Endogenous human cytomegalovirus gB is efficiently presented by MHC class II molecules to CD4+ CTL

Human cytomegalovirus (HCMV) infects endothelial, epithelial, and glial cells in vivo. These cells can express MHC class II proteins, but are unlikely to play important roles in priming host immunity. Instead, it seems that class II presentation of endogenous HCMV antigens in these cells allows recognition of virus infection. We characterized class II presentation of HCMV glycoprotein B (gB), a membrane protein that accumulates extensively in endosomes during virus assembly. Human CD4+ T cells specific for gB were both highly abundant in blood and cytolytic in vivo. gB-specific CD4+ T cell clones recognized gB that was expressed in glial, endothelial, and epithelial cells, but not exogenous gB that was fed to these cells. Glial cells efficiently presented extremely low levels of endogenous gB--expressed by adenovirus vectors or after HCMV infection--and stimulated CD4+ T cells better than DCs that were incubated with exogenous gB. Presentation of endogenous gB required sorting of gB to endosomal compartments and processing by acidic proteases. Although presentation of cellular proteins that traffic into endosomes is well known, our observations demonstrate for the first time that a viral protein sorted to endosomes is presented exceptionally well, and can promote CD4+ T cell recognition and killing of biologically important host cells

The Mycobacterium tuberculosis Phagosome Is a HLA-I Processing Competent Organelle

Mycobacterium tuberculosis (Mtb) resides in a long-lived phagosomal compartment that resists maturation. The manner by which Mtb antigens are processed and presented on MHC Class I molecules is poorly understood. Using human dendritic cells and IFN-γ release by CD8+ T cell clones, we examined the processing and presentation pathway for two Mtb–derived antigens, each presented by a distinct HLA-I allele (HLA-Ia versus HLA-Ib). Presentation of both antigens is blocked by the retrotranslocation inhibitor exotoxin A. Inhibitor studies demonstrate that, after reaching the cytosol, both antigens require proteasomal degradation and TAP transport, but differ in the requirement for ER–golgi egress and new protein synthesis. Specifically, presentation by HLA-B8 but not HLA-E requires newly synthesized HLA-I and transport through the ER–golgi. Phenotypic analysis of the Mtb phagosome by flow organellometry revealed the presence of Class I and loading accessory molecules, including TAP and PDI. Furthermore, loaded HLA-I:peptide complexes are present within the Mtb phagosome, with a pronounced bias towards HLA-E:peptide complexes. In addition, protein analysis also reveals that HLA-E is enriched within the Mtb phagosome compared to HLA-A2. Together, these data suggest that the phagosome, through acquisition of ER–localized machinery and as a site of HLA-I loading, plays a vital role in the presentation of Mtb–derived antigens, similar to that described for presentation of latex bead-associated antigens. This is, to our knowledge, the first description of this presentation pathway for an intracellular pathogen. Moreover, these data suggest that HLA-E may play a unique role in the presentation of phagosomal antigens

Endosomal MR1 Trafficking Plays a Key Role in Presentation of Mycobacterium tuberculosis Ligands to MAIT Cells

Mucosal-Associated Invariant T (MAIT) cells, present in high frequency in airway and other mucosal tissues, have Th1 effector capacity positioning them to play a critical role in the early immune response to intracellular pathogens, including Mycobacterium tuberculosis (Mtb). MR1 is a highly conserved Class I-like molecule that presents vitamin B metabolites to MAIT cells. The mechanisms for loading these ubiquitous small molecules are likely to be tightly regulated to prevent inappropriate MAIT cell activation. To define the intracellular localization of MR1, we analyzed the distribution of an MR1-GFP fusion protein in antigen presenting cells. We found that MR1 localized to endosomes and was translocated to the cell surface upon addition of 6-formyl pterin (6-FP). To understand the mechanisms by which MR1 antigens are presented, we used a lentiviral shRNA screen to identify trafficking molecules that are required for the presentation of Mtb antigen to HLA-diverse T cells. We identified Stx18, VAMP4, and Rab6 as trafficking molecules regulating MR1-dependent MAIT cell recognition of Mtb-infected cells. Stx18 but not VAMP4 or Rab6 knockdown also resulted in decreased 6-FP-dependent surface translocation of MR1 suggesting distinct pathways for loading of exogenous ligands and intracellular mycobacterially-derived ligands. We postulate that endosome-mediated trafficking of MR1 allows for selective sampling of the intracellular environment.Career Development Award: (#IK2 CX000538); U.S. Department of Veterans Affairs Clinical Sciences Research and Development Program (MJH); U.S.Department of Veterans Affairs Biomedical Laboratory Research and Development Program (DML) Merit Award: (#I01 BX000533); American Lung Association: (RT-350058)

Human Neonatal Dendritic Cells Are Competent in MHC Class I Antigen Processing and Presentation

Neonates are clearly more susceptible to severe disease following infection with a variety of pathogens than are adults. However, the causes for this are unclear and are often attributed to immunological immaturity. While several aspects of immunity differ between adults and neonates, the capacity of dendritic cells in neonates to process and present antigen to CD8+ T cells remains to be addressed. We used human CD8+ T cell clones to compare the ability of neonatal and adult monocyte-derived dendritic cells to present or process and present antigen using the MHC class I pathway. Specifically, we assessed the ability of dendritic cells to present antigenic peptide, present an HLA-E–restricted antigen, process and present an MHC class I-restricted antigen through the classical MHC class I pathway, and cross present cell-associated antigen via MHC class I. We found no defect in neonatal dendritic cells to perform any of these processing and presentation functions and conclude that the MHC class I antigen processing and presentation pathway is functional in neonatal dendritic cells and hence may not account for the diminished control of pathogens

Interpretation of ambiguous situations: evidence for a dissociation between social and physical threat in Williams syndrome

There is increasing evidence that Williams syndrome (WS) is associated with elevated anxiety that is non-social in nature, including generalised anxiety and fears. To date very little research has examined the cognitive processes associated with this anxiety. In the present research, attentional bias for non-social threatening images in WS was examined using a dot-probe paradigm. Participants were 16 individuals with WS aged between 13 and 34 years and two groups of typically developing controls matched to the WS group on chronological age and attentional control ability respectively. The WS group exhibited a significant attention bias towards threatening images. In contrast, no bias was found for group matched on attentional control and a slight bias away from threat was found in the chronological age matched group. The results are contrasted with recent findings suggesting that individuals with WS do not show an attention bias for threatening faces and discussed in relation to neuroimaging research showing elevated amygdala activation in response to threatening non-social scenes in WS

The Adolescent Depression Rating Scale (ADRS): a validation study

BACKGROUND: To examine the psychometric properties of the Adolescent Depression Rating Scale (ADRS), a new measure was specifically designed to evaluate adolescent depression. METHODS: The 11-item clinician-report and 44-item self-report versions of the ADRS were developed from a qualitative phase involving interviews of experts and adolescents. These two instruments were then administered to 402 French speaking adolescents with and without depressive disorders. Item distribution, internal consistency, convergent validity, discriminant validity and factorial structure were assessed. RESULTS: After reduction procedures, a 10-item clinician version and a 10-item self-report version were obtained. The ADRS demonstrated good internal consistency (alpha Cronbach coefficient >.70). It also discriminated better between adolescents with and without depression than the Hamilton Depressive Rating Scale and the Beck Depression Inventory (BDI-13). CONCLUSION: The ADRS is a useful, short, clinician-report and self-report scale to evaluate adolescent depression. Further studies to replicate our findings and evaluate ADRS sensitivity to effects of treatment and psychometric properties in populations of adolescents with several psychiatric disorders are warranted

QuantiFERON®-TB gold in-tube performance for diagnosing active tuberculosis in children and adults in a high burden setting.

To determine whether QuantiFERON®-TB Gold In-Tube (QFT) can contribute to the diagnosis of active tuberculosis (TB) in children in a high-burden setting and to assess the performance of QFT and tuberculin skin test (TST) in a prospective cohort of TB suspect children compared to adults with confirmed TB in Tanzania. Sensitivity and specificity of QFT and TST for diagnosing active TB as well as indeterminate QFT rates and IFN-γ levels were assessed in 211 TB suspect children in a Tanzanian district hospital and contrasted in 90 adults with confirmed pulmonary TB. Sensitivity of QFT and TST in children with confirmed TB was 19% (5/27) and 6% (2/31) respectively. In adults sensitivity of QFT and TST was 84% (73/87) and 85% (63/74). The QFT indeterminate rate in children and adults was 27% and 3%. Median levels of IFN-γ were lower in children than adults, particularly children <2 years and HIV infected. An indeterminate result was associated with age <2 years but not malnutrition or HIV status. Overall childhood mortality was 19% and associated with an indeterminate QFT result at baseline. QFT and TST showed poor performance and a surprisingly low sensitivity in children. In contrast the performance in Tanzanian adults was good and comparable to performance in high-income countries. Indeterminate results in children were associated with young age and increased mortality. Neither test can be recommended for diagnosing active TB in children with immature or impaired immunity in a high-burden setting

Rapid Immunomagnetic Negative Enrichment of Neutrophil Granulocytes from Murine Bone Marrow for Functional Studies In Vitro and In Vivo

Polymorphonuclear neutrophils (PMN) mediate early immunity to infection but can also cause host damage if their effector functions are not controlled. Their lack or dysfunction is associated with severe health problems and thus the analysis of PMN physiology is a central issue. One prerequisite for PMN analysis is the availability of purified cells from primary organs. While human PMN are easily isolated from peripheral blood, this approach is less suitable for mice due to limited availability of blood. Instead, bone marrow (BM) is an easily available reservoir of murine PMN, but methods to obtain pure cells from BM are limited. We have developed a novel protocol allowing the isolation of highly pure untouched PMN from murine BM by negative immunomagnetic isolation using a complex antibody cocktail. The protocol is simple and fast (∼1 h), has a high yield (5–10*106 PMN per animal) and provides a purity of cells equivalent to positive selection (>80%). Most importantly, cells obtained by this method are non-activated and remain fully functional in vitro or after adoptive transfer into recipient animals. This method should thus greatly facilitate the study of primary murine PMN in vitro and in vivo

Depressive symptom trajectories among girls in the juvenile justice system: 24-month outcomes of an RCT of Multidimensional Treatment Foster Care

Youth depression is a significant and growing international public health problem. Youth who engage in high levels of delinquency are at particularly high risk for developing problems with depression. The present study examined the impact of a behavioral intervention designed to reduce delinquency (Multidimensional Treatment Foster Care; MTFC) compared to a group care intervention (GC; i.e., services as usual) on trajectories of depressive symptoms among adolescent girls in the juvenile justice system. MTFC has documented effects on preventing girls' recidivism, but its effects on preventing the normative rise in girls' depressive symptoms across adolescence have not been examined. This indicated prevention sample included 166 girls (13-17 years at T1) who had at least one criminal referral in the past 12 months and who were mandated to out-of-home care; girls were randomized to MTFC or GC. Intent-to-treat analyses examined the main effects of MTFC on depression symptoms and clinical cut-offs, and whether benefits were greatest for girls most at risk. Depressive symptom trajectories were specified in hierarchical linear growth models over a 2 year period using five waves of data at 6 month intervals. Depression clinical cut-off scores were specified as nonlinear probability growth models. Results showed significantly greater rates of deceleration for girls in MTFC versus GC for depressive symptoms and for clinical cut-off scores. The MTFC intervention also showed greater benefits for girls with higher levels of initial depressive symptoms. Possible mechanisms of effect are discussed, given MTFC's effectiveness on targeted and nontargeted outcomes. © 2013 Society for Prevention Research