- Zentrum für Arzneimittelforschung, Entwicklung und Sicherheit (33)
A characterization of four B16 murine melanoma cell sublines molecular fingerprint and proliferation behavior
Simona Cîntă Pînzaru
Melania F. Munteanu
- Background: One of the most popular and versatile model of murine melanoma is by inoculating B16 cells in the syngeneic C57BL6J mouse strain. A characterization of different B16 modified cell sub-lines will be of real practical interest. For this aim, modern analytical tools like surface enhanced Raman spectroscopy/scattering (SERS) and MTT were employed to characterize both chemical composition and proliferation behavior of the selected cells.
Methods: High quality SERS signal was recorded from each of the four types of B16 cell sub-lines: B164A5, B16GMCSF, B16FLT3, B16F10, in order to observe the differences between a parent cell line (B164A5) and other derived B16 cell sub-lines. Cells were incubated with silver nanoparticles of 50–100 nm diameter and the nanoparticles uptake inside the cells cytoplasm was proved by transmission electron microscopy (TEM) investigations. In order to characterize proliferation, growth curves of the four B16 cell lines, using different cell numbers and FCS concentration were obtained employing the MTT proliferation assay. For correlations doubling time were calculated.
Results: SERS bands allowed the identification inside the cells of the main bio-molecular components such as: proteins, nucleic acids, and lipids. An "on and off" SERS effect was constantly present, which may be explained in terms of the employed laser power, as well as the possible different orientations of the adsorbed species in the cells in respect to the Ag nanoparticles. MTT results showed that among the four tested cell sub-lines B16 F10 is the most proliferative and B164A5 has the lower growth capacity. Regarding B16FLT3 cells and B16GMCSF cells, they present proliferation ability in between with slight slower potency for B16GMCSF cells.
Conclusion: Molecular fingerprint and proliferation behavior of four B16 melanoma cell sub-lines were elucidated by associating SERS investigations with MTT proliferation assay.
Biglycan: a multivalent proteoglycan providing structure and signals
Madalina V. Nastase
Marian F. Young
- Research over the past few years has provided fascinating results indicating that biglycan, besides being a ubiquitous structural component of the extracellular matrix (ECM), may act as a signaling molecule. Proteolytically released from the ECM, biglycan acts as a danger signal signifying tissue stress or injury. As a ligand of innate immunity receptors and activator of the inflammasome, biglycan stimulates multifunctional proinflammatory signaling linking the innate to the adaptive immune response. By clustering several types of receptors on the cell surface and orchestrating their downstream signaling events, biglycan is capable to autonomously trigger sterile inflammation and to potentiate the inflammatory response to microbial invasion. Besides operating in a broad biological context, biglycan also displays tissue-specific affinities to certain receptors and structural components, thereby playing a crucial role in bone formation, muscle integrity, and synapse stability at the neuromuscular junction. This review attempts to provide a concise summary of recent data regarding the involvement of biglycan in the regulation of inflammation and the musculoskeletal system, pointing out both a signaling and a structural role for this proteoglycan. The potential of biglycan as a novel therapeutic target or agent for the treatment of inflammatory diseases and skeletal muscular dystrophies is also addressed.
Do non-genomically encoded fusion transcripts cause recurrent chromosomal translocations?
- We among others have recently demonstrated that normal cells produce “fusion mRNAs”. These fusion mRNAs do not derive from rearranged genomic loci, but rather they are derived from “early-terminated transcripts” (ETTs). Premature transcriptional termination takes place in intronic sequences that belong to “breakpoint cluster regions”. One important property of ETTs is that they exhibit an unsaturated splice donor site. This results in: (1) splicing to “cryptic exons” present in the final intron; (2) Splicing to another transcript of the same gene (intragenic trans-splicing), resulting in “exon repetitions”; (3) splicing to a transcript of another gene (intergenic trans-splicing), leading to “non-genomically encoded fusion transcripts” (NGEFTs). These NGEFTs bear the potential risk to influence DNA repair processes, since they share identical nucleotides with their DNA of origin, and thus, could be used as “guidance RNA” for DNA repair processes. Here, we present experimental data about four other genes. Three of them are associated with hemato-malignancies (ETV6, NUP98 and RUNX1), while one is associated with solid tumors (EWSR1). Our results demonstrate that all genes investigated so far (MLL, AF4, AF9, ENL, ELL, ETV6, NUP98, RUNX1 and EWSR1) display ETTs and produce transpliced mRNA species, indicating that this is a genuine property of translocating genes.
Heavy ions and X-rays in brain tumor treatment : a comparison of their biological effects on tissue slice cultures
Michel Guy André Mittelbronn
Patrick Nikolaus Harter
- Background: In this interdisciplinary project, the biological effects of heavy ions are compared to those of X-rays using tissue slice culture preparations from rodents and humans. Advantages of this biological model are the conservation of an organotypic environment and the independency from genetic immortalization strategies used to generate cell lines. Its open access allows easy treatment and observation via live-imaging microscopy. Materials and methods: Rat brains and human brain tumor tissue are cut into 300 micro m thick tissue slices. These slices are cultivated using a membrane-based culture system and kept in an incubator at 37°C until treatment. The slices are treated with X-rays at the radiation facility of the University Hospital in Frankfurt at doses of up to 40 Gy. The heavy ion irradiations were performed at the UNILAC facility at GSI with different ions of 11.4 A MeV and fluences ranging from 0.5–10 x 106 particles/cm². Using 3D-confocal microscopy, cell-death and immune cell activation of the irradiated slices are analyzed. Planning of the irradiation experiments is done with simulation programs developed at GSI and FIAS. Results: After receiving a single application of either X-rays or heavy ions, slices were kept in culture for up to 9d post irradiation. DNA damage was visualized using gamma H2AXstaining. Here, a dose-dependent increase and time-dependent decrease could clearly be observed for the X-ray irradiation. Slices irradiated with heavy ions showed less gamma H2AX-positive cells distributed evenly throughout the slice, even though particles were calculated to penetrate only 90–100 micro m into the slice. Conclusions: Single irradiations of brain tissue, even at high doses of 40 Gy, will result neither in tissue damage visible on a macroscopic level nor necrosis. This is in line with the view that the brain is highly radio-resistant. However, DNA damage can be detected very well in tissue slices using gamma H2AX-immuno staining. Thus, slice cultures are an excellent tool to study radiation-induced damage and repair mechanisms in living tissues.
Inhibition of the soluble epoxide hydrolase promotes albuminuria in mice with progressive renal disease
Rainer U. Pliquett
Sung H. Hwang
Bruce D. Hammock
Ralf Peter Louis Brandes
- Epoxyeicotrienoic acids (EETs) are cytochrome P450-dependent anti-hypertensive and anti-inflammatory derivatives of arachidonic acid, which are highly abundant in the kidney and considered reno-protective. EETs are degraded by the enzyme soluble epoxide hydrolase (sEH) and sEH inhibitors are considered treatment for chronic renal failure (CRF). We determined whether sEH inhibition attenuates the progression of CRF in the 5/6-nephrectomy model (5/6-Nx) in mice. 5/6-Nx mice were treated with a placebo, an ACE-inhibitor (Ramipril, 40 mg/kg), the sEH-inhibitor cAUCB or the CYP-inhibitor fenbendazole for 8 weeks. 5/6-Nx induced hypertension, albuminuria, glomerulosclerosis and tubulo-interstitial damage and these effects were attenuated by Ramipril. In contrast, cAUCB failed to lower the blood pressure and albuminuria was more severe as compared to placebo. Plasma EET-levels were doubled in 5/6 Nx-mice as compared to sham mice receiving placebo. Renal sEH expression was attenuated in 5/6-Nx mice but cAUCB in these animals still further increased the EET-level. cAUCB also increased 5-HETE and 15-HETE, which derive from peroxidation or lipoxygenases. Similar to cAUCB, CYP450 inhibition increased HETEs and promoted albuminuria. Thus, sEH-inhibition failed to elicit protective effects in the 5/6-Nx model and showed a tendency to aggravate the disease. These effects might be consequence of a shift of arachidonic acid metabolism into the lipoxygenase pathway.
Consequences of altered eicosanoid patterns for nociceptive processing in mPGES-1-deficient mice
Carlo Federico Angioni
Rolf M. Nusing
- Cyclooxygenase-2 (COX-2)-dependent prostaglandin (PG) E2 synthesis in the spinal cord plays a major role in the development of inflammatory hyperalgesia and allodynia. Microsomal PGE2 synthase-1 (mPGES-1) isomerizes COX-2-derived PGH2 to PGE2. Here, we evaluated the effect of mPGES-1-deficiency on the noci-ceptive behavior in various models of nociception that depend on PGE2 synthesis. Surprisingly, in the COX-2-dependent zymosan-evoked hyperalgesia model, the nociceptive behavior was not reduced in mPGES-1-deficient mice despite a marked decrease of the spinal PGE2 synthesis. Similarly, the nociceptive behavior was unaltered in mPGES-1-deficient mice in the formalin test. Importantly, spinal cords and primary spinal cord cells derived from mPGES-1-deficient mice showed a redirection of the PGE2 synthesis to PGD2, PGF2α and 6-keto-PGF1α (stable metabolite of PGI2). Since the latter prostaglandins serve also as mediators of noci-ception they may compensate the loss of PGE2 synthesis in mPGES-1-deficient mice.
Pro-inflammatory signaling by IL-10 and IL-22: bad habit stirred up by interferons?
- Interleukin (IL)-10 and IL-22 are key members of the IL-10 cytokine family that share characteristic properties such as defined structural features, usage of IL-10R2 as one receptor chain, and activation of signal transducer and activator of transcription (STAT)-3 as dominant signaling mode. IL-10, formerly known as cytokine synthesis inhibitory factor, is key to deactivation of monocytes/macrophages and dendritic cells. Accordingly, pre-clinical studies document its anti-inflammatory capacity. However, the outcome of clinical trials assessing the therapeutic potential of IL-10 in prototypic inflammatory disorders has been disappointing. In contrast to IL-10, IL-22 acts primarily on non-leukocytic cells, in particular epithelial cells of intestine, skin, liver, and lung. STAT3-driven proliferation, anti-apoptosis, and anti-microbial tissue protection is regarded a principal function of IL-22 at host/environment interfaces. In this hypothesis article, hidden/underappreciated pro-inflammatory characteristics of IL-10 and IL-22 are outlined and related to cellular priming by type I interferon. It is tempting to speculate that an inherent inflammatory potential of IL-10 and IL-22 confines their usage in tissue protective therapy and beyond that determines in some patients efficacy of type I interferon treatment.
The CYP2D6 animal model: how to induce autoimmune hepatitis in mice
- Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to generate an animal model that reflects the characteristics of the human disease 3-5. However, in various models the induction of disease was rather complex and often hepatitis was only transient3-5. Therefore, we have developed a straightforward mouse model that uses the major human autoantigen in type 2 autoimmune hepatitis (AIH-2), namely hCYP2D6, as a trigger6. Type 1 liver-kidney microsomal antibodies (LKM-1) antibodies recognizing hCYP2D6 are the hallmark of AIH-27,8. Delivery of hCYP2D6 into wildtype FVB or C57BL/6 mice was by an Adenovirus construct (Ad-2D6) that ensures a direct delivery of the triggering antigen to the liver. Thus, the ensuing local inflammation generates a fertile field9 for the subsequent development of autoimmunity. A combination of intravenous and intraperitoneal injection of Ad-2D6 is the most effective route to induce a long-lasting autoimmune damage to the liver (section 1). Here we provide a detailed protocol on how autoimmune liver disease is induced in the CYP2D6 model and how the different aspects of liver damage can be assessed. First, the serum levels of markers indicating hepatocyte destruction, such as aminotransferases, as well as the titers of hCYP2D6 antibodies are determined by sampling blood retroorbitaly (section 2). Second, the hCYP2D6-specific T cell response is characterized by collecting lymphocytes from the spleen and the liver. In order to obtain pure liver lymphocytes, the livers are perfused by PBS via the portal vein (section 3), digested in collagen and purified over a Percoll gradient (section 4). The frequency of hCYP2D6-specific T cells is analyzed by stimulation with hCYP2D6 peptides and identification of IFNγ-producing cells by flow cytometry (section 5). Third, cellular infiltration and fibrosis is determined by immunohistochemistry of liver sections (section 6). Such analysis regimen has to be conducted at several times after initiation of the disease in order to prove the chronic nature of the model. The magnitude of the immune response characterized by the frequency and activity of hCYP2D6-specific T and/or B cells and the degree of the liver damage and fibrosis have to be assessed for a subsequent evaluation of possible treatments to prevent, delay or abrogate the autodestructive process of the liver.
Wound healing in mice with high-fat diet- or ob gene-induced diabetes-obesity syndromes: a comparative study
Josef Martin Pfeilschifter
- In the past, the genetically diabetic-obese diabetes/diabetes (db/db) and obese/obese (ob/ob) mouse strains were used to investigate mechanisms of diabetes-impaired wound healing. Here we determined patterns of skin repair in genetically normal C57Bl/6J mice that were fed using a high fat diet (HFD) to induce a diabetes-obesity syndrome. Wound closure was markedly delayed in HFD-fed mice compared to mice which had received a standard chow diet (CD). Impaired wound tissue of HFD mice showed a marked prolongation of wound inflammation. Expression of vascular endothelial growth factor (VEGF) was delayed and associated with the disturbed formation of wound margin epithelia and an impaired angiogenesis in the reduced granulation tissue. Normal wound contraction was retarded and disordered. Wound disorders in obese C57Bl/6J mice were paralleled by a prominent degradation of the inhibitor of NFκB (IκB-α) in the absence of an Akt activation. By contrast to impaired wound conditions in ob/ob mice, late wounds of HFD mice did not develop a chronic inflammatory state and were epithelialized after 11 days of repair. Thus, only genetically obese and diabetic ob/ob mice finally developed chronic wounds and therefore represent a better suited experimental model to investigate diabetes-induced wound healing disorders.
The human operculo-insular cortex is pain-preferentially but not pain-exclusively activated by trigeminal and olfactory stimuli
Bruno Georg Oertel
- Increasing evidence about the central nervous representation of pain in the brain suggests that the operculo-insular cortex is a crucial part of the pain matrix. The pain-specificity of a brain region may be tested by administering nociceptive stimuli while controlling for unspecific activations by administering non-nociceptive stimuli. We applied this paradigm to nasal chemosensation, delivering trigeminal or olfactory stimuli, to verify the pain-specificity of the operculo-insular cortex. In detail, brain activations due to intranasal stimulation induced by non-nociceptive olfactory stimuli of hydrogen sulfide (5 ppm) or vanillin (0.8 ppm) were used to mask brain activations due to somatosensory, clearly nociceptive trigeminal stimulations with gaseous carbon dioxide (75% v/v). Functional magnetic resonance (fMRI) images were recorded from 12 healthy volunteers in a 3T head scanner during stimulus administration using an event-related design. We found that significantly more activations following nociceptive than non-nociceptive stimuli were localized bilaterally in two restricted clusters in the brain containing the primary and secondary somatosensory areas and the insular cortices consistent with the operculo-insular cortex. However, these activations completely disappeared when eliminating activations associated with the administration of olfactory stimuli, which were small but measurable. While the present experiments verify that the operculo-insular cortex plays a role in the processing of nociceptive input, they also show that it is not a pain-exclusive brain region and allow, in the experimental context, for the interpretation that the operculo-insular cortex splay a major role in the detection of and responding to salient events, whether or not these events are nociceptive or painful.