Saliency processing and obesity: a preliminary imaging study of the stop signal task

Obesity has been associated with altered cerebral functions including cognitive control. The stop signal task (SST) has been widely used to study cognitive control by producing high conflict stop trials among many low conflict go trials. Contrasting these stop trials with go trials provides a measure of saliency processing and response inhibition. By comparing functional magnetic resonance images of obese (BMI >30) and lean (BMI <22) females performing the SST, we observed differences in regional brain activations despite similar behavioral performance between groups. Specifically, lean females had greater activations in the insula, inferior parietal cortex, cuneus, and supplementary motor area than obese females during stop as compared to go trials. This difference was caused by diminished brain activations in obese females in stop as compared to go trials. Furthermore, the brain activations in these regions inversely correlated to BMI across subjects. These preliminary findings suggest altered neural processes of cognitive control in obesity.     

Drug susceptibility in Leishmania isolates following miltefosine treatment in cases of visceral leishmaniasis and post kala-azar dermal leishmaniasis

Background

With widespread resistance to antimonials in Visceral Leishmaniasis (VL) in the Indian subcontinent, Miltefosine (MIL) has been introduced as the first line therapy. Surveillance of MIL susceptibility in natural populations of Leishmania donovani is vital to preserve it and support the VL elimination program.

Methodology and Principal Findings

We measured in vitro susceptibility towards MIL and paromomycin (PMM) in L. donovani isolated from VL and PKDL, pre- and post-treatment cases, using an amastigote-macrophage model. MIL susceptibility of post-treatment isolates from cured VL cases (n = 13, mean IC₅₀±SD = 2.43±1.44 µM), was comparable (p>0.05) whereas that from relapses (n = 3, mean IC₅₀ = 4.72±1.99 µM) was significantly higher (p = 0.04) to that of the pre-treatment group (n = 6, mean IC₅₀ = 1.86±0.75 µM). In PKDL, post-treatment isolates (n = 3, mean IC₅₀ = 16.13±2.64 µM) exhibited significantly lower susceptibility (p = 0.03) than pre-treatment isolates (n = 5, mean IC₅₀ = 8.63±0.94 µM). Overall, PKDL isolates (n = 8, mean IC₅₀ = 11.45±4.19 µM) exhibited significantly higher tolerance (p<0.0001) to MIL than VL isolates (n = 22, mean IC₅₀ = 2.58±1.58 µM). Point mutations in the miltefosine transporter (LdMT) and its beta subunit (LdRos3) genes previously reported in parasites with experimentally induced MIL resistance were not present in the clinical isolates. Further, the mRNA expression profile of these genes was comparable in the pre- and post-treatment isolates. Parasite isolates from VL and PKDL cases were uniformly susceptible to PMM with respective mean IC₅₀ = 7.05±2.24 µM and 6.18±1.51 µM.

Conclusion

The in vitro susceptibility of VL isolates remained unchanged at the end of MIL treatment; however, isolates from relapsed VL and PKDL cases had lower susceptibility than the pre-treatment isolates. PKDL isolates were more tolerant towards MIL in comparison with VL isolates. All parasite isolates were uniformly susceptible to PMM. Mutations in the LdMT and LdRos3 genes as well as changes in the expression of these genes previously correlated with experimental resistance to MIL could not be verified for the field isolates.     

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents ³. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells ¹¹ are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period ⁴. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.     

Mom—It Helps When You're Right Here! Attenuation of Neural Stress Markers in Anxious Youths Whose Caregivers Are Present during fMRI

Close proximity to an attachment figure, such as a caregiver, has been shown to attenuate threat-related activity in limbic regions such as the hypothalamus in healthy individuals. We hypothesized that such features might be similarly attenuated by proximity during a potentially stressful situation in a clinically anxious population of youths. Confirmation of this hypothesis could support the role of attachment figures in the management of anxiety among children and adolescents. Three groups were analyzed: anxious children and adolescents who requested that their caregiver accompany them in the scanner room, anxious children and adolescents without their caregiver in the scanner room and healthy controls (each of N = 10). The groups were matched for age and, among the two anxious groups, for diagnosis (mean age 9.5). The children and adolescents were exposed to physical threat words during an fMRI assessment. Results indicate that activity in the hypothalamus, ventromedial, and ventrolateral prefrontal cortex were significantly reduced in anxious children and adolescents who requested that their caregiver accompany them in the scanner room compared to those without their caregiver in the scanner room. Mean activity in these regions in anxious children and adolescents with their caregiver in the scanner room was comparable to that of healthy controls. These data suggest links between social contact and neural mechanisms of emotional reactivity; specifically, presence of caregivers moderates the increase in anxiety seen with stressful stimuli. Capitalizing on the ability of anxious youths to manifest low levels of anxiety-like information processing in the presence of a caregiver could help in modeling adaptive function in behavioral treatments.     

HIV Protease Inhibitors Do Not Cause the Accumulation of Prelamin A in PBMCs from Patients Receiving First Line Therapy: The ANRS EP45 “Aging” Study

Background

The ANRS EP45 “Aging” study investigates the cellular mechanisms involved in the accelerated aging of HIV-1 infected and treated patients. The present report focuses on lamin A processing, a pathway known to be altered in systemic genetic progeroid syndromes.

Methods

35 HIV-1 infected patients being treated with first line antiretroviral therapy (ART, mean duration at inclusion: 2.7±1.3 years) containing boosted protease inhibitors (PI/r) (comprising lopinavir/ritonavir in 65% of patients) were recruited together with 49 seronegative age- and sex-matched control subjects (http://clinicaltrials.gov/, {"type":"clinical-trial","attrs":{"text":"NCT01038999","term_id":"NCT01038999"}}NCT01038999). In more than 88% of patients, the viral load was <40 copies/ml and the CD4+ cell count was >500/mm³. Prelamin A processing in peripheral blood mononuclear cells (PBMCs) from patients and controls was analysed by western blotting at inclusion. PBMCs from patients were also investigated at 12 and 24 months after enrolment in the study. PBMCs from healthy controls were also incubated with boosted lopinavir in culture medium containing various concentrations of proteins (4 to 80 g/L).

Results

Lamin A precursor was not observed in cohort patient PBMC regardless of the PI/r used, the dose and the plasma concentration. Prelamin A was detected in PBMC incubated in culture medium containing a low protein concentration (4 g/L) but not in plasma (60–80 g/L) or in medium supplemented with BSA (40 g/L), both of which contain a high protein concentration.

Conclusions

Prelamin A processing abnormalities were not observed in PBMCs from patients under the PI/r first line regimen. Therefore, PI/r do not appear to contribute to lamin A-related aging in PBMCs. In cultured PBMCs from healthy donors, prelamin A processing abnormalities were only observed when the protein concentration in the culture medium was low, thus increasing the amount of PI available to enter cells.ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01038999","term_id":"NCT01038999"}}NCT01038999 http://clinicaltrials.gov/ct2/show/{"type":"clinical-trial","attrs":{"text":"NCT01038999","term_id":"NCT01038999"}}NCT01038999.     

C. elegans BLOC-1 Functions in Trafficking to Lysosome-Related Gut Granules

The human disease Hermansky-Pudlak syndrome results from defective biogenesis of lysosome-related organelles (LROs) and can be caused by mutations in subunits of the BLOC-1 complex. Here we show that C. elegans glo-2 and snpn-1, despite relatively low levels of amino acid identity, encode Pallidin and Snapin BLOC-1 subunit homologues, respectively. BLOC-1 subunit interactions involving Pallidin and Snapin were conserved for GLO-2 and SNPN-1. Mutations in glo-2 and snpn-1,or RNAi targeting 5 other BLOC-1 subunit homologues in a genetic background sensitized for glo-2 function, led to defects in the biogenesis of lysosome-related gut granules. These results indicate that the BLOC-1 complex is conserved in C. elegans. To address the function of C. elegans BLOC-1, we assessed the intracellular sorting of CDF-2::GFP, LMP-1, and PGP-2 to gut granules. We validated their utility by analyzing their mislocalization in intestinal cells lacking the function of AP-3, which participates in an evolutionarily conserved sorting pathway to LROs. BLOC-1(-) intestinal cells missorted gut granule cargo to the plasma membrane and conventional lysosomes and did not have obviously altered function or morphology of organelles composing the conventional lysosome protein sorting pathway. Double mutant analysis and comparison of AP-3(-) and BLOC-1(-) phenotypes revealed that BLOC-1 has some functions independent of the AP-3 adaptor complex in trafficking to gut granules. We discuss similarities and differences of BLOC-1 activity in the biogenesis of gut granules as compared to mammalian melanosomes, where BLOC-1 has been most extensively studied for its role in sorting to LROs. Our work opens up the opportunity to address the function of this poorly understood complex in cell and organismal physiology using the genetic approaches available in C. elegans.     

Skin TLR7 Triggering Promotes Accumulation of Respiratory Dendritic Cells and Natural Killer Cells

The TLR7 agonist imiquimod has been used successfully as adjuvant for skin treatment of virus-associated warts and basal cell carcinoma. The effects of skin TLR7 triggering on respiratory leukocyte populations are unknown. In a placebo-controlled experimental animal study we have used multicolour flow cytometry to systematically analyze the modulation of respiratory leukocyte subsets after skin administration of imiquimod. Compared to placebo, skin administration of imiquimod significantly increased respiratory dendritic cells (DC) and natural killer cells, whereas total respiratory leukocyte, alveolar macrophages, classical CD4+ T helper and CD8+ T killer cell numbers were not or only moderately affected. DC subpopulation analyses revealed that elevation of respiratory DC was caused by an increase of respiratory monocytic DC and CD11b(hi) DC subsets. Lymphocyte subpopulation analyses indicated a marked elevation of respiratory natural killer cells and a significant reduction of B lymphocytes. Analysis of cytokine responses of respiratory leukocytes after stimulation with Klebsiella pneumonia indicated reduced IFN-γ and TNF-α expression and increased IL-10 and IL-12p70 production after 7 day low dose skin TLR7 triggering. Additionally, respiratory NK cytotoxic activity was increased after 7d skin TLR7 triggering. In contrast, lung histology and bronchoalveolar cell counts were not affected suggesting that skin TLR7 stimulation modulated respiratory leukocyte composition without inducing overt pulmonary inflammation. These data suggest the possibility to modulate respiratory leukocyte composition and respiratory cytokine responses against pathogens like Klebsiella pneumonia through skin administration of a clinically approved TLR7 ligand. Skin administration of synthetic TLR7 ligands may represent a novel, noninvasive means to modulate respiratory immunity.     

PI3Kδ is essential for tumor clearance mediated by cytotoxic T lymphocytes

Background

PI3Kδ is a lipid kinase of the phosphoinositide 3-kinase class 1A family and involved in early signaling events of leukocytes regulating proliferation, differentiation and survival. Currently, several inhibitors of PI3Kδ are under investigation for the treatment of hematopoietic malignancies. In contrast to the beneficial effect of inhibiting PI3Kδ in tumor cells, several studies reported the requirement of PI3Kδ for the function of immune cells, such as natural killer and T helper cells. Cytotoxic T lymphocytes (CTLs) are essential for tumor surveillance. The scope of this study is to clarify the potential impact of PI3Kδ inhibition on the function of CTLs with emphasis on tumor surveillance.

Principal Findings

PI3Kδ-deficient mice develop significantly bigger tumors when challenged with MC38 colon adenocarcinoma cells. This defect is accounted for by the fact that PI3Kδ controls the secretory perforin-granzyme pathway as well as the death-receptor pathway of CTL-mediated cytotoxicity, leading to severely diminished cytotoxicity against target cells in vitro and in vivo in the absence of PI3Kδ expression. PI3Kδ-deficient CTLs express low mRNA levels of important components of the cytotoxic machinery, e.g. prf1, grzmA, grzmB, fasl and trail. Accordingly, PI3Kδ-deficient tumor-infiltrating CTLs display a phenotype reminiscent of naïve T cells (CD69^lowCD62L^high). In addition, electrophysiological capacitance measurements confirmed a fundamental degranulation defect of PI3Kδ−/− CTLs.

Conclusion

Our results demonstrate that CTL-mediated tumor surveillance is severely impaired in the absence of PI3Kδ and predict that impaired immunosurveillance may limit the effectiveness of PI3Kδ inhibitors in long-term treatment.