Congenital adrenal hyperplasia, cyp21a2 deficiency : clinical and physiological aspects of pregnancy, screening and growth

The subjects dealt with in this thesis are clinical aspects of congenital adrenal hyperplasia (CAH), such as neonatal screening, growth and the incidence of CAH during the last centu ry in Sweden. In addition, we have used CAH as a model system to study possible prenatal effects of androgen exposure on growth and gestational length. Gestational age at birth correlated with CYP21A2 genotype in girls (P < 0.01), but not in boys with CAH (n = 109; 62 females, 47 males) (Paper I). The exact number of gestational days was known in 66 patients (37 females, 29 males). The pregnancy was longer for females with the most severe form, null genotype, 285.7 days, than for I172N, 273.9 days (P < 0.01 ) or V281L, 274.7 days (P < 0.05), indicating that higher androgen levels in severe forms could explain this effect. No differences between genotypes were seen in CAH males, possibly because testicular androgen production is high in normal male foetuses an d adrenal androgens therefore may not have an additional effect. The cortisol deficiency is equal in CAH girls and boys, making this deficiency a less likely explanation. Birth weight standard deviation score (SDS) corrected for gestational age in children with CAH (n = 73; 43 females, 30 males) did not differ from that of the reference population (mean, CI 95%: 0.0, - 0.3 to 0.3, and 0.2, − 0.2 to 0.6, for boys and girls, respectively) (Paper II). Nor did the birth weight differ between CYP21A2 genotype grou ps (P > 0.05). In 29 46,XY females with complete androgen insensitivity syndrome (CAIS), the mean birth weight SDS was similar to that of reference boys (mean, CI 95%: 0.1, - 0.2 to 0.4) and higher than the reference of females (mean, CI 95%: 0.4, 0.1 to 0. 7, P = 0.02). Hence, these results indicate that gestational age at birth, but not prenatal growth, is affected by androgen exposure. In a retrospective, population - based cohort study we investigated the apparent incidence of CAH in Sweden between 1910 and 2011 (Paper III). We identified 606 patients with known CYP21A2 genotype in 490 cases (81%). The female:male ratio was 1.25:1 for the whole cohort, but close to 1 in patients detected in the screening. The number of diagnosed patients increased dramatical ly in the 1960s and 1970s. The proportion of salt - wasting (SW) CAH compared to milder forms increased in both sexes after the introduction of neonatal screening from 114/242 to 165/292 (P < 0.05). The milder forms were diagnosed more often in females. This means that both boys and girls with SW CAH were missed before screening and that screening for CAH does not only increase the number of detected boys with SW CAH as previously thought, but also of girls. The neonatal screening for CAH in Sweden was studie d from the start in 1986 to 2011 (Paper IV). A total of 2 737 932 neonates (99.8% of all live births) had been screened. No cases with evident SW CAH had been missed, sensitivity 100%. The sensitivity was lower in the simple virilising form, 79%, and non - c lassical CAH, 32%. The positive predictive value was higher in full - term infants, 25.1%, than in pre - terms, 1.4% (P < 0.001). The recall rate was lower in full - terms, 0.03%, than in pre - term infants, 0.57% (P < 0.001). An analysis of all publications descr ibing neonatal screening programmes since 1996 revealed that the screening sensitivity correlated negatively with the duration of follow - up (P = 0.034). In contrast to current reports, our study shows that neonatal screening is effective in identifying SW CAH. Growth in CAH was studied in a prospective, observational cohort study including all children born or diagnosed with CAH between 1989 and 1994, 80 patients (46 females, 34 males). Most children were treated with a glucocorticoid dose within the recomm ended 10 – 15 mg/m2 body surface area. Corrected final height correlated with CYP21A2 genotype (P = 0.012). An important finding was that the corrected final height SDS was lower in patients who had been treated with the addition of prednisolone, - 1.1 ± 1.0, than in those who had been treated with cortisone acetate and/or h ydrocortisone alone, - 0.60 ± 1.0 (P < 0.05). Furthermore, body mass index at 18 years of age was higher in patients treated with prednisolone, 25.3 ± 4.7 kg/m2, compared to 23.4 ± 4.5 kg/m2 (P < 0.05). Hence, the results suggest that treatment with prednis olone should be avoided in growing subjects with CAH

Inhibition of MicroRNA-125a Promotes Human Endothelial Cell Proliferation and Viability through an Antiapoptotic Mechanism.

The microRNA-125a (miR-125a) is highly expressed in endothelial cells, but its role in vascular biology is not known. Endothelial cell proliferation and viability play an important role in endothelial healing, and we hypothesize that miR-125a regulates this process. The aim of the present study was to investigate if miR-125a controls human endothelial cell proliferation, viability and endothelial healing, and to assess the mechanisms involved. We showed that overexpression of miR-125a by transfection with miR-125a mimic reduced human umbilical vein endothelial cell (HUVEC) proliferation and viability, and stimulated apoptosis as demonstrated by a miR-125a-induced increase of the proportion of annexin V-positive cells monitored by flow cytometry. Moreover, we showed that the miR-125a mimic downregulated the antiapoptotic Bcl2 protein and upregulated caspase 3, suggesting that these two proteins represent molecular targets for miR-125a. Accordingly, transfection with miR-125a inhibitor, downregulating miR-125a expression, promoted HUVEC proliferation and viability, and reduced apoptosis. Importantly, transfection with miR-125a inhibitor promoted HUVEC tube formation in Matrigel, suggesting that reduction of miR-125a has a proangiogenic effect. In conclusion, downregulation of miR-125a through local transfection with miR-125a inhibitor might be a new way to enhance endothelial cell proliferation and viability, thereby promoting the reendothelialization observed in response to intimal injury. © 2014 S. Karger AG, Basel

Social Insecurity &amp; Games : Games for self-reflection

This is a study of how games can be used to encourage self-reflection. The study uses Everyday-Social Anxiety to establish a base point from which to make a game. During the study a game was developed using the research found. The game uses mechanics in order to link the player to the games protagonist. This link is then used in an attempt to encourage the player to self-reflect. Known design methods are used as guidelines of how the study and game is made. These methods originate from both game design and design of informative systems (such as servers). Some psychology sources are used in order to gather an understanding of what Social Anxiety is and how it affects people, the focus however, lies on the subject of Media technology and game development. The study concludes with results and a discussion. In the discussion the entirety of the study is motivate and reflected over by the authors. The results are specifically presented in a conclusion, presenting what was done, and what the study led to.LIVET.ex

Mesenchymal Stromal Cells Are More Immunosuppressive In Vitro If They Are Derived from Endometriotic Lesions than from Eutopic Endometrium

Endometriosis is an inflammatory disease with predominance of immunosuppressive M2 macrophages in the pelvic cavity that could be involved in the pathology through support and immune escape of ectopic lesions. Mesenchymal stromal cells (MSC) are found in ectopic lesions, and MSC from nonendometriosis sources are known to induce M2 macrophages. Therefore, MSC were hypothesized to play a role in the pathology of endometriosis. The aim was to characterize the functional phenotype of MSC in ectopic and eutopic endometrium from women with endometriosis. Stromal cells from endometriotic ovarian cysts (ESCcyst) and endometrium (ESCendo) were examined if they exhibited a MSC phenotype. Then, ESC were phenotypically examined for protein and gene expression of immunosuppressive and immunostimulatory molecules. Finally, ESC were functionally examined for their effects on monocyte differentiation into macrophages. ESCcyst and ESCendo expressed MSC markers, formed colonies, and differentiated into osteoblasts and adipocytes. Phenotypically, ESCcyst were more immunosuppressive, with significantly higher expression of immunosuppressive molecules. Functionally, ESCcyst induced more spindle-shaped macrophages, with significantly higher expression of CD14 and CD163, both features of M2 macrophages. The results suggest that ESCcyst may be more immunosuppressive than ESCendo and may promote immunosuppressive M2 macrophages that may support growth and reduce immunosurveillance of ectopic lesions

Mesenchymal Stromal Cells Support Endometriotic Stromal Cells In Vitro

Endometriosis is an inflammatory disease marked by ectopic growth of endometrial cells. Mesenchymal stromal cells (MSC) have immunosuppressive properties that have been suggested as a treatment for inflammatory diseases. Therefore, the aim herein was to examine effects of allogeneic MSC on endometriosis-derived cells in vitro as a potential therapy for endometriosis. MSC from allogeneic adipose tissue (Ad-MSC) and stromal cells from endometrium (ESCendo) and endometriotic ovarian cysts (ESCcyst) from women with endometriosis were isolated. The effects of Ad-MSC on ESCendo and ESCcyst were investigated using in vitro proliferation, apoptosis, adhesion, tube formation, migration, and invasion assays. Ad-MSC significantly increased proliferation of ESC compared to untreated controls. Moreover, Ad-MSC significantly decreased apoptosis and increased survival of ESC. Ad-MSC significantly increased adhesion of ESCendo and not ESCcyst on fibronectin. Conditioned medium from cocultures of Ad-MSC and ESC significantly increased tube formation of human umbilical vein endothelial cells on matrigel. Ad-MSC may significantly increase migration of ESCcyst and did not increase invasion of both cell types. The data suggest that allogeneic Ad-MSC should not be considered as a potential therapy for endometriosis, because they may support the pathology by maintaining and increasing growth of ectopic endometrial tissue

Maternal Adaptive Immune Cells in Decidua Parietalis Display a More Activated and Coinhibitory Phenotype Compared to Decidua Basalis

The maternal part of the placenta, the decidua, consists of maternal immune cells, decidual stromal cells, and extravillous fetal trophoblasts. In a successful pregnancy, these cell compartments interact to provide an intricate balance between fetal tolerance and antimicrobial defense. These processes are still poorly characterized in the two anatomically different decidual tissues, basalis and parietalis. We examined immune cells from decidua basalis and parietalis from term placentas (n=15) with flow cytometry. By using multivariate discriminant analysis, we found a clear separation between the two decidual compartments based on the 81 investigated parameters. Decidua parietalis lymphocytes displayed a more activated phenotype with a higher expression of coinhibitory markers than those isolated from basalis and contained higher frequencies of T regulatory cells. Decidua basalis contained higher proportions of monocytes, B cells, and mucosal-associated invariant T (MAIT) cells. The basalis B cells were more immature, and parietalis MAIT cells showed a more activated phenotype. Conventional T cells, NK cells, and MAIT cells from both compartments potently responded with the production of interferon-γ and/or cytotoxic molecules in response to stimulation. To conclude, leukocytes in decidua basalis and parietalis displayed remarkable phenotypic disparities, indicating that the corresponding stromal microenvironments provide different immunoregulatory signals

Comparison of relative levels in the presence or absence of FOXO1 inhibitor in decidualizing endometrial stromal cells.

<p>Comparison of relative levels in the presence or absence of FOXO1 inhibitor in decidualizing endometrial stromal cells.</p

Micrographs and forward and side scatter analysis of undifferentiated stromal and decidualized cells in the absence or presence of insulin.

<p>Representative micrographs of undifferentiated stromal and decidualized cells (MPA + db-cAMP) in the absence or presence of insulin were taken using an inverted microscope with 40x magnification. Forward vs side scatter (FCS/SSC) dot plot generated by flow cytometer showing the cell distribution of undifferentiated stromal and decidualized cells in the absence or presence of insulin.</p

A model on <i>FOXO1</i> regulation via insulin-induced phosphorylation-dependent degradation.

<p>A model on <i>FOXO1</i> regulation through insulin-induced phosphorylation-dependent degradation as suggested in some other cell types: <i>PI3K</i> is activated by insulin, the <i>FOXO</i> proteins are phosphorylated, resulting in their transcriptional inactivation through their nuclear export and cytoplasmic retention followed by a multistep negative regulation including ubiquitination and proteasomal degradation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171004#pone.0171004.ref026" target="_blank">26</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171004#pone.0171004.ref031" target="_blank">31</a>].</p