Expression and localization pattern of ABCA1 in diverse human placental primary cells and tissues

The ATP-binding cassette transporter A1 (ABCA1) mediates the transport of cholesterol, phospholipids, and other lipophilic molecules across cellular membranes. Recent data provide evidence that ABCA1 plays an important role in placental function but the exact cellular sites of ABCA1 action in the placenta remain controversial. To clarify this issue, we analyzed the cellular and subcellular localization of ABCA1 with immunocytochemistry, immunofluorescence and subsequent confocal or immunofluorescence microscopy in different types of isolated primary placenta cells: cytotrophoblast cells, amnion epithelial cells, villous macrophages (Hofbauer cells), and mesenchymal cells isolated from chorionic membrane and placental villi. After 12 h of cultivation, primary cytotrophoblast cells showed intensive membrane and cytoplasmic staining for ABCA1. After 24 h, with progressive syncytium formation, ABCA1 staining intensity was markedly reduced and ABCA1 was dispersed in the cytoplasm of the forming syncytial layer. In amnion epithelial cells, placental macrophages and mesenchymal cells, ABCA1 was predominantly localized at the cell membrane and cytoplasmic compartments partially corresponding to the endoplasmic reticulum. In these cell types, the ABCA1 staining intensity was not dependent on the cultivation time. In conclusion, ABCA1 shows marked expression levels in diverse placental cell types. The multitopic localization of ABCA1 in diverse human placental cells not all directly involved in materno-fetal exchange suggests that this protein may not only participate in transplacental lipid transport but could have additional regulatory functions.

Human neural stem cells enhance structural plasticity and axonal transport in the ischaemic brain

Stem cell transplantation promises new hope for the treatment of stroke although significant questions remain about how the grafted cells elicit their effects. One hypothesis is that transplanted stem cells enhance endogenous repair mechanisms activated after cerebral ischaemia. Recognizing that bilateral reorganization of surviving circuits is associated with recovery after stroke, we investigated the ability of transplanted human neural progenitor cells to enhance this structural plasticity. Our results show the first evidence that human neural progenitor cell treatment can significantly increase dendritic plasticity in both the ipsi- and contralesional cortex and this coincides with stem cell-induced functional recovery. Moreover, stem cell-grafted rats demonstrated increased corticocortical, corticostriatal, corticothalamic and corticospinal axonal rewiring from the contralesional side; with the transcallosal and corticospinal axonal sprouting correlating with functional recovery. Furthermore, we demonstrate that axonal transport, which is critical for both proper axonal function and axonal sprouting, is inhibited by stroke and that this is rescued by the stem cell treatment, thus identifying another novel potential mechanism of action of transplanted cells. Finally, we established in vitro co-culture assays in which these stem cells mimicked the effects observed in vivo. Through immunodepletion studies, we identified vascular endothelial growth factor, thrombospondins 1 and 2, and slit as mediators partially responsible for stem cell-induced effects on dendritic sprouting, axonal plasticity and axonal transport in vitro. Thus, we postulate that human neural progenitor cells aid recovery after stroke through secretion of factors that enhance brain repair and plasticity.

Frog oocytes to unveil the structure and supramolecular organization of human transport proteins

Structural analyses of heterologously expressed mammalian membrane proteins remain a great challenge given that microgram to milligram amounts of correctly folded and highly purified proteins are required. Here, we present a novel method for the expression and affinity purification of recombinant mammalian and in particular human transport proteins in Xenopus laevis frog oocytes. The method was validated for four human and one murine transporter. Negative stain transmission electron microscopy (TEM) and single particle analysis (SPA) of two of these transporters, i.e., the potassium-chloride cotransporter 4 (KCC4) and the aquaporin-1 (AQP1) water channel, revealed the expected quaternary structures within homogeneous preparations, and thus correct protein folding and assembly. This is the first time a cation-chloride cotransporter (SLC12) family member is isolated, and its shape, dimensions, low-resolution structure and oligomeric state determined by TEM, i.e., by a direct method. Finally, we were able to grow 2D crystals of human AQP1. The ability of AQP1 to crystallize was a strong indicator for the structural integrity of the purified recombinant protein. This approach will open the way for the structure determination of many human membrane transporters taking full advantage of the Xenopus laevis oocyte expression system that generally yields robust functional expression.

iTRAQ identification of candidate serum biomarkers associated with metastatic progression of human prostate cancer

A major challenge in the management of patients with prostate cancer is identifying those individuals at risk of developing metastatic disease, as in most cases the disease will remain indolent. We analyzed pooled serum samples from 4 groups of patients (n = 5 samples/group), collected prospectively and actively monitored for a minimum of 5 yrs. Patients groups were (i) histological diagnosis of benign prostatic hyperplasia with no evidence of cancer 'BPH', (ii) localised cancer with no evidence of progression, 'non-progressing' (iii) localised cancer with evidence of biochemical progression, 'progressing', and (iv) bone metastasis at presentation 'metastatic'. Pooled samples were immuno-depleted of the 14 most highly abundant proteins and analysed using a 4-plex iTRAQ approach. Overall 122 proteins were identified and relatively quantified. Comparisons of progressing versus non-progressing groups identified the significant differential expression of 25 proteins (p<0.001). Comparisons of metastatic versus progressing groups identified the significant differential expression of 23 proteins. Mapping the differentially expressed proteins onto the prostate cancer progression pathway revealed the dysregulated expression of individual proteins, pairs of proteins and 'panels' of proteins to be associated with particular stages of disease development and progression. The median immunostaining intensity of eukaryotic translation elongation factor 1 alpha 1 (eEF1A1), one of the candidates identified, was significantly higher in osteoblasts in close proximity to metastatic tumour cells compared with osteoblasts in control bone (p = 0.0353, Mann Whitney U). Our proteomic approach has identified leads for potentially useful serum biomarkers associated with the metastatic progression of prostate cancer. The panels identified, including eEF1A1 warrant further investigation and validation.

Gene expression by human monocytes from peripheral blood in response to exposure to metals

With increasing life expectancy and active lifestyles, the longevity of arthroplasties has become an important problem in orthopaedic surgery and will remain so until novel approaches to joint preservation have been developed. The sensitivity of the recipient to the metal alloys may be one of the factors limiting the lifespan of implants. In the present study, the response of human monocytes from peripheral blood to an exposure to metal ions was investigated, using the method of real-time polymerase chain reaction (PCR)-based low-density arrays. Upon stimulation with bivalent (Co2+ and Ni2+) and trivalent (Ti3+) cations and with the calcium antagonist LaCl3, the strength of the elicited monocytic response was in the order of Co2+ > or = Ni2+ > Ti3+ > or = LaCl3. The transcriptional regulation of the majority of genes affected by the exposure of monocytes to Co2+ and Ni2+ was similar. Some genes critically involved in the processes of inflammation and bone resorption, however, were found to be differentially regulated by these bivalent cations. The data demonstrate that monocytic gene expression is adapted in response to metal ions and that this response is, in part, specific for the individual metals. It is suggested that metal alloys used in arthroplasties may affect the extent of inflammation and bone resorption in the peri-implant tissues in dependence of their chemical composition.

Lipopolysaccharides from atherosclerosis-associated bacteria antagonize TLR4, induce formation of TLR2/1/CD36 complexes in lipid rafts and trigger TLR2-induced inflammatory responses in human vascular endothelial cells

Infection with bacteria such as Chlamydia pneumonia, Helicobacter pylori or Porphyromonas gingivalis may be triggering the secretion of inflammatory cytokines that leads to atherogenesis. The mechanisms by which the innate immune recognition of these pathogens could lead to atherosclerosis remain unclear. In this study, using human vascular endothelial cells or HEK-293 cells engineered to express pattern-recognition receptors (PRRs), we set out to determine Toll-like receptors (TLRs) and functionally associated PRRs involved in the innate recognition of and response to lipopolysaccharide (LPS) from H. pylori or P. gingivalis. Using siRNA interference or recombinant expression of cooperating PRRs, we show that H. pylori and P. gingivalis LPS-induced cell activation is mediated through TLR2. Human vascular endothelial cell activation was found to be lipid raft-dependent and to require the formation of heterotypic receptor complexes comprising of TLR2, TLR1, CD36 and CD11b/CD18. In addition, we report that LPS from these bacterial strains are able to antagonize TLR4. This antagonistic activity of H. pylori or P. gingivalis LPS, as well as their TLR2 activation capability may be associated with their ability to contribute to atherosclerosis.

Are full or empty beer bottles sturdier and does their fracture-threshold suffice to break the human skull?

Beer bottles are often used in physical disputes. If the bottles break, they may give rise to sharp trauma. However, if the bottles remain intact, they may cause blunt injuries. In order to investigate whether full or empty standard half-litre beer bottles are sturdier and if the necessary breaking energy surpasses the minimum fracture-threshold of the human skull, we tested the fracture properties of such beer bottles in a drop-tower. Full bottles broke at 30 J impact energy, empty bottles at 40 J. These breaking energies surpass the minimum fracture-threshold of the human neurocranium. Beer bottles may therefore fracture the human skull and therefore serve as dangerous instruments in a physical dispute.

Long-term follow-up of metabolic activity in human alveolar echinococcosis using FDG-PET

AIM: [(18)F]fluoro-deoxyglucose positron-emission-tomography (FDG-PET) detects metabolic activity in alveolar echinococcosis (AE). The slow changes in metabolic and morphological characteristics require long-term follow-up of patients. This is the first study to evaluate metabolic activity over may years, hereby assessing the utility of FDG-PET for the evaluation of disease progression and response to treatment. PATIENTS, METHODS: 15 patients received a follow-up FDG-PET combined with computed tomography (integrated PET/CT) with a median of 6.5 years after the first PET in 1999. Number and location of enhanced metabolic activity in the area of AE lesions was determined. Quantification of intensity of metabolic activity was assessed by calculation of mean standardized uptake values. RESULTS: AE lesions in 11/15 patients had been metabolically inactive initially, but only two showed permanent inactivity over the course of 81 months. Interestingly, in two patients metabolic activity was newly detected after 80 and 82 months. Benzimidazole treatment was intermittently discontinued in seven cases. Persisting activity at FDG-PET demanded continued benzimidazole treatment in four patients. Neither treatment duration, lesional size, calcifications nor regressive changes correlated with metabolic activity. CONCLUSION: Treatment responses are heterogeneous and vary from progressive disease despite treatment to long-term inactive disease with discontinued treatment. Lack of metabolic activity indicates suppressed parasite activity and is not equivalent to parasite death. However, metabolic activity may remain suppressed for years, allowing for temporary treatment discontinuation. Relapses are reliably detected with PET and restarting benzimidazole treatment prevents parasite expansion.

Mapping of direction and muscle representation in the human primary motor cortex controlling thumb movements

Larger body parts are somatotopically represented in the primary motor cortex (M1), while smaller body parts, such as the fingers, have partially overlapping representations. The principles that govern the overlapping organization of M1 remain unclear. We used transcranial magnetic stimulation (TMS) to examine the cortical encoding of thumb movements in M1 of healthy humans. We performed M1 mapping of the probability of inducing a thumb movement in a particular direction and used low intensity TMS to disturb a voluntary thumb movement in the same direction during a reaction time task. With both techniques we found spatially segregated representations of the direction of TMS-induced thumb movements, thumb flexion and extension being best separated. Furthermore, the cortical regions corresponding to activation of a thumb muscle differ, depending on whether the muscle functions as agonist or as antagonist for flexion or extension. In addition, we found in the reaction time experiment that the direction of a movement is processed in M1 before the muscles participating in it are activated. It thus appears that one of the organizing principles for the human corticospinal motor system is based on a spatially segregated representation of movement directions and that the representation of individual somatic structures, such as the hand muscles, overlap.

Differential regulation of human 3β-hydroxysteroid dehydrogenase type 2 for steroid hormone biosynthesis by starvation and cyclic AMP stimulation: studies in the human adrenal NCI-H295R cell model

Human steroid biosynthesis depends on a specifically regulated cascade of enzymes including 3β-hydroxysteroid dehydrogenases (HSD3Bs). Type 2 HSD3B catalyzes the conversion of pregnenolone, 17α-hydroxypregnenolone and dehydroepiandrosterone to progesterone, 17α-hydroxyprogesterone and androstenedione in the human adrenal cortex and the gonads but the exact regulation of this enzyme is unknown. Therefore, specific downregulation of HSD3B2 at adrenarche around age 6-8 years and characteristic upregulation of HSD3B2 in the ovaries of women suffering from the polycystic ovary syndrome remain unexplained prompting us to study the regulation of HSD3B2 in adrenal NCI-H295R cells. Our studies confirm that the HSD3B2 promoter is regulated by transcription factors GATA, Nur77 and SF1/LRH1 in concert and that the NBRE/Nur77 site is crucial for hormonal stimulation with cAMP. In fact, these three transcription factors together were able to transactivate the HSD3B2 promoter in placental JEG3 cells which normally do not express HSD3B2. By contrast, epigenetic mechanisms such as methylation and acetylation seem not involved in controlling HSD3B2 expression. Cyclic AMP was found to exert differential effects on HSD3B2 when comparing short (acute) versus long-term (chronic) stimulation. Short cAMP stimulation inhibited HSD3B2 activity directly possibly due to regulation at co-factor or substrate level or posttranslational modification of the protein. Long cAMP stimulation attenuated HSD3B2 inhibition and increased HSD3B2 expression through transcriptional regulation. Although PKA and MAPK pathways are obvious candidates for possibly transmitting the cAMP signal to HSD3B2, our studies using PKA and MEK1/2 inhibitors revealed no such downstream signaling of cAMP. However, both signaling pathways were clearly regulating HSD3B2 expression.

Models of hybridization during range expansions and their application to recent human evolution

Several lines of genetic, archeological and paleontological evidence suggest that anatomically modern humans (Homo sapiens) colonized the world in the last 60,000 years by a series of migrations originating from Africa (e.g. Liu et al., 2006; Handley et al., 2007; Prugnolle, Manica, and Balloux, 2005; Ramachandran et al. 2005; Li et al. 2008; Deshpande et al. 2009; Mellars, 2006a, b; Lahr and Foley, 1998; Gravel et al., 2011; Rasmussen et al., 2011). With the progress of ancient DNA analysis, it has been shown that archaic humans hybridized with modern humans outside Africa. Recent direct analyses of fossil nuclear DNA have revealed that 1–4 percent of the genome of Eurasian has been likely introgressed by Neanderthal genes (Green et al., 2010; Reich et al., 2010; Vernot and Akey, 2014; Sankararaman et al., 2014; Prufer et al., 2014; Wall et al., 2013), with Papua New Guineans and Australians showing even larger levels of admixture with Denisovans (Reich et al., 2010; Skoglund and Jakobsson, 2011; Reich et al., 2011; Rasmussen et al., 2011). It thus appears that the past history of our species has been more complex than previously anticipated (Alves et al., 2012), and that modern humans hybridized several times with local hominins during their expansion out of Africa, but the exact mode, time and location of these hybridizations remain to be clarifi ed (Ibid.; Wall et al., 2013). In this context, we review here a general model of admixture during range expansion, which lead to some predictions about expected patterns of introgression that are relevant to modern human evolution.

Xenotransplantation of cryopreserved human ovarian tissue--a systematic review of MII oocyte maturation and discussion of it as a realistic option for restoring fertility after cancer treatment.

OBJECTIVE To systematically review the reporting of MII (MII) oocyte development after xenotransplantation of human ovarian tissue. DESIGN Systematic review in accordance with the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA). SETTING Not applicable. PATIENT(S) Not applicable. INTERVENTION(S) Formation of MII oocytes after xenotransplantation of human ovarian tissue. MAIN OUTCOME MEASURE(S) Any outcome reported in Pubmed. RESULT(S) Six publications were identified that report on formation of MII oocytes after xenotransplantation of human ovarian tissue. CONCLUSION(S) Xenografting of human ovarian tissue has proved to be a useful model for examining ovarian function and follicle development in vivo. With human follicles that have matured through xenografting, the possibility of cancer transmission and relapse can also be eliminated, because cancer cells are not able to penetrate the zona pellucida. The reported studies have demonstrated that xenografted ovarian tissue from a range of species, including humans, can produce antral follicles that contain mature (MII) oocytes, and it has been shown that mice oocytes have the potential to give rise to live young. Although some ethical questions remain unresolved, xenotransplantation may be a promising method for restoring fertility. This review furthermore describes the value of xenotransplantation as a tool in reproductive biology and discusses the ethical and potential safety issues regarding ovarian tissue xenotransplantation as a means of recovering fertility.

Regulation of human (adrenal) androgen biosynthesis-New insights from novel throughput technology studies

Androgens are precursors for sex steroids and are predominantly produced in the human gonads and the adrenal cortex. They are important for intrauterine and postnatal sexual development and human reproduction. Although human androgen biosynthesis has been extensively studied in the past, exact mechanisms underlying the regulation of androgen production in health and disease remain vague. Here, the knowledge on human androgen biosynthesis and regulation is reviewed with a special focus on human adrenal androgen production and the hyperandrogenic disorder of polycystic ovary syndrome (PCOS). Since human androgen regulation is highly specific without a good animal model, most studies are performed on patients harboring inborn errors of androgen biosynthesis, on human biomaterials and human (tumor) cell models. In the past, most studies used a candidate gene approach while newer studies use high throughput technologies to identify novel regulators of androgen biosynthesis. Using genome wide association studies on cohorts of patients, novel PCOS candidate genes have been recently described. Variant 2 of the DENND1A gene was found overexpressed in PCOS theca cells and confirmed to enhance androgen production. Transcriptome profiling of dissected adrenal zones established a role for BMP4 in androgen synthesis. Similarly, transcriptome analysis of human adrenal NCI-H295 cells identified novel regulators of androgen production. Kinase p38α (MAPK14) was found to phosphorylate CYP17 for enhanced 17,20 lyase activity and RARB and ANGPTL1 were detected in novel networks regulating androgens. The discovery of novel players for androgen biosynthesis is of clinical significance as it provides targets for diagnostic and therapeutic use.