New Insights into the CD133 (Prominin-1) Expression in Mouse and Human Colon Cancer Cells.

Following its discovery as a cancer stem cell marker, CD133 has been widely studied for its role in colorectal tumorigenesis. Indeed, colon cancer remains one of the major causes of cancer-related disease and death worldwide, and there is a strong need for an improvement of current diagnostic, prognostic, and therapeutic strategies. Thus, efforts have been devoted to try to understand whether CD133 might play a role in human colorectal tumorigenesis and might contribute to a better management of colon cancer patients. This chapter reviews the current knowledge on CD133 expression in normal and cancer colon tissues, both in humans and mice, discussing apparently conflicting data reported in the two species. Moreover, a great attention is devoted to the available information regarding the functional role of CD133 in colon cancer cells. Finally, the proposed clinical applications of CD133, as a prognostic and/or predictive marker as well as a target for novel antineoplastic strategies in colorectal cancer, are discussed. Overall, the available data support a potential important role of CD133 as cancer stem cell marker in colon cancer cells and warrant future studies to verify its potential use in the routine clinical management of colon cancer patients.

Identification of Endothelin-1 and NR4A2 as CD133-regulated genes in colon cancer cells.

Several in vitro assays have been proposed to identify cancer stem cells (CSCs), including immunophenotyping, sphere assay and side population (SP) assay. CD133 antigen has been proposed as a CSC marker in colon cancer (CC). However, no functional data are available to date and conflicting results have been reported regarding its role as true CSC marker. Here we set out to identify a molecular signature associated with potential CSC. CD133(+) cells isolated from the CaCo-2 CC cell line were analysed by microarray molecular profiling compared to CD133(-) counterparts. Various differentially expressed genes were identified and the most relevant transcripts found to be over-expressed in CD133(+) cells were evaluated by quantitative RT-PCR in the CD133(+) fractions isolated from several CC cell lines. In the attempt to find a correlation between putative CSCs, isolated by means of CD133 immunophenotyping and the SP approach, we demonstrated a significant enrichment of CD133(+) cells within the SP fraction of CC cells, and comparison of the gene expression profiles revealed that Endothelin-1 (END-1) and nuclear receptor subfamily 4, group A, member 2 (NR4A2) transcripts are highly expressed in both CD133(+) and SP fractions of CC cells. Moreover, depletion of CD133 by siRNA induced a significant attenuation of END-1 and NR4A2 expression levels in CaCo-2 cells, while expression of all three molecules decreased during sodium butyrate-induced differentiation. In conclusion, we have identified a molecular signature associated with potential CSCs and showed for the first time the existence of a functional relationship between CD133, END-1 and NR4A2 expression in colon cancer cells.

Effects of sub-acute exposure to rhodium (as Rh (III) chloride hydrate) on cytokines in female Wistar rats.

Quantitative changes in different cytokines were determined in serum of female Wistar rats exposed to Rhodium (III) chloride hydrate to evaluate its early effects on the immune system. Findings revealed an inhibitory effect of Rh salt since each cytokine, with the exceptions of IL-1α and IL-2 levels observed at the highest doses of exposure, was reduced compared to the controls and interestingly, the lowest doses induced the greatest inhibition. This generalized decrease of cytokine levels was not related to a specific cytokine pathway, and may suggest an anti-inflammatory role of Rh salt.

Evaluation of in vitro toxic effects of cement dusts: a preliminary study.

Cement is widely used for construction and several reports have suggested a potential toxicity of cement dusts although it has never been definitively assessed. To determine the cytotoxic and bioactive effects of cement dusts, cultures of normal rat fibroblasts were exposed to different types of cements and cell growth parameters, apoptosis and the occurrence of DNA damage (both in terms of DNA breaks and oxidative damage) were analyzed. Cells were exposed to cement extracts or cultured in direct contact with cement dusts and the results obtained were compared to cells cultured in fresh medium. A dose-dependent decrease in viable cells was observed with all tested cements. Different results were obtained in the cell-cement direct contact tests compared to the indirect contact tests performed using extracts. Inhibition of cell growth was associated in most cases with an accumulation of cells in the S-phase of the cell-cycle and the appearance of an apoptotic peak. DNA strand breaks, assessed by comet test, and increase in the levels of 8-OHdG, an important marker of DNA oxidative damage, always occurred by incubating cells in the presence of cement extracts or dusts. However, after removal of cement, a rapid damage repair was generally observed with an almost complete recovery within 12 hours. In conclusion, all cements analyzed in this study displayed a limited toxicity in vitro without significant differences amongst them. Overall, the results obtained indicate that cements should be treated as hazardous materials but they do not allow to make accurate predictions regarding the in vivo effects. Further studies are warranted to reach a better understanding of the potential toxic effects of cements, to identify the responsible mechanisms and to evaluate the possibility of modulating and/ or preventing them.

Characterization of mucosal cytokine profile in ulcerative colitis patients under conventional and anti-TNF-a treatment.

OBJECTIVES: Cytokines play a pivotal role in inflammatory bowel disease (IBD). We investigated the expression of inflammatory and regulatory cytokines in inflamed and uninflamed mucosal samples of ulcerative colitis patients. METHODS: Twenty-five ulcerative colitis patients were enrolled. Bioptic samples from inflamed and not inflamed intestinal areas were obtained. Multiplex analysis for inflammatory and regulatory cytokines was performed. Serum C-reactive protein (CRP) was assessed. Endoscopic Mayo score and histological simplified Geboes score were calculated. RESULTS: Interleukin (IL)-1Ra, IL-6, IL-8, IL-17, induced Protein (IP)-10, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1a, MIP-1b resulted increased in ulcerative colitis inflamed vs ulcerative colitis not inflamed areas. No differences were registered between conventional and anti-tumor necrosis factor-a regimens. No difference with CRP levels was found. IL-7 resulted reduced in patients with endoscopic Mayo score ≥2. All the not inflamed samples had a Geboes score <2A, while all the inflamed specimens had a Geboes score ≥2B. IL-1Ra resulted increased in the group with a Geboes score ≥4. CONCLUSIONS: Inflamed and adjacent not inflamed mucosal areas in ulcerative colitis patients share detailed inflammatory molecular pathways, but can be differentiated endoscopically and histologically on the basis of specific cytokines levels. This underlines the complexity of the mucosal cytokine network in ulcerative colitis and highlights the major limitations of a single proinflammatory target therapeutic strategy in IBD.

Muscle Microdialysis to Investigate Inflammatory Biomarkers in Facioscapulohumeral Muscular Dystrophy.

Recent progresses in the understanding of facioscapulohumeral muscular dystrophy (FSHD) genetics opened the way to the development of targeted therapies. However, knowledge about pathophysiology of muscle damage is still limited and there is increasing need to identify biomarkers of disease activity in the perspective of clinical trial readiness.We analyzed inflammatory mediators in the interstitial fluid of muscles with different MRI signal in FSHD patients, comparing muscles displaying early lesions on short-tau inversion recovery (STIR) sequences with normal ones. Patients with one T1-weighted normal and STIR hyperintense (STIR+) and contralateral T1-weighted and STIR normal (STIR-) lower limb muscle were asked to enter the study. Twelve consecutive patients, five controls, and one non-penetrant gene carrier underwent prolonged muscle microdialysis with high cut-off membranes. Microdialysates were analyzed using xMAP technology with a wide panel for cytokines, chemokines, and growth factors. A small number of inflammatory mediators were dysregulated in STIR+ versus STIR- and control muscles: CXCL13, upregulated in STIR+ muscles compared with controls (p < 0.01); CXCL5, downregulated in STIR+ compared with STIR- muscles (p < 0.05); and G-CSF, downregulated in STIR+ muscles compared with controls (p < 0.05). CXCL13 was also upregulated in the STIR+ muscles compared with the contralateral STIR- muscles of the same patient (p < 0.01).These results support the evidence of a selective inflammatory process taking place in STIR+ FSHD muscles. The application of microdialysis could provide insights on novel mechanisms involved in muscle damage in FSHD and in other myopathies. Further studies are needed to validate these investigated molecules as tissue and circulating biomarkers.

Toll-Like Receptor 2 Mediates In Vivo Pro- and Anti-inflammatory Effects of Mycobacterium Tuberculosis and Modulates Autoimmune Encephalomyelitis.

Mycobacteria display pro- and anti-inflammatory effects in human and experimental pathology. We show here that both effects are mediated by Toll-like receptor 2 (Tlr2), by exploiting a previously characterized Tlr2 variant (Met82Ile). Tlr2 82ile promoted self-specific proinflammatory polarization as well as expansion of ag-specific FoxP3(+) Tregs, while Tlr2 82met impairs the expansion of Tregs and reduces the production of IFN-γ and IL-17 proinflammatory cytokines. Preferential dimerization with Tlr1 or Tlr6 could not explain these differences. In silico, we showed that Tlr2 variant Met82Ile modified the binding pocket for peptidoglycans and participated directly to a putative binding pocket for sugars and cadherins. The distinct pro- and anti-inflammatory actions impacted severity, extent of remission, and distribution of the lesions within the central nervous system of experimental autoimmune encephalomyelitis. Thus, Tlr2 has a janus function in vivo as mediator of the role of bacterial products in balancing pro- and anti-inflammatory immune responses.

Exposure to Palladium Nanoparticles Affects Serum Levels of Cytokines in Female Wistar Rats.

BACKGROUND: Information currently available on the impact of palladium on the immune system mainly derives from studies assessing the biological effects of palladium salts. However, in the last years, there has been a notable increase in occupational and environmental levels of fine and ultrafine palladium particles released from automobile catalytic converters, which may play a role in palladium sensitization. In this context, the evaluation of the possible effects exerted by palladium nanoparticles (Pd-NPs) on the immune system is essential to comprehensively assess palladium immunotoxic potential. AIM: Therefore, the aim of this study was to investigate the effects of Pd-NPs on the immune system of female Wistar rats exposed to this xenobiotic for 14 days, by assessing possible quantitative changes in a number of cytokines: IL-1α, IL-2, IL-4, IL-6, IL-10, IL-12, GM-CSF, INF-γ and TNF-α. METHODS: Twenty rats were randomly divided into four exposure groups and one of control. Animals were given a single tail vein injection of vehicle (control group) and different concentrations of Pd-NPs (0.012, 0.12, 1.2 and 12 µg/kg). A multiplex biometric enzyme linked immunosorbent assay was used to evaluate cytokine serum levels. RESULTS: The mean serum concentrations of all cytokines decreased after the administration of 0.012 µg/kg of Pd-NPs, whereas exceeded the control levels at higher exposure doses. The highest concentration of Pd-NPs (12 µg/kg) induced a significant increase of IL-1α, IL-4, IL-6, IL-10, IL-12, GM-CSF and INF-γ compared to controls. DISCUSSION AND CONCLUSIONS: These results demonstrated that Pd-NP exposure can affect the immune response of rats inducing a stimulatory action that becomes significant at the highest administered dose. Our findings did not show an imbalance between cytokines produced by CD4+ T helper (Th) cells 1 and 2, thus suggesting a generalized stimulation of the immune system with a simultaneous activation and polarization of the naïve T cells towards Th1 and Th2 phenotype.

Laparoscopic surgery for colorectal cancer is not associated with an increase in the circulating levels of several inflammation-related factors.

It has been hypothesized that inflammatory response triggered by surgery might induce the release of molecules that could promote proliferation, invasion and metastasis of surviving cancer cells. To test this hypothesis, the levels of multiple inflammation-related circulating factors were analyzed in patients undergoing surgery for colorectal cancer. A Luminex xMAP system was used to simultaneously assess levels of IL-1ß, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, FGF, eotaxin, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, MIP-1ß, PDGF-BB, RANTES, TNF-α and VEGF in 20 colorectal cancer patients and 10 age-matched non-neoplastic patients. In cancer patients analyses were performed at baseline (before surgery) and at different time points (up to 30 days) following laparoscopic surgery. Significantly higher levels of IL-1ß, IL-7, IL-8, G-CSF, IFN-γ and TNF-α were detected in colorectal cancer patients compared to controls at baseline. In colorectal cancer patients, circulating levels decreased progressively following surgery and after day 30 post-surgery were no longer different from controls. These findings suggest that expression levels of several cytokines are higher in colorectal cancer patients compared to control subjects and no significant increase in several inflammation-related circulating factors is observed following laparoscopic surgery for cancer. Confirmation and validation in a different and larger cohort of patients are warranted.

Cancer stem cells in colorectal cancer from pathogenesis to therapy: controversies and perspectives.

Colorectal cancer remains one of the most common and lethal malignancies worldwide despite the use of various therapeutic strategies. A better understanding of the mechanisms responsible for tumor initiation and progression is essential for the development of novel, more powerful therapies. The traditional, so-called "stochastic model" of tumor development, which assumes that each cancer cell is tumorigenic, has been deeply challenged during the past decade by the identification of cancer stem cells (CSCs), a biologically distinct subset of cells within the bulk of tumor mass. This discovery led to the development of the hierarchical model of tumorigenesis which assumes that only CSCs have the ability to initiate tumor growth, both at primary and metastatic sites. This model implies that the elimination of all CSCs is fundamental to eradicate tumors and that failure to do so might be responsible for the occurrence of relapses and/or metastases frequently observed in the clinical management of colorectal cancer patients. Identification and isolation of CSCs is essential for a better understanding of their role in the tumorigenetic process and for the development of CSC-specific therapies. Several methods have been used for this purpose and many efforts have been focused on the identification of specific CSC-surface markers. This review provides an overview of the proposed roles of CSC in human colorectal tumorigenesis focusing on the most important molecules identified as CSC-specific markers in colorectal cancer and on the potential strategies for the development of CSC-targeted therapy.

HIV-infected patients show impaired cellular immune response to influenza vaccination compared to healthy subjects.

Detailed data on cellular immune response to influenza vaccination in HIV-infected patients are lacking. We analyzed cellular (IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IFN-γ, TNF-α, GM-CSF) and humoral (IgG and IgM) immune response in 81 HIV-infected and 30 HIV-negative subjects, before (T0) and 4 weeks (T1) after receiving a single dose of trivalent MF59-adjuvanted influenza vaccine. No difference in humoral response (IgG or IgM) was demonstrated between the two groups. While an increase in most cytokines from T0 to T1 was observed in HIV-uninfected subjects, cytokines production did not significantly increased in HIV-infected patients. Exploring Th1 response, higher CD8 cells count was significantly associated with lower post-vaccination IFNγ levels, while a higher CD4 cells count was associated with a greater response. Exploring Th2 response, higher HIV viral load was significantly associated with reduced post-vaccination IL-10 levels. In conclusion, in HIV-infected patients influenza vaccination could have good efficacy in sustaining humoral response but cellular response appeared impaired.

Rhodium and iridium salts inhibit proliferation and induce DNA damage in rat fibroblasts in vitro.

Environmental concentration of the platinum group elements is increased in the last years due to their use in automobile catalytic converters. Limited data are available on the effects of such elements at a cellular level and on their toxicity, especially for rhodium and iridium which have been more recently introduced in use. The toxic effects of rhodium and iridium salts were analyzed on a normal diploid rat fibroblast cell line in vitro. Both salts halted cell growth in a dose- and time-dependent fashion by inhibiting cell cycle progression, inducing apoptosis and modulating the expression of cell cycle regulatory proteins. In fact, they both caused an accumulation of cells in the G2/M phase of the cell cycle and affected the expression levels of pRb, cyclins D1 and E, p21(Waf1) and p27(Kip1). DNA strand breaks, as assessed by comet test, and an increase in the intracellular levels of reactive oxygen species also occurred in exposed cell cultures. These findings suggest a potential toxicity of both iridium and rhodium salts and emphasize the need for further studies to understand their effects at a cellular level to enable a better assessment of their toxic effects and to identify ways for their modulation and/or prevention.