Christine Hackbarth

Non-thermal plasma-treated solution demonstrates antitumor activity against pancreatic cancer cells in vitro and in vivo

Pancreatic cancer is associated with a high mortality rate. In advanced stage, patients often experience peritoneal carcinomatosis. Using a syngeneic murine pancreatic cancer cell tumor model, the effect of non-thermal plasma (NTP) on peritoneal metastatic lesions was studied. NTP generates reactive species of several kinds which have been proven to be of relevance in cancer. In vitro, exposure to both plasma and plasma-treated solution significantly decreased cell viability and proliferation of 6606PDA cancer cells, whereas mouse fibroblasts were less affected. Repeated intraperitoneal treatment of NTP-conditioned medium decreased tumor growth in vivo as determined by magnetic resonance imaging, leading to reduced tumor mass and improved median survival (61 vs 52 days; p < 0.024). Tumor nodes treated by NTP-conditioned medium demonstrated large areas of apoptosis with strongly inhibited cell proliferation. Contemporaneously, no systemic effects were found. Apoptosis was neither present in the liver nor in the gut. Also, the concentration of different cytokines in splenocytes or blood plasma as well as the distribution of various hematological parameters remained unchanged following treatment with NTP-conditioned medium. These results suggest an anticancer role of NTP-treated solutions with little to no systemic side effects being present, making NTP-treated solutions a potential complementary therapeutic option for advanced tumors.

Further insights into the anti-PF4/heparin IgM immune response

Anti-platelet factor 4 (PF4)/heparin antibodies are not only the cause of heparin-induced thrombocytopenia but might also play a role in the antibacterial host defence. Recently, marginal zone (MZ) B cells were identified to be crucial for anti-PF4/heparin IgG antibody production in mice. Combining human studies and a murine model of polymicrobial sepsis we further characterised the far less investigated anti-PF4/heparin IgM immune response. We detected anti-PF4/heparin IgM antibodies in the sera of paediatric patients < 6 months of age after cardiac surgery and in sera of splenectomised mice subjected to polymicrobial sepsis. In addition, PF4/heparin-specific IgM B cells were not only found in murine spleen, but also in peritoneum and bone marrow upon in vitro stimulation. Together, this indicates involvement of additional B cell populations, as MZ B cells are not fully developed in humans until the second year of life and are restricted to the spleen in mice. Moreover, PF4/heparin-specific B cells were detected in human cord blood upon in vitro stimulation and PF4-/- mice produced anti-PF4/heparin IgM antibodies after polymicrobial sepsis. In conclusion, the anti-PF4/heparin IgM response is a potential innate immune reaction driven by a B cell population distinct from MZ B cells.

Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a diverting enterostomy. The etiology of this disease remains ill-defined but appears to differ from that of classical inflammatory bowel diseases such as Crohns disease and ulcerative colitis. Research aimed to decipher the pathophysiological mechanisms leading to the development of this disease has been severely hampered by the lack of an appropriate murine model. This protocol generates a murine model of DC that facilitates the study of the immune systems role and its interaction with the microbiome in the development of DC. In this model using C57BL/6 animals, distal parts of the colon are excluded from the fecal stream by creating a distal colostomy, triggering the development of mild to moderate inflammation in the excluded bowel segments and reproducing the hallmark lesions of human DC with a moderate systemic inflammatory response. In contrast to the rat model, a large number of genetically-modified murine models on the C57BL/6 background are available. The combination of these animals with our model allows the potential roles of individual cytokines, chemokines, or receptors of bioactive molecules (e.g., interleukin (IL)-17; IL-10, chemokine CXCL13, chemokine receptors CXCR5 and CCR7, and the sphingosine-1-phosphate receptor 4) to be assessed in the pathogenesis of DC. The availability of congenic mouse strains on the C57BL/6 background largely facilitates transfer experiments to establish the roles of distinct cell types involved in the etiology of DC. Finally, the model offers the opportunity to assess the influences of local interventions (e.g., modification of the local microbiome or local anti-inflammatory therapy) on mucosal immunity in affected and non-affected bowel segments and the on systemic immune homeostasis.

Cold physical plasma treated buffered saline solution as effective agent against pancreatic cancer cells

BACKGROUND Cold physical plasma has been suggested as a new anticancer tool recently. However, direct use of plasma is limited to visible tumors and in some clinical situations, is not feasible. This includes repetitive treatment of peritoneal metastases, which commonly occur in advanced gastrointestinal cancer and in pancreatic cancer in particular. In case of diffuse intraperitoneal metastatic spread, Hyperthermic Intraperitoneal Intraoperative Chemotherapy (HIPEC) is used as a therapeutic approach. Plasma-treated solutions may combine non-toxic characteristics with the anticancer effects of HIPEC. Previous work has provided evidence for an anticancer efficacy of plasma-treated cell culture medium but the clinical relevance of such an approach is low due to its complex formulation and lack of medical accreditation. OBJECTIVE Plasma-treated Phosphate-Buffered Saline (PBS), which closely resembles medically certified solutions, was investigated for its cytotoxic effect on 2D monolayer murine pancreatic cancer cells in vitro. METHODS Toxicity studies of primary murine fibroblasts, PDA6606 murine pancreatic cancer cells, and COLO 357 human pancreatic cancer cells exposed to plasma-treated PBS were performed. RESULTS Plasma-treated PBS significantly decreased cancer cell metabolisms and proliferation whereas plasma-treated Dulbeccos Modified Eagle Medium had no effect. Moreover, tumor cell growth attenuation was significantly higher when compared to syngeneic primary murine fibroblasts. Both results were confirmed in a human pancreatic cancer cell line. Finally, plasma-treated PBS also decreased the size of pancreatic tumors in a three-dimensional manner, and induction of apoptosis was found to be responsible for all anticancer effects identified. CONCLUSION Plasma-treated PBS inhibited cell growth in 2D and 3D models of cancer. These results may help facilitate the development of new plasma-derived anticancer agent with clinical relevance in the future.

Deviation of the Fecal Stream in Colonic Bowel Segments Results in Increased Numbers of Isolated Lymphoid Follicles in the Submucosal Compartment in a Novel Murine Model of Diversion Colitis

Introduction Diversion colitis is a significant health problem due to its high incidence in patients with diverting enterostomy. This mucosal inflammation presents characteristic histopathological features allowing for the differentiation of this entity from other inflammatory bowel diseases. The pathophysiology of this disease remains ill-defined, in part due to the lack of appropriate animal models. The present study was performed in order to develop and characterize a murine model of diversion colitis. Methods A diverting loop colostomy was performed in C57BL/6 mice either in the ascending colon or in the transverse colon. Animals were assessed for clinical and histopathological parameters during short-term and long-term survival. Results Animals with a colostomy in the transverse colon showed a good long-term survival and developed a mild colitis in the bypassed bowel closely resembling the human pathology on a histopathological level. Conclusion This model is a promising tool to further elucidate the pathomechanism leading to impaired mucosal homeostasis in bypassed colonic segments. Moreover, the establishment of the model in the C57BL/6 background allows the combination of this colitis model with various transgenic mouse strains to investigate the effect of locally deregulated mucosal immunity on systemic immune homeostasis and to develop specific therapeutic strategies.