CARD9-Dependent Neutrophil Recruitment Protects against Fungal Invasion of the Central Nervous System

Candida is the most common human fungal pathogen and causes systemic infections that require neutrophils for effective host defense. Humans deficient in the C-type lectin pathway adaptor protein CARD9 develop spontaneous fungal disease that targets the central nervous system (CNS). However, how CARD9 promotes protective antifungal immunity in the CNS remains unclear. Here, we show that a patient with CARD9 deficiency had impaired neutrophil accumulation and induction of neutrophil-recruiting CXC chemokines in the cerebrospinal fluid despite uncontrolled CNS Candida infection. We phenocopied the human susceptibility in Card9 ^-/- mice, which develop uncontrolled brain candidiasis with diminished neutrophil accumulation. The induction of neutrophil-recruiting CXC chemokines is significantly impaired in infected Card9 ^-/- brains, from both myeloid and resident glial cellular sources, whereas cell-intrinsic neutrophil chemotaxis is Card9-independent. Taken together, our data highlight the critical role of CARD9-dependent neutrophil trafficking into the CNS and provide novel insight into the CNS fungal susceptibility of CARD9-deficient humans.     

Effective Treatment of Established GL261 Murine Gliomas through Picornavirus Vaccination-Enhanced Tumor Antigen-Specific CD8+ T Cell Responses

Glioblastoma (GBM) is among the most invasive and lethal of cancers, frequently infiltrating surrounding healthy tissue and giving rise to rapid recurrence. It is therefore critical to establish experimental model systems and develop therapeutic approaches that enhance anti-tumor immunity. In the current study, we have employed a newly developed murine glioma model to assess the efficacy of a novel picornavirus vaccination approach for the treatment of established tumors. The GL261-Quad system is a variation of the GL261 syngeneic glioma that has been engineered to expresses model T cell epitopes including OVA_257–264. MRI revealed that both GL261 and GL261-Quad tumors display characteristic features of human gliomas such as heterogeneous gadolinium leakage and larger T2 weighted volumes. Analysis of brain-infiltrating immune cells demonstrated that GL261-Quad gliomas generate detectable CD8+ T cell responses toward the tumor-specific K^b:OVA_257–264 antigen. Enhancing this response via a single intracranial or peripheral vaccination with picornavirus expressing the OVA_257–264 antigen increased anti-tumor CD8+ T cells infiltrating the brain, attenuated progression of established tumors, and extended survival of treated mice. Importantly, the efficacy of the picornavirus vaccination is dependent on functional cytotoxic activity of CD8+ T cells, as the beneficial response was completely abrogated in mice lacking perforin expression. Therefore, we have developed a novel system for evaluating mechanisms of anti-tumor immunity in vivo, incorporating the GL261-Quad model, 3D volumetric MRI, and picornavirus vaccination to enhance tumor-specific cytotoxic CD8+ T cell responses and track their effectiveness at eradicating established gliomas in vivo.     

Self-Reported Trait Mindfulness and Affective Reactivity: A Motivational Approach Using Multiple Psychophysiological Measures

As a form of attention, mindfulness is qualitatively receptive and non-reactive, and is thought to facilitate adaptive emotional responding. One suggested mechanism is that mindfulness facilitates disengagement from an affective stimulus and thereby decreases affective reactivity. However, mindfulness has been conceptualized as a state, intervention, and trait. Because evidence is mixed as to whether self-reported trait mindfulness decreases affective reactivity, we used a multi-method approach to study the relationship between individual differences in self-reported trait mindfulness and electrocortical, electrodermal, electromyographic, and self-reported responses to emotional pictures. Specifically, while participants (N = 51) passively viewed pleasant, neutral, and unpleasant IAPS pictures, we recorded high-density (128 channels) electrocortical, electrodermal, and electromyographic data to the pictures as well as to acoustic startle probes presented during the pictures. Afterwards, participants rated their subjective valence and arousal while viewing the pictures again. If trait mindfulness spontaneously reduces general emotional reactivity, then for individuals reporting high rather than low mindfulness, response differences between emotional and neutral pictures would show relatively decreased early posterior negativity (EPN) and late positive potential (LPP) amplitudes, decreased skin conductance responses, and decreased subjective ratings for valence and arousal. High mindfulness would also be associated with decreased emotional modulation of startle eyeblink and P3 amplitudes. Although results showed clear effects of emotion on the dependent measures, in general, mindfulness did not moderate these effects. For most measures, effect sizes were small with rather narrow confidence intervals. These data do not support the hypothesis that individual differences in self-reported trait mindfulness are related to spontaneous emotional responses during picture viewing.     

Body Composition QTLs Identified in Intercross Populations Are Reproducible in Consomic Mouse Strains

Genetic variation contributes to individual differences in obesity, but defining the exact relationships between naturally occurring genotypes and their effects on fatness remains elusive. As a step toward positional cloning of previously identified body composition quantitative trait loci (QTLs) from F₂ crosses of mice from the C57BL/6ByJ and 129P3/J inbred strains, we sought to recapture them on a homogenous genetic background of consomic (chromosome substitution) strains. Male and female mice from reciprocal consomic strains originating from the C57BL/6ByJ and 129P3/J strains were bred and measured for body weight, length, and adiposity. Chromosomes 2, 7, and 9 were selected for substitution because previous F₂ intercross studies revealed body composition QTLs on these chromosomes. We considered a QTL confirmed if one or both sexes of one or both reciprocal consomic strains differed significantly from the host strain in the expected direction after correction for multiple testing. Using these criteria, we confirmed two of two QTLs for body weight (Bwq5-6), three of three QTLs for body length (Bdln3-5), and three of three QTLs for adiposity (Adip20, Adip26 and Adip27). Overall, this study shows that despite the biological complexity of body size and composition, most QTLs for these traits are preserved when transferred to consomic strains; in addition, studying reciprocal consomic strains of both sexes is useful in assessing the robustness of a particular QTL.     

Targeting Ergosterol Biosynthesis in Leishmania donovani: Essentiality of Sterol 14alpha-demethylase

Leishmania protozoan parasites (Trypanosomatidae family) are the causative agents of cutaneous, mucocutaneous and visceral leishmaniasis worldwide. While these diseases are associated with significant morbidity and mortality, there are few adequate treatments available. Sterol 14alpha-demethylase (CYP51) in the parasite sterol biosynthesis pathway has been the focus of considerable interest as a novel drug target in Leishmania. However, its essentiality in Leishmania donovani has yet to be determined. Here, we use a dual biological and pharmacological approach to demonstrate that CYP51 is indispensable in L. donovani. We show via a facilitated knockout approach that chromosomal CYP51 genes can only be knocked out in the presence of episomal complementation and that this episome cannot be lost from the parasite even under negative selection. In addition, we treated wild-type L. donovani and CYP51-deficient strains with 4-aminopyridyl-based inhibitors designed specifically for Trypanosoma cruzi CYP51. While potency was lower than in T. cruzi, these inhibitors had increased efficacy in parasites lacking a CYP51 allele compared to complemented parasites, indicating inhibition of parasite growth via a CYP51-specific mechanism and confirming essentiality of CYP51 in L. donovani. Overall, these results provide support for further development of CYP51 inhibitors for the treatment of visceral leishmaniasis.     

Social Behavioral Deficits Coincide with the Onset of Seizure Susceptibility in Mice Lacking Serotonin Receptor 2c

The development of social behavior is strongly influenced by the serotonin system. Serotonin 2c receptor (5-HT_2cR) is particularly interesting in this context considering that pharmacological modulation of 5-HT_2cR activity alters social interaction in adult rodents. However, the role of 5-HT_2cR in the development of social behavior is unexplored. Here we address this using Htr2c knockout mice, which lack 5-HT_2cR. We found that these animals exhibit social behavior deficits as adults but not as juveniles. Moreover, we found that the age of onset of these deficits displays similar timing as the onset of susceptibility to spontaneous death and audiogenic-seizures, consistent with the hypothesis that imbalanced excitation and inhibition (E/I) may contribute to social behavioral deficits. Given that autism spectrum disorder (ASD) features social behavioral deficits and is often co-morbid with epilepsy, and given that 5-HT_2cR physically interacts with Pten, we tested whether a second site mutation in the ASD risk gene Pten can modify these phenotypes. The age of spontaneous death is accelerated in mice double mutant for Pten and Htr2c relative to single mutants. We hypothesized that pharmacological antagonism of 5-HT_2cR activity in adult animals, which does not cause seizures, might modify social behavioral deficits in Pten haploinsufficient mice. SB 242084, a 5-HT_2cR selective antagonist, can reverse the social behavior deficits observed in Pten haploinsufficient mice. Together, these results elucidate a role of 5-HT_2cR in the modulation of social behavior and seizure susceptibility in the context of normal development and Pten haploinsufficiency.     

INTROGRESSION IN THE DROSOPHILA SUBOBSCURA—D. MADEIRENSIS SISTER SPECIES: EVIDENCE OF GENE FLOW IN NUCLEAR GENES DESPITE MITOCHONDRIAL DIFFERENTIATION

Species hybridization, and thus the potential for gene flow, was once viewed as reproductive mistake. However, recent analysis based on large datasets and newly developed models suggest that gene exchange is not as rare as originally suspected. To investigate the history and speciation of the closely related species Drosophila subobscura, D. madeirensis, and D. guanche, we obtained polymorphism and divergence data for 26 regions throughout the genome, including the Y chromosome and mitochondrial DNA. We found that the D. subobscura X/autosome ratio of silent nucleotide diversity is significantly smaller than the 0.75 expected under neutrality. This pattern, if held genomewide, may reflect a faster accumulation of beneficial mutations on the X chromosome than on autosomes. We also detected evidence of gene flow in autosomal regions, while sex chromosomes remain distinct. This is consistent with the large X effect on hybrid male sterility seen in this system and the presence of two X chromosome inversions fixed between species. Overall, our data conform to chromosomal speciation models in which rearrangements are proposed to serve as gene flow barriers. Contrary to other observations in Drosophila, the mitochondrial genome appears resilient to gene flow in the presence of nuclear exchange.