Nitric Oxide Regulation during Anti-tumor Immunotherapy

During tumor progression a strong immunosuppression is developed and is believed to be the major reason why immunotherapy fails. This suppression is induced in response to factors produced from tumor cells, but can also be induced after immunotherapy. NO has been shown to play a major role in this suppression both in patients with gliomas and colon carcinomas. The aim of this thesis is to clarify the role of the NO mediated immunosuppression in rats with intra-hepatic colon carcinomas and intra-cerebral gliomas, and to clarify whether inhibition of NO inducing enzyme, iNOS, would diminish this suppression and improve the anti-tumor immunotherapy. In study I-IV we investigated both unspecific and specific iNOS inhibitors, and we could show that the specific iNOS inhibitors were superior in enhancing immune responses, including lymphocyte proliferation and cytokine production, as well as nitrite reduction. The specific iNOS inhibitor mercaptoethylguanidine, MEG, was the most effective inhibitor. MEG in combination with IFN-? based immunotherapy prolonged the survival and increased the cure rate three fold of rats with intra-cerebral gliomas. Interestingly, the inhibition of NO by MEG was time dependent, and the prolonged survival was achieved only when MEG was administered after more than one immunization. The prolonged survival seemed to be dependent on enhanced local and systemic immune responses. MEG treatment significantly increased the lymphocyte numbers both in the spleen and in the tumor draining lymph nodes (the deep cervical lymph nodes). In the deep cervical lymph nodes, MEG treatment increased CD4+ T cells but not the B cells, suggesting a skewing toward a Th1 immunity, which is important during tumor eradication. Furthermore, MEG treatment decreased the auto-reactive and the suppressive CD8+CD25+ T cells. In the colon carcinoma model, we showed that additional suppressive mechanism exists, which is mediated by the enzyme cyclooxygenase (COX). The inhibition of both NOS and COX increased immune responses six times as compared to inhibition of each enzyme alone. Finally, treatment with MEG in combination IL-18/IFN-? reduced tumor volume by 50 % in rats with colon carcinomas. MEG has been shown to inhibit production of both iNOS and COX, which might explain its superior effects compared to the other NOS inhibitors. Thus we have been able to show that immunotherapy using tumor cells genetically modified to produce cytokines could be used to eradicate established tumors, however, only when combined with therapies that minimize the immune suppression developed during tumor progression

Hur upplever gymnasieelever digitala simuleringar under kemilektionen

I relation till den ökade digitaliseringen av samhället och verktygen för undervisning finns det relativt lite information om användning av simuleringar i svenska gymnasieskolor. Därför syftar denna studie till att undersöka hur gymnasieelever i årskurs 1 använder och utvärderar digitala simuleringar i kemiundervisningen. Genom ett webbaserat enkätformulär ställdes generella frågor kring elevernas användning av digitala läromedel. Resultaten visade att majoriteten av gymnasieeleverna i denna klass använde den analoga boken men kände till digitala simuleringar. Enkätundersökningen efterföljdes med gruppintervjuer för att ta reda på: a. hur eleverna upplevde utövandet av den digitala simuleringen som tilldelades under kemilektionen, b. ifall simuleringen kunde påverka förståelsen av kemiska begreppet mättnad och c. vilka för- och nackdelar eleverna kunde identifiera.Resultat visade också att majoriteten av eleverna upplevde utövandet av simuleringen positivt. Den positiva upplevelsen var kopplad till att simuleringen var lättbegriplig med möjligheten att lätt kunna göra om momenten. Vidare förklarade eleverna att enkla strukturen, smidigheten samt tidsbesparingen var andra fördelaktiga faktorer. Till nackdelarna identifierades problematik med att få exakta mått samt att simuleringen upplevdes begränsad. Eleverna önskade fler utmaningar med ökad svårighetsgrad. När det gäller begreppsförståelse urskildes två grupper i denna klass, varav den ena fann att användningen av simuleringen underlättade förståelse av begreppet mättnad, medan den andra gruppen inte kopplade förändringar i simuleringen och tyckte att skärmen bara ändrade färg. Vidare lyftes fram överdimensionering av digitala läromedel som orsakat tydlig trötthet på skärmen och i samband med det skulle simuleringar enbart komplettera undervisningen och aldrig ersätta laborationerna. Slutligen visade studien tydligt att, även om simuleringen är digital, används den bäst i lärarens närvaro

Hur upplever gymnasieelever digitala simuleringar under kemilektionen

I relation till den ökade digitaliseringen av samhället och verktygen för undervisning finns det relativt lite information om användning av simuleringar i svenska gymnasieskolor. Därför syftar denna studie till att undersöka hur gymnasieelever i årskurs 1 använder och utvärderar digitala simuleringar i kemiundervisningen. Genom ett webbaserat enkätformulär ställdes generella frågor kring elevernas användning av digitala läromedel. Resultaten visade att majoriteten av gymnasieeleverna i denna klass använde den analoga boken men kände till digitala simuleringar. Enkätundersökningen efterföljdes med gruppintervjuer för att ta reda på: a. hur eleverna upplevde utövandet av den digitala simuleringen som tilldelades under kemilektionen, b. ifall simuleringen kunde påverka förståelsen av kemiska begreppet mättnad och c. vilka för- och nackdelar eleverna kunde identifiera. Resultat visade också att majoriteten av eleverna upplevde utövandet av simuleringen positivt. Den positiva upplevelsen var kopplad till att simuleringen var lättbegriplig med möjligheten att lätt kunna göra om momenten. Vidare förklarade eleverna att enkla strukturen, smidigheten samt tidsbesparingen var andra fördelaktiga faktorer. Till nackdelarna identifierades problematik med att få exakta mått samt att simuleringen upplevdes begränsad. Eleverna önskade fler utmaningar med ökad svårighetsgrad. När det gäller begreppsförståelse urskildes två grupper i denna klass, varav den ena fann att användningen av simuleringen underlättade förståelse av begreppet mättnad, medan den andra gruppen inte kopplade förändringar i simuleringen och tyckte att skärmen bara ändrade färg. Vidare lyftes fram överdimensionering av digitala läromedel som orsakat tydlig trötthet på skärmen och i samband med det skulle simuleringar enbart komplettera undervisningen och aldrig ersätta laborationerna. Slutligen visade studien tydligt att, även om simuleringen är digital, används den bäst i lärarens närvaro

Hur upplever gymnasieelever digitala simuleringar under kemilektionen

I relation till den ökade digitaliseringen av samhället och verktygen för undervisning finns det relativt lite information om användning av simuleringar i svenska gymnasieskolor. Därför syftar denna studie till att undersöka hur gymnasieelever i årskurs 1 använder och utvärderar digitala simuleringar i kemiundervisningen. Genom ett webbaserat enkätformulär ställdes generella frågor kring elevernas användning av digitala läromedel. Resultaten visade att majoriteten av gymnasieeleverna i denna klass använde den analoga boken men kände till digitala simuleringar. Enkätundersökningen efterföljdes med gruppintervjuer för att ta reda på: a. hur eleverna upplevde utövandet av den digitala simuleringen som tilldelades under kemilektionen, b. ifall simuleringen kunde påverka förståelsen av kemiska begreppet mättnad och c. vilka för- och nackdelar eleverna kunde identifiera.Resultat visade också att majoriteten av eleverna upplevde utövandet av simuleringen positivt. Den positiva upplevelsen var kopplad till att simuleringen var lättbegriplig med möjligheten att lätt kunna göra om momenten. Vidare förklarade eleverna att enkla strukturen, smidigheten samt tidsbesparingen var andra fördelaktiga faktorer. Till nackdelarna identifierades problematik med att få exakta mått samt att simuleringen upplevdes begränsad. Eleverna önskade fler utmaningar med ökad svårighetsgrad. När det gäller begreppsförståelse urskildes två grupper i denna klass, varav den ena fann att användningen av simuleringen underlättade förståelse av begreppet mättnad, medan den andra gruppen inte kopplade förändringar i simuleringen och tyckte att skärmen bara ändrade färg. Vidare lyftes fram överdimensionering av digitala läromedel som orsakat tydlig trötthet på skärmen och i samband med det skulle simuleringar enbart komplettera undervisningen och aldrig ersätta laborationerna. Slutligen visade studien tydligt att, även om simuleringen är digital, används den bäst i lärarens närvaro

The Dual Role of Nitric Oxide in Glioma

Malignant gliomas bear the most dismal prognosis of all human cancers despite the progress in therapy of many other tumors. The search for alternative and complementary treatments has therefore a high priority. Emerging knowledge of the dual and diverging role of nitric oxide in glioma biology has focused on possibilities to achieve anti-glioma effects by modulation of nitric oxide (NO) release and function in these tumors. NO has been shown to influence proliferation of glioma cells, vascular function in glioams, invasive capacity of gliomas, effects of chemo and radiotherapy and also immune reactivity against these tumors. The mechanisms behind the reported diverse and dual effects of NO in glioma biology are multiple. Some of the diversity can be explained by different experimental setups as in vitro versus in vivo models but the cellular sources, timing, absolute levels and gradients play a decisive role for the effects of NO on glioma biology. Current research in this field is hampered by the lack of inhibitors and donors approved for clinical use

Low-dose combretastatin A4 phosphate enhances the immune response of tumor hosts to experimental colon carcinoma

Purpose: Although there is a need to enhance the therapeutic efficiency in cancer by combining immunotherapeutic procedures with other therapy, combination with chemotherapy is complicated due to immunosuppressive effects of most chemotherapeutic drugs. The purpose of this investigation was to study whether combining tumor cell immunization with the vascular targeting drug combretastatin A4 phosphate (CA4P) would enhance tumor retardation and/or affect the antitumor immune response. Experimental Design: Rats with intrahepatic colon carcinoma were immunized weekly with IL-18/IFN gamma-transfected tumor cells, starting day 9, and were treated with a low-dose CA4P (2 mg/kg, 5 days a week starting day 7). The effect of CA4P was studied on tumor growth and on immune reactivity in vitro. Results: Rats with preexisting tumor, immunized and treated with low-dose CA4P, had a significantly retarded tumor growth compared with rats receiving CA4P or immunization alone. Splenocytes from rats treated with this combination had a significantly enhanced antitumor immune response compared with splenocytes from control rats. Exposure of nonadherent splenocytes to CA4P in vitro did not enhance their proliferation. However, 3-hour pretreatment of adherent splenocytes with 0.3 mu g/mL CA4P significantly enhanced proliferation and IFN gamma production of admixed nonadherent splenocytes, partly due to nitric oxide reduction. Combining the nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester with CA4P and immunization further retarded tumor growth. Conclusion: Concomitant treatment of rats with progressively growing tumor with immunization and low-dose CA4P significantly enhances the therapeutic effect as compared with either treatment alone and results in an enhanced antitumor immune reactivity

Postimmunization with IFN-gamma-secreting glioma cells combined with the inducible nitric oxide synthase inhibitor mercaptoethylguanidine prolongs survival of rats with intracerebral tumors

High-grade gliomas are one of the most aggressive human tumors with <1% of patients surviving 5 years after surgery. Immunotherapy could offer a possibility to eradicate remnant tumor cells after conventional therapy. Experimental immunotherapy can induce partial cure of established intracerebral tumors in several rodent models. One reason for the limited therapeutic effects could be immunosuppression induced by both the growing tumor and the induced immune reaction. NO has been implicated in tumor-derived immune suppression in tumor-bearing hosts, and unspecific inhibitors of NO synthase have been shown to boost antitumor immunity. In this study, we show that the inducible NO synthase (iNOS)-specific inhibitor mercaptoethylguanidine (MEG) superiorly enhanced lymphocyte reactivity after polyclonal stimulation compared with the iNOS-specific inhibitor L-NIL and the unspecific NO synthase inhibitor L-NAME. Both iNOS inhibitors increased the number and proliferation of T cells but not of B cells. When combined during postimmunization with IFN-gamma-secreting N32 rat glioma cells of rats harboring intracerebral tumors, only MEG increased the cure rate. However, this was only achieved when MEG was administered after immunizations. These findings implicate that NO has both enhancing and suppressive effects after active immunotherapy

Synergism between GM-CSF and IFNgamma: Enhanced immunotherapy in mice with glioma.

Glioblastoma multiforme is the most common malignant primary brain tumor and also one of the most therapy-resistant tumors. Because of the dismal prognosis, various therapies modulating the immune system have been developed in experimental models. Previously, we have shown a 37-70% cure in a rat glioma model where rats were peripherally immunized with tumor cells producing IFN. On the basis of these results, we wanted to investigate whether a combination of GM-CSF and IFN could improve the therapeutic effect in a mouse glioma model, GL261 (GL-wt). Three biweekly intraperitoneal (i.p.) immunizations with irradiated GM-CSF-transduced GL261 cells (GL-GM) induced a 44% survival in mice with intracranial glioma. While treatment of GL-wt and GL-GM with IFN in vitro induced upregulation of MHC I and MHC II on the tumor cells, it could not enhance survival after immunization. However, immunizations with GL-GM combined with recombinant IFN at the immunization site synergistically enhanced survival with a cure rate of 88%. Tumors from mice receiving only 1 immunization on Day 10 after tumor inoculation were sectioned on Day 20 for analysis of leukocyte infiltration. Tumor volume was reduced and the infiltration of macrophages was denser in mice immunized with GL-GM combined with IFN compared with that of both wildtype and nonimmunized mice. To our knowledge, this is the first study to demonstrate a synergy between GM-CSF and IFN in experimental immunotherapy of tumors, by substantially increasing survival as well as inducing a potent anti-tumor response after only 1 postponed immunization

Recruited brain tumor-derived mesenchymal stem cells contribute to brain tumor progression.

The identity of the cells that contribute to brain tumor structure and progression remains unclear. Mesenchymal stem cells (MSCs) have recently been isolated from normal mouse brain. Here, we report the infiltration of MSC-like cells into the GL261 murine glioma model. These Brain Tumor derived Mesenchymal Stem Cells (BT-MSCs) are defined with the phenotype (Lin-Sca-1+CD9+CD44+CD166+/-) and have multipotent differentiation capacity. We show that the infiltration of BT-MSCs correlates to tumor progression; furthermore, BT-MSCs increased the proliferation rate of GL261 cells in vitro. For the first time, we report that the majority of GL261 cells expressed mesenchymal phenotype under both adherent and sphere culture conditions in vitro, and that the non-MSC population is non-tumorigenic in vivo. Although the GL261 cell line expressed mesenchymal phenotype markers in vitro, most BT-MSCs are recruited cells from host origin in both wild type GL261 inoculated into GFP-transgenic mice and GL261-GFP cells inoculated into wild type mice. We show the expression of chemokine receptors CXCR4 and CXCR6 on different recruited cell populations. In vivo, the GL261 cells change marker profile, and acquire a phenotype that is more similar to cells growing in sphere culture conditions. Finally, we identify a BT-MSC population in human glioblastoma that is CD44+CD9+CD166+ both in freshly isolated and culture-expanded cells. Our data indicate that cells with MSC-like phenotype infiltrate into the tumor stroma and play an important role in tumor cell growth in vitro and in vivo. Thus we suggest that targeting BT-MSCs could be a possible strategy for treating glioblastoma patients. Stem Cells 2013