Protein pUL128 of human cytomegalovirus is necessary for monocyte infection and blocking of migration

We have previously shown that only endotheliotropic strains of human cytomegalovirus (HCMV), such as TB40E, infect monocytes and impair their chemokine-driven migration. The proteins encoded by the UL128-131A region (UL128, UL130, and UL131A) of the HCMV genome, which assemble into a pentameric gH-gL-UL128-UL130-UL131A envelope complex, have been recognized as determinants for HCMV endothelial cell tropism. The genes for these proteins are typically inactivated by mutations in all fibroblast-adapted strains that have lost the diversified tropism of clinical isolates. By using mutant HCMV reconstituted from TB40E-derived bacterial artificial chromosomes (BAC) encoding a wild-type (wt) or mutated form of UL128, we show here that UL128-131A products are essential determinants of infection in monocytes and that pUL128, in particular, can block chemokine-driven motility. The virus BAC4, encoding wt UL128, established infection in monocytes, induced the intracellular retention of several chemokine receptors, and rendered monocytes unresponsive to different chemokines. In contrast, the virus BAC1, encoding a mutated UL128, failed to infect monocytes and to downregulate chemokine receptors. BAC1-exposed monocytes did not express immediate-early (IE) products, retained virions in cytoplasmic vesicles, and exhibited normal chemokine responsiveness. A potential role of second-site mutations in the observed phenotype was excluded by using the revertant viruses BAC1rep and BAC4mut. By incubating noninfected monocytes with soluble recombinant pUL128, we observed both the block of migration and the chemokine receptor internalization. We propose that among the gH-gL-UL128-UL130-UL131A complex subunits, the UL128 protein is the one that triggers monocyte paralysis.     

Human recombinant domain antibodies against multiple sclerosis antigenic peptide CSF114(Glc)

Multiple sclerosis (MS) is a chronic auto‐immune disease characterized by a damage to the myelin component of the central nervous system. Self‐antigens created by aberrant glycosylation have been described to be a key component in the formation of auto‐antibodies. CSF114(Glc) is a synthetic glucopeptide detecting in vitro MS‐specific auto‐antibodies, and it is actively used in diagnostics and research to monitor and quantify MS‐associated Ig levels. We reasoned that antibodies raised against this probe could have been relevant for MS. We therefore screened a human Domain Antibody library against CSF114(Glc) using magnetic separation as a panning method. We obtained and described several clones, and the one with the highest signals was produced as a 6×His‐tagged protein to properly study the binding properties as a soluble antibody. By surface plasmon resonance measurements, we evidenced that our clone recognized CSF114(Glc) with high affinity and specific for the glucosylated peptide. Kinetic parameters of peptide–clone interaction were calculated obtaining a value of K_D in the nanomolar range. Harboring a human framework, this antibody should be very well tolerated by human immune system and may represent a valuable tool for MS diagnosis and therapy, paving the way to new research strategies. Copyright © 2014 John Wiley & Sons, Ltd.     

The anti‐apoptotic Bcl‐2 protein regulates hair follicle stem cell function

Maintaining the architecture, size and composition of an intact stem cell (SC) compartment is crucial for tissue homeostasis and regeneration throughout life. In mammalian skin, elevated expression of the anti‐apoptotic Bcl‐2 protein has been reported in hair follicle (HF) bulge SCs (BSCs), but its impact on SC function is unknown. Here, we show that systemic exposure of mice to the Bcl‐2 antagonist ABT‐199/venetoclax leads to the selective loss of suprabasal BSCs (sbBSCs), thereby disrupting cyclic HF regeneration. RNAseq analysis shows that the pro‐apoptotic BH3‐only proteins BIM and Bmf are upregulated in sbBSCs, explaining their addiction to Bcl‐2 and the marked susceptibility to Bcl‐2 antagonism. In line with these observations, conditional knockout of Bcl‐2 in mouse epidermis elevates apoptosis in BSCs. In contrast, ectopic Bcl‐2 expression blocks apoptosis during HF regression, resulting in the accumulation of quiescent SCs and delaying HF growth in mice. Strikingly, Bcl‐2‐induced changes in size and composition of the HF bulge accelerate tumour formation. Our study identifies a niche‐instructive mechanism of Bcl‐2‐regulated apoptosis response that is required for SC homeostasis and tissue regeneration, and may suppress carcinogenesis.     

Type-1, but Not Type-5, Metabotropic Glutamate Receptors are Coupled to Polyphosphoinositide Hydrolysis in the Retina

mGlu1 and mGlu5 metabotropic glutamate receptors are expressed in the vertebrate retina, and are co-localized in some retinal neurons. It is believed that both receptors are coupled to polyphosphoinositide (PI) hydrolysis in the retina and their function may diverge in some cells because of a differential engagement of downstream signaling molecules. Here, we show that it is only the mGlu1 receptor that is coupled to PI hydrolysis in the retina. We used either bovine retinal slices or intact mouse retinas challenged with the mixed mGlu1/5 receptor agonist, DHPG. In both models, DHPG-stimulated PI hydrolysis was abrogated by the selective mGlu1 receptor antagonist, JNJ16259685, but was insensitive to the mGlu5 receptor antagonist, MPEP. In addition, the PI response to DHPG was unchanged in the retina of mGlu5^?/? mice but was abolished in the retina of crv4 mice lacking mGlu1 receptors. Stimulation of the mitogen-activated protein kinase pathway by DHPG in intact mouse retinas were also entirely mediated by mGlu1 receptors. Our data provide the first example of a tissue in which a biochemically detectable PI response is mediated by mGlu1, but not mGlu5, receptors. Hence, bovine retinal slices might be used as a model for the functional screening of mGlu1 receptor ligands. In addition, the mGlu1 receptor caters the potential as a drug target in the experimental treatment of degenerative disorders of the retina.

     

DNA damage in eelpout (Zoarces viviparus) from Göteborg harbour

The relationship between DNA damage and the exposure of marine organisms to environmental contaminants was examined in the G?teborg harbour area. This research is part of a wider ecotoxicological study planned to evaluate the biological impact of chemical contamination in the River G?ta estuary, following a bunker oil (10-100 tonnes) spill occurred in June 2003. Here we present data on the DNA strand breaks derived using the comet assay and the presence of apoptotic cells using the diffusion assay in nucleated erythrocytes of the eelpout (Zoarces viviparus) from the study area and at a clean reference site. Polycyclic aromatic hydrocarbon metabolites were also analyzed in the bile of exposed fish. The results showed a high level of damaged DNA, paralleled by a peak in bile PAH metabolites, in fish from the most impacted site, 3 weeks after the oil spill. A significant recovery was observed in specimens from the spill site, 5 months later, but not in fish caught in the middle part of G?teborg harbour, which is chronically subjected to heavy chemical pollution. The levels of apoptic cells did not show any marked variations, but a significant recovery was observed in fish from the oil impacted site 5 months after the spill.     

Thioredoxin specifically cross-desensitizes monocytes to MCP-1

Thioredoxin (Trx) is a protein disulfide oxidoreductase which can be secreted and acts as a cytokine. As we recently reported that Trx is chemotactic, we investigated whether it desensitizes monocytes or PMN to other chemokines. Preincubation for 15 min with Trx inhibited the chemotactic response of monocytes to MCP-1, but not to fMLP. This effect was independent of whether Trx was present during the chemotaxis assay or only during the preincubation. Preincubation (5 min) with Trx also inhibited the increase in intracellular Ca(2+) induced by MCP-1 in monocytes, but not that induced by fMLP. Preincubation with Trx did not affect the chemotactic response induced in PMN by IL-8. The inhibition of chemotactic and Ca(2+) responses to MCP-1 in monocytes was not due to a down-regulation of the MCP-1 receptor, as shown by receptor binding studies. The Ca(2+) response to MCP-1 was also inhibited by Trx in a CCR2-transfected cell line. It is suggested that Trx inhibits monocyte responses to chemokines by acting downstream of the chemokine receptors. Since there are high concentrations of circulating Trx in infection and inflammatory diseases, this might act as an inhibitor of monocyte migration in vivo.     

Right Limbic FDG-PET Hypometabolism Correlates with Emotion Recognition and Attribution in Probable Behavioral Variant of Frontotemporal Dementia Patients

The behavioural variant of frontotemporal dementia (bvFTD) is a rare disease mainly affecting the social brain. FDG-PET fronto-temporal hypometabolism is a supportive feature for the diagnosis. It may also provide specific functional metabolic signatures for altered socio-emotional processing. In this study, we evaluated the emotion recognition and attribution deficits and FDG-PET cerebral metabolic patterns at the group and individual levels in a sample of sporadic bvFTD patients, exploring the cognitive-functional correlations. Seventeen probable mild bvFTD patients (10 male and 7 female; age 67.8±9.9) were administered standardized and validated version of social cognition tasks assessing the recognition of basic emotions and the attribution of emotions and intentions (i.e., Ekman 60-Faces test-Ek60F and Story-based Empathy task-SET). FDG-PET was analysed using an optimized voxel-based SPM method at the single-subject and group levels. Severe deficits of emotion recognition and processing characterized the bvFTD condition. At the group level, metabolic dysfunction in the right amygdala, temporal pole, and middle cingulate cortex was highly correlated to the emotional recognition and attribution performances. At the single-subject level, however, heterogeneous impairments of social cognition tasks emerged, and different metabolic patterns, involving limbic structures and prefrontal cortices, were also observed. The derangement of a right limbic network is associated with altered socio-emotional processing in bvFTD patients, but different hypometabolic FDG-PET patterns and heterogeneous performances on social tasks at an individual level exist.     

New clues on the origin of the Friedreich ataxia expanded alleles from the analysis of new polymorphisms closely linked to the mutation

Friedreichs ataxia (FRDA) is an autosomal recessive neurodegenerative disorder commonly caused by large expansions of a GAA repeat in the first intron of the frataxin gene, FRDA. The expansion of the triplet repeat is localized within an Alu sequence. FRDA GAA-repeat alleles can be divided into three classes depending on their lengths: short normal alleles (SN), long normal alleles (LN) and expanded pathological alleles (E). We made an accurate analysis of the Alu sequence containing the GAA repeat. We found a new single-nucleotide polymorphism (SNP) that is the closest one to the GAA repeat. We studied this new SNP and the polymorphic polyA region contiguous to the GAA triplets in two populations with different frequencies of FRDA. We found that, while both E and LN alleles seem to be genetically homogeneous and likely related, SN represents a more heterogeneous class of alleles. Indeed, one SNP variation (T) was more frequently associated with (GAA)₈ alleles, whereas the other one (C) with (GAA)₉ repeat(s). The long normal and expanded alleles presented the C haplotype. The same correlation was described for polyA-tract polymorphisms. Thus, 14A was commonly associated with (GAA)₈ alleles and 17A with (GAA)₉ alleles. The long normal alleles more frequently showed the 17A haplotype. Our data seem to suggest that all the E alleles come from LN alleles, while LN alleles come from a defined subclass of SN alleles.