Neutralization of the chemokine CXCL10 reduces inflammatory cell invasion and demyelination and improves neurological function in a viral model of multiple sclerosis

Intracerebral infection of mice with mouse hepatitis virus (MHV) results in an acute encephalomyelitis followed by a chronic demyelinating disease with clinical and histological similarities with the human demyelinating disease multiple sclerosis (MS). Following MHV infection, chemokines including CXC chemokine ligand (CXCL)10 (IFN inducible protein 10 kDa), CXCL9 (monokine induced by IFN-gamma), and CC chemokine ligand 5 (RANTES) are expressed during both acute and chronic stages of disease suggesting a role for these molecules in disease exacerbation. Previous studies have shown that during the acute phase of infection, T lymphocytes are recruited into the CNS by the chemokines CXCL10 and CXCL9. In the present study, MHV-infected mice with established demyelination were treated with antisera against these two chemokines, and disease severity was assessed. Treatment with anti-CXCL10 reduced CD4+ T lymphocyte and macrophage invasion, diminished expression of IFN-gamma and CC chemokine ligand 5, inhibited progression of demyelination, and increased remyelination. Anti-CXCL10 treatment also resulted in an impediment of clinical disease progression that was characterized by a dramatic improvement in neurological function. Treatment with antisera against CXCL9 was without effect, demonstrating a critical role for CXCL10 in inflammatory demyelination in this model. These findings document a novel therapeutic strategy using Ab-mediated neutralization of a key chemokine as a possible treatment for chronic human inflammatory demyelinating diseases such as MS.     

Molecular Diversity of Anthracnose Pathogen Populations Associated with UK Strawberry Production Suggests Multiple Introductions of Three Different Colletotrichum Species

Fragaria × ananassa (common name: strawberry) is a globally cultivated hybrid species belonging to Rosaceae family. Colletotrichum acutatum sensu lato (s.l.) is considered to be the second most economically important pathogen worldwide affecting strawberries. A collection of 148 Colletotrichum spp. isolates including 67 C. acutatum s.l. isolates associated with the phytosanitary history of UK strawberry production were used to characterize multi-locus genetic variation of this pathogen in the UK, relative to additional reference isolates that represent a worldwide sampling of the diversity of the fungus. The evidence indicates that three different species C. nymphaeae, C. godetiae and C. fioriniae are associated with strawberry production in the UK, which correspond to previously designated genetic groups A2, A4 and A3, respectively. Among these species, 12 distinct haplotypes were identified suggesting multiple introductions into the country. A subset of isolates was also used to compare aggressiveness in causing disease on strawberry plants and fruits. Isolates belonging to C. nymphaeae, C. godetiae and C. fioriniae representative of the UK anthracnose pathogen populations showed variation in their aggressiveness. Among the three species, C. nymphaeae and C. fioriniae appeared to be more aggressive compared to C. godetiae. This study highlights the genetic and pathogenic heterogeneity of the C. acutatum s.l. populations introduced into the UK linked to strawberry production.     

BCL-2 is dispensable for thrombopoiesis and platelet survival

Navitoclax (ABT-263), an inhibitor of the pro-survival BCL-2 family proteins BCL-2, BCL-X_L and BCL-W, has shown clinical efficacy in certain BCL-2-dependent haematological cancers, but causes dose-limiting thrombocytopaenia. The latter effect is caused by Navitoclax directly inducing the apoptotic death of platelets, which are dependent on BCL-X_L for survival. Recently, ABT-199, a selective BCL-2 antagonist, was developed. It has shown promising anti-leukaemia activity in patients whilst sparing platelets, suggesting that the megakaryocyte lineage does not require BCL-2. In order to elucidate the role of BCL-2 in megakaryocyte and platelet survival, we generated mice with a lineage-specific deletion of Bcl2, alone or in combination with loss of Mcl1 or Bclx. Platelet production and platelet survival were analysed. Additionally, we made use of BH3 mimetics that selectively inhibit BCL-2 or BCL-X_L. We show that the deletion of BCL-2, on its own or in concert with MCL-1, does not affect platelet production or platelet lifespan. Thrombocytopaenia in Bclx-deficient mice was not affected by additional genetic loss or pharmacological inhibition of BCL-2. Thus, BCL-2 is dispensable for thrombopoiesis and platelet survival in mice.Platelets are anucleate blood cells that play essential roles in haemostasis, wound healing and a range of other processes, including inflammation and immunity.¹ They are produced by megakaryocytes, large polyploid cells that develop primarily in the bone marrow, spleen and foetal liver.² Recent work has demonstrated that the survival of megakaryocytes and platelets is governed by the BCL-2 family proteins.³ Both cell types possess a classical BAK/BAX-mediated intrinsic apoptosis pathway that must be restrained in order for them to develop and survive.In platelets, BCL-X_L is the critical pro-survival BCL-2 family member required to keep BAK and BAX in check. The first evidence of this came from Wagner et al.,⁴ who reported severe thrombocytopaenia in mice after MMTV-Cre-mediated deletion of Bclx in the haematopoietic system, skin and various secretory tissues. It has since been shown that megakaryocyte-restricted deletion of Bclx in mice reduces platelet lifespan from ~5 days to ~5 h, with a concomitant decrease in platelet counts to ~2% of wild-type levels.^{5, 6} Pharmacological inhibition of BCL-X_L with the BH3 mimetics ABT-737⁷ or Navitoclax (ABT-263)⁸ (which both also inhibit BCL-2 and BCL-W) triggers BAK/BAX-mediated platelet apoptosis.^{9, 10, 11} As a result, these drugs cause dose-dependent thrombocytopaenia in mice, dogs and humans.^{9, 11, 12, 13, 14} Indeed, thrombocytopaenia is the dose-limiting toxicity for Navitoclax.^{12, 13, 14} This fact provided additional impetus for the development of agents that specifically target BCL-2, beginning with ABT-199,¹⁵ a BCL-2-selective antagonist currently in clinical trials for the treatment of a range of haematological malignancies including chronic lymphocytic leukaemia, non-Hodgkin''s lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma and acute myeloid leukaemia. ABT-199 has already shown very promising anti-tumour activity, with little to no impact on platelet counts.^{15, 16} These data suggest that BCL-2 is dispensable for the development and survival of platelets.In megakaryocytes, BCL-X_L is also critical for survival. Although not absolutely required for their growth and maturation, BCL-X_L is essential for megakaryocytes to proceed safely through pro-platelet formation and platelet shedding.⁵ In addition to BCL-X_L, megakaryocytes also depend on the pro-survival activity of MCL-1. Conditional deletion of Mcl1 alone has no effect on this lineage. In contrast, combined megakaryocyte-specific loss of Bclx and Mcl1 results in the failure of megakaryopoiesis, systemic haemorrhage and embryonic lethality.^{5, 17, 18} These defects are rescued by deletion of Bak and Bax.¹⁸Consistent with the genetic studies, administration of ABT-737 to Mcl1^Pf4Δ/Pf4Δ mice, which lack MCL-1 in megakaryocytes and platelets, induces acute, fulminant BAK/BAX-dependent megakaryocyte apoptosis. Given that, in addition to BCL-X_L, ABT-737 also targets BCL-2,⁷ these data suggested that BCL-2 might also contribute to the development and survival of the megakaryocyte lineage. This is supported by recent studies demonstrating that neonatal human platelets contain increased levels of BCL-2 relative to adult counterparts,¹⁹ and that platelet lifespan is extended in transgenic mice expressing BCL-2 under the control of the pan-haematopoietic Vav promoter.²⁰ In light of these observations, and intense ongoing activity surrounding the development of novel BH3 mimetics,²¹ we set out to elucidate the role of BCL-2 in megakaryocytes and platelets. Mice with a megakaryocyte-specific deletion of Bcl2, either alone or in combination with deletion of Mcl1 or Bclx, were generated. The effect of these mutations, and of BCL-2 or BCL-X_L-selective BH3 mimetics, on the megakaryocyte lineage was assessed.     

Overexpression of GLUT4 in mice causes up-regulation of UCP3 mRNA in skeletal muscle

Mitochondrial uncoupling protein 3 (UCP3) is expressed in skeletal muscles. We have hypothesized that increased glucose flux in skeletal muscles may lead to increased UCP3 expression. Male transgenic mice harboring insulin-responsive glucose transporter (GLUT4) minigenes with differing lengths of 5'-flanking sequence (-3237, -2000, -1000 and -442 bp) express different levels of GLUT4 protein in various skeletal muscles. Expression of the GLUT4 transgenes caused an increase in UCP3 mRNA that paralleled the increase of GLUT4 protein in gastrocnemius muscle. The effects of increased intracellular GLUT4 level on the expression of UCP1, UCP2 and UCP3 were compared in several tissues of male 4 month-old mice harboring the -1000 GLUT4 minigene transgene. In the -1000 GLUT4 transgenic mice, expression of GLUT4 mRNA and protein in skeletal muscles, brown adipose tissue (BAT), and white adipose tissue (WAT) was increased by 1.4 to 4.0-fold. Compared with non-transgenic littermates, the -1000 GLUT4 mice exhibited about 4- and 1.8-fold increases of UCP3 mRNA in skeletal muscle and WAT, respectively, and a 38% decrease of UCP1 mRNA in BAT. The transgenic mice had a 16% increase in oxygen consumption and a 14% decrease in blood glucose and a 68% increase in blood lactate, but no change in FFA or beta-OHB levels. T3 and leptin concentrations were decreased in transgenic mice. Expression of UCP1 in BAT of the -442 GLUT4 mice, which did not overexpress GLUT4 in this tissue, was not altered. These findings indicate that overexpression of GLUT4 up-regulates UCP3 expression in skeletal muscle and down-regulates UCP1 expression in BAT, possibly by increasing the rate of glucose uptake into these tissues.     

He hears,she hears: Are there sex differences in auditory processing?

Songbirds learn individually unique songs through vocal imitation and use them in courtship and territorial displays. Previous work has identified a forebrain auditory area, the caudomedial nidopallium (NCM), that appears specialized for discriminating and remembering conspecific vocalizations. In zebra finches (ZFs), only males produce learned vocalizations, but both sexes process these and other signals. This study assessed sex differences in auditory processing by recording extracellular multiunit activity at multiple sites within NCM. Juvenile female ZFs (n = 46) were reared in individual isolation and artificially tutored with song. In adulthood, songs were played back to assess auditory responses, stimulus‐specific adaptation, neural bias for conspecific song, and memory for the tutor's song, as well as recently heard songs. In a subset of females (n = 36), estradiol (E2) levels were manipulated to test the contribution of E2, known to be synthesized in the brain, to auditory responses. Untreated females (n = 10) showed significant differences in response magnitude and stimulus‐specific adaptation compared to males reared in the same paradigm (n = 9). In hormone‐manipulated females, E2 augmentation facilitated the memory for recently heard songs in adulthood, but neither E2 augmentation (n = 15) nor E2 synthesis blockade (n = 9) affected tutor song memory or the neural bias for conspecific song. The results demonstrate subtle sex differences in processing communication signals, and show that E2 levels in female songbirds can affect the memory for songs of potential suitors, thus contributing to the process of mate selection. The results also have potential relevance to clinical interventions that manipulate E2 in human patients. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 302–314, 2015     

Actinomycin D induced DNase I hypersensitivity and asymmetric structure transmission in a DNA hexadecamer.

DNase I cleavage rates and nmr chemical shifts are shown to change for DNA sequences distal to an intercalated actinomycin D molecule in a duplex hexadecamer upon drug binding. Both sets of observations suggest that the source of these changes is a DNA-mediated structural response. The nmr results imply the response is transmitted preferentially in a 5'-to-3' direction from the drug binding site. An inequivalent response of the two strands to a ligand-induced conformational change immediately suggests a mechanism for distinguishing the sense and antisense strands of DNA.     

Urogenital syndrome (us): a developmental mutation on Chromosome 2 of the mouse

Urogenital syndrome (us) is a recessive mutation in mice characterized primarily by abnormalities of the axial skeleton and urogenital organs. We established linkage of us with the centromeric end of Chromosome (Chr) 2, using the Robertsonian Chr Rb(2.8)2Lub. Analysis of progeny from crosses using the Chr 2 markers Danforth's short tail (Sd) and ulnaless (Ul) positioned us near two loci that have recently been mapped by RFLPs, nonerythroid -spectrin (Spna-2) and the paired-box-containing-gene-8 (Pax-8). The position of us relative to these loci was established by analysis of progeny from interspecific backcrosses between the us strains and Mus spretus. The estimated map distances and most likely gene order are centromere-Pax-8-2.1±1.2-us-0.7±0.7-Spna-2; however, the reverse order cannot be ruled out. Our data make it unlikely that us is a mutation in either Spna-2 or Pax-8. Spna-2 is close enough to us, however, to be a useful marker for positional cloning of the us gene. The human mutation Nail-patella-syndrome (NPS1) maps to the region of human Chr 9 (9q34) that is homologous to the us region of mouse Chr 2. Phenotypic similarities between the two syndromes suggest the possibility that they are caused by mutations at homologous loci.