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Adjuvants boost vaccine responses, enhancing protective immunity against infections that are most common among the very young. Many adjuvants activate innate immunity, some via Toll-Like Receptors (TLRs), whose activities varies with age. Accordingly, characterization of age-specific adjuvant-induced immune responses may inform rational adjuvant design targeting vulnerable populations. In this study, we employed proteomics to characterize the adjuvant-induced changes of secretomes from human newborn and adult monocytes in response to Alum, the most commonly used adjuvant in licensed vaccines; Monophosphoryl Lipid A (MPLA), a TLR4-activating adjuvant component of a licensed Human Papilloma Virus vaccine; and R848 an imidazoquinoline TLR7/8 agonist that is a candidate adjuvant for early life vaccines. Monocytes were incubated in vitro for 24 h with vehicle, Alum, MPLA, or R848 and supernatants collected for proteomic analysis employing liquid chromatography-mass spectrometry (LC-MS) (data available via ProteomeXchange, ID PXD003534). 1894 non-redundant proteins were identified, of which approximate to 30 - 40% were common to all treatment conditions and approximate to 5% were treatment-specific. Adjuvant-stimulated secretome profiles, as identified by cluster analyses of over-represented proteins, varied with age and adjuvant type. Adjuvants, especially Alum, activated multiple innate immune pathways as assessed by functional enrichment analyses. Release of lactoferrin, pentraxin 3, and matrix metalloproteinase-9 was confirmed in newborn and adult whole blood and blood monocytes stimulated with adjuvants alone or adjuvanted licensed vaccines with distinct clinical reactogenicity profiles. MPLA-induced adult monocyte secretome profiles correlated in silico with transcriptome profiles induced in adults immunized with the MPLA-adjuvanted RTS,S malaria vaccine (Mosquirix). Overall, adjuvants such as Alum, MPLA and R848 give rise to distinct and age-specific monocyte secretome profiles, paralleling responses to adjuvant-containing vaccines in vivo. Age-specific in vitro modeling coupled with proteomics may provide fresh insight into the ontogeny of adjuvant action thereby informing targeted adjuvanted vaccine development for distinct age groups.

It is well accepted that junctophilin (JPHs) isoforms act as a physical bridge linking plasma membrane and endoplasmic reticulum (ER) for channel crosstalk in excitable cells. Our purpose is to investigate whether JPHs are involved in the proper communication between Ca2+ influx and subsequent Ca2+ amplification in pancreatic beta cells, thereby participating in regulating insulin secretion. The expression of JPH isoforms was examined in human and mouse pancreatic tissues, and JPH3 expression was found in both the beta cells. In mice, knockdown of Jph3 (si-Jph3) in islets decreased glucose-stimulated insulin secretion (GSIS) accompanied by mitochondrial function impairment. Si-Jph3 lowered the insulin secretory response to Ca2+ signaling in the presence of glucose, and reduced [Ca2+] c transient amplitude triggered by caffeine. Si-Jph3 also attenuated mitofusin 2 expression, thereby disturbing the spatial organization of ER-mitochondria contact in islets. These results suggest that the regulation of GSIS by the KATP channel-independent pathways is partly impaired due to decrease of JPH3 expression in mouse islets. JPH3 also binds to type 2 ryanodine receptors (RyR2) in mouse and human pancreatic tissues, which might contribute to Ca2+ release amplification in GSIS. This study demonstrates some previously unrecognized findings in pancreatic tissues: (1) JPH3 expresses in mouse and human beta cells; (2) si-Jph3 in mouse primary islets impairs GSIS in vitro; (3) impairment in GSIS in si-Jph3 islets is due to changes in RyR2-[Ca2+] c transient amplitude and ER-mitochondria contact.

Vocalizations produced by developing young early in life have simple acoustic features and are thought to be innate. Complex forms of early vocal learning are less likely to evolve in young altricial songbirds because the forebrain vocal-learning circuit is underdeveloped during the period when early vocalizations are produced. However, selective pressure experienced in early postnatal life may lead to early vocal learning that is likely controlled by a simpler brain circuit. We found the food begging calls produced by fledglings of the brown-headed cowbird (Molothrus ater), a generalist avian brood parasite, induced the expression of several immediate early genes and early circuit innervation in a forebrain vocal-motor pathway that is later used for vocal imitation. The forebrain neural activity was correlated with vocal intensity and variability of begging calls that appears to allow cowbirds to vocally match host nestmates. The begging-induced forebrain circuits we observed in fledgling cowbirds were not detected in nonparasitic passerines, including species that are close relatives to the cowbird. The involvement of forebrain vocal circuits during fledgling begging and its association with vocal learning plasticity may be an adaptation that provides young generalist brood parasites with a flexible signaling strategy to procure food from a wide range of heterospecific host parents. (c) 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 615-625, 2016

Background: Inborn errors of interferon-gamma (IFN-gamma)-mediated immunity underlie disseminated disease caused by Mycobacterium bovis Bacillus Calmette-Guerin (BCG) live vaccines. We hypothesized that some patients with osteitis after BCG vaccination may have an impaired IFN-gamma immunity. Our aim was to investigate interleukin (IL)-12 and IFN-gamma ex vivo production stimulated with BCG and BCG + IFN-gamma or BCG + IL-12, respectively, in BCG osteitis survivors. Methods: Fresh blood samples were collected from 132 former BCG osteitis Finnish patients now aged 21-49 years, and IL-12 and IFN-gamma were measured in cell cultures with and without stimulation with BCG and with BCG + IFN-gamma or BCG + IL-12, respectively. As a pilot study, known disease-causing genes controlling IFN-gamma immunity (IFNGR1, IFNGR2, STAT1, IL12B, IL12RB1, ISG15, IRF8, NEMO and CYBB) were investigated in 20 selected patients by whole exome sequencing. Results: By the limit of <5th percentile, ex vivo IL-12 concentration and increase in concentration was low in 5 and ex vivo IFN-gamma concentration and increase in concentration was low in 6 patients (including 2 samples with both IL-12 and IFN-gamma findings). By the limit of <10th percentile, an additional 6 and 4 patients were, respectively, detected (including 2 samples with both findings). With 2 exceptions, low concentrations and low increases in concentrations picked-up the same cases. Mutations in known disease-causing IFN-gamma-related genes were not found in any of these patients. Conclusion: These findings call for searching of mutations in new genes governing IFN-gamma-dependent immunity to live BCG vaccine.

Staphylococcus haemolyticus is one of the most common pathogens associated with medical-device related infections, but its molecular epidemiology is poorly explored. In the current study, we aimed to better understand the genetic mechanisms contributing to S. haemolyticus diversity in the hospital environment and their impact on the population structure and clinical relevant phenotypic traits. The analysis of a representative S. haemolyticus collection by multilocus sequence typing (MLST) has identified a single highly prevalent and diverse genetic lineage of nosocomial S. haemolyticus clonal complex (CC) 29 accounting for 91% of the collection of isolates disseminated worldwide. The examination of the sequence changes at MLST loci during clonal diversification showed that recombination had a higher impact than mutation in shaping the S. haemolyticus population. Also, we ascertained that another mechanism contributing significantly to clonal diversification and adaptation was mediated by insertion sequence (IS) elements. We found that all nosocomial S. haemolyticus, belonging to different STs, were rich in IS1272 copies, as determined by Southern hybridization of macrorestriction patterns. In particular, we observed that the chromosome of a S. haemolyticus strain within CC29 was highly unstable during serial growth in vitro which paralleled with IS1272 transposition events and changes in clinically relevant phenotypic traits namely, mannitol fermentation, susceptibility to beta-lactams, biofilm formation and hemolysis. Our results suggest that recombination and IS transposition might be a strategy of adaptation, evolution and pathogenicity of the major S. haemolyticus prevalent lineage in the hospital environment.

Parasites have long been known to influence host responses to infection through the secretion of virulence factors. Extracellular vesicles are emerging as important mediators of these manipulations, and a new study by Szempruch et al, suggests they could play a crucial role in host responses to African trypanosome infections.

Cerebellar degeneration-related protein 2 (cdr2) is expressed in the central nervous system, and its ectopic expression in tumor cells of patients with gynecological malignancies elicits immune responses by cdr2-specific autoantibodies and T lymphocytes, leading to neurological symptoms. However, little is known about the regulation and function of cdr2 in neurodegenerative diseases. Because we found that cdr2 is highly expressed in the midbrain, we investigated the role of cdr2 in experimental models of Parkinson's disease (PD). We found that cdr2 levels were significantly reduced after stereotaxic injection of 1-methyl-4-phenylpyridinium (MPP+) into the striatum. cdr2 levels were also decreased in the brains of post-mortem PD patients. Using primary cultures of mesencephalic neurons and MN9D cells, we confirmed that MPP+ reduces cdr2 in tyrosine hydroxylase-positive dopaminergic neuronal cells. The MPP+-induced decrease of cdr2 was primarily caused by calpain-and ubiquitin proteasome system-mediated degradation, and cotreatment with pharmacological inhibitors of these enzymes or overexpression of calcium-binding protein rendered cells less vulnerable to MPP+-mediated cytotoxicity. Consequently, overexpression of cdr2 rescued cells from MPP+-induced cytotoxicity, whereas knockdown of cdr2 accelerated toxicity. Collectively, our findings provide insights into the novel regulatory mechanism and potentially protective role of onconeural protein during dopaminergic neurodegeneration.

Uveal melanomas are molecularly distinct from cutaneous melanomas and lack mutations in BRAF, NRAS, KIT, and NF1. Instead, they are characterized by activating mutations in GNAQ and GNA11, two highly homologous alpha subunits of G(alpha q/11) heterotrimeric G proteins, and in PLCB4 (phospholipase C beta 4), the downstream effector of G(alpha q) signaling(1-3). We analyzed genomics data from 136 uveal melanoma samples and found a recurrent mutation in CYSLTR2 (cysteinyl leukotriene receptor 2) encoding a p.Leu129Gln substitution in 4 of 9 samples that lacked mutations in GNAQ, GNA11, and PLCB4 but in 0 of 127 samples that harbored mutations in these genes. The Leu129Gln CysLT(2)R mutant protein constitutively activates endogenous G(alpha q) and is unresponsive to stimulation by leukotriene. Expression of Leu129Gln CysLT(2)R in melanocytes enforces expression of a melanocyte-lineage signature, drives phorbol ester-independent growth in vitro, and promotes tumorigenesis in vivo. Our findings implicate CYSLTR2 as a uveal melanoma oncogene and highlight the critical role of G(alpha q) signaling in uveal melanoma pathogenesis.

Antimetabolites that affect nucleotide metabolism are frontline chemotherapy agents in several cancers and often successfully target one carbon metabolism. However, the precise mechanisms and resulting determinants of their therapeutic value are unknown. We show that 5-fluorouracil (5-FU), a commonly used antimetabolite therapeutic with varying efficacy, induces specific alterations to nucleotide metabolism by disrupting pyrimidine homeostasis. An integrative metabolomics analysis of the cellular response to 5-FU reveals intracellular uracil accumulation, whereas deoxyuridine levels exhibited increased flux into the extracellular space, resulting in an induction of overflow metabolism. Subsequent analysis from mice bearing colorectal tumors treated with 5-FU show specific secretion of metabolites in tumor-bearing mice into serum that results from alterations in nucleotide flux and reduction in overflowmetabolism. Together, these findings identify a determinant of an antimetabolite response that may be exploited to more precisely define the tumors that could respond to targeting cancer metabolism.

The human brain has enormously complex cellular diversity and connectivities fundamental to our neural functions, yet difficulties in interrogating individual neurons has impeded understanding of the underlying transcriptional landscape. We developed a scalable approach to sequence and quantify RNA molecules in isolated neuronal nuclei from a postmortem brain, generating 3227 sets of single-neuron data from six distinct regions of the cerebral cortex. Using an iterative clustering and classification approach, we identified 16 neuronal subtypes that were further annotated on the basis of known markers and cortical cytoarchitecture. These data demonstrate a robust and scalable method for identifying and categorizing single nuclear transcriptomes, revealing shared genes sufficient to distinguish previously unknown and orthologous neuronal subtypes as well as regional identity and transcriptomic heterogeneity within the human brain.