Paramedics' experiences and educational needs when participating end-of-life care at home: A mixed method study.

BACKGROUND: Paramedics face end-of-life care patients during emergency calls and more recently through planned protocols. However, paramedics experiences and educational needs concerning preplanned end-of-life care at home remain largely unknown. AIM: To describe experiences and educational needs of the paramedics included in the end-of-life care protocol. DESIGN: A mixed method study with a questionnaire including open ended questions and numeric evaluations on a Likert scale. SETTING/PARTICIPANTS: The questionnaire was delivered to and answered by all the 192 paramedics working in North Karelia fire and rescue department during the time of the data collection in 2017. RESULTS: Over 80% of the paramedics agreed that the protocol helped them to take care of the patients and to improve the quality of end-of-life care. Visits to the patients were considered useful and the end-of-life care as a meaningful work by 76.5% and 62.5% of the paramedics, respectively. The paramedics expressed challenges in psychosocial aspects, communication, symptom management, and their role in end-of-life care. Encountering and communication with the families as well as managing the most common symptoms were emphasized as educational needs. Using a patient controlled analgesia device emerged as an example of practical educational aspect. CONCLUSIONS: Paramedics considered end-of-life care at home meaningful but called for more competency in supporting and encountering the families and in symptom management. Our results can be utilized when developing end-of-life care protocols and education for the paramedics. Patients' and families' views on the paramedics' participation in end-of-life care should be evaluated in the future.

Tissue distribution and subcellular localization of hyaluronan synthase isoenzymes.

Hyaluronan synthases (HAS) are unique plasma membrane glycosyltransferases secreting this glycosaminoglycan directly to the extracellular space. The three HAS isoenzymes (HAS1, HAS2, and HAS3) expressed in mammalian cells differ in their enzymatic properties and regulation by external stimuli, but clearly distinct functions have not been established. To overview the expression of different HAS isoenzymes during embryonic development and their subcellular localization, we immunostained mouse embryonic samples and cultured cells with HAS antibodies, correlating their distribution to hyaluronan staining. Their subcellular localization was further studied by GFP-HAS fusion proteins. Intense hyaluronan staining was observed throughout the development in the tissues of mesodermal origin, like heart and cartilages, but also for example during the maturation of kidneys and stratified epithelia. In general, staining for one or several HASs correlated with hyaluronan staining. The staining of HAS2 was most widespread, both spatially and temporally, correlating with hyaluronan staining especially in early mesenchymal tissues and heart. While epithelial cells were mostly negative for HASs, stratified epithelia became HAS positive during differentiation. All HAS isoenzymes showed cytoplasmic immunoreactivity, both in tissue sections and cultured cells, while plasma membrane staining was also detected, often in cellular extensions. HAS1 had brightest signal in Golgi, HAS3 in Golgi and microvillous protrusions, whereas most of the endogenous HAS2 immunoreactivity was localized in the ER. This differential pattern was also observed with transfected GFP-HASs. The large proportion of intracellular HASs suggests that HAS forms a reserve that is transported to the plasma membrane for rapid activation of hyaluronan synthesis.

Hyaluronan production enhances shedding of plasma membrane-derived microvesicles.

Many cell types secrete plasma membrane-bound microvesicles, suggested to play an important role in tissue morphogenesis, wound healing, and cancer spreading. However, the mechanisms of their formation have remained largely unknown. It was found that the tips of long microvilli induced in cells by overexpression of hyaluronan synthase 3 (HAS3) were detach into the culture medium as microvesicles. Moreover, several cell types with naturally active hyaluronan synthesis released high numbers of plasma membrane-derived vesicles, and inhibition of hyaluronan synthesis reduced their formation. The vesicles contained HAS, and were covered with a thick hyaluronan coat, a part of which was retained even after purification with high-speed centrifugation. HAS3 overexpressing MDCK cells cultured in a 3-D matrix as epithelial cysts released large amounts of HAS- and hyaluronan-positive vesicles from their basal surfaces into the extracellular matrix. As far as we know, hyaluronan synthesis is one of the first molecular mechanisms shown to stimulate the production of microvesicles. The microvesicles have a potential to deliver the hyaluronan synthase machinery and membrane and cytoplasmic materials to other cells, influencing tissue regeneration, inflammation and tumor progression.

HAS3-induced accumulation of hyaluronan in 3D MDCK cultures results in mitotic spindle misorientation and disturbed organization of epithelium.

The amount of hyaluronan (HA) is low in simple epithelia under normal conditions, but during tumorigenesis, trauma or inflammation HA is increased on the epithelial cells and surrounding stroma. Excessive HA in epithelia is suggested to interfere with cell-cell adhesions, resulting in disruption of the epithelial barrier function. In addition, stimulated HA synthesis has been correlated with epithelial-to-mesenchymal transition and invasion of cancer cells. However, the effects of HA overload on normal epithelial morphogenesis have not been characterized in detail. Madin-Darby canine kidney (MDCK) cells form polarized epithelial cysts, when grown in a 3-dimensional (3D) matrix. These cells were used to investigate whether stimulated HA synthesis, induced by stable overexpression of GFP-HAS3, influences cell polarization and epithelial morphogenesis. GFP-HAS3 expression in polarized MDCK cells resulted in active HA secretion at apical and basolateral membrane domains. HA-deposits interfered with the formation of cell-cell junctions, resulting in impaired barrier function. In 3D cyst cultures, HA accumulated into apical lumina and was also secreted from the basal side. The HAS3-expressing cysts failed to form a single lumen and instead displayed multiple small lumina. This phenotype was correlated with aberrant mitotic spindle orientation in dividing cells. The results of this study indicate that excess pericellular HA disturbs the normal cell-cell and cell-ECM interactions in simple epithelia, leading to aberrant epithelial morphogenesis. The morphological abnormalities observed in 3D epithelial cultures upon stimulated HAS3 expression may be related to premalignant changes, including intraluminal invasion and deregulated epithelialization, probably mediated by the mitotic spindle orientation defects.

Regulation of the hyaluronan synthase 2 gene by convergence in cyclic AMP response element-binding protein and retinoid acid receptor signaling.

The human hyaluronan synthase 2 (HAS2) gene encodes for an enzyme making hyaluronan, altered concentrations of which are associated with many pathological situations including wounding, several inflammatory conditions, and malignant tumors. In this study we showed that HAS2 is a primary target of the cAMP activator forskolin and the nuclear hormone all-trans-retinoic acid (RA). The first 2250 bp of the promoter contain three response elements (REs) for the transcription factor CREB1 as well as two REs for the nuclear receptor RAR. Chromatin immunoprecipitation and re-chromatin immunoprecipitation assays using selected fragments of the promoter containing the putative REs showed that forskolin and all-trans-RA modulate the formation of complexes between CREB1 and RAR with various co-regulators at the predicted sites. Interestingly, CREB1 complexes are regulated by all-trans-RA as are RAR complexes by forskolin. Reporter gene assays using nested promoter fragments supported these findings. Forskolin and all-trans-RA co-stimulation reduced the binding of CREB1, RAR, and the co-repressor nuclear receptor co-repressor 1 (NCoR1), but enhanced the association of co-activators MED1 and CREB-binding protein (CBP). RNA interference experiments suggested that MED1 and NCoR1 are central for the all-trans-RA induction of the HAS2 gene and CBP dominates its forskolin response. In general, our findings suggest a convergence of CREB1 and RAR signaling, and demonstrate the individual character of each RE in terms of co-regulator use.

Mesotheliomas show higher hyaluronan positivity around tumor cells than metastatic pulmonary adenocarcinomas.

Hyaluronan is a unique glycosaminoglycan of the extracellular matrix, abundant in normal connective tissues but highly increased in many pathological conditions like cancer. Mesothelioma, one of the most malignant cancer types, is associated with high content of hyaluronan, with elevated levels of hyaluronan in pleural effusions and serum of the patients. Metastatic lung adenocarcinomas are typically less aggressive and have a better prognosis as compared to mesotheliomas, a reason why it is highly important to find reliable tools to differentiate these cancer types. The main purpose of this study was to evaluate the amount of hyaluronan, hyaluronan producing synthases (HAS's) and hyaluronan receptor CD44, in mesothelioma and metastatic lung adenocarcinomas. Furthermore, we wanted to clarify the role of hyaluronan, CD44 and HAS's as putative markers for differentiating malignant mesothelioma from metastatic lung adenocarcinomas. The main finding of this study was that mesotheliomas are significantly more positive for hyaluronan staining than metastatic adenocarcinomas. Unexceptionally, a trend of CD44 positivity of stromal cells was higher in adenocarcinomas as compared to mesotheliomas. However, no statistically significant differences were found between the staining of any of the HAS isoenzymes either in tumor cells or stromal cells of different groups of cases. The results show that there are significant differences in hyaluronan content between metastatic lung adenocarcinomas and mesotheliomas. However, as previous studies have suggested, hyaluronan alone is not a sufficient independent marker for diagnostic differentiation of these cancer types, but could be utilized as a combination together with other specific markers.

cDNA array reveals mechanosensitive genes in chondrocytic cells under hydrostatic pressure.

Hydrostatic pressure (HP) has a profound effect on cartilage metabolism in normal and pathological conditions, especially in weight-bearing areas of the skeletal system. As an important component of overall load, HP has been shown to affect the synthetic capacity and well-being of chondrocytes, depending on the mode, duration and magnitude of pressure. In this study we examined the effect of continuous HP on the gene expression profile of a chondrocytic cell line (HCS-2/8) using a cDNA array containing 588 well-characterized human genes under tight transcriptional control. A total of 51 affected genes were identified, many of them not previously associated with mechanical stimuli. Among the significantly up-regulated genes were immediate-early genes, and genes involved in heat-shock response (hsp70, hsp40, hsp27), and in growth arrest (GADD45, GADD153, p21(Cip1/Waf1), tob). Markedly down-regulated genes included members of the Id family genes (dominant negative regulators of basic helix-loop-helix transcription factors), and cytoplasmic dynein light chain and apoptosis-related gene NIP3. These alterations in the expression profile induce a transient heat-shock gene response and activation of genes involved in growth arrest and cellular adaptation and/or differentiation.

EGF upregulates, whereas TGF-beta downregulates, the hyaluronan synthases Has2 and Has3 in organotypic keratinocyte cultures: correlations with epidermal proliferation and differentiation.

Hyaluronan, a major extracellular matrix molecule in the vital cell layers of skin epidermis, has been suggested to support proliferation and migration of keratinocytes, during challenges like wounding and inflammation. An organotypic keratinocyte culture originated from continuous rat epidermal keratinocyte cell line was subjected to the proliferative and antiproliferative growth factors epidermal growth factor and transforming growth factor beta, respectively, to study their influence on hyaluronan synthesis and epidermal morphology. Epidermal growth factor induced a 4-fold increase of epidermal hyaluronan concentration. This was associated with upregulation of the hyaluronan synthases Has2 and Has3, and the hyaluronan receptor CD44. 5-Bromo-2'-deoxyuridine labeling, basal cell height, and the thickness of vital epidermis were increased, reflecting the hyperplastic effects of epidermal growth factor. The expression of keratin 10 and the maturation of filaggrin were inhibited, and epidermal permeability barrier became less efficient, indicating compromised terminal differentiation by epidermal growth factor. In contrast, transforming growth factor beta reduced the content of hyaluronan and the mRNA of Has2 and Has3. At the same time, transforming growth factor beta suppressed keratinocyte proliferation and epidermal thickness, but retained intact differentiation. The results suggest that epidermal hyaluronan synthesis, controlled by epidermal growth factor and transforming growth factor beta through changes in the expression of Has2 and Has3, correlates with epidermal proliferation, thickness, and differentiation.

Reticulon 4 in chondrocytic cells: barosensitivity and intracellular localization.

Members of the reticulon gene family are endoplasmic reticulum (ER)-related proteins expressed in various human tissues, but their molecular functions are not understood. The reticulon 4 subfamily consists of three members, reticulon 4/Nogo-A, -B and -C. Reticulon 4-A is under intense investigation because of its inhibitory effect on neurite outgrowth, and reticulon 4-B has been suggested to induce apoptosis. Reticulon 4-C, the shortest member of this subfamily, is the least characterized. Reticulons are presumably guided to endoplasmic reticulum by a putative N-terminal retention motif. In this study the expressions of reticulon 4 subtypes in human chondrosarcoma cell line and in primary bovine chondrocytes were analyzed on mRNA level. These cell types, exposed to strong mechanical forces in vivo, were subjected to high hydrostatic pressure and mechanical stretch to study the possible mechanosensitivity of reticulon 4 genes. In addition, a green fluorescent protein-tagged reticulon 4-C and a fusion protein with mutated endoplasmic reticulum retention signal were used to study the significance of the C-terminal translocation signal (the di-lysine motif). As the result, both cell types expressed the three main isoforms of reticulon 4 family. The steady-state level of reticulon 4-B mRNA was shown to be up-regulated by pressure, but not by mechanical stretch indicating transcriptional barosensitivity. The reticular distribution pattern of reticulon 4-C was observed indicating a close association with endoplasmic reticulum. Interestingly, this pattern was maintained despite of the disruption of the putative localization signal. This suggests the presence of another, yet unidentified endoplasmic reticulum retention mechanism.

High pressure effects on cellular expression profile and mRNA stability. A cDNA array analysis.

Hydrostatic pressure has a profound effect on cartilage tissue and chondrocyte metabolism. Depending on the type and magnitude of pressure various responses can occur in the cells. The mechanisms of mechanotransduction at cellular level and the events leading to specific changes in gene expression are still poorly understood. We have previously shown that induction of stress response in immortalized chondrocytes exposed to high static hydrostatic pressure increases the stability of heat shock protein 70 mRNA. In this study, our aim was to examine the effect of high pressure on gene expression profile and to study whether stabilization of mRNA molecules is a general phenomenon under this condition. For this purpose a cDNA array analysis was used to compare mRNA expression profile in pressurized vs. non-pressurized human chondrosarcoma cells (HCS 2/8). mRNA stability was analyzed using actinomycin-treated and nontreated samples collected after pressure treatment. A number of immediate-early genes, and genes regulating cell cycle and growth were up-regulated due to high pressure. Decrease in osteonectin, fibronectin, and collagen types VI and XVI mRNAs was observed. Also bikunin, cdc37 homologue and Tiam1, genes linked with hyaluronan metabolism, were down-regulated. In general, stability of down-regulated mRNA species appeared to increase. However, no increase in mRNA above control level due to stabilization was noticed in the genes available in the array. On the other hand, mRNAs of certain immediate-early genes, like c-jun, jun-B and c-myc, became destabilized under pressure treatment. Increased accumulation of mRNA on account of stabilization under high pressure conditions seems to be a tightly regulated, specific phenomenon.

Chronic UVR causes increased immunostaining of CD44 and accumulation of hyaluronan in mouse epidermis.

Chronic intense UV radiation is the main cause of epidermal tumors. Because hyaluronan (HA), a large extracellular polysaccharide, is known to promote malignant growth, hyaluronan expression was studied in a model in which long-term UV radiation (UVR) induces epidermal tumors. Mouse back skin was exposed three times a week for 10.5 months to UVR corresponding to one minimal erythema dose, processed for histology, and stained for hyaluronan and the hyaluronan receptor CD44. This exposure protocol caused epidermal hyperplasia in most of the animals; tumors, mainly squamous cell carcinomas (SCCs), were found in ~20% of the animals. Specimens exposed to UVR showed increased hyaluronan and CD44 staining throughout the epidermal tissue. In hyperplastic areas, hyaluronan and CD44 stainings correlated positively with the degree of hyperplasia. Well-differentiated SCCs showed increased hyaluronan and CD44 staining intensities, whereas poorly differentiated tumors and dysplastic epidermis showed areas where HA and CD44 were locally reduced. The findings indicate that HA and CD44 increase in epidermal keratinocytes in the premalignant hyperplasia induced by UV irradiation and stay elevated in dysplasia and SCC, suggesting that the accumulation of hyaluronan and CD44 is an early marker for malignant transformation and may be a prerequisite for tumor formation.

All-trans retinoic acid-induced hyaluronan production and hyperplasia are partly mediated by EGFR signaling in epidermal keratinocytes.

All-trans retinoic acid (RA) compromises epidermal differentiation and causes keratinocyte hyperproliferation through mechanisms not completely understood, but may involve the regulatory matrix molecule hyaluronan. In this work, the influences of all-trans RA on epidermal morphology and hyaluronan metabolism were examined in organotypic and monolayer cultures of rat epidermal keratinocytes (REKs). All-trans RA treatment of organotypic REK cultures (10 days) increased the synthesis of hyaluronan, the expression of hyaluronan synthases Has2 and Has3, and the CD44 receptor, with hyperplasia of the epidermis. The hyperplasia and hyaluronan production induced by all-trans RA were blocked with (1) AG1478, an inhibitor of the EGFR; (2) UO126, an inhibitor of the MAPK/ERK kinase, and (3) GM6001, an inhibitor of the matrix metalloproteinases. These effects were consistent with the findings that all-trans RA upregulated heparin-binding epidermal growth factor-like growth factor mRNA expression and increased the phosphorylation of EGFR and extracellular signal-regulated kinase 1/2 (ERK1/2). Interestingly, the activation of EGFR and ERK1/2 was seen already 30 minutes after all-trans RA treatment, suggesting that the activation of this signaling pathway is a primary response to all-trans RA. These results indicate that the effects of all-trans RA on keratinocyte proliferation and hyaluronan synthesis are partly mediated through EGFR signaling.

Changed lamellipodial extension, adhesion plaques and migration in epidermal keratinocytes containing constitutively expressed sense and antisense hyaluronan synthase 2 (Has2) genes.

Hyaluronan is a major component of the epidermal extracellular matrix, is actively synthesized by keratinocytes and shows fast matrix turnover in the stratified epithelium. We probed the importance of hyaluronan synthesis in keratinocytes by establishing cell lines carrying the exogenous hyaluronan synthase 2 (Has2) gene in sense and antisense orientations to increase and decrease their hyaluronan synthesis, respectively. Compared with cell lines transfected with the vector only, most clones containing the Has2 sense gene migrated faster in an in vitro wounding assay, whereas Has2 antisense cells migrated more slowly. Has2 antisense clones showed delayed entry into the S phase of cell cycle following plating, smaller lamellipodia and less spreading on the substratum. The decrease of hyaluronan on the undersurface of Has2 antisense cells was associated with an increased area of adhesion plaques containing vinculin. Exogenous hyaluronan added to the keratinocyte cultures had a minor stimulatory effect on migration after wounding but did not restore the reduced migratory ability of Has2 antisense cells. Hyaluronan decasaccharides that displace receptor bound hyaluronan in keratinocytes, and Streptomyces hyaluronidase sufficient to remove most cell surface hyaluronan had little effect on cell migration. The results suggest that the dynamic synthesis of hyaluronan directed by Has2, rather than the abundance of pericellular hyaluronan, controls keratinocyte migration, a cell function vital for the repair of squamous epithelia following wounding.

Keratinocyte growth factor stimulates migration and hyaluronan synthesis in the epidermis by activation of keratinocyte hyaluronan synthases 2 and 3.

Keratinocyte growth factor (KGF) activates keratinocyte migration and stimulates wound healing. Hyaluronan, an extracellular matrix glycosaminoglycan that accumulates in wounded epidermis, is known to promote cell migration, suggesting that increased synthesis of hyaluronan might be associated with the KGF response in keratinocytes. Treatment of monolayer cultures of rat epidermal keratinocytes led to an elongated and lifted cell shape, increased filopodial protrusions, enhanced cell migration, accumulation of intermediate size hyaluronan in the culture medium and within keratinocytes, and a rapid increase of hyaluronan synthase 2 (Has2) mRNA, suggesting a direct influence on this gene. In stratified, organotypic cultures of the same cell line, both Has2 and Has3 with the hyaluronan receptor CD44 were up-regulated and hyaluronan accumulated in the epidermis, the spinous cell layer in particular. At the same time the expression of the early differentiation marker keratin 10 was inhibited, whereas filaggrin expression and epidermal permeability were less affected. The data indicate that Has2 and Has3 belong to the targets of KGF in keratinocytes, and support the idea that enhanced hyaluronan synthesis acts an effector for the migratory response of keratinocytes in wound healing, whereas it may delay keratinocyte terminal differentiation.