Novel regulatory SNPs in the promoter region of the TNFRSF18 gene in a Gabonese population

Parasites are accountable for driving diversity within immune gene families. We identified and investigated regulatory single nucleotide polymorphisms (SNPs) in the promoter regions of the tumor necrosis factor receptor superfamily member 18 (TNFRSF18) gene by direct sequencing in a group of male Gabonese individuals exposed to a wide array of parasitic diseases such as malaria, filariasis and schistosomiasis. Two new promoter variants were identified in 40 individuals. Both novel variants were heterozygous and were linked to SNP #rs3753344 (C/T), which has been described. One of the SNP variants (ss2080581728) was close to the general transcription factor site, the TATA box. We further validated these new promoter variants for their allelic gene expression using transient transfection assays. One new promoter variant with two base changes (C/T - ss2080581728/rs3753344) displayed an altered expression of the marker gene. Both novel variants remained less active at the non-induced state in comparison to the major allele. The allele frequencies observed in this study were consistent with data for other African populations. The detection and analysis of these human immune gene polymorphisms contribute to a better understanding of the interaction between host-parasite and expression of Treg activity.

Essential regulatory elements for NHE3 gene transcription in renal proximal tubule cells

The objectives of the present study were to identify the cis-elements of the promoter absolutely required for the efficient rat NHE3 gene transcription and to locate positive and negative regulatory elements in the 5’-flanking sequence (5’FS), which might modulate the gene expression in proximal tubules, and to compare this result to those reported for intestinal cell lines. We analyzed the promoter activity of different 5’FS segments of the rat NHE3 gene, in the OKP renal proximal tubule cell line by measuring the activity of the reporter gene luciferase. Because the segment spanning the first 157 bp of 5’FS was the most active it was studied in more detail by sequential deletions, point mutations, and gel shift assays. The essential elements for gene transcription are in the region -85 to -33, where we can identify consensual binding sites for Sp1 and EGR-1, which are relevant to NHE3 gene basal transcription. Although a low level of transcription is still possible when the first 25 bp of the 5’FS are used as promoter, efficient transcription only occurs with 44 bp of 5’FS. There are negative regulatory elements in the segments spanning -1196 to -889 and -467 to -152, and positive enhancers between -889 and -479 bp of 5’FS. Transcription factors in the OKP cell nuclear extract efficiently bound to DNA elements of rat NHE3 promoter as demonstrated by gel shift assays, suggesting a high level of similarity between transcription factors of both species, including Sp1 and EGR-1.

Effect of thymosin alpha-1 on subpopulations of Th1, Th2, Th17, and regulatory T cells (Tregs) in vitro

Thymosin alpha 1 (Tα1) has been shown to have beneficial effects on numerous immune system parameters, but little is known about the effects of Tα1 on patients with gastric carcinoma. The objective of this study was to determine the effect of Tα1 on subpopulations of Th1, Th2, Th17, and regulatory T cells (Tregs) in vitro, and to evaluate its efficacy as an immunoregulatory factor in patients with gastric carcinoma. We compared the effect of Tα1 on the frequency of CD4+ and CD8+ T cells, especially the CD4+CD25+Foxp3+ Tregs in peripheral blood mononuclear cells (PBMCs) from gastric carcinoma patients (N = 35) and healthy donors (N = 22). We also analyzed the changes in the proliferation of PBMCs in response to treatment with Tα1, and examined the production of Th1, Th2, and Th17 cytokines by PBMCs and tumor-infiltrating lymphocytes. The treatment of PBMCs from gastric cancer patients, with Tα1 (50 µg/mL) alone increased the percentage of CD4+CD25+Foxp3+ (suppressive antitumor-specific Tregs) from 1.68 ± 0.697 to 2.19 ± 0.795% (P < 0.05). Our results indicate that Tα1 increases the percentage of Tregs and IL-1β, TNF-α, and IL-6 in vitro.

Prolonged acceptance of skin grafts induced by B cells places regulatory T cells on the histopathology scene

The participation of regulatory T (Treg) cells in B cell-induced T cell tolerance has been claimed in different models. In skin grafts, naive B cells were shown to induce graft tolerance. However, neither the contribution of Treg cells to B cell-induced skin tolerance nor their contribution to the histopathological diagnosis of graft acceptance has been addressed. Here, using male C57BL/6 naive B cells to tolerize female animals, we show that skin graft tolerance is dependent on CD25+ Treg cell activity and independent of B cell-derived IL-10. In fact, B cells from IL-10-deficient mice were able to induce skin graft tolerance while Treg depletion of the host inhibited 100% graft survival. We questioned how Treg cell-mediated tolerance would impact on histopathology. B cell-tolerized skin grafts showed pathological scores as high as a rejected skin from naive, non-tolerized mice due to loss of skin appendages, reduced keratinization and mononuclear cell infiltrate. However, in tolerized mice, 40% of graft infiltrating CD4+ cells were FoxP3+ Treg cells with a high Treg:Teff (effector T cell) ratio (6:1) as compared to non-tolerized mice where Tregs comprise less than 8% of total infiltrating CD4 cells with a Treg:Teff ratio below 1:1. These results render Treg cells an obligatory target for histopathological studies on tissue rejection that may help to diagnose and predict the outcome of a transplanted organ.

BAFF promotes regulatory T-cell apoptosis and blocks cytokine production by activating B cells in primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is a chronic and slowly progressive cholestatic liver disease of autoimmune etiology. A number of questions regarding its etiology are unclear. CD4+CD25+ regulatory T cells (Tregs) play a critical role in self-tolerance and, for unknown reasons, their relative number is reduced in PBC patients. B-cell-activating factor (BAFF) is a key survival factor during B-cell maturation and its concentration is increased in peripheral blood of PBC patients. It has been reported that activated B cells inhibit Treg cell proliferation and there are no BAFF receptors on Tregs. Therefore, we speculated that excessive BAFF may result in Treg reduction via B cells. To prove our hypothesis, we isolated Tregs and B cells from PBC and healthy donors. BAFF and IgM concentrations were then analyzed by ELISA and CD40, CD80, CD86, IL-10, and TGF-β expression in B cells and Tregs were measured by flow cytometry. BAFF up-regulated CD40, CD80, CD86, and IgM expression in B cells. However, BAFF had no direct effect on Treg cell apoptosis and cytokine secretion. Nonetheless, we observed that BAFF-activated B cells could induce Treg cell apoptosis and reduce IL-10 and TGF-β expression. We also showed that BAFF-activated CD4+ T cells had no effect on Treg apoptosis. Furthermore, we verified that bezafibrate, a hypolipidemic drug, can inhibit BAFF-induced Treg cell apoptosis. In conclusion, BAFF promotes Treg cell apoptosis and inhibits cytokine production by activating B cells in PBC patients. The results of this study suggest that inhibition of BAFF activation is a strategy for PBC treatment.

Imbalanced expression of functional surface molecules in regulatory and effector T cells in systemic lupus erythematosus

Regulatory T (TREG) cells play an important role in maintaining immune tolerance and avoiding autoimmunity. We analyzed the expression of membrane molecules in TREG and effector T cells in systemic lupus erythematosus (SLE). TREG and effector T cells were analyzed for the expression of CTLA-4, PD1, CD28, CD95, GITR, HLA-DR, OX40, CD40L, and CD45RO in 26 patients with active disease, 31 with inactive disease, and 26 healthy controls. TREG cells were defined as CD25+/highCD127Ø/lowFoxP3+, and effector T cells were defined as CD25+CD127+FoxP3Ø. The ratio of TREG to effector T cells expressing GITR, PD1, HLA-DR, OX40, CD40L, and CD45RO was determined in the three groups. The frequency of TREG cells was similar in patients with SLE and controls. However, SLE patients had a decreased frequency of CTLA-4+TREG and CD28+TREG cells and an increased frequency of CD40L+TREG cells. There was a decrease in the TREG/effector-T ratio for GITR+, HLA-DR+, OX40+, and CD45RO+ cells, and an increased ratio of TREG/effector-T CD40L+ cells in patients with SLE. In addition, CD40L+TREG cell frequency correlated with the SLE disease activity index (P=0.0163). In conclusion, our findings showed several abnormalities in the expression of functionally critical surface molecules in TREG and effector T cells in SLE that may be relevant to the pathogenesis of this disease.

Comparison of totally tubeless percutaneous nephrolithotomy and standard percutaneous nephrolithotomy for kidney stones: a randomized, clinical trial

This study aimed to compare the totally tubeless percutaneous nephrolithotomy and standard percutaneous nephrolithotomy techniques regarding their rates of success and complications in patients with kidney stones. Patients were randomly assigned to two groups. Forty-four patients (24 men; mean age: 50.40±2.02 years) received totally tubeless percutaneous nephrolithotomy (PCNL; no nephrostomy catheter or ureteral catheter after PCNL) and 40 patients (18 men; mean age: 49.95±13.38 years) underwent standard PCNL (a nephrostomy catheter and ureteral catheter were used after PCNL). All surgeries were performed by one surgeon. Postoperative changes in hemoglobin, the blood transfusion rate, changes in creatinine levels, operation time, analgesic need, hospitalization time, and complication rate were compared between the groups. No significant differences were observed in age, gender, stone size, and surgery side between the groups (P<0.05). The operation time was significantly lower in the totally tubeless PCNL group than in the standard PCNL group (P=0.005). Pethidine requirements were significantly higher in the standard PCNL group than the totally tubeless PCNL group (P=0.007). Hospitalization time was significantly higher in the standard PCNL group than in the totally tubeless PCNL group (P<0.0001). The complication rate was 15% in the standard PCNL group and 9.1% in the totally tubeless PCNL group (P=0.73). The totally tubeless PCNL technique is safe and effective, even for patients with staghorn stones. This technique is associated with decreased pain, analgesic needs, and operative and hospitalization time. We believe that a normal peristaltic ureter is the best drainage tube.

Effect of standard and neutral-pH peritoneal dialysis solutions upon fibroblasts proliferation

Introduction: Continuous exposition of the peritoneal membrane to conventional dialysis solutions is an important risk factor for inducing structural and functional alterations. Objective: To compare in vitro mouse fibroblast NIH-3T3 cell viability after exposition to a neutral pH dialysis solution in comparison to cells exposed to a standard solution. Methods: Experimental study to compare the effects of a conventional standard or a neutral-pH, low-glucose degradation products peritoneal dialysis solution on the viability of exposed fibroblasts in cell culture. Both solutions were tested in all the commercially available glucose concentrations. Cell viability was evaluated with tetrazolium salt colorimetric assay. Results: Fibroblast viability was significantly superior in the neutral pH solution in comparison to control, in all three glucose concentrations (Optical density in nm-means ± SD: 1.5% 0.295 ± 0.047 vs. 0.372 ± 0.042, p < 0.001; 2.3% 0.270 ± 0.036 vs. 0.337 ± 0.051, p < 0.001; 4.25% 0.284 ± 0.037 vs. 0.332 ± 0.032, p < 0.001; control vs. neutral pH respectively, Student t Test). There was no significant difference in cell viability between the three concentrations of glucose when standard solution was used (ANOVA p = 0.218), although cell viability was higher after exposition to neutral pH peritoneal dialysis fluid at 1.5% in comparison to 2.3 and 4.25% glucose concentrations (ANOVA p = 0.008: Bonferroni 1.5% vs. 2.3% p = 0.033, 1.5% vs. 4.25% p = 0.014, 2.3% vs. 4.25% p = 1.00). Conclusion: Cell viability was better in neutral pH dialysis solution, especially in the lower glucose concentration. A more physiological pH and lower glucose degradation products may be responsible for such results.

Analysis and guidelines for implementation of EN 1090-2 Standard to assist execution class assignment of steel structures

Finnish design and consulting companies are delivering robust and cost-efficient steel structures solutions to a large number of manufacturing companies worldwide. Recently introduced EN 1090-2 standard obliges these companies to specify the execution class of steel structures for their customers. This however, requires clarifying, understanding and interpreting the sophisticated procedure of execution class assignment. The objective of this research is to provide a clear explanation and guidance through the process of execution class assignment for a given steel structure and to support the implementation of EN 1090-2 standard in Rejlers Oy, one of Finnish design and consulting companies. This objective is accomplished by creating a guideline for designers that elaborates on the four-step process of the execution class assignment for a steel structure or its part. Steps one to three define the consequence class (projected consequences of structure failure), the service category (hazards associated with the service use exploitation of steel structure) and the production category (manufacturing process peculiarities), based on the ductility class (capacity of structure to withstand deformations) and the behaviour factor (corresponds to structure seismic behaviour). The final step is the execution class assignment taking into account results of previous steps. Main research method is indepth literature review of European standards family for steel structures. Other research approach is a series of interviews of Rejlers Oy representatives and its clients, results of which have been used to evaluate the level of EN 1090-2 awareness. Rejlers Oy will use the developed novel coherent standard implementation guideline to improve its services and to obtain greater customer satisfaction.

Pienten yhtiöiden tilintarkastusstandardin arvioitu vaikutus tilintarkastuksen hyötyihin – Standard for Audits of Small Entities, SASE

Suomessa ja muissa Pohjoismaissa on tunnistettu haasteita kansainvälisten tilintarkastusstandardien eli ISA-standardien soveltamisessa pienten yhtiöiden tilintarkastuksiin. ISA-standardien on kritisoitu sisältävän liian yksityiskohtaisia ja jopa epärealistisia vaatimuksia ajatellen pienien yhtiöiden tilintarkastuksia. Pohjoismaisella yhteistyöllä onkin alettu kehittää omaa standardia pienten yhtiöiden tilintarkastuksiin. Niin sanotun SASE-standardin on raportoitu muun muassa korostavan tilintarkastajan ammatillista harkintaa ja keventävän pienten yhtiöiden tilintarkastuksen vaatimuksia. Koska kuitenkin aikaisemmin sääntelyn lisäämisellä on pyritty muun muassa lisääntyneeseen tilintarkastuksen luotettavuuteen, josta myös pienten yhtiöiden on itse tutkittu hyötyvän, on tutkimuksessa haluttu selvittää, miten kehitteillä olevan SASE-standardin arvioidaan vaikuttavan yleisesti pienten yhtiöiden tilintarkastuksella saavutettaviin hyötyihin. Tutkimus on toteutettu toiminta-analyyttisena laadullisena tutkimuksena, ja tutkimusmetodina on käytetty tilintarkastajien ja yliopiston tutkijatohtorin haastatteluja. Tutkimuksen tulosten mukaan kehitteillä olevan SASE-standardin arvioidaan vaikuttavan pienten yhtiöiden tilintarkastuksen hyötyihin kaksisuuntaisesti. Toisaalta SASE-standardi voi lisätä tilintarkastuksen hyötyjä, koska tilintarkastaja voisi keskittyä tarkastuskohteen kannalta olennaisempiin asioihin, hyödyntää hiljaista tietoaan sekä lisätä vuorovaikutusta yhtiön omistajajohdon kanssa. SASE-standardin ei arvioida muuttavan juurikaan käytännön tilintarkastustyötä ja periaatteessa olennaisuuksiin keskittymisen ja riskien tehokkaamman tunnistamisen tulisi hyödyttää myös tilintarkastuskohteen ulkoisia sidosryhmiä, kuten rahoittajia. Kuitenkin tutkimuksessa nousi esiin SASE-standardin mahdollinen heikko maine. Mikäli SASE-standardia pidettäisiin vähäpätöisempänä tai epäluotettavampana kuin aikaisempaa käytäntöä, luottamus tilintarkastusta ja tarkastuskohdetta kohtaan heikkenisi ja sitä kautta myös tilintarkastuksen hyödyt pienenisivät. Tutkimuksen johtopäätöksenä voidaan esittää, että tilintarkastuksen sääntelyn keventämisen vaikutus tilintarkastuksen hyötyihin ei niinkään synny käytännön työn kautta. Tilintarkastuksen hyötyihin pikemminkin vaikuttavat maineeseen, viestintään ja vuorovaikutukseen liittyvät seikat. Toimivalla vuorovaikutuksella yhtiön omistajajohdon ja tilintarkastajan välillä voidaan tilintarkastuksen hyötyjä korostaa, ja toisaalta taas riittävällä ulkoisiin sidosryhmiin kohdistuvalla tiedottamisella voidaan hyötyjen heikkenemistä ehkäistä. Keskeiseksi seikaksi tutkimuksessa nousee se, että tilintarkastuksen luotettavuuden ja tason säilyminen on pystyttävä todistamaan.

Novel cancer-related regulatory targets for the signalling sphingolipids sphingosine-1-phosphate and sphingosylphosphorylcholine

The signalling sphingolipid sphingosine-1-phosphate (S1P) is necessary for development of the immune system and vasculature and on a cellular level regulates migration, proliferation and survival. Due to these traits S1P has an important role in cancer biology. It is considered a primarily cancer-promoting factor and the enzyme which produces it, sphingosine kinase (SphK), is often over-expressed in tumours. S1P is naturally present in the blood, lymph, tissue fluids and cell cytoplasm and functions through its cell surface receptors (S1P1-5) and as an intracellular second messenger. Sphingosylphosphorylcholine (SPC) is closely related to S1P and has similar regulatory functions but has not been extensively studied. Both S1P and SPC are able to evoke either stimulatory or inhibitory effects on cancer cells depending on the context. The aim of this thesis work was to study novel regulatory targets of S1P and SPC, which mediate the effects of S1P/SPC signalling on cancer cell behaviour. The investigated targets are the transcription factor hypoxia-inducible factor 1 (HIF-1), the intermediate filament protein vimentin and components of the Hippo signalling pathway. HIF-1 has a central role in cancer biology, as it regulates a multitude of cancer-related genes and is potently activated by intratumoural hypoxia through stabilization of the regulatory subunit HIF-1α. Tumours typically harbour high HIF-1α levels and HIF-1, in turn, facilitates tumour angiogenesis and metastasis and regulates cancer cell metabolism. We found S1P to induce follicular thyroid cancer cell migration in normal oxygen conditions by increasing HIF-1α synthesis and stability and subsequently HIF-1 activity. Vimentin is a central regulator of cell motility and is also commonly over-expressed in cancers. Vimentin filaments form a cytoskeletal network in mesenchymal cells as well as epithelial cancer cells which have gone through epithelial-mesenchymal transition (EMT). Vimentin is heavily involved in cancer cell invasion and gives tumours metastatic potential. We saw both S1P and SPC induce phosphorylation of vimentin monomers and reorganization of the vimentin filament network in breast and anaplastic thyroid cancer cells. We also found vimentin to mediate the anti-migratory effect of S1P/SPC on these cells. The Hippo pathway is a novel signalling cascade which controls cancer-related processes such as cellular proliferation and survival in response to various extracellular signals. The core of the pathway consists of the transcriptional regulators YAP and TAZ, which activate predominantly cancer-promoting genes, and the tumour suppressive kinases Lats1 and Lats2 which inhibit YAP/TAZ. Increased YAP expression and activity has been reported for a wide variety of cancers. We found SPC to regulate Hippo signalling in breast cancer cells in a two-fold manner through effects on phosphorylation status, activity and/or expression of YAP and Lats2. In conclusion, this thesis reveals new details of the signalling function of S1P and SPC and regulation of the central oncogenic factors HIF-1 and vimentin as well as the novel cancer-related pathway Hippo.

Interoperability for IoT Health Systems Using HL7 Standard

Internet of Things or IoT is revolutionizing the world we are living in, similarly the way Internet and the web did few decades ago. It is changing how we interact with the things surrounding us. Electronic health and remote patient monitoring are the ways of utilizing these technological improvements towards the healthcare. There are many applications of IoT in eHealth such as, it will open the gate to provide healthcare to the remote areas of the world, where healthcare through traditional hospital systems cannot be provided. To connect these new eHealth IoT systems with the existing healthcare information systems, we can use the existing interoperability standards commonly used in healthcare information systems. In this thesis we implemented an eHealth IoT system based on Health Level 7 interoperability standard for continuous data transmission. There is not much previous work done in implementing the HL7 for continuous sensor data transmission. Some of the previous work was limited to sensors which are not continuous in nature and some of it is only theatrical architecture. This thesis aims to prove that it is possible to implement an eHealth IoT system by using sensors which require continues data transmission, such as respiratory sensors, and to connect it with the existing eHealth information system semantically by using HL7 interoperability standard. This system will be beneficial in implementing eHealth IoT systems for those patients, who requires continuous healthcare personal monitoring. This includes elderly people and patients, whose health need to be monitored constantly. To implement the architecture, HL7 v2.5 is selected due to its ease of implementation and low size. We selected some open source technologies because of their open licenses and large developer community. We will also review the most efficient technology available in every layer of eHealth IoT system and will propose an efficient system.