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Deehan M.,NovImmune | Garces S.,Gulbenkian Institute of Science | Garces S.,Garcia Of Orta Hospital | Kramer D.,Sanofi S.A. | And 4 more authors.
Autoimmunity Reviews | Year: 2015

All protein drugs (biologicals) have an immunogenic potential and we are armed with multiple guidelines, regulatory documents and white papers to assist us in assessing the level of risk for unwanted immunogenicity of new biologicals. However, for certain biologicals, significant immunogenicity becomes only apparent after their use in patients. Causes of immunogenicity are multifactorial but not yet fully understood. Within the pharmaceutical industry there are only a few opportunities to openly discuss the causes and consequences of immunogenicity with regard to the development of new biologicals. The annual Open Scientific Symposium of the European Immunogenicity Platform (EIP) is one such meeting that brings together scientists and clinicians from academia and industry to build know-how and expertise in the field of immunogenicity. The critical topics discussed at the last EIP meeting (February 2014) will be reviewed here. The current opinion of this expert group is that the assessment of unwanted immunogenicity can be improved by using prediction tools, optimizing the performance of immunogenicity assays and learning from the clinical impact of other biologicals that have already been administered to patients. A multidisciplinary approach is warranted to better understand and minimize drug immunogenicity and its clinical consequences. However, this prediction does not directly translate to the immunogenicity observed in clinical practice. In parallel, such immune-monitoring will provide important information to help us understand the human immune response to biologic therapies. © 2015 Elsevier B.V.


Murta D.,University of Lisbon | Batista M.,University of Lisbon | Silva E.,University of Lisbon | Trindade A.,University of Lisbon | And 5 more authors.
PLoS ONE | Year: 2013

The transcription and expression patterns of Notch pathway components (Notch 1-3, Delta1 and 4, Jagged1) and effectors (Hes1, Hes2, Hes5 and Nrarp) were evaluated (through RT-PCR and IHC) in the mouse testis at key moments of post-natal development, and along the adult spermatogenic cycle. Notch pathway components and effectors are transcribed in the testis and expressed in germ, Sertoli and Leydig cells, and each Notch component shows a specific cell-type and time-window expression pattern. This expression at key testis developmental events prompt for a role of Notch signaling in pre-pubertal spermatogonia quiescence, onset of spermatogenesis, and regulation of the spermatogenic cycle. © 2013 Murta et al.


Gutermuth T.,Gulbenkian Institute of Science | Gutermuth T.,University of Würzburg | Lassig R.,Free University of Berlin | Portes M.-T.,Gulbenkian Institute of Science | And 9 more authors.
Plant Cell | Year: 2013

Apical growth in pollen tubes (PTs) is associated with the presence of tip-focused ion gradients and fluxes, implying polar localization or regulation of the underlying transporters. The molecular identity and regulation of anion transporters in PTs is unknown. Here we report a negative gradient of cytosolic anion concentration focused on the tip, in negative correlation with the cytosolic Ca2+ concentration. We hypothesized that a possible link between these two ions is based on the presence of Ca2+-dependent protein kinases (CPKs). We characterized anion channels and CPK transcripts in PTs and analyzed their localization. Yellow fluorescent protein (YFP) tagging of a homolog of SLOW ANION CHANNEL-ASSOCIATED1 (SLAH3:YFP) was widespread along PTs, but, in accordance with the anion efflux, CPK2/CPK20/CPK17/CPK34:YFP fluorescence was strictly localized at the tip plasma membrane. Expression of SLAH3 with either CPK2 or CPK20 (but not CPK17/CPK34) in Xenopus laevis oocytes elicited S-type anion channel currents. Interaction of SLAH3 with CPK2/CPK20 (but not CPK17/CPK34) was confirmed by Förster-resonance energy transfer fluorescence lifetime microscopy in Arabidopsis thaliana mesophyll protoplasts and bimolecular fluorescence complementation in living PTs. Compared with wild-type PTs, slah3-1 and slah3-2 as well as cpk2-1 cpk20-2 PTs had reduced anion currents. Doublemutant cpk2-1 cpk20-2 and slah3-1 PTs had reduced extracellular anion fluxes at the tip. Our studies provide evidence for a Ca2+-dependent CPK2/CPK20 regulation of the anion channel SLAH3 to regulate PT growth. © 2013 American Society of Plant Biologists. All rights reserved.


Sato T.,Institute Pasteur Paris | Rocancourt D.,Institute Pasteur Paris | Marques L.,University of Lisbon | Marques L.,Gulbenkian Institute of Science | And 3 more authors.
PLoS Genetics | Year: 2010

All skeletal muscle progenitor cells in the body derive from the dermomyotome, the dorsal epithelial domain of developing somites. These multipotent stem cells express Pax3, and this expression is maintained in the myogenic lineage where Pax3 plays an important role. Identification of Pax3 targets is therefore important for understanding the mechanisms that underlie the onset of myogenesis. In a microarray screen of Pax3-GFP sorted cells, with analysis on Pax3 gain and loss of function genetic backgrounds, we identify Dmrt2, expressed in the dermomyotome, as a Pax3 target. In vitro gel shift analysis and chromatin immunoprecipitation with in vivo extracts show that Pax3 binds to a conserved 286 bp sequence, situated at 218 kb from Dmrt2. This sequence directs reporter transgene expression to the somite, and this is severely affected when the Pax3 site is mutated in the context of the locus. In Dmrt2 mutant embryos, somite maturation is perturbed and the skeletal muscle of the myotome is abnormal. We now report that the onset of myogenesis is also affected. This depends on activation, in the epaxial dermomyotome, of the myogenic determination gene, Myf5, through its early epaxial enhancer. This sequence contains sites that bind Dmrt2, which belongs to the DM class of DNA-binding proteins. Mutation of these sites compromises activity of the enhancer in transgenic embryos where the reporter transgene is under the control of the Myf5 epaxial enhancer. Transactivation of this site by Dmrt2 is demonstrated in vitro, and conditional overexpression of Dmrt2 in Pax3 expressing cells in the somite confirms the role of this factor in the activation of Myf5. These results reveal a novel genetic network, comprising a Pax3/Dmrt2/Myf5 regulatory cascade that operates in stem cells of the epaxial dermomyotome to initiate skeletal muscle formation. © 2010 Sato et al.


ten Broek C.M.A.,University of Antwerp | ten Broek C.M.A.,Naturalis Biodiversity Center | Bakker A.J.,John Innes Center | Bakker A.J.,University of East Anglia | And 3 more authors.
Evolutionary Biology | Year: 2012

Homeotic transformations of vertebrae are particularly common in humans and tend to come associated with malformations in a wide variety of organ systems. In a dataset of 1,389 deceased human foetuses and infants a majority had cervical ribs and approximately half of these individuals also had missing twelfth ribs or lumbar ribs. In ~10 % of all cases there was an additional shift of the lumbo-sacral boundary and, hence, homeotic transformations resulted in shifts of at least three vertebral boundaries. We found a strong coupling between the abnormality of the vertebral patterns and the amount and strength of associated malformations, i. e., the longer the disturbance of the vertebral patterning has lasted, the more associated malformations have developed and the more organ systems are affected. The germ layer of origin of the malformations was not significantly associated with the frequency of vertebral patterns. In contrast, we find significant associations with the different developmental mechanisms that are involved in the causation of the malformations, that is, segmentation, neural crest development, left-right patterning, etc. Our results, thus, suggest that locally perceived developmental signals are more important for the developmental outcome than the origin of the cells. The low robustness of vertebral A-P patterning apparent from the large number of homeotic transformations is probably caused by the strong interactivity of developmental processes and the low redundancy of involved morphogens during early organogenesis. Additionally, the early irreversibility of the specification of the A-P identity of vertebrae probably adds to the vulnerability of the process by limiting the possibility for recovery from developmental disturbances. The low developmental robustness of vertebral A-P patterning contrasts with a high robustness of the A-P patterning of the vertebral regions. Not only the order is invariable, also the variation in the number of vertebrae per region is small. This robustness is in agreement with the evolutionary stability of vertebral regions in tetrapods. Finally, we propose a new hypothesis regarding the constancy of the presacral number of vertebrae in mammals. © 2012 The Author(s).


Ten Broek C.M.A.,University of Antwerp | Ten Broek C.M.A.,Naturalis Biodiversity Center | Bots J.,University of Antwerp | Varela-Lasheras I.,Gulbenkian Institute of Science | And 2 more authors.
PLoS ONE | Year: 2013

Fluctuating asymmetry (FA), as an indirect measure of developmental instability (DI), has been intensively studied for associations with stress and fitness. Patterns, however, appear heterogeneous and the underlying causes remain largely unknown. One aspect that has received relatively little attention in the literature is the consequence of direct mechanical effects on asymmetries. The crucial prerequisite for FA to reflect DI is that environmental conditions on both sides should be identical. This condition may be violated during early human development if amniotic fluid volume is deficient, as the resulting mechanical pressures may increase asymmetries. Indeed, we showed that limb bones of deceased human fetuses exhibited increased asymmetry, when there was not sufficient amniotic fluid (and, thus, space) in the uterine cavity. As amniotic fluid deficiency is known to cause substantial asymmetries and abnormal limb development, these subtle asymmetries are probably at least in part caused by the mechanical pressures. On the other hand, deficiencies in amniotic fluid volume are known to be associated with other congenital abnormalities that may disturb DI. More specifically, urogenital abnormalities can directly affect/reduce amniotic fluid volume. We disentangled the direct mechanical effects on FA from the indirect effects of urogenital abnormalities, the latter presumably representing DI. We discovered that both factors contributed significantly to the increase in FA. However, the direct mechanical effect of uterine pressure, albeit statistically significant, appeared less important than the effects of urogenital abnormalities, with an effect size only two-third as large. We, thus, conclude that correcting for the relevant direct factors allowed for a representative test of the association between DI and stress, and confirmed that fetuses form a suitable model system to increase our understanding in patterns of FA and symmetry development. © 2013 ten Broek et al.


PubMed | Aix - Marseille University, University Katyavala Bwila, University Paris Diderot and Gulbenkian Institute of Science
Type: Journal Article | Journal: The American journal of pathology | Year: 2015

Tissue pantetheinase, encoded by the VNN1 gene, regulates response to stress, and previous studies have shown that VNN genes contribute to the susceptibility to malaria. Herein, we evaluated the role of pantetheinase on erythrocyte homeostasis and on the development of malaria in patients and in a new mouse model of pantetheinase insufficiency. Patients with cerebral malaria have significantly reduced levels of serum pantetheinase activity (PA). In mouse, we show that a reduction in serum PA predisposes to severe malaria, including cerebral malaria and severe anemia. Therefore, scoring pantetheinase in serum may serve as a severity marker in malaria infection. This disease triggers anacute stress in erythrocytes, which enhances cytoadherence and hemolysis. We speculated thatserum pantetheinase might contribute to erythrocyte resistance to stress under homeostatic conditions. We show that mutant mice with a reduced serum PA are anemic and prone to phenylhydrazine-induced anemia. A cytofluorometric and spectroscopic analysis documented an increased frequency of erythrocytes with an autofluorescent aging phenotype. This is associated with an enhanced oxidative stress and shear stress-induced hemolysis. Red blood cell transfer and bone marrow chimera experiments show that the aging phenotype is not cell intrinsic but conferred by the environment, leading to a shortening of red blood cell half-life. Therefore, serum pantetheinase level regulates erythrocyte life span and modulates the risk of developing complicated malaria.


PubMed | University of Vigo, University Vila Velha and Gulbenkian Institute of Science
Type: | Journal: Computers in biology and medicine | Year: 2015

We describe a set of new algorithms and a software tool, StabiTissue, for stabilizing in vivo intravital microscopy images that suffer from soft-tissue background movement. Because these images lack predetermined anchors and are dominated by noise, we use a pixel weighted image alignment together with a correction for nonlinear tissue deformations. We call this correction a poor mans diffeomorphic map since it ascertains the nonlinear regions of the image without resorting to a full integral equation method. To determine the quality of the image stabilization, we developed an ensemble sampling method that quantifies the coincidence between image pairs from randomly distributed image regions. We obtain global stabilization alignment through an iterative constrained simulated annealing optimization procedure. To show the accuracy of our algorithm with existing software, we measured the misalignment error rate in datasets taken from two different organs and compared the results to a similar and popular open-source solution. Present open-source stabilization software tools perform poorly because they do not treat the specific needs of the IV-2pM datasets with soft-tissue deformation, speckle noise, full 5D inter- and intra-stack motion error correction, and undefined anchors. In contrast, the results of our tests demonstrate that our method is more immune to noise and provides better performance for datasets possessing nonlinear tissue deformations. As a practical application of our software, we show how our stabilization improves cell tracking, where the presence of background movement would degrade track information. We also provide a qualitative comparison of our software with other open-source libraries/applications. Our software is freely available at the open source repository http://sourceforge.net/projects/stabitissue/.


Fonseca R.,Gulbenkian Institute of Science
Neuropsychopharmacology | Year: 2013

Fear conditioning, a form of associative learning is thought to involve the induction of an associative long-term potentiation of cortical and thalamic inputs to the lateral amygdala. Here, we show that stimulation of the thalamic input can reinforce a transient form of plasticity (E-LTP) induced by weak stimulation of the cortical inputs. This synaptic cooperation occurs within a time window of 30 min, suggesting that synaptic integration at amygdala synapses can occur within large time windows. Interestingly, we found that synaptic cooperation is not symmetrical. Reinforcement of a thalamic E-LTP by subsequent cortical stimulation is only observed within a shorter time window. We found that activation of endocannabinoid CB1 receptors is involved in the time restriction of thalamic and cortical synaptic cooperation in an activity-dependent manner. Our results support the hypothesis that synaptic cooperation can underlie associative learning and that synaptic tagging and capture is a general mechanism in synaptic plasticity. © 2013 American College of Neuropsychopharmacology. All rights reserved.


Fonseca R.,Gulbenkian Institute of Science
Synaptic Tagging and Capture: From Synapses to Behavior | Year: 2015

Activity-dependent plasticity of synaptic connections is a hallmark of the mammalian brain and represents a key mechanism for rewiring neural circuits during development, experience-dependent plasticity, and brain disorders. Understanding the rules that determine how different neuronal inputs interact with each other, allow us to gain insight on the cellular and molecular mechanisms involved in memory establishment and maintenance. One of the most intriguing aspects of memory formation is the observation that past and ongoing activity can infl uence how information is processed and maintained in the brain. At the cellular level, the synaptic tagging and capture (STC) theory states that the maintenance of activity-dependent synaptic changes is based on the interaction between synaptic-specific tags and the capture of plasticity-related proteins. The STC has provided a solid framework to account for the input specifi city of synaptic plasticity but also provides a working model to understand the heterosynaptic interaction between different groups of synapses. In this chapter, I will discuss the evidence regarding the cooperative and competitive interactions between different groups of synapses. In particular, I will address the properties of synaptic cooperation and competition that contribute to the refi nement of neuronal connections during development. Later, I will address the evidence that similar rules operate during the induction and maintenance of synaptic plasticity. Due to the intricate relationship between synaptic plasticity and memory formation, understanding the cellular rules of cooperative and competitive interactions between synapses, will allow us to further dissect the rules underlying associative learning. © Springer Science+Business Media New York 2015.

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