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Purpose: In HT29 colon cancer cells, a close interplay between self-DNA-induced TLR9 signaling and autophagy response was found, with remarkable effects on cell survival and differentiation. IGF1R activation drives the development and malignant progression of colorectal cancer. IGF1R inhibition displays a controversial effect on autophagy. The interrelated roles of IGF1R inhibition and TLR9/autophagy signaling in HT29 cancer cells have not yet been clarified. In our study, we aimed to investigate the complex interplay of IGF1R inhibition and TLR9/autophagy signaling in HT29 cells. Methods: HT29 cells were incubated with tumor-originated self-DNA with or without inhibitors of IGF1R (picropodophyllin), autophagy (chloroquine), and TLR9 (ODN2088), respectively. Cell proliferation and metabolic activity measurements, direct cell counting, NanoString and Taqman gene expression analyses, immunocytochemistry, WES Simple Western blot, and transmission electron microscopy investigations were performed. Results: The concomitant use of tumor-derived self-DNA and IGF1R inhibitors displays anti-proliferative potential, which can be reversed by parallel TLR9 signaling inhibition. The distinct effects of picropodophyllin, ODN2088, and chloroquine per se or in combination on HT29 cell proliferation and autophagy suggest that either the IGF1R-associated or non-associated autophagy machinery is "Janus-faced" regarding its actions on cell proliferation. Autophagy, induced by different combinations of self-DNA and inhibitors is not sufficient to rescue HT29 cells from death but results in the survival of some CD133-positive stem-like HT29 cells. Conclusion: The creation of new types of combined IGF1R, autophagy, and/or TLR9 signaling inhibitors would play a significant role in the development of more personalized anti-tumor therapies for colorectal cancer., Competing Interests: Author BW was employed by the company Lufthansa Systems Hungária. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sipos, Bohusné Barta, Simon, Nagy, Dankó, Raffay, Petővári, Zsiros, Wichmann, Sebestyén and Műzes.)

In relation of immunobiology, the consequence of the crosstalk between TLR9-signaling and autophagy is poorly documented in HT29 cancer cells. To assess the TLR9-mediated biologic effects of modified self-DNA sequences on cell kinetics and autophagy response HT29 cells were incubated separately with intact genomic (g), hypermethylated (m), fragmented (f), and hypermethylated/fragmented (m/f) self-DNAs. Cell viability, apoptosis, cell proliferation, colonosphere-formation were determined. Moreover, the relation of TLR9-signaling to autophagy response was assayed by real-time RT-PCR, immunocytochemistry and transmission electron microscopy (TEM). After incubation with g-, m-, and m/f-DNAs cell viability and proliferation decreased, while apoptosis increased. F-DNA treatment resulted in an increase of cell survival. Methylation of self-DNA resulted in decrease of TLR9 expression, while it did not influence the positive effect of DNA fragmentation on MyD88 and TRAF6 overexpression, and TNFα downregulation. Fragmentation of DNA abrogated the positive effect of methylation on IRAK2, NFκB and IL-8 mRNA upregulations. In case of the autophagy genes and proteins, g- and f-DNAs caused significant upregulation of Beclin1, Atg16L1, and LC3B. According to TEM analyses, autophagy was present in each group of tumor cells, but to a varying degree. Incubation with m-DNA suppressed tumor cell survival by inducing features of apoptotic cell death, and activated mitophagy. F-DNA treatment enhanced cell survival, and activated macroautophagy and lipophagy. Colonospheres were only present after m-DNA incubation. Our data provided evidence for a close existing interplay between TLR9-signaling and the autophagy response with remarkable influences on cell survival in HT29 cells subjected to modified self-DNA treatments.

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.

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.

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.

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.

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.

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 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.

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.

It has been proposed that matrix metalloproteinases (MMPs) play a role in tumor invasion. We determined protein expression of matrix metalloproteinase-9 (MMP-9) in colorectal cancer (CRC), corresponding normal mucosa and colorectal adenomas. For confirmation of immunohistochemical results MMP-9 TaqMan RT-PCR analysis was performed. Expression of MMP-9 was determined on paraffin embedded biopsy sections by immunohistochemistry in 31 CRC patients (from cancer tissue and corresponding normal mucosa) and in 30 patients with adenoma (nine adenomas with high grade of dysplasia). MMP-9 immunostaining was determined semi-quantitatively. For Taqman RT-PCR analyses normal mucosa (n = 5), adenoma without (n = 6) and with high grade dysplasia (n = 7) and CRC (n = 10) were investigated. Statistical analysis with ANOVA, LSD test and correlation analysis were performed. P value of <0.05 was considered significant. The MMP-9 expression in CRC was significantly higher compared to adenomas or the normal mucosa (P = 0.001). Significantly higher expression of MMP-9 has been observed in adenomas with high grade dysplasia compared to other adenomas or normal colon (P < 0.001). Diffuse strong MMP-9 expression was present in tumor as well as in stromal cells. In adenoma samples, dysplastic epithelial cells showed moderate intensive cytoplasmic MMP-9 expression, with a clear-cut differentiation between dysplastic and non-dysplastic areas. Staining intensity correlated with the grade of CRC. We demonstrate a significantly higher expression of MMP-9 in adenoma with high grade dysplasia-CRC sequence as compared to normal tissue. The over-expression of MMP-9 strongly suggests its association with colorectal carcinogenesis.