Molecular Mechanisms of Nasal Epithelium in Rhinitis and Rhinosinusitis

Peer reviewe

Cytomegalovirus infection of human kidney cells in vitro

Cytomegalovirus infection of human kidney cells in vitro. To study which structures of a kidney allograft are the main targets for cytomegalovirus (CMV), human glomerular epithelial and mesangial cells, as well as tubular epithelial and endothelial cells were isolated by steel meshes of different pore sizes and enzymatic treatments. The various cultured cell types were characterized by morphology and specific antibodies. Human CMV was inoculated onto cell monolayers using two different culture methods: conventional tissue culture and rapid shell vial culture. To analyze whether CMV had a direct effect on the immunologic properties of kidney parenchymal cells, MHC class I and class II antigen expression was estimated before and after the infection. CMV infected all kidney cells identically. All cells expressed class I strongly after the infection, but they were class I positive prior to infection. Class II antigens were not expressed on the cell surface either before or after the infection. In conclusion, human kidney cells of glomerular, tubular and vascular origin were all infected by CMV without any difference. CMV had no significant direct effects on the antigenic properties of the cells

Analysis of human brain tissue derived from DBS surgery

Background: Transcriptomic and proteomic profiling of human brain tissue is hindered by the availability of fresh samples from living patients. Postmortem samples usually represent the advanced disease stage of the patient. Furthermore, the postmortem interval can affect the transcriptomic and proteomic profiles. Therefore, fresh brain tissue samples from living patients represent a valuable resource of metabolically intact tissue. Implantation of deep brain stimulation (DBS) electrodes into the human brain is a neurosurgical treatment for, e.g., movement disorders. Here, we describe an improved approach to collecting brain tissues from surgical instruments used in implantation of DBS device for transcriptomics and proteomics analyses. Methods: Samples were extracted from guide tubes and recording electrodes used in routine DBS implantation procedure to treat patients with Parkinson's disease, genetic dystonia and tremor. RNA sequencing was performed in tissues extracted from the recording microelectrodes and liquid chromatography-mass spectrometry (LC-MS) performed in tissues from guide tubes. To assess the performance of the current approach, the obtained datasets were compared with previously published datasets representing brain tissues. Results: Altogether, 32,034 RNA transcripts representing the unique Ensembl gene identifiers were detected from eight samples representing both hemispheres of four patients. By using LC-MS, we identified 734 unique proteins from 31 samples collected from 14 patients. The datasets are available in the BioStudies database (accession number S-BSST667). Our results indicate that surgical instruments used in DBS installation retain brain material sufficient for protein and gene expression studies. Comparison with previously published datasets obtained with similar approach proved the robustness and reproducibility of the protocol. Conclusions: The instruments used during routine DBS surgery are a useful source for obtaining fresh brain tissues from living patients. This approach overcomes the issues that arise from using postmortem tissues, such as the effect of postmortem interval on transcriptomic and proteomic landscape of the brain, and can be used for studying molecular aspects of DBS-treatable diseases.Peer reviewe

Extensive shift in placental transcriptome profile in preeclampsia and placental origin of adverse pregnancy outcomes

One in five pregnant women suffer from gestational complications, prevalently driven by placental malfunction. Using RNASeq, we analyzed differential placental gene expression in cases of normal gestation, late-onset preeclampsia (LO-PE), gestational diabetes (GD) and pregnancies ending with the birth of small-for-gestational-age (SGA) or large-for-gestational-age (LGA) newborns (n = 8/group). In all groups, the highest expression was detected for small noncoding RNAs and genes specifically implicated in placental function and hormonal regulation. The transcriptome of LO-PE placentas was clearly distinct, showing statistically significant (after FDR) expressional disturbances for hundreds of genes. Taqman RT-qPCR validation of 45 genes in an extended sample (n = 24/group) provided concordant results. A limited number of transcription factors including LRF, SP1 and AP2 were identified as possible drivers of these changes. Notable differences were detected in differential expression signatures of LO-PE subtypes defined by the presence or absence of intrauterine growth restriction (IUGR). LO-PE with IUGR showed higher correlation with SGA and LO-PE without IUGR with LGA placentas. Whereas changes in placental transcriptome in SGA, LGA and GD cases were less prominent, the overall profiles of expressional disturbances overlapped among pregnancy complications providing support to shared placental responses. The dataset represent a rich catalogue for potential biomarkers and therapeutic targets.Peer reviewe

Fast growth associated with aberrant vasculature and hypoxia in fibroblast growth factor 8b (FGF8b) over-expressing PC-3 prostate tumour xenografts

Background: Prostate tumours are commonly poorly oxygenated which is associated with tumour progression and development of resistance to chemotherapeutic drugs and radiotherapy. Fibroblast growth factor 8b (FGF8b) is a mitogenic and angiogenic factor, which is expressed at an increased level in human prostate tumours and is associated with a poor prognosis. We studied the effect of FGF8b on tumour oxygenation and growth parameters in xenografts in comparison with vascular endothelial growth factor (VEGF)-expressing xenografts, representing another fast growing and angiogenic tumour model. Methods: Subcutaneous tumours of PC-3 cells transfected with FGF8b, VEGF or empty (mock) vectors were produced and studied for vascularity, cell proliferation, glucose metabolism and oxygenation. Tumours were evaluated by immunohistochemistry (IHC), flow cytometry, use of radiolabelled markers of energy metabolism ([F-18] FDG) and hypoxia ([F-18] EF5), and intratumoral polarographic measurements of pO(2). Results: Both FGF8b and VEGF tumours grew rapidly in nude mice and showed highly vascularised morphology. Perfusion studies, pO(2) measurements, [F-18] EF5 and [F-18] FDG uptake as well as IHC staining for glucose transport protein (GLUT1) and hypoxia inducible factor (HIF) 1 showed that VEGF xenografts were well-perfused and oxygenised, as expected, whereas FGF8b tumours were as hypoxic as mock tumours. These results suggest that FGF8b-induced tumour capillaries are defective. Nevertheless, the growth rate of hypoxic FGF8b tumours was highly increased, as that of well-oxygenised VEGF tumours, when compared with hypoxic mock tumour controls. Conclusion: FGF8b is able to induce fast growth in strongly hypoxic tumour microenvironment whereas VEGF-stimulated growth advantage is associated with improved perfusion and oxygenation of prostate tumour xenografts

Alendronate decreases orthotopic PC-3 prostate tumor growth and metastasis to prostate-draining lymph nodes in nude mice

<p>Abstract</p> <p>Background</p> <p>Metastatic prostate cancer is associated with a high morbidity and mortality but the spreading mechanisms are still poorly understood. The aminobisphosphonate alendronate, used to reduce bone loss, has also been shown to inhibit the invasion and migration of prostate cancer cells <it>in vitro</it>. We used a modified orthotopic PC-3 nude mouse tumor model of human prostate cancer to study whether alendronate affects prostate tumor growth and metastasis.</p> <p>Methods</p> <p>PC-3 cells (5 × 10⁵) were implanted in the prostates of nude mice and the mice were treated with alendronate (0.5 mg/kg/day in PBS, s.c.) or vehicle for 4 weeks. After sacrifice, the sizes of tumor-bearing prostates were measured and the tumors and prostate-draining regional iliac and sacral lymph nodes were excised for studies on markers of proliferation, apoptosis, angiogenesis and lymphangiogenesis, using histomorphometry and immunohistochemistry.</p> <p>Results</p> <p>Tumor occurrence in the prostate was 73% in the alendronate-treated group and 81% in the control group. Mean tumor size (218 mm³, range: 96–485 mm³, <it>n </it>= 11) in the alendronate-treated mice was 41% of that in the control mice (513 mm³, range: 209–1350 mm³, <it>n </it>= 13) (<it>p </it>< 0.05). In the iliac and sacral lymph nodes of alendronate-treated mice, the proportion of metastatic area was only about 10% of that in control mice (<it>p </it>< 0.001). Immunohistochemical staining of tumor sections showed that alendronate treatment caused a marked decrease in the number of CD34-positive endothelial cells in tumors (<it>p </it>< 0.001) and an increase in that of ISEL positive apoptotic cells in tumors as well as in lymph node metastases (<it>p </it>< 0.05) compared with those in the vehicle-treated mice. The density of m-LYVE-1-stained lymphatic capillaries was not changed.</p> <p>Conclusion</p> <p>Our results demonstrate that alendronate treatment opposes growth of orthotopic PC-3 tumors and decreases tumor metastasis to prostate-draining lymph nodes. This effect could be at least partly explained by decreased angiogenesis and increased apoptosis. The results suggest that bisphosphonates have anti-tumoral and anti-invasive effects on primary prostate cancer.</p

Androgen and fibroblast growth factor 8 (FGF8) downregulation of thrombospondin 1 (TSP1) in mouse breast cancer cells

In the search for androgen target genes responsible for malignant growth in S115 mouse mammary tumor cells we found that thrombospondin 1 (TSP1) expression was strongly downregulated by testosterone (Te). Experiments with cycloheximide suggested that Te repression of TSP1 was dependent on de novo protein synthesis. TSPI repression by Te was preceded by the induction of fibroblast growth factor 8 (FGF8) expression. FGF8 has previously been shown to mediate androgen effects on proliferation of S 115 cells by autocrine/paracrine mechanisms. It has also been shown to increase breast cancer cell growth as tumors in nude mice and to stimulate tumor angiogenesis. We studied here the possibility that FGF8 belonged to the Te-induced de novo synthesized proteins that mediate the effect of Te on TSP1 expression in these cells. We found that addition of FGF8b to in vitro cultures or ectopic expression of FGF8b in S115 cells repressed TSP1 expression at mRNA and protein levels even in the absence of Te. FGF2, another angiogenic member of FGF family, also downregulated TSPI mRNA level in the in vitro cultures of S115 cells. The antisense oligonucleotides for FGF8 did not, however, prevent Te-repression of TSP1 mRNA expression and a neutralizing anti-FGF8b antibody only partially opposed Te induced downregulation of TSP1. These results suggest that both androgen and FGF8 inhibit TSP1 expression independently. They also suggest that opposite to many other androgen-induced responses in S115 cells, the effect of Te on the expression TSP1 is not mediated by FGF8