Ülevaade Ravimiameti 30. aastapäeva konverentsil „Ravimid, patsient ja innovatsioon“ käsitletud teemadest

Eesti Arst 2021; 100(10):535–53

EXPRESSION OF cAMP AND CREB IN THE HUMAN PENIS

The aim of this study is to investigate the expression of adenosine 3',5'-cyclic monophosphate (cAMP) and cAMP-response element-binding protein (CREB) in the human penis as it is known that luteinizing hormone (LH) regulates cellular function mostly through the cAMP signaling pathway and LH receptors are expressed by the penile endothelium. Penile tissue was obtained from three patients during partial or total penectomy due to a rectal cancer with secondary penile metastasis or squamous cell carcinoma of the penis. Immunohistochemistry was used for the detection of cAMP and CREB. Positive immunoreaction for cAMP was present in most cells of superficial, intermedial, and basal layer of urethral epithelium and in fibroblast-like cells (FLC) of interstitial tissue and endothelial cells (EC) of cavernous spaces in corpus spongiosum penis. Positive staining for cAMP was also visible in EC of cavernous spaces and in FLC of interstitial tissue in corpus cavernosum penis. Positive immunoreaction for CREB was present in the superficial and intermedial layer of urethral epithelium, and some positive immunoreaction was also noticed in EC of cavernous spaces and in FLC of interstitial tissue in corpus spongiosum penis. Positive staining was also visible in the EC of cavernous spaces and in fibroplast-like cells of the interstitial tissue in the corpus cavernosum penis. Our results show the presence of cAMP and CREB in the human penis. While LH exerts its actions through cAMP signaling system and our previous studies have shown the expression of luteinizing hormone/choriogonadotropin (LHCG) receptor in the mouse and human penis, this finding may support the hypothesis that LH could affect the spongious and cavernous tissue of the human penis and thereby influence the development of erectile dysfunction among aging men.Peer reviewe

TGF-β isovormide ja luukoe eelrakkude roll heterotoopse ossifikatsiooni patogeneesis : eksperimentaalne ja kliiniline uuring puusaliigese endoproteesimisest

Väitekirja elektroonilisest versioonist puuduvad varem avaldatud publikatsioonide täistekstid.Dissertation focuses on the changes in the content and expression of transforming growth factor beta isoforms (TGF-β1, TGF-β2 and TGF-β3) in heterotopic ossification (HO) during its formation. Since the HOs formed after total hip arthroplasty (THA) are clinically frequent (with approximately 37% of THA patients being affected), material was collected from patients who had undergone the above mentioned operation and came for revision surgery and material was also collected from an animal model mimicking the situation after THA in order to study initial and early changes of HO formation (days 3 and 21). TGF-β isoforms were chosen as these growth factors have many functions in the body and in the bone, but they have hardly been studied in the context of HO formation. Also the changes in the morphology of HO were followed during its formation. The main findings of this study were the specific changes in the content and expression of the less spread isoforms TGF-β2 and TGF-β3. However, this kind of dynamics was not found for the more spread isoform TGF-β1 (content in normal bone tissue about 200 µg/kg). The similar features in the dynamics of TGF-β isoforms in humans and in the animal model suggest that this model can be used to evaluate the dynamics of these growth factors and initial and early stages of HO formation. In addition it was found that by allowing the access of the cells from the open femoral canal to the site of HO formation in our animal model the HO formation was not significantly affected, which suggest that local biochemical signals play the most important role.Dissertatsioon keskendub muutustele transformeeriva kasvufaktori beeta isovormide (TGF-β1,TGF-β2 ja TGF-β3) sisalduses ja ekspressioonis skeletivälises luus e heterotoopses ossifikatsioonis (HO) selle moodustumise käigus. Kuna kliiniliselt on kõige levinumaks HO-d, mis on tekkinud pärast puusaliigese endoproteesimist (esineb ligikaudu 37% patsientidest), koguti materjali nii patsientidelt, kes olid nimetatud operatsiooni läbinud ja tulid kordusoperatsioonile kui ka loommudelist, milles rottidel jäljendati puusaliigese endoproteesimisel tekkivat olukorda, et uurida HO tekke varajasi etappe (3 ja 21 päeva). TGF-β isovormidele keskenduti kui organismis ja luukoes erinevaid funktsioone täitvatele, kuid HO tekke kontekstis vähe uuritud kasvufaktoritele. Samuti jälgiti muutusi HO morfoloogias selle kasvamise ajal. Uurimustöö olulisemateks leidudeks olid spetsiifilised muutused vähemlevinud TGF-β isovormide (TGF-β2 ja TGF-β3) sisalduses ja ekspressioonis, kuna luukoes enimlevinud isovormi TGF-β1 puhul (sisaldus luukoes 200 µg/kg) sellist dünaamikat ei täheldatud. Sarnased jooned TGF-β isovormide dünaamikas nii inimestel kui loommudelis viitavad sellele, et antud mudel sobib nimetatud kasvufaktorite ja HO tekke varajaste etappide uurimiseks. Lisaks ilmnes, et luuüdikanali rakkude juurdepääsu lubamine HO tekkekohale luuüdikanali avamise teel, ei mõjutanud loommudelis HO teket olulisel määral, millest võib järeldada, et siin on suurem roll lokaalsetel biokeemilistel signaalidel

Mast Cells Differentiated in Synovial Fluid and Resident in Osteophytes Exalt the Inflammatory Pathology of Osteoarthritis

Introduction: Osteophytes are a prominent feature of osteoarthritis (OA) joints and one of the clinical hallmarks of the disease progression. Research on osteophytes is fragmentary and modes of its contribution to OA pathology are obscure. Aim: To elucidate the role of osteophytes in OA pathology from a perspective of molecular and cellular events. Methods: RNA-seq of fully grown osteophytes, collected from tibial plateau of six OA patients revealed patterns corresponding to active extracellular matrix re-modulation and prominent participation of mast cells. Presence of mast cells was further confirmed by immunohistochemistry, performed on the sections of the osteophytes using anti-tryptase alpha/beta-1 and anti-FC epsilon RI antibodies and the related key up-regulated genes were validated by qRT-PCR. To test the role of OA synovial fluid (SF) in mast cell maturation as proposed by the authors, hematopoietic stem cells (HSCs) and ThP1 cells were cultured in a media supplemented with 10% SF samples, obtained from various grades of OA patients and were monitored using specific cell surface markers by flow cytometry. Proteomics analysis of SF samples was performed to detect additional markers specific to mast cells and inflammation that drive the cell differentiation and maturation. Results: Transcriptomics of osteophytes revealed a significant upregulation of mast cells specific genes such as chymase 1 (CMA1; 5-fold) carboxypeptidase A3 (CPA3; 4-fold), MS4A2/FCERI (FCERI; 4.2-fold) and interleukin 1 receptor-like 1 (IL1RL1; 2.5-fold) indicating their prominent involvement. (In IHC, anti-tryptase alpha/beta-1 and anti- FC epsilon RI-stained active mast cells were seen populated in cartilage, subchondral bone, and trabecular bone.) Based on these outcomes and previous learnings, the authors claim a possibility of mast cells invasion into osteophytes is mediated by SF and present in vitro cell differentiation assay results, wherein ThP1 and HSCs showed differentiation into HLA-DR+/CD206+ and FCERI+ phenotype, respectively, after exposing them to medium containing 10% SF for 9 days. Proteomics analysis of these SF samples showed an accumulation of mast cell-specific inflammatory proteins. Conclusions: RNA-seq analysis followed by IHC study on osteophyte samples showed a population of mast cells resident in them and may further accentuate inflammatory pathology of OA. Besides subchondral bone, the authors propose an alternative passage of mast cells invasion in osteophytes, wherein OA SF was found to be necessary and sufficient for maturation of mast cell precursor into effector cells