with fatal meningitis were not a focus of our study because including survivors of MM allows us to obtain detailed information on follow-up and long term consequences of the disease which are of particular clinical relevance. In addition, the number of patients with fatal meningitis is very low in The Netherlands. The effect of treatment of BM was not incorporated in this study. This effect may not be of great influence since comparable protocols for treatment of BM were used nationwide in the period these children have been admitted to Dutch hospitals, and timing of treatment is divided equally between groups and independent of genotype distribution.Multidisciplinary efforts are needed in order to bundle and translate genetic studies into beneficial interventions  enforced by the field called Public Health Genomics .TLR9 haplotype I and N. meningitidis). Although the multiple testing corrections reduce the number of statistically significant associations of the exploratory study, the trends that remain give indications for potential roles of the studied variables in the severity of bacterial meninigitis.We have performed several statistical analyses and therefore also performed corrections for multiple testing. Using the rough false discovery rate (rFDR) would shift the threshold for statistical significance from <0.05 to <0.026, reducing the number of significant associations described in this paper. Using the more conservative Holm-Bonferroni corrections, shifts the threshold to <0.002, resulting in one remaining significant association (TLR9 SNPs, functional consequences and long term effects in the CNS are needed to reveal the exact mechanism causing the differences in clinical course of MM and to obtain genetic traits which can be used for patient profiling and management of meningitis patients.More association and functional studies on TLR9 -1237 and TLR9 +2824 mutant genotypes were associated with protection against meningococcemia, an essential prerequisite for meningeal invasion. We also associated these SNPs with enhanced local inflammatory responses inside the CNS. Carriers of the TLR9 + 2848 mutant genotype have a lower chance of developing meningitis when colonized with N. meningitidis. When they eventually do so, they will have more efficient bacterial killing inside the CNS but may also result in a higher chance of developing neurological sequelae.In this study, All authors declare to have no competing interests of any kind regarding this study.in silico analysis, and critically revised the manuscript. AMvF and SAM designed and supervised the study and critically revised the manuscript. All authors have read and approved the final version of the manuscript.GTJvW and MSS collected the patient data and samples, performed the genotype and statistical analysis and wrote the manuscript. SO supervised the genotype and statistical analysis, performed the The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/112/prepub


Our previous studies have suggested that the primary impact of immune cell infiltration into the normal or pre-invasive tissue component is associated with the physical destruction of epithelial capsules, which may promote tumor progression and invasion. Our current study attempted to further verify our previous observations and determine the primary type(s) of infiltrating immune cells and the possible mechanism associated with physical destructions of the epithelial capsules.In total, the study was conducted with 250 primary breast and prostate tumors, the primary immune cell of cytotoxic T-lymphocytes (CTL), Natural killer cells (NK) and Mast cells were analyzed by immunohistochemistry, fluorescent labeling and apoptosis assay. qRT-PCR was used for gene expression analysis. Our current study assessed the physical disruption of these immune cells and potential impact on the epithelial capsule of human breast and prostate tumors.Our study yield several clinically-relevant findings that have not been studied before. (1) A vast majority of these infiltrating immune cells are distributed in the normal-appearing or pre-invasive tissue components rather than in invasive cancer tissues. (2) These cells often form rings or semilunar structures that either surround focally-disrupted basal cell layers or physically attach to the basal cells. (3) Basal cells physically associated with these immune cells generally displayed distinct signs of degeneration, including substantially elevated apoptosis, necrosis, and reduced tumor suppressor p63 expression. In contrast, luminal cells overlying focally disrupted basal cell layers had a substantially increased proliferation rate and elevated expression of stem cell markers compared to their adjacent morphologically similar counterparts that overlie a non-disrupted capsule.Our findings suggest that at the early stage of tumor invasion, CTL, NK and Mast cells are the main types of tumor infiltrating immune cells involved in focal degenerative products in the tumor capsules. The primary impact of these infiltrating immune cells is that they are associated with focal disruptions of the tumor capsule, which selectively favor tumor stem cells proliferation and invasion. A large number of publications have reported that the direct physical contact between infiltrated immune cells and tumor cells is associated with the physical destruction of associated tumor cells, reduction of the tumor size, and significantly improved clinical prognoses -6. Howev vessels ,11, (b) colonies , (c) T-lrophages ; (d) infmotility ,15.in vitro assays cannot fully mimic the intrinsic events in human carcinogenesis [The contradictory observations regarding the impact of tumor-infiltrating immune cells have caused confusions in judging the clinical implications of aberrant infiltration of immune cells within tumor tissues. In addition, as immune cell-mediated lysis or cytotoxic assays are almost exclusively conducted on cell cultures or animal models, these ogenesis -18. It hogenesis ,20.We have speculated that these contradictory reports and statements may result from different tumor stages, in which infiltrating immune cells may be associated with differential consequences depending on the cell type. To validate our speculation, our previous studies compared the pattern and frequency of physical association of tumor-infiltrating immune cells with basal and luminal cells of breast and prostate tumors that harbor both pre-invasive and invasive components. Our studies revealed that: (1) over 90% of infiltrating immune cells were distributed in the normal or pre-invasive tissue component, while fewer than 10% were in the invasive tissue component, (2) within the normal or pre-invasive tissue component, over 90% of the epithelial structures with a focally disrupted epithelial capsule were associated with infiltrating immune cells, compared to about 30% in epithelial structures with a non-disrupted capsule, (3) a vast majority of infiltrating immune cells were located on or near the site of focally disrupted epithelial capsules, and (4) epithelial cells overlying focally disrupted capsules often show a substantially increased proliferation rate and often form finger- or tongue-like projections invading the adjacent stroma -25. BaseOur current study attempted to further verify our previous observations and determine the primary type(s) of infiltrating immune cells and the possible mechanism(s) associated with physical destructions of the epithelial capsules. As it has been well documented that: (1) CTL, natural killer (NK) and Mast cells are the primary immune cell types for detecting and eliminating degenerated and altered host cells and (2) these three cell types harbor similar cytotoxic granules and share the same mechanism for the exocytosis of their granules -31, we hFormalin-fixed, paraffin-embedded human breast tissue (N = 150) were retrieved from the files of the Department of Pathology and Department of Oncology, the Affiliated Nanjing Maternity and Child Health Care Hospital, Nanjing Medical University. Human prostate (N = 100) tumor tissue blocks were obtained from Department of Pathology, Affiliated Jiangsu people’s hospital, Nanjing Medical University with an IRB approval from Nanjing Medical University.Serial 5-7 μm sections were made from the breast and prostate tumor tissue blocks. The first and last sections from each block were stained with Hematoxylin & Eosin (H&E) for morphological classification using published criteria. Double immunohistochemistry was applied to assess the potential impact of infiltrating immune cells on other cell types and structures using previously published protocol . The secp < 0.05.Immune cell aggregates and tumor infiltrating immune cells were elucidated with leukocyte common antigen , CD8 (clone: C8144B), CD16 (clone: DJ130c) and Mast cell tryptase (clone: AA1) (Dako). The breast myoepithelial and prostate basal cells were identified with smooth muscle actin  and cytokeratin  respectively. The focal capsule disruptions were defined as the presence of a physical gap in a given myoepithelial or basal cell layer that is larger than the combined size of at least three epithelial cells and in at least three consecutive sections. Physical signs of degeneration-related changes were defined as the loss of expression of phenotypic markers, vacuolation, fragmentation, swelling, nuclear membrane breakage, chromatin condensation, atrophy, and necrosis. To assess the potential impact of physical association between infiltrated immune cells and basal or luminal cells, immunostained sections and photographs were independently reviewed by three investigators. A given cell was considered immunoreactive if distinct immunoreactivity was consistently present in its cytoplasm, membrane or nucleus while absent in all negative controls. All infiltrated immune cell aggregates in each case were counted, the frequencies of these aggregates within pre-invasive and invasive tissue components were statistically compared by the Pearson’s Chi-square test. To compare the frequencies of cytological signs of degeneration in basal and luminal cells that are physically associated with infiltrated immune cells, digital images were taken in 3–5 randomly selected basal cell layers and associated luminal cells subjacent to or surrounded by infiltrated immune-cells. Digital images were enlarged and viewed on a computer to detect potential signs of degeneration defined as above. The total numbers of cells counted and cells with cytological signs of degeneration in each group were added and averaged, the averaged frequencies were statistically compared by the Pearson’s Chi-square test. Statistical significance was defined as To assess the biological and molecular changes along with potential mechanisms caused by the physical association between infiltrating immune cells and the basal or luminal cells, the following technical approaches were utilized:Apoptosis assay. Tissue sections from 10 cases with a high frequency of degeneration-related changes in the basal cell layers were subjected to apoptosis detection with Apoptosis Detection Kit  according to the manufacturer’ protocol. After wash, the sections were staining for CK34βE12 to determine the histological origin of apoptotic cells.(A) The expression status of tumor suppressor p63 in the basal cell layers: p63 expression in basal cells within non-disrupted and focally disrupted residual layers were compared by double immunostaining of CK34βE12 and p63. To verify the sub-cellular localization of basal cell phenotypic makers CK34βE12 and p63, sets of adjacent human prostate tissue sections from 10 selected cases were double immunostained for CK34βE12 and p63 . The antigen-antibody complexes were distinguished by different secondary antibodies labeled with different fluorophores  according to the manufacturer’s instructions. Immunostained sections were examined and digital images were taken in a fluorescent microscope .(B) Proliferation index and lymphatic duct density: To further assess the potential impact of infiltrating immune cells on cell proliferation and angiogenesis, tissue sections from 30-selected cases were double immunostained for CK34βE12 and a cell proliferation marker, Ki-67 . The proliferation status in epithelial cells surrounded by infiltrating immune cells were compared to those cells that were distant from infiltrating immune cells. In addition, special attention was paid to identify whether proliferating cells were preferentially located on the site of focally disrupted basal cell layers.(C) Microdisection and Quantitative Real-Time PCR (qRT-PCR): Five unstained adjacent sections or immunostained sections with pre-invasive tumors showing extensive focal capsule disruptions and immune cell infiltration were deparaffinized and lightly stained with hematoxylin. Guided by immunostained sections, morphologically similar pre-invasive lesions with and without extensive focal capsule disruptions and immune cell infiltration were microdissected and subjected to RNA extraction by RNeasy FFPE Kit (Qiagen) according to the manufacturer’s instructions. Total RNA (0.5 μg) was reverse transcribed in a total volume of 20 μl using the Omniscript RT kit (Qiagen). Quantitative PCR was performed in triplicate in an ABI-Prism 7700 instrument  using QuantiTect Sybr green  according to the manufacturer’s protocol. The appropriate primer sets were listed in Additional file -ΔCt, where Ct is the threshold cycle and ΔCt = CtTarget - Ct GAPDH.(D) Student’s test was performed for two group comparisons. The data presented represent mean ± SEM. P-value of less than 0.05 was considered to be statistically significant.The sub-cellular localization and cell types that reacted to the antibodies were consistent with those of published data and specifications of manufactures. All negative controls were consistently devoid of distinct immunoreactivity. The results were highly consistent in duplicates or triplicates. The basal and luminal cells were distinguishable by morphology and by immunohistochemistry in all study cases. The basal cell population is characterized by its spindle or elongated shape with densely stained nucleus, CK34βE12 staining in the cytoplasm, and p63 staining in nucleus. The luminal cell population is characterized by its round or oval shape with lightly stained nucleus harboring distinct nucleoli. It is devoid of CK34βE12 and p63 expression. CD16, CD56, Fox3p, Mast, and neutrophils cells are primarily known as lymphocytes; thus, they have been collectively referred as infiltrated lymphocytes below. Both the prostate basal and breast myoepithelial cells are referred to as basal cells below.p < 0.01).Similar to our previous studies, lymphocyte aggregates were almost exclusively in some normal appearing or pre-invasive breast and prostate epithelial structures, in which many infiltrated lymphocytes were physically attached to the basal cell layers that were often focally disrupted or substantially attenuated Figure . In sharin situ breast and prostate cancer tissue component, infiltrating immune cells often formed ring or semilunar structures, completely or partially surrounding the epithelial tissue. As shown in Figure Within the In normal or hyperplastic epithelial structures with a focally disrupted epithelial capsule, a majority of infiltrating immune cells was located on or near the site of focal capsule disruptions. This observation is shown in Figure In pre-invasive cancerous epithelial structures with a focally disrupted tumor capsule, infiltrating immune cells were also preferentially located on the site of focally disrupted capsules. As shown in Figure The predominant type of infiltrating immune cells associated with focal capsule disruptions appears to be CTL, which can be easily recognized by their morphology and immunoreactivities to the anti-CD8 antibody. As shown in Figure Similar NK and Mast cell infiltration was also seen in pre-invasive cancerous epithelial structures. As shown in Figure Focal capsule disruptions appear to result from focal degenerative alterations in the constitutional elements of the epithelial capsule. As shown in Figure Compared to their non-disrupted counterparts, the residual basal cells in focally disrupted capsule have significantly lower expression of tumor suppressor p63. As shown in Figure In contrast to these degenerative changes the epithelial cells overlying focally disrupted capsules often showed substantially increased cell proliferation. As shown in Figure Our qRT-PCR results revealed that microdissected luminal cells overlying focal capsule disruptions have significantly higher expression levels of four stem cell markers , which were 6-, 4-, 2- and 6-fold higher, respectively, than their undisrupted counterparts at a distance from the site of focal capsule disruptions Figure .The preferential association of tumor-infiltrating lymphocytes with the basal cell population appears to result primarily from focal degeneration and disruption of the basal cell layers. Our previous studies have consistently shown that compared to its morphologically clear-cut and undisrupted counterpart, a focally disrupted basal cell layer has significantly lower expressions of tumor suppressor p63 and proliferating cell nuclear antigen, while it has significantly higher frequencies of apoptosis and degeneration . As showa) local loss of tumor suppressors and paracrine inhibitory functions, which confer epithelial cell growth advantages allowing them to escape from programmed cell death [(b) local increase of immune cells infiltration, could increase of permeability to oxygen, nutrients, and growth factors, which selectively favors the proliferation of progenitor/stem cells [c) direct epithelial stromal cell contact, which augments the expression of stromal matrix metalloproteinase (MMP) or other cell adhesion molecules , which facilitate epithelial to mesenchymal transition (EMT) and cell motility [d) direct exposure of the epithelial cells to different cytokines, which facilitates vasculogenic mimicry and tumor angiogenesis [e) direct physical contact between newly formed cell clusters and stromal cells, which stimulates the production of tenascin and other host factors such as hepatocyte growth factor(HGF) that facilitate stromal tissue remodeling and drug resisiance [As the breast and prostate epithelium is normally devoid of both blood vessels and lymphatic ducts and the my epithelial or basal cell layer is the sole source of several tumor suppressors -37, a foll death ; (b) locem cells ,40; (c) motility ; (d) dirogenesis , and (e)sisiance -45. Thessisiance ,47, Nanosisiance . Elevate(1) mammary ductal intraepithelial neoplasia (DIN)-flat type is a subtle epithelial alteration characterized by one or a few layer(s) of atypical cells replacing the native epithelium, but loss of heterozygosity (LOH) was detected in 17 of 22 (77%) lesions, and monoclonality was established in the 2 cases analyzed [(2) LOH was detected in morphologically normal lobules adjacent to breast cancers. In 10 cases with LOH at chromosome 3p22-25 in the carcinoma, 6 displayed the same LOH in adjacent normal lobules [(3) the prostate tissues in a subset of aged men or normal-appearing prostate tissues adjacent to prostate cancer harbored a DNA phenotype that is identical to invasive and metastasized prostate cancer [Our recent studies have consistently revealed that 15-30% of the patients with non-invasive breast or prostate tumors harbor epithelial structures that are morphologically normal or hyperplastic in H&E stained sections, but they show frequent focal capsule disruptions with budding cells in immunohistochemically stained sections -31. The analyzed ; (2) LOH lobules , and (3)e cancer -53. TogeThe infiltrated host immune cell classification in combine with some other marker may have a prognostic value for tumor invasion and metastasis . As the (1) 154 total lymphocyte aggregates detected were preferentially associated with pre-invasive structures that have focally disrupted capsules; (2) infiltrated lymphocytes were predominantly seen at the site of focally disrupted tumor capsules; (3) a vast majority of the basal cells were immediately subjacent to or physically associated with infiltrating lymphocytes and displayed a wide variety of cytological signs of degeneration; and (4) focal tumor capsule disruptions coupled with lymphocyte infiltration with the stem cell signature appear to facilitate proliferation and dissociation of the overlying luminal cells. Together, our findings are consistent with our hypothesis: tumor-infiltrating lymphocytes are exclusively or preferentially associated with degenerated basal cells in the initial or early stage of tumor invasion. To our knowledge, this is the first report, which suggests that the primary impact of tumor infiltrating CTL, NK and Mast cells may be associated with the physical disruptions of the tumor capsule. In addition, our findings could offer a reasonable explanation for the contradictory reports and statements regarding the impact and clinical significance of immune cell infiltration into tumor tissues.Our current study in breast and prostate tumors has revealed: We declare that we have no competing interests.Y, H, S and S proposed the hypothesis. Y and H designed and performed the experiments. W, S and S analyzed and interpreted the data. Y, H, Y, S and S wrote the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/13/258/prepubList of primer sequences for RT-PCR.Click here for file


Tumor-associated macrophages (TAMs) contribute substantially to the tumor mass of gliomas and have been shown to play a major role in the creation of a tumor microenvironment that promotes tumor progression. Shortcomings of attempts at antiglioma immunotherapy may result from a failure to adequately address these effects. Emerging evidence supports an independent categorization of glioma TAMs as alternatively activated M2-type macrophages, in contrast to classically activated proinflammatory M1-type macrophages. These M2-type macrophages exert glioma-supportive effects through reduced anti-tumor functions, increased expression of immunosuppressive mediators, and nonimmune tumor promotion through expression of trophic and invasion-facilitating substances. Much of our work has demonstrated these features of glioma TAMs, and together with the supporting literature will be reviewed here. Additionally, the dynamics of glioma cell-TAM interaction over the course of tumor development remain poorly understood; our efforts to elucidate glioma cell-TAM dynamics are summarized. Finally, the molecular pathways which underlie M2-type TAM polarization and gene expression similarly require further investigation, and may present the most potent targets for immunotherapeutic intervention. Highlighting recent evidence implicating the transcription factor STAT3 in immunosuppressive tumorigenic glioma TAMs, we advocate for gene array-based approaches to identify yet unappreciated expression regulators and effector molecules important to M2-type glioma TAMs polarization and function within the glioma tumor microenvironment. Additioin vitro , indicatThe immune response to tumor-associated antigens and mechanisms of immunosuppression by tumor cells have been the most actively investigated areas of cancer immunotherapy research. These efforts have led to FDA-approved immunotherapy-based treatment protocols that have been shown to reduce tumor burden and prolong survival in patients with many different systemic tumors  includinWe and others have shown that tumor-associated monocytes and microglia (TAMs) are the predominant infiltrating immune cell in malignant glioma and can account for up to 40% of the tumor cell mass –21. Becaα, IL-1Β, and IL-12), upregulation of cell surface molecules necessary for antigen presentation (including MHC II and costimulatory molecules CD80 and CD86), and an overall enhanced ability to phagocytose pathogenic material [Β or TNF-α [β, downregulation of cell surface molecules necessary for antigen presentation including MHC II, CD80, and CD86, decreased phagocytic capacity, and upregulation of cell surface antigens FasL and B7-H1 both known to stimulate programmed cell death in lymphocytes, among other effects [Classically activated M1-type macrophages participate in the coordinated response to immunogenic antigens primarily through production of proinflammatory mediators  under pathological conditions. TAMs in human glioma are generally believed to originate from at least two distinct sources. Principal among them are resident microglia, believed to monitor their local neural tissue environment through extensive ramifications, and subsequently to activate a phagocytic phenotype, nearly identical to activated macrophage phenotypes, upon stimulation . A recenric rats . Still, As previously mentioned, the overwhelming predominance of TAMs in the immune infiltrate of both murine and human malignant gliomas has heightened awareness of the influential role these cells may have on both creation of an immunosuppressive tumor microenvironment and facilitation of glioma cell progression , 47. Cumα and a significant upregulation of IL-10 secretion by stimulated naïve human monocytes in vitro [in vivo when we used our murine glioma model to demonstrate a reduction of TNF-α expression by TAMs during late stages of tumor growth [Despite clear evidence of chemotaxis to glioma tumor tissue and subsequent contact with glioma-specific antigens known to be classically immunogenic , TAMs inin vitro . These fr growth .  FigureThe mature M1 macrophage marker CD14 serves as a coreceptor of TLR4 and is upregulated in nearly all CNS pathologies . Howeverγ or LPS [in vivo, as we have found that monocytes reisolated following coculture with malignant glioma cells demonstrate an inability to activate allogenic CD4+ T cells [γ from CD4+ T cells cultured with GBM-treated monocytes [Other deficiencies of glioma TAMs thought to contribute to local immunosuppression are reduction in the expression of HLA and costimulatory molecules. For instance, our group has previously shown significantly reduced CD80 and HLA-DR expression on stimulated naïve human monocytyes when cocultured with GBM cells . This fiγ or LPS . These p T cells . In addionocytes . Similaronocytes , a knownex vivo human GBM specimens [Glioma TAMs have also been shown to be deficient in phagocytosis. Rodrigues and colleagues demonstrated a significant reduction in the ability to phagocytose bacterial cell wall particles following stimulation in glioma cell-conditioned monocytes, as compared with both astrocyte-conditioned and unconditioned monocytes . In anotpecimens . This grpecimens . β, and cell surface antigens B7-H1, and FasL [In addition to the antitumor effector function deficiencies described earlier, accumulating evidence suggests that glioma TAMs are actively immunosuppressive. Glioma TAMs are now thought to represent an altered phenotype resulting from the directed influence of tumor cells upon immune cells in efforts to produce a favorable tumor microenvironment. Among the immunosuppressive mediators upregulated in glioma TAMs, those that appear to exert a predominate effect include cytokines IL-10, TGF-and FasL , 33, 34.ex vivo were compared with TAMs from meningiomas and these findings are presented in ex vivo GBM tumor specimens overwhelmingly to TAMs, though also present in glioma cells to a much lesser extent [β following co-culture with glioma cell lines, as compared to isolated culture as well as co-culture with NHA [Our group has previously demonstrated that when cocultured with malignant glioma cells, stimulated naïve human monocytes significantly upregulate expression of IL-10 . Quantitr extent . Furtherwith NHA . Hence, with NHA , 34. Both glioma cells  and TAMsB7-homolog 1 (B7-H1) is a homolog of the costimulatory surface antigens CD80 and CD86 (B7.1 and B7.2) that has been shown to attenuate T-cell receptor function through engagement of the programmed death receptor (PD-1) on the surface of T cells . PD-1 acα in response to stimulation [Although the mechanisms are unknown, there is evidence that malignant gliomas are able to alter monocytes so they become tolerogenic. When human monocytes previously cultured with malignant glioma cells are co-cultured with naïve monocytes, naïve monocytes had a dramatically reduced ability to secrete TNF-mulation . These fβ, EGF, and HGF/SF have drawn the most attention, though dissecting the precise role of TAMs in the production of these trophic factors remains to be accomplished. Glioma cells are known to produce a number of self-supportive factors concurrently with their corresponding cell surface receptors, together acting to promote their own proliferation, migration, angiogenesis, and subsequently tumor extension –62. Indeβ) is an extremely potent immunosuppressive and transformative cytokine whose expression is mostly associated with glioma cells themselves [β through induction of VEGF and FGFs [β may also promote tumor cell migration and invasion through induction of MMP expression in conjunction with suppression of tissue inhibitor of metalloproatease expression [β isoform 1 (TGF-β1) under certain pathological conditions including neuritis and trauma [in situ hybridization, Kiefer et al. localized the expression of the TGF-β1 isoform to activated glioma TAMs in a murine model, suggesting to the authors this isoform's involvement in a mutually reinforcing paracrine loop with glioma cells [β exerts immunosuppression by driving alternative polarization in TAMs, TGF-β produced by the glioma TAMs may promote tumor growth and invasion by stimulating the upregulation of its own cognate receptors TBRI and TBRII on glioma cells [β proposed to exist in the glioma microenvironment.Tissue growth factor beta  have emerged as a pivotal signaling mechanism in high grade glioma. EGFR amplification is seen in approximately 50% of GBM, and in approximately 50% of those tumors the glioma cells express EGFRvIII, a mutant receptor that persistently activates downstream immunosuppressive pathways including those involving STAT3 . In two ex vivo human glioma and TAM cells [in situ hybridization with fluorescence immunohistochemistry to demonstrate expression of both HGF/SF and c-Met in a majority of TAMs isolated from human ex vivo GBM specimens [in vitro that glioma-derived HGF/SF is a potent chemotactic agent on microglia [Hepatocyte growth factor/scatter factor acts exclusively through the tyrosine kinase receptor c-Met and expression of both the soluble ligand and receptor has been demonstrated in both AM cells , 75. Kunicroglia  postulaticroglia  and furticroglia , yet it icroglia , 78. Altex vivo human samples taken at the time of surgical resection, or similarly upon murine specimens harvested at a late-stage time point when glioma tumor mass is grossly apparent. Both of these scenarios likely represent end-stage tumors wherein the tumor has advanced to aggressive behavior, abetted by immunosuppressive glioma-promoting TAMs, as this review has demonstrated. Little data is available to suggest if the partnership between glioma cells and alternatively activated M2 TAMs arises at tumor onset and therefore contributes early in tumor formation, or if TAMs initially manifest a classically activated M1 phenotype to combat tumor development until some critical point when tumor-derived mediators overwhelmingly direct M2 polarization, followed by rapid tumor progression and clinical presentation. Recent efforts by our group have sought to address these questions through kinetic studies of the infiltration and function of immune cells in a murine glioma model, both within the tumor microenvironment and peripherally [α secretion. Of great interest, no change was observed in the proportion of TAMs secreting TNF-α between the early and intermediate time points, though a 2.5-fold reduction in the TNF-α -secreting TAMs was evident by the final time point. These findings are summarized in α. A major shortcoming of the efforts to understand glioma-associated macrophages remains a paucity of data describing the dynamics of glioma cell and macrophage interactions. Most of the findings reviewed to this point are based upon pherally . Using mγ—producing CD4+ T cells and regulatory T cells (CD4+FoxP3+). Between the early and intermediate time points, the proportion of IFN-γ—producing CD4+ T cells, increased modestly and sustained this elevated frequency through the late time point. Interestingly, the proportion of regulatory T cells increased significantly between the early and intermediate time points and also remained elevated, though slightly decreased from peak frequency, through the late time point. Moreover, the proportion of these T-reg cells that expressed IL-10 mirror the overall frequency, increasing significantly between early and intermediate time points and exhibiting a sustained effect through the late time point. These findings suggest that peripheral immunosuppressive changes may precede those in the tumor microenvironment. Efforts of our group and others have established regulatory T cells and others as major immunosuppressive cellular mediators implicated both locally and systemically in glioma patients [Peripheral immune changes were investigated with splenic preparations, evaluated for presence of IFN-patients –81. The As this review suggests, the creation of a potent immunosuppressive, glioma conducive, tumor microenvironment at least in part results from the complex interplay of glioma cells and alternatively activated TAMs that involve many immune mediators with pleiotropic affects. While extensive efforts to characterize glioma TAMs have established many of the individual immunosuppressive and tumor permissive features specific to these cells, an understanding of the molecular pathways and signaling molecules affecting gene expression which lead to the altered phenotype of glioma TAMs is not known. Intracellular mediators at points of signaling convergence within glioma TAMs and the molecular products which result from their activation present potential targets for immunotherapy-based strategies directed against malignant gliomas. Furthermore, recent evidence highlighting prognostic differences among distinct molecular subtypes of glioma, including those with 1p/19q codeletion, isocitrate dehydrogenase (IDH) mutations, and differential MGMT methylation status , raises At this point, recognition of differential activation of the signal transducer and activator of transcription 3 protein (STAT3) within both glioma cells  and glioin vivo, we then isolated TAMs from patients with primary and recurrent malignant gliomas and confirmed their differential expression patterns ex vivo. Our next step is to use siRNA to block these candidate genes in our GBM: monocyte co-culture model to determine if monocyte function can be restored. Importantly, preliminary analysis confirms that STAT3 is not among our candidate genes, suggesting that multiple pathways contribute to TAM dysfunction in gliomas. We hope that the identification of genes differentially expressed in glioma TAMs may give us a better understanding of the pathways driving M2-type polarization and lead to new, more effective targets for glioma based immunotherapy.Though STAT3 activation appears to play a key role in generating and perpetuating the M2-type TAMs in gliomas, it is unclear whether a single dominant molecule or a complex network of molecules is responsible for the immunosuppressive phenotype of glioma TAMs. Our group therefore conducted a microarray-based approach of sorted human monocytes after co-culturing with malignant gliomas in an attempt to better characterize these pathways. Through extensive pathway analyses and network exploration, we identified a small subset of novel candidate genes that could be responsible for glioma-induced dysfunction of TAMs. To demonstrate that these candidate genes identified by the microarray analysis were important Are tumor-associated monocytes/microglia in malignant gliomas friends or foes? Although a comprehensive answer to this question remains elusive, the considerable efforts described in this review seem to cast TAMs in glioma as a formidable foe, espousing an altered activation state within the local tumor microenvironment characterized by deficiencies in antitumor effector functions, upregulation of potent immunosuppressive mediators, and participation in tumorigenic loops of paracrine signaling involving expression of trophic factors and their cognate receptors. Although the dynamics of this malign partnership between glioma cells and TAMs remain unclear over the course of tumor progression, a turning point seems to occur late in tumor development, perhaps providing a better opportunity for clinically based immunotherapy. Given the compelling evidence that TAMs contribute significantly to the creation and maintenance of immunosuppression and tumor progression, it is unlikely that clinically effective immunotherapy against malignant gliomas will be achieved until we gain a better understanding of how to influence TAM function in the local tumor microenvironment.


Acute respiratory illness is the leading cause of asthma exacerbations yet the mechanisms underlying this association remain unclear. To address the deficiencies in our understanding of the molecular events characterizing acute respiratory illness-induced asthma exacerbations, we undertook a transcriptional profiling study of the nasal mucosa over the course of acute respiratory illness amongst individuals with a history of asthma, allergic rhinitis and no underlying respiratory disease.Transcriptional profiling experiments were performed using the Agilent Whole Human Genome 4X44K array platform. Time point-based microarray and principal component analyses were conducted to identify and distinguish acute respiratory illness-associated transcriptional profiles over the course of our study. Gene enrichment analysis was conducted to identify biological processes over-represented within each acute respiratory illness-associated profile, and gene expression was subsequently confirmed by quantitative polymerase chain reaction.We found that acute respiratory illness is characterized by dynamic, time-specific transcriptional profiles whose magnitudes of expression are influenced by underlying respiratory disease and the mucosal repair signature evoked during acute respiratory illness. Most strikingly, we report that people with asthma who experience acute respiratory illness-induced exacerbations are characterized by a reduced but prolonged inflammatory immune response, inadequate activation of mucosal repair, and the expression of a newly described exacerbation-specific transcriptional signature.Findings from our study represent a significant contribution towards clarifying the complex molecular interactions that typify acute respiratory illness-induced asthma exacerbations. Acute respiratory illness (ARI) is a leading cause of morbidity across all demographic and socioeconomic strata worldwide and the primary trigger for exacerbations of chronic lower airway diseases including asthma ,2. WhileTranscriptional profiling studies have revealed that the pathophysiology, phenotypes and therapeutic response of asthma are accompanied by distinct gene expression profiles -14. HoweComparative profiling studies between exacerbation and a ‘stable’ asthmatic baseline have revealed that asthma exacerbations involve complex interactions between acute and chronic and/or allergic states of inflammation -20. HoweTo address the deficiencies in our understanding of the molecular events that characterize ARI and ARI-induced asthma exacerbations, we undertook a transcriptional profiling study of the nasal mucosa over the course of ARI in individuals with asthma, allergic rhinitis and no underlying respiratory disease. We report that ARI is characterized by dynamic, time-specific transcriptional profiles whose magnitudes of expression are influenced by underlying respiratory disease and the mucosal repair signature evoked during ARI. Most strikingly, people with asthma who experience ARI-induced exacerbations are characterized by a reduced but prolonged inflammatory immune response, inadequate activation of mucosal repair, and the expression of a newly described exacerbation-specific transcriptional signature.This report encompassed samples from the Naturally Acquired Upper Respiratory Illness study conducted at University of California, San Francisco (UCSF) from November 2001 to November 2004. The study design was approved by the UCSF Committee of Human Research. This study conformed to the Helsinki Declaration. Written informed consent was obtained for all participants prior to inclusion in the study.Participants with a history of asthma, hay fever or no underlying airway disease were recruited for the study via community advertisements. Upon the onset of ARI or ‘common cold’ symptoms, recruited participants were instructed to attend the first of three required visits to the study clinic. The first and second visits were designed to obtain samples and clinical data during the peak and reduction/resolution stages of symptomatic illness, whereas the third visit would occur when volunteers were asymptomatic and serve as a prospective baseline (BL) for the study. The study population represented attended the first, second and third clinic visits on average 2 days , 6 days  and 89 days (range 29 to 297 days) after the onset of ARI symptoms.U-test for comparisons between groups. Correlations were analyzed using Spearman’s rank correlation.At each visit, we obtained nasal scrapings  from the lower turbinates  after two nasal lavages  to remove overlying mucus. We also measured at each visit exhaled nitric oxide (eNO) and forced expiratory flow in 1 second (FEV1) by performing spirometry. At the BL visit, participants underwent a methacholine test to assess airway hyper-reactivity and an allergy skin test to determine if they were atopic . Particia priori as either:Asthma exacerbation during ARI was defined 1. an increase in asthma symptoms that made the participant increase or start inhaled or oral corticosteroids, or seek medical care in a doctor’s office, urgent care clinic or emergency department or2. an increase in chest or asthma symptom score by ≥10 points for ≥2 days over average daily scores during baseline week plus at least one of these objective changes: use of ≥4 puffs of albuterol above the average daily use in the baseline week for ≥2 days; decline of ≥10% in FEV1 during ARI (first or second visit) compared to FEV1 at BL; or decline of ≥20% in PEF for ≥2 days during the cold week compared with best PEF achieved in the BL week.Total RNA was extracted from nasal mucosa samples using RNeasy Mini Kit  with the addition of a DNAse treatment  and re-purification phase. Extracted RNA was further purified using the RNA Clean and Concentrator  and quality assessed by RNA 6000 Pico Kit and 2100 Bioanalyzer . From those samples exhibiting high RNA integrity, 650 ng of RNA was submitted to the UCSF Shared Microarray Core Facilities for microarray experiments employing the Whole Human Genome 4X44K array platform . Microarray data has been deposited in the Gene Expression Omnibus [GEO:GSE46171].2 normalized gene expression data to identify overall temporal transcriptional profiles of ARI . A 4×3 analysis of variance linear model was fitted to the comparisons to estimate the mean expression values and differentially expressed genes identified by calculation of moderated t-statistic, B statistic, false discovery rate and raw P-value for each comparison of interest. In addition, a mixed linear model was fitted for each gene to estimate the within-subject correlation. A single robust average of this correlation was used as the within-subject correlation when fitting the linear model for all genes. Adjusted P-values were produced by the method proposed by Holm [P <0.05). All procedures were carried out using functions in the R package limma in Bioconductor[Because small group numbers limited the statistical power of group-based analyses, time point-based comparative analyses were conducted using log by Holm . To encoconductor.P <0.05). All PCAs were performed on covariance matrices of gene expression data represented as log2 normalized probe intensities  using PAST (v2.15) [Principal component analysis (PCA) was used to identify the primary source of variation driving the differentially expressed genes identified in time point-based microarray analysis  .T-test (matched samples) or Mann–Whitney U-test (unmatched samples).Initial PCA included all matched time point microarray samples from each study group to encompass all potential sources of variation over the course of our study . Additional group-based PCAs encompassing three time point transitions  were then conducted using all available microarray samples for each group. To statistically confirm the influence of disease status on the ARI-induced changes in gene expression found by PCA, the separation of time point clusters along the principal component 1 (PC1) axis was determined by analysis of PC1 scores between samples by paired Since we regarded PC1 to represent the ARI-associated variation within our dataset, we determined ARI-associated transcriptional profiles by ranking the n = 2,456 genes based on their PC1 loadings. Gene rankings were generated for each group-based BL to D2, D2 to D6, and D6 to BL PCA and were used to identify the ARI-associated profiles that characterized the peak, reduction and resolution stages of each group respectively.Since no consistent component loading cutoff could be found within the literature, the top n = 100 most positive or negative loading genes were then selected from each group and subjected to comparative analyses  using VENNY . For the2) were scaled so that the maximum intensity of each gene across time points and study groups was 1 and the minimum was 0. Hierarchical cluster analysis of microarray data (log2 fold change in expression) was performed with Euclidean distance and average linkage using MEV [Kinetics of gene expression were determined by plotting gene probe intensities at each time point. Probe intensities  was performed using both MetaCore™  and Gene Enrichment Profiler . MetaCorU-test. Primer and probe sequences are available upon request.Total RNA from participants with three  or two  matched samples were selected for quantitative real-time PCR analysis. Missing samples were due to either participant failing to attend one of the three clinic visits or sample RNA quantity being minimal. Genes were selected for expression confirmation based on PC1 loadings. Depending on the stage of ARI analyzed, the highest ranked  or lowest (D6 of reduction) genes were selected. Gene expression in each sample was normalized to glyceraldehyde-3-phosphate dehydrogenase and peptidylprolyl isomerase A as outlined previously . SignifiA summary of the study design is depicted in Figure P <0.05; data not shown). PCA confirmed that the primary source of variation driving the differential expression  was time-dependent  indicated that 33 genes were common Figure . QuantitComparative analysis of the ARI-associated D6 profiles revealed no common genes existed across study groups. Rather, 66 genes were shared amongst the Healthy, Allrg Rhin and Asm NoEx groups and the Asm Ex group was characterized almost exclusively by a set of 91 distinct genes Figure .We focused initially on the 66 ARI-associated genes shared between the Healthy, Allrg Rhin and Asm NoEx groups. Using GEA, we determined that these shared-D6 genes were associated with processes and pathways consistent with mucosal repair and recruitment of immune cells  and the focus exclusively on the upper airways may have been limitations of our study, these findings represent a significant contribution towards clarifying the complex molecular interactions that characterize ARI-induced asthma exacerbations.Allrg Rhin: patients with allergic rhinitis; ARI: acute respiratory illness; Asm Ex: patients with asthma and exacerbation; Asm NoEx: patients with asthma without exacerbation; BL: baseline; CCL: Chemokine (C-C motif) ligand; CXCL: Chemokine (C-X-C motif) ligand; D2: day 2; D6: day 6; eNO: exhaled nitric oxide; FEV1: forced expiratory flow in 1 second; GEA: gene enrichment analysis; ICS: inhaled corticosteroids; NS: nasal steroids; PC1: principal component 1; PCA: principal component analysis; PCR: polymerase chain reaction; PEF: peak expiratory flow rate; TMEM: transmembrane protein.The authors declare that they have no competing interests.PCA and HAB conceived and designed the study. TW was involved in study recruitment and sample collection. SF, JS, PM and AsB processed samples. DJE, AnB, RB and CE conducted the micorarray experiments. SB conducted principal components analysis. PM conducted data analysis and qPCR experiments. PM, SB, PCA and RPS drafted the manuscript. All authors read and approved the final manuscript.Contains recruitment and numbers of participants represented in the current study and PCA.Click here for fileContains lists of common, shared or distinct genes identified in comparative analysis of ARI-associated profiles for the peak, reduction and resolution stages of ARI.Click here for fileContains results of GEA of gene sets.Click here for file


PUF3 deleted strain exhibits more robust growth than wild-type cells and the shift in translation from polysomes to monosomes is attenuated, suggesting puf3Δ cells perceive less stress. In agreement, the ratio of reduced:oxidized glutathione, a major antioxidant and indicator of cellular redox state, is increased in unstressed puf3Δ cells but remains lower under stress. In untreated conditions, Puf3p migrates with polysomes rather than ribosome-free fractions, but this is lost under stress. Finally, reverse transcriptase-polymerase chain reaction (RT-PCR) of Puf3p targets following affinity purification shows Puf3p-mRNA associations are maintained or increased under oxidative stress. Collectively, these results point to Puf3p acting as a translational repressor in a manner exceeding the global translational response, possibly by temporarily limiting synthesis of new mitochondrial proteins as cells adapt to the stress.In response to stress, the translation of many mRNAs in yeast can change in a fashion discordant with the general repression of translation. Here, we use machine learning to mine the properties of these mRNAs to determine specific translation control signals. We find a strong association between transcripts acutely translationally repressed under oxidative stress and those associated with the RNA-binding protein Puf3p, a known regulator of cellular mRNAs encoding proteins targeted to mitochondria. Under oxidative stress, a  Cells maintain homeostasis by eliciting a variety of responses to environmental stress. In the postgenomic era, it is common to measure these responses at the transcriptional level using microarrays or more recently RNA-seq . These di to the ribosome, can be inhibited by specific eIF2α kinases  in the mRNA sequence  indicates that Puf3p remains bound to its target mRNAs after oxidative stress. Collectively, these data highlight a novel function for Puf3p in the repression of translation and/or induced mRNA decay of specific mRNAs in S. cerevisiae in response to oxidative stress. A large fraction of Puf3p mRNA targets encode proteins targeted to mitochondria, in line with its role in oxidative stress  and 2 mM H202 (high peroxide). The translation state for each mRNA was determined by calculating the log2 ratio of intensities in the polysome compared with the monosome fractions for both control and stress conditions [log2(polysomecontrol:monosomecontrol) and log2(polysomestress:monosomestress)]. A second ratio of these two values gives the change in translation state [log2(polysomecontrol:monosomecontrol):log2(polysomestress:monosomestress)]. Transcripts were defined as being translationally up-regulated or down-regulated if the change in translation state was >1.0 (a 2-fold change in absolute ratios). To ensure the statistical significance of these transcripts, a two-factor ANOVA model using Limma  in the processed form [details of initial array normalization are given in ]. The dang Limma  functionRandom forests are an ensemble classifier with high accuracy, based on the output from many classification/regression trees , which hTraining and testing of the input data was performed with the RandomForest package in R . PerformSupplementary Table S1.When generating the training set each transcript was subdivided into three sections: 5′UTR, 3′UTR and ORF . Funding for open access charge: Biotechnology and Biological Sciences Research Council (BBSRC) [BBG012571].Conflict of interest statement. None declared.


Dictyostelium has a single tip that acts as an organising centre patterning the rest of the slug. High adenosine levels at the tip are believed to be responsible for this tip dominance and the adenosine antagonist, caffeine overrides this dominance promoting multiple tip formation.The multicellular slug in Dictyostelium discoideum. Pharmacological inhibition of cAMP phosphodiesterase also resulted in multiple tips. Caffeine reduces cAMP levels by 16.4, 2.34, 4.71 and 6.30 folds, respectively in D. discoideum, D. aureostipes, D. minutum and Polysphondylium pallidum. We propose that altered cAMP levels, perturbed cAMP gradient and impaired signalling may be the critical factors for the origin of multiple tips in other Dictyostelids as well. In the presence of caffeine, slug cell movement gets impaired and restricted. The cell type specific markers, ecmA  and pspA (prespore) cells are not equally contributing during additional tip formation. During additional tip emergence, prespore cells transdifferentiate to compensate the loss of prestalk cells.Caffeine induced multiple tip effect is conserved in all the Dictyostelids tested. Two key components of cAMP relay namely, cAMP phosphodiesterase (Pde4) and adenyl cyclase-A (AcaA) levels get reduced during secondary tip formation in Caffeine decreases adenyl cyclase–A (AcaA) levels and as a consequence low cAMP is synthesised altering the gradient. Further if cAMP phosphodiesterase (Pde4) levels go down in the presence of caffeine, the cAMP gradient breaks down. When there is no cAMP gradient, directional movement is inhibited and might favour re-differentiation of prespore to prestalk cells. D. discoideum slug consists of two prominent cell types: the anterior prestalk cells and the posterior prespore cells , and 5 mM K4 [Fe (CN6)] solution and incubated at 37°C for 45 minutes before observation.Slugs were fixed in 3.7% formaldehyde solution in Z-buffer  was procured from Cell Signaling, USA. This kit contains a cAMP XPTM rabbit mAB coated 96 well plate and HRP linked cAMP, substrate (TMB) and other necessary reagents. The slugs were lysed in a 100 μl of 1X lysis buffer containing 1 mM PMSF (phenyl methyl sulfonyl fluoride) and the lysed sample was incubated in ice for 10 minutes. 50 μl of the test sample and 50 μl of the HRP-linked cAMP solution were added on to the assay plate and was incubated at room temperature for 3 hours on a horizontal orbital shaker. The supernatant was discarded and the wells were washed thrice with 200 μl of 1X wash buffer and thereafter 100 μl of TMB substrate was added to the wells. Subsequently, the plate was incubated at room temperature for 10 minutes and the reaction was terminated by adding 100 μl of stop solution. The absorbance was measured at 450 nm. The cAMP standard curved was used to calculate the absolute amount of cAMP in the test samples.To quantify cAMP levels in the slugs, a cAMP XPNikon SMZ-1000 stereo zoom microscope with epi-fluorescence optics was used for monitoring and taking the pictures. The fluorescence images were taken using a Nikon 80i eclispse upright microscope.D. discoideum amobae with neutral red and allowed them to develop as slugs which were transferred to a non-nutrient plates containing 5 mM caffeine. Neutral red stains the anterior prestalk region of the slug and during ectopic tip formation the staining gets redistributed from prestalk to prespore region of the slug after 1 h of development  also gets reduced.Adenyl cyclase-A (AcaA) is known to express in the slug tip  and catag Figure C. The les Figure D. DuringBesides reduced cAMP levels, a perturbed cAMP gradient may also result in secondary tips. If extracellular cAMP phosphodiesterase (Pde4) is absent or its activity inhibited, then cAMP synthesized at the slug tip will not hydrolyze to AMP and therefore the gradient will not get established. Using a specific chemical inhibitor of extracellular cAMP phosphodiesterase (Pde4) ,35 Iso bpde4- cells on non-nutrient agar plates. pde4- mound breaks and form tips after 9 hours of development  developed in caffeine containing plates were prominent than the ones developed in the presence of IBMX Figure C. Thus oD. aureostipes (group-1), D. minutum (group-3) and P. pallidum (group-2). Slugs with additional tips show reduced cAMP levels in all the species tested  in the presence of 5 mM caffeine. The fluorescent ecmA-GFP expressing region of the slug was surgically removed and thereafter we checked the expression patterns during multiple tip formation. Faint fluorescent patch of expressing ecmA-GFP was observed 2 hours after removal Figure  and the The proportion of prespore to prestalk cells in the slug is tightly controlled in slime molds  but in cDictyostelium, the differentiation of cell types and its fate is regulated by morphogens which are DIF (Differentiation Inducing Factor), cAMP, ammonia and adenosine [D. discoideum, cAMP acts as a chemoattractant as well as regulating cell differentiation [D. minutum, P. pallidum and D. aureostipes, cAMP is involved in cell differentiation only and not chemotaxis [D. discoideum, adenosine is known to prevent competing tip formation by favouring tip dominance [Polysphondylium the primary tip is known to inhibit secondary tip formation [Polysphondylium goes through a spontaneous spherical to radial symmetry breaking event and during this transition many tip arise around the equator of a spherical mass of cells equidistant from each other suggesting that that one tip inhibits the other [Polysphondylium is akin to caffeine induced multiple tip effect. Multiple tip phenotype induced by caffeine was observed in all the slime molds species we investigated suggesting a conserved mechanism regulating secondary tip formation in slime molds. The formation of secondary tips is controlled by relayed cAMP signal strength and suppression of tip dominance [In denosine . In D. dntiation . Howeveremotaxis ,9,13. Themotaxis . In D. dominance . In the ormation  and surghe other . Probablominance . In thisominance  and in tominance .Dictyostelium genome and hence caffeine could possibly target cAMP phosphodiesterases (PdsA and Pde4) or ryanodine receptor. It is not known if caffeine targets ryanodine receptors in Dictyostelium but caffeine does not affect PdsA levels. It is likely that caffeine by impairing cAMP relay and altering intracellular calcium levels cause pleiotropic effect on signaling, motility and gene expression all leading to multiple tip formation.Few known mechanisms of caffeine action include inhibition of cyclic nucleotide phosphodiesterase, competitive inhibition of adenosine receptors, inhibition of ryanodine receptors  and inac2 it did not result in secondary tip formation (data not shown) the slugs rounded up similar to a mound seen in controls (without drug). Similarly, when slugs were transferred to a plate containing 5 mM EGTA, there was arrested development (data not shown). Studies with caffeine analogs and supplementing calcium with a specific ionophore did not result in multiple tip formation suggesting that an increase of cytosolic calcium alone is not responsible for additional tip effect and a caffeine specific pool of calcium reservoirs is activated during additional tip formation.Theophylline , theobromine  and paraxanthine  are caffeine analogs, sharing a common structural xanthine backbone ,42. Thes(CRAC) and Target of Rapamycin complex-2 (TORC2) [Dictyostelium TOR is a part of both TORC1 and TORC2 so it is likely that caffeine can inactivate both TORC1 and TORC2. TORC2 regulates chemotaxis and multicellular organization by monitoring the expression levels of adenyl cyclase-A (acaA) and cAMP signal relay. The reduced expression of adenyl cyclses and decreased cAMP levels in slugs with additional tips (in the presence of caffeine) can be due to impaired activity of TORC2. However, in the presence of rapamycin, we did not observe multiple tips in the slugs (data not shown). It is not known if rapamycin affects pde4 levels. Also the specific calcium pool mobilized by caffeine may not be responding to rapamycin though both could impair TORC2 activity. Though caffeine and rapamycin are known to induce identical set of genes in yeast [During post aggregate stages of development, the tip of the slug continues to be a source of cAMP signalling . Adenyl  (TORC2) . In yeas (TORC2) . Howeverin yeast , structuIn vitro experiments suggest that PdsA levels are not inhibited by caffeine and so it is likely that Pde4 is a key component responsible for maintaining gradients of cAMP as well as adenosine. cAMP phosphodiesterase (Pde4) hydrolyses cAMP into AMP thus generating cAMP gradient from anterior to the posterior region of the slug. Overproduction of the phosphodiesterase Pde4 is known to arrest development at the tight mound stage, prior to tip formation. During multiple tip formation, cAMP phosphodiesterase (Pde4) levels go down and adenosine levels and cAMP gradient also gets perturbed. Inhibition of cAMP phosphodiesterase (Pde4) activity with iso butyl methyl xanthine (IBMX) results in secondary tips in slugs and fruiting bodies. Though application of IBMX, a proven inhibitor of Pde4 gives rise to multiple tips, the phenotype is prominent with caffeine. Further, many pleiotropic pathways converge to give multiple tip effect whereas IBMX effect may be specific to Pde4 alone.Polysphondylium in the presence of extracellular cAMP suggests the disruption of cAMP gradient leading to tip formation elsewhere in the slug [Adenosine is the breakdown product of cyclic AMP, and known to act as an antagonist to cyclic AMP, providing an intrinsic negative feedback loop ,47. Adenthe slug . PkaC isthe slug . It is n3 and cAMP determines the thickness of the sheath [It is known that a positional signal in isolated prestalk or prespore part induces localized transdifferentiation of the appropriate cell type to form the prestalk and prespore pattern. During secondary tip formation, we observed the prestalk marker EcmA-GFP expression in the prespore region indicating a position–dependent mechanism of regulation. Cells in the prespore region of the slug move in periodic fashion with a speed of 20 μm / minute . If the e sheath . The shee sheath . During Soon after transferring the slugs to caffeine containing plates, the cell migration stops within the slug and impaired cell movement alone may not be responsible for giving the multi-tipped slug. The multiple tip effect gets severe with time suggesting that the phenotype that we observe depends on changes in gene expression also. Prolonged observation with intense beam of light for microscopic observations prevented multiple tip formation in the presence of caffeine (data not shown).P. pallidum slugs, affects fruiting body branching with tertiary branches coming out of the secondary stalks , Council of Scientific and Industrial Research (CSIR), New Delhi, Government of India.Movie 1A. Cell movement in control slug.Click here for fileMovie 1B. Cell movement in slug in the presence of caffeine (during multiple tip formation).Click here for fileFigure S1. Effect of caffeine analogs (5 mM) on secondary tip formation. Theophylline, paraxanthine, theobromine and adenosine were checked for the secondary tip formation in slugs D. discoideum. Slugs in the presence of these compounds culminated except caffeine which induced secondary tip formation. Scale bar = 200 μm.Click here for fileFigure S2. Effect on caffeine and adenosine on TipA and TipB mutants at mound stage. Both compounds showed no response in promoting multiple tips. Scale bar = 200 μm. (PDF 101 kb)Click here for fileFigure S3. Effect of 1 mM caffeine on fruiting bodies of P. pallidum. Scale bar=1000 μm.Click here for file


The interplay between olefin metathesis and dendrimers and other nano systems is addressed in this mini review mostly based on the authors’ own contributions over the last decade. Two subjects are presented and discussed: (i) The catalysis of olefin metathesis by dendritic nano-catalysts via either covalent attachment (ROMP) or, more usefully, dendrimer encapsulation – ring closing metathesis (RCM), cross metathesis (CM), enyne metathesis reactions (EYM) – for reactions in water without a co-solvent and (ii) construction and functionalization of dendrimers by CM reactions. Olefin metathesis reactions –7 have btrans-bis-phosphine complexes [cis-bis-phosphine ruthenium benzylidene catalysts. We therefore used Reetz’s bis-phosphines derived from the commercial polyamine DSM dendrimers [3(CO)12] catalyzed by [FeICp(η6-C6Me6)] leading to the substitution of a carbonyl of the [Ru3(CO)12] by a dendritic phosphine on each tether , 3. Activation of mesitylene by the CpFe+ moiety in 4, followed by a one-pot perallylation yielded [CpFe+][PF6-], 5, from which the free arene derivative 6 was obtained on visible-light photolytic decomplexation , 7,  –33. Thereactions –35. When[+ group –38. Thesticinium –41. The lexation –3542. Fictive CM  43–44].–44.+ grend generation Grubbs metathesis catalyst 7 proceeded selectively to produce dendrimers whose tethers were terminated by carboxylic acid groups 3 and iterations , generated in the catalytic cycle of RCM reactions involving terminal olefins, is usually highly susceptible to dimerization and decomposition in CH2Cl2 or C6H6 [11 is used for the RCM reactions. This strongly argues in favor of dendritic protection (probably by encapsulation) of the reactive species. RCM reactions need less catalyst 7 in organic solvents [11. Thus the hydrophobic dendrimer interior should indeed favor the protection this intermediate ruthenium-methylene species from side reactions occurring in the presence of water.Another key feature of the system is that the aqueous solution of the water-soluble dendrimer Olefin metathesis reactions are powerful methods that can be used for the construction of dendrimers and their functionalization with water-solubilizing carboxylate groups and other termini. In turn, water-soluble dendrimers can be used as molecular micelles as exemplified here. The implication of dendrimers in olefin metathesis reactions has mainly been focused on recovering the catalyst by loading the dendrimer with a functionalized catalyst. This strategy has been of very little success, because the % of catalyst used in metathesis reactions was rather high. This is due to the reactivity of methylene-metal intermediates that leads to side reactions. Consequently, another strategy involves protecting the catalytic intermediate in nanoreactors. Dendrimers are shown here to be excellent reactors achieving the goal of decreasing the catalytic amount when water is used as solvent. The success of using water as a reaction medium, even without co-solvent, is important in avoiding polluting organic solvents. Moreover, a very low Ru catalyst loading is possible in RCM with the fully recyclable water solution of the dendritic nanoreactor.


X-ray repair cross-complementing group 1 (XRCC1) is one of the DNA repair genes encoding a scaffolding protein that participate in base excision repair (BER) pathway. However, studies on the association between polymorphisms in this gene and glioma have yielded conflicting results. This meta-analysis was performed to derive a more precise estimation between XRCC1 polymorphisms  and glioma risk.Data were collected from several electronic databases, with the last search up to November 28, 2012. Meta-analysis was performed by critically reviewing 9 studies for Arg399Gln polymorphism (3146 cases and 4296 controls), 4 studies for Arg194Trp polymorphism (2557 cases and 4347 controls), and 4 studies for Arg280His polymorphism (1936 cases and 2895 controls). All of the statistical analyses were performed using the software programs STATA (version 11.0).The combined results showed that Arg399Gln polymorphism was significantly associated with glioma risk , whereas Arg194Trp/Arg280His polymorphisms were all not significantly associated with glioma risk. As for ethnicity, Arg399Gln polymorphism was associated with increased risk of glioma among Asians , but not among Caucasians. Stratified analyses by histological subtype indicated that the Gln allele of Arg399Gln polymorphism showed borderline association with the risk of glioblastoma among Caucasians. However, no evidence was observed in subgroup analyses for Arg194Trp/Arg280His polymorphisms.Our meta-analysis suggested that Arg399Gln polymorphism was associated with increased risk of glioma among Asians and borderline increased risk for glioblastoma among Caucasians, whereas Arg194Trp/Arg280His polymorphisms might have no influence on the susceptibility of glioma in different ethnicities. Gliomas are the most common type of primary brain tumors, including astrocytomas, oligodendrogliomas, oligoastrocytomas, and glioblastoma, which can be classified into four clinical grades  based on histopathological characteristics and biological behaviors. Among them, the glioblastoma (GBM) is the most frequent and malignant histologic type with worse prognosis DNA damage or reduced DNA repair capacity is viewed as an important mechanism in genetic instability and carcinogenesis caused by ionizing radiation and environmental chemical agents. There are four major DNA repair pathways, including base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR) and double strand break repair (DSBR) A comprehensive literature search was performed using PubMed and EMBASE to identify studies that evaluated the association between XRCC1 polymorphisms and the risk of glioma up to November 28, 2012 with the following terms and keywords: (XRCC1 or ‘X-ray repair cross-complementation group 1’ or ‘DNA repair gene’) and (polymorphism or variant or variation) and (glioma or ‘brain tumor’). The search was limited to human studies. Additional studies were identified by hand searching references in original articles and review articles. The following criteria were used for the study selection: 1) a case–control study evaluating at least one polymorphism in the XRCC1 gene; 2) studies with full text articles; 3) sufficient data for estimating an odds ratio (OR) with 95% confidence interval (CI); 4) genotype distribution of control population must be in Hardy–Weinberg equilibrium (HWE); 5) no overlapping data. If studies had the same or overlapping data, only the largest study should be included in the final analysis.Information was carefully extracted from all the eligible studies independently by three investigators according to the selection criteria listed above. The following data were collected from each study: first author, publication year, country, racial descent , source of controls, genotyping method, numbers of cases and controls, genotype frequency of cases and controls, and the result of Hardy–Weinberg equilibrium test. For conflicting evaluation, a consensus was reached by discussion. We did not define any minimum number of patients for inclusion in our meta-analysis.http://ihg2.helmholtz-muenchen.de/cgi-bin/hw/hwa1.pl) was applied to determine if observed frequencies of genotypes in controls conformed to HWE (P<0.05 was considered significant). Crude ORs together with their corresponding 95% CIs were used to assess the strength of association between these three XRCC1 polymorphisms  and glioma risk for each study. The pooled ORs were performed for additive model , codominant model , dominant model (aa+Aa versus AA), recessive model (aa versus Aa+AA) respectively. Heterogeneity assumption was checked by a chi-square-based Q test I2 statistics was calculated to quantify the proportion of the total variation across studies due to heterogeneity p-value of >0.05 for the Q-test indicated a lack of heterogeneity among studies, so that the pooled OR estimate of each study was calculated by the fixed-effects model (the Mantel–Haenszel method) A Chi-square test using a web-based program Click here for additional data file.