Role of P-selectin in platelet sequestration in pulmonary capillaries during endotoxemia

Background: There is growing evidence that platelets accumulate in the lung and contribute to the pathogenesis of acute lung injury during endotoxemia. The aims of the present study were to localize platelet sequestration in the pulmonary microcirculation and to investigate the role of P-selectin as a molecular mechanism of platelet endothelial cell interaction. Methods: We used in vivo fluorescence microscopy to quantify the kinetics of fluorescently labeled erythrocytes and platelets in alveolar capillary networks in rabbit lungs. Results: Six hours after onset of endotoxin infusion we observed a massive rolling along and firm adherence of platelets to lung capillary endothelial cells whereas under control conditions no platelet sequestration was detected. P-selectin was expressed on the surface of separated platelets which were incubated with endotoxin and in lung tissue. Pretreatment of platelets with fucoidin, a P-selectin antagonist, significantly attenuated the endotoxin-induced platelet rolling and adherence. In contrast, intravenous infusion of fucoidin in endotoxin-treated rabbits did not inhibit platelet sequestration in pulmonary capillaries. Conclusion: We conclude that platelets accumulate in alveolar capillaries following endotoxemia. P-selectin expressed on the surface of platelets seems to play an important role in mediating this platelet-endothelial cell interaction. Copyright (c) 2006 S. Karger AG, Basel

Abnormal Intracellular Accumulation and Extracellular Aβ Deposition in Idiopathic and Dup15q11.2-q13 Autism Spectrum Disorders

<div><h3>Background</h3><p>It has been shown that amyloid ß (Aβ), a product of proteolytic cleavage of the amyloid β precursor protein (APP), accumulates in neuronal cytoplasm in non-affected individuals in a cell type–specific amount.</p> <h3>Methodology/Principal Findings</h3><p>In the present study, we found that the percentage of amyloid-positive neurons increases in subjects diagnosed with idiopathic autism and subjects diagnosed with duplication 15q11.2-q13 (dup15) and autism spectrum disorder (ASD). In spite of interindividual differences within each examined group, levels of intraneuronal Aβ load were significantly greater in the dup(15) autism group than in either the control or the idiopathic autism group in 11 of 12 examined regions (p<0.0001 for all comparisons; Kruskall-Wallis test). In eight regions, intraneuronal Aβ load differed significantly between idiopathic autism and control groups (p<0.0001). The intraneuronal Aβ was mainly N-terminally truncated. Increased intraneuronal accumulation of Aβ_17–40/42 in children and adults suggests a life-long enhancement of APP processing with α-secretase in autistic subjects. Aβ accumulation in neuronal endosomes, autophagic vacuoles, Lamp1-positive lysosomes and lipofuscin, as revealed by confocal microscopy, indicates that products of enhanced α-secretase processing accumulate in organelles involved in proteolysis and storage of metabolic remnants. Diffuse plaques containing Aβ_1–40/42 detected in three subjects with ASD, 39 to 52 years of age, suggest that there is an age-associated risk of alterations of APP processing with an intraneuronal accumulation of a short form of Aβ and an extracellular deposition of full-length Aβ in nonfibrillar plaques.</p> <h3>Conclusions/Significance</h3><p>The higher prevalence of excessive Aβ accumulation in neurons in individuals with early onset of intractable seizures, and with a high risk of sudden unexpected death in epilepsy in autistic subjects with dup(15) compared to subjects with idiopathic ASD, supports the concept of mechanistic and functional links between autism, epilepsy and alterations of APP processing leading to neuronal and astrocytic Aβ accumulation and diffuse plaque formation.</p> </div

Agent-Based Model of Therapeutic Adipose-Derived Stromal Cell Trafficking during Ischemia Predicts Ability To Roll on P-Selectin

Intravenous delivery of human adipose-derived stromal cells (hASCs) is a promising option for the treatment of ischemia. After delivery, hASCs that reside and persist in the injured extravascular space have been shown to aid recovery of tissue perfusion and function, although low rates of incorporation currently limit the safety and efficacy of these therapies. We submit that a better understanding of the trafficking of therapeutic hASCs through the microcirculation is needed to address this and that selective control over their homing (organ- and injury-specific) may be possible by targeting bottlenecks in the homing process. This process, however, is incredibly complex, which merited the use of computational techniques to speed the rate of discovery. We developed a multicell agent-based model (ABM) of hASC trafficking during acute skeletal muscle ischemia, based on over 150 literature-based rules instituted in Netlogo and MatLab software programs. In silico, trafficking phenomena within cell populations emerged as a result of the dynamic interactions between adhesion molecule expression, chemokine secretion, integrin affinity states, hemodynamics and microvascular network architectures. As verification, the model reasonably reproduced key aspects of ischemia and trafficking behavior including increases in wall shear stress, upregulation of key cellular adhesion molecules expressed on injured endothelium, increased secretion of inflammatory chemokines and cytokines, quantified levels of monocyte extravasation in selectin knockouts, and circulating monocyte rolling distances. Successful ABM verification prompted us to conduct a series of systematic knockouts in silico aimed at identifying the most critical parameters mediating hASC trafficking. Simulations predicted the necessity of an unknown selectin-binding molecule to achieve hASC extravasation, in addition to any rolling behavior mediated by hASC surface expression of CD15s, CD34, CD62e, CD62p, or CD65. In vitro experiments confirmed this prediction; a subpopulation of hASCs slowly rolled on immobilized P-selectin at speeds as low as 2 µm/s. Thus, our work led to a fundamentally new understanding of hASC biology, which may have important therapeutic implications

Absence of Positive Selection on Centromeric Histones in Tetrahymena Suggests Unsuppressed Centromere-Drive in Lineages Lacking Male Meiosis

Centromere-drive is a process where centromeres compete for transmission through asymmetric "female" meiosis for inclusion into the oocyte. In symmetric "male" meiosis, all meiotic products form viable germ cells. Therefore, the primary incentive for centromere-drive, a potential transmission bias, is believed to be missing from male meiosis. In this article, we consider whether male meiosis also bears the primary cost of centromere-drive. Because different taxa carry out different combinations of meiotic programs (symmetric + asymmetric, symmetric only, asymmetric only), it is possible to consider the evolutionary consequences of centromere-drive in the context of these differing systems. Groups with both types of meiosis have large, rapidly evolving centromeric regions, and their centromeric histones (CenH3s) have been shown to evolve under positive selection, suggesting roles as suppressors of centromere-drive. In contrast, taxa with only symmetric male meiosis have shown no evidence of positive selection in their centromeric histones. In this article, we present the first evolutionary analysis of centromeric histones in ciliated protozoans, a group that only undergoes asymmetric "female" meiosis. We find no evidence of positive selection acting on CNA1, the CenH3 of Tetrahymena species. Cytological observations of a panel of Tetrahymena species are consistent with dynamic karyotype evolution in this lineage. Our findings suggest that defects in male meiosis, and not mitosis or female meiosis, are the primary selective force behind centromere-drive suppression. Our study raises the possibility that taxa like ciliates, with only female meiosis, may therefore undergo unsuppressed centromere drive

Mapping genetic variations to three- dimensional protein structures to enhance variant interpretation: a proposed framework

The translation of personal genomics to precision medicine depends on the accurate interpretation of the multitude of genetic variants observed for each individual. However, even when genetic variants are predicted to modify a protein, their functional implications may be unclear. Many diseases are caused by genetic variants affecting important protein features, such as enzyme active sites or interaction interfaces. The scientific community has catalogued millions of genetic variants in genomic databases and thousands of protein structures in the Protein Data Bank. Mapping mutations onto three-dimensional (3D) structures enables atomic-level analyses of protein positions that may be important for the stability or formation of interactions; these may explain the effect of mutations and in some cases even open a path for targeted drug development. To accelerate progress in the integration of these data types, we held a two-day Gene Variation to 3D (GVto3D) workshop to report on the latest advances and to discuss unmet needs. The overarching goal of the workshop was to address the question: what can be done together as a community to advance the integration of genetic variants and 3D protein structures that could not be done by a single investigator or laboratory? Here we describe the workshop outcomes, review the state of the field, and propose the development of a framework with which to promote progress in this arena. The framework will include a set of standard formats, common ontologies, a common application programming interface to enable interoperation of the resources, and a Tool Registry to make it easy to find and apply the tools to specific analysis problems. Interoperability will enable integration of diverse data sources and tools and collaborative development of variant effect prediction methods

Comparative Genomics of the Apicomplexan Parasites Toxoplasma gondii and Neospora caninum: Coccidia Differing in Host Range and Transmission Strategy

Toxoplasma gondii is a zoonotic protozoan parasite which infects nearly one third of the human population and is found in an extraordinary range of vertebrate hosts. Its epidemiology depends heavily on horizontal transmission, especially between rodents and its definitive host, the cat. Neospora caninum is a recently discovered close relative of Toxoplasma, whose definitive host is the dog. Both species are tissue-dwelling Coccidia and members of the phylum Apicomplexa; they share many common features, but Neospora neither infects humans nor shares the same wide host range as Toxoplasma, rather it shows a striking preference for highly efficient vertical transmission in cattle. These species therefore provide a remarkable opportunity to investigate mechanisms of host restriction, transmission strategies, virulence and zoonotic potential. We sequenced the genome of N. caninum and transcriptomes of the invasive stage of both species, undertaking an extensive comparative genomics and transcriptomics analysis. We estimate that these organisms diverged from their common ancestor around 28 million years ago and find that both genomes and gene expression are remarkably conserved. However, in N. caninum we identified an unexpected expansion of surface antigen gene families and the divergence of secreted virulence factors, including rhoptry kinases. Specifically we show that the rhoptry kinase ROP18 is pseudogenised in N. caninum and that, as a possible consequence, Neospora is unable to phosphorylate host immunity-related GTPases, as Toxoplasma does. This defense strategy is thought to be key to virulence in Toxoplasma. We conclude that the ecological niches occupied by these species are influenced by a relatively small number of gene products which operate at the host-parasite interface and that the dominance of vertical transmission in N. caninum may be associated with the evolution of reduced virulence in this species

Differential endocytotic characteristics of a novel human B/DC cell line HBM-Noda: effective macropinocytic and phagocytic function rather than scavenging function

In order to characterize a novel human B cell-lineage dendritic cell line (B/DC line) as an antigen-presenting cell (APC), we compared three types of endocytosis (micropinocytosis via a clathrin-coated pit, macropinocytosis via membrane ruffling, and phagocytosis) among myeloid-related, macrophage (Mφ) cell lines and a B/DC line. In the present examination, we used a unique human dendritic cell (DC) line, HBM-Noda (Noda). Flow cytometric and immunocytochemical analyses revealed that Noda not only expresses some DC markers, but also it expresses some B-cell associated markers. Noda shows strong capacities to stimulate allogenic T cells, to produce immunoglobulin G (IgG), and to perform immunoglobulin gene rearrangment. These data strongly suggest that Noda is a B-cell lineage DC line. The endocytic differences among these cell lines were as follows. (1) The level of micropinocytosis of Noda was significantly less than that of conventional human Mφ cell lines, and the formation of a clathrin-coated pit was not observed in Noda. (2) The level of macropinocytosis of Noda was also smaller than that of conventional Mφ cells indicating that the active membrane ruffling of Noda induces rapid recycling. (3) Phagocytosis of opsonized sheep red blood cells (SRBC) was performed more efficiently in Noda than in other Mφ cell lines. Collectively, these data suggest that in human bone marrow cells, we can identify a unique DC subtype, B/DC line, which develops through a lymphoid DC-differentiation pathway, and DC in this lineage plays an important role in the host immune response because of its effective uptake of a variety of size of antigens by using the skilful membrane ruffling and surface receptor