Dengue virus (DENV) is the most prevalent arthropod-borne viral disease in humans. DENV causes a spectrum of illness ranging from mild to potentially severe complications. Dendritic cells (DCs) play a critical role in initiating and regulating highly effective antiviral immune response that include linking innate and adaptive immune responses. This study was conducted to comparatively characterize in detail the relative proportion, phenotypic changes, and maturation profile of subsets of both myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) in children with dengue fever (DF), dengue hemorrhagic fever (DHF) and for purposes of control healthy individuals. The mDCs (Lin-CD11c+CD123lo), the pDCs (Lin-CD11c-CD123+) and the double negative (DN) subset (Lin-/HLA-DR+/CD11c-CD123-) were analyzed by polychromatic flow cytometry. The data were first analyzed on blood samples collected from DENV-infected patients at various times post-infection. Results showed that the relative proportion of mDCs were significantly decreased which was associated with an increase in disease severity in samples from DENV-infected patients. While there was no significant difference in the relative proportion of pDCs between healthy and DENV-infected patients, there was a marked increase in the DN subset. Analysis of the kinetics of changes of pDCs showed that there was an increase but only during the early febrile phase. Additionally, samples from patients during acute disease showed marked decreases in the relative proportion of CD141+and CD16+mDC subsets that were the major mDC subsets in healthy individuals. In addition, there was a significant decrease in the level of CD33-expressing mDCs in DENV patients. While the pDCs showed an up-regulation of maturation profile during acute DENV infection, the mDCs showed an alteration of maturation status. This study suggests that different relative proportion and phenotypic changes as well as alteration of maturation profile of DC subsets may play a critical role in the dengue pathogenesis and disease outcome.
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Diane G. Carnathan;
Katherine S. Wetzel;
Joana Yu;
S. Thera Lee;
Brent A. Johnson;
Mirko Paiardini;
Jian Yan;
Matthew P. Morrow;
Niranjan Y. Sardesai;
David B. Weiner;
Hildegund C. J. Ertl;
Guido Silvestri
An effective T-cell-based AIDS vaccine should induce strong HIV-specific CD8+T cells in mucosal tissues without increasing the availability of target cells for the virus. Here, we evaluated five immunization strategies that include Human adenovirus-5 (AdHu5), Chimpanzee adenovirus-6 (AdC6) or -7 (AdC7), Vaccinia virus (VV), and DNA given by electroporation (DNA/EP), all expressing Simian immunodeficiency virus group specific antigen/transactivator of transcription (SIVmac239Gag/Tat). Five groups of six rhesus macaques (RMs) each were vaccinated with DNA/EP-AdC6-AdC7, VV-AdC6-AdC7, DNA/-EPVV- AdC6, DNA/EP-VV-AdC7, or AdHu5-AdHu5-AdHu5 and were challenged repeatedly with low-dose intrarectal SIVmac239. Upon challenge, there were no significant differences among study groups in terms of virus acquisition or viral load after infection. When taken together, the immunization regimens did not protect against SIV acquisition compared with controls but did result in an ∼1.6-log decline in set-point viremia. Although all immunized RMs had detectable SIVspecific CD8+ T cells in blood and rectal mucosa, we found no correlation between the number or function of these SIV-specific CD8+ T cells and protection against SIV acquisition. Interestingly, RMs experiencing breakthrough infection showed significantly higher prechallenge levels of CD4+C-C chemokine receptor type 5 (CCR5)+HLA-DR+ T cells in the rectal biopsies (RB) than animals that remained uninfected. In addition, among the infected RMs, the percentage of CD4+CCR5+Ki- 67+ T cells in RBs prechallenge correlated with higher early viremia. Overall, these data suggest that the levels of activated CD4+CCR5+ target T cells in the rectal mucosa may predict the risk of SIV acquisition in RMs vaccinated with vectors that express SIVGag/Tat.
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Jian Bao;
Min Qin;
Yacoubou Abdoul Razak Mahaman;
Bin Zhang;
Fang Huang;
Kuan Zeng;
Yiyuan Xia;
Dan Ke;
Qun Wang;
ROng Liu;
Lian-Zhi Wang;
Keqiang Ye;
Xiaochuan Wang
Amyloid beta (Aβ) is a major pathological marker in Alzheimer's disease (AD), which is principally regulated by the rate-limiting β-secretase (i.e., BACE1) cleavage of amyloid precursor protein (APP). However, how BACE1 activity is posttranslationally regulated remains incompletely understood. Here, we show that BACE1 is predominantly SUMOylated at K501 residue, which escalates its protease activity and stability and subsequently increases Aβ production, leading to cognitive defect seen in the AD mouse model. Compared with a non-SUMOylated K501R mutant, injection of wild-type BACE1 significantly increases Aβ production and triggers cognitive dysfunction. Furthermore, overexpression of wild-type BACE1, but not non-SUMOylated K501R mutant, facilitates senile plaque formation and aggravates the cognitive deficit seen in the APP/PS1 AD mouse model. Together, our data strongly suggest that K501 SUMOylation on BACE1 plays a critical role in mediating its stability and enzymatic activity.
Ribonucleoprotein (RNP) granules are higher order assemblies of RNA, RNA-binding proteins, and other proteins, that regulate the transcriptome and protect RNAs from environmental challenge. There is a diverse range of RNP granules, many cytoplasmic, which provide various levels of regulation of RNA metabolism. Here we present evidence that the yeast transcription termination factor, Nab3, is targeted to intranuclear granules in response to glucose starvation by Nab3's proline/glutamine-rich, prion-like domain (PrLD) which can assemble into amyloid in vitro. Localization to the granule is reversible and sensitive to the chemical probe 1,6 hexanediol suggesting condensation is driven by phase separation. Nab3's RNA recognition motif is also required for localization as seen for other PrLD-containing RNA-binding proteins that phase separate. Although the PrLD is necessary, it is not sufficient to localize to the granule. A heterologous PrLD that functionally replaces Nab3's essential PrLD, directed localization to the nuclear granule, however a chimeric Nab3 molecule with a heterologous PrLD that cannot restore termination function or viability, does not form granules. The Nab3 nuclear granule shows properties similar to well characterized cytoplasmic compartments formed by phase separation, suggesting that, as seen for other elements of the transcription machinery, termination factor condensation is functionally important.
The assembly and release of retroviruses from the host cells requires a coordinated series of interactions between viral structural proteins and cellular trafficking pathways. Although a number of cellular factors involved in retrovirus assembly have been identified, it is likely that retroviruses utilize additional trafficking factors to expedite their assembly and budding that have not yet been defined. We performed a screen using an siRNA library targeting host membrane trafficking genes in order to identify new host factors that contribute to retrovirus assembly or release. We utilized two retroviruses that follow very distinct assembly pathways, HIV-1 and Mason-Pfizer monkey virus (M-PMV) in order to identify host pathways that are generally applicable in retrovirus assembly versus those that are unique to HIV or M-PMV. Here we report the identification of 24 host proteins identified in the screen and subsequently validated in follow-up experiments as contributors to the assembly or release of both viruses. In addition to identifying a number of previously unsuspected individual trafficking factors, we noted multiple hits among proteins involved in modulation of the actin cytoskeleton, clathrin-mediated transport pathways, and phosphoinositide metabolism. Our study shows that distant genera of retroviruses share a number of common interaction strategies with host cell trafficking machinery, and identifies new cellular factors involved in the late stages of retroviral replication.
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Flora Mikaeloff;
Sara Svensson Akusjarvi;
George M Ikomey;
Shuba Krishnan;
Maike Sperk;
Soham Gupta;
Gustavo Daniel Vega Magdaleno;
Alejandra Escos;
Emilia Lyonga;
Marie Claire Okomo;
Claude Tayou Tagne;
Hemalatha Babu;
Christian L Lorson;
Ákos Vegvari;
Akhil C Banerjea;
Julianna Kele;
Luke Elizabeth Hanna;
Kamal Singh;
João Pedro de Magalhaes;
Rui Benfeitas;
Ujjwal Neogi
Despite successful combination antiretroviral therapy (cART), persistent low-grade immune activation together with inflammation and toxic antiretroviral drugs can lead to long-lasting metabolic flexibility and adaptation in people living with HIV (PLWH). Our study investigated alterations in the plasma metabolic profiles by comparing PLWH on long-term cART(>5 years) and matched HIV-negative controls (HC) in two cohorts from low- and middle-income countries (LMIC), Cameroon, and India, respectively, to understand the system-level dysregulation in HIV-infection. Using untargeted and targeted LC-MS/MS-based metabolic profiling and applying advanced system biology methods, an altered amino acid metabolism, more specifically to glutaminolysis in PLWH than HC were reported. A significantly lower level of neurosteroids was observed in both cohorts and could potentiate neurological impairments in PLWH. Further, modulation of cellular glutaminolysis promoted increased cell death and latency reversal in pre-monocytic HIV-1 latent cell model U1, which may be essential for the clearance of the inducible reservoir in HIV-integrated cells.
Vaccines for many important diseases remain elusive, and those for others need to be updated frequently. Vaccine efficacy has been hindered by existing sequence diversity in proteins and by newly-acquired mutations that enable escape from vaccine-induced immune responses. To address these limitations, we developed an approach for nanopatterning protein antigens that combines the site-specific incorporation of non-canonical amino acids with chemical modification to focus the immune response on conserved protein regions. We demonstrated the approach using green fluorescent protein (GFP) as a model antigen and with a promising malaria vaccine candidate, Merozoite surface protein 119 (MSP119). Immunization of mice with nanopatterned MSP119 elicited antibodies that recognized MSP119 from heterologous strains, differing in sequence at as many as 21 of 96 residues. Nanopatterning should enable the elicitation of broadly protective antibodies against a wide range of pathogens and toxins.
Prevention of HIV acquisition and replication requires long lasting and effective immunity. Given the state of HIV vaccine development, innovative vectors and immunization strategies are urgently needed to generate safe and efficacious HIV vaccines. Here, we developed a novel lentivirus-based DNA vector that does not integrate in the host genome and undergoes a single-cycle of replication. Viral proteins are constitutively expressed under the control of Tat-independent LTR promoter from goat lentivirus. We immunized six macaques once only with CAL-SHIV-IN- DNA using combined intramuscular and intradermal injections plus electroporation. Antigen-specific T cell responses were monitored for 47 weeks post-immunization (PI). PBMCs were assessed directly ex vivo or after 6 and 12 days of in vitro culture using antigenic and/or homeostatic proliferation. IFN-γ ELISPOT was used to measure immediate cytokine secretion from antigen specific effector cells and from memory precursors with high proliferative capacity (PHPC). The memory phenotype and functions (proliferation, cytokine expression, lytic content) of specific T cells were tested using multiparametric FACS-based assays. All immunized macaques developed lasting peripheral CD8+ and CD4+ T cell responses mainly against Gag and Nef antigens. During the primary expansion phase, immediate effector cells as well as increasing numbers of proliferating cells with limited effector functions were detected which expressed markers of effector (EM) and central (CM) memory phenotypes. These responses contracted but then reemerged later in absence of antigen boost. Strong PHPC responses comprising vaccine-specific CM and EM T cells that readily expanded and acquired immediate effector functions were detected at 40/47 weeks PI. Altogether, our study demonstrated that a single immunization with a replication-limited DNA vaccine elicited persistent vaccine-specific CM and EM CD8+ and CD4+ T cells with immediate and readily inducible effector functions, in the absence of ongoing antigen expression. Copyright:
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Lotte C. Houtepen;
Christiaan H. Vinkers;
Tania Carrillo-Roa;
Marieke Hiemstra;
Pol A. van Lier;
Wim Meeus;
Susan Branje;
Christine M. Heim;
Charles B. Nemeroff;
Jonathan Mill;
Leonard C Schalkwyk;
Menno P. Creyghton;
René S. Kahn;
Marian Joëls;
Elisabeth Binder;
Marco P.M. Boks
DNA methylation likely plays a role in the regulation of human stress reactivity. Here we show that in a genome-wide analysis of blood DNA methylation in 85 healthy individuals, a locus in the Kit ligand gene (KITLG; cg27512205) showed the strongest association with cortisol stress reactivity (P=5.8 × 10-6). Replication was obtained in two independent samples using either blood (N=45, P=0.001) or buccal cells (N=255, P=0.004). KITLG methylation strongly mediates the relationship between childhood trauma and cortisol stress reactivity in the discovery sample (32% mediation). Its genomic location, a CpG island shore within an H3K27ac enhancer mark, and the correlation between methylation in the blood and prefrontal cortex provide further evidence that KITLG methylation is functionally relevant for the programming of stress reactivity in the human brain. Our results extend preclinical evidence for epigenetic regulation of stress reactivity to humans and provide leads to enhance our understanding of the neurobiological pathways underlying stress vulnerability.
Due to their autosynchronous roles in shaping the anti-tumor immune response, complex immune regulatory networks acting both locally within the tumor microenvironment as well as in its draining lymph nodes play critical roles in the cancer immunotherapy response. We describe herein a thermosensitive co-polymer hydrogel system formed from biocompatible polymers gelatin and Pluronic® F127 that are widely used in humans to enable the sustained release of a nitric oxide donor and antibody blocking immune checkpoint cytotoxic T-lymphocyte-associated protein-4 for efficient and durable anti-tumor immunotherapy. By virtue of its unique gel formation and degradation properties that sustain drug retention at the tumor tissue site for triggered release by the tumor microenvironment and formation of in situ micelles optimum in size for lymphatic uptake, this rationally designed thermosensitive hydrogel facilitates modulation of two orthogonal immune signaling networks relevant to the regulation of the anti-tumor immune response to improve local and abscopal effects of cancer immunotherapy.