Ward De Spiegelaere
Ghent University, Internal Medicine, Faculty Member
An appropriate normalization strategy is crucial for data analysis from real time reverse transcription polymerase chain reactions (RT-qPCR). It is widely supported to identify and validate stable reference genes, since no single... more
An appropriate normalization strategy is crucial for data analysis from real time reverse transcription polymerase chain reactions (RT-qPCR). It is widely supported to identify and validate stable reference genes, since no single biological gene is stably expressed between cell types or within cells under different conditions. Different algorithms exist to validate optimal reference genes for normalization. Applying human cells, we here compare the three main methods to the online available RefFinder tool that integrates these algorithms along with R-based software packages which include the NormFinder and GeNorm algorithms. 14 candidate reference genes were assessed by RT-qPCR in two sample sets, i.e. a set of samples of human testicular tissue containing carcinoma in situ (CIS), and a set of samples from the human adult Sertoli cell line (FS1) either cultured alone or in co-culture with the seminoma like cell line (TCam-2) or with equine bone marrow derived mesenchymal stem cells ...
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BACKGROUND: The HepaRG cell line is widely used as an alternative for primary human hepatocytes for numerous applications, including drug screening, and is progressively gaining importance as a human-relevant cell source. Consequently,... more
BACKGROUND: The HepaRG cell line is widely used as an alternative for primary human hepatocytes for numerous applications, including drug screening, and is progressively gaining importance as a human-relevant cell source. Consequently, increasing numbers of experiments are being performed with this cell line, including real-time quantitative PCR (RT-qPCR) experiments for gene expression studies. CONTENT: When RT-qPCR experiments are performed, results are reliable only when attention is paid to several critical aspects, including a proper normalization strategy. Therefore, in 2011 we determined the most optimal reference genes for gene expression studies in the HepaRG cell system, according to the MIQUE (Minimum Information for Publication of Quantitative
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Abacavir is a nucleoside reverse transcriptase inhibitor used as part of combination antire-troviral therapy in HIV-1-infected patients. Because this drug can cause a hypersensitivity reaction that is correlated with the presence of the... more
Abacavir is a nucleoside reverse transcriptase inhibitor used as part of combination antire-troviral therapy in HIV-1-infected patients. Because this drug can cause a hypersensitivity reaction that is correlated with the presence of the HLA-B*57:01allotype, screening for the presence of HLA-B*57:01 is recommended before abacavir initiation. Different genetic as-says have been developed for HLA-B*57:01 screening, each with specific sensitivity, turn-around time and assay costs. Here, a new real-time PCR (qPCR) based analysis is described and compared to sequence specific primer PCR with capillary electrophoresis (SSP PCR CE) on 149 patient-derived samples, using sequence specific oligonucleotide hybridization combined with high resolution SSP PCR as gold standard. In addition to these PCR based methods, a complementary approach was developed using flow cytometry with an HLA-B17 specific monoclonal antibody as a pre-screening assay to diminish the number of samples for genetic testing...
Sabine Kinloch-de Loes, Lucy Dorrell, Hongbing Yang, Gareth A. D. Hardy, Sabine Yerly, Cristina Cellerai, Linos Vandekerckhove, Ward De Spiegelaere, Eva Malatinkova, Willie Wee Lee Koh, and Margaret A. Johnson Division of Infection and... more
Sabine Kinloch-de Loes, Lucy Dorrell, Hongbing Yang, Gareth A. D. Hardy, Sabine Yerly, Cristina Cellerai, Linos Vandekerckhove, Ward De Spiegelaere, Eva Malatinkova, Willie Wee Lee Koh, and Margaret A. Johnson Division of Infection and Immunity, Royal Free Campus, University College London, Royal Free Hospital, London, Department of Immunology, University College London Medical School, Royal Free Campus, Nuffield Department of Medicine, Oxford National Institute of Health Research Biomedical Research Centre, University of Oxford, Centre for Immunology and Virology, Imperial College London, and Chelsea and Westminster Hospital, London, United Kingdom; Laboratory of Virology, Geneva University Hospital, and Division of Immunology and Allergy, Department of Medicine, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland; HIV Translational Research Unit, Ghent University, University Hospital Ghent, Belgium; and Department of Immunology and Molecular Pathology, University Colle...
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Studying the effects of HIV infection on the host transcriptome has typically focused on protein-coding genes. However, recent advances in the field of RNA sequencing revealed that long non-coding RNAs (lncRNAs) add an extensive... more
Studying the effects of HIV infection on the host transcriptome has typically focused on protein-coding genes. However, recent advances in the field of RNA sequencing revealed that long non-coding RNAs (lncRNAs) add an extensive additional layer to the cell’s molecular network and are crucial for normal cellular function. lncRNAs exert the ability to control a wide range of (post-)transcriptional processes and offer unique possibilities for pathogens like HIV to hijack the cellular machinery and reshape gene expression in their favor. Therefore, lncRNA discovery can result in new insights into the HIV-host interplay. We performed transcriptome profiling throughout a characterized primary HIV infection in vitro to investigate lncRNA expression at the different HIV replication cycle processes (reverse transcription, integration and particle production). Subsequently, guilt-by-association, transcription factor and co-expression analysis were performed to infer biological roles for the ...
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Background: There is an increasing interest in characterisation of the viral reservoir in patients on long-term antiretroviral therapy in the context of HIV cure studies. The main question remains which assay is most relevant to... more
Background: There is an increasing interest in characterisation of the viral reservoir in patients on long-term antiretroviral therapy in the context of HIV cure studies. The main question remains which assay is most relevant to accurately predict the size of the replication competent viral reservoir. Although both PCR and viral outgrowth assays have been proposed, mainly PCR based assays have been validated in clinical trials. Conflicting data exists about the correlation of viral outgrowth assays and PCR based assays. In addition, within individual patients, the long term variability of PCR reservoir markers of total HIV DNA, 2LTR circles and full length cell-associated (CA) RNA is poorly addressed. Materials and Methods: We set-up a study with a well-defined patient cohort (N=25) to characterize the longitudinal kinetics of the viral reservoir by PCR based methods and to assess the correlation of the viral reservoir markers with the viral outgrowth assay. Blood samples were drawn...
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Droplet digital PCR (ddPCR) technology can be used to quantify nucleic acid concentrations by measuring the fluorescence intensity for each single droplet and subsequently calling the droplet as positive or negative depending on its... more
Droplet digital PCR (ddPCR) technology can be used to quantify nucleic acid concentrations by measuring the fluorescence intensity for each single droplet and subsequently calling the droplet as positive or negative depending on its fluorescence intensity. Thus, setting a correct fluorescence level threshold is needed to accurately identify the presence (absence) of target nucleic acid. Currently available methods assume a normal distribution of the fluorescence readout. We show that this assumption does not hold in a large majority of assessed experiments and that wrongfully relying on this assumption typically results in an increase of false positives. By relying on extreme value theory (EVT) we do not make any assumptions on the droplet fluorescence intensity distribution. Using this EVT framework we develop a methodology for modeling the maximum fluorescence intensity of the negative droplets using negative control samples (no target nucleic acid present). We show how we subsequ...
Training-induced follow-up of multiple muscle plasticity parameters in postural stability vs. locomotion muscles provides an integrative physiological view on shifts in the muscular metabolic machinery. It can be expected that not all... more
Training-induced follow-up of multiple muscle plasticity parameters in postural stability vs. locomotion muscles provides an integrative physiological view on shifts in the muscular metabolic machinery. It can be expected that not all muscle plasticity parameters show the same expression time profile across muscles. This knowledge is important to underpin results of metabolomic studies. Twelve non-competing Standardbred mares were subjected to standardized harness training. Muscle biopsies were taken on a non-training day before and after 8 weeks. Shifts in muscle fiber type composition and muscle fiber cross-sectional area (CSA) were compared in the m. pectoralis, the m. vastus lateralis, and the m. semitendinosus. In the m. vastus lateralis, which showed most pronounced training-induced plasticity, two additional muscle plasticity parameters (capillarization and mitochondrial density) were assessed. In the m. semitendinosus, additionally the mean minimum Feret's diameter was a...
Equine bioenergetics have predominantly been studied focusing on glycogen and fatty acids. Combining omics with conventional techniques allows for an integrative approach to broadly explore and identify important biomolecules. Friesian... more
Equine bioenergetics have predominantly been studied focusing on glycogen and fatty acids. Combining omics with conventional techniques allows for an integrative approach to broadly explore and identify important biomolecules. Friesian horses were aquatrained (n = 5) or dry treadmill trained (n = 7) (8 weeks) and monitored for: evolution of muscle diameter in response to aquatraining and dry treadmill training, fiber type composition and fiber cross-sectional area of the M. pectoralis, M. vastus lateralis and M. semitendinosus and untargeted metabolomics of the M. pectoralis and M. vastus lateralis in response to dry treadmill training. Aquatraining was superior to dry treadmill training to increase muscle diameter in the hindquarters, with maximum effect after 4 weeks. After dry treadmill training, the M. pectoralis showed increased muscle diameter, more type I fibers, decreased fiber mean cross sectional area, and an upregulated oxidative metabolic profile: increased β-oxidation (...
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Background The HIV-1 proviral genome harbors multiple CpG islands (CpGIs), both in the promoter and intragenic regions. DNA methylation in the promoter region has been shown to be heavily involved in HIV-1 latency regulation in cultured... more
Background The HIV-1 proviral genome harbors multiple CpG islands (CpGIs), both in the promoter and intragenic regions. DNA methylation in the promoter region has been shown to be heavily involved in HIV-1 latency regulation in cultured cells. However, its exact role in proviral transcriptional regulation in infected individuals is poorly understood or characterized. Moreover, methylation at intragenic CpGIs has never been studied in depth. Results A large, well-characterized HIV-1 patient cohort (n = 72), consisting of 17 long-term non-progressors and 8 recent seroconverters (SRCV) without combination antiretroviral therapy (cART), 15 early cART-treated, and 32 late cART-treated patients, was analyzed using a next-generation bisulfite sequencing DNA methylation method. In general, we observed low level of promoter methylation and higher levels of intragenic methylation. Additionally, SRCV showed increased promoter methylation and decreased intragenic methylation compared with the o...
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Macrophages are primarily known as phagocytic immune cells, but they also play a role in diverse processes, such as morphogenesis, homeostasis and regeneration. In this review, we discuss the influence of macrophages on angiogenesis, the... more
Macrophages are primarily known as phagocytic immune cells, but they also play a role in diverse processes, such as morphogenesis, homeostasis and regeneration. In this review, we discuss the influence of macrophages on angiogenesis, the process of new blood vessel formation from the pre‐existing vasculature. Macrophages play crucial roles at each step of the angiogenic cascade, starting from new blood vessel sprouting to the remodelling of the vascular plexus and vessel maturation. Macrophages form promising targets for both pro‐ and anti‐angiogenic treatments. However, to target macrophages, we will first need to understand the mechanisms that control the functional plasticity of macrophages during each of the steps of the angiogenic cascade. Here, we review recent insights in this topic. Special attention will be given to the TIE2‐expressing macrophage (TEM), which is a subtype of highly angiogenic macrophages that is able to influence angiogenesis via the angiopoietin–TIE pathway.
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RTL1 (retrotransposon Gag-like 1) is an essential gene in the development of the human and murine placenta. Several fetal and placental abnormalities such as intrauterine growth restriction (IUGR) and hydrops conditions have been... more
RTL1 (retrotransposon Gag-like 1) is an essential gene in the development of the human and murine placenta. Several fetal and placental abnormalities such as intrauterine growth restriction (IUGR) and hydrops conditions have been associated with altered expression of this gene. However, the function of RTL1 has not been identified. RTL1 is located on a highly conserved region in eutherian mammals. Therefore, the genetic and molecular analysis in horses could hold important implications for other species, including humans. Here, we demonstrated that RTL1 is paternally expressed and is localized within the endothelial cells of the equine (Equus caballus) chorioallantois. We developed an equine placental microvasculature primary cell culture and demonstrated that RTL1 knockdown leads to loss of the sprouting ability of these endothelial cells. We further demonstrated an association between abnormal expression of RTL1 and development of hydrallantois. Our data suggest that RTL1 may be e...
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Exuberant granulation tissue (EGT) is a frequently encountered complication during second intention healing in equine distal limb wounds. Although it is still unknown what exactly triggers the formation of this tissue, previous research... more
Exuberant granulation tissue (EGT) is a frequently encountered complication during second intention healing in equine distal limb wounds. Although it is still unknown what exactly triggers the formation of this tissue, previous research has revealed a persistent inflammatory response in these wounds. In this preliminary study we examined this inflammatory response in EGT-developing wounds as well as in experimental induced wounds. Immunohistological stainings were performed to detect primary inflammatory immune cells (MAC387 staining) as well as pro-resolution immune cells (CD163 staining). Our results show a significantly higher amount of MAC387+ and CD163+ cells in the fibrotic regions of EGT compared with the 19-day-old experimental wounds. This persistent high amount of fibrosis-promoting CD163+ cells in EGT suggests that the wound healing processes in EGT-developing wounds are arrested at the level of the proliferation phase.
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DNA methylation is one of the most important epigenetic modifications in the regulation of gene transcription. The current gold standard to study this modification is bisulfite sequencing. Although multiple commercial bisulfite treatment... more
DNA methylation is one of the most important epigenetic modifications in the regulation of gene transcription. The current gold standard to study this modification is bisulfite sequencing. Although multiple commercial bisulfite treatment kits provide good conversion efficiencies, DNA loss and especially DNA fragmentation remain troublesome. This hampers DNA methylation profiling of long DNA sequences. Here, we explored the performance of twelve commercial bisulfite kits by an in-depth comparison of DNA fragmentation using gel electrophoresis, qPCR and digital PCR, DNA recovery by spectroscopic measurements and digital PCR and conversion efficiency by next generation sequencing. The results show a clear performance difference between the bisulfite kits, and depending on the specific goal of the study, the most appropriate kit might differ. Moreover, we demonstrated that digital PCR is a valuable method to monitor both DNA fragmentation as well as DNA recovery after bisulfite treatment.
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The HIV Cure Research Center (HCRC) in Ghent organised the first HIV Reservoir Characterization Symposium, and brought together virologists, molecular biologists, immunologists and clinicians to discuss the most recent developments in HIV... more
The HIV Cure Research Center (HCRC) in Ghent organised the first HIV Reservoir Characterization Symposium, and brought together virologists, molecular biologists, immunologists and clinicians to discuss the most recent developments in HIV reservoir characterisation with a view to achieving an HIV cure. The one-day symposium covered new developments in the field of HIV reservoir and HIV cure research, with the latest news on the European HIV cure trials. This report summarises the major themes discussed during the symposium.
Research Interests: Genetics, Gene expression, Quality Control, Internal Control, Cell line, and 15 moreIn Vitro, Humans, Legislation, Female, Hepatocellular Carcinoma, Normalization, Selection, Ribosomal proteins, Biochemical, Gene, Gen, BIOCHEMICAL PHARMACOLOGY, Reference Values, Gene expression profiling, and Safety Evaluation
Quantitative real-time PCR (qPCR) is implemented in many molecular laboratories worldwide for the quantification of viral nucleic acids. However, over the last two decades, there has been renewed interest in the concept of digital PCR... more
Quantitative real-time PCR (qPCR) is implemented in many molecular laboratories worldwide for the quantification of viral nucleic acids. However, over the last two decades, there has been renewed interest in the concept of digital PCR (dPCR) as this platform offers direct quantification without the need for standard curves, a simplified workflow and the possibility to extend the current detection limit. These benefits are of great interest in terms of the quantification of low viral levels in HIV reservoir research because changes in the dynamics of residual HIV reservoirs will be important to monitor HIV cure efforts. Here, we have implemented a systematic literature screening and text mining approach to map the use of droplet dPCR (ddPCR) in the context of HIV quantification. In addition, several technical aspects of ddPCR were compared with qPCR: accuracy, sensitivity, precision and reproducibility, to determine its diagnostic utility. We have observed that ddPCR was used in diff...
The mechanisms for the establishment and the persistence of the latent HIV-1 reservoir remain to be completely defined. HIV-1 infection is characterised by the integration of the reverse transcribed proviral DNA into the host's... more
The mechanisms for the establishment and the persistence of the latent HIV-1 reservoir remain to be completely defined. HIV-1 infection is characterised by the integration of the reverse transcribed proviral DNA into the host's genome. This integrated proviral DNA can remain replication silent, but a small part of it is fully competent to restart viral replication when treatment is interrupted. Hence, this replication-competent provirus is the cause of viral rebound and is called the viral reservoir. The exact site of proviral integration within the host's cellular chromosome may affect the transcriptional activity of HIV. Thanks to recent technological advances, HIV-1 integration site analysis has been used to assess HIV-1 reservoirs in HIV-infected individuals. Analysis of HIV-1 integration sites in infected individuals undergoing suppressive ART led to identification of expanded clonal cell populations, indicating that clonal proliferation of the proviral reservoir may co...
Driven by its potential benefits over currently available methods, and the recent development of commercial platforms, digital polymerase chain reaction (dPCR) has received increasing attention in virology research and diagnostics as a... more
Driven by its potential benefits over currently available methods, and the recent development of commercial platforms, digital polymerase chain reaction (dPCR) has received increasing attention in virology research and diagnostics as a tool for the quantification of nucleic acids. The current technologies are more precise and accurate, but may not be much more sensitive, compared with quantitative PCR (qPCR) applications. The most promising applications with the current technology are the analysis of mutated sequences, such as emerging drug-resistant mutations. Guided by the recent literature, this review focuses on three aspects that demonstrate the potential of dPCR for virology researchers and clinicians: the applications of dPCR within both virology research and clinical virology, the benefits of the technique over the currently used real-time qPCR, and the importance and availability of specific data analysis approaches for dPCR. Comments are provided on current drawbacks and often overlooked pitfalls that need further attention to allow widespread implementation of dPCR as an accurate and precise tool within the field of virology.
The persistence of a reservoir of latently infected CD4 T cells remains one of the major obstacles to cure HIV. Numerous strategies are being explored to eliminate this reservoir. To translate these efforts into clinical trials, there is... more
The persistence of a reservoir of latently infected CD4 T cells remains one of the major obstacles to cure HIV. Numerous strategies are being explored to eliminate this reservoir. To translate these efforts into clinical trials, there is a strong need for validated biomarkers that can monitor the reservoir over time in vivo. A comprehensive study was designed to evaluate and compare potential HIV-1 reservoir biomarkers. A cohort of 25 patients, treated with suppressive antiretroviral therapy was sampled at three time points, with median of 2.5 years (IQR: 2.4-2.6) between time point 1 and 2; and median of 31 days (IQR: 28-36) between time point 2 and 3. Patients were median of 6 years (IQR: 3-12) on ART, and plasma viral load (<50 copies/ml) was suppressed for median of 4 years (IQR: 2-8). Total HIV-1 DNA, unspliced (us) and multiply spliced HIV-1 RNA, and 2LTR circles were quantified by digital PCR in peripheral blood, at 3 time points. At the second time point, a viral outgrowt...
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[This corrects the article DOI: 10.1371/journal.ppat.1005472.].
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Persistent reservoirs remain the major obstacles to achieve an HIV-1 cure. Prolonged early antiretroviral therapy (ART) may reduce the extent of reservoirs and allow for virological control after ART discontinuation. We compared HIV-1... more
Persistent reservoirs remain the major obstacles to achieve an HIV-1 cure. Prolonged early antiretroviral therapy (ART) may reduce the extent of reservoirs and allow for virological control after ART discontinuation. We compared HIV-1 reservoirs in a cross-sectional study using PCR-based techniques in blood and tissue of early treated seroconverters, late treated patients, ART-naïve seroconverters and long-term non-progressors (LTNPs) who have spontaneous virological control without treatment. A decade of early ART reduced total and integrated HIV-1 DNA levels compared to later treatment initiation, but not reaching the low levels of LTNPs. Total HIV-1 DNA in rectal biopsies did not differ between cohorts. Importantly, lower viral transcription (unspliced RNA) and enhanced immune preservation (CD4/CD8) reminiscent of LTNPs were found in early compared to late treated patients. This suggests that early treatment is associated with some immuno-virological features of LTNPs that may im...
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Whether ART regimens differ in their propensity to allow persistent HIV-1 detection remains unclear. To investigate this, we performed a cross-sectional study to characterize HIV-1 persistence in peripheral blood during suppressive... more
Whether ART regimens differ in their propensity to allow persistent HIV-1 detection remains unclear. To investigate this, we performed a cross-sectional study to characterize HIV-1 persistence in peripheral blood during suppressive therapy with NRTIs plus a PI or nevirapine. Residual plasma HIV-1 RNA was quantified by real-time PCR. Cell-associated proviral total HIV-1 DNA, unspliced and multiply spliced HIV-1 RNA and 2-long terminal repeat (2-LTR) circles were quantified by digital PCR. Comparing PI with nevirapine recipients, residual plasma HIV-1 RNA detection rates were 47/80 (58.8%) versus 37/81 (45.7%), with median (IQR) levels of 4 (3-8) versus 4 (3-7) copies/mL (P = 0.207); detection was less likely with longer duration of suppressive ART (P = 0.020), independently of treatment. HIV-1 DNA was detected in all patients, with median levels of 2.3 (IQR 2.0-2.7) versus 2.5 (IQR 2.1-2.7) log10 copies/10(6) PBMCs, respectively; HIV-1 DNA levels were associated with pre-ART viral load (P = 0.004) and with residual HIV-1 RNA (P = 0.034), unspliced HIV-1 RNA (P = 0.001) and 2-LTR circles (P = 0.005), independently of treatment. No significant differences were revealed in levels of residual plasma HIV-1 RNA, total HIV-1 DNA or intracellular markers of ongoing virus replication (unspliced and multiply spliced HIV-1 RNA and 2-LTR circles) between treatment groups.
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Digital PCR is rapidly gaining interest in the field of molecular biology for absolute quantification of nucleic acids. However, the first generation of platforms still needs careful validation and requires a specific methodology for data... more
Digital PCR is rapidly gaining interest in the field of molecular biology for absolute quantification of nucleic acids. However, the first generation of platforms still needs careful validation and requires a specific methodology for data analysis to distinguish negative from positive signals by defining a threshold value. The currently described methods to assess droplet digital PCR (ddPCR) are based on an underlying assumption that the fluorescent signal of droplets is normally distributed. We show that this normality assumption does not likely hold true for most ddPCR runs, resulting in an erroneous threshold. We suggest a methodology that does not make any assumptions about the distribution of the fluorescence readouts. A threshold is estimated by modelling the extreme values in the negative droplet population using extreme value theory. Furthermore, the method takes shifts in baseline fluorescence between samples into account. An R implementation of our method is available, allowing automated threshold determination for absolute ddPCR quantification using a single fluorescent reporter.
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The introduction of new tools for molecular analysis, such as RT-qPCR and microarrays, has provided researchers with powerful applications to study renal disease and development. However, the high cellular heterogeneity of the renal... more
The introduction of new tools for molecular analysis, such as RT-qPCR and microarrays, has provided researchers with powerful applications to study renal disease and development. However, the high cellular heterogeneity of the renal tissue complicates the molecular analysis of specific cells and cell groups such as glomerular or tubular cells. In the past, glomerular sieving and manual dissection were used for the isolation of glomeruli. However, these techniques cannot be used for the isolation of specific glomeruli or for the co-isolation of additional tissue fractions. In recent decades, new microdissection techniques such as laser-assisted microdissection have been developed. These applications allow the isolation of small cell groups from heterogeneous tissue for molecular analysis, including microarray and RT-qPCR. Although very promising, some drawbacks are associated with these techniques. The isolated sample material is generally small and requires sensitive assays. In addi...
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Shadow of prion protein is a gene potentially involved in the pathogenesis of prion diseases. However, the Shadoo protein encoded by this gene has not yet been studied in sheep, an important species in prion matters. Therefore, we... more
Shadow of prion protein is a gene potentially involved in the pathogenesis of prion diseases. However, the Shadoo protein encoded by this gene has not yet been studied in sheep, an important species in prion matters. Therefore, we developed a polyclonal antibody against ovine Shadoo and assessed the presence and distribution of this protein in the ovine brain by immunohistochemistry. The strongest staining level was found in the cerebellum (especially in the Purkinje cells) and in the pons, but cerebrum, hippocampus, pituitary gland, medulla oblongata, thalamus and hypothalamus were also immunopositive. Remarkably, a typical granular pattern was seen in most of the tested brain tissues, which might indicate that Shadoo is primarily expressed at synapses. The results of this study and the availability of an ovine anti-Shadoo antibody can contribute to future research on the function of Shadoo and on its potential involvement in prion diseases.
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Due to the scarcity of HIV-1 latently infected cells in patients, in vitro primary latency models are now commonly used to study the HIV-1 reservoir. To this end, a number of experimental systems have been developed. Most of these models... more
Due to the scarcity of HIV-1 latently infected cells in patients, in vitro primary latency models are now commonly used to study the HIV-1 reservoir. To this end, a number of experimental systems have been developed. Most of these models differ based on the nature of the primary CD4+ T-cell type, the used HIV strains, activation methods, and latency assessment strategies. Despite these differences, most models share some common characteristics. Here, we provide a systematic review covering the primary HIV latency models that have been used to date with the aim to compare these models and identify minimal requirements for such experiments. A systematic search on PubMed and Web of Science databases generated a short list of 17 unique publications that propose new in vitro latency models. Based on the described methods, we propose and discuss a generalized workflow, visualizing all the necessary steps to perform such an in vitro study, with the key choices and validation steps that nee...
Erythropoietin (EPO) is known to be a hematopoietic growth factor and a regulator of red blood cell production. Recently, EPO has also been reported to function as a tissue-protective cytokine and as an angiogenesis promoting factor. EPO... more
Erythropoietin (EPO) is known to be a hematopoietic growth factor and a regulator of red blood cell production. Recently, EPO has also been reported to function as a tissue-protective cytokine and as an angiogenesis promoting factor. EPO is mainly regulated by hypoxia through the action of hypoxia inducible factors (HIF-1alpha and HIF-2alpha). The localization of the EPO protein and the HIF-2alpha protein were immunohistochemically analyzed in developing porcine embryos. Both proteins were localized in developing cartilage tissue. HIF-2alpha and EPO protein were expressed in the peripheral chondrocytes of cartilage anlagen, in the perichondrium and in the cell condensations that will eventually differentiate into cartilage tissue. The results of this study reveal that EPO might play a role as a survival factor or as a mitogen in developing cartilage tissue. Moreover, the presence of both proteins at the same locations supports the hypothesis that EPO expression is regulated by HIF-2alpha.