[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) play a critical role in many biological processes and are aberrantly expressed in human cancers. Particular miRNAs function either as tumor suppressors or oncogenes and appear to have diagnostic and prognostic significance. Although numerous miRNAs are dys-regulated in colorectal cancer (CRC) only a small fraction has been characterized functionally. Using high-throughput functional screening and miRNA profiling of clinical samples the present study aims at identifying miRNAs important for the control of cellular growth and/or apoptosis in CRC. The high-throughput functional screening was carried out in six CRC cell lines transfected with a pre-miR library including 319 synthetic human pre-miRs. Phenotypic alterations were evaluated by immunostaining of cleaved cPARP (apoptosis) or MKI67 (proliferation). Additionally, TaqMan Human MicroRNA Array Set v2.0 was used to profile the expression of 667 miRNAs in 14 normal colon mucosa and 46 microsatellite stable stage II CRC patients. Among the miRNAs that induced growth arrest and apoptosis in the CRC cell lines, and at same time were dys-regulated in the clinical samples, miR-375 was selected for further analysis. Independent in vitro analysis of transient and stable transfected CRC cell lines confirmed that miR-375 reduces cell viability through the induction of apoptotic death. We identified YAP1 as a direct miR-375 target in CRC and show that HELLS and NOLC1 are down-stream targets. Knock-down of YAP1 mimicked the phenotype induced by miR-375 over-expression indicating that miR-375 most likely exerts its pro-apoptotic role through YAP1 and its anti-apoptotic down-stream targets BIRC5 and BCL2L1. Finally, in vivo analysis of mouse xenograft tumors showed that miR-375 expression significantly reduced tumor growth. We conclude that the high-throughput screening successfully identified miRNAs that induce apoptosis and/or inhibit proliferation in CRC cells. Finally, combining the functional screening with profiling of CRC tissue samples we identified clinically relevant miRNAs and miRNA targets in CRC.
PLoS ONE 01/2014; 9(6):e96767. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: RNAi-based strategies provide a great therapeutic potential for treatment of various human diseases including kidney disorders, but face the challenge of in vivo delivery and specific targeting. The chitosan delivery system has previously been shown to target siRNA specifically to the kidneys in mice when administered intravenously. Here we confirm by 2D and 3D bioimaging that chitosan formulated siRNA is retained in the kidney for more than 48 hours where it accumulates in proximal tubule epithelial cells (PTECs), a process that was strongly dependent on the molecular weight of chitosan. Chitosan/siRNA nanoparticles, administered to chimeric mice with conditional knockout of the megalin gene, distributed almost exclusively in cells that expressed megalin, implying that the chitosan/siRNA particle uptake was mediated by a megalin-dependent endocytotic pathway. Knockdown of the water channel aquaporin 1 (AQP1) by up to 50% in PTECs was achieved utilizing the systemic i.v. delivery of chitosan/AQP1 siRNA in mice. In conclusion, specific targeting PTECs with the chitosan nanoparticle system may prove to be a useful strategy for knockdown of specific genes in PTECs, and provides a potential therapeutic strategy for treating various kidney diseases.
[Show abstract][Hide abstract] ABSTRACT: Bacterial biofilms causing implant-associated osteomyelitis is a severe complication with limited antimicrobial therapy options. We designed an animal model, in which implant associated osteomyelitis arise from a Staphylococcus aureus biofilm on a tibia implant. Two bioluminescently engineered (luxA-E transformed), strains of S. aureus were utilized, Xen29 and Xen31. Biofilm formation was assessed with epifluorescence microscopy. Quantitative measurements were performed day 4, 6, 8, 11 and 15 post-surgery. Bacteria were extracted from the biofilm by sonication and the bacterial load quantified by culturing. Biofilm formation was evident from day 6 post-implantation. Mean bacterial load from implants was ∼1×104 CFU/implant, while mean bacterial load from infected tibias were 1×106 CFU/bone. Bioluminesence imaging revealed decreasing activity throughout the 15-day observation period, with signal intensity for both strains reaching that of the negative control by day 15 while there was no significant reduction in bacterial load. The model is suitable for testing antimicrobial treatment options for implant associated OM, as treatment efficacy on both biofilm and viable counts can be assessed.
PLoS ONE 01/2014; 9(10):e103688. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In patients with acute kidney injury (AKI) mortality remains high, despite the fact that the patients are treated with continuous renal replacement therapy. The interaction between the kidney and the immune system might explain the high mortality observed in AKI. In order to elucidate the interaction between the kidney and immune system we developed a two-hit model of AKI and endotoxemia. Our hypothesis was that ischemia/reperfusion (I/R) of the kidney simultaneously with endotoxemia would generate a more extensive inflammatory response compared to I/R of the hind legs. Our expectation was that elevated levels of cytokines would be found in both blood and in organs distant to the kidneys. Forty mice were divided into five groups. The mice were subjected to the following operations: A: Sham only, no lipopolysaccharide (LPS); B: I/R of both kidneys + LPS; C: LPS only; D: Nephrectomy + LPS; E: I/R of both hind legs + LPS. In groups B and E, I/R times were identical. All mice were kept alive for 24 h and then sacrificed. Levels of interleukin (IL)-1β, IL-6, IL-10, and tumor necrosis factor-α were measured in the blood. The activity of myeloperoxidase (MPO) in lungs, kidneys, and liver was evaluated as an indirect measurement of accumulation of granulocytes. In this study, significantly higher amount of IL-6 and IL-10 in the plasma was observed following renal I/R compared to hind leg I/R. The elevated levels of cytokine in plasma were observed following nephrectomy and endotoxemia. The neutrophil infiltration of distant organs measured by the levels of MPO in the lung and liver also showed a significantly higher level in renal I/R compared to hind leg I/R. Renal I/R is associated with a more pronounced inflammatory response in blood and distant organs. The high cytokine levels measured following nephrectomy might be explained by compromised elimination of cytokines by the kidney in AKI.
[Show abstract][Hide abstract] ABSTRACT: We describe a simple method for bone engineering using biodegradable scaffolds with mesenchymal stem cells derived from human induced-pluripotent stem cells (hiPS-MSCs). The hiPS-MSCs expressed mesenchymal markers (CD90, CD73, and CD105), possessed multipotency characterized by tri-lineages differentiation: osteogenic, adipogenic, and chondrogenic, and lost pluripotency - as seen with the loss of markers OCT3/4 and TRA-1-81 - and tumorigenicity. However, these iPS-MSCs are still positive for marker NANOG. We further explored the osteogenic potential of the hiPS-MSCs in synthetic polymer polycaprolactone (PCL) scaffolds or PCL scaffolds functionalized with natural polymer hyaluronan and ceramic TCP (PHT) both in vitro and in vivo. Our results showed that these iPS-MSCs are functionally compatible with the two 3D scaffolds tested and formed typically calcified structure in the scaffolds. Overall, our results suggest the iPS-MSCs derived by this simple method retain fully osteogenic function and provide a new solution towards personalized orthopedic therapy in the future.
[Show abstract][Hide abstract] ABSTRACT: Background:The tyrosine kinase receptor HER4 is a member of the epidermal growth factor receptor (EGFR) family. It plays diverse roles in cancer development and cancer progression and can both exert oncogenic and tumour-suppressive activities. Alternatively spliced isoforms of HER4 are critical to the different signalling possibilities of HER4.Methods:We use a splice-switching oligonucleotide (SSO) to direct the alternative splicing of HER4 from the CYT1 to the CYT2 isoform in HER4-expressing breast cancer cells.Results:Treatment with a target-specific SSO was accompanied by a decreased growth of the cells (P<0.0001). In addition, the SSO treatment induced a decreased activity of Akt. We confirmed the SSO-dependent switching of the HER4 isoform CYT1 to CYT2 expression in a xenografted mouse tumour model driven by subcutaneously injected MCF7 cells. We hence demonstrated the feasibility of SSO-directed splice-switching activity in vivo. Furthermore, the SSO treatment efficiently decreased the growth of the xenografted tumour (P=0.0014).Conclusion:An SSO directing the splicing of HER4 towards the CYT2 isoform has an inhibitory effect of cancer cell growth in vitro and in vivo. These results may pave the way for the development of new anticancer drugs in HER4-deregulated cancers in humans.British Journal of Cancer advance online publication, 21 May 2013; doi:10.1038/bjc.2013.247 www.bjcancer.com.
British Journal of Cancer 05/2013; · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mitochondria, the powerhouses of our cells, are remnants of a eubacterial endosymbiont. Notwithstanding the evolutionary time that has passed since the initial endosymbiotic event, mitochondria have retained many hallmarks of their eubacterial origin. Recent studies have indicated that during perturbations of normal homeostasis, such as following acute trauma leading to massive necrosis and release of mitochondria, the immune system might mistake symbiont for enemy and initiate an inappropriate immune response. The innate immune system is the first line of defense against invading microbial pathogens, and as such is the primary suspect in the recognition of mitochondria-derived danger-associated molecular patterns (DAMPs) and initiation of an aberrant response. Conversely, innate immune mechanisms are also central to non-inflammatory clearance of innocuous agents. Here we investigated the role of a central humoral component of innate immunity, the lectin pathway of complement, in recognition of mitochondria in vitro and in vivo. We found that the soluble pattern-recognition molecules (PRMs), mannan-binding lectin (MBL), L-ficolin and M-ficolin were able to recognize mitochondria. Furthermore, MBL in complex with MBL-associated serine protease 2 (MASP-2) was able to activate the lectin pathway and deposit C4 onto mitochondria, suggesting that these molecules are either involved in homeostatic clearance of mitochondria or induction of untoward inflammatory reactions. We found that following mitochondrial challenge, C3 was consumed in vivo in the absence of overt inflammation, indicating a potential role of complement in non-inflammatory clearance of mitochondria. Thus, we report here the first indication of involvement of the lectin pathway in mitochondrial immune handling.
Journal of Biological Chemistry 02/2013; · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Circulating mannan-binding lectin (MBL) levels are elevated in type 1 diabetes. Further, high MBL levels are associated with the development of diabetic nephropathy. In animals, a direct effect of MBL on diabetic kidney changes is observed. We hypothesized that MBL levels and detrimental complement activation increase as a consequence of diabetes. We measured plasma MBL before and seven weeks after inducing diabetes by streptozotocin. Mice have two MBLs, MBL-A and MBL-C. Diabetes-induction led to an increase in MBL-C concentration whereas no change during the study was found in the control group. The increase in MBL-C was associated with the increasing plasma glucose levels. In accordance with the observed changes in circulating MBL levels, liver expression of Mbl2 mRNA (encoding MBL-C) was increased in diabetes. Mbl1 expression (encoding MBL-A) did not differ between diabetic and control animals. The estimated half-life of recombinant human MBL was significantly prolonged in mice with diabetes compared with control mice. Complement activation in plasma and glomeruli did not differ between groups. We demonstrate for the first time that MBL levels increase after induction of diabetes and in parallel with increasing plasma glucose. Our findings support the previous clinical observations of increased MBL in type 1 diabetes. This change may be explained by alternations both in MBL production and turnover.
Scandinavian Journal of Immunology 01/2013; · 2.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Harnessing the RNA interference pathway offers a new therapeutic modality; however, solutions to overcome biological barriers to small interfering RNA (siRNA) delivery are required for clinical translation. This work demonstrates, by direct northern and quantitative PCR (qPCR) detection, stability, gastrointestinal (GI) deposition, and translocation into peripheral tissue of nonmodified siRNA after oral gavage of chitosan/siRNA nanoparticles in mice. In contrast to naked siRNA, retained structural integrity and deposition in the stomach, proximal and distal small intestine, and colon was observed at 1 and 5 hours for siRNA within nanoparticles. Furthermore, histological detection of fluorescent siRNA at the apical regions of the intestinal epithelium suggests mucoadhesion provided by chitosan. Detection of intact siRNA in the liver, spleen, and kidney was observed 1 hour after oral gavage, with an organ distribution pattern influenced by nanoparticle N:P ratio that could reflect differences in particle stability. This proof-of-concept work presents an oral delivery platform that could have the potential to treat local and systemic disorders by siRNA.Molecular Therapy - Nucleic Acids (2013) 2, e76; doi:10.1038/mtna.2013.2; published online 5 March 2013.
[Show abstract][Hide abstract] ABSTRACT: Some of the main concerns with application of naked small interfering RNA are rapid degradation and urinary excretion resulting in a short plasma half-life. In this study we investigated how conjugation of polyethylene glycol (PEG) with variable chain length affects siRNA pharmacokinetics and biodistribution. The PEG chains were conjugated to chemically stabilized siRNA at the 5' terminal end of the passenger strand using click chemistry. The siRNA conjugate remained functionally active and showed significantly prolonged circulation in the blood stream after intravenous injection. siRNA conjugated with 20kDa PEG (PEG20k-siRNA) was most persistent, approximately 50% PEG20k-siRNA remained 1h post-injection, while the uncoupled siRNA was rapidly removed >90% at 15min. fluorescent imaging of the living animal showed increased concentration of siRNA in peripheral tissue and delayed urine excretion when coupled to PEG 20k. Biodistribution studies by northern blotting revealed equal distribution of conjugated siRNA in liver, kidney, spleen and lung without significant degradation 24 h post-injection. Our study demonstrates that PEG conjugated siRNA can be applied as a delivery system to improve siRNA bioavailability and may potentially increase the efficiency of siRNA in therapeutic applications.
[Show abstract][Hide abstract] ABSTRACT: Gene electrotransfer is an effective non-viral technique for delivery of plasmid DNA into tissues. From a clinical perspective muscle is an attractive target tissue as long-term high-level transgenic expression can be achieved. Spatial distribution of the transgenic protein following gene electrotransfer to muscle in a large animal model has not yet been investigated. In this study 17 different doses of plasmid DNA (1-1500 µg firefly luciferase pCMV-Luc) were delivered in vivo to porcine gluteal muscle using electroporation. Forty-eight hours post treatment several biopsies were obtained from each transfection site in order to examine the spatial distribution of the transgenic product. We found a significantly higher luciferase activity in biopsies from the center of the transfection site compared to biopsies taken adjacent to the center 1 and 2 cm along muscle fibre orientation (p<0.05 and p<0.0001, respectively). On average, 43 % of the total luciferase activity was localized in the center biopsy. In conclusion, we found that gene electrotransfer to muscle in a large animal model led to localized gene expression corresponding to the area delineated by the electrodes. High doses of plasmid DNA did not lead to a larger area of the muscle expressing the transgenic protein.
Human Gene Therapy Methods 11/2012; · 1.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Targeted transgenesis using site-specific recombinases is an attractive method to create genetically modified animals as it allows for integration of the transgene in a pre-selected transcriptionally active genomic site. Here we describe the application of recombinase-mediated cassette exchange (RMCE) in cells from a Göttingen minipig with four RMCE acceptor loci, each containing a green fluorescence protein (GFP) marker gene driven by a human UbiC promoter. The four RMCE acceptor loci segregated independent of each other, and expression profiles could be determined in various tissues. Using minicircles in RMCE in fibroblasts with all four acceptor loci and followed by SCNT, we produced piglets with a single copy of a transgene incorporated into one of the transcriptionally active acceptor loci. The transgene, consisting of a cDNA of the Alzheimer's disease-causing gene PSEN1M146I driven by an enhanced human UbiC promoter, had an expression profile in various tissues similar to that of the GFP marker gene. The results show that RMCE can be done in a pre-selected transcriptionally active acceptor locus for targeted transgenesis in pigs.
[Show abstract][Hide abstract] ABSTRACT: Background. Radiation-induced fibrosis (RIF) is a dose-limiting complication of cancer radiotherapy and causes serious problems, i.e. restricted tissue flexibility, pain, ulceration or necrosis. Recently, we have successfully treated RIF in a mouse model by intraperitoneal administration of chitosan/siRNA nanoparticles directed towards silencing TNF alpha in local macrophage populations, but the mechanism for the therapeutic effect at the lesion site remains unclear. Methods. Using the same murine RIF model we utilized an optical imaging technique and fluorescence microscopy to investigate the uptake of chitosan/fluorescently labeled siRNA nanoparticles by peritoneal macrophages and their subsequent migration to the inflamed tissue in the RIF model. Results. We observed strong accumulation of the fluorescent signal in the lesion site of the irradiated leg up to 24 hours using the optical imaging system. We further confirm by immunohistochemical staining that Cy3 labeled siRNA resides in macrophages of the irradiated leg. Conclusion. We provide a proof-of-concept for host macrophage trafficking towards the inflamed region in a murine RIF model, which thereby suggests that the chitosan/siRNA nanoparticle may constitute a general treatment for inflammatory diseases using the natural homing potential of macrophages to inflammatory sites.
[Show abstract][Hide abstract] ABSTRACT: The aim was to further characterize the SKG model of rheumatoid arthritis (RA) and its potential for studying intervention treatments, with special focus on bone targeting therapies. Three individual studies were conducted, using a total of 71 SKG mice, comparing arthritis induction with mannan versus zymosan A, female versus male mice, and the effect of dexamethasone intervention treatment initiated at different time points after arthritis induction. Hind paws were embedded undecalcified in methyl methacrylate, and sections were stained with Masson-Goldner trichrome. Areal Bone Mineral Density (aBMD) of the femora was determined with pDXA. RNA was extracted from the hind paws followed by the quantification by reverse transcriptase PCR. SKG mice stimulated with mannan presented a higher arthritis score than mice stimulated with zymosan A. Female SKG mice developed a more severe arthritis than male SKG mice. Dexamethasone inhibited arthritis clinically as well as histologically when the treatment was initiated prophylactically or within the first week of arthritis. Femoral aBMD was lower in animals with arthritis than in control animals. The RANKL RNA expression was elevated in arthritic mice, whereas OPG RNA expression was unchanged. The results suggest mannan as arthritis inductor and female instead of male mice in experiments as well as an optimal time window for the initiation of treatment. Systemic bone loss as well as local up regulation of RANKL was present early in SKG arthritis. These results demonstrate that SKG arthritis is a suitable new model for evaluating therapies in RA.
Rheumatology International 09/2012; · 2.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Alzheimer's disease (AD) is a disabling, fatal disease, where animal models potentially can enable investigation of aetiology and treatment. The first litter of Göttingen minipigs carrying a mutation for human AD was born in 2007, showing transgene expression. In human AD patients, memory impairment is the most striking and consistent feature. The aim of the present study was to examine effects of the APPsw transgene on memory of AD minipigs compared with non-transgenic controls at two ages (1-2 years) using the spontaneous object recognition test (SORT), which is based on behavioural discrimination of familiar and novel objects. No significant difference between AD minipigs and controls was found when comparing object recognition as a measure of memory. The minipigs did explore the novel object significantly more than the familiar, indicating the expected recognition of the familiar object. Two different inter-phase intervals were used (IPI: 10-40 min). For both ages, object recognition was evident using 10 min IPI. When using 40 min IPI, object recognition was evident only at age 1 year. Comparing memory of a relatively small group of AD minipigs and controls at two rather young ages using the SORT, we were not able to show memory impairment in APPsw carrying minipigs. Being an age-dependent disease, the transgene is expected to cause AD-like symptoms in this porcine model, and the SORT should be repeated at older ages.
[Show abstract][Hide abstract] ABSTRACT: Synthetic glucocorticoids are potent anti-inflammatory drugs but serious side effects such as bone mobilization, muscle mass loss, immunosuppression, and metabolic alterations make glucocorticoid therapy a difficult balance. The therapeutic anti-inflammatory effect of glucocorticoids relies largely on the suppressed release of tumor-necrosis factor-α and other cytokines by macrophages at the sites of inflammation. We have now developed a new biodegradable anti-CD163 antibody-drug conjugate that specifically targets the glucocorticoid, dexamethasone to the hemoglobin scavenger receptor CD163 in macrophages. The conjugate, that in average contains four dexamethasone molecules per antibody, exhibits retained high functional affinity for CD163. In vitro studies in rat macrophages and in vivo studies of Lewis rats showed a strong anti-inflammatory effect of the conjugate measured as reduced lipopolysaccharide-induced secretion of tumor-necrosis factor-α. The in vivo potency of conjugated dexamethasone was about 50-fold that of nonconjugated dexamethasone. In contrast to a strong systemic effect of nonconjugated dexamethasone, the equipotent dose of the conjugate had no such effect, measured as thymus lymphocytes apoptosis, body weight loss, and suppression of endogenous cortisol levels. In conclusion, the study shows antibody-drug conjugates as a future approach in anti-inflammatory macrophage-directed therapy. Furthermore, the data demonstrate CD163 as an excellent macrophage target for anti-inflammatory drug delivery.
[Show abstract][Hide abstract] ABSTRACT: Vascular endothelial growth factor (VEGF) is an angiogenic growth factor that plays a critical role in several diseases, including cancer, rheumatoid arthritis and diseases of the eye. Persistent regulation of VEGF by expression of small interfering RNAs targeting VEGF represents a potential future strategy for treatment of such diseases. As a step toward this goal, the present study combines the potency of VEGF-targeted miRNA mimics, produced from a miRNA cluster, with delivery by adeno-associated virus (AAV)-based vectors.
Nine different engineered tri-cistronic miRNA clusters encoding anti-VEGF effectors were generated and tested in adult human retinal pigment epithelial (ARPE-19) cells using Renilla luciferase screening, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), western blotting and immunostaining analysis. In vivo efficacy was tested by the injection of scAAV2/8 vectors expressing the most effective miRNA cluster into murine hindlimb muscles, followed by quantitative RT-PCR.
Plasmids containing anti-VEGF miRNA clusters showed efficient silencing of VEGF and demonstrated a combined gene silencing effect for miRNA clusters composed of multiple miRNA-mimicked RNA interference effectors. The most potent molecule, miR-5,10,7, resulted in a knockdown of VEGF by approximately 75%. Injection of scAAV2/8 vectors expressing miR-5,10,7 into murine hindlimb muscles, resulted in a 44% reduction of endogenous VEGF.
We have developed miRNA clusters encoding anti-VEGF effectors and shown, in a mouse model, that VEGF is efficiently down-regulated by scAAV2/8-delivered miRNA clusters, allowing potent attenuation of VEGF. These findings may contribute to the development of gene therapy based on AAV-mediated delivery of miRNA clusters.
The Journal of Gene Medicine 03/2012; 14(5):328-38. · 2.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Herpes simplex viruses (HSVs) are highly prevalent neurotropic viruses. While they can replicate lytically in cells of the epithelial lineage, causing lesions on mucocutaneous surfaces, HSVs also establish latent infections in neurons, which act as reservoirs of virus for subsequent reactivation events. Immunological control of HSV involves activation of innate immune pattern-recognition receptors such as TLR3, which detects double-stranded RNA and induces type I IFN expression. Humans with defects in the TLR3/IFN pathway have an elevated susceptibility to HSV infections of the CNS. However, it is not known what cell type mediates the role of TLR3 in the immunological control of HSV, and it is not known whether TLR3 sensing occurs prior to or after CNS entry. Here, we show that in mice TLR3 provides early control of HSV-2 infection immediately after entry into the CNS by mediating type I IFN responses in astrocytes. Tlr3-/- mice were hypersusceptible to HSV-2 infection in the CNS after vaginal inoculation. HSV-2 exhibited broader neurotropism in Tlr3-/- mice than it did in WT mice, with astrocytes being most abundantly infected. Tlr3-/- mice did not exhibit a global defect in innate immune responses to HSV, but astrocytes were defective in HSV-induced type I IFN production. Thus, TLR3 acts in astrocytes to sense HSV-2 infection immediately after entry into the CNS, possibly preventing HSV from spreading beyond the neurons mediating entry into the CNS.
The Journal of clinical investigation 03/2012; 122(4):1368-76. · 15.39 Impact Factor