Selective blockade of glycoprotein VI clustering on collagen helices

Platelet activation by collagen relies on the interaction of the receptor glycoprotein VI (GPVI) with collagen helices. We have previously generated two recombinant single chain human antibodies (scFvs) to human GPVI. The first, 10B12, binds to the collagen-binding site on the apical surface between the two immunoglobulin-like domains (D1D2) of the receptor and so directly inhibits GPVI function. The second, 1C3, binds D1D2 independently of 10B12 and has been shown to have a more subtle effect on platelet responses to collagen. Here we have shown that 1C3 potentiates the effect of 10B12 on platelet aggregation induced by collagen and cross-linked collagen-related peptide (CRP-XL). We investigated this by measuring the effect of both scFvs on the binding of D1D2 to immobilized collagen and CRP. As expected, 10B12 completely inhibited binding of GPVI to each ligand in a dose-dependent manner. However, 1C3 inhibited only a proportion of GPVI binding to its ligands, implying that it interferes with another aspect of ligand recognition by GPVI. To further understand the mode of inhibition, we used a unique set of CRPs in which the content of critical glycine-proline-hydroxyproline (GPO) triplets was varied in relation to an "inert" scaffold sequence of GPP motifs. We observed that a stepwise increase in D1D2 binding with (GPO)(2) content was blocked by 1C3. Together these results indicate that 1C3 inhibits clustering of the immunoglobulin-like domains of GPVI on collagen/CRPs, a conclusion that is supported by mapping the 1C3 epitope to the region including isoleucine 148 in D2.     

Inactivation of IL11 signaling causes craniosynostosis, delayed tooth eruption, and supernumerary teeth

Craniosynostosis and supernumerary teeth most often occur as isolated developmental anomalies, but they are also separately manifested in several malformation syndromes. Here, we describe a human syndrome featuring craniosynostosis, maxillary hypoplasia, delayed tooth eruption, and supernumerary teeth. We performed homozygosity mapping in three unrelated consanguineous Pakistani families and localized the syndrome to a region in chromosome 9. Mutational analysis of candidate genes in the region revealed that all affected children harbored homozygous missense mutations (c.662C>G [p.Pro221Arg], c.734C>G [p.Ser245Cys], or c.886C>T [p.Arg296Trp]) in IL11RA (encoding interleukin 11 receptor, alpha) on chromosome 9p13.3. In addition, a homozygous nonsense mutation, c.475C>T (p.Gln159X), and a homozygous duplication, c.916_924dup (p.Thr306_Ser308dup), were observed in two north European families. In cell-transfection experiments, the p.Arg296Trp mutation rendered the receptor unable to mediate the IL11 signal, indicating that the mutation causes loss of IL11RA function. We also observed disturbed cranial growth and suture activity in the Il11ra null mutant mice, in which reduced size and remodeling of limb bones has been previously described. We conclude that IL11 signaling is essential for the normal development of craniofacial bones and teeth and that its function is to restrict suture fusion and tooth number. The results open up the possibility of modulation of IL11 signaling for the treatment of craniosynostosis.     

Low-dose/dose-rate γ radiation depresses neural differentiation and alters protein expression profiles in neuroblastoma SH-SY5Y cells and C17.2 neural stem cells

The effects of low doses of ionizing radiation on cellular development in the nervous system are presently unclear. The focus of the present study was to examine low-dose γ-radiation-induced effects on the differentiation of neuronal cells and on the development of neural stem cells to glial cells. Human neuroblastoma SH-SY5Y cells were exposed to (137)Cs γ rays at different stages of retinoic acid-induced neuronal differentiation, and neurite formation was determined 6 days after exposure. When SH-SY5Y cells were exposed to low-dose-rate γ rays at the onset of differentiation, the number of neurites formed per cell was significantly less after exposure to either 10, 30 or 100 mGy compared to control cells. Exposure to 10 and 30 mGy attenuated differentiation of immature C17.2 mouse-derived neural stem cells to glial cells, as verified by the diminished expression of glial fibrillary acidic protein. Proteomic analysis of the neuroblastoma cells by 2D-PAGE after 30 mGy irradiation showed that proteins involved in neuronal development were downregulated. Proteins involved in cell cycle and proliferation were altered in both cell lines after exposure to 30 mGy; however, the rate of cell proliferation was not affected in the low-dose range. The radiation-induced attenuation of differentiation and the persistent changes in protein expression is indicative of an epigenetic rather than a cytotoxic mechanism.     

Prevalence of Disease and Relationships between Laboratory Phenotype and Bleeding Severity in Platelet Primary Secretion Defects

Background

The prevalence of platelet primary secretion defects (PSD) among patients with bleeding diathesis is unknown. Moreover, there is paucity of data on the determinants of bleeding severity in PSD patients.

Objective

To determine the prevalence of PSD in patients with clinical bleeding and to study the relationships between the type of platelet defect and bleeding severity.

Methods

Data on patients referred for bleeding to the Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Milan (Italy) in the years between 2008 and 2012 were retrieved to study the prevalence of PSD. Demographic, clinical and laboratory information on 32 patients with a diagnosis of PSD was used to compare patients with or without associated medical conditions and to investigate whether or not the type and extension of platelet defects were associated with the bleeding severity score (crude and age-normalized) or with the age at first bleeding requiring medical attention.

Results

The estimated prevalence of PSD among 207 patients with bleeding diathesis and bleeding severity score above 4 was 18.8% (95% confidence interval [CI]: 14.1–24.7%). Patients without associated medical conditions had earlier age of first bleeding (18 vs 45 years; difference: -27 years; 95% CI: -46 to -9 years) and different platelet functional defect patterns (Fisher''s exact test of the distribution of patterns, P = 0.007) than patients with accompanying medical conditions. The type and extension of platelet defect was not associated with the severity of bleeding.

Conclusions

PSD is found in approximately one fifth of patients with clinical bleeding. In patients with PSD, the type and extension of laboratory defect was not associated with bleeding severity.     

Identification of the minimal glycopeptide core recognized by T cells in a model for rheumatoid arthritis

Collagen induced arthritis (CIA) is a common mouse model for rheumatoid arthritis. Two sets of truncated peptides derived from type II collagen have been prepared and tested for binding to A(q), a MHC-II molecule associated with development of CIA. Binding to A(q) correlated well with predictions from a computer-based model. T-cell hybridomas, obtained in CIA, were also used to study the ability of A(q) bound peptides to trigger a T-cell response. The minimal peptide epitope required for binding, as well as for giving a T-cell response, was determined to be CII260-267. In collagen this epitope is often glycosylated at hydroxylysine 264 and glycosylation has been shown to be an immunodominant feature in CIA. Synthesis and evaluation of CII260-267 carrying a beta-D-galactosyl moiety at position 264 revealed that this glycopeptide stimulated representative members from a panel of carbohydrate-specific T-cell hybridomas obtained in CIA.     

Molecular and morphological evidence for multiple species within Paranoplocephala omphalodes (Cestoda, Anoplocephalidae) in Microtus voles (Arvicolinae)

Haukisalmi, V., Wickström, L. M., Henttonen, H., Hantula, J. & Gubányi, A. (2004). Molecular and morphological evidence for multiple species within Paranoplocephala omphalodes (Cestoda, Anoplocephalidae) in Microtus voles (Arvicolinae). —Zoologica Scripta, 33, 277–290. The present study was designed to test the hypothesis that the anoplocephalid cestode Paranoplocephala omphalodes (Hermann, 1783), a Holarctic parasite of Microtus voles, is a complex of host‐specific species, rather than a single host‐generalist species, using uni‐ and multivariate morphometrics and DNA sequence data from the mitochondrial cytochrome oxidase I gene. The phylogenetic methods applied to the mtDNA sequence data showed consistently that the cestodes morphologically recognizable as P. omphalodes include four well‐supported monophyletic groups, representing at least three distinct, largely host‐specific species. Multivariate morphometrics (discriminant analysis) successfully distinguished the four main mtDNA clades of P. omphalodes‐like cestodes. The true P. omphalodes is shown to be a parasite of Microtus arvalis, M. agrestis and Clethrionomys glareolus in Europe. Microtus oeconomus harbours two host‐specific, allopatric and possibly conspecific clades, one with a Holarctic and another with an (eastern) Beringian (Alaskan) distribution. The eastern Beringian endemic M. miurus is also parasitized with a host‐specific, morphologically divergent species of Paranoplocephala. The cestode clades recognized in M. oeconomus and M. miurus represent 2–3 undescribed species. Molecular phylogenetic analyses supported the monophyly of the ‘northern clade’ of Paranoplocephala spp., an assemblage including P. kalelai from Clethrionomys spp., P. macrocephala from Microtus spp. and all clades of P. omphalodes‐like cestodes except those representing the true P. omphalodes from Europe. The intra‐ and interspecific phylogeny within the northern clade is compared tentatively with the known evolutionary history of the hosts.     

Morphological and molecular characterisation of Paranoplocephala buryatiensis n. sp. and P. longivaginata Chechulin & Gulyaev, 1998 (Cestoda: Anoplocephalidae) in voles of the genus Clethrionomys

A new species, Paranoplocephala buryatiensis n. sp. (Cestoda:Anoplocephalidae), is described from the grey-sided vole Clethrionomys rufocanus (Sundevall) in the Republic of Buryatia (Russian Federation) and compared with P. longivaginata Chechulin & Gulyaev, 1998, a parasite of the red vole C. rutilus (Pallas) in the same region. P. buryatiensis n. sp. and P. longivaginata both have an exceptionally long vagina and cirrus, unique features among known species of Paranoplocephala Lühe, 1910. The new species differs from P. longivaginata primarily by its wider and more robust body, lower length/width ratio of mature proglottides, tendency of testes to occur in two separate groups, seminal receptacle of a different shape and the position of the cirrus-sac with respect to the ventral longitudinal osmoregulatory canal. The cytochrome oxidase subunit I (COI) sequence data support the independent status of these species, and show that they form a monophyletic assemblage within Paranoplocephala (sensu lato). Assuming cospeciation, an indirect calibration using host speciation dates estimated a rate of mtDNA substitution of 1.0–1.7% pairwise (0.5–0.85% per lineage) sequence divergence per million years. A faunistic review of Paranoplocephala species in C. rufocanus and C. rutilus in the Holarctic region is presented.     

The role of hypothalamic peptide gene expression in alcohol self-administration behavior

Self-administration of ethanol and food share many common features and Richter hypothesized that an increase in ethanol consumption would decrease feeding to balance the excess calories contained in the ethanol. Previously, we have shown that individual alcohol consumption correlates with neurotransmitter gene expression, especially in the prefrontal cortex. To test the hypothesis of Richter, we measured hypothalamic gene expression of receptors or neuropeptides of known relevance for the regulation of food intake using qPCR and correlated this to individual ethanol consumption in Wistar rats. For validation, gene expression was first correlated with body weight. We found a correlation of dynorphin, somatostatin, melanocortin-4 receptor and serotonin 5-HT_2C with body weight and trends to correlation for CART, thus confirming the established role of the hypothalamus in the regulation of weight. For ethanol consumption, correlations were found for CRH receptors 1 and 2 and vasopressin while strong trends were observed for galanin receptor 1, orexin receptor 1, MCH and adrenoceptor _1B. Therefore, alcohol consumption does seem to involve several hypothalamic systems which also mediate feeding responses and suggests that the hypothalamus, together with the prefrontal cortex, may determine the ‘stopping point’ of an individual.