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Carcinoma-associated fibroblast (CAF) as prominent cell type of the tumour microenvironment has complex interaction with both the cancer cells and other non-neoplastic surrounding cells. The CAF-derived regulators and extracellular matrix proteins can support cancer progression by providing a protective microenvironment for the cancer cells via reduction of chemotherapy sensitivity. On the other hand, these proteins may act as powerful prognostic markers as well as potential targets of anticancer therapy. In this review, we summarise the clinical importance of the major CAF-derived signals influencing tumour behaviour and determining the outcome of chemotherapy., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Background: Somatostatin (SST) has anti-proliferative and pro-apoptotic effects. Our aims were to analyze and compare the SST expression during normal aging and colorectal carcinogenesis at mRNA and protein levels. Furthermore, we tested the methylation status of SST in biopsy samples, and the cell growth inhibitory effect of the SST analogue octreotide in human colorectal adenocarcinoma cell line., Methods: Colonic samples were collected from healthy children (n1 = 6), healthy adults (n2 = 41) and colorectal cancer patients (CRCs) (n3 = 34) for SST mRNA expression analysis, using HGU133 Plus2.0 microarrays. Results were validated both on original (n1 = 6; n2 = 6; n3 = 6) and independent samples ((n1 = 6; n2 = 6; n3 = 6) by real-time PCR. SST expressing cells were detected by immunohistochemistry on colonic biopsy samples (n1 = 14; n2 = 20; n3 = 23). The effect of octreotide on cell growth was tested on Caco-2 cell line. SST methylation percentage in biopsy samples (n1 = 5; n2 = 5; n3 = 9) was defined using methylation-sensitive restriction enzyme digestion., Results: In case of normal aging SST mRNA expression did not alter, but decreased in cancer (p < 0.05). The ratio of SST immunoreactive cells was significantly higher in children (0.70% ± 0.79%) compared to CRC (0% ± 0%) (p < 0.05). Octreotide significantly increased the proportion of apoptotic Caco-2 cells. SST showed significantly higher methylation level in tumor samples (30.2% ± 11.6%) compared to healthy young individuals (3.5% ± 1.9%) (p < 0.05)., Conclusions: In cancerous colonic mucosa the reduced SST production may contribute to the uncontrolled cell proliferation. Our observation that in colon cancer cells octreotide significantly enhanced cell death and attenuated cell proliferation suggests that SST may act as a regulator of epithelial cell kinetics. The inhibition of SST expression in CRC can be epigenetically regulated by promoter hypermethylation.

Epithelial layer of the intestine relies upon stem cells for maintaining homeostasis and regeneration. Two types of stem cells are currently defined in intestinal crypts: the cycling crypt base columnar cells and quiescent cells. Though several candidate markers and regulators of rapidly cycling and quiescent stem cells have been identified so far, the exact nature of quiescent cells is still questionable since investigations mainly focused on candidate markers rather than the label-retaining population itself. Recent results, however, have strengthened the argument for functional plasticity. Using a lineage tracing strategy label-retaining cells (LRCs) of the intestinal epithelium were marked, then followed by a pulse-chase analysis it was found that during homeostasis, LRCs were Lgr5-positive and were destined to become Paneth and neuroendocrine cells. Nevertheless, it was demonstrated that LRCs are capable of clonogenic growth by recall to the self-renewing pool of stem cells in case of epithelial injury. These new findings highlight on the hierarchical and spatial organization of intestinal epithelial homeostasis and the important plasticity of progenitors during tissue regeneration, moreover, provide a motivation for studying their role in disorders like colorectal cancer.

Cancer represents a major health problem worldwide, therefore on the basis of current research results constantly more effective therapeutic strategies are expected. Chronic, unchecked inflammation has widely been suggested to trigger carcinogenesis. The innate immune system ensures a first line host defense in which the inflammasome is essential maintaining a delicate balance betweeen pro- and anti-inflammatory signals in order to generate an appropriate immune response without harming the host. Studies have revealed a remarkable, but contradictory link of host inflammatory responses to tumorigenesis. Indeed, activation of the multiprotein complex inflammasome by danger signals seems to play diverse and sometimes conflicting, suppressive or stimulatory role in cancer development and progression with a significant context-dependency. The pleitropic inflammasomes may act at cell-autonomous level to eliminate malignant cells via the programmed cell death type of inflammatory pyroptosis, but on the contrary, may favor the production of gowth and trophic factors for tumor cells and their microenvironment. Further, upon caspase-1 activation the inflammasome can provoke sterile inflammation, and thus facilitate carcinogenesis, though in antigen-presenting cells it can elicit anti-tumor immune responses. Clarifying the exact, context-specific impact of inflammasomes on tumorigenesis represents a new research area with the potential to introduce promising novel targets for cancer therapeutics.

Background: Determination of methylated Septin 9 (mSEPT9) in plasma has been shown to be a sensitive and specific biomarker for colorectal cancer (CRC). However, the relationship between methylated DNA in plasma and colon tissue of the same subjects has not been reported., Methods: Plasma and matching biopsy samples were collected from 24 patients with no evidence of disease (NED), 26 patients with adenoma and 34 patients with CRC. Following bisulfite conversion of DNA a commercial RT-PCR assay was used to determine the total amount of DNA in each sample and the fraction of mSEPT9 DNA. The Septin-9 protein was assessed using immunohistochemistry., Results: The percent of methylated reference (PMR) values for SEPT9 above a PMR threshold of 1% were detected in 4.2% (1/24) of NED, 100% (26/26) of adenoma and 97.1% (33/34) of CRC tissues. PMR differences between NED vs. adenoma and NED vs. CRC comparisons were significant (p<0.001). In matching plasma samples using a PMR cut-off level of 0.01%, SEPT9 methylation was 8.3% (2/24) of NED, 30.8% (8/26) of adenoma and 88.2% (30/34) of CRC. Significant PMR differences were observed between NED vs. CRC (p<0.01) and adenoma vs. CRC (p<0.01). Significant differences (p<0.01) were found in the amount of cfDNA (circulating cell-free DNA) between NED and CRC, and a modest correlation was observed between mSEPT9 concentration and cfDNA of cancer (R2 = 0.48). The level of Septin-9 protein in tissues was inversely correlated to mSEPT9 levels with abundant expression in normals, and diminished expression in adenomas and tumors., Conclusions: Methylated SEPT9 was detected in all tissue samples. In plasma samples, elevated mSEPT9 values were detected in CRC, but not in adenomas. Tissue levels of mSEPT9 alone are not sufficient to predict mSEPT9 levels in plasma. Additional parameters including the amount of cfDNA in plasma appear to also play a role.

Background: Presence of cell-free-circulating DNA (fcDNA) sequences in sera of patients with inflammatory bowel diseases (IBD) is a well-established phenomenon. Potential roles of fcDNA in diagnosis, prognosis and therapy monitoring of chronic inflammatory colonic disorders have already been examined, albeit its actual biological function still remains unclear., Aims and Methods: In the present experiment, we studied the immunobiological effects of isolated fcDNA of normal and inflammatory origin administered intravenously to mice prior to induction of dextran sulfate sodium (DSS)-colitis. In addition to evaluate the current disease and histological activity, changes of the gene expression profile in isolated lamina propria cells upon TLR9 ligation were assayed., Results: A single intravenous dose of fcDNA pretreatment with colitic fcDNA exhibited beneficial response concerning the clinical and histological severity of DSS-colitis as compared to effects of normal fcDNA. Pretreatment with colitic fcDNA substantially altered the expression of several TLR9-related and inflammatory cytokine genes in a clinically favorable manner., Conclusions: During the process of acute colitis, the subsequent inflammatory environment presumably results in changes of fcDNA with the potential to facilitate the downregulation of inflammation and improvement of regeneration. Thus, preconditioning of mice with colitis-derived fcDNA via TLR9 signaling could exert a tissue-protective effect and influence beneficially the course of DSS-colitis. Elucidating mechanisms of immune response alterations by nucleic acids may provide further insight into the etiology of IBD and develop the basis of novel immunotherapies.

Carcinoma-associated fibroblast (CAF) as prominent cell type of the tumour microenvironment has complex interaction with both the cancer cells and other non-neoplastic surrounding cells. The CAF-derived regulators and extracellular matrix proteins can support cancer progression by providing a protective microenvironment for the cancer cells via reduction of chemotherapy sensitivity. On the other hand, these proteins may act as powerful prognostic markers as well as potential targets of anticancer therapy. In this review, we summarise the clinical importance of the major CAF-derived signals influencing tumour behaviour and determining the outcome of chemotherapy., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Epigenetic changes of stromal-epithelial interactions are of key importance in the regulation of colorectal carcinoma (CRC) cells and morphologically normal, but genetically and epigenetically altered epithelium in normal adjacent tumor (NAT) areas. Here we demonstrated retained protein expression of well-known Wnt inhibitor, secreted frizzled-related protein 1 (SFRP1) in stromal myofibroblasts and decreasing epithelial expression from NAT tissues towards the tumor. SFRP1 was unmethylated in laser microdissected myofibroblasts and partially hypermethylated in epithelial cells in these areas. In contrast, we found epigenetically silenced myofibroblast-derived SFRP1 in CRC stroma. Our results suggest that the myofibroblast-derived SFRP1 protein might be a paracrine inhibitor of epithelial proliferation in NAT areas and loss of this signal may support tumor proliferation in CRC.

In inflammatory bowel diseases the presence of free-circulating DNA (fcDNA) sequences in the sera is an established phenomenon, albeit its real biological function still remains unclear. In our study the immunobiologic effects of a single-dose, intravenously administered fcDNA of normal and colitic origin were assayed in DSS-colitic and control mice. In parallel with disease and histological activity evaluations changes of the TLR9 and inflammatory cytokine signaling gene expression profiles were assayed in isolated cells of the lamina propria. Intravenously administered colitis-derived fcDNA displayed a more prominent beneficial action regarding the clinical and histological severity of DSS-colitis than that of fcDNA of normal origin. Systemic administration of colitis-derived fcDNA significantly altered the expression of certain TLR9-related and proinflammatory cytokine genes in a clinically favorable manner. Presumably due to induction of severe colitis, the subsequent marked inflammatory environment may result changes in fcDNA with a potential to promote the downregulation of inflammation and improvement of tissue regeneration. Elucidating mechanisms of innate immune alterations by nucleic acids may provide further insight into the etiology of inflammatory bowel diseases, and develop the basis of novel nucleic acid-based immunotherapies.

Semaphorins and plexins represent a highly conserved group of proteins that have recently been found to exert widespread regulatory functions outside the nervous system, including angiogenesis and immune regulation. Furthermore, these molecules are definitely implicated in the etiology of carcinogenesis and immune disorders. Their expression patterns and levels are deregulated in cancer cells and in cells of the tumor milieu. During the multistep development of cancer, its characteristic features include sustained tumor cell proliferation, resistance to cell death, limitless replicative capacity, activation of angiogenesis along with invasion and metastatic spread, cancer-related smoldering inflammation, and evasion of antitumor immune responses. The diversity of the semaphorin/plexin complexes and, thus, the multiple stimulated molecular interactions allow varied and diverse cell signaling events. The elicited transduction pathways might be involved in modifying the intricate mechanisms of tumorigenesis. Indeed, these pleiotropic signals may influence not only the intrinsic properties of cancer cells but they could also represent a possible link in mediating the cross-talk between tumor cells and the surrounding multiple stromal cells. In tumorigenesis, however, a dual role of different semaphorins is proposed, as some of them may elicit tumor regression, whereas others definitely promote cancer cell survival and progression. The current antitumoral or prosurvival responsiveness to semaphorins is mainly cell context dependent; nevertheless, their precise relation to cancer networks has not yet been fully elucidated. Here, we survey the many faces of a subset of the large semaphorin family, termed immune semaphorins, in carcinogenesis.

Early molecular detection of the colorectal dysplasia-carcinoma transition may augment the accuracy of diagnosis in case of biopsy orientation errors. The combination of high-throughput microarray-based biomarker screening with tissue microarray-based prospective protein biomarker expression analysis could represent an additional test in routine automated diagnostic procedures. Our aim was to test and select protein markers to identify protein expression profile alterations, focusing on the dysplasia-carcinoma transition in sporadic colorectal tumors. Dysplasia-carcinoma transition-specific transcript sets were previously identified using HGU133plus2 microarrays and Taqman RT-PCR cards. Here, 26 potential dysplasia-carcinoma transition-specific markers were tested by immunohistochemistry at the protein level using tissue microarrays in a total of 168 independent colonic biopsy samples. A set of 26 transcripts [including matrix metalloproteinase-3 (MMP3) and chemokine (C-X-C motif) ligand 1 (CXCL1)] has been determined recently, indicating a linear expression correlation with the adenoma-dysplasia-carcinoma sequence, thereby having the potential to discriminate between dysplasia and early malignancy. Currently, we find that high-grade dysplastic sessile adenomatous-stage and early-stage colorectal cancer conditions can be differentiated correctly by the stromal expression of MMP3 and CXCL1, respectively, on tissue microarray-based analysis. Furthermore, in cases of sporadic colorectal tumors, MMP3 protein expression in the lamina propria itself seems to be highly specific for the detection of tumorous transition. Our current and recent results indicate that appropriate antibody marker combinations are highly suitable for tissue microarray-based and digital microscopy-based, automated, high-capacity diagnostic application in tumorous colonic diseases.

Intratumoral heterogeneity including genetic and nongenetic mechanisms refers to biological differences amongst malignant cells originated within the same tumor. Both, cell differentiation hierarchy and stochasticity in gene expression and signaling pathways may result in phenotypic differences of cancer cells. Since a tumor consists of cancer cell clones that display distinct behaviours, changes in clonal proliferative behavior may also contribute to the phenotypic variability of tumor cells. There is a need to reveal molecular actions driving chemotherapeutic resistance in colon cancer cells. In general, it is widely hypothesized that therapeutic resistance in colorectal cancer is a consequence of the preferential survival of cancer stem cells. However, recent data regarding colorectal cancer suggest that resistance to anticancer therapy and post-therapeutic tumor reappearence could be related to variations of clonal dynamics. Understanding the interaction of genetic and nongenetic determinants influencing the functional diversity and therapy response of tumors should be a future direction for cancer research.

Accumulating evidence indicates that the aberrantly altered process of autophagy is definitely involved in carcinogenesis. Nonetheless, Toll-like receptors (TLRs) sensing cell-derived pattern/danger-associated molecules also have the capacity to promote tumor development and immune escape. TLRs are usually expressed in immunocompetent cells, though several types of cancer cells have also been reported to display these innate immune receptors. On the other hand, however, both TLR- and autophagy-related signals may exert tumor suppressor mechanisms mainly in a cell-specific and context-dependent manner. The role of autophagy has been radically expanded, and now this machinery is considered as a fundamental eukaryotic cellular homeostatic process and integral component of the immune system influencing infection, inflammation and immunity. Recent studies have documented that TLRs and autophagy are interrelated in response to danger signals, furthermore there is a controling cross-talk among them to avoid deficient or excessive immunological effects. Although the potential interaction of autophagy and TLRs in cancer cells has not yet been clarified, it seems to be a critical aspect of cancer development and progression. Upon translation of basic knowledge into practice it is reasonable to speculate that modulation of the TLR-autophagy regulatory loop might be relevant for cancer treatment by providing further possible therapeutic targets.

To understand the biologic role of self-DNA bound to Toll-like Receptor 9 (TLR9), we assayed its effect on gene and methyltransferase expressions and cell differentiation in HT29 cells. HT29 cells were incubated separately with type-1 (normally methylated/nonfragmented), type-2 (normally methylated/fragmented), type-3 (hypermethylated/nonfragmented), or type-4 (hypermethylated/fragmented) self-DNAs. Expression levels of TLR9-signaling and proinflammatory cytokine-related genes were assayed by qRT-PCR. Methyltransferase activity and cell differentiation were examined by using DNA methyltransferase (DNMT1, -3A, -3B) and cytokeratin (CK) antibodies. Treatment with type-1 DNA resulted in significant increase in TLR9 expression. Type-2 treatment resulted in the overexpression of TLR9-related signaling molecules (MYD88A, TRAF6) and the IL8 gene. In the case of type-3 treatment, significant overexpression of NFkB, IRAK2, and IL8 as well as downregulation of TRAF6 was detected. Using type-4 DNA, TRAF6 and MYD88A gene expression was upregulated, while MYD88B, IRAK2, IL8, and TNFSF10 were all underexpressed. CK expression was significantly higher only after type-1 DNA treatment. DNMT3A expression could also be induced by type-1 DNA treatment. DNA structure may play a significant role in activation of the TLR9-dependent and even independent proinflammatory pathways. There may be a molecular link between TLR9 signaling and DNMT3A. The mode of self-DNA treatment may influence HT29 cell differentiation.

Background and Aims: Sporadic colorectal cancer (CRC) development is a sequential process showing age-dependency, uncontrolled epithelial proliferation and decreased apoptosis. During juvenile growth cellular proliferation and apoptosis are well balanced, which may be perturbed upon aging. Our aim was to correlate proliferative and apoptotic activities in aging human colonic epithelium and colorectal cancer. We also tested the underlying molecular biology concerning the proliferation- and apoptosis-regulating gene expression alterations., Materials and Methods: Colorectal biopsies from healthy children (n1 = 14), healthy adults (n2 = 10), adult adenomas (n3 = 10) and CRCs (n4 = 10) in adults were tested for Ki-67 immunohistochemistry and TUNEL apoptosis assay. Mitosis- and apoptosis-related gene expression was also studied in healthy children (n1 = 6), adult (n2 = 41) samples and in CRC (n3 = 34) in HGU133plus2.0 microarray platform. Measured alterations were confirmed with RT-PCR both on dependent and independent sample sets (n1 = 6, n2 = 6, n3 = 6)., Results: Mitotic index (MI) was significantly higher (p<0.05) in intact juvenile (MI = 0.33±0.06) and CRC samples (MI = 0.42±0.10) compared to healthy adult samples (MI = 0.15±0.06). In contrast, apoptotic index (AI) was decreased in children (0.13±0.06) and significantly lower in cancer (0.06±0.03) compared to healthy adult samples (0.17±0.05). Eight proliferation- (e.g. MKI67, CCNE1) and 11 apoptosis-associated genes (e.g. TNFSF10, IFI6) had altered mRNA expression both in the course of normal aging and carcinogenesis, mainly inducing proliferation and reducing apoptosis compared to healthy adults. Eight proliferation-associated genes including CCND1, CDK1, CDK6 and 26 apoptosis-regulating genes (e.g. SOCS3) were differently expressed between juvenile and cancer groups mostly supporting the pronounced cell growth in CRC., Conclusion: Colorectal samples from children and CRC patients can be characterized by similarly increased proliferative and decreased apoptotic activities compared to healthy colonic samples from adults. Therefore, cell kinetic alterations during colorectal cancer development show uncontrolled rejuvenescence as opposed to the controlled cell growth in juvenile colonic epithelium.

Our bloodstream is considered to be an environment well separated from the outside world and the digestive tract. According to the standard paradigm large macromolecules consumed with food cannot pass directly to the circulatory system. During digestion proteins and DNA are thought to be degraded into small constituents, amino acids and nucleic acids, respectively, and then absorbed by a complex active process and distributed to various parts of the body through the circulation system. Here, based on the analysis of over 1000 human samples from four independent studies, we report evidence that meal-derived DNA fragments which are large enough to carry complete genes can avoid degradation and through an unknown mechanism enter the human circulation system. In one of the blood samples the relative concentration of plant DNA is higher than the human DNA. The plant DNA concentration shows a surprisingly precise log-normal distribution in the plasma samples while non-plasma (cord blood) control sample was found to be free of plant DNA.

Crohn's disease representing a clinical phenotype of inflammatory bowel disease is a polygenic immune disorder with complex multifactor etiology. Recent genome-wide association studies of susceptibility loci have highlighted on the importance of the autophagy pathway, which previously had not been implicated in disease pathology. Autophagy represents an evolutionarily highly conserved multi-step process of cellular self-digestion due to sequestration of excessive, damaged, or aged proteins and intracellular organelles in double-membranous vesicles of autophagosomes, terminally self-digested in lysosomes. Autophagy is deeply involved in regulation of cell development and differentiation, survival and senescence, and it also fundamentally affects the inflammatory pathways, as well as the innate and adaptive arms of immune responses. Autophagy is mainly activated due to sensors of the innate immunity, i.e., by pattern recognition receptor signaling. The interplay of genes regulating immune functions is strongly influenced by the environment, especially gut resident microbiota. The basic challenge for intestinal immune recognition is the requirement of a simultaneous delicate balance between tolerance and responsiveness towards microbes. On the basis of autophagy-related risk genetic polymorphisms (ATG16L1, IRGM, NOD2, XBP1) impaired sensing and handling of intracellular bacteria by innate immunity, closely interrelated with the autophagic and unfolded protein pathways seem to be the most relevant immunobiologic events. Autophagy is now widely considered as a key regulator mechanism with the capacity to integrate several aspects of Crohn's disease pathogenesis. In this review, recent advances in the exciting crosstalk of susceptibility coding variants-related autophagy and innate immunity are discussed.

Toll-like receptors (TLRs) recognize specific motifs which are frequently present in bacteria, fungi, prokaryotes and viruses. Amongst TLRs, TLR9 can be activated by such bacterial or viral DNA fragments, immunoglobulin-DNA complexes or synthetic oligonucleotides, which all contain unmethylated cytosine-guanine nucleotide sequences (CpGs). Emerging data indicate that TLR9 signaling has a role in, and may influence, colorectal carcinogenesis and colonic inflammation. CpGs are classified into three groups according to their influence on both the antigen-specific humoral- and cellular immunity, and the production of type 1 interferons and proinflammatory cytokines. TLR9 activation via CpGs may serve as a new therapeutic target for several cancerous and various inflammatory conditions. Due to its probable anti-cancer effects, the application possibilities of TLR9-signaling modulation may be extremely diverse even in colorectal tumors. In this review we aimed to summarize the current knowledge about TLR-signaling in the pathogenesis and therapy of inflammatory bowel diseases and colorectal cancer. Due to the species-specific differences in TLR9 expression, however, one must be careful in translating the animal model data into the human system, because of the differences between CpG-oligodeoxynucleotide-responsive cells. TLR9 agonist DNA-based immunomodulatory sequences could also represent a promising therapeutic alternative in systemic inflammatory conditions and chronic colonic inflammations as their side effects are not significant.

Multicentric Castleman's disease (MCD) is a sytemic disorder with flares of non-specific symptoms suggestive of a chronic inflammatory syndrome. It is typically accompanied by generalized lymphadenopathy and multiorgan involvement. Histologically, two main variants of Castleman's disease exist, the hyalin vascular type and the plasma cell variant. Upon localization unicentric (localized), and multicentric (diffuse, systemic) subtypes can be distinguished with more different disease outcomes. Patients often exhibit acute phase reactions and several autoimmune phenomena, and are at high risk for developing malignancies. Both the idiopathic and the HHV-8-driven infectious forms of MCD represent distinct disease entities with a less favorable prognosis. The induction of human IL-6 excess via yet unknown upstream mechanisms, and overexpression of viral IL-6 by HHV-8 can pivotally influence MCD biology. Based on the role of IL-6 in pathogenesis, MCD is also designated as IL-6 lymphadenopathy. To date there are no direct therapeutic evidences, but having been translated to daily practice the main regulatory factors may serve as promising therapeutic targets.

We present and discuss the case of a HIV-negative female finally diagnosed upon histopathologic and molecular biologic evaluations with human herpesvirus 8 (HHV8)-positive multicentric Castleman's disease (MCD) of plasma cell type, but with no detectable HHV8-DNA in peripheral blood. She failed to respond to combination immunosuppressive therapeutic trials of corticosteroids and azathioprine, and neither an immunochemotherapy of rituximab-CVP (R-CVP) induced disease resolution. However, monoclonal anti-IL-6R antibody (tocilizumab) immunotherapy resulted in beneficial disease stabilization. A control lymph node biopsy indicated mild polyclonal plasmacytosis, and a negative HHV8 determination. The patient is still receiving tocilizumab. This case emphasizes the individual nature of MCD requiering more personalized disease management., (© 2012 APMIS. Published by John Wiley & Sons Ltd.)

DNA methylation analysis methods have undergone an impressive revolution over the past 15 years. Regarding colorectal cancer (CRC), the localization and distribution of several differently methylated genes have been determined by genome-wide DNA methylation assays. These genes do not just influence the pathogenesis of CRC, but can be used further as diagnostic or prognostic markers. Moreover, the identified four DNA methylation-based subgroups of CRC have important clinical and therapeutic merit. Since genome-wide DNA methylation analyzes result in a large amount of data, there is a need for complex bioinformatic and pathway analysis. Future challenges in epigenetic alterations of CRC include the demand for comprehensive identification and experimental validation of gene abnormalities. By introduction of genome-wide DNA methylation profiling into clinical practice not only the patients' risk stratification but development of targeted therapies will also be possible.

Autophagy or self-digestion of cells is activated upon various stressful stimuli and has been found to be a survival and drug resistance pathway in cancer. However, genetic studies support that autophagy can act as a tumor suppressor. Furthermore, defective autophagy is implicated in tumorigenesis, as well. The precise impact of autophagy on malignant transformation has not yet been clarified, but recent data suggest that this complex process is mainly directed by cell types, phases, genetic background and microenvironment. Relation of autophagy to anticancer immune responses may indicate a novel aspect in cancer chemotherapy.

Cytokines are indispensable signals of the mucosa-associated immune system for maintaining normal gut homeostasis. An imbalance of their profile in favour of inflammation initiation may lead to disease states, such as that is observed in inflammatory bowel diseases (IBD). Although Crohn's disease (CD) is often described as a prototype of T-helper 1-type diseases, and ulcerative colitis (UC) is traditionally viewed as a T-helper 2-mediated condition, the classic paradigm, which categorises cytokines into pro- and anti-inflammatory groups, has recently been changed. The inflammation regulatory pathways may not be mutually exclusive as individual cytokines can have diverse and even opposing functions in various clinical and immunological settings. None the less there are many common immunological responses in IBD that are mediated by cytokines. Although they regulate and influence the development, course and recurrence of the inflammatory process, the concrete pathogenic role of these small signaling molecules is sometimes not unambiguous in the subtypes of the disease. Our aim is to review the current information about pro- and anti-inflammatory effects of traditionally studied and recently discovered cytokines in the pathogenesis of UC and CD. The better understanding of their production and functional activity may lead to the development of new therapeutic modalities.

Regulatory T cells (T(regs)) are key elements in immunological self-tolerance. The number of T(regs) may alter in both peripheral blood and in colonic mucosa during pathological circumstances. The local cellular, microbiological and cytokine milieu affect immunophenotype and function of T(regs). Forkhead box P3+ T(regs) function shows altered properties in inflammatory bowel diseases (IBDs). This alteration of T(regs) function can furthermore be observed between Crohn's disease and ulcerative colitis, which may have both clinical and therapeutical consequences. Chronic mucosal inflammation may also influence T(regs) function, which together with the intestinal bacterial flora seem to have a supporting role in colitis-associated colorectal carcinogenesis. T(regs) have a crucial role in the immunoevasion of cancer cells in sporadic colorectal cancer. Furthermore, their number and phenotype correlate closely with the clinical outcome of the disease, even if their contribution to carcinogenesis has previously been controversial. Despite knowledge of the clinical relationship between IBD and colitis-associated colon cancer, and the growing number of immunological aspects encompassing sporadic colorectal carcinogenesis, the molecular and cellular links amongst T(regs), regulation of the inflammation, and cancer development are still not well understood. In this paper, we aimed to review the current data surrounding the role of T(regs) in the pathogenesis of IBD, colitis-associated colon cancer and sporadic colorectal cancer.

Our aim was to examine cell transition events by detecting the frequency of intrapithelial α-smooth muscle actin (SMA)(+)/cytokeratin (CK)(+) cells during colorectal adenoma-carcinoma sequence, in relation to E-cadherin expression. Our further aim was to determine the proliferative activity of intraepithelial α-SMA(+) cells. Histologically healthy, adenoma, and colorectal cancer (CRC) biopsy samples were taken during routine colonoscopy and were included into tissue microarrays (TMAs). Slides immunostained for Ki-67, α-SMA, E-cadherin and pan-cytokeratin were digitalized and analyzed by using a digital microscope software. The proportion of α-SMA(+)/CK(+) cells was significantly higher in CRC samples (3.34 ± 1.01%) compared to healthy (1.94 ± 0.69%) or adenoma (1.62 ± 0.78%) samples (p < 0.01). E-cadherin expression negatively correlated with the number of α-SMA(+) cells. The majority of intraepithelial α-SMA(+) cells were in the proliferative phase. During tumor progression, the appearance of dot-like α-SMA staining in CK positive cells may indicate the initial phase of the epithelial-to-mesenchymal transition (EMT). The high proportion of intraepithelial α-SMA(+) proliferating cells may refer to their increased plasticity compared to differentiated cells. The negative correlation between E-cadherin and intraepithelial α-SMA expression suggests that EMT is facilitated by a loss of epithelial cell contact.

The latest avenue of research is revealing the existence of and role for the colonic stem cells in the physiological renewal of the mucosa and in pathological circumstances where they have both positive and negative effects. In the case of human colon, different levels of stem cell compartments exist. First, the crypt epithelial stem cells, which have a role in the normal crypt epithelial cell dynamics and in colorectal carcinogenesis. Close to the crypts, the second layer of stem cells can be found; the local subepithelial stem cell niche, including the pericryptic subepithelial myofibroblasts that regulate the epithelial cell differentiation and have a crucial role in cancer progression and chronic inflammation-related fibrosis. The third level of stem cell compartment is the immigrating bone-marrow-derived stem cells, which have an important role in wound healing after severe mucosal inflammation, but are also involved in cancer invasion. This paper focuses on stem cell biology in the context of physiological and pathological processes in the human colon.

Tumorigenesis is accompanied by changes in the DNA methylation pattern. Our aim was to test a novel approach for identification of transcripts at whole transcript level which are regulated by DNA methylation. Our approach is based on comparison of data obtained from transcriptome profiling of primary human samples and in vitro cell culture models. Epithelial cells were collected by LCM from normal, adenoma, and tumorous colonic samples. Using gene expression analysis, we identified downregulated genes in the tumors compared to normal tissues. In parallel 3000 upregulated genes were determined in HT-29 colon adenocarcinoma cell culture model after DNA demethylation treatment. Of the 2533 transcripts showing reduced expression in the tumorous samples, 154 had increased expression as a result of DNA demethylation treatment. Approximately 2/3 of these genes had decreased expression already in the adenoma samples. Expression of five genes (GCG, NMES-1, LRMP, FAM161B and PTGDR), was validated using RT-PCR. PTGDR showed ambiguous results, therefore it was further studied to verify the extent of DNA methylation and its effect on the protein level. Results confirmed that our approach is suitable for genome-wide screening of genes which are regulated or inactivated by DNA methylation. Activity of these genes possibly interferes with tumor progression, therefore genes identified can be key factors in the formation and in the progression of the disease.

Background: The early molecular detection of the dysplasia-carcinoma transition may enhance the strength of diagnosis in the case of colonic biopsies. Our aims were to identify characteristic transcript sets in order to develop diagnostic mRNA expression patterns for objective classification of benign and malignant colorectal diseases and to test the classificatory power of these markers on an independent sample set., Methodology/principal Findings: Colorectal cancer (CRC) and adenoma specific transcript sets were identified using HGU133plus2 microarrays and 53 biopsies (22 CRC, 20 adenoma and 11 normal). Ninety-four independent biopsies (27 CRC, 29 adenoma and 38 normal) were analyzed on microarrays for testing the classificatory power of the discriminatory genes. Array real-time PCR validation was done on 68 independent samples (24 CRC, 24 adenoma and 20 normal). A set of 11 transcripts (including CXCL1, CHI3L1 and GREM1) was determined which could correctly discriminate between high-grade dysplastic adenoma and CRC samples by 100% sensitivity and 88.9% specificity. The discriminatory power of the marker set was proved to be high on independent samples in both microarray and RT-PCR analyses. 95.6% of original and 94.1% of cross-validated samples was correctly classified in discriminant analysis., Conclusions/significance: The identified transcripts could correctly characterize the dysplasia-carcinoma transition in biopsy samples, also on a large independent sample set. These markers can establish the basis of gene expression based diagnostic classification of colorectal cancer. Diagnostic RT-PCR cards can become part of the automated routine procedure.

Matrix metalloproteinases play an important role in extracellular matrix remodelling. It has been proposed that matrix metalloproteinase-9 (MMP-9) is involved in epithelial damage in ulcerative colitis (UC). However, to our knowledge, no data are available in terms of MMP-9 expression in microscopic colitis. Determination of mucosal protein expression levels of MMP-9 in lymphocytic colitis (LC), collagenous colitis (CC) and UC. MMP-9 immunohistochemical expressions were analyzed in paraffin-embedded tissue samples by immunohistochemistry including patients with LC, CC, UC, active diverticulitis, inactive diverticular disease and healthy control subjects. UC was also subgrouped according to the severity of inflammation. Immunostaining was determined semiquantitatively. Independent colonic biopsies from healthy and severe UC cases were used for gene expression analyses. For further comparison MMP-9 serum antigen levels were also determined in patients with UC and control patients without macroscopic or microscopic changes during colonoscopy. MMP-9 mucosal expression was significantly higher in UC (26.7 ± 19.5%) compared to LC (6.6 ± 9.3%), CC (6.4 ± 7.6%), active diverticulitis (5.33 ± 2.4%), inactive diverticular disease (5.0 ± 2.2%) and controls (6.3 ± 2.6%) (P < 0.001). The immunohistochemical expression of MMP-9 in LC and CC was similar as compared to controls. MMP-9 expression was significantly higher in each inflammatory group of UC compared to controls (mild: 11.0 ± 2.8%, moderate: 23.9 ± 3.7%, severe UC: 52.6 ± 3.9% and 6.3 ± 2.6%, respectively, P < 0.005). The gene expression microarray data and RT-PCR results demonstrated a significantly higher expression of MMP-9 in severely active UC compared to healthy controls (P < 0.001). Significantly higher MMP-9 serum antigen concentrations were observed in UC patients compared with the control group (P < 0.05). MMP-9 seems to play no role in the inflammatory process of LC and CC. In contrast, the mucosal up-regulation of MMP-9 correlated with the severity of inflammation in UC. The increased MMP-9 expression could contribute to the severity of mucosal damage in active UC.

Background: Methylated Septin 9 (SEPT9) is a sensitive biomarker for colorectal cancer (CRC) from peripheral blood. However, its relationship to cancer localization, guaiac-based fecal occult blood test (gFOBT) and carcinoembryonic antigen (CEA) have not been described., Methodology/principal Findings: Plasma samples were collected for SEPT9 analysis from patients with no evidence of disease (NED) (n=92) before colonoscopy and CRC (n=92) before surgical treatment. DNA was isolated and bisulfite-converted using Epi proColon kit 2.0. Qualitative determination was performed using Epi proColon 2.0 RT-PCR assay. Samples for gFOBT and CEA analysis were collected from NED (n=17 and 27, respectively) and CRC (n=22 and 27, respectively). SEPT9 test was positive in 15.2% (14/92) of NED and 95.6% (88/92) of CRC, including 100% (67/67) from stage II to stage IV CRC and 84% (21/25) of stage I CRC when a sample was called positive if 1 out of 3 PCR replicates was positive. In a second analysis (2 out of 3 PCR replicates) specificity improved to 99% (91/92) of NEDs, at a sensitivity of 79.3% (73/92) of SEPT9 positives in CRC. gFOBT was positive in 29.4% (5/17) of NED and 68.2% (15/22) of CRC and elevated CEA levels were detected in 14.8% (4/27) of NED and 51.8% (14/27) of CRC. Both SEPT9 (84.8%) and CEA (85.2%) showed higher specificity than gFOBT (70.6%). SEPT9 was positive in 96.4% (54/56) of left-sided colon cancer (LSCC) cases and 94.4% (34/36) of right-sided colon cancer (RSCC) cases. gFOBT was positive in 83.3% (10/12) of cases with LSCC and 50% (5/10) of cases with RSCC, elevated CEA was detected 60% (9/15) of LSCC and 41.7% (5/12) of RSCC., Conclusions/significance: The high degree of sensitivity and specificity of SEPT9 in plasma makes it a better method to detect CRC than gFOBT and CEA, even for the more difficult to detect RSCC.

Colorectal cancer is one of the leading death causes in the world. Specificity and sensitivity of the present screening methods are unsuitable and their compliance is too low. Nowadays the most effective method is the colonoscopy, because it gives not only macroscopic diagnosis but therapeutic possibility as well, however the compliance of the patients is very low. Hence development of new diagnostic methods is needed. Altered expression of septin 9 was found in several tumor types including colorectal cancer. The aim of this study was to detect the methylation related mRNA and protein expression changes of septin 9 in colorectal adenoma-dysplasia-carcinoma sequence and to analyze its reversibility by demethylation treatment. Septin 9 protein expression showed significant difference between normal and colorectal cancer (CRC) samples (p < 0,001). According to biopsy microarray results, septin 9 mRNA expression decreased in the progression of colon neoplastic disease (p < 0,001). In laser microdissected epithelial cells, septin 9 significantly underexpressed in CRC compared to healthy controls (p < 0,001). The expression of septin9&#95;v1 region was higher in the healthy samples, while septin9&#95;v2, v4, v4*, v5 overexpression were detected in cancer epithelial cells compared to normal. The septin 9 mRNA and protein levels of HT29 cells increased after demethylation treatment. The increasing methylation of septin 9 gene during colorectal adenoma-dysplasia-carcinoma sequence progression is reflected in the decreasing mRNA and protein expression, especially in the epithelium. These changes can be reversed by demethylation agents converting this screening marker gene into therapeutic target.

Bone marrow derived mesenchymal stem cells (BM-MSCs) take part in the colonic mucosal regeneration. They are multipotent cells, which can be identified with both negative (i.e. CD13, CD 14, CD45, c-Kit, major histocompatibility complex /MHC class I and II) and positive (i.e. CD54 (ICAM1), CD133, CD146 (MCAM), CD166, Flk-1, Sca-1, Thy-1, stage-specific antigen I /SSEA-I and Musashi-1, HLA class I) markers. These cells can repopulate the gastrointestinal mucosa as they may differentiate into stromal- (i.e. myofi-broblast) or epithelial-like (Paneth-, epithel-, goblet or enteroendocrin) cells without proliferation. During the mesenchymal to epithelial transition (MET) stem cells enter the epithelial layer and take up epithelial cell-like properties. Rarely BM-MSCs may retain their stem cell characteristics and are capable of producing progeny. The isolated lymphoid aggregates may serve as a platform from where BM-MSCs migrate to the nearby crypts as mediated by several chemoattractant proteins, which are expressed in injured tissue. The number of BM-MSCs is influenced by the degree of inflammation. In this review we summarize the current information about the role of BM-MSCs in the repair progress of injured colonic epithelium and their potential clinical applications.

A molecular diagnostic assay using easily accessible peripheral blood would greatly assist in the screening and diagnosis of ulcerative colitis (UC) and Crohn's disease (CD). Transcriptional profiles in blood/biopsy samples from 12 UC (6/12), 9 CD (5/9), 6 non-inflammatory bowel disease (non-IBD) colitis (6/0), and 11 healthy (11/11) patients were assessed by Affymetrix HGU133Plus2.0 microarrays. Prediction analysis of microarrays, discriminant and ROC analyses were performed, the results were validated by RT-PCR and immunohistochemistry using also an independent set of samples (15 blood samples, 45 biopsies). A set of 13 transcripts was differentially expressed in IBD, non-IBD controls and healthy blood samples (100% specificity and sensitivity). Validated difference was found in 16 transcripts between UC, non-IBD and normal blood, and 4 transcripts between CD, non-IBD and normal samples. UC and CD blood cases could be also distinguished by 5 genes with 100% specificity and sensitivity. Some disease associated alterations in blood transcripts were also detected in colonic tissue. IBD subtypes may be discriminated from non-IBD (diverticulitis, infective and ischemic colitis) in vitro from peripheral blood by screening for differential gene expression revealed in this study. Transcriptional profile alterations in peripheral blood can be located in diseased colon.

Colorectal cancer progression is characterized by altered epithelial proliferation and apoptosis and by changed expression of tumor development regulators. Our aims were to determine the proliferative/apoptotic epithelial cell ratio (PAR) in the adenoma-dysplasia-carcinoma sequence (ADCS), and to examine its association with osteopontin (OPN), a previously identified protein product related to cancer development. One mm diameter cores from 13 healthy colons, 13 adenomas and 13 colon carcinoma samples were included into a tissue microarray (TMA) block. TUNEL reaction and Ki-67 immunohistochemistry were applied to determine the PAR. The osteopontin protein was also immunodetected. Stained slides were semiquantitatively evaluated using digital microscope and statistically analyzed with logistic regression and Fisher's exact test. The PAR continuously increased along the ADCS. It was significantly (p < 0.001) higher in cancer epithelium (8.84 ± 7.01) than in adenomas (1.40 ± 0.78) and in normal controls (0.89 ± 0.21) (p < 0.001). Also, significant positive correlation was observed between elevated PAR and the expression of osteopontin. Cytoplasmic OPN expression was weak in healthy samples. In contrast, cytoplasmic immunoreaction was moderately intensive in adenomas, while in colon cancer strong, diffuse cytoplasmic immune staining was detected. Increasing PAR and OPN expression along ADCS may help monitoring colorectal cancer progression. The significantly elevated OPN protein levels we found during normal epithelium to carcinoma progression may contribute to the increased fibroblast-myofibroblast transition determining stem cell niche in colorectal cancer.

The gastrointestinal effect of aging, the recognition of its molecular background and the mapping its connections with several diseases like sporadic colorectal cancer of elder people are a new and promising area of molecular gastroenterology. Nowadays, it is a well-known fact that some age-related molecular changes (e.g.: DNA methylation, telomere shortening) can be detected in several types of colorectal cancers. The known epidemiologic and molecular biologic features of sporadic colorectal cancer are not enough to explain the genetic, gene expression or epigenetic changes that may be involved in the increase of the disease over 45-50 age years. The connections of these alterations to the process of aging are also unclear. The understanding and custom-tailored modification of these mechanisms are of great clinical importance regarding of prevention and modern therapeutic strategies. In this review, we aimed to summarize the age-related microscopic and molecular changes of the human colon, as well as their role in the development of colorectal cancer of the elder people.

Unlabelled: Changes of the DNA methylation pattern are proven to be an important process during tumorigenesis. This event can occur in several manners in the tumor microenvironment and there are still not any effective and high-throughput methods for genome-wide analysis of this phenomenon., Aims: Our aim was to identify colorectal cancer development and progression specific marker genes regulated by DNA methylation using gene expression analysis. In this study we present a gene expression-based method combined with a cell culture model, which can be used for a genome-wide analysis of the methylation events during the colorectal tumorigenesis., Materials and Methods: Genes, which expression increased after the demethylation were determined in HT-29 colon adenocarcinoma cells treated with 10 microM 5-aza-2'-deoxycitidine. In parallel, 5000 epithelial cells were collected with laser microdissection (LCM) from normal, adenoma and tumorous colonic samples. The genes with gradually decreasing expression along the adenoma-carcinoma sequence were identified. By comparing the two groups, the transcripts, which are supposed to be regulated by methylation, could be determined. Finally, the identified gene set was validated on independent samples using RT-PCR., Conclusion: The regulation of the identified genes showing decreased expression during the adenoma-carcinoma sequence, can be associated with DNA methylation. On the basis of our results, the set of genes including tumorsuppressors can be determined genome-widely, which can be key factors in the formation and the prognosis of the disease. The identified genes showing colorectal cancer specific methylation pattern can be potential therapeutic targets in the future.

Objective: Mesenchymal-epithelial transition may have crucial role in mucosal regeneration, hence we assayed epithelial growth factor receptor (EGFR), insulin-like growth factor receptor-1 (IGF1R), hepatocyte-derived growth factor receptor (HGFR), CDX2 and cytokeratin (CK) expression in lymphoid aggregates (LA) of ulcerative colitis (UC)., Material and Methods: Tissue microarrays (TMAs) made of biopsy samples from 20 mildly, 20 moderately and 20 severely active UC, 12 non-specific colitis (NSC) and 20 healthy colon were prepared, and immunolabelled with anti-EGFR, -IGF1R, -HGFR, -CDX2, -CK antibodies. After virtual microscopic evaluation, one-way ANOVA and correlation analysis were performed. For validation, TaqMan real-time RT-PCR was performed by using RNA from laser microdissected LA from 10 healthy colon and 10 endoscopically active UC biopsies., Results: The number of LA was in tight positive correlation with the severity of inflammation (r=0.9). The number of EGFR/HGFR positive subepithelial cells was found to be significantly elevated in severe (21.6+/-2.1%/21.3+/-1.9%), moderate (14.3+/-1.7%/14.6+/-1.6%) and mild (7.2+/-1.6%/7.4+/-1.3%) inflammation compared to healthy colon mucosa (2.6+/-1.4%/2.4+/-1.03%) (p < 0.005). Some alterations were found between UC and NSC samples regarding EGFR and HGFR expression. IGF1R immunoreactive cells were only found in a trace number in all cases. Increasing trend of CDX2 and CK positive subepithelial cells was found in active UC, but it was not in significant correlation with the severity of inflammation., Conclusion: EGFR and HGFR positive subepithelial cells in LA may be involved in the induction of the regenerative mucosal processes. The presence of CDX2/CK positive subepithelial cells suggests that mesenchymal-to-epithelial transition may be located to lymphoid aggregates.

The continuous reformation and rapid repair of the colonic mucosa is essential for avoiding the aggregation of pernicious mutations induced by bacterial, toxic, or mitogenic factors. Gut-associated lymphoid tissue is supposed to play a central role in the organization of the repair mechanisms. In inflammatory conditions, the number, the diameter and the density of isolated lymphoid follicles (ILFs) are increasing. They are involved not just in immune surveillance, but their presence is also indispensable in normal mucosal regeneration of the colon. The relation of ILFs to the components of mucosal renewal such as bone marrow derived stem cells, follicular dendritic cells, subepithelial myofibroblasts or crypt formation has not been directly studied, and data about their putative organizer role are scattered in scientific literature. Whether they act as a regenerative pool containing stem cells in case of mucosal damage, or they are responsible only for the optimal cytokine milieu for the differentiation of immigrating stem cells is a question under debate. Our aim is to review the relation of ILFs to the different elements of colonic mucosal repair.

The exact molecular background and the connection between protein and mRNA expression in colorectal cancer (CRC) development and progression are not completely elucidated. Our purposes were the identification of protein markers of colorectal carcinogenesis and progression using protein arrays and validation on tissue microarrays. The connection between antibody and mRNA expression array results was also examined. Using cancerous and adjacent normal samples from 10 patients with early and 6 with advanced CRC, 67 differentially expressed genes were identified between normal and cancerous samples. A marker set containing 6 proteins (CCNA1, AR, TOP1, TGFB, HSP60, ERK1) was developed which could differentiate normal specimen, early and late stage CRC with high sensitivity and specificity. Dukes D stage samples were analyzed on HGU133plus2.0 microarrays. In these samples, mRNA and protein expression of 143 genes showed strong positive correlations (R2>0.8), while a negative correlation (R2>0.9) was found in case of 95 genes. Based on our results a correlation could be established between transcriptome and antibody array results, hence the former may be used as a high-capacity screening method before applying antibody arrays containing already planned targets. Antibody microarrays may have a fundamental importance in testing of marker combinations and future application in diagnostics of tumorous diseases.

Helicobacter pylori is one of the most common pathogens affecting humankind, infecting approximately 50% of the world's population. Of those infected, many will develop asymptomatic gastritis, but 10% develop gastric or duodenal ulcers. The clinical outcome of the infection may involve a combination of bacterial factors, host factors and environmental factors. In the process of development of gastritis, ulceration and cancer, several cellular and molecular steps follow each other. Infection, acid survival, adhesion, cytotoxicity, epithelial cell turnover changes, inflammation, regeneration or pathological alteration towards erosions, ulceration, and cancer can be observed on the cellular level. Bacterial factors like urease, AmiE, AmiF, hydrogenase and arginase are needed for survival in the acidic gastric environment. The bacterial flagellae are essential to move the bacteria towards the epithelial surface. Adhesive factors like BabA, SabA and ureaseA are necessary for adhesion against MHC-II complexes and Le antigens. The bacteria VacA and CagA are cytotoxic factors. The Cag type IV secretion system delivers these proteins inside the epithelial cells. After disruption of epithelial cell junctions, the bacteria can pass through the gastric wall facing direct immune response from neutrophils, lymphocytes, mast cells and dendritic cells. This review describes and summarizes our present molecular biological information and knowledge about the Helicobacter infective component, cell functions and processes. The possible role of host counter responses and interactions with gastric epithelia and immune cells are also detailed., (Copyright © 2010 S. Karger AG, Basel.)

Unlabelled: In the process of regeneration following colon inflammation mesenchymal stem cells (MSC) of bone marrow origin may also take part besides their local counterparts. These cells migrate in the colon epithelium where they may contribute to epithelial regeneration or form progeny for keeping up local stem cell pool. MSC cells probably leave circulation around lymphoid aggregates to then migrate into nearby crypts. During migration they change their phenotype upon the influence of local microenvironment., Aims: In this study epithelial migration and transition of bone marrow stem cells were examined. Samples from normal healthy individuals and from aspecific inflammation were used. The possible role of lymphoid aggregates in the epithelial regeneration was also studied., Materials and Methods: Samples of normal colon (2) and those showing mild aspecific colitis (3) from female patients who were initially transplanted with male bone marrow were studied. First we detected gender chromosomes with fluorescent in situ hybridization (FISH) and the samples were archived with digital scanning. Then CD45 and cytokeratin (CK) double immunofluorescent reactions (IF) were made followed by digitalization again. Digitalized samples were estimated simultaneously with virtual microscopy (Mirax Viewer)., Results: Significant elevation of CD45 negative/Y-FISH positive potential MSCs were found in crypts locating to the neighborhood of lymphoid aggregates (1,075%) compared with both normal (0,027%, p = 0,002) and mild colitis (0,045%, p = 0,004) samples., Conclusion: Local stem cells probably have enough regeneration capacity in case of minor inflammation. However, in aspecific inflammation the number of MSCs contributing to epithelial regeneration was elevated, suggesting their facilitated contribution to the repair process with less probable forming of local stem cell progeny.

Background: The exact molecular biological background of colorectal cancer development and progression are not hitherto known. Using microarray systems, hundreds or thousands of parameters could be examined simultaneously for answering the mentioned questions., Aim: To identify possible protein markers of colorectal cancer development and progression using antibody array, and the validation of these markers on tissue microarrays done with colorectal cancer samples. Furthermore, to determine colorectal cancer diagnostic marker combination in protein level., Materials and Methods: Surgically resected samples from ten Dukes B and six Dukes D stage patients containing both diseased and un-involved parts of the colon were freshly frozen. The samples were homogenized and the extracted proteins were used for Clontech AB500 arrays. Twelve selected genes were validated on tissue microarrays., Results: The expression of 67 proteins was altered (p < 0.05) between the normal colon and cancerous samples. These genes are related to apoptosis (n = 5), cell cycle regulation (n = 7), transcription (n = 4), DNA replication (n = 6) and other cell functions, such as transport and cell adhesion (n = 45). Twelve potential markers were immunohistochemically validated on morphological level by using tissue microarrays (CYCA1, HSP60, TOP1, APC, CBP, ERK, EGFR, C-myc, Cald, DARPP32, MRE11A, AR, EPS8)., Conclusion: Based on these results, validated colorectal cancer development related protein markers are involved in a wide spectrum of cell functions such as apoptosis, cell cycle regulation, and signal transduction. A set of six proteins has been determined, which helps to differentiate between normal specimen, early and late stage colorectal cancer with high sensitivity and specificity.

R(P)- and S(P)-diastereomers of 5'-dimethoxytrityl-thymidine-3'-O-[O-(2-cyanoethyl)-N,N-diisopropyl]-phosphoramidite (T-CED) were separated by silica gel chromatography. Oxidation of both isomers with H(2)O(2), elemental sulfur and selenium, respectively, resulted in the corresponding oxidized analogues in nearly quantitative yields. All reactions were found to proceed with retention of P-configuration. This was confirmed by thorough NMR analysis which, in addition, aimed to study the spectral properties of the diastereomers with special respect to differences in the heteroatom effect of the O, S and Se atoms, double-bonded to the phosphorus, on the vicinal carbon-phosphorus couplings. It was found that the changes in the DeltaJ (=(3)J(P,C4') - (3)J(P,C2')) values were basically induced by the electronegativity of the heteroatoms, rather than differences in the rotational preferences about the C3'-O3' bond. The impact of the benzene solvent on the above couplings is also discussed. The effect of these heteroatoms on the chromatographic (normal and reverse phase HPLC) behavior of the compounds was also investigated and the reverse phase HPLC profiles showed an unambiguous correlation between the electronegativity of the heteroatoms and the chromatographic mobility of the analogues., (2008 Wiley-Liss, Inc.)

Neoplastic progression in Barrett's esophagus (BE) occurs by a multistep process associated with early molecular and morphological changes. This study evaluated cell proliferation and p53 expression and their correlation in the development and progression of esophageal adenocarcinoma. PCNA and p53 expressions were analyzed in biopsy samples by immunohistochemistry including patients with reflux esophagitis, BE, BE with concomitant esophagitis, Barrett's dysplasia, esophageal adenocarcinoma and a control group without any histological changes. Progressive increase in cell proliferation and p53 expression was found in the sequence of malignant transformation of the esophageal mucosa. While cell proliferation was significantly lower in the control group compared with all other groups, there was no increase in p53 expression of esophageal tissues that were negative for dysplasia. Dysplastic BE tissues revealed significantly higher cell proliferation and p53 expression levels compared to BE, reflux esophagitis or BE with concomitant esophagitis. Both, cell proliferation and p53 expression were significantly higher in adenocarcinoma compared to BE or Barrett's dysplasia. Interestingly, while just BE with concomitant esophagitis showed significantly higher p53 expression levels than BE, both, BE with concomitant esophagitis and reflux esophagitis revealed significantly higher cell proliferation levels compared to BE. Alterations of cell proliferation and p53 expression showed a strong correlation. Simultaneous activation of cell proliferation and p53 expression strongly suggest their association with esophageal epithelial tumor genesis and particularly, their specific role in the biology of esophageal adenocarcinoma. Quantification of these parameters in BE is thought to be useful to identify patients at higher risk for progression to adenocarcinoma.

DNA methylation acts in early tumorigenesis. Its detection is possible either from tissue, stool or peripheral blood. Septin 9 is a sensitive methylation marker, which has been studied in several cancers such as breast and ovarian tumors and in neurological or hematological diseases. Septin proteins have an important role from cytoskeleton organisation to development of embryonal pattern. Nowadays intensive researches are going on about the relation between the septin 9 gene hypermethylation and colorectal cancer development.