Differentiation Potential of Pancreatic Fibroblastoid Cells/Stellate Cells: Effects of Peroxisome Proliferator-Activated Receptor Gamma Ligands

Pancreatic stellate cells have been investigated mostly for their activation process, supposed to support the development of pancreatic disease. Few studies have been presented on reversal of the activation process in vitro. Thiazolidinediones (TZDs) have been used as antidiabetics and have now been reported to exert antifibrotic activity. We tested effects of natural and synthetic ligands of peroxisome proliferator-activated receptor gamma (PPARγ) on human pancreatic fibroblastoid cells (hPFCs) in search for specificity of action. Ciglitazone, as a prototype of TZDs, was shown to have reversible growth inhibitory effects on human pancreatic fibroblastoid cells/stellate cells. Cells treated with ciglitazone for three days showed enhanced lipid content and induction of proteins involved in lipid metabolism. Collagen synthesis was reduced in hPFC. Interaction of PPARγ with DNA binding sites upon ligand binding was shown by gel shift analysis. These findings point toward a potential for adipocyte differentiation in human pancreatic fibroblastoid cells

Randomized controlled phase I/II study to investigate immune stimulatory effects by low dose radiotherapy in primarily operable pancreatic cancer

<p>Abstract</p> <p>Background</p> <p>The efficiencies of T cell based immunotherapies are affected by insufficient migration and activation of tumor specific effector T cells in the tumor. Accumulating evidence exists on the ability of ionizing radiation to modify the tumor microenvironment and generate inflammation. The aim of this phase I/II clinical trial is to evaluate whether low dose single fraction radiotherapy can improve T cell associated antitumor immune response in patients with pancreatic cancer.</p> <p>Methods/Design</p> <p>This trial has been designed as an investigator initiated; prospective randomised, 4-armed, controlled Phase I/II trial. Patients who are candidates for resection of pancreatic cancer will be randomized into 4 arms. A total of 40 patients will be enrolled. The patients receive 0 Gy, 0.5 Gy, 2 Gy or 5 Gy radiation precisely targeted to their pancreatic carcinoma. Radiation will be delivered by external beam radiotherapy using a 6 MV Linac with IMRT technique 48 h prior to the surgical resection. The primary objective is the determination of an active local external beam radiation dose, leading to tumor infiltrating T cells as a surrogate parameter for antitumor activity. Secondary objectives include local tumor control and recurrence patterns, survival, radiogenic treatment toxicity and postoperative morbidity and mortality, as well as quality of life. Further, frequencies of tumor reactive T cells in blood and bone marrow as well as whole blood cell transcriptomics and plasma-proteomics will be correlated with clinical outcome. An interim analysis will be performed after the enrolment of 20 patients for safety reasons. The evaluation of the primary endpoint will start four weeks after the last patient's enrolment.</p> <p>Discussion</p> <p>This trial will answer the question whether a low dose radiotherapy localized to the pancreatic tumor only can increase the number of tumor infiltrating T cells and thus potentially enhance the antitumor immune response. The study will also investigate the prognostic and predictive value of radiation-induced T cell activity along with transcriptomic and proteomic data with respect to clinical outcome.</p> <p>Trial registration</p> <p>ClinicalTrials.gov - <a href="http://www.clinicaltrials.gov/ct2/show/NCT01027221">NCT01027221</a></p

A randomized controlled trial to investigate the influence of low dose radiotherapy on immune stimulatory effects in liver metastases of colorectal cancer

<p>Abstract</p> <p>Background</p> <p>Insufficient migration and activation of tumor specific effector T cells in the tumor is one of the main reasons for inadequate host anti-tumor immune response. External radiation seems to induce inflammation and activate the immune response. This phase I/II clinical trial aims to evaluate whether low dose single fraction radiotherapy can improve T cell associated antitumor immune response in patients with colorectal liver metastases.</p> <p>Methods/Design</p> <p>This is an investigator-initiated, prospective randomised, 4-armed, controlled Phase I/II trial. Patients undergoing elective hepatic resection due to colorectal cancer liver metastasis will be enrolled in the study. Patients will receive 0 Gy, 0.5 Gy, 2 Gy or 5 Gy radiation targeted to their liver metastasis. Radiation will be applied by external beam radiotherapy using a 6 MV linear accelerator (Linac) with intensity modulated radiotherapy (IMRT) technique two days prior to surgical resection. All patients admitted to the Department of General-, Visceral-, and Transplantion Surgery, University of Heidelberg for elective hepatic resection are consecutively screened for eligibility into this trial, and written informed consent is obtained before inclusion. The primary objective is to assess the effect of active local external beam radiation dose on, tumor infiltrating T cells as a surrogate parameter for antitumor activity. Secondary objectives include radiogenic treatment toxicity, postoperative morbidity and mortality, local tumor control and recurrence patterns, survival and quality of life. Furthermore, frequencies of systemic tumor reactive T cells in blood and bone marrow will be correlated with clinical outcome.</p> <p>Discussion</p> <p>This is a randomized controlled patient blinded trial to assess the safety and efficiency of low dose radiotherapy on metastasis infiltrating T cells and thus potentially enhance the antitumor immune response.</p> <p>Trial registration</p> <p>ClinicalTrials.gov: <a href="http://www.clinicaltrials.gov/ct2/show/NCT01191632">NCT01191632</a></p

Inhibition of tyrosine kinase receptors by SU6668 promotes abnormal stromal development at the periphery of carcinomas

Dynamic contrast-enhanced (albumin-Gd-DTPA) magnetic resonance imaging, performed during 2 weeks of daily administration of an inhibitor of tyrosine kinase receptors (SU6668) in an HT-29 colon carcinoma model, revealed the onset of a hyper-enhancing rim, not observed in untreated tumours. To account for tissue heterogeneity in the quantitative analysis, we segmented tumours into three subunits automatically identified by cluster analysis of the enhancement curves using a k-means algorithm. Transendothelial permeability (Kps) and fractional plasma volume (fPV) were calculated in each subunit. An avascular and necrotic region, an intermediate zone and a well-vascularised periphery were reliably identified. During untreated tumour growth, the identified sub-regions did not substantially change their enhancement pattern. Treatment with SU6668 induced major changes at tumour periphery where a significant increase of Kps and fPV was observed with respect to control tumours. Histology revealed a sub-capsular layer composed of hyper-dense viable tumour cells in the periphery of untreated tumours. The rim of viable neoplastic cells was reduced in treated tumours, and replaced by loose connective tissue characterised by numerous vessels, which explains the observed hyper-enhancement. The present data show a peripheral abnormal development of cancer-associated stroma, indicative of an adaptive response to anti-angiogenic treatment

Treatment of non-small cell lung cancer with intensity-modulated radiation therapy in combination with cetuximab: the NEAR protocol (NCT00115518)

BACKGROUND: Even today, treatment of Stage III NSCLC still poses a serious challenge. So far, surgical resection is the treatment of choice. Patients whose tumour is not resectable or who are unfit to undergo surgery are usually referred to a combined radio-chemotherapy. However, combined radio-chemotherapeutic treatment is also associated with sometimes marked side effects but has been shown to be more efficient than radiation therapy alone. Nevertheless, there is a significant subset of patients whose overall condition does not permit administration of chemotherapy in a combined-modality treatment. It could be demonstrated though, that NSCLCs often exhibit over-expression of EGF-receptors hence providing an excellent target for the monoclonal EGFR-antagonist cetuximab (Erbitux(®)) which has already been shown to be effective in colorectal as well as head-and-neck tumours with comparatively mild side-effects. METHODS/DESIGN: The NEAR trial is a prospective phase II feasibility study combining a monoclonal EGF-receptor antibody with loco-regional irradiation in patients with stage III NSCLC. This trial aims at testing the combination's efficacy and rate of development of distant metastases with an accrual of 30 patients. Patients receive weekly infusions of cetuximab (Erbitux(®)) plus loco-regional radiation therapy as intensity-modulated radiation therapy. After conclusion of radiation treatment patients continue to receive weekly cetuximab for 13 more cycles. DISCUSSION: The primary objective of the NEAR trial is to evaluate toxicities and feasibility of the combined treatment with cetuximab (Erbitux(®)) and IMRT loco-regional irradiation. Secondary objectives are remission rates, 3-year-survival and local/systemic progression-free survival

Randomized phase II – study evaluating EGFR targeting therapy with Cetuximab in combination with radiotherapy and chemotherapy for patients with locally advanced pancreatic cancer – PARC: study protocol [ISRCTN56652283]

BACKGROUND: Pancreatic cancer is the fourth commonest cause of death from cancer in men and women. Advantages in surgical techniques, radiation therapy techniques, chemotherapeutic regimes, and different combined-modality approaches have yielded only a modest impact on the prognosis of patients with pancreatic cancer. Thus there is clearly a need for additional strategies. One approach involves using the identification of a number of molecular targets that may be responsible for the resistance of cancer cells to radiation or to other cytotoxic agents. As such, these molecular determinants may serve as targets for augmentation of the radiotherapy or chemotherapy response. Of these, the epidermal growth factor receptor (EGFR) has been a molecular target of considerable interest and investigation, and there has been a tremendous surge of interest in pursuing targeted therapy of cancers via inhibition of the EGFR. METHODS/DESIGN: The PARC study is designed as an open, controlled, prospective, randomized phase II trial. Patients in study arm A will be treated with chemoradiation using intensity modulated radiation therapy (IMRT) combined with gemcitabine and simultaneous cetuximab infusions. After chemoradiation the patients receive gemcitabine infusions weekly over 4 weeks. Patients in study arm B will be treated with chemoradiation using intensity modulated radiation therapy (IMRT) combined with gemcitabine and simultaneous cetuximab infusions. After chemoradiation the patients receive gemcitabine weekly over 4 weeks and cetuximab infusions over 12 weeks. A total of 66 patients with locally advanced adenocarcinoma of the pancreas will be enrolled. An interim analysis for patient safety reasons will be done one year after start of recruitment. Evaluation of the primary endpoint will be performed two years after the last patient's enrolment. DISCUSSION: The primary objective of this study is to evaluate the feasibility and the toxicity profile of trimodal therapy in pancreatic adenocarcinoma with chemoradiation therapy with gemcitabine and intensity modulated radiation therapy (IMRT) and EGFR-targeted therapy using cetuximab and to compare between two different methods of cetuximab treatment schedules (concomitant versus concomitant and sequential cetuximab treatment). Secondary objectives are to determine the role and the mechanism of cetuximab in patient's chemoradiation regimen, the response rate, the potential of this combined modality treatment to concert locally advanced lesions to potentially resectable lesions, the time to progression interval and the quality of life

Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Combined Species Identification and Drug Sensitivity Testing in Mycobacteria.

&lt;p&gt;Species identification and drug susceptibility testing (DST) of mycobacteria are important yet complex processes traditionally reserved for reference laboratories. Recent technical improvements in matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has started to facilitate routine mycobacterial identifications in clinical laboratories. In this paper, we investigate the possibility of performing phenotypic MALDI-based DST in mycobacteriology using the recently described MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA). We randomly selected 72 clinical Mycobacterium tuberculosis and nontuberculous mycobacterial (NTM) strains, subjected them to MBT-ASTRA methodology, and compared its results to current gold-standard methods. Drug susceptibility was tested for rifampin, isoniazid, linezolid, and ethambutol (M. tuberculosis, n = 39), and clarithromycin and rifabutin (NTM, n = 33). Combined species identification was performed using the Biotyper Mycobacteria Library 4.0. Mycobacterium-specific MBT-ASTRA parameters were derived (calculation window, m/z 5,000 to 13,000, area under the curve [AUC] of &gt;0.015, relative growth [RG] of &lt;0.5; see the text for details). Using these settings, MBT-ASTRA analyses returned 175/177 M. tuberculosis and 65/66 NTM drug resistance profiles which corresponded to standard testing results. Turnaround times were not significantly different in M. tuberculosis testing, but the MBT-ASTRA method delivered on average a week faster than routine DST in NTM. Databases searches returned 90.4% correct species-level identifications, which increased to 98.6% when score thresholds were lowered to 1.65. In conclusion, the MBT-ASTRA technology holds promise to facilitate and fasten mycobacterial DST and to combine it directly with high-confidence species-level identifications. Given the ease of interpretation, its application in NTM typing might be the first in finding its way to current diagnostic workflows. However, further validations and automation are required before routine implementation can be envisioned.&lt;/p&gt;</p

Geraniol and Geranial Dehydrogenases Induced in Anaerobic Monoterpene Degradation by Castellaniella defragrans

Castellaniella defragrans is a Betaproteobacterium capable of coupling the oxidation of monoterpenes with denitrification. Geraniol dehydrogenase (GeDH) activity was induced during growth with limonene in comparison to growth with acetate. The N-terminal sequence of the purified enzyme directed the cloning of the corresponding open reading frame (ORF), the first bacterial gene for a GeDH (geoA, for geraniol oxidation pathway). The C. defragrans geraniol dehydrogenase is a homodimeric enzyme that affiliates with the zinc-containing benzyl alcohol dehydrogenases in the superfamily of medium-chain-length dehydrogenases/reductases (MDR). The purified enzyme most efficiently catalyzes the oxidation of perillyl alcohol (k cat/K m = 2.02 × 106 M−1 s−1), followed by geraniol (k cat/K m = 1.57 × 106 M−1 s−1). Apparent K m values of <10 μM are consistent with an in vivo toxicity of geraniol above 5 μM. In the genetic vicinity of geoA is a putative aldehyde dehydrogenase that was named geoB and identified as a highly abundant protein during growth with phellandrene. Extracts of Escherichia coli expressing geoB demonstrated in vitro a geranial dehydrogenase (GaDH) activity. GaDH activity was independent of coenzyme A. The irreversible formation of geranic acid allows for a metabolic flux from β-myrcene via linalool, geraniol, and geranial to geranic acid