Quantification of A. fumigatus-specific CD154+ T-cells-preanalytic considerations.

Fungal specific CD154+ T-cells have been described as a biomarker in invasive aspergillosis. The influence of sample storage on the detection of these cells was assessed. Six-hour delay prior to PBMC isolation is associated with an 18% decrease of cell viability and alterations of the cellular composition of the sample. This results in 87% reduction of CD154+ A. fumigatus specific cells due to reduced assay sensitivity and increased background values in unstimulated samples. If prompt cell measurement is not feasible, isolated PBMCs can be frozen (at -20°C and -80°C) and processed later with comparable assay reliability (mean value fresh vs. thawing: 0.126, 0.133; Pearson-Coefficient: 0.962).

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

AIM: 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. METHOD: GITR and MMP-9 were analysed at protein [immunohistochemistry/immunofluorescence (IF)] and messenger RNA level (real-time polymerase chain reaction) in surgical specimens with complicated and non-complicated diverticulitis (n=101). IF double staining and regression analysis were performed for both markers. GITR expression was correlated with clinical data and its usefulness as a diagnostic test was investigated. RESULTS: High GITR expression (≥41%) was observed in the inflammatory infiltrate in complicated diverticulitis, in contrast to non-complicated diverticulitis where GITR expression was low (P<0.001). High GITR expression was significantly associated with steroid use and pulmonary diseases (both P<0.001). MMP-9 expression correlated with GITR expression (R(2) =0.7268, P<0.0001, r=0.85) as demonstrated with IF double-staining experiments. Co-labelling of GITR with CD68, but not CD15, suggested that GITR-expressing cells in diverticulitis are macrophages. GITR expression was superior to C-reactive protein (CRP), white cell count and temperature in distinguishing complicated and non-complicated diverticulitis. CONCLUSIONS: Our results suggest that GITR expression in inflammatory cells might potentially indicate a molecular link between steroid use and complicated forms of acute sigmoid diverticulitis. Increased MMP-9 expression by GITR signalling might explain the morphological changes in the colonic wall of perforated and phlegmonous diverticulitis. Analysis of soluble GITR might be a promising strategy for future research.

Clinical relevance of a new pre-treatment laboratory prognostic index in patients with oral squamous cell carcinoma.

The purpose of this retrospective study was to develop a pre-treatment laboratory prognostic index (LPI) based on laboratory results that might serve as an extension to clinicopathological parameters for prognosis and treatment in patients with oral squamous cell carcinoma (OSCC). Pre-treatment LPI was calculated from C-reactive protein (CRP), hemoglobin (Hb) levels, and count of white blood cells (WBCs) due to significant (P < 0.05) association with locoregional recurrence measured for each parameter by receiver operating characteristic (ROC) curves in 187 patients with OSCC. Positive predictive values (+PV, precision rate) and negative predictive values (-PV) of LPI were measured. Likelihood ratios (LRs) were used to assess how good the pre-treatment LPI diagnostic test is to determine locoregional recurrence of the disease. CRP expression by cancer cells was confirmed by immunocytochemistry and FACS analysis. ROC analysis determined cutoff values for CRP levels, Hb levels, and WBC count and showed significant differences between nonrecurrent and recurrent group of OSCC. On univariate analysis, patients with high pre-treatment LPI (LPI ≥ 2, hazard ratio (HR) = 3.8670, 95% confidence interval (CI) = 2.2518-6.6407, P < 0.0001) had a significant poorer prognosis. Multivariate analysis showed that the most important independent prognostic factor was high pre-treatment LPI (LPI ≥ 2, HR = 3.6450, 95% CI = 2.3964-5.5441, P < 0.0001). Moreover, pre-treatment LPI ≥ 2 showed high probability that locoregional recurrence will be present later (+PV, LPI ≥ 2, 86.4%, 95% CI = 65.1-97.1). High +LR gave an excellent indication for a good quality of the test (LR+, LPI ≥ 2, 12.77, 95% CI = 8.8-18.6). Immunohistochemistry and FACS analysis confirmed inflammatory CRP expression by cancer cells. This study highlights the combination of inflammatory CRP levels, Hb levels, and WBC count as the most important independent prognostic factor in predicting disease recurrence of patients with OSCC. LPI can be used as a pre-treatment inflammatory biomarker that may identify OSCC with a more aggressive biological phenotype of the disease and might be helpful for guiding further post-operative treatment in OSCC.

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 (т = 0.794). Immunofluorescence double staining experiments revealed increased TNFR-1 expression by CD8+ cells. CONCLUSIONS: 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.

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. CONCLUSIONS: 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.