Establishment of an in vitro 3D model for neuroblastoma enables preclinical investigation of combined tumor-stroma drug targeting.

The majority of anti-cancer therapies target the proliferating tumor cells, while the tumor stroma, principally unaffected, survives, and provide a niche for surviving tumor cells. Combining tumor cell and stroma-targeting therapies thus have a potential to improve patient outcome. The neuroblastoma stroma contains cancer-associated fibroblasts expressing microsomal prostaglandin E synthase-1 (mPGES-1). mPGES-1-derived prostaglandin E2 (PGE2 ) is known to promote tumor growth through increased proliferation and survival of tumor cells, immune suppression, angiogenesis, and therapy resistance, and we, therefore, hypothesize that mPGES-1 constitutes an interesting stromal target. Here, we aimed to develop a relevant in vitro model to study combination therapies. Co-culturing of neuroblastoma and fibroblast cells in 3D tumor spheroids mimic neuroblastoma tumors with regard to the cyclooxygenase/mPGES-1/PGE2 pathway. Using the spheroid model, we show that the inhibition of fibroblast-derived mPGES-1 enhanced the cytotoxic effect of doxorubicin and vincristine and significantly reduced tumor cell viability and spheroid growth. Cyclic treatment with vincristine in combination with an mPGES-1 inhibitor abrogated cell repopulation. Moreover, inhibition of mPGES-1 potentiated the cytotoxic effect of vincristine on established neuroblastoma allografts in mice. In conclusion, we established a 3D neuroblastoma model, highlighting the potential of combining stromal targeting of mPGES-1 with tumor cell targeting drugs like vincristine.

Malondialdehyde and 4-hydroxy-2-hexenal are formed during dynamic gastrointestinal in vitro digestion of cod liver oils.

Marine long-chain polyunsaturated fatty acids (LC n-3 PUFA) are associated with reduced risk for inflammatory diseases, such as cardiovascular diseases and rheumatoid arthritis. These fatty acids, however, are rapidly oxidized, generating highly reactive malondialdehyde (MDA), 4-hydroxy-2-hexenal (HHE) and 4-hydroxy-2-nonenal (HNE). These oxidation products may interact with DNA and proteins, thus possibly leading to impaired cell functions. Little is known about the formation of MDA, HHE and HNE in fish oil in the gastrointestinal (GI) tract. In this study, the effect of dynamic in vitro digestion of cod liver oil on the generation of MDA, HHE and HNE was evaluated using the TNO Gastro-Intestinal Model (tiny-TIM). Effects of pre-formed oxidation products, pre-emulsification of the oil, and addition of oxidants (EDTA and hemoglobin, Hb) on GI oxidation were evaluated. Formation of aldehydes occurred during GI digestion. However, only emulsified oil fortified with 11.5 µM Hb oxidized to a degree that overcame the dilution induced by gastric secretion, which caused increased aldehyde concentrations in gastric lumen up to 90 min. The maximum levels of aldehydes generated in this study were 24.5 µM MDA, 1.6 µM HHE and 0.07 µM HNE. Oils containing different amounts of pre-formed lipid oxidation products maintained the same oxidation ranking order during digestion, even though the relative changes were not directly proportional. Emulsification of the oil had an unclear effect in the gastric phase, but a pro-oxidative effect in the intestinal phase. In general, higher aldehyde levels were reached in the intestinal lumen than in the initial meal, demonstrating that GI digestion promotes oxidation. Hence, epithelial cells may be exposed to elevated amounts of reactive aldehydes for several hours after a meal containing fish oil.

Formation of reactive aldehydes (MDA, HHE, HNE) during the digestion of cod liver oil: comparison of human and porcine in vitro digestion models.

In this work, we investigated lipid oxidation of cod liver oil during gastrointestinal (GI) digestion using two types of in vitro digestion models. In the first type of model, we used human GI juices, while we used digestive enzymes and bile from porcine origin in the second type of model. Human and porcine models were matched with respect to factors important for lipolysis, using a standardized digestion protocol. The digests were analysed for reactive oxidation products: malondialdehyde (MDA), 4-hydroxy-trans-2-nonenal (HNE), and 4-hydroxy-trans-2-hexenal (HHE) by liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (LC/APCI-MS), and for free fatty acids (FFA) obtained during the digestion by gas chromatography-mass spectrometry (GC-MS). The formation of the oxidation products MDA, HHE, and HNE was low during the gastric digestion, however, it increased during the duodenal digestion. The formation of the oxidation products reached higher levels when digestive juices of human origin were used (60 µM of MDA, 9.8 µM of HHE, and 0.36 µM of HNE) [corrected] compared to when using enzymes and bile of porcine origin (0.96, and 1.6 µM of MDA; 0.16, and 0.23 µM of HHE; 0.026, [corrected] and 0.005 µM of HNE, respectively, in porcine models I and II). In all models, FFA release was only detected during the intestinal step, and reached up to 31% of total fatty acids (FA). The findings in this work may be of importance when designing oxidation oriented lipid digestion studies.

An organic electronic biomimetic neuron enables auto-regulated neuromodulation.

Current therapies for neurological disorders are based on traditional medication and electric stimulation. Here, we present an organic electronic biomimetic neuron, with the capacity to precisely intervene with the underlying malfunctioning signalling pathway using endogenous substances. The fundamental function of neurons, defined as chemical-to-electrical-to-chemical signal transduction, is achieved by connecting enzyme-based amperometric biosensors and organic electronic ion pumps. Selective biosensors transduce chemical signals into an electric current, which regulates electrophoretic delivery of chemical substances without necessitating liquid flow. Biosensors detected neurotransmitters in physiologically relevant ranges of 5-80 µM, showing linear response above 20 µm with approx. 0.1 nA/µM slope. When exceeding defined threshold concentrations, biosensor output signals, connected via custom hardware/software, activated local or distant neurotransmitter delivery from the organic electronic ion pump. Changes of 20 µM glutamate or acetylcholine triggered diffusive delivery of acetylcholine, which activated cells via receptor-mediated signalling. This was observed in real-time by single-cell ratiometric Ca(2+) imaging. The results demonstrate the potential of the organic electronic biomimetic neuron in therapies involving long-range neuronal signalling by mimicking the function of projection neurons. Alternatively, conversion of glutamate-induced descending neuromuscular signals into acetylcholine-mediated muscular activation signals may be obtained, applicable for bridging injured sites and active prosthetics.

Effect of in vitro digested cod liver oil of different quality on oxidative, proteomic and inflammatory responses in the yeast Saccharomyces cerevisiae and human monocyte-derived dendritic cells.

BACKGROUND: Upon oxidation of the polyunsaturated fatty acids in fish oil, either before ingestion or, as recently shown, during the gastro-intestinal passage, a cascade of potentially cytotoxic peroxidation products, such as malondialdehyde and 4-hydroxy-2-hexenal, can form. In this study, we digested fresh and oxidised cod liver oils in vitro, monitored the levels of lipid peroxidation products and evaluated oxidative, proteomic and inflammatory responses to the two types of digests in the yeast Saccharomyces cerevisiae and human monocyte-derived dendritic cells. RESULTS: Digests of cod liver oil with 22-53 µmol L(-1) malondialdehyde and 0.26-3.7 µmol L(-1) 4-hydroxy-2-hexenal increased intracellular oxidation and cell energy metabolic activity compared to a digested blank in yeast cells and the influence of digests on mitochondrial protein expression was more pronounced for oxidised cod liver oil than fresh cod liver oil. The four differentially expressed and identified proteins were related to energy metabolism and oxidative stress response. Maturation of dendritic cells was affected in the presence of digested fresh cod liver oil compared to the digested blank, measured as lower CD86 expression. The ratio of secreted cytokines, IL-12p40/IL-10, suggested a pro-inflammatory effect of the digested oils in relation to the blank (1.47-1.67 vs. 1.07). CONCLUSION: Gastro-intestinal digestion of cod liver oil increases the amount of oxidation products and resulting digests affect oxidation in yeast and immunomodulation of dendritic cells.

Oxidation of cod liver oil during gastrointestinal in vitro digestion.

Oxidation of cod liver oil rich in long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) was investigated during a gastrointestinal (GI) in vitro digestion. The digestion stimulated TBA-reactive substances (TBARS) formation in both the gastric and intestinal steps, whereas levels of lipid hydroperoxides remained nearly constant. The presence of digestive compounds was decisive for the TBARS development because TBARS did not change when the cod liver oil was subjected only to the temperature and pH gradient of the GI model. Preformed oxidation products in the cod liver oil resulted in further elevated TBARS levels during the digestion. Addition of hemoglobin (11.5 µM) to emulsified cod liver oil dramatically increased TBARS and lipid hydroperoxide levels during GI digestion, whereas 1 mg α-tocopherol/g oil did not show any protection against oxidation. Specific concern thus needs to be taken in the design of foods containing LC n-3 PUFA to preserve these lipids and avoid harmful oxidation, both before and after consumption.

Inhibition of hemoglobin-mediated oxidation of regular and lipid-fortified washed cod mince by a white grape dietary fiber.

The effectiveness of a white grape dietary fiber concentrate (WGDF) against hemoglobin-mediated oxidation of washed cod mince, with and without 10% added herring oil, was evaluated during ice storage. WGDF was added at two different levels: 2 and 4% based on final weight. An ethanol extract with the ethanol extractable polyphenols (EPP) and the ethanol-extracted grape dietary fiber residue were also tested as antioxidants in the washed cod mince. The addition of WGDF to the model system completely and significantly (p

Infliximab attenuates immunoreactivity of brain-derived neurotrophic factor in a rat model of herniated nucleus pulposus.

STUDY DESIGN: The effect of infliximab, a chimeric monoclonal antibody to TNF-alpha, on induction of brain-derived neurotrophic factor (BDNF) was examined using an experimental herniated nucleus pulposus (NP) model. OBJECTIVES: To investigate whether treatment of infliximab could attenuate an induction of BDNF, which functions as a modulator of pain, following NP application to the nerve root. SUMMARY OF BACKGROUND DATA: Evidence from basic scientific studies proposes that TNF-alpha is involved in the development of NP-induced nerve injuries. However, the therapeutic mechanisms of infliximab against pain have not been elucidated experimentally. METHODS: Twenty rats were used in this study. In the test groups, the animals underwent application of NP to the L4 nerve roots and received a single systemic (intraperitoneal) injection of infliximab at the time of surgery (Infli-0 group, n = 5) or at 1 day after operation (Infli-1 group, n = 5). As a control treatment, sterile water was administered intraperitoneally to 5 rats with NP application (NP group) and to 5 sham-operated rats (sham group). On day 3 after surgery, the L4 dorsal root ganglion (DRG) and L4 spinal segment were harvested and assessed regarding BDNF immunoreactivity. RESULTS.: Application of NP induced a marked increase of BDNF immunoreactivity in number in the DRG neurons and within the superficial layer in the dorsal horn compared with the sham group (P < 0.01). Infliximab treatment in the Infli-0 and Infli-1 groups reduced the BDNF induction in both DRG and spinal cord (P < 0.05). CONCLUSION: These findings indicate that infliximab attenuates the elevated BDNF levels induced by NP. The present study therefore further indicates the importance of TNF-alpha in sciatica due to disc herniation and the possible therapeutic use of a TNF-alpha inhibitor for this condition.