Fatty Acid Fingerprints and Hyaluronic Acid in Extracellular Vesicles from Proliferating Human Fibroblast-like Synoviocytes

Extracellular vesicles (EVs) function as conveyors of fatty acids (FAs) and other bioactive lipids and can modulate the gene expression and behavior of target cells. EV lipid composition influences the fluidity and stability of EV membranes and reflects the availability of lipid mediator precursors. Fibroblast-like synoviocytes (FLSs) secrete EVs that transport hyaluronic acid (HA). FLSs play a central role in inflammation, pannus formation, and cartilage degradation in joint diseases, and EVs have recently emerged as potential mediators of these effects. The aim of the present study was to follow temporal changes in HA and EV secretion by normal FLSs, and to characterize the FA profiles of FLSs and EVs during proliferation. The methods used included nanoparticle tracking analysis, confocal laser scanning microscopy, sandwich-type enzyme-linked sorbent assay, quantitative PCR, and gas chromatography. The expression of hyaluronan synthases 1–3 in FLSs and HA concentrations in conditioned media decreased during cell proliferation. This was associated with elevated proportions of 20:4n-6 and total n-6 polyunsaturated FAs (PUFAs) in high-density cells, reductions in n-3/n-6 PUFA ratios, and up-regulation of cluster of differentiation 44, tumor necrosis factor α, peroxisome proliferator-activated receptor (PPAR)-α, and PPAR-γ. Compared to the parent FLSs, 16:0, 18:0, and 18:1n-9 were enriched in the EV fraction. EV counts decreased during cell growth, and 18:2n-6 in EVs correlated with the cell count. To conclude, FLS proliferation was featured by increased 20:4n-6 proportions and reduced n-3/n-6 PUFA ratios, and FAs with a low degree of unsaturation were selectively transferred from FLSs into EVs. These FA modifications have the potential to affect membrane fluidity, biosynthesis of lipid mediators, and inflammatory processes in joints, and could eventually provide tools for translational studies to counteract cartilage degradation in inflammatory joint diseases

Metformin decreases hyaluronan synthesis by vascular smooth muscle cells

Metformin is the first-line drug in the treatment of type 2 diabetes worldwide based on its effectiveness and cardiovascular safety. Currently metformin is increasingly used during pregnancy in women with gestational diabetes mellitus, even if the long-term effects of metformin on offspring are not exactly known. We have previously shown that high glucose concentration increases hyaluronan (HA) production of cultured human vascular smooth muscle cells (VSMC) via stimulating the expression of hyaluronan synthase 2 (HAS2). This offers a potential mechanism whereby hyperglycemia leads to vascular macroangiopathy. In this study, we examined whether gestational metformin use affects HA content in the aortic wall of mouse offspring in vivo. We also examined the effect of metformin on HA synthesis by cultured human VSMCs in vitro. We found that gestational metformin use significantly decreased HA content in the intima-media of mouse offspring aortas. In accordance with this, the synthesis of HA by VSMCs was also significantly decreased in response to treatment with metformin. This decrease in HA synthesis was shown to be due to the reduction of both the expression of HAS2 and the amount of HAS substrates, particularly UDP-N-acetylglucosamine. As shown here, gestational metformin use is capable to program reduced HA content in the vascular wall of the offspring strongly supporting the idea, that metformin possesses long-term vasculoprotective effects.</p

Isolation of extracellular vesicles from the bronchoalveolar lavage fluid of healthy and asthmatic horses

Extracellular vesicles (EVs) are membrane-bound particles that engage in inflammatory reactions by mediating cell–cell interactions. Previously, EVs have been isolated from the bronchoalveolar lavage fluid (BALF) of humans and rodents. The aim of this study was to investigate the number and size distribution of EVs in the BALF of asthmatic horses (EA, n = 35) and healthy horses (n = 19). Saline was injected during bronchoscopy to the right lung followed by manual aspiration. The retrieved BALF was centrifuged twice to remove cells and biological debris. The supernatant was concentrated and EVs were isolated using size-exclusion chromatography. Sample fractions were measured with nanoparticle tracking analysis (NTA) for particle number and size, and transmission electron microscopy and confocal laser scanning microscopy were used to visualize EVs. The described method was able to isolate and preserve EVs. The mean EV size was 247 ± 35 nm (SD) in the EA horses and 261 ± 47 nm in the controls by NTA. The mean concentration of EVs was 1.38 × 1012 ± 1.42 × 1012 particles/mL in the EA horses and 1.33 × 1012 ± 1.07 × 1012 particles/mL in the controls with no statistically significant differences between the groups. With Western blotting and microscopy, these particles were documented to associate with EV protein markers (CD63, TSG101, HSP70, EMMPRIN, and actin) and hyaluronan. Equine BALF is rich in EVs of various sizes, and the described protocol is usable for isolating EVs. In the future, the role of EVs can be studied in horses with airway inflammation.Peer reviewe

Counts of hyaluronic acid-containing extracellular vesicles decrease in naturally occurring equine osteoarthritis

Osteoarthritis (OA) is a degenerative joint disease with inadequately understood pathogenesis leading to pain and functional limitations. Extracellular vesicles (EVs) released by synovial joint cells can induce both pro- and anti-OA effects. Hyaluronic acid (HA) lubricates the surfaces of articular cartilage and is one of the bioactive molecules transported by EVs. In humans, altered EV counts and composition can be observed in OA synovial fluid (SF), while EV research is in early stages in the horse-a well-recognized OA model. The aim was to characterize SF EVs and their HA cargo in 19 horses. SF was collected after euthanasia from control, OA, and contralateral metacarpophalangeal joints. The SF HA concentrations and size distribution were determined with a sandwich-type enzyme-linked sorbent assay and size-exclusion chromatography. Ultracentrifugation followed by nanoparticle tracking analysis (NTA) were utilized to quantify small EVs, while confocal laser scanning microscopy (CLSM) and image analysis characterized larger EVs. The number and size distribution of small EVs measured by NTA were unaffected by OA, but these results may be limited by the lack of hyaluronidase pre-treatment of the samples. When visualized by CLSM, the number and proportion of larger HA-containing EVs (HA-EVs) decreased in OA SF (generalized linear model, count: p = 0.024, %: p = 0.028). There was an inverse association between the OA grade and total EV count, HA-EV count, and HA-EV % (r(s) = - 0.264 to - 0.327, p = 0.012-0.045). The total HA concentrations were also lower in OA (generalized linear model, p = 0.002). To conclude, the present study discovered a potential SF biomarker (HA-EVs) for naturally occurring equine OA. The roles of HA-EVs in the pathogenesis of OA and their potential as a joint disease biomarker and therapeutic target warrant future studies.Peer reviewe

HAS3-induced extracellular vesicles from melanoma cells stimulate IHH mediated c-Myc upregulation via the hedgehog signaling pathway in target cells.

Intercellular communication is fundamental to the survival and maintenance of all multicellular systems, whereas dysregulation of communication pathways can drive cancer progression. Extracellular vesicles (EVs) are mediators of cell-to-cell communication that regulate a variety of cellular processes involved in tumor progression. Overexpression of a specific plasma membrane enzyme, hyaluronan synthase 3 (HAS3), is one of the factors that can induce EV shedding. HAS3, and particularly its product hyaluronan (HA), are carried by EVs and are known to be associated with the tumorigenic properties of cancer cells. To elucidate the specific effects of cancerous, HAS3-induced EVs on target cells, normal human keratinocytes and melanoma cells were treated with EVs derived from GFP-HAS3 expressing metastatic melanoma cells. We found that the HA receptor CD44 participated in the regulation of EV binding to target cells. Furthermore, GFP-HAS3-positive EVs induced HA secretion, proliferation and invasion of target cells. Our results suggest that HAS3-EVs contains increased quantities of IHH, which activates the target cell hedgehog signaling cascade and leads to the activation of c-Myc and regulation of claspin expression. This signaling of IHH in HAS3-EVs resulted in increased cell proliferation. Claspin immunostaining correlated with HA content in human cutaneous melanocytic lesions, supporting our in vitro findings and suggesting a reciprocal regulation between claspin expression and HA synthesis. This study shows for the first time that EVs originating from HAS3 overexpressing cells carry mitogenic signals that induce proliferation and epithelial-to-mesenchymal transition in target cells. The study also identifies a novel feedback regulation between the hedgehog signaling pathway and HA metabolism in melanoma, mediated by EVs carrying HA and IHH.Peer reviewe

Kylmään veteen : kyselytutkimus uuden työtekijän saamasta perehdytyksestä

Tämän opinnäytetyön aihe löytyi liiketalouden tunnilta, jossa keskusteltiin perehdyttämisestä. Opiskelijoilla ei tuntunut olevan kovin positiivisia kokemuksia perehdyttämisestä, josta syntyi halu tutkia asiaa laajemmin. Tämän tutkimuksen tarkoitus oli tutkia Tampereen ammattikorkeakoulun liiketalouden opiskelijoiden edellisessä palkkatyössään saamaa perehdytystä. Tutkimuskysymyksiä oli useita liittyen vastaajien kokemaan perehdyttämistarpeeseen, vastaajien kokemiseen itsensä tervetulleina, perehdytyksessä käsiteltäviin asioihin ja käytettyihin menetelmiin, perehdyttäjään, prosessin kestoon, molemminpuoliseen palautteeseen ja prosessin onnistumiseen. Tutkimusmenetelmänä käytettiin määrällistä kyselytutkimusta, joka toteutettiin sähköpostitse lähetetyllä e-lomakkeella. Tulosten perusteella kaikki vastaajat kokivat tarvitsevansa perehdytystä aloittaessaan uudessa työssä. Perehdytyksen tarpeella oli selvä käänteinen yhteys työkokemuksen määrään. Pääsääntöisesti vastaajat kokivat itsensä tervetulleiksi uudella työpaikalla. Tulosten perusteella perehdytyksessä tulisi käsitellä enemmän työterveyteen ja -turvallisuuteen, työhyvinvointiin ja työn päättymiseen liittyviä asioita. Sopivasti käsiteltiin työympäristöä, toimintatapoja, omaa työtä ja organisaatiota. Käytetyimmät menetelmät olivat tutustumiskierros yrityksen tiloissa, ”vierihoito” jossa seurataan kokeneemman työntekijän työskentelyä sekä perehdyttämisopas. Useimmiten perehdyttämisestä vastasi kollega, ja koko prosessi kesti selvästi alle kuukauden. Vastaajat olisivat kaivanneet enemmän palautetta edistymisestään. Perehdytettävältä itseltään ei myöskään pyydetty palautetta prosessin onnistumisesta. Peräti neljännes vastaajista piti perehdytystään melko tai täysin epäonnistuneena. Mitä lyhemmän perehdytyksen vastaaja oli saanut, sitä tyytymättömämpi hän oli. Avoimen kysymyksen kehitysehdotuksissa mainittiin suunnitelmallisuuden ja selkeyden puute, ajan puute sekä se, että asioista joutui ottamaan itse selvää. Suunnitelmallisuuden ja selkeyden puute saa niin perehdyttäjän kuin perehdytettävän kokemaan olonsa epävarmaksi. Perehdyttämiseen tulisikin panostaa niin ajallisesti kuin sisällöllisesti. Jokaisen perehdytettävän yksilöllinen tilanne tulisi myös huomioida perehdyttämistä suunniteltaessa. Kaikkia ei voi eikä kannata opettaa samalla tavalla. Myös molemminpuoliseen palautteeseen kannattaa panostaa, sillä vain palautteen avulla kumpikin osapuoli pystyy kehittymään ja oppimaan.The topic for this thesis arose in a business class, where students discussed the orientation of an employee. Very few positive experiences came up during the discussion, which brought about the curiosity to study the topic more. The purpose of this thesis was to study the orientation process of the business students of Tampere University of Applied Sciences in their previous workplace. There were several research problems concerning the recipients’ need for orientation, the recipients’ feeling of being welcome, the topics and methods of orientation, the person responsible for orientation, the length of the process, mutual feedback and the overall success of the orientation process. The survey was conducted as a quantitative survey, which was carried out with an electronic questionnaire via e-mail. The results of the survey showed that all recipients felt that they needed orientation when starting a new job. This had a clear reverse connection with the amount of the recipient’s work experience. The majority of recipients felt themselves welcome in their new place of work. The results indicated that occupational health and safety, the employees’ well-being and the termination of the employment should be addressed more during orientation. Work environment, codes of conduct, the employee’s own work and organizational issues were adequately covered. The most widely used methods were a tour of the premises, observing a more experienced co-worker and an orientation manual. In the majority of the cases, a colleague was in charge of the orientation and the whole process took clearly less than a month. The recipients would have wanted more feedback on their progress. In most cases they did not have the opportunity to give feedback on the orientation process. No less than a quarter of the recipients felt that the orientation process was either fairly or totally unsuccessful. The shorter the orientation, the more unsatisfied the recipient. An open question asked for suggestions for improving the process. The topics most often mentioned were the lack of orderliness and clarity, the shortage of time and having to find out about things on their own. The lack of orderliness and clarity makes both the employee and the person responsible for the orientation feel themselves uneasy and uncertain. Organizations should allocate time for the orientation and invest in the contents of the orientation. The individual situation of each employee should be taken into consideration while planning the orientation. Not everybody learns in the same way. Organizations should also focus on mutual feedback. Only feedback can help both sides to improve and learn

Aivoinfarktipotilaan kuntoutus sairaanhoitajan näkökulmasta : Opas omaisille

Opinnäytetyön tarkoitus oli tehdä opas aivoinfarktipotilaan omaisille Hä-meenlinnan kuntoutuslaitos Ilveskotiin. Pyyntö oppaan tarpeellisuudesta tuli Ilveskodin osastonhoitajalta ja henkilökunnalta. Oppaassa tuodaan esille omaisten osallistumisen tärkeyttä aivoinfarktipotilaan kuntoutukseen. Opas antaa tietoa siitä, kuinka omainen voi aktiivisesti osallistua läheisensä kuntoutukseen ja auttaa omaista kuntoutumaan parhaalla mahdollisella tavalla. Oppaassa kerrottiin, miten aivoinfarktipotilaan kuntoutus etenee ja mitä kaikkea siihen sisältyy. Tarkoituksena oli tuoda esille arkipäiväisiä ja konkreettisia esimerkkejä siitä, kuinka omainen voi tukea läheisensä kuntoutusta. Aivoinfarktipotilaan kuntoutus valittiin opinnäytetyön aiheeksi siksi, että aihe on kiinnostava ja uutta tietoa sen hoidosta ja kuntoutuksesta tulee ko-ko ajan. Aivoverenkiertohäiriöt ovat kasvava ongelma Suomessa ja on tär-keää jakaa tietoa siitä kaikille. Opinnäytetyö kertoo yleisesti aivoverenkiertohäiriöistä, mutta perehtyy teoreettisessa viitekehyksessä aivoinfarktiin ja sen kuntoutukseen. Kun-toutusprosessia tarkasteltiin sairaanhoitajan näkökulmasta. Pohdinnan taustaksi tehtiin kysely Ilveskodin hoitajille, joilta kysyttiin heidän tieto-jaan ja kokemuksiaan aivoinfarktipotilaan hoidosta. Opinnäytetyön yhtenä tarkoituksena on myös valistaa ihmisiä aivoveren-kiertohäiriöistä ja sen aiheuttamista elämänmuutoksista, jotta ihmiset osai-sivat kiinnittää paremmin huomiota elämäntapoihinsa ja näin ennaltaeh-käistä sairastumista. Aivoinfarktiin sairastuminen muuttaa sairastuneen ja hänen läheistensä loppuelämän. Kuntoutuminen on vaikea ja pitkä prosessi, mutta kuntoutuksella saadaan aikaan hyviä tuloksia. Monipuolinen ja yksilöllinen kuntoutus mahdollistaa elämän, joka on elämisen arvoinen.The purpose of this thesis was to provide Ilveskoti, a rehabilitation centre located in Hämeenlinna, with a guide for the families of cerebral infarction patients. The request for the need of the guide came from the charge nurse and the staff. The guide highlights the importance of relatives’ participa-tion in the rehabilitation of cerebral infarction patients. The guide explains how the relatives can take active part in the rehabilitation and help the pa-tient rehabilitate the best way possible. The guide describes the process of rehabilitation and everything it includes. The aim was to bring out practical examples on how the relatives can support the rehabilitation. Rehabilitation of cerebral infarction patients was chosen as the topic for the thesis because the subject is interesting. Cerebrovascular diseases are a growing problem in Finland and it is important to share the information on it to everyone. The thesis explains cerebrovascular diseases in general but focuses in the theoretical framework on the cerebral infarction and its rehabilitation. The rehabilitation process is discussed from a nurse’s point of view. For the background of the discussion, a survey was conducted among the nurses of Ilveskoti. They were asked about their knowledge and experiences in the treatment of cerebral infarction patients. One of the purposes of the thesis was to educate people on the cerebrovas-cular diseases and the changes in life it causes so that people would know how to pay more attention to their way of life and thus prevent ill health. Cerebral infarction changes the life of the patient and their families per-manently. Rehabilitation is a difficult and long process but it can achieve excellent results. Versatile, individual rehabilitation enables a life worth living

The Density and Length of Filopodia Associate with the Activity of Hyaluronan Synthesis in Tumor Cells

Filopodia are multifunctional finger-like plasma membrane protrusions with bundles of actin filaments that exist in virtually all cell types. It has been known for some time that hyaluronan synthesis activity induces filopodial growth. However, because of technical challenges in the studies of these slender and fragile structures, no quantitative analyses have been performed so far to indicate their association with hyaluronan synthesis. In this work we comprehensively address the direct quantification of filopodial traits, covering for the first time length and density measurements in a series of human cancer cell lines with variable levels of hyaluronan synthesis. The synthesis and plasma membrane binding of hyaluronan were manipulated with hyaluronan synthase 3 (HAS3) and hyaluronan receptor CD44 overexpression, and treatments with mannose, 4-methylumbelliferone (4-MU), and glucosamine. The results of this work show that the growth of filopodia was associated with the levels of hyaluronan synthesis but was not dependent on CD44 expression. The results confirm the hypothesis that abundance and length of filopodia in cancer cells is associated with the activity of hyaluronan synthesis