Stefan Mehrle

Bortezomib is ineffective in an orthotopic mouse model of pancreatic adenocarcinoma

The purpose of the present study was to evaluate the potency of the proteasome inhibitor bortezomib ± gemcitabine in vitro and in vivo in pancreatic carcinoma. It could be shown that bortezomib induced apoptosis and inhibited proliferation of pancreatic carcinoma very efficiently in vitro. In contrast, in an orthotopic pancreatic adenocarcinoma mouse model, gemcitabine treatment inhibited tumor growth, whereas bortezomib promoted it. Bortezomib-treated animals showed significantly higher tumor burden compared with gemcitabine-treated and control animals, although bortezomib was locally active and induced a decrease of proteasome activity, which was most pronounced following the simultaneous administration of gemcitabine. Also, tumor progression was not caused by immunosuppression as a result of proteasome inhibition. Interestingly, anti-CD31 staining of tumors showed that angiogenesis was significantly increased in the tumors of bortezomib-treated mice compared with the tumors of control animals. In addition, bortezomib resulted an increase of pericytes, vascular endothelial growth factor, RGS-5, and hypoxia-inducible factor-1α in the tumor. Although this study supports efficacy of bortezomib against pancreatic carcinoma in vitro, it strongly indicates that bortezomib therapy has a significant tumor-promoting effect in vivo by induction of angiogenesis. The data are in accordance with the complete failure of bortezomib in a phase II trial for this indication. Choosing the right schedule of gemcitabine and bortezomib showed some synergistic effects, but the gain might not be big enough to compensate the potentially detrimental effects. [Mol Cancer Ther 2008;7(11):3624–31]

SAP and SLAM expression in anti-CD3 activated lymphocytes correlates with cytotoxic activity

Signalling lymphocyte activation molecule (SLAM)‐associated protein (SAP) is a small protein that is mutant in humans with X‐linked lymphoproliferative (XLP) disease. Patients with XLP disease are affected by fatal EBV infection and malignant B‐cell lymphomas. The increased risk for B‐cell lymphomas is suggested to result from impaired immunosurveillance of B‐cell proliferation by T cells. In this study, we investigated the role of SLAM and SAP in activation of effector cells with cytotoxic activity, cytokine‐induced killer (CIK) cells, which are generated by non‐specific stimulation of the TCR and addition of exogenous IL‐2. Agonistic TCR activation 1 day after preparation (day +1) resulted in cell activation, with a peak of SLAM on day +6 visible at both the protein and mRNA level as well as membrane detectable SLAM. This increase in SLAM expression correlated significantly with SAP expression at the mRNA level as well as at the protein level. Cytotoxic activity peaked 1 day after the observed SAP and SLAM peaks. At that point in time, IL‐10 secretion, which was high during the early days of culture, decreased. In conclusion, activation of peripheral blood cells with agonistic anti‐CD3 antibody and exogenous IL‐2, as used for generation of CIK cells, results in significant SLAM and SAP activation 5 days after TCR stimulation. This peak correlates with cytotoxic activity against tumour cells. Expression of SLAM and SAP seems to be important in the activation of cytotoxic effector cells.

Liver Imaging with a Novel Hepatitis B Surface Protein Derived SPECT-Tracer

Diagnostic imaging of the liver by ultrasound, computed tomography (CT) and magnetic resonance tomography (MRT) is generally limited to the visualization of the morphology. In order to exploit the intriguing liver tropism of the human hepatitis B virus (HBV) for molecular imaging of the liver, peptidic tracers derived from the HBV large envelope protein (L) were studied. An N-terminally stearoylated tracer comprising amino acids 2-48 of the PreS1-domain of the L protein was synthesized by solid phase peptide synthesis. Mercaptoacetyltriglycerin (MAG3) was linked to this peptide to enable (99m)Tc labeling. Biodistribution studies in mice showed an excellent liver accumulation of this novel class of radiotracer with 84%, 84%, 65%, and 16% of the injected dose in the liver after 10 min, 1 h, 4 h, and 24 h, respectively. Imaging studies on a gamma camera showed a clear visualization of the liver already 10 min post intravenous injection. These studies confirmed the exclusive accumulation of the tracer in the liver with negligible background in other organs. Owing to a significant biliary clearance rate of the (99m)Tc bound via a linker, the tracer enabled imaging of the bile ducts starting 30 min after injection. Analysis of the route of excretion revealed complete clearance within 24 h post injection. Clearance was predominantly via renal secretion. In conclusion, the novel class of tracers shows excellent pharmacokinetic, biodistribution, and clearance kinetics. It provides unique biological information different from the current imaging modalities. Its primary application is likely to be the evaluation of liver cancer patients. Specific indications may include tumor staging, the differentiation of malignant versus benign hepatic lesions, and liver tumors of nonhepatocellular origin.

Comparison of phenotype of γδ T cells generated using various cultivation methods

It has been demonstrated, that gammadelta T cells play an important role in the development of immune responses to many pathogens. gammadelta T cells play a role in the clearance of viral and microbiological infections, anti-tumor responses, but also in autoimmune diseases. Many different protocols for the isolation and cultivation of gammadelta T cells can be found in the literature. Here we compare three common cultivation protocols for gammadelta T cells derived from peripheral blood with a newly developed protocol depending on SLAM (Signaling Lymphocyte Activation Molecule) stimulation. We demonstrate that the cultivation protocol chosen to raise gammadelta T cells has direct impact on the resulting gammadelta T cell phenotype. We show differences in gammadelta TCR composition, memory phenotype formation, CD8 receptor expression and the expression of NK cell markers depending on the stimulation protocol used. As such, the cultivation protocol chosen for a series of experiments might have significant impact on the outcome of the experiments and should be considered carefully.

Multi-Drug Resistance: Induktion und Inhibition beim Pankreaskarzinom

Wir konnten zeigen, dass beim experimentellen Pankreaskarzinom die vermehrte Expression von MDR-assoziierten Proteinen durch die Behandlung mit 5-FU, Cisplatin und Gemcitabine induziert wird. Der Peptidchemosensitizer R121 kann die therapieinduzierte MRP1 und MRP3 Expression senken, scheint jedoch den klinischen Verlauf nicht zu beeinflussen.