Accumulating evidence suggests that somatic stem cells undergo mutagenic transformation into cancer initiating cells. The serous subtype of ovarian adenocarcinoma in humans has been hypothesized to arise from at least two possible classes of progenitor cells: the ovarian surface epithelia (OSE) and/or an as yet undefined class of progenitor cells residing in the distal end of the fallopian tube.Comparative gene expression profiling analyses were carried out on OSE removed from the surface of normal human ovaries and ovarian cancer epithelial cells (CEPI) isolated by laser capture micro-dissection (LCM) from human serous papillary ovarian adenocarcinomas. The results of the gene expression analyses were randomly confirmed in paraffin embedded tissues from ovarian adenocarcinoma of serous subtype and non-neoplastic ovarian tissues using immunohistochemistry. Differentially expressed genes were analyzed using gene ontology, molecular pathway, and gene set enrichment analysis algorithms.Consistent with multipotent capacity, genes in pathways previously associated with adult stem cell maintenance are highly expressed in ovarian surface epithelia and are not expressed or expressed at very low levels in serous ovarian adenocarcinoma. Among the over 2000 genes that are significantly differentially expressed, a number of pathways and novel pathway interactions are identified that may contribute to ovarian adenocarcinoma development.inconsistent with claims that ovarian surface epithelia cannot serve as the origin of ovarian cancer initiating cells.Our results are consistent with the hypothesis that human ovarian surface epithelia are multipotent and capable of serving as the origin of ovarian adenocarcinoma. While our findings do not rule out the possibility that ovarian cancers may also arise from other sources, they are  Ovarian cancer is the most lethal of all gynecological cancers . In the dedifferentiation origin of adenocarcinomas from more differentiated cells [e.g. endosalpingeal, endometrial and endocervical cells). The lack of morphological similarity between OSE and ovarian adenocarcinomas has led to claims that at least the serous subtype of ovarian adenocarcinoma is not derived from OSE but rather from an as yet to be defined, more differentiated cell residing in the distal end of the fallopian tube [BRCA1 or BRCA2 genes that have been estimated to account for more than 80% of inherited breast and ovarian cancers [in vivo label retention and in vitro clonogenicity [initiating cells [It has been estimated that up to 90% of ovarian adenocarcinomas are derived from ovarian surface (coelomic) epithelia (OSE) ,4. Howeved cells , OSE areian tube -9. Indee cancers . However cancers . For exagenicity . These fgenicity . The facng cells . Indeed,ng cells  and datang cells -18. The ng cells ,20.in vivo and 12 samples of laser capture microdissected cancer epithelia (CEPI) from serous papillary ovarian adenocarcinomas collected from flash frozen tissue. We find that over 2000 genes are significantly differentially expressed between the OSE and CEPI samples. Consistent with a multipotent phenotype, we found that genes previously associated with adult stem cell maintenance are highly expressed in OSE. Pathway analysis implicates key signaling molecules and novel pathway interactions in ovarian cancer development.To test the hypothesis that human OSE retain properties of relatively uncommitted multipotent progenitor cells until undergoing neoplastic transformation, we conducted gene expression profiling analyses on 12 OSE samples collected ® Plus (Medscand), immediately suspended in RNA later (Ambion), and stored at -20°C. Indications for removal of healthy ovaries included other gynecologic pathologies as indicated in Table All tissues were collected by the Ovarian Cancer Institute following approved Institutional Review Board protocols from Northside Hospital and Georgia Institute of Technology, Atlanta, GA. Normal ovarian surface epithelial (OSE) cells were collected from ovaries at time of surgery using a CytobrushRNA was extracted from LCM cells on the MacroCaps in 25 μL of extraction buffer and isolated following PicoPure RNA Isolation Kit (Arcturus) protocols. OSE cells were pelleted from RNAlater and RNA was isolated with Trizol (Invitrogen) and purified with the PicoPure RNA Isolation Kit (Arcturus). RNA quality was verified on the Bioanalyzer RNA Pico Chip (Agilent Technologies).Total RNA from each of the above 24 extractions was amplified using the RiboAmp OA or HS kit (Arcturus) that maintain the original mRNA representation after two amplification rounds, enabling accurate gene expression profiles from ultra small samples. The amplified mRNA was subsequently labeled using the IVT Labeling Kit (Affymetrix), to produce biotin-labeled mRNA suitable for hybridizing to GeneChip Probe Arrays (Affymetrix U133 Plus 2.0).http://www.ncbi.nlm.nih.gov/projects/geo/ under the series number GSE14407. Probe sets that were called absent by default MAS5 criteria in all 24 samples were removed before further processing. Probe set results were further evaluated using Spotfire DecisionSite software Probes were considered differentially expressed if they had a fold change value of ≥ 3 and a p-value < .005 (Student's t-test). This resulted in 2915 probe sets differentially expressed between the twelve OSE and twelve CEPI samples. These probe sets were filtered for redundant gene titles  to yield 2320 unique genes being represented by the probe sets. Groups of .CEL files from previous studies were processed in a similar fashion to identify differentially expressed genes. U133 Plus 2.0 probesets were converted to all possible U95 Set probesets using the ID converter application of Babelomics [http://www.r-project.org/.Affymetrix.CEL files were processed using the Affymetrix Expression Console (EC) Software Version 5.0. Files were processed using the default MAS5 3' expression workflow. All reported microarray data are described in accordance with MIAME guidelines. The processed and raw data files for the 24 samples used in this study have been deposited in the Gene Expression Omnibus (GEO) belomics  for compbelomics . The tweArchival formalin fixed, paraffin embedded tissues from ovarian adenocarcinoma of serous subtype and non-neoplastic ovarian tissues were obtained from the files of Emory University Hospital and Crawford Long Hospital, Atlanta, GA. Emory University's Institutional Review Board (IRB) approved the immunohistochemistry screen of these tissues. The sections (5 microns) were deparaffinized and rehydrated. Antigen retrieval was in citrate buffer (pH 6) using an electric pressure cooker for 5 min at 120°C with cooling for 10 min before immunostaining. All tissues were exposed to 3% hydrogen peroxide for 5 min, primary antibodies for 30 min, DAKO ENVISION system (DAKO Corp) HRP labeled polymer conjugated with secondary antibody for 30 min, diaminobenzidine as chromogen for 5 min and DAKO automated (DAKO AUTOSTAINER) hematoxylin and counterstain for 15 min. Primary antibodies used in this study were anti-ALDH1A2 , LHX9 (Abcam), and SFRP1 (GenWay). All incubations were performed at room temperature. Between incubations, sections were washed with tris-buffered saline (TBS) buffer. Cover slipping was performed using the Tissue-Tek SCA  automatic cover slipper. Slides were scored by a board certified pathologist (S.L.). Slides were photographed with an Olympus C5050 digital camera attached to the optical port of an Olympus BX60 compound microscope.We generated 24 individual gene expression profiles (Affymetrix Human Genome U133 Plus 2.0 Arrays) from 12 OSE brushings and 12 CEPI samples isolated by laser capture microdissection. Relevant histopathologies of the 24 samples are listed in Table To assess the correspondence between our data and other independently gathered ovarian cancer microarray data sets, we compared differentially expressed probes (DEPs) for significant overlaps using the Bonferonni corrected hypergeometric distribution probability. We first compared our DEPs to those generated after performing an identical differential expression analysis  on five normal ovarian surface epithelial brushings and eleven flash frozen bulk tissue serous ovarian cancers that were previously characterized with the Affymetrix Human Genome U95 Set of 3' Expression Arrays . Our anahttp://www.intgen.org/ Expression Project For Oncology  using the Affymetrix Human Genome U133 Plus 2.0 Arrays. Comparison of our OSE versus the exp O bulk tissue data resulted in 9942 DEPs. When compared to our 2915 DEPs, a statistically significant overlap  again was detected.We also compared our DEPs to findings from a differential expression analysis of our OSE to twelve serous papillary ovarian cancer bulk tissue microarrays produced independently by the International Genomics Consortium The DEPs from each of these analyses are presented in Additional file Genomica software package [In order to provide a global view of the biological processes associated with genes differentially expressed between OSE and CEPI, we searched for enrichment of functional annotations within the Gene Ontology (GO) database using the  package . To provCDKN1B) are highly expressed in OSE. In contrast, genes known to be involved in the transition from G1 to the S phase of the cell cycle are highly expressed in CEPI. Examples are the cyclins E1, E2, B2 and A2 , as well as, members of the Origin Recognition (ORC6L) and Mini-Chromosome Maintenance  complexes. The results are consistent with the hypothesis that OSE are arrested in the G0/G1 phase of the cell cycle while CEPI are actively replicating.Figure Pathway Express [With the aid of  Express , we idenTGFB2), the TGFB2 receptors II and III , disabled homolog 2 mitogen-responsive phosphoprotein (Drosophila) (DAB2), bone morphogenic protein 2 (BMP2), as well as, the BMP receptors BMPR1A, and BMPR2 are all significantly reduced in CEPI while SMAD6 (mothers against decapentaplegic homolog 6 (Drosophila)), an inhibitory SMAD [SNAI2) [DCN)[CHRDL1) are also expressed at high levels in OSE [The TGFB/BMP pathway is known to be an important growth inhibitor of epithelial cells . The resory SMAD , is high [SNAI2) -29 is ex [SNAI2) . ExtraceI2) [DCN), and chos in OSE .WNT5A, DAB2, [SFRP1) and secreted frizzled related protein 2 (SFRP2) in OSE indicates that major components of WNT signaling are attenuated on the surface of the ovary. The high expression of WNT7A and several FZD receptors in CEPI alternatively suggests that various components of WNT signaling are activated in CEPI.The WNT (wingless-type MMTV integration site family members) signaling pathway has been implicated in a variety of normal and disrupted developmental processes such as stem cell maintenance , embryonA, DAB2,  secretedZIC1), an activator of WNT signaling [APOE) [ZIC1 in CEPI is significantly correlated with APOE expression (Pearson's r = +0.65).Among the most highly expressed genes in CEPI is Zic family member 1  - a geneg [APOE) . Over-exJAG1) produced by mesothelial cells of the intraperitoneal cavity [NOTCH3 is highly expressed in CEPI and expressed at significantly lower levels in OSE.The complex interactions that exist between signaling pathways are readily evident in our data. For example, the WNT pathway is known to have recurrent and consistent interactions with the NOTCH signaling pathway . The NOTl cavity ,47. NOTCl cavity . Our resIGF2) is up-regulated while SFRP1 and SFRP2 are down-regulated) [Activation of the Hedgehog (Hh) signaling pathway has previously been implicated in multiple cancers ,34. Consgulated) , are likALDH1A2). This gene encodes an enzyme responsible for the conversion of retinaldehyde to retinoic acid - a known marker of lineage specific stem cells [CRABPs) [CYP26A1) [Retinoids are vitamin A-derived morphogens that can directly modulate WNT signaling during normal development . The genem cells . The low[CRABPs) ,55 and cCYP26A1) ,57). TheFigure cis-regulatory motifs, etc.) [In order to provide a comparative gene expression perspective to the differentially expressed genes identified in our study, we employed Gene Set Enrichment Analysis (GSEA) to compare our results to a large collection of previously curated biological experiments  . The Mols, etc.) . Statist-23) to OSE was BOQUEST_CD31PLUS_VS_CD31 MINUS_DN  and differentiated (CD31+) adipose tissue cells [The gene set with the highest GSEA normalized enrichment score , and KIT+  [CD44 is highly expressed in OSE and expressed at significantly lower levels in CEPI, KIT expression was found to be low in all of our samples and thus not differentially expressed. However, the default Affymetrix MAS5 absent/present calls detected the presence of KIT in 5 of our 12 OSE samples but absent in all 12 CEPI samples suggesting that KIT is expressed in OSE. In addition, we found that KIT ligand (KITLG), is highly expressed in OSE and significantly lower expressed in CEPI. These findings indicate that OCSC and OSE express a number of overlapping genes and support the notion that OSE could give rise to OCSC.The cancer stem cell model posits that only a specific subset of a cancer cell population is able to sustain tumor growth . Putativhomolog) ,62. WhilLHX1, LHX2, LHX6 and LHX9) are differentially expressed between OSE and CEPI. LHX2 and 9 are highly expressed in OSE and not expressed in CEPI, whereas LHX1 and LHX6 are expressed in CEPI and not in OSE.The capacity for self-renewal in multiple adult stem cell lineages has been associated with expression of the LIM homeobox genes ,64. Our -64) to CEPI was SERUM_ FIBROBLAST_ CELLCYLE  and have been previously associated with stage III epithelial ovarian cancers [The gene set with the highest positive GSEA normalized enrichment score . The fact that many of the genes highly expressed in OSE have previously been associated with the maintenance of stem cells in a quiescent state is relevant to hypotheses on the cellular origin of ovarian cancer.Under the dedifferentiation hypothesis of cancer development, cancer cells are postulated to be less differentiated than their progenitor cells but often resemble their tissue of origin phenotypically . The facLHX2 and LHX9 are expressed in OSE supports the notion that asymmetric cellular division is occurring in OSE. Progenitor cells can subsequently go through several rounds of cell division before terminating into a mature differentiated cell. Whether or not stem cells self-renew or differentiate is regulated by the microenvironment. A microenvironment that is conducive to stem cell self-renewal is referred to as a stem cell niche [The processes by which stem cells self-renew and differentiate are accomplished by a combination of cellular division strategies known as symmetric and asymmetric division ,70. Symmll niche ,68. Stemll niche . In contThe above description of stem cells and the niches that control their division is relevant to OSE because during the period between ovulations, OSE are quiescent. While arrested cell growth and division are associated with terminal differentiation, at least some OSE must not terminally differentiate because they reactivate their cell cycle and proliferate in response to ovulation. Evidence recently has been presented showing that cells on the surface of the macaque ovary transition from quiescent to a replicating phenotype in response to ovulation . Similarinterovulatory or facultative stem cell niche and this suggests that all or many of the resting cells on the surface of the ovary are not terminally differentiated but arrested in a quiescent state characteristic of adult stem cell populations [facultative stem cell niche is our finding that transcription factors previously implicated in self-renewal and asymmetric division are expressed in OSE [CCNE) in CEPI. It has recently been shown that the constitutive expression of CCNE in mouse embryonic stem cells results in an almost nonexistent G0/G1 phase of the cell cycle [CCNE in CEPI may contribute to cancer growth in a similar fashion.Our results are consistent with the hypothesis that the surface of the ovary is an ulations ,71. We fulations ,71,76,77d in OSE ,78. In cd in OSE -84. Of pll cycle . The lacThe complex molecular processes underlying the onset and development of epithelial ovarian cancer is only beginning to be unraveled. Our results indicate that a number of key developmental pathways are involved in the establishment and development of ovarian cancer. While many of these pathways have previously been either directly or indirectly implicated in ovarian cancer, detailed network analyses of our gene expression data led to the identification of linkages between these pathways attributable to the altered expression of key regulatory genes. We believe that the type of detailed network analyses of gene expression data presented in this paper when combined with next generation sequencing for mutation analyses of individual ovarian adenocarcinoma genomes will help expand our understanding of the origins of ovarian cancer and facilitate the development of more effective therapies.inconsistent with claims that ovarian surface epithelia cannot serve as the origin of ovarian cancer initiating cells.Accumulating evidence suggests that somatic stem cells undergo mutagenic transformation into cancer initiating cells. Our results indicate that OSE express many genes involved in somatic stem cell maintenance. These findings are consistent with the hypothesis that human OSE retain properties of relatively uncommitted multipotent progenitor cells until undergoing neoplastic transformation. The multipotent nature of OSE may contribute to the complex histological subtypes of epithelial ovarian cancer . While our findings do not rule out the possibility that ovarian cancers may also arise from other sources, they are The authors declare that they have no competing interests.LW and LM carried out the microarray experiments. SL performed and interpreted the immunohistochemistry. BB and KT aided in the coordination of the study and the collection of samples for study. NB participated in the design of the study, the analysis and interpretation of the microarray data and drafted the manuscript. JM conceived of the study, participated in its design and coordination and drafted 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/1755-8794/2/71/prepubsupplemental_table_1_2915.xls. differentially expressed Affymetrix probe sets.Click here for filesupplemental_table_2_2320.xls. differentially expressed genes.Click here for filesupplemental_table_3_oci_expo_mda_intersections.xls. OCI, EXPO, and MD Anderson data intersection probe set lists.Click here for filesupplemental_table_4_go_p_05_genomica.xls. Gene Ontology List.Click here for filesupplemental_figure_1.tif. heat map of genes in figure Click here for filesupplemental_figure_1_legend.doc. supplemental_figure_1_legend.Click here for filesupplemetal_table_5_63_overlap_genes.xls. 63 Gene Overlap between OSE gene set and BOQUEST_CD31PLUS_VS_CD31 MINUS_DN gene set from MSigDB.Click here for filesupplemetal_table_6_65_overlap_genes.xls. 65 Gene Overlap between CEPI gene set and SERUM_FIBROBLAST_CELLCYCLE gene set from MSigDB.Click here for file


Panthera leo) coincided with the death of a third of the population, and a second high-mortality CDV epidemic struck the nearby Ngorongoro Crater lion population in 2001. The extent of adult mortalities was unusual for CDV and prompted an investigation into contributing factors. Serological analyses indicated that at least five “silent” CDV epidemics swept through the same two lion populations between 1976 and 2006 without clinical signs or measurable mortality, indicating that CDV was not necessarily fatal. Clinical and pathology findings suggested that hemoparsitism was a major contributing factor during fatal epidemics. Using quantitative real-time PCR, we measured the magnitude of hemoparasite infections in these populations over 22 years and demonstrated significantly higher levels of Babesia during the 1994 and 2001 epidemics. Babesia levels correlated with mortalities and extent of CDV exposure within prides. The common event preceding the two high mortality CDV outbreaks was extreme drought conditions with wide-spread herbivore die-offs, most notably of Cape buffalo (Syncerus caffer). As a consequence of high tick numbers after the resumption of rains and heavy tick infestations of starving buffalo, the lions were infected by unusually high numbers of Babesia, infections that were magnified by the immunosuppressive effects of coincident CDV, leading to unprecedented mortality. Such mass mortality events may become increasingly common if climate extremes disrupt historic stable relationships between co-existing pathogens and their susceptible hosts.Extreme climatic conditions may alter historic host-pathogen relationships and synchronize the temporal and spatial convergence of multiple infectious agents, triggering epidemics with far greater mortality than those due to single pathogens. Here we present the first data to clearly illustrate how climate extremes can promote a complex interplay between epidemic and endemic pathogens that are normally tolerated in isolation, but with co-infection, result in catastrophic mortality. A 1994 canine distemper virus (CDV) epidemic in Serengeti lions ( Epidemics are usually presumed to be caused by single pathogens, a premise that may in some circumstances be overly simplistic. Temporal and spatial convergence of several infectious agents under environmental conditions that favor their transmission and propagation could create a “perfect storm” of pathogens, resulting in significantly greater mortality, as was suspected in the widespread collapse of honeybee colonies Panthera leo) coincided with the death of a third of the populationA 1994 canine distemper virus (CDV) epidemic in Serengeti lions  of CDV infection from the Serengeti (as indicated by negative serum titers) punctuated by short periods with widespread (60–95%) CDV exposure. The duration of these epidemics could only be estimated from the two CDV outbreaks with coincident increases in mortality (6–7 mo in the Serengeti study area in 1994 and 10 wks in Ngorongoro in 2001). Stomoxys fly infestations, and some were clinically dehydrated. All 10 lions had macrocytic normochromic anemia with hematocrits ranging from 18–32% [normal lion ranges being 35–45% ], total red cell numbers ranging from 3.8–4.8×1012/l , and hemoglobin ranging from 7–12 g/l , an anemia consistent with ongoing intravascular hemolysis and not CDV, and a dying cub was observed with bloody urine, consistent with hemolysis from hemoparasitismThe Crater epidemic in 2001 provided a unique opportunity to investigate the linkage between severe drought and CDV-related mortality. Ten anesthetized lions were examined over a 5-day period in the 10-wk span when 24 of the 61 Crater lions died. Five of these 10 lions were in poor physical condition and eight had fevers >39° C, as well as enlarged lymph nodes and spleens. Many had skin ulcers associated with Although nearly 40% of the population was lost during the epidemic, only two lion carcasses were located for necropsy, an adult found dead and a 5-mo old orphaned cub that was killed by hyenas. The adult suffered from severe systemic babesiosis with low numbers of red blood cells in most organs (suggestive of anemia), sludging of parasitized red blood cells in intestinal capillaries , and widBabesia is a tick-borne intraerythrocytic protozoan (hemoparasite) that usually infects African lions at low levels without compromising their health Babesia were noted in blood smears collected from lions. To quantify these hemoparasite infections and determine if levels increased during high-mortality CDV epidemics, we measured hemoparasite burdens by quantitative real-time PCR using primers designed to detect Babesia, Theileria, Cytauxzoon, and Hepatozoon. We assayed a total of 346 blood samples from 301 individuals collected from 1984–2004, comparing hemoparasite levels during CDV epidemics with typical infection levels in these populations. We found that hemoparasite burdens were significantly higher during the high-mortality 1994 and 2001 lion die-offs (n = 6), had hemoparasite levels that were among the highest measured (fold differences of 37–60). Also, the orphaned cub that was killed by hyenas similarly had high hemoparasite levels (fold difference of 34) consistent with the histologic findings of severe babesiosis.die-offs . All of Babesia  and Hepatozoon felis  infection was ruled out as a co-factor because nearly all of the adult Serengeti and Crater lions have been FIV+ each year since at least 1984, and FIV-infection in lions has never been linked with mortalityBabesia co-infection correlated with mortality trends.We then investigated whether the increased load of hemoparasites was due to the introduction of novel pathogens during the fatal CDV epidemics. Four species of on felis  were detBabesia: prides with higher average CDV titers had a greater proportion of individuals with high levels of Babesia , and prides with the highest levels of Babesia suffered the highest mortality rates with many prides suffering >67% mortality   implicate an interactive effect of CDV and  P<0.01) . The onlBabesia hyperinfection during fatal CDV epidemics, because immunosuppression would also have occurred during silent CDV epidemics. Thus the severity of babesiosis during the high fatality epidemics must be attributable in part to the environmental conditions in 1994 and 2001 that strongly favored the propagation of tick vectors for hemoparasites in the lions.In other species, death of CDV-infected animals has been attributed to overwhelming infections with co-pathogens, such as toxoplasmosis, that result from CDV-induced immunosuppressionDiceros bicornis) in the Crater died during 2001 due to fulminating babesiosis that was attributed to the extensive tick infestationsRhipicephalus appendiculatus, Rhipicephalus simus, and Amblyomma hebraeum are shared between wild herbivores and lions B. felis, B. leo, and B. gibsoniBabesia infectivity on the dying buffalo. In support of that hypothesis, hemoparasite levels were significantly higher in lions that fed on buffalo during the 1994 Serengeti die-off than in lions from prides with low buffalo exposure . Among buffalo-eating lions, hemoparasite levels were significantly higher in lions that had been infected with CDV (P = 0.0099), demonstrating the permissive effect of viral immunosuppression. Two prides that were exposed to CDV but did not prey on buffalo, had lower levels of Babesia infection and no increased mortality in 1994, evidence that exposure to buffalo influenced disease outcome in lions.Tick levels in the Serengeti ecosystem during the 1993–4 and 2001 droughts were at unusually high levels due to the general debility of the herbivores and to pasture management in the CraterBabesia in the Crater than Serengeti lions may have resulted from a variety of ecological and genetic factors. The Crater floor supports a high density of large mammalian herbivores and associated carnivores that can serve as hosts to Babesia vectors. Anthropogenic landscape alterations also may have contributed to heavy tick burdens. Grass fire on the Crater floor was actively suppressed from the 1970s until the high tick infestations of 2001Stomoxys flies also flourished in the rains following the Crater drought, causing pruritic skin ulcers on lions similar to those seen during a severe (but undiagnosed) lion die-off in the Crater in 1962Stomoxys flies collected from lions in 2001 and successfully amplified Babesia sp. and Hepatozoon sp., but could not confirm whether the flies served as additional biological Babesia vectors during the epidemic.The higher burden of Babesia infection resulting from climatic events that increased the lions' exposure to tick-infested prey. The combination of high tick exposure and CDV-related immunosuppression caused the hemoparasite infections to become fulminating. If extreme weather events become increasingly frequent owing to global climate change, the consequent synchronization of proliferating pathogens or their vectors may cause disease to become a major threat to historically stable populations that had previously coexisted with multiple viral and parasitic pathogens.Our findings show that high-mortality CDV epidemics coincided with unusually high levels of Blood samples  were collected from African lions  every year between 1984–2004 except 1988 and 1997. Blood was stored either as whole blood in EDTA or as packed red blood cells and serum. All samples were first preserved in liquid nitrogen before being stored at −70°C. Serum was tested for CDV antibodies by a viral neutralization assay using Onderstepoort CDV strain inoculated onto Vero cells.4, embedded in Epon, sectioned at 50 nm, stained with lead citrate and uranyl acetate, and examined on a Phillips 301 electron microscope.Histopathology and immunohistochemistry to detect CDV antigens in tissues were performed by published methods.Babesia sp., Cytauxzoon sp., Theileria sp., and Hepatozoon sp. DNA was isolated from blood samples and flies using a commercially available kit . To quantify levels of selected hemoparasites, generic PCR primers (Bab1: 5′CAG TTG GGG GCA TTC GTA T 3′ and Bab3: 5′ CTC CCC CCA GAA CCC AAA G 3′) were designed that amplified a 246 bp fragment of the 18 s rRNA gene in known Babesia sp., Cytauxzoon sp., Theileria sp., and Hepatozoon sp. Total hemoparasite DNA was quantified by real-time PCR . Reaction mixture (25 µl) contained 2.5 pmol forward and reverse primers and 12.5 µl iQ SYBR Green Supermix (Bio-Rad) used according to manufacturers' directions. PCR conditions were 94°C 5 min; 40 cycles of 94°C for 2 min, 55°C for 30 s, 72°C for 30 s; and a dissociation stage. Appropriate positive  and negative controls were included in all assays. All samples were run in triplicate and threshold values averaged. Assay efficiency is 93.2%.Total hemoparasite DNA was quantified from whole blood or red blood cells (RBC) by real-time PCR using generic PCR primers designed to amplify a 246 bp fragment of the 18 s rRNA gene in Babesia sp. do not have a white blood cell stage, it was biologically relevant to determine relative hemoparasitemia in relation to RBC concentration. As mammalian RBC do not contain nuclei, genes typically used to normalize between samples for quantitative PCR (such as GAPDH) could not be used. Archived blood samples had been frozen, so RBC were lysed and RBC concentrations could not be determined. Therefore, samples were normalized using hemoglobin concentrations  as a proxy for RBC number. Quantification of hemoglobin in frozen whole blood and red blood cell pellets were shown to correlate with hemoglobin concentrations in fresh whole canine blood (data not shown). Relative quantity of hemoparasite DNA was then calculated as average threshold cycle normalized by hemoglobin concentration and expressed as the fold difference greater than the sample with the smallest quantity of hemoparasite DNA.Because 5′ GAG TAG/T CAA TTG GA G 3′ and Bab7: 5′ GGC AAA TGC TTT CGC AGT AG 3′) with a GC-clamp (CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCGCCGC-) attached to the 5′ end of the forward primer to improve separation of closely related sequences. DNA extracts were thawed, and 2.5 µl was added to a 25 µl reaction volume containing standard amounts of GeneAmp reagents , 2.5 pmol of each primer and 1.5 mM MgCl2. Amplification  was performed in a thermocycler (Applied Biosystems model 9700). Amplified products were separated and visualized on a 12% polyacrilamide gel with a 0–40% urea-formamide gradient  run in TAE buffer at 60°C and 300V for 4 ½ hours in a BIORAD D-GENE apparatus . Positive controls , as well as no DNA-negative controls were included with each electrophoresis. DNA was stained with a fluorescent nucleic acid stain  and examined under ultraviolet light. Seventy-four representative bands from 31 lions were sequenced by cutting the band out of the gel and soaking in dH2O overnight, re-amplifying the DNA from the band using a new forward primer (Bab8: 5′ATT GGA GGG CAA GTC TGG 3′) with Bab7 primer (459 bp). Amplicons were purified  and nucleotide sequences of both strands were determined using an automated capillary sequencer  at the University of Chicago Cancer Research Center DNA Sequencing Facility. Sequences were compared to published sequences of confirmed strains in the GenBank database to confirm their identity.To determine if more than one type of hemoparasite was present within individual blood samples, a 477 bp fragment of the 18 s rRNA gene was amplified by PCR using primers  and reverse (Hep2: 5′ CCT CTC TTA TGC TGT TAG AAT TGG 3′) primers. Blood samples with a total fold difference greater than 10 that also had Hepatozoon sp. identified within the sample by DGGE were quantified using this PCR. Amplification conditions were 94°C 5 min; 40 cycles of 94°C for 2 min, 55°C for 30 s, 72°C for 30 s; and a dissociation stage. Positive controls were included with each plate to standardize across reactions, and no DNA-negative controls were included in all assays. All samples were run in triplicate and threshold values averaged. Assay efficiency was 96.3%.To determine the extent to which total hemoparasite loads were due to Mycoplasma sp. were calculated using previously published methodsRelative quantities of Percent bone marrow fat is directly related to the percent dry weight of marrow measured from earlier calibrations using Soxhlet fat extraction. Long bone marrow samples were collected, dried, and the % marrow fat calculated by this method. The details of these techniques are provided in Sinclair & Arcese


Chlamydia is an obligate intracellular pathogen that causes a wide range of diseases in humans. Attachment and entry are key processes in infectivity and subsequent pathogenesis of Chlamydia, yet the mechanisms governing these interactions are unknown. It was recently shown that a cell line, CHO6, that is resistant to attachment, and thus infectivity, of multiple Chlamydia species has a defect in protein disulfide isomerase (PDI) N–terminal signal sequence processing. Ectopic expression of PDI in CHO6 cells led to restoration of Chlamydia attachment and infectivity; however, the mechanism leading to this recovery was not ascertained. To advance our understanding of the role of PDI in Chlamydia infection, we used RNA interference to establish that cellular PDI is essential for bacterial attachment to cells, making PDI the only host protein identified as necessary for attachment of multiple species of Chlamydia. Genetic complementation and PDI-specific inhibitors were used to determine that cell surface PDI enzymatic activity is required for bacterial entry into cells, but enzymatic function was not required for bacterial attachment. We further determined that it is a PDI-mediated reduction at the cell surface that triggers bacterial uptake. While PDI is necessary for Chlamydia attachment to cells, the bacteria do not appear to utilize plasma membrane–associated PDI as a receptor, suggesting that Chlamydia binds a cell surface protein that requires structural association with PDI. Our findings demonstrate that PDI has two essential and independent roles in the process of chlamydial infectivity: it is structurally required for chlamydial attachment, and the thiol-mediated oxido-reductive function of PDI is necessary for entry. Chlamydia is a large burden on global health. It is the most common cause of infectious blindness, and the CDC (Centers for Disease Control and Prevention) estimates that in the United States alone there are more than 2 million people with sexually transmitted Chlamydia infections. Chlamydia is an obligate intracellular bacteria; thus, attachment and subsequent invasion of cells are key steps in Chlamydia pathogenesis. While strides have been made in understanding the molecular mechanism of Chlamydia infection, fundamental aspects of this process still remain elusive. We have identified a host protein, protein disulfide isomerase (PDI), that is essential for Chlamydia attachment as well as for entry into cells. Cell-surface PDI-mediated disulfide reduction is required for Chlamydia entry into cells, whereas bacterial attachment is independent of PDI enzymatic activity. Although PDI is necessary for Chlamydia attachment, the bacteria apparently does not bind directly to cell-associated PDI, suggesting that Chlamydia attaches to a host protein(s) associated with PDI. This study advances our understanding of Chlamydia pathogenesis by the characterization of a host factor essential for independent stages of bacterial attachment and entry. Fundamental to understanding of intracellular bacterial pathogenesis is knowledge of the mechanism of bacterial attachment and subsequent entry into cells. There are two main processes by which bacteria stimulate their entry into nonphagocytic cells: by bacterial contact mediated activation of a cell surface receptor (the “zipper” mechanism) or by injecting bacterial proteins into the cell cytosol (the “trigger” mechanism) Chlamydia is an obligate intracellular bacteria that can infect a number of different eukaryotic cells. Human chlamydial infection causes a wide range of pathologies. Chlamydia is the most common bacterial sexually transmitted disease Chlamydia infects cells as a metabolically inactive elementary body (EB) and then once within cells differentiates into the metabolically active but noninfectious form known as the reticulate body (RB). The EB are small (0.3-µm) and have a rigid outer membrane consisting of a mesh of disulfide cross-linked cysteine-rich proteins Chlamydia attachment as infectivity is lost following mild trypsin treatment of cells Chlamydia attachment. However, only one mammalian protein, PDI, has been demonstrated to be involved in attachment of multiple species and serovars of ChlamydiaAttachment and entry into cells are key steps in chlamydial development and pathogenesis, yet the mechanism governing these interactions is still unknown. A number of bacterial ligands, including the major outer membrane protein Chlamydia infection was originally elucidated by proteomic analysis of CHO6 cells. CHO6 cells were generated by chemical mutagenesis of Chlamydia-susceptible CHOK1 cells and the mutant cells are resistant to attachment of C. trachomatis, C. pneumoniae, and C. psittaciThe role for PDI in PDI is a multi-functional protein: it catalyzes the reduction, oxidation, and isomerization of disulfide bonds, it can act as a chaperone or anti-chaperone Chlamydia infectivity. We have determined that although cellular PDI is required for both Chlamydia attachment and entry the requirement is mechanistically different in the two processes. PDI cell surface enzymatic activity was necessary for entry of bacteria into cells. In contrast Chlamydia attachment to host cells required PDI but was independent of cell surface PDI enzymatic activity.It is known that PDI is involved in chlamydial infection ChlamydiaChlamydia attachment is unknown. PDI is essential for cell viability, thus gene disruption approaches cannot be used to test if PDI is necessary for Chlamydia attachment or if additional mutations play a role in the phenotype of the CHO6 cell line. In contrast, siRNA has been used to transiently knockdown expression of PDI in mammalian cells Chlamydia infectivity. HeLa cells were transfected with PDI-targeting siRNA and PDI knockdown was assessed by immunoblot. Approximately 80–90% reduction in PDI expression was achieved  as cell surface PDI enzymatic activity is required for DT-mediated killing of cells. PDI reduces interchain disulfide bonds in DT triggering chain separation that then allows for translocation of DT chain A across the membrane and subsequent toxin-induced cell death Chlamydia infectivity.Prior to examination of Chlamydia attachment as cells transfected with native PDI phosphine hydrochloride) was then added and the cells were incubated at 37° C for 2 h. The addition of TCEP was able to overcome bacitracin inhibition of Chlamydia entry, confirming that the stimulus necessary for bacterial entry into cells is a PDI-mediated reduction occurring at the cell surface  was analyzed. Chlamydia are able to attach to CHO6 cells expressing PDI-4CS but the bacteria are unable to enter  is necessary for DT mediated cell death observed . These rChlamydia to PDI, but the possibility that the gpi-anchor was causing a structural change in PDI that led to the lack of bacterial attachment could not be ruled out. To address this possibility the effect of polyclonal PDI-specific antibody of Chlamydia attachment was analyzed. The effect of antibody to PDI on Chlamydia infection has previously been evaluated by Davis et al.Chlamydia infection. We further developed those findings by specifically addressing inhibition of bacterial attachment as well as entry. When PDI antibody was present prior to and during bacterial attachment no significant change in the number of bacteria attached to cells was observed  that is associated with PDI.A polyclonal PDI antibody was used in these experiments, and it is likely that this antibody would inhibit PDI enzymatic activity by steric interference. To directly assess the level of PDI enzymatic activity a turbidimetric assay of insulin disulfide reduction was performed . We deteChlamydia entry. Independent of PDI enzymatic function, PDI was additionally essential for bacterial attachment to host cells.Cell surface PDI-mediated disulfide bond reduction is involved in the infectious entry of a number of viruses. Upon binding of human immunodeficiency virus (HIV) envelope protein to CD4 receptor and co-receptor (CCR5 or CXCR4) PDI reduces disulfide bonds in HIV gp120, exposing the gp41 fusion peptide et al.et al.C. trachomatis serovar E. These results can now be understood in terms of the direct enzymatic role for PDI in bacterial entry. Raulston et al.et al.et al.et al.et al.et al.et al.Chlamydia.The role for protein disulfide exchange in chlamydial infection has been previously explored using inhibitors. Davis Chlamydia attachment is independent of cell surface PDI enzymatic activity. Testing of gpi-anchored PDI tethered to the cell surface suggested that chlamydiae do not directly bind PDI as the sole target for attachment and this implicates an interaction with other proteins that require PDI. Consistent with characterized functions of PDI, it may be PDI's function as a chaperone or structural component of a host protein or protein complex Chlamydia binds may be a multiprotein complex that includes PDI.A requirement for PDI in chlamydial attachment was implicated by complementation of the PDI gene in attachment-deficient mutant CHO6 cells C. trachomatis L2 EB prior to infection leads to a decrease in inclusion forming units In addition to a requirement of PDI for chlamydial attachment, it was shown that PDI enzymatic activity was necessary and sufficient to stimulate chlamydial entry into the host cell. Given the ability of simple chemical reduction to replace the enzymatic function of PDI for cell-adherent chlamydiae, it appears that reduction rather than disulfide exchange is minimally required for chlamydial entry. It is not known whether PDI functions to reduce a host or bacterial component to initiate entry. It has been previously shown that reduction of 2β1 and L-selectin resulting in receptor activation and ligand binding One can speculate that following initial bacterial attachment, PDI enzymatic activity mediates the establishment of a functional contact between the chlamydial organism and the host cell leading to bacterial uptake. It is known that plasma membrane PDI interacts with a number of host surface proteins. For example, PDI modifies the adhesion receptors integrin αChlamydia infectivity, these include epithelial membrane protein 2 Chlamydia-host cell receptor interactions. PDI is involved in protein folding in the endoplasmic reticulum and PDI expression can be correlated to the level of secretion of a number of proteins Multiple cell surface proteins have been previously implicated in Chlamydia type III secretion into the cell membrane and subsequent Tarp translocation. The type III secretion system needle protein of C. trachomatis, CdsF, was recently identified Alternative to acting on a host protein, PDI could be targeting the chlamydial organism. The highly disulfide cross-linked structure of the chlamydial EB surface proteins that are only present in the infectious form of the chlamydial organism Chlamydia-host cell interaction remain enigmatic these findings illuminate important new details of the molecular mechanisms involved. Chlamydia attachment and entry into cells are separable processes that both have a unique requirement for PDI. Determination of the target of PDI enzymatic activity that leads to chlamydial entry may be expected to provide targets for generation of new anti-Chlamydia therapies and illuminate fundamental cell biological processes exploited by Chlamydia to mediate pathogen attachment and infectivity.While the full intricacies of the 2. C. trachomatis L2/434/Bu EB and C. psittaci PF6 BC were purified from L929 cells on 30% and 30–44% discontinuous Renografin gradients  CHOK1 and CHO6 cells were maintained in RPMI 1640  supplemented with 10% heat-inactivated fetal bovine serum (FBS) , 2 mM glutamine (Invitrogen), and 1 mM HEPES (Invitrogen). HeLa and L929 cells were maintained in RPMI 1640 supplemented with 10% FBS. Cells were grown at 37°C in an atmosphere containing 5% CO5 cells were plated on 12 mm glass coverslips in 24 well plates. Cells were washed with Hanks buffered saline solution (HBSS) (Invitrogen) and then incubated with C. trachomatis L2/434/Bu or C. psittaci PF6 BC in RPMI 1640 with 10% FBS for 1 h at 24°C 2. For entry analysis cells were fixed with methanol or with 4% paraformaldehyde, for infectivity assessment cells were fixed with methanol. Following fixation coverslips were incubated 15 min in blocking solution ) . Blocking solution was removed and coverslips were incubated 1 h with mouse anti-Chlamydia MOMP antibody for C. trachomatis or anti-Chlamydia LPS antibody  for C. psittaci and then washed with HBSS. The wash was followed with a 30 min incubation with goat anti-mouse AlexaFluor 488 (Invitrogen) and Evans Blue. Coverslips were mounted in VectaShield Hard Set mounting media (Fisher) and evaluated on a Nikon Eclipse E800 microscope. When used bacitracin  was added 20 min prior to infection at 3 mM, infection was performed in media containing 3 mM bacitracin and following infection cells were maintained in culture media with 3 mM bacitracin. When used 50 mM TCEP  in cell culture media was added following bacterial attachment. For attachment and entry inhibition analysis cells were incubated for 20 min with polyclonal rabbit anti-PDI antibody  or goat anti-bovine IgG antibody , the bacterial inoculum was then added to the antibody containing media and cell were incubated for 1 h at 24°C. When Stressgen rabbit anti-PDI antibody was used for PDI visualization, PDI staining was performed similarly to Chlamydia staining and goat anti-rabbit AlexaFluor 594 (Invitrogen) was used as a secondary.On the night prior to analysis 1×10Chlamydia but were larger due to aggregation were enumerated separately when separate organisms could be discerned or when distinction was not possible were counted as one to provide a conservative estimate of bound organisms per cell.For quantification the number of cell-associated apple-green fluorescing particles of size and shape consistent with 300 nM organisms were counted in two planes in 8 separate fields of view containing at least 10 cells. Fluorescent particles that appeared to be 5′ - TGC CCC GTG GTC TGG CCA CTC CAA AGC TCT GG - 3′), Cys-55,58-Ser R (5′ - CCA GAG CTT TGG AGT GGC CAG ACC ACG GGG CA - 3′), Cys-399,402-Ser F (5′ - TAT GCC CCC TGG TCT GGC CAC TCC AAG CAG CT - 3′), and Cys-399,402-Ser R (5′ - AGC TGC TTG GAG TGG CCA GAC CAG GGG GCA TA - 3′). The gpi-anchor for PDI was amplified from human folate receptor 1 using primers GPI F (5′ - ATT GCC CGG GGC TGC AGC CAT GAG TGG - 3′) and GPI R (5′ – CAA TGT CGA CTC AGC TGA GCA GCC ACA GCA – 3′). The gpi-anchor was than ligated to PDI lacking a stop codon and the PDI-gpi construct was ligated into the pBICEP-CMV-1 vector. PDI vectors were transfected into CHO cells with Effectene  following manufacturer's instructions. Chlamydia attachment and entry were evaluated 48 h after transfection.The gene encoding protein disulfide isomerase was cloned from CHOK1 cDNA and inserted into the expression vector pBICEP-CMV-1 (Sigma) yielding PDI with a N-terminal FLAG tag. PDI protein lacking enzymatic function (PDI-4CS) was generated by mutating 4 essential cysteine residues to serine with the Stratagene  QuikChange Kit and primers Cys-55,58-Ser F (5′- GAC CTC CCC TTC AAA GTT GTT –3′)  PDI was silenced in HeLa cells with PDI-specific siRNA  (0 or 100 ng ml−1) in HBSS containing 15% FBS. Following incubation cells were washed three times with HBSS and were than maintain in RPMI 1640 without phenol red supplemented with 10% FBS, 2 mM glutamine, and 1 mM Hepes at 37°C in an atmosphere containing 5% CO2. 72 h after toxin treatment cell viability was determined by Promega  CellTiter 96 Aqueous One Cell Proliferation Assay as directed by the manufacturer's instructions. Cell viability is linearly proportional to absorbance.24 h prior to toxin treatment cells were plated in 96-well plates at 1×10Chlamydia per cell was determined using a quantitative PCR based method previously described by Hybiske and Stephens, C. trachomatis was isolated using the High-Pure PCR template preparation kit . Purified genomic DNA was used as a template in quantitative PCR to determine the relative levels of chlamydial (16S) and CHOK1 (ß-globin) genomic equivalents.The number of http://www.ncbi.nlm.nih.gov/sites/entrez?db=protein) accession number for genes discussed in this paper are Homo sapiens PDI (NP_006840) and Cricetulus griseus PDI (AAM_00284).The NCBI Entrez . Bacterial infection was analyzed 24 h after initial inoculation. In cells cultured without bacitracin, the development of a productive infection, as indicated by the presence of large bacteria containing vacuoles, was observed (bottom panel). When bacitracin was present throughout the course of the infection, the bacteria remained persistently attached to cells and no productive infection occurred (bottom panel). These results are similar to our analysis of the effect of bacitracin on Chlamydia infection of CHOK1 cells (Bacitracin does not affect K1 cells .(2.88 MB TIF)Click here for additional data file.Figure S2Polyclonal PDI antibody inhibits PDI enzymatic activity. PDI enzymatic activity was evaluated by measuring the rate of insulin reduction spectrophotometrically at 650 nm as turbidity formation from the precipitation of the insulin B chain following insulin reduction. A control reaction (Control) was performed without PDI. A reaction with PDI (PDI) and with PDI preincubated with PDI-specific antibody (PDI+Ab) was also done. Readings were taken every 5 min for 1 h. Error bars indicate the standard of deviation from three separate experiments performed on the same day.(0.57 MB TIF)Click here for additional data file.Protocol S1Supporting Protocol(0.03 MB DOC)Click here for additional data file.


Patients with UICC stage II colorectal cancer (CRC) have a risk of approximately 20% to develop disease recurrence after tumour resection. The presence and significance of micrometastases for locoregional recurrence in these patients lacking histopathological lymph node involvement on routine stained HE sections is undefined. Oestrogen receptor (ER) promoter methylation has earlier been identified in CRC. Therefore, we evaluated the methylation status of the ER promoter in lymph nodes from 49 patients with CRC UICC stage I and II as a molecular marker of micrometastases and predictor of local recurrence. DNA from 574 paraffin-embedded lymph nodes was isolated and treated with bisulphite. For the detection of methylated ER promoter sequences, quantitative real-time methylation-specific PCR was used. Of the 49 patients tested, 15 (31%) had ER methylation-positive lymph nodes. Thirteen of those (86%) remained disease free and two (14%) developed local recurrence. In the resected lymph nodes of 34 of the 49 patients (69%), no ER promoter methylation could be detected and none of these patients experienced a local relapse. The methylation status of the ER promoter in lymph nodes of UICC stage I and II CRC patients may be a useful marker for the identification of patients at a high risk for local recurrence. Colorectal cancer (CRC) is the third most common cancer in western countries with an incidence of 45–60 per 100 000 per year in Europe and the United States  were available for analysis. Case-specific clinicopathological and experimental parameters, including the number of lymph nodes analysed by rt-MSP and clinical follow-up, are given in Table 2.μm) and one section (3 μm) for HE staining. The lymph nodes were manually microdissected and pooled for DNA isolation to yield one sample per patient. Tissue sections were deparaffinised in xylene and rehydrated using ethanol in decreasing concentrations. Rehydrated tissue sections were digested using proteinase K dissolved in TE buffer diluted at 1 : 10 in distilled water. Genomic DNA was isolated using a commercially available DNA extraction kit . The DNA concentration of each patient sample was measured  with a mean concentration of 157 ng μl−1 (±82 ng μl−1). Two micrograms of each DNA sample were treated with sodium bisulphite converting unmethylated cytosine to uracil and leaving methylated cytosine intact, as described by μl H2O.Six serial sections were used for DNA isolation  and negative controls (MCF-7 cell line). The cell lines used have been well characterised regarding the methylation status of their ER promoter (ACTB. Dilution series showed linearity of amplification down to 1 : 10 000 for rt-MSP for methylated ER promoter as well as for ACTB.Two microlitres of the eluate containing the bisulphite-treated DNA has been used for each rt-MSP. Amplifications were carried out in 96-well plates in a 7000 Sequence detector (Applied Biosystems) in triplets using Universal Mastermix® (Applied Biosystems). The reaction volume was 25 n=37) and pT4pN0 (n=1) tumours, respectively. In all, 9 (18%) patients had cancer of the rectum, 12 (24%) of the sigmoid colon, 7 (14%) of the transverse and descending and 21 (43%) of the ascending colon, respectively. At the last follow-up as of December 2006, the average follow-up was 64 months (range 47–79). Two patients with rectal cancer had received adjuvant radiochemotherapy.To validate our approach and to confirm the findings by Ten of 49 patients died during the follow-up period, three from disease recurrence, one from an accident and six from cardiovascular disease. In total, four patients developed recurrence . One patient developed local and distant recurrence, one patient developed solely local recurrence and two patients developed solely distant metastases. Patients who died and did not develop tumour recurrence were treated as censored by statistical survival analysis.Both patients with local recurrence were positive by rt-MSP . They had well and moderately differentiated tumours of the rectum and sigmoid, respectively, and both had been staged as pT3 pN0 cM0 (UICC stage II). In both patients with local recurrence, six and seven lymph nodes had been examined. Local recurrence was diagnosed 11 and 33 months after initial diagnosis. Both patients died of their tumour recurrence.Of the 49 patients tested, 15 (31%) had ER methylation-positive lymph nodes. Thirteen of those (86%) remained disease free and two (14%) developed local recurrence. In the resected lymph nodes of 34 of the 49 patients (69%), no ER promoter methylation could be detected and none of these patients experienced a local relapse. Of the 13 patients with positive ER promoter methylation in their resected lymph nodes and a disease-free follow-up, three had CRC UICC stage I and 10 UICC stage II. Regarding age, gender, tumour localisation, UICC stage, grading and the number of resected lymph nodes, there were no statistically significant differences between the patients with and without positive lymph nodes detected by ER promoter methylation.None of the rt-MSP-negative patients (0 out of 34) developed local recurrence , but 2 out of 34 (6%) rt-MSP-negative patients developed metastases in the liver and lung without evidence for lymph node or local recurrence. In these patients there was no methylation of the ER promoter in the lymph nodes removed at surgery. In 27 out of the 49 patients with CRC UICC stage I/II, MSI status, as previously assessed ; 7 out oAccording to clinical staging systems , the presence of lymph node metastases at the time of diagnosis predicts a poor prognosis for CRC patients. But even among patients with lymph nodes negative by histopathologic criteria (pN0), 10–20% will develop local tumour recurrence, which has been attributed to micrometastases outside the resected lymphatic drainage area. The most sensitive methods to detect micrometastases are immunohistochemistry (IHC) and nucleic acid-based methods. Only few studies could show micrometastases detected by IHC in lymph nodes of UICC stage I and II patients to correlate with disease recurrence and survival (, who found CK-positive cells on histological examination exclusively on the outside of lymph nodes in almost 30% of RT–PCR-positive specimens. Other markers, including CEA, MUC2, GCC, matrilysin, β-HCG and others, have been explored to detect lymph node micrometastases by RT–PCR and yielded inconsistent results (In addition, there have been several reports on the detection of CK mRNA by RT–PCR. Some studies showed a correlation between CK 20 expression detected by RT–PCR and disease recurrence as well as overall survival, whereas other studies did not (Rosenberg The high stability of DNA compared with RNA is one of the main advantages of DNA-based detection methods, which therefore can be applied to the analysis of paraffin-embedded tissues. We demonstrated rt-MSP of DNA from paraffin-embedded lymph node tissue as a robust and practicable method to detect micrometastases.The high negative predictive value was achieved, because none of the 33 patients negative for ER promoter methylation in their lymph nodes developed local recurrence. On the other hand, not all MSP-positive patients developed local recurrence during follow-up. This limitation might in part be explained by a too short follow-up period, the heterogeneity of the study population in terms of the number of investigated lymph nodes or other molecular factors, respectively. The small number of recurrences is another limitation that does not allow a statistical multivariable analysis and results in a low positive predictive value.Molecular factors have recently been shown to predict disease outcome as distinct genetic subtypes of CRC are defined (Another reason for the high rate of detected micrometastases by rt-MSP compared with recurrences might be the high sensitivity of rt-MSP, which may detect single tumour cells in a lymph node that might not have the potential to develop to a clinically relevant tumour. This obviously raises the question of the clinical significance of micrometastases for the individual patient. However, the most likely explanation for the lower positive predictive value seems to lie in a potentially complete resection of all micrometastases bearing lymph nodes at surgery.Lymph node and distant metastases represent two different routes of CRC progression. Whereas the first is probably due to a spread of tumour cells through the local lymph system, the latter arises from circulating tumour cells. Histopathological lymph node involvement is a predictor of disease recurrence. Generally, the route of tumour spread is dependant on the overall tumour load. This interpretation is supported by the close correlation between the quantitative level of CEA transcripts in serum and tumour stage (In summary, rt-MSP for ER promoter methylation may represent a sensitive and robust method to identify patients with UICC stage I and II CRC at risk for local recurrence. Positive patients have a risk for local recurrence similar to UICC stage III.


Japanese encephalitis virus (JEV) genotypes in Thailand were studied in pigs and mosquitoes collected near houses of confirmed human JEV cases in 2003–2005. Twelve JEV strains isolated belonged to genotype I, which shows a switch from genotype III incidence that started during the 1980s. The origin of Japanese encephalitis virus (JEV) was recognized before 1935, and JEV was isolated in Japan in 1935. The virus has since spread from India to Indonesia and within the past 3 decades has reached previously unaffected parts of Asia and northern Australia  and envelope (E) genes, 5 virus genotypes have been identified, including genotypes I to III . These have been found distributed all over southern Asia; a GIV strain was isolated from eastern Indonesia, and an isolate originating in Malaysia may represent a fifth genotype  of JEV incidence in 7 provinces representative of the 4 regions of Thailand . Pig farms and rice fields within a 2-km radius around houses of confirmed human cases of Japanese encephalitis were targeted for sample collection. Ten healthy sentinel piglets (10 weeks of age) were surveyed in each province, and blood samples were collected weekly for 14 weeks. Adult mosquitoes were collected on a monthly basis according to the targeted pig farm and availability of breeding sites for vectors  by usingFifty microliters each from pig serum specimens and from filtered suspension of crushed mosquitoes were used for virus isolation on C6/36 cells. We tested JEV propagation by immunofluorescent assay. RNA extraction was done from a supernatant of JEV-positive cell culture, after first passage, according to manufacturer’s protocol as well as RNA reverse transcription–PCR (RT-PCR) . RT-PCR was performed on 4 μL of cDNA template by using 2.5 units of AmpliTaq Gold DNA Polymerase . Overlapping JEV E gene fragments were amplified with 2 sets of primers: Ea forward primer (5′-ATA GTA GCT ATG TGT GCA AAC AAG G 5-3′), Ea reverse primer (5′-GAA TTC RGT YGT GCC YTT CAG AGC-3′); and Eb forward primer (5′-AGC TCA GTR AAG TTR ACA TCA GG-3′), Eb reverse primer (5′-GAA TTC AAT GGC ACA KCC WGT GTC-3′), respectively .Twelve JEV strains were isolated, 3 from mosquitoes in 2003 and 10 from pigs in 2004 and 2005. The new JEV sequences were analyzed with a group of 22 previously published JEV strain sequences, including 6 from Thailand and 16 from other Asian countries. A phylogenetic tree was generated, and all 12 JEV new isolates fit into the same GI cluster, as did 3 other Thai strains previously isolated in 1982, 1984, and 1992 . Eleven GI strains appeared to cluster phylogenetically but not geographically, which suggests virus strains were transported over noncontiguous domains at variable geographic distances. Major environmental changes have occurred since the early 1950s with the increase in local and international transportation systems. Some researchers (Although GIII strains were historically reported to circulate mostly in northern Thailand in the early 1980s, GI and GIII were found co-circulating from the north to the south; thereafter, only the GI strain was isolated in Thailand (In Thailand, the epidemiologic pattern of Japanese encephalitis first showed a visible decline in incidence with the development of immunization programs, but this decline also corresponded to the late 1980s when the practice of raising pigs in the backyard evolved into industrialized pig farming and the high rate of piglet seroconversion showed an intense virus circulation. The dramatic increase of industrial pig farming and trading must have played a major role in the dispersion of JEV genotypes within past decades in Asia. Concurrently with pig farming, the culicid main vectors have changed (


To further elucidate the potential immuno-modulatory functions of flavanol-rich cocoa, the present investigation examined whether isolated CFP fractions (monomers through decamers) influence the secretion of tumor necrosis factor-α (TNF-α) from resting and phytohemagluttinin (PHA)-stimulated human peripheral blood mononuclear cells (PBMC). We used an in vitro culture system where PBMC from 14 healthy subjects were introduced to individual CFP fractions for 72 h prior to measuring the levels of TNF-α released. The intermediate-sized CFP fractions (tetramers through octamers) were the most active on resting cells, causing a 3–4 fold increase in TNF-α relative to media baseline. The monomers and dimers were the least stimulatory of the fractions tested, displaying a 42 and 31% increase, respectively, over media control, whereas the trimers, nonamers and decamers showed an intermediate stimulation of this cytokine. In the presence of PHA, the intermediate-sized CFP fractions again were the most active, enhancing TNF-α secretion in the range of 48–128% relative to the PHA control. The monomers and dimers were slightly inhibitory , while trimers, nonamers and decamers stimulated moderate increases in TNF-α levels . The above results lend support to the concept that CFP can be immunomodulatory. The stimulation of TNF-α secretion may contribute to the putative beneficial effects of dietary flavanoids against microbial infection and tumorigenesis.Epidemiological reports have suggested that the consumption of foods rich in flavonoids is associated with a lower incidence of certain degenerative diseases, including cardiovascular disease. Flavanols and their related oligomers, the procyanidins CFP, isolated from cocoa can modulate the production and level of several signaling molecules associated with immune function and inflammation


P < 0.001). With the exception of the putative alternate splice site, aberrant transcripts were not detected in matched normal mucosa. These results suggest that members of the HIT family of genes are only selectively involved in tumorigenesis and that perturbation of FHIT gene expression is an early event in colorectal tumorigenesis. © 1999 Cancer Research CampaignForty-five colorectal adenocarcinomas were examined for alterations in the HIT family genes FHIT and PKCI-1/HINT by a combination of reverse transcriptase polymerase chain reaction and DNA sequencing. In all cases a single transcript corresponding to the reported sequence was detected using primers specific for the PKCI-1/HINT gene. In contrast multiple transcripts were detected using primers specific for the FHIT gene transcript. 6% (3/45) of tumours evinced no detectable expression of any FHIT transcript and a further 12% (6/45) produced only the normal full length transcripts. Ninety-six aberrant transcripts were characterized from the remaining tumours. Deviations from the normal full length sequence characterized included deletions, insertions of novel sequences, a point mutation as well as the usage of a putative alternate splice site in exon 10. Message variants were detected with approximately equal frequency in all tumour stages with the exception that templates with insertions were found solely in Dukes’ stage B tumours (


Immunological tolerance or functional unresponsiveness to a transplant is arguably the only approach that is likely to provide long-term graft survival without the problems associated with life-long global immunosuppression. Over the past 50 years, rodent models have become an invaluable tool for elucidating the mechanisms of tolerance to alloantigens. Importantly, rodent models can be adapted to ensure that they reflect more accurately the immune status of human transplant recipients. More recently, the development of genetically modified mice has enabled specific insights into the cellular and molecular mechanisms that play a key role in both the induction and maintenance of tolerance to be obtained and more complex questions to be addressed. This review highlights strategies designed to induce alloantigen specific immunological unresponsiveness leading to transplantation tolerance that have been developed through the use of experimental models. Classic experiments reported by Billingham, Brent and Medawar more than 50 years ago  demonstrExperimental models have proven extremely useful for the field of transplantation to progress to its current state; however, it is necessary to revisit these models to investigate methods of tolerance induction in situations that are more relevant to the clinical setting. This review discusses various methods utilized in experimental models to achieve alloantigen-specific immunologic unresponsiveness as well as the future direction of experimental models in the field of transplantation.It is well known that the thymus is essential for both T-cell maturation and the induction of tolerance to self antigens. During maturation, thymocytes are positively selected provided that their TCR has a ‘low’ intrinsic affinity for self MHC. The process of negative selection results in the elimination of thymocytes that react to self-MHC+ peptide with too high an avidity. The aim of these two processes is to ensure that mature T cells emigrating from the thymus will be able to recognize and respond to peptides derived from the external environment bound to self MHC molecules when they are presented in the periphery but unable to respond to self peptides presented by MHC molecules of the host. Two of the ways in which this natural process of deletion of self reactive T cells has been exploited to try and induce tolerance to donor alloantigens are described below.et al. who confirmed that the thymus was a suitable transplant site. This study, in the rat model, demonstrated the principle that intrathymic injection of allogeneic islets together with lymphocyte depletion in the periphery reversed diabetes and induced normoglycaemia [One approach to achieve central deletion of donor antigen reactive T cells has involved intrathymic administration of alloantigen. Studies using this approach were initiated in the 1960s and developed further by Posselt lycaemia . Limitinlycaemia . Since tlycaemia ; howeveret al. investigated the safety and tolerability of an intrathymic injection of donor splenocytes peri-operatively [In a clinical study, Remuzzi ratively . Prelimiet al. revealed that irradiated mice reconstituted with a mixture of T-cell depleted host and donor bone marrow accepted donor skin grafts permanently, rejected third party grafts and did not develop graft versus host disease (GVHD) [Early work by Sachs e (GVHD) . The suce (GVHD) .The requirement for pretransplant host conditioning with sub-lethal irradiation and/or myeloablative agents have limited the development and clinical application of this approach to its fullest extent. Nevertheless, data from rodent as well a large animal studies and more recently clinical studies demonstrate that mixed chimaerism is an effective approach for inducing tolerance to a defined set of donor alloantigens –17. To pet al. revealed that lasting chimaerism and donor specific transplantation tolerance could be achieved through a protocol, which combined the administration of a high dose of fully MHC mismatched bone marrow with a single injection of anti-CD40L and cytotoxic T-lymphocyte associated antigen 4-Ig (CTLA-4 Ig) [et al. were able to develop a protocol using busulfan, an alkylating agent that preferentially depletes early haematopoietic stem cells, in conjunction with bone marrow administration and simultaneous blockade of the CD40 and CD28 co stimulatory pathways (using anti-CD40L and CTLA-4 Ig respectively) to establish for titratable levels of haematopoietic chimaerism that could result in donor specific tolerance in a mouse skin allograft model [An elegant study by Wekerle LA-4 Ig) . An alteLA-4 Ig) . Mice trLA-4 Ig) . By exteft model .Recent clinical data suggest that long lasting mixed chimaerism may not be essential for the induction of tolerance to donor alloantigens. In recent patient studies, the majority of recipients of HLA-haploidentical stem cell transplantation with nonmyeloablative conditioning and immunosuppressive GVHD prophylaxis were able to achieve 100% donor cell engraftment and maintained graft function without the onset of GVHD with a mismatch of 2–3 HLA antigens ,30.in vitro studies using RESCs. Thus, it may be possible to harness FasL dependent mechanism to avoid rejection when donor cells are injected into a nonmyeloablated host [Data from the experimental rat model have also demonstrated the possibility that pretransplant conditioning of the host may not always be required to attain the long-term survival of donor derived cells. Injection of rat embryonic stem like cells (RESCs) via the portal vein was found to result in a state of mixed chimaerism in which 5–8% of donor cells resided in the white blood cell population of recipient rats. Furthermore, rats with surviving RESCs were able to accept cardiac allografts permanently in a donor alloantigen specific manner . One queted host .in vitro [in vitro, MSCs have been quite controversial in vivo. Recent data on the effects of MSCs on immunomodulation in the rat allogeneic cardiac transplant transplant model suggest that MSCs may suppress T-cell proliferative responses in vitro; however, translation in vivo was not achieved as allograft survival was not prolonged and rejection responses were, in fact, accelerated [In a recent work, multipotent bone-marrow derived stromal cells, or mesenchymal stem cells (MSC) have been shown to possess an immunoregulatory capacity, at high doses, by suppressing the activation and proliferation of both naïve and memory T cells in vitro –34. Furtin vitro ,36. Althelerated . More woCentral deletion of auto or self reactive T cells in the thymus is a relatively incomplete process. Therefore, the immune system has developed additional strategies for regulating the functional capacity of T cells with potential autoreactivity that escape deletion in the thymus and emerge into the periphery. Peripheral tolerance is the term applied to these naturally arising mechanisms that lead to anergy, deletion or suppression of self reactive T cells in the periphery. Investigations in rodent models have sought to develop these mechanisms to obtain peripheral tolerance to alloantigens. Costimulation blockade is one approach that has been shown to induce peripheral tolerance to alloantigens and is discussed in the section below.in vitro, inhibits alloresponses and induces T-cell anergy [Blockade of co stimulatory molecules at the time of alloantigen recognition has been shown in experimental models to be a potential strategy for inducing peripheral tolerance. Engagement of B7.1/B7.2 (CD80/CD86 found on APCs) with CD28 (found on T cells) at the time of antigen recognition induces T cells to produce IL-2, a cytokine involved in their growth and proliferation . Blockadl anergy . In rodeet al. reported that treatment with CTLA-4Ig either pre or post-transplantation resulted in skin allograft rejection in mice pretreated with a tolerizing protocol of anti-CD40L/DST, which led the authors to conclude that signalling through CTLA-4 is required to achieve permanent graft acceptance [CTLA-4Ig has been used to treat recipients at the time of transplantation with promising results . Interesceptance .ex vivo with CTLA-4Ig could reconstitute haematopoiesis in vivo with a reduced risk of GVHD [CTLA-4-Ig therapy has been explored in clinical trials in solid organ and bone marrow transplantation. In the latter setting, donor bone marrow, which was mismatched with the recipient for one HLA haplotype, was cocultured with irradiated recipient cells for 36 h in the presence of CTLA-4Ig. Transfusion of these donor cells into the recipient led to a reduction in the frequency of donor specific alloreactive T cells and engraftment of the bone marrow. In addition, only three of 11 transplant recipients showed any evidence of GVHD thus suggesting that treatment of donor bone marrow  of GVHD . Further of GVHD .in vitro when compared with CTLA-4Ig [Experiment models using CTLA-4Ig laid the groundwork for further pharmacotherapeutic developments targeted at the B7:CD28/CTLA4 pathway. The most promising of these developments was the introduction of Belatacept (LEA29Y), a derivative of CTLA-4Ig ,47. BelaCTLA-4Ig . OriginaCTLA-4Ig . These aCTLA-4Ig . AdditioCTLA-4Ig . Althoug+ T-cell responses [+ T cells [A second co stimulatory pathway of importance is the CD40 (found on APCs)/CD40L (CD154) (found on T cells) pathway [esponses . Attemptesponses ,51. For  T cells ,51.+ T cells are more susceptible to co stimulatory blockade than CD8+ T cells; therefore, in some rodent models the allograft survival can only occur if the co stimulatory molecule blocking agent is used in conjunction with an agent that depleted CD8+ T cells [In general, CD4 T cells –54. In a T cells .et al. has investigated the agonistic role that human chimaeric antibodies to CD40 (Chi220) have in abrogating immune responses. In these nonhuman primate models, the use of Chi220 alone was not impressive in the prolongation of renal and islet allografts; however, when combined with belatacept therapy allograft survival was markedly improved. These data suggest that future investigations of tolerance induction via costimulation blockade are necessary [Long-term acceptance of cardiac, renal and islet allografts in several murine and nonhuman primate models was achieved with CD40 blockade using anti-CD154 monoclonal antibody as monotherapy or in conjunction with CD28 blockade –60. Howeecessary ,66.−/−) showed prolonged allograft survival. Additionally, blockade of ICOS using an anti-ICOS antibody in conjunction with cyclosporine administration led to sustained allograft survival without the development of transplant arteriosclerosis [−/− mice treated with cyclosporine showed prolonged survival of cardiac allografts, decreased intragraft expression of IFN-γ and IFN-γ induced chemokine inducible protein -10 [−/− mice treated with a PD-L1 Ig fusion protein showed prolonged allograft survival, and in some cases permanent survival [Some of the newly discovered co stimulatory molecules may also be targets for transplantation tolerance induction. In a mouse cardiac allograft model, mice deficient in the induced co stimulatory molecule – ICOS  expression could be used as a tool to enrich a sub population of CD4+ T cells, which demonstrated powerful regulatory activity [+ T cells and <1% of peripheral CD8+ T cells. Although many studies within the last decade have demonstrated that CD25+ CD4+ T cells can mediate tolerance to self antigens [+ CD4+ T cells have also been found to regulate responses to alloantigens.Sakaguchi activity . CD25 isantigens ,79–84, m+ CD4+ T cells capable of regulating responses to alloantigens in vivo /DST tolerized mice were unable to prevent rejection of fully MHC mismatched skin allografts [Graca antigens . CD25+ Cat model . Data frlografts . Further+, CD8+ CD28−, TCR+ CD4− CD8− (‘double negative’) and natural killer T cells, thus CD25+ CD4+ cells may only represent one subset. The context in which the regulatory activity arises may impact the phenotypic and functional characteristics the regulatory populations possess. For example, in our system, CD25− CD4+ T cells isolated from anti-CD4 antibody/DST treated mice were unable to prevent skin allograft rejection in our studies [− CD4+ T cells from tolerant mice were able to prevent skin graft rejection mediated by unsorted spleen cells. This latter observation has been supported by data from Chiffoleau et al. demonstrated that tolerance could be transferred by thymic and splenic CD25+ CD4+ T cells, but in 50% of the cases, this transferable tolerance was mediated by splenic CD25− CD4+ T cells [+ CD4+ T reg in their low levels of CD25 expression [At present, a definitive marker to enrich regulatory cells is under active investigation. Other populations of regulatory cells clearly exist, including CD8 studies ,86, wher T cells . Taken tpression .+ CD4+ regulatory T cells control responses to alloantigens are still under investigation. Recently, cytokines have been found to play an important role in mediating suppression in some systems in vivo. IL-10 and TGF-β play key roles in the suppressive activity of alloantigen specific CD25+ CD4+ regulatory T cells [in vivo from day 0 to day 4 post-transplantation abrogated tolerance, as six of six animals rejected their cardiac allografts.The mechanisms by which CD25 T cells . As depi T cells  Fig. 2)+ CD4+ re T cells . Similar T cells . Moreove+ CD4+ T cells have been shown in mice to express constitutively surface and cytoplasmic CTLA-4 (CD152) [+ CD4+ T cells, which suppress skin allograft rejection are also dependent on signalling through CTLA-4, as blockade of CTLA-4 with anti-CTLA-4 antibody led to acute rejection of skin allografts [In addition to cytokines, cell associated molecules have been found to be involved in immunoregulation in some situations. CD25 (CD152) . Althoug (CD152) . Data frlografts .The interplay among IL-10, TGF-β and CTLA-4 in the suppression of alloresponses is still not fully understood. Recent data have demonstrated a link between IL-10 and TGF-β, with IL-10 enhancing the expression of TGF-β receptor on activated and resting cells . As cros+ CD4+ regulatory T cells in the thymus and periphery have been found to express the glucocorticoid induced tumor necrosis factor receptor (GITR) [+ CD4+ T cells, we might suggest that this molecule or its ligand could be of therapeutic interest in the generation of tolerance to both self and alloantigens. Investigations into the role of signalling through GITR in the induction and maintenance phases of tolerance have shown that their pathways may play a differential role, abrogating the induction of unresponsiveness but not affecting immunoregulation once it is established [An increasing list of molecules have found to be expressed by T cells with regulatory activity, including cell surface molecules such as CD62, CD103 and GITR (see below) and the transcription factor Foxp3. CD25r (GITR) ,100. Sigablished .Overall, the models presented may have to be revisited, in the light of recent theories regarding the impact that memory T cells (Tm) may have on the induction of tolerance. The concept of ‘heterologous immunity’ proposed by Larsen’s group refers to chronic immunologic activation by various environmental stimuli leading to a population of Tm which can cross react with alloantigens. These Tm seem to be resistant to conventional induction therapies and may prove to be a hurdle for newer therapeutic approaches ,103. AltIt has become increasingly clear that research in experimental models has allowed for greater insight into the mechanisms of transplantation tolerance. These models have also established the principles in which new therapeutic approaches can be devised to enable robust tolerance to alloantigens to be achieved for the future of clinical transplantation . Great c+ CD4+ T cells may be a strategy to induce donor specific transplantation tolerance, it has been well documented in a mouse tumour model that CD25+ regulatory T cells suppress tumour specific responses, leading to tumour growth. It is only when this subset is eliminated that tumour immunity can be restored [Furthermore, it is imperative to remember that manipulation of one aspect of the immune response may have a deleterious effect on other important immune pathways. Although the generation and expansion of CD25restored –114. Conrestored .So what is the future direction of research into transplantation tolerance using experimental models? The identification of new co stimulatory pathways and the current interest in chemokines and their receptors may offer new targets for immune intervention that need to be fully explored in experimental models before such strategies can be considered and selected appropriately for further development. Although there is much information regarding the role regulatory T cells play in suppressing responses to self antigens, more research is needed to examine the role these cells play in regulating responses to alloantigens. One aim of transplant immunologists is to determine definitive markers of transplantation tolerance. Studies in animal systems may enable this goal to be achieved.


Sulindac is an FDA-approved non-steroidal anti-inflammatory drug (NSAID) that affects prostaglandin production by inhibiting cyclooxygenases (COX) 1 and 2. Sulindac has also been of interest for more than decade as a chemopreventive for adenomatous colorectal polyps and colon cancer.Pretreatment of human colon and lung cancer cells with sulindac enhances killing by an oxidizing agent such as tert-butyl hydroperoxide (TBHP) or hydrogen peroxide. This effect does not involve cyclooxygenase (COX) inhibition. However, under the conditions used, there is a significant increase in reactive oxygen species (ROS) within the cancer cells and a loss of mitochondrial membrane potential, suggesting that cell death is due to apoptosis, which was confirmed by Tunel assay. In contrast, this enhanced killing was not observed with normal lung or colon cells.These results indicate that normal and cancer cells handle oxidative stress in different ways and sulindac can enhance this difference. The combination of sulindac and an oxidizing agent could have therapeutic value. Sulindac was one of the early non-steroidal anti-inflammatory drugs (NSAIDs), which affect prostaglandin production by inhibiting cyclooxygenases (COX) 1 and 2 Sulindac is a pro-drug that must be converted to the active COX-inhibitor, sulindac sulfide Since sulindac is a substrate for MsrA In the present studies we extended these earlier results using cancer cell lines derived from lung and colon tissue. We provide further evidence that the enhanced killing observed with sulindac and oxidative stress involves mitochondrial dysfunction leading to cell death via apoptosis. These new data strengthen the potential for specifically enhancing the therapeutic application of sulindac and its derivatives for cancer treatment by using them in conjunction with a compound that produces reactive oxygen species (ROS).Human lung and colon cancer cell lines were preincubated in the presence or absence of sulindac for 48 hours. Excess sulindac was removed by washing prior to the 2 hr incubation with TBHP as described in the Each cancer cell line had a marked decrease in cell viability in the presence of TBHP following pretreatment with 500 µM sulindac . ViabiliSeveral lines of evidence indicate that the enhanced killing of cancer cells by sulindac and oxidative stress does not involve COX inhibition. Two other NSAIDs, acetylsalicylic acid (aspirin) and ibuprofen, at 500 µM, failed to increase the sensitivity of cancer cells to oxidative stress , followeIt was important to determine whether the enhanced killing effect of sulindac and sulindac sulfone on cancer cells in the presence of TBHP  also occSimilar experiments were performed using the normal colon cells. There was no effect on cell viability in the presence of TBHP when the normal colon cells were pretreated with either 500 µM sulindac or 250 µM sulindac sulfone (data not shown). Thus, neither normal lung nor normal colon cells showed enhanced killing by TBHP following treatment with sulindac or sulindac sulfone, as was observed with the two cancer cell lines.Lung cancer cells were used to gain more information on the mechanism of the enhanced killing effect of sulindac in the presence of TBHP. To investigate whether the enhanced killing of the cancer cells observed with sulindac and oxidative stress might involve mitochondrial dysfunction, changes in the level of intracellular ROS were determined. For these experiments lung cancer cells were treated with sulindac for 48 hours, exposed to TBHP and then the intracellular ROS level was visualized using a fluorescent dye as described in the To further explore the mechanism of killing cancer cells exposed to oxidative stress after pretreatment with sulindac, we investigated whether there is a concomitant loss of mitochondrial membrane potential, which is known to initiate apoptotic cell death. Effects on mitochondrial membrane potential were evaluated using changes in the fluorescence of the JC-1 dye as described in Sulindac and its metabolites, such as sulindac sulfide and sulindac sulfone, have been shown to have anti-cancer activity It appears likely that the mechanism of the selective killing of cancer cells seen in these studies involves mitochondrial dysfunction, possibly as a result of increased ROS production. While treatment of cancer cells with sulindac or TBHP individually leads to a modest increase in the level of ROS , there iThe present results indicate a fundamental difference in the way normal and cancer cells respond to oxidative stress. Sulindac and its metabolites can accentuate this difference, which leads to enhanced killing of cancer cells, but not normal cells, by oxidative stress. It is well established that cancer and normal cells differ in their oxidative metabolism and that cancer cells have a higher rate of glycolysis than normal cells, a phenomenon first described by Warburg The results described provide further evidence that a combination of sulindac and an oxidizing agent might have clinical therapeutic value in treating a variety of cancers. In a previous preliminary study we reported that the results of a limited proof of concept human clinical trial using sulindac (1–5%) and hydrogen peroxide (25%) gels applied daily for three weeks on actinic keratoses (AK) involving the upper extremities Sulindac, acetylsalicylic acid (aspirin), (S)-(+)-ibuprofen, and tert butyl-hydroperoxide (TBHP) were obtained from Sigma . Sulindac sulfone was synthesized by Custom Synthesis Inc. . All tissue culture media including fetal bovine serum and other supplements were purchased from American Type Culture Collection .A colon cancer cell line (RKO), a lung cancer cell line (A549), and fibroblast cell lines derived from normal human colon tissue (CCD-18Co) and normal human lung (MRC-5) were obtained from ATCC . All cell lines were maintained in the recommended culture medium. The normal cell lines were not immortalized and early passage cells were used for the experiments reported here. Cell lines were determined to be free of mycoplasma using the VenorGeM® Mycoplasma Detection Kit (Sigma-Aldrich), which is a highly sensitive PCR-based assay.2 incubator. The culture medium was then removed and the cells washed once with fresh culture medium with serum. After removal of the wash solution, fresh culture medium with serum that contained the indicated final concentration of TBHP or hydrogen peroxide was added to the cells, and the cells were incubated an additional 2 hours. Similar results were obtained when sulindac was included during the 2 hr treatment with the oxidizing agent.Unless otherwise indicated, the cells were pretreated with sulindac, sulindac sulfone or another NSAID for 48 hr prior to exposure to TBHP for 2 hr. Cell suspensions  containing the indicated supplement were plated in 96 well microtiter plates using 100 µl of the indicated cell suspension. The plates were incubated for 48 hr at 37°C in a 5% CO384; Molecular Devices). Background absorbance was subtracted from each sample. It has been reported that some anticancer drugs can cause changes in absorbance in MTS-based assays for cell viability in the absence of cells Cell viability was determined by the CellTiter 96 Aqueous One Cell Proliferation Assay  according to the manufacture's instructions. The assay utilizes a novel tetrazolium compound that metabolically active cells convert to a water-soluble formazan by the action of cellular dehydrogenases, which is measured by absorbance at 490 nm using a colorimetric microtiter plate reader (SpectraMax PlusIntracellular ROS levels were determined by using the Reactive Oxygen Species (ROS) Detection Reagents from Molecular Probes  as described elsewhere Loss of mitochondrial membrane potential was determined using the JC-1 dye from Molecular Probes . Loss of mitochondrial membrane potential leads to increased green fluorescence in the cytosol and a corresponding decrease in mitochondrial red fluorescence. Thus, changes in mitochondrial membrane potential were determined by following the red to green staining shift using an FITC filter (Zeiss inverted microscope-Axiovert 40 CFL). Quantitation of the fluorescence signals used both standard densitometric methods and a Photoshop  based image analysis Apoptotic cells were detected using the Deadend (Promega) colorimetric TUNEL assay that end labels fragmented DNA. Cells were fixed with 4% formaldehyde before being permeabilized with 0.2% triton-X-100. Cells were then washed with PBS prior to incubation with recombinant terminal deoxynucleotidyl transferase(TdT) and biotinylated nucleotides for 1 h at 37°C, which incorporates the biotinylated nucleotides at 3′ ends of fragmented DNA. Cells were washed with PBS and then incubated with horseradish peroxidase-streptavidin (HRP-Streptavidin) at room temperature for 30 min. HRP-streptavidin labeled cells were detected by hydrogen peroxide and diaminobenzidine (DAB). Apoptotic cells are visualized by dark brown nuclear staining.Results of cell viability experiments are expressed as the mean of four replicates of a representative experiment. The error bars indicate the standard error of the mean (SEM). Means were compared using standard t-tests and the P-values are indicated in the figure legends. P values<0.05 were considered statistically significant. Quantitation of the fluorescence signals used both standard densitometric methods and a Photoshop  based image analysis Figure S1Cell Death due to Apoptosis. A TUNEL assay was used to detect apoptosis of lung cancer cells. (A) untreated cells; (B) cells treated with only 500 µM sulindac; (C) cells treated with only 180 µM TBHP; (D) cells treated with both 500 µM sulindac and 180 µM TBHP. Increased levels of apoptosis are indicated by enhanced formation of brown coloration. The experimental design is described in the legends of (0.22 MB TIF)Click here for additional data file.