Browsing by Subject "TISSUE"

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  • Radhakrishnan, Dhanya; Shanmukhan, Anju Pallipurath; Kareem, Abdul; Aiyaz, Mohammed; Varapparambathu, Vijina; Toms, Ashna; Kerstens, Merijn; Valsakumar, Devisree; Landge, Amit N.; Shaji, Anil; Mathew, Mathew K.; Sawchuk, Megan G.; Scarpella, Enrico; Krizek, Beth A.; Efroni, Idan; Mähönen, Ari Pekka; Willemsen, Viola; Scheres, Ben; Prasad, Kalika (2020)
    Aerial organs of plants, being highly prone to local injuries, require tissue restoration to ensure their survival. However, knowledge of the underlying mechanism is sparse. In this study, we mimicked natural injuries in growing leaves and stems to study the reunion between mechanically disconnected tissues. We show that PLETHORA (PLT) and AINTEGUMENTA (ANT) genes, which encode stem cell-promoting factors, are activated and contribute to vascular regeneration in response to these injuries. PLT proteins bind to and activate the CUC2 promoter. PLT proteins and CUC2 regulate the transcription of the local auxin biosynthesis gene YUC4 in a coherent feed-forward loop, and this process is necessary to drive vascular regeneration. In the absence of this PLT-mediated regeneration response, leaf ground tissue cells can neither acquire the early vascular identity marker ATHB8, nor properly polarise auxin transporters to specify new venation paths. The PLT-CUC2 module is required for vascular regeneration, but is dispensable for midvein formation in leaves. We reveal the mechanisms of vascular regeneration in plants and distinguish between the wound-repair ability of the tissue and its formation during normal development.
  • Rajala, Kristiina; Lindroos, Bettina; Hussein, Samer M.; Lappalainen, Riikka S.; Pekkanen-Mattila, Mari; Inzunza, Jose; Rozell, Bjorn; Miettinen, Susanna; Narkilahti, Susanna; Kerkela, Erja; Aalto-Setälä, Katriina; Otonkoski, Timo; Suuronen, Riitta; Hovatta, Outi; Skottman, Heli (2010)
  • Rotgers, E.; Cisneros-Montalvo, S.; Jahnukainen, K.; Sandholm, J.; Toppari, J.; Nurmio, M. (2015)
    Accurate analysis and quantification of different testicular cell populations are of central importance in studies of male reproductive biology. The traditional histomorphometric and immunohistochemical methods remain the gold standard in studying the complex dynamics of the testicular tissue. Through past years advances have been made in the application of flow cytometry for the rapid analysis of testicular cell populations. Detection of DNA content and of surface antigens and fluorescent reporters have been widely used to analyze and sort cells. Detection of intracellular antigens can broaden the possibilities of applying flow cytometry in studies of male reproduction. Here, we report a detailed protocol for the preparation of rat testicular tissue for detection of intracellular antigens by flow cytometry, and a pipeline for subsequent data analysis and troubleshooting. Rat testicular ontogenesis was chosen as the experimental model to validate the performance of the assay using vimentin and gamma H2AX as intracellular markers for the somatic and spermatogenic cells, respectively. The results show that the assay is reproducible and recapitulates the rat testis ontogenesis.
  • Ding, Yaping; Li, Wei; W. Schubert, Dirk; R. Boccaccini, Aldo; A. Roether, Judith; Santos, Hélder A. (2021)
    Electrospun organic/inorganic hybrid scaffolds have been appealing in tissue regeneration owing to the integrated physiochemical and biological performances. However, the conventional electrospun scaffolds with non-woven structures usually failed to enable deep cell infiltration due to the densely stacked layers among the fibers. Herein, through self-assembly-driven electrospinning, a polyhydroxybutyrate/poly(ε-caprolactone)/58S sol-gel bioactive glass (PHB/PCL/58S) hybrid scaffold with honeycomb-like structures was prepared by manipulating the solution composition and concentration during a one-step electrospinning process. Here, the mechanisms enabling the formation of self-assembled honeycomb-like structures were investigated through comparative studies using Fourier-transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) between PHB/PCL/58S and PHB/PCL/sol-gel silica systems. The obtained honeycomb-like structure was built up from nanofibers with an average diameter of 370 nm and showed a bimodal distribution of pores: large polygonal pores up to hundreds of micrometers within the honeycomb-cells and irregular pores among the nanofibers ranging around few micrometers. The cell-materials interactions were further studied by culturing MG-63 osteoblast-like cells for 7 days. Cell viability, cell morphology and cell infiltration were comparatively investigated as well. While cells merely proliferated on the surface of non-woven structures, MG-63 cells showed extensive proliferation and deep infiltration up to 100~200 μm into the honeycomb-like structure. Moreover, the cellular spatial organization was readily regulated by the honeycomb-like pattern as well. Overall, the newly obtained hybrid scaffold may integrate the enhanced osteogenicity originating from the bioactive components, and the improved cell-material interactions brought by the honeycomb-like structure, making the new scaffold a promising candidate for tissue regeneration.
  • Salo, Raimo A.; Belevich, Ilya; Jokitalo, Eija; Gröhn, Olli; Sierra, Alejandra (2021)
    Validation and interpretation of diffusion magnetic resonance imaging (dMRI) requires detailed understanding of the actual microstructure restricting the diffusion of water molecules. In this study, we used serial block-face scanning electron microscopy (SBEM), a three-dimensional electron microscopy (3D-EM) technique, to image seven white and grey matter volumes in the rat brain. SBEM shows excellent contrast of cellular membranes, which are the major components restricting the diffusion of water in tissue. Additionally, we performed 3D structure tensor (3D-ST) analysis on the SBEM volumes and parameterised the resulting orientation distributions using Watson and angular central Gaussian (ACG) probability distributions as well as spherical harmonic (SH) decomposition. We analysed how these parameterisations described the underlying orientation distributions and compared their orientation and dispersion with corresponding parameters from two dMRI methods, neurite orientation dispersion and density imaging (NODDI) and constrained spherical deconvolution (CSD). Watson and ACG parameterisations and SH decomposition captured well the 3D-ST orientation distributions, but ACG and SH better represented the distributions due to its ability to model asymmetric dispersion. The dMRI parameters corresponded well with the 3D-ST parameters in the white matter volumes, but the correspondence was less evident in the more complex grey matter. SBEM imaging and 3D-ST analysis also revealed that the orientation distributions were often not axially symmetric, a property neatly captured by the ACG distribution. Overall, the ability of SBEM to image diffusion barriers in intricate detail, combined with 3D-ST analysis and parameterisation, provides a step forward toward interpreting and validating the dMRI signals in complex brain tissue microstructure.
  • Mazzoni, Annalisa; Maravic, Tatjana; Tezvergil-Mutluay, Arzu; Tjäderhane, Leo; Scaffa, Polliana Mendes Candia; Seseogullari-Dirihan, Roda; Bavelloni, Alberto; Gobbi, Pietro; Pashley, David H.; Tay, Franklin R.; Breschi, Lorenzo (2018)
    Objectives: Matrix metalloproteinases (MMPs) are dentinal endogenous enzymes claimed to have a vital role in dentin organic matrix breakdown. The aim of the study was to investigate presence, localization and distribution of MMP-7 in sound human dentin. Methods: Dentin was powdered, demineralized and dissolved in isoelectric focusing buffer. Resolved proteins were transferred to nitrocellulose membranes for western blotting (WB) analyses. For the zymographic analysis, aliquots of dentin protein were electrophoresed in 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis containing fluorescently labeled gelatin. Further, the concentrations of dentinal MMPs were measured using Fluorescent Microsphere Immunoassay with a human MMP-MAP multiplex kit. Pre- and post-embedding immunolabeling technique was used to investigate the localization and distribution of MMP-7 in dentin. Dentin was cryo-fractured, the fragments partially decalcified and labeled with a primary monoclonal anti-MMP-7 and a secondary antibody conjugated with gold nanoparticles. MMP-7 labelings were identified in the demineralized dentin matrix as highly electron-dense dispersed gold particles. Results: WB and zymographic analysis of extracted dentin proteins showed presence of MMP-7 (similar to 20-28 KDa). Further, MMP-7 was found in the supernatants of the incubated dentin beams using Fluorescent Microsphere Immunoassay. FEI-SEM and TEM analyses established MMP-7 as an intrinsic constituent of the human dentin organic matrix. Conclusion: This study demonstrated that MMP-7 is an endogenous component of the human dentin fibrillar network. Clinical significance: It is pivotal to understand the underlying processes behind dentin matrix remodeling and degradation in order to develop the most optimal clinical protocols and ensure the longevity of dental restorations.
  • Toppinen, M.; Perdomo, Maria; Palo, J. U.; Simmonds, P.; Lycett, S. J.; Soderlund-Venermo, M.; Sajantila, A.; Hedman, K. (2015)
    DNA in human skeletal remains represents an important historical source of host genomic information and potentially of infecting viruses. However, little is known about viral persistence in bone. We searched ca. 70-year-old long bones of putative Finnish casualties from World War II for parvovirus B19 (B19V) DNA, and found a remarkable prevalence of 45%. The viral sequences were exclusively of genotypes 2 (n = 41), which disappeared from circulation in 1970's, or genotype 3 (n = 2), which has never been reported in Northern Europe. Based on mitochondrial and Y-chromosome profiling, the two individuals carrying B19V genotype 3 were likely from the Soviet Red Army. The most recent common ancestor for all genotypes was estimated at early 1800s. This work demonstrates the forms of B19V that circulated in the first half of the 20th century and provides the first evidence of the suitability of bone for exploration of DNA viruses.
  • Pitkänen, S.; Paakinaho, K.; Pihlman, H.; Ahola, N.; Hannula, M.; Asikainen, S.; Manninen, M.; Morelius, M.; Keränen, P.; Hyttinen, J.; Kellomäki, M.; Laitinen-Vapaavuori, O.; Miettinen, S. (2019)
    Most synthetic bone grafts are either hard and brittle ceramics or paste-like materials that differ in applicability from the gold standard autologous bone graft, which restricts their widespread use. Therefore, the aim of the study was to develop an elastic, highly porous and biodegradable beta-tricalciumphosphate/poly(L-lactide-co-epsilon-caprolactone) (beta-TCP/PLCL) composite for bone applications using supercritical CO2 foaming. Ability to support osteogenic differentiation was tested in human adipose stem cell (hASC) culture for 21 d. Biocompatibility was evaluated for 24 weeks in a rabbit femur-defect model. Foamed composites had a high ceramic content (50 wt%) and porosity (65-67 %). After 50 % compression, in an aqueous environment at 37 degrees C, tested samples returned to 95 % of their original height. Hydrolytic degradation of beta-TCP/PLCL composite, during the 24-week follow-up, was very similar to that of porous PLCL scaffold both in vitro and in vivo. Osteogenic differentiation of hASCs was demonstrated by alkaline phosphatase activity analysis, alizarin red staining, soluble collagen analysis, immunocytochemical staining and qRT-PCR. In vitro, hASCs formed a pronounced mineralised collagen matrix. A rabbit femur defect model confirmed biocompatibility of the composite. According to histological Masson-Goldner's trichrome staining and micro-computed tomography, beta-TCP/PLCL composite did not elicit infection, formation of fibrous capsule or cysts. Finally, native bone tissue at 4 weeks was already able to grow on and in the beta-TCP/PLCL composite. The elastic and highly porous beta-TCP/PLCL composite is a promising bone substitute because it is osteoconductive and easy-to-use and mould intraoperatively.
  • Makinen, Netta; Kampjarvi, Kati; Frizzell, Norma; Butzow, Ralf; Vahteristo, Pia (2017)
    Uterine smooth muscle tumors range from benign leiomyomas to malignant leiomyosarcomas. Based on numerous molecular studies, leiomyomas and leiomyosarcomas mostly lack shared mutations and the majority of tumors are believed to develop through distinct mechanisms. To further characterize the molecular variability among uterine smooth muscle tumors, and simultaneously insinuate their potential malignant progression, we examined the frequency of known genetic leiomyoma driver alterations (MED12 mutations, HMGA2 overexpression, biallelic FH inactivation) in 65 conventional leiomyomas, 94 histopathological leiomyoma variants (18 leiomyomas with bizarre nuclei, 22 cellular, 29 highly cellular, and 25 mitotically active leiomyomas), and 51 leiomyosarcomas. Of the 210 tumors analyzed, 107 had mutations in one of the three driver genes. No tumor had more than one mutation confirming that all alterations are mutually exclusive. MED12 mutations were the most common alterations in conventional and mitotically active leiomyomas and leiomyosarcomas, while leiomyomas with bizarre nuclei were most often FH deficient and cellular tumors showed frequent HMGA2 overexpression. Highly cellular leiomyomas displayed the least amount of alterations leaving the majority of tumors with no known driver aberration. Our results indicate that based on the molecular background, histopathological leiomyoma subtypes do not only differ from conventional leiomyomas, but also from each other. The presence of leiomyoma driver alterations in nearly one third of leiomyosarcomas suggests that some tumors arise through leiomyoma precursor lesion or that these mutations provide growth advantage also to highly aggressive cancers. It is clinically relevant to understand the molecular background of various smooth muscle tumor subtypes, as it may lead to improved diagnosis and personalized treatments in the future.
  • Salonius, Eve; Rieppo, Lassi; Nissi, Mikko J.; Pulkkinen, Hertta J.; Brommer, Harold; Bruenott, Anne; Silvast, Tuomo S.; Van Weeren, P. Rene; Muhonen, Virpi; Brama, Pieter A. J.; Kiviranta, Ilkka (2019)
    Aim: The horse joint, due to its similarity with the human joint, is the ultimate model for translational articular cartilage repair studies. This study was designed to determine the critical size of cartilage defects in the equine carpus and serve as a benchmark for the evaluation of new cartilage treatment options. Material and Methods: Circular full-thickness cartilage defects with a diameter of 2, 4, and 8 mm were created in the left middle carpal joint and similar osteochondral (3.5 mm in depth) defects in the right middle carpal joint of 5 horses. Spontaneously formed repair tissue was examined macroscopically, with MR and mu CT imaging, polarized light microscopy, standard histology, and immunohistochemistry at 12 months. Results: Filling of 2 mm chondral defects was good (77.8 +/- 8.5%), but proteoglycan depletion was evident in Safranin-O staining and gadolinium-enhanced MRI (T-1Gd). Larger chondral defects showed poor filling (50.6 +/- 2.7% in 4 mm and 31.9 +/- 7.3% in 8 mm defects). Lesion filling in 2, 4, and 8 mm osteochondral defects was 82.3 +/- 3.0%, 68.0 +/- 4.6% and 70.8 +/- 15.4%, respectively. Type II collagen staining was seen in 9/15 osteochondral defects but only in 1/15 chondral defects. Subchondral bone pathologies were evident in 14/15 osteochondral samples but only in 5/15 chondral samples. Although osteochondral lesions showed better neotissue quality than chondral lesions, the overall repair was deemed unsatisfactory because of the subchondral bone pathologies. Conclusion: We recommend classifying 4 mm as critical osteochondral lesion size and 2 mm as critical chondral lesion size for cartilage repair research in the equine carpal joint model.
  • Nieminen, H. J.; Ylitalo, T.; Karhula, S.; Suuronen, J. -P.; Kauppinen, S.; Serimaa, R.; Haeggstrom, E.; Pritzker, K. P. H.; Valkealahti, M.; Lehenkari, P.; Finnila, M.; Saarakkala, S. (2015)
    Objective: Collagen distribution within articular cartilage (AC) is typically evaluated from histological sections, e.g., using collagen staining and light microscopy (LM). Unfortunately, all techniques based on histological sections are time-consuming, destructive, and without extraordinary effort, limited to two dimensions. This study investigates whether phosphotungstic acid (PTA) and phosphomolybdic acid (PMA), two collagen-specific markers and X-ray absorbers, could (1) produce contrast for AC X-ray imaging or (2) be used to detect collagen distribution within AC. Method: We labeled equine AC samples with PTA or PMA and imaged them with micro-computed tomography (micro-CT) at pre-defined time points 0, 18, 36, 54, 72, 90, 180, 270 h during staining. The micro-CT image intensity was compared with collagen distributions obtained with a reference technique, i.e., Fourier-transform infrared imaging (FTIRI). The labeling time and contrast agent producing highest association (Pearson correlation, BlandeAltman analysis) between FTIRI collagen distribution and micro-CT -determined PTA distribution was selected for human AC. Results: Both, PTA and PMA labeling permitted visualization of AC features using micro-CT in non-calcified cartilage. After labeling the samples for 36 h in PTA, the spatial distribution of X-ray attenuation correlated highly with the collagen distribution determined by FTIRI in both equine (mean +/- S.D. of the Pearson correlation coefficients, r = 0.96 +/- 0.03, n = 12) and human AC (r = 0.82 +/- 0.15, n = 4). Conclusions: PTA-induced X-ray attenuation is a potential marker for non-destructive detection of AC collagen distributions in 3D. This approach opens new possibilities in development of non-destructive 3D histopathological techniques for characterization of OA. (C) 2015 The Authors. Published by Elsevier Ltd and Osteoarthritis Research Society International.
  • Huttala, Outi; Mysore, R.; Sarkanen, J. R.; Heinonen, T.; Olkkonen, Vesa; Ylikomi, T. (2016)
    Adipose tissue-related diseases such as obesity and type 2 diabetes are worldwide epidemics. In order to develop adipose tissue cultures in vitro that mimic more faithfully the in vivo physiology, new well-characterized and publicly accepted differentiation methods of human adipose stem cells are needed. The aims of this study are (1) to improve the existing natural adipose tissue extract (ATE)-based induction method and (2) to study the effects of a differentiation method on insulin responsiveness of the resulting adipocytes. Different induction media were applied on human adipose stromal cell (hASC) monocultures to study the differentiation capacity of the induction media and the functionality of the differentiated adipocytes. Cells were differentiated for 14 days to assess triglyceride accumulation per cell and adipocyte-specific gene expression (PPAR gamma, adiponectin, AP2, leptin, Glut4, Prdm16, CIDEA, PGC1-alpha, RIP140, UCP and ADCY5). Insulin response was studied by measuring glucose uptake and inhibition of lipolysis after incubation with 100 or 500 nM insulin. The selected differentiation method included a 3-day induction with ATE, 6 days in serum-free medium supplemented with 1.15 mu M insulin and 9.06 mu M Troglitazone, followed by 4 days in a defined serum- and insulin-free stimulation medium. This protocol induced prominent general adipocyte gene expression, including markers for both brown and white adipocytes and triglyceride accumulation. Moreover, the cells were sensitive to insulin as observed from increased glucose uptake and inhibition of lipolysis. This differentiation protocol provides a promising approach for the induction of hASC adipogenesis to obtain functional and mature human adipocytes.
  • Lagus, Heli; Klaas, Mariliis; Juteau, Susanna; Elomaa, Outi; Kere, Juha; Vuola, Jyrki; Jaks, Viljar; Kankuri, Esko (2019)
    Because molecular memories of past inflammatory events can persist in epidermal cells, we evaluated the long-term epidermal protein expression landscapes after dermal regeneration and in psoriatic inflammation. We first characterized the effects of two dermal regeneration strategies on transplants of indicator split-thickness skin grafts (STSGs) in ten adult patients with deep burns covering more than 20% of their body surface area. After fascial excision, three adjacent areas within the wound were randomized to receive a permanent dermal matrix, a temporary granulation-tissue-inducing dressing or no dermal component as control. Control areas were covered with STSG immediately, and treated areas after two-weeks of dermis formation. Epidermis-dermis-targeted proteomics of one-year-follow-up samples were performed for protein expression profiling. Epidermal expression of axonemal dynein heavy chain 10 (DNAH10) was increased 20-fold in samples having had regenerating dermis vs control. Given the dermal inflammatory component found in our dermal regeneration samples as well as in early psoriatic lesions, we hypothesized that DNAH10 protein expression also would be affected in psoriatic skin samples. We discovered increased DNAH10 expression in inflammatory lesions when compared to unaffected skin. Our results associate DNAH10 expression with cell proliferation and inflammation as well as with the epidermal memory resulting from the previous regenerative signals of dermis. This study (ISRCTN14499986) was funded by the Finnish Ministry of Defense and by government subsidies for medical research.
  • Hellinen, Laura; Hongisto, Heidi; Ramsay, Eva; Kaarniranta, Kai; Vellonen, Kati-Sisko; Skottman, Heli; Ruponen, Marika (2020)
    The retinal pigment epithelial (RPE) cell monolayer forms the outer blood-retinal barrier and has a crucial role in ocular pharmacokinetics. Although several RPE cell models are available, there have been no systematic comparisons of their barrier properties with respect to drug permeability. We compared the barrier properties of several RPE secondary cell lines (ARPE19, ARPE19mel, and LEPI) and both primary (hfRPE) and stem-cell derived RPE (hESC-RPE) cells by investigating the permeability of nine drugs (aztreonam, ciprofloxacin, dexamethasone, fluconazole, ganciclovir, ketorolac, methotrexate, voriconazole, and quinidine) across cell monolayers. ARPE19, ARPE19mel, and hfRPE cells displayed a narrow P-app value range, with relatively high permeation rates (5.2-26 x 10(-6) cm/s. In contrast, hESC-RPE and LEPI cells efficiently restricted the drug flux, and displayed even lower P-app values than those reported for bovine RPE-choroid, with the range of 0.4-32 cm(-6)/s (hESC-RPE cells) and 0.4-29 x 10(-6) cm/s, (LEPI cells). Therefore, ARPE19, ARPE19mel, and hfRPE cells failed to form a tight barrier, whereas hESC-RPE and LEPI cells restricted the drug flux to a similar extent as bovine RPE-choroid. Therefore, LEPI and hESC-RPE cells are valuable tools in ocular drug discovery.
  • Patrikoski, Mimmi; Lee, Michelle Hui Ching; Makinen, Laura; Ang, Xiu Min; Mannerström, Bettina; Raghunath, Michael; Miettinen, Susanna (2017)
    Microenvironment plays an important role for stem cell proliferation and differentiation. Macromolecular crowding (MMC) was recently shown to assist stem cells in forming their own matrix microenvironment in vitro. The ability of MMC to support adipose stem cell (ASC) proliferation, metabolism, and multilineage differentiation was studied under different conditions: fetal bovine serum- (FBS-) and human serum- (HS-) based media and xeno- and serum-free (XF/SF) media. Furthermore, the immunophenotype of ASCs under MMC was evaluated. The proliferative capacity of ASCs under MMC was attenuated in each condition. However, osteogenic differentiation was enhanced under MMC, shown by increased deposition of mineralized matrix in FBS and HS cultures. Likewise, significantly greater lipid droplet accumulation and increased collagen IV deposition indicated enhanced adipogenesis under MMC in FBS and HS cultures. In contrast, chondrogenic differentiation was attenuated in ASCs expanded under MMC. The ASC immunophenotype was maintained under MMC with significantly higher expression of CD54. However, MMC impaired metabolic activity and differentiation capacity of ASCs in XF/SF conditions. Both the supportive and inhibitory effects of MMC on ASC are culture condition dependent. In the presence of serum, MMC maintains ASC immunophenotype and enhances adipogenic and osteogenic differentiation at the cost of reduced proliferation.
  • Salonius, Eve; Muhonen, Virpi; Lehto, Kalle; Järvinen, Elina; Pyhältö, Tuomo; Hannula, Markus; Aula, Antti S.; Uppstu, Peter; Haaparanta, Anne-Marie; Rosling, Ari; Kellomäki, Minna; Kiviranta, Ilkka (2019)
    Deep osteochondral defects may leave voids in the subchondral bone, increasing the risk of joint structure collapse. To ensure a stable foundation for the cartilage repair, bone grafts can be used for filling these defects. Poly(lactide-co-glycolide) (PLGA) is a biodegradable material that improves bone healing and supports bone matrix deposition. We compared the reparative capacity of two investigative macroporous PLGA-based biomaterials with two commercially available bone graft substitutes in the bony part of an intra-articular bone defect created in the lapine femur. New Zealand white rabbits (n = 40) were randomized into five groups. The defects, 4 mm in diameter and 8 mm deep, were filled with neat PLGA; a composite material combining PLGA and bioactive glass fibres (PLGA-BGf); commercial beta-tricalcium phosphate (beta-TCP) granules; or commercial bioactive glass (BG) granules. The fifth group was left untreated for spontaneous repair. After three months, the repair tissue was evaluated with X-ray microtomography and histology. Relative values comparing the operated knee with its contralateral control were calculated. The relative bone volume fraction ( increment BV/TV) was largest in the beta-TCP group (p
  • Yang, Yaohua; Wu, Lang; Shu, Xiang; Lu, Yingchang; Shu, Xiao-Ou; Cai, Qiuyin; Beeghly-Fadiel, Alicia; Li, Bingshan; Ye, Fei; Berchuck, Andrew; Anton-Culver, Hoda; Banerjee, Susana; Benitez, Javier; Bjorge, Line; Brenton, James D.; Butzow, Ralf; Campbell, Ian G.; Chang-Claude, Jenny; Chen, Kexin; Cook, Linda S.; Cramer, Daniel W.; defazio, Anna; Dennis, Joe; Doherty, Jennifer A.; Doerk, Thilo; Eccles, Diana M.; Edwards, Digna Velez; Fasching, Peter A.; Fortner, Renee T.; Gayther, Simon A.; Giles, Graham G.; Glasspool, Rosalind M.; Goode, Ellen L.; Goodman, Marc T.; Gronwald, Jacek; Harris, Holly R.; Heitz, Florian; Hildebrandt, Michelle A.; Hogdall, Estrid; Hogdall, Claus K.; Huntsman, David G.; Kar, Siddhartha P.; Karlan, Beth Y.; Kelemen, Linda E.; Kiemeney, Lambertus A.; Kjaer, Susanne K.; Koushik, Anita; Lambrechts, Diether; Le, Nhu D.; Levine, Douglas A.; Massuger, Leon F.; Matsuo, Keitaro; May, Taymaa; McNeish, Iain A.; Menon, Usha; Modugno, Francesmary; Monteiro, Alvaro N.; Moorman, Patricia G.; Moysich, Kirsten B.; Ness, Roberta B.; Nevanlinna, Heli; Olsson, Hakan; Onland-Moret, N. Charlotte; Park, Sue K.; Paul, James; Pearce, Celeste L.; Pejovic, Tanja; Phelan, Catherine M.; Pike, Malcolm C.; Ramus, Susan J.; Riboli, Elio; Rodriguez-Antona, Cristina; Romieu, Isabelle; Sandler, Dale P.; Schildkraut, Joellen M.; Setiawan, Veronica W.; Shan, Kang; Siddiqui, Nadeem; Sieh, Weiva; Stampfer, Meir J.; Sutphen, Rebecca; Swerdlow, Anthony J.; Szafron, Lukasz M.; Teo, Soo Hwang; Tworoger, Shelley S.; Tyrer, Jonathan P.; Webb, Penelope M.; Wentzensen, Nicolas; White, Emily; Willett, Walter C.; Wolk, Alicja; Woo, Yin Ling; Wu, Anna H.; Yan, Li; Yannoukakos, Drakoulis; Chenevix-Trench, Georgia; Sellers, Thomas A.; Pharoah, Paul D. P.; Zheng, Wei; Long, Jirong (2019)
    DNA methylation is instrumental for gene regulation. Global changes in the epigenetic landscape have been recognized as a hallmark of cancer. However, the role of DNA methylation in epithelial ovarian cancer (EOC) remains unclear. In this study, high-density genetic and DNA methylation data in white blood cells from the Framingham Heart Study (N = 1,595) were used to build genetic models to predict DNA methylation levels. These prediction models were then applied to the summary statistics of a genome-wide association study (GWAS) of ovarian cancer including 22,406 EOC cases and 40,941 controls to investigate genetically predicted DNA methylation levels in association with EOC risk. Among 62,938 CpG sites investigated, genetically predicted methylation levels at 89 CpG were significantly associated with EOC risk at a Bonferroni-corrected threshold of P <7.94 x 10(-7). Of them, 87 were located at GWAS-identified EOC susceptibility regions and two resided in a genomic region not previously reported to be associated with EOC risk. Integrative analyses of genetic, methylation, and gene expression data identified consistent directions of associations across 12 CpG, five genes, and EOC risk, suggesting that methylation at these 12 CpG may influence EOC risk by regulating expression of these five genes, namely MAPT, HOXB3, ABHD8, ARHGAP27, and SKAP1. We identified novel DNA methylation markers associated with EOC risk and propose that methylation at multiple CpG may affect EOC risk via regulation of gene expression. Significance: Identification of novel DNA methylation markers associated with EOC risk suggests that methylation at multiple CpG may affect EOC risk through regulation of gene expression.
  • Hanifeh, Mohsen; Rajamäki, Minna Marjaana; Syrjä, Pernilla; Mäkitalo, Laura; Kilpinen, Susanne; Spillmann, Thomas (2018)
    Background: Matrix metalloproteinases (MMPs) 2 and 9 are zinc-and calcium-dependent endopeptidases involved in the breakdown and reconstitution of extracellular matrix under both physiological and pathological conditions. Mucosal MMP-2 and -9 activities have been reported to be upregulated in the intestine of humans with inflammatory bowel disease (IBD), and in animal models of IBD. However, their involvement in the pathogenesis of canine chronic enteropathies (CE) is unknown. This study investigated mucosal pro-and active MMP-2 and -9 activities in dogs with CE and healthy dogs using gelatin zymography, and also to determine the association of their activities in dogs with CE with the canine IBD activity index (CIBDAI), histopathologic findings, the clinical outcome, and hypoalbuminemia. Intestinal mucosal samples from duodenum, ileum, colon, and cecum were collected from 40 dogs with CE and 18 healthy Beagle dogs. Results: In dogs with CE, the number of samples positive for mucosal pro-and active MMP-2 was significantly higher in the duodenum (P <0.0001 and P = 0.011, respectively), ileum (P = 0.002 and P = 0.018, respectively), and colon (P <0.0001 and P = 0.002, respectively), compared with healthy controls. Mucosal pro-MMP-9-positive samples in the duodenum and colon were significantly more frequent in dogs with CE than in healthy dogs (P = 0.0004 and P = 0.001, respectively). Despite the presence of mucosal samples positive for active MMP-9 in the intestinal segments of dogs with CE, the difference compared to healthy controls did not reach statistical significance. None of the intestinal mucosal samples in healthy dogs showed gelatinolytic activity corresponding to the control bands of active MMP-2 and -9. Mucosal active MMP-9 activities displayed a significant positive association with the severity of neutrophil infiltration in the duodenum (P = 00.040), eosinophils in the cecum (P = 00.037), and the CIBDAI score for ileum samples (P = 0.023). There was no significant association of pro-and active MMP-2 and -9 levels with the clinical outcome or hypoalbuminemia. Conclusions: This study is the first to demonstrate upregulation of mucosal pro-and active MMP-2 and pro-MMP-9 in the intestine of dogs with CE compared to healthy dogs. The results provide supporting evidence for the possible involvement of MMP-2 and -9 in the pathogenesis of canine CE.
  • Tommola, Paivi; Unkila-Kallio, Leila; Paetau, Anders; Meri, Seppo; Kalso, Eija; Paavonen, Jorma (2016)
    BACKGROUND: Provoked vestibulodynia manifests as allodynia of the vulvar vestibular mucosa. The exact mechanisms that result in altered pain sensation are unknown. Recently, we demonstrated the presence of secondary lymphoid tissue, which is the vestibule-associated lymphoid tissue in the vestibular mucosa, and showed that this tissue becomes activated in provoked vestibulodynia. OBJECTIVE: The purpose of this study was to examine whether expression of intraepithelial nerve fibers and nerve growth factor are related to immune activation in provoked vestibulodynia. STUDY DESIGN: Vestibular mucosal specimens were obtained from 27 patients with severe provoked vestibulodynia that was treated by vestibulectomy and from 15 control subjects. We used antibodies against the protein gene product 9.5, the neuron specific neurofilament, and nerve growth factor for immunohistochemistry to detect intraepithelial nerve fibers and nerve growth factor expressing immune cells in the vestibular mucosa. For intraepithelial nerve fibers, we determined their linear density (fiber counts per millimeter of the outer epithelial surface, protein gene product 9.5) or presence (neuron specific neurofilament). Nerve growth factor was analyzed by counting the staining-positive immune cells. Antibodies against CD20 (B lymphocytes) and CD3 (T lymphocytes) were used to identify and locate mucosal areas with increased density of lymphocytes and the presence of germinal centers (ie, signs of immune activation). B-cell activation index was used to describe the overall intensity of B-cell infiltration. RESULTS: We found more protein gene product 9.5-positive intraepithelial fibers in vestibulodynia than in the control samples (6.3/mm [range, 0.0-15.8] vs 2.0/mm [range, 0.0-12.0]; P = .006). Neuron specific neurofilament -positive intraepithelial fibers were found in 17 of 27 vestibulodynia cases (63.0%) and in none of the control cases. Protein gene product 9.5-positive intraepithelial fibers were more common in samples with more pronounced immune activation. The density of these fibers was higher in samples with than without germinal centers (6.1/mm [range, 4.3-15.8] vs 3.0/mm [range, 0.0-13.4]; P = .020). A positive correlation between the fiber density and B-cell activation index score of the sample was found (Spearman's Rho, 0.400; P = .004; R-2 = 0.128). No significant difference, however, was found in the density or presence of nerve fibers between samples with high and low T-cell densities. We identified areas of minor and major vestibular glands in 16 of the patient samples and in 1 control sample. Protein gene product 9.5-positive nerve fibers were found more often in glandular epithelium surrounded by B-cell infiltration than in glands without B cells (P = .013). Also, the presence of neuron specific neurofilament-positive fibers in glandular epithelium was associated with B-cell infiltrates (P = .053). Nerve growth factor-positive immune cells were more common in mucosal areas with than without B-cell infiltration and intraepithelial nerve fibers. CONCLUSION: Excessive epithelial nerve growth in provoked vestibulodynia is associated with increased B-cell infiltration and the presence of germinal centers. This supports the fundamental role of immune activation in provoked vestibulodynia.
  • Pampanini, Valentina; Wagner, Magdalena; Asadi-Azarbaijani, Babak; Oskam, Irma C.; Sheikhi, Mona; Sjödin, Marcus O. D.; Lindberg, Johan; Hovatta, Outi; Sahlin, Lena; Bjorvang, Richelle D.; Otala, Marjut; Damdimopoulou, Pauliina; Jahnukainen, Kirsi (2019)
    STUDY QUESTION: Does first-line chemotherapy affect the quality of ovarian pre-antral follicles and stromal tissue in a population of young patients? SUMMARY ANSWER: Exposure to first-line chemotherapy significantly impacts follicle viability, size of residual intact follicles, steroid secretion in culture and quality of the stromal compartment. WHAT IS KNOWN ALREADY: First-line chemotherapy is considered to have a low gonadotoxic potential, and as such, does not represent an indication for fertility preservation. Studies investigating the effects of chemotherapy on the quality of ovarian tissue stored for fertility preservation in young patients are limited and the results sometimes contradictory. STUDY DESIGN, SIZE, DURATION: We conducted a retrospective cohort study including young patients referred to three centers (Helsinki, Oslo and Tampere) to perform ovarian tissue cryopreservation for fertility preservation between 2003 and 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 43 patients (age 1-24 years) were included in the study. A total of 25 were exposed to first-line chemotherapy before cryopreservation, whereas 18 patients were not. Density and size of follicles divided by developmental stages, prevalence of atretic follicles, health of the stromal compartment and functionality of the tissue in culture were evaluated and related to age and chemotherapy exposure. Activation of dormant follicles and DNA damage were also assessed. MAIN RESULTS AND THE ROLE OF CHANCE: Patients exposed to first-line chemotherapy showed a significantly higher density of atretic primordial and intermediary follicles than untreated patients. The intact primordial and intermediary follicles were significantly smaller in size in patients exposed to chemotherapy. Production of steroids in culture was also significantly impaired and a higher content of collagen and DNA damage was observed in the stromal compartment of treated patients. Collectively, these observations may indicate reduced quality and developmental capacity of follicles as a consequence of first-line chemotherapy exposure. Neither increased activation of dormant follicles nor elevated levels of DNA damage in oocyte nuclei were found in patients exposed to chemotherapy. LIMITATIONS, REASONS FOR CAUTION: The two groups were not homogeneous in terms of age and the patients were exposed to different treatments, which did not allow us to distinguish the effect of specific agents. The limited material availability did not allow us to perform all the analyses on the entire set of patients. WIDER IMPLICATION OF THE FINDINGS: This study provides for the first time a comprehensive analysis of the effects of first-line chemotherapy on the health, density and functionality of follicles categorized according to the developmental stage in patients under 24 years of age. When exposed to these treatments, patients were considered at low/medium risk of infertility. Our data suggest a profound impact of these relatively safe therapies on ovarian health and encourages further exploration of this effect in follow-up studies in order to optimize fertility preservation for young cancer patients.