Maria Lazariotou

Expression of Foxp3 in Colorectal Cancer but Not in Treg Cells Correlates with Disease Progression in Patients with Colorectal Cancer

Background Regulatory T cells (Treg) expressing the transcription factor forkhead-box protein P3 (Foxp3) have been identified to counteract anti-tumor immune responses during tumor progression. Besides, Foxp3 presentation by cancer cells itself may also allow them to evade from effector T-cell responses, resulting in a survival benefit of the tumor. For colorectal cancer (CRC) the clinical relevance of Foxp3 has not been evaluated in detail. Therefore the aim of this study was to study its impact in colorectal cancer (CRC). Methods and Findings Gene and protein analysis of tumor tissues from patients with CRC was performed to quantify the expression of Foxp3 in tumor infiltrating Treg and colon cancer cells. The results were correlated with clinicopathological parameters and patients overall survival. Serial morphological analysis demonstrated Foxp3 to be expressed in cancer cells. High Foxp3 expression of the cancer cells was associated with poor prognosis compared to patients with low Foxp3 expression. In contrast, low and high Foxp3 level in tumor infiltrating Treg cells demonstrated no significant differences in overall patient survival. Conclusions Our findings strongly suggest that Foxp3 expression mediated by cancer cells rather than by Treg cells contribute to disease progression.

LgR5 expression and cancer stem cell hypothesis: clue to define the true origin of esophageal adenocarcinomas with and without Barrett's Esophagus?

BackgroundInvestigation of the expression of an intestinal stem cell marker in esophageal adenocarcinomas (EAC) with and without Barretts Esophagus (BE), with respect to a cancer stem cell (CSC) hypothesis.Materials and methodsExpression of a putative intestinal stem cell marker LgR5 was analyzed in esophageal cancer specimen (n = 70: 41 EAC with BE, 19 EAC without BE, and n = 10 esophageal squamous-cell carcinomas, ESCC) and in the adenocarcinoma cell line OE-33. Ki-67 and Cdx-2 were co-labelled with LgR5 in double staining experiments. Immunhistochemical expression results were confirmed by RT-PCR and correlated with tumor stage and five-year survival rates.ResultsLgR5was found expressed in 35 of 41 (85%) EAC with BE and in 16 of 19 (81%) EAC without BE. By contrast, LgR5 was not found to be expressed in ESCC. Quantification of immunolabeling showed 15% LgR5+ cells in EAC with BE, 32% LgR5+ cells in adjacent BE and 13% in EAC without BE. Immunofluorescence double staining experiments with LgR5 and Ki-67 revealed a subpopulation (~5%) of proliferating LgR+/Ki-67+ cells. On mRNA-level, expression of LgR5 was higher in BE in comparison to EAC (p = 0.0159). High levels of LgR5 expression in BE associated EAC were associated with poorer survival in univariate analysis.ConclusionThe stem cell marker LgR5 is expressed in EAC, irrespective of association with BE, and appears to have negative impact on survival. The subset of proliferating LgR5+ cells (<5%) might resemble rapidly cycling CSCs, which needs to be substantiated in further investigations.

Tumor necrosis factor-α is associated with positive lymph node status in patients with recurrence of colorectal cancer-indications for anti-TNF-α agents in cancer treatment.

Introduction: The progressive growth of malignancies is accompanied by a decline in the immune response through mechanisms which are poorly understood. Apoptosis and induction of inflammation by tumor released cytokines as tumor escape mechanisms have been proposed to play an important role in colorectal carcinogenesis. Methods: Expression of Tumor necrosis factor-alpha (TNF-α) was analyzed in colorectal cancer specimen and the cancer cell line HT-29 by immunohistochemistry and RT-PCR. TNF-α expression on protein and mRNA level were correlated with clinical characteristics and impact on survival. TNFR-1 was co-labelled with TNF-α and CD8+ cytotoxic T cells in immunofluorescence double staining experiments. Results: 94% (n=98/104) of the patients with CRC expressed TNF-α. High TNF-α expression was significantly associated with positive lymph node stage and recurrence of the tumor. Multivariate analysis revealed high TNF-α expression as an independent prognostic factor. Immunohistochemistry was correlated with RT-PCR results (τ=0.794). Immunofluorescence double staining experiments revealed increased TNFR-1 expression by CD8+ cells. Conclusion: TNF-α expression by tumor cells may be an efficient immunological escape mechanism by inflammation-enhanced metastases and probably by induction of apoptosis in tumor-infiltrating CD8+ immune cells resulting in a down regulation of the tumoral immune response. Our data support the role of tumor-derived TNF-α expression as an important promoter of tumoral immune escape mechanisms and malignant progression, and suggest that analysis on either protein (immunohistochemistry) or RNA level (RT-PCR) can be used effectively in this respect. Targeting TNF-α may be a promising option, especially in cases with high TNF-α expression and positive lymph node metastases.

MMP-1 is a (pre-)invasive factor in Barrett-associated esophageal adenocarcinomas and is associated with positive lymph node status

BackgroundEsophageal adenocarcinomas (EACs) arise due to gastroesophageal reflux, with Barretts esophagus (BE) regarded as precancerous lesion. Matrix metalloproteinases (MMPs) might play a role during the multistep carcinogenetic process.MethodsExpression of MMP-1 and -13 was analyzed in esophageal cancer (n = 41 EAC with BE, n = 19 EAC without BE, and n = 10 esophageal squamous-cell carcinomas, ESCC), furthermore in BE without intraepithelial neoplasia (IN) (n = 18), and the cell line OE-33. MMP-1 was co-labelled with Ki-67 (proliferation), Cdx-2 (marker for intestinal metaplasia, BE) and analyzed on mRNA level. MMP-1 staining results were correlated with clinicopatholocical parameters.ResultsOn protein level, MMP-1 expression was found in 39 of 41 (95%) EAC with BE, in 19 of 19 (100%) EAC without BE, in 6 of 10 (60%) ESCC, and in 10 of 18 (56%) BE without IN. No expression of MMP-13 was found in these specimens. Quantification showed 48% MMP-1 positive cells in EAC with BE, compared to 35% in adjacent BE (p < 0.05), 44% in EAC without BE, 32% in ESCC, and 4% in BE without IN. Immunofluorescence double staining experiments revealed increased MMP-1 expressing in proliferating cells (MMP-1+/Ki-67+) (r = 0.943 for BE and r = 0.811 for EAC). On mRNA-level, expression of MMP-1 was significantly higher in EAC compared to BE (p = 0.01) and confirmed immunohistochemical staining results. High MMP-1 levels were associated with lymph node metastases but not with poorer survival (p = 0.307).ConclusionsOur findings suggest that MMP-1 plays a role as preinvasive factor in BE-associated EAC. Expression of MMP-1 in proliferating BE and EAC cells suggest malignant proliferation following the clonal expansion model.

Hyperthermic Intraperitoneal Chemotherapy in Patients with Peritoneal Carcinomatosis: Role of Heat Shock Proteins and Dissecting Effects of Hyperthermia

BackgroundIn patients with isolated peritoneal carcinomatosis (PC) of gastrointestinal cancer, hyperthermic intraperitoneal chemotherapy (HIPEC) represents a promising treatment option integrated into multimodal concepts. Heat shock proteins (HSP) seem to play a major role in cellular stress during HIPEC therapy. We analyzed differentially hyperthermic conditions and HSPs responsible for cell stress–mediated repair mechanisms in tumor tissues from patients who underwent HIPEC therapy and in an in vitro hyperthermic model.MethodsTumor tissues from our patient cohort with isolated PC were selected for further analysis when representative material was available before and after HIPEC therapy. To further dissect the role of HSPs under conditions of hyperthermia, gene and protein expression was additionally determined, together with cellular apoptosis and proliferation in human HT-29 colon cancer cells.ResultsDifferently up-regulated HSP70/72 and HSP90 gene and protein expression was found in all investigated patient tumors. In vitro studies confirmed observations from clinical tumor analysis as underlying HSP-mediated cell stress mechanisms. Moreover, results from proliferation and apoptosis assays combined with differentiated HSP expression analysis demonstrated the relevance of preselecting specific target temperatures to achieve optimal toxic effects on remaining tumor cells in vivo.ConclusionsTherapeutic approaches like HIPEC to achieve antiproliferative and apoptosis-inducing cellular effects in patients with PC are negatively influenced by highly conserved HSP mechanisms in tumor cells. This study shows for the first time that specific hyperthermic conditions are necessary to be established to achieve optimal toxic effects on tumor cells during HIPEC therapy, a finding that opens potentially new therapeutic strategies.

Allergic Predisposition, Histamine and Histamine Receptor Expression (H1R, H2R) Are Associated with Complicated Courses of Sigmoid Diverticulitis

BackgroundWe aimed to evaluate our hypothesis that allergic predisposition and expression of histamine receptors might contribute to complicated courses of sigmoid diverticulitis.MethodsExpression of histamine and histamine receptors (H1R, H2R) was analysed on protein level (immunohistochemistry/immunofluorescence (IF)) as well as mRNA level (reverse transcription–PCR (RT-PCR) in surgical specimen of patients (nu2009=u2009101) having undergone resection for sigmoid diverticulits (nu2009=u200957 complicated diverticulitis/nu2009=u200944 non-complicated diverticulitis).ResultsThe mean number of comorbid diseases per patient was 1.76u2009±u20091.25. Thirty-nine of 101 patients (38.6%) exhibited allergic predisposition (grass poll, food, drug, pets, etc.). Comorbid diseases were significantly associated with complicated diverticulitis (pu2009=u20090.027). Complicated sigmoid diverticulitis was significantly associated with high H1R and H2R expression (pu2009<u20090.001). Furthermore, an association of complicated diverticulitis with allergic predisposition was found (odds ratiou2009=u20093.2, pu2009=u20090.0097). IF double-labelling experiments showed a strong correlation of increased histamine expression with expression of H1R and H2R on intestinal enterocytes (histamine/H1R, rhou2009=u20090.841, pu2009<u20090.0001 and histamine/H2R, rhou2009=u20090.806, pu2009<u20090.0001). The results of increased H1R and H2R expression in complicated sigmoid diverticulitis were also detected on mRNA level in a subset of patients (RT-PCR, pu2009=u20090.009).ConclusionsOur findings suggest that allergic predisposition might be another important risk factor for complicated courses of acute sigmoid diverticulitis and linked with histamine receptor expression. Supportive therapies with antihistaminic drugs might become an option. Allergic predisposition might be worth considering when indicating surgery for sigmoid diverticulitis.

Glucocorticoid-induced tumour necrosis factor receptor expression: a potential molecular link between steroid intake and complicated diverticulitis?

Aimu2002 Immunosuppression and steroid medication have been identified as risk factors for complicated sigmoid diverticulitis. The underlying molecular mechanisms have not yet been elucidated. We hypothesized that glucocorticoid‐induced tumour necrosis factor receptor (GITR) and matrix metalloproteinase‐9 (MMP‐9) might play a role.

Preoperative serum h-FABP concentration is associated with postoperative incidence of acute kidney injury in patients undergoing cardiac surgery

BackgroundFatty acid binding protein (FABP) is an intracellular transport protein associated with myocardial damage size in patients undergoing cardiac surgery. Furthermore, elevated FABP serum concentrations are related to a number of common comorbidities, such as heart failure, chronic kidney disease, diabetes mellitus, and metabolic syndrome, which represent important risk factors for postoperative acute kidney injury (AKI). Data are lacking on the association between preoperative FABP serum level and postoperative incidence of AKI.MethodsThis prospective cohort study investigated the association between preoperative h-FABP serum concentrations and postoperative incidence of AKI, hospitalization time and length of ICU treatment. Blood samples were collected according to a predefined schedule. The AKI Network definition of AKI was used as primary endpoint. All associations were analysed using descriptive and univariate analyses.ResultsBetween 05/2009 and 09/2009, 70 patients undergoing cardiac surgery were investigated. AKI was observed in 45 patients (64%). Preoperative median (IQR) h-FABP differed between the AKI group (2.9 [1.7–4.1] ng/ml) and patients without AKI (1.7 [1.1–3.3] ng/ml; pu2009=u20090.04), respectively. Patients with AKI were significantly older. No statistically significant differences were found for gender, type of surgery, operation duration, CPB-, or X-Clamp time, preoperative cardiac enzymes, HbA1c, or CRP between the two groups. Preoperative h-FABP was also correlated with the length of ICU stay (rsu2009=u20090.32, pu2009=u20090.007).ConclusionsWe found a correlation between preoperative serum h-FABP and the postoperative incidence of AKI. Our results suggest a potential role for h-FABP as a biomarker for AKI in cardiac surgery.

Inhibition of nuclear translocation of calcineurin suppresses T-cell activation and prevents acute rejection of donor hearts.

Background. Inhibition of calcineurin (CnA) activity by cyclosporine A (CsA) is the mainstay in immunosuppressive therapy. CsA inhibits the phosphatase activity of the cytosolic phosphatase CnA and, therefore, prevents the dephosphorylation and subsequently nuclear translocation of the transcription factor nuclear factor of activated T cells (NFAT). However, CsA has multiple other targets within the cell and is, therefore, not specific. We developed a new approach to inhibit CnA/NFAT signaling. This synthetic peptide prevented CnA nuclear translocation in vitro. The purpose of this study was to demonstrate that this novel approach could potentially inhibit T-cell function in vitro and in vivo. Methods. T-cell activation (Jurkat T cells, naïve rat T cells, and peripheral human T cells) was assessed by protein synthesis, interleukin (IL)-2 promoter activity, and IL-2 levels after T-cell activation. Immunohistological stainings for CnA were performed to investigate nuclear localization of CnA. The immunosuppressive effects in vivo of the synthetic peptide were investigated in rats with heterotopic transplanted hearts. Results. The nuclear localization signal peptide significantly decreased alloantigen-specific T-lymphocyte proliferation, IL-2 promoter activity, and IL-2 production (338%±27% vs. 149%±11%, n=8, P<0.05) in cultured T cells by inhibition of CnA nuclear translocation. The synthetic peptide also significantly decreased the number of graft infiltrating CD8+ T lymphocytes. Moreover, treatment with the synthetic inhibitory inhibited acute graft rejection (5±0.6 days vs. 12±2 days, n=10, P<0.05). Conclusions. Inhibition of nuclear translocation of CnA is a novel approach to inhibit the activation of the CnA/NFAT signaling cascade. Further studies have to demonstrate the long-term use of this principle in vivo.

Heterotopic Rat Heart Transplantation (Lewis to F344): Early ICAM-1 Expression After 8 Hours of Cold Ischemia

BACKGROUNDnPrimary graft dysfunction is a still poorly understood complication after cardiac transplantation. Ischemia/reperfusion injury contributes to different disorders resulting in impaired graft function.nnnMETHODSnIn a heterotopic rat heart transplantation model we extended graft ischemic time up to 8 hours.nnnRESULTSnUsing immunohistochemistry we detected an up to 4-fold increase in intracellular adhesion molecule-1 (ICAM-1) expression during 4 hours of reperfusion, independent of ischemic time (30-minute ischemia: 7.65 +/- 2.15 without reperfusion, 19.46 +/- 4.6 after 4-hour reperfusion; 240-minute ischemia: 5.6 +/- 1.99 and 22.3 +/- 3.77; 480-minute ischemia: 3.7 +/- 1.56 and 13.1 +/- 2.2). Eight-hour ischemic allografts had an increase in CD8-positive cells (1.37 +/- 0.5 and 2.3 +/- 0.77) and a significant increase in MHC II expression (11.48 +/- 2.1 and 18.27 +/- 1.34) during 4 hours of reperfusion.nnnCONCLUSIONSnWe hypothesize that these findings reflect an early inflammatory reaction in the allograft possibly triggered by oxidative stress. During therapeutic interventions, both of these pathways must be considered.