Blood-Based Proteomics for Adult-Onset Focal Dystonias.

OBJECTIVES: The adult-onset focal dystonias are characterized by over-active muscles leading to abnormal movements. For most cases, the etiology and pathogenesis remain unknown. In the current study, unbiased proteomics methods were used to identify potential changes in blood plasma proteins. METHODS: A large-scale unbiased proteomics screen was used to compare proteins (N = 6,345) in blood plasma of normal healthy controls (N = 49) with adult-onset focal dystonia (N = 143) consisting of specific subpopulations of cervical dystonia (N = 45), laryngeal dystonia (N = 49), and blepharospasm (N = 49). Pathway analyses were conducted to identify relevant biological pathways. Finally, protein changes were used to build a prediction model for dystonia. RESULTS: After correction for multiple comparisons, 15 proteins were associated with adult-onset focal dystonia. Subgroup analyses revealed some proteins were shared across the dystonia subgroups while others were unique to 1 subgroup. The top biological pathways involved changes in the immune system, metal ion transport, and reactive oxygen species. A 4-protein model showed high accuracy in discriminating control individuals from dystonia cases [average area under the curve (AUC) = 0.89]. INTERPRETATION: These studies provide novel insights into the etiopathogenesis of dystonia, as well as novel potential biomarkers. ANN NEUROL 2024;96:110-120.

Chronic SIV-Induced neuroinflammation disrupts CCR7+ CD4+ T cell immunosurveillance in the rhesus macaque brain.

CD4+ T cells survey and maintain immune homeostasis in the brain, yet their differentiation states and functional capabilities remain unclear. Our approach, combining single-cell transcriptomic analysis, ATAC-Seq, spatial transcriptomics, and flow cytometry, revealed a distinct subset of CCR7+ CD4+ T cells resembling lymph node central memory (TCM) cells. We observed chromatin accessibility at the CCR7, CD28, and BCL-6 loci, defining molecular features of TCM. Brain CCR7+ CD4+ T cells exhibited recall proliferation and interleukin-2 production ex vivo, showcasing their functional competence. We identified the skull bone marrow as a local niche for these cells alongside CNS border tissues. Sequestering TCM cells in lymph nodes using FTY720 led to reduced CCR7+ CD4+ T cell frequencies in the cerebrospinal fluid, accompanied by increased monocyte levels and soluble markers indicating immune activation. In macaques chronically infected with SIVCL757 and experiencing viral rebound due to cessation of antiretroviral therapy, a decrease in brain CCR7+ CD4+ T cells was observed, along with increased microglial activation and initiation of neurodegenerative pathways. Our findings highlight a role for CCR7+ CD4+ T cells in CNS immune surveillance, and their decline during chronic SIV highlights their responsiveness to neuroinflammation.

Exploration of potential immune mechanisms in cervical dystonia.

BACKGROUND: Although there are many possible causes for cervical dystonia (CD), a specific etiology cannot be identified in most cases. Prior studies have suggested a relationship between autoimmune disease and some cases of CD, pointing to possible immunological mechanisms. OBJECTIVE: The goal was to explore the potential role of multiple different immunological mechanisms in CD. METHODS: First, a broad screening test compared neuronal antibodies in controls and CD. Second, unbiased blood plasma proteomics provided a broad screen for potential biologic differences between controls and CD. Third, a multiplex immunoassay compared 37 markers associated with immunological processes in controls and CD. Fourth, relative immune cell frequencies were investigated in blood samples of controls and CD. Finally, sequencing studies investigated the association of HLA DQB1 and DRB1 alleles in controls versus CD. RESULTS: Screens for anti-neuronal antibodies did not reveal any obvious abnormalities. Plasma proteomics pointed towards certain abnormalities of immune mechanisms, and the multiplex assay pointed more specifically towards abnormalities in T lymphocytes. Abnormal immune cell frequencies were identified for some CD cases, and these cases clustered together as a potential subgroup. Studies of HLA alleles indicated a possible association between CD and DRB1*15:03, which is reported to mediate the penetrance of autoimmune disorders. CONCLUSIONS: Altogether, the association of CD with multiple different blood-based immune measures point to abnormalities in cell-mediated immunity that may play a pathogenic role for a subgroup of individuals with CD.

An Exploratory, Randomized, Double-Blind Clinical Trial of Dipraglurant for Blepharospasm.

BACKGROUND: Blepharospasm is treated with botulinum toxin, but obtaining satisfactory results is sometimes challenging. OBJECTIVE: The aim is to conduct an exploratory trial of oral dipraglurant for blepharospasm. METHODS: This study was an exploratory, phase 2a, randomized, double-blind, placebo-controlled trial of 15 participants who were assigned to receive a placebo or dipraglurant (50 or 100 mg) and assessed over 2 days, 1 and 2 hours following dosing. Outcome measures included multiple scales rated by clinicians or participants, digital video, and a wearable sensor. RESULTS: Dipraglurant was well tolerated, with no obvious impact on any of the measurement outcomes. Power analyses suggested fewer subjects would be required for studies using a within-subject versus independent group design, especially for certain measures. Some outcome measures appeared more suitable than others. CONCLUSION: Although dipraglurant appeared well tolerated, it did not produce a trend for clinical benefit. The results provide valuable information for planning further trials in blepharospasm. © 2024 International Parkinson and Movement Disorder Society.

Spiny projection neurons exhibit transcriptional signatures within subregions of the dorsal striatum.

The dorsal striatum is organized into functional territories defined by corticostriatal inputs onto both direct and indirect spiny projection neurons (SPNs), the major cell types within the striatum. In addition to circuit connectivity, striatal domains are likely defined by the spatially determined transcriptomes of SPNs themselves. To identify cell-type-specific spatiomolecular signatures of direct and indirect SPNs within dorsomedial, dorsolateral, and ventrolateral dorsal striatum, we used RNA profiling in situ hybridization with probes to >98% of protein coding genes. We demonstrate that the molecular identity of SPNs is mediated by hundreds of differentially expressed genes across territories of the striatum, revealing extraordinary heterogeneity in the expression of genes that mediate synaptic function in both direct and indirect SPNs. This deep insight into the complex spatiomolecular organization of the striatum provides a foundation for understanding both normal striatal function and for dissecting region-specific dysfunction in disorders of the striatum.

CCR7+ CD4 T Cell Immunosurveillance Disrupted in Chronic SIV-Induced Neuroinflammation in Rhesus Brain.

CD4 T cells survey and maintain immune homeostasis in the brain, yet their differentiation states and functional capabilities remain unclear. Our approach, combining single-cell transcriptomic analysis, ATAC-seq, spatial transcriptomics, and flow cytometry, revealed a distinct subset of CCR7+ CD4 T cells resembling lymph node central memory (T CM ) cells. We observed chromatin accessibility at the CCR7, CD28, and BCL-6 loci, defining molecular features of T CM . Brain CCR7+ CD4 T cells exhibited recall proliferation and interleukin-2 production ex vivo, showcasing their functional competence. We identified the skull bone marrow as a local niche for these cells alongside other CNS border tissues. Sequestering T CM cells in lymph nodes using FTY720 led to reduced CCR7+ CD4 T cell frequencies in the cerebrospinal fluid, accompanied by increased monocyte levels and soluble markers indicating immune activation. In macaques chronically infected with SIVCL57 and experiencing viral rebound due to cessation of antiretroviral therapy, a decrease in brain CCR7+ CD4 T cells was observed, along with increased microglial activation and initiation of neurodegenerative pathways. Our findings highlight a role for CCR7+ CD4 T cells in CNS immune surveillance and their decline during chronic SIV-induced neuroinflammation highlights their responsiveness to neuroinflammatory processes. In Brief: Utilizing single-cell and spatial transcriptomics on adult rhesus brain, we uncover a unique CCR7+ CD4 T cell subset resembling central memory T cells (T CM ) within brain and border tissues, including skull bone marrow. Our findings show decreased frequencies of this subset during SIV- induced chronic neuroinflammation, emphasizing responsiveness of CCR7+ CD4 T cells to CNS disruptions. Highlights: CCR7+ CD4 T cells survey border and parenchymal CNS compartments during homeostasis; reduced presence of CCR7+ CD4 T cells in cerebrospinal fluid leads to immune activation, implying a role in neuroimmune homeostasis. CNS CCR7+ CD4 T cells exhibit phenotypic and functional features of central memory T cells (T CM ) including production of interleukin 2 and the capacity for rapid recall proliferation. Furthermore, CCR7+ CD4 T cells reside in the skull bone marrow. CCR7+ CD4 T cells are markedly decreased within the brain parenchyma during chronic viral neuroinflammation.

Live-cell invasive phenotyping uncovers the ALK2/BMP6 iron homeostasis pathway as a therapeutic vulnerability in LKB1-mutant lung cancer.

The acquisition of invasive properties is a prerequisite for tumor progression and metastasis. Molecular subtypes of KRAS-driven lung cancer exhibit distinct modes of invasion that likely contribute to unique growth properties and therapeutic susceptibilities. Despite this, pre-clinical discovery strategies designed to exploit invasive phenotypes are lacking. To address this, we designed an experimental system to screen for targetable signaling pathways linked to active early invasion phenotypes in the two most prominent molecular subtypes, TP53 and LKB1, of KRAS-driven lung adenocarcinoma (LUAD). By combining live-cell imaging of human bronchial epithelial cells in a 3D invasion matrix with RNA transcriptome profiling, we identified the LKB1-specific upregulation of bone morphogenetic protein 6 (BMP6). Examination of early-stage lung cancer patients confirmed upregulation of BMP6 in LKB1-mutant lung tumors. At the molecular level, we find that the canonical iron regulatory hormone Hepcidin is induced via BMP6 signaling upon LKB1 loss, where intact LKB1 kinase activity is necessary to maintain signaling homeostasis. Furthermore, pre-clinical studies in a novel Kras/Lkb1-mutant syngeneic mouse model show that potent growth suppression was achieved by inhibiting the ALK2/BMP6 signaling axis with single agents that are currently in clinical trials. We show that alterations in the iron homeostasis pathway are accompanied by simultaneous upregulation of ferroptosis protection proteins. Thus, LKB1 is sufficient to regulate both the 'gas' and 'breaks' to finely tune iron-regulated tumor progression.

Abnormalities of neural stem cells in Lesch-Nyhan disease.

Lesch-Nyhan disease (LND) is a neurodevelopmental disorder caused by variants in the HPRT1 gene, which encodes the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGprt). HGprt deficiency provokes numerous metabolic changes which vary among different cell types, making it unclear which changes are most relevant for abnormal neural development. To begin to elucidate the consequences of HGprt deficiency for developing human neurons, neural stem cells (NSCs) were prepared from 6 induced pluripotent stem cell (iPSC) lines from individuals with LND and compared to 6 normal healthy controls. For all 12 lines, gene expression profiles were determined by RNA-seq and protein expression profiles were determined by shotgun proteomics. The LND lines revealed significant changes in expression of multiple genes and proteins. There was little overlap in findings between iPSCs and NSCs, confirming the impact of HGprt deficiency depends on cell type. For NSCs, gene expression studies pointed towards abnormalities in WNT signaling, which is known to play a role in neural development. Protein expression studies pointed to abnormalities in the mitochondrial F0F1 ATPase, which plays a role in maintaining cellular energy. These studies point to some mechanisms that may be responsible for abnormal neural development in LND.

Neuroinflammatory transcriptional programs induced in rhesus pre-frontal cortex white matter during acute SHIV infection.

BACKGROUND: Immunosurveillance of the central nervous system (CNS) is vital to resolve infection and injury. However, immune activation within the CNS in the setting of chronic viral infections, such as HIV-1, is strongly linked to progressive neurodegeneration and cognitive decline. Establishment of HIV-1 in the CNS early following infection underscores the need to delineate features of acute CNS immune activation, as these early inflammatory events may mediate neurodegenerative processes. Here, we focused on elucidating molecular programs of neuroinflammation in brain regions based on vulnerability to neuroAIDS and/or neurocognitive decline. To this end, we assessed transcriptional profiles within the subcortical white matter of the pre-frontal cortex (PFCw), as well as synapse dense regions from hippocampus, superior temporal cortex, and caudate nucleus, in rhesus macaques following infection with Simian/Human Immunodeficiency Virus (SHIV.C.CH505). METHODS: We performed RNA extraction and sequenced RNA isolated from 3 mm brain punches. Viral RNA was quantified in the brain and cerebrospinal fluid by RT-qPCR assays targeting SIV Gag. Neuroinflammation was assessed by flow cytometry and multiplex ELISA assays. RESULTS: RNA sequencing and flow cytometry data demonstrated immune surveillance of the rhesus CNS by innate and adaptive immune cells during homeostasis. Following SHIV infection, viral entry and integration within multiple brain regions demonstrated vulnerabilities of key cognitive and motor function brain regions to HIV-1 during the acute phase of infection. SHIV-induced transcriptional alterations were concentrated to the PFCw and STS with upregulation of gene expression pathways controlling innate and T-cell inflammatory responses. Within the PFCw, gene modules regulating microglial activation and T cell differentiation were induced at 28 days post-SHIV infection, with evidence for stimulation of immune effector programs characteristic of neuroinflammation. Furthermore, enrichment of pathways regulating mitochondrial respiratory capacity, synapse assembly, and oxidative and endoplasmic reticulum stress were observed. These acute neuroinflammatory features were substantiated by increased influx of activated T cells into the CNS. CONCLUSIONS: Our data show pervasive immune surveillance of the rhesus CNS at homeostasis and reveal perturbations of important immune, neuronal, and synaptic pathways within key anatomic regions controlling cognition and motor function during acute HIV infection. These findings provide a valuable framework to understand early molecular features of HIV associated neurodegeneration.

Uric Acid in Parkinson's Disease: What Is the Connection?

Numerous studies have linked Parkinson's disease (PD) with low levels of uric acid (UA). Low UA has been associated with the risk of developing PD, and its progression and severity. The biological mechanisms underlying these relationships have never been firmly established. The most frequently proposed mechanism is that UA is an antioxidant. Low UA is thought to predispose to oxidative stress, which contributes to dopamine neuron degeneration, and leads to initial appearance of symptoms of PD and its worsening over time. Several recent studies have questioned this explanation. In this review, we describe the biology of UA, its many links with PD, evidence regarding UA as an antioxidant, and we question whether UA causes PD or contributes to its progression. We also address the possibility that something about PD causes low UA (reverse causation) or that low UA is a biomarker of some other more relevant mechanism in PD. We hope the evidence provided here will stimulate additional studies to better understand the links between UA and PD. Elucidating these mechanisms remains important, because they may provide new insights into the pathogenesis of PD or novel approaches to treatments. © 2022 International Parkinson and Movement Disorder Society.

Anti-inflammatory effects of recreational marijuana in virally suppressed youth with HIV-1 are reversed by use of tobacco products in combination with marijuana.

BACKGROUND: Marijuana's putative anti-inflammatory properties may benefit HIV-associated comorbidities. How recreational marijuana use affects gene expression in peripheral blood cells (PBC) among youth with HIV-1 (YWH) is unknown. APPROACH: YWH with defined substance use (n = 54) receiving similar antiretroviral therapy (ART) were assigned to one of four analysis groups: YWH with detectable plasma HIV-1 (> 50 RNA copies/ml) who did not use substances (H+V+S-), and YWH with undetectable plasma HIV-1 who did not use substances (H+V-S-), or used marijuana alone (H+V-S+[M]), or marijuana in combination with tobacco (H+V-S+[M/T]). Non-substance using youth without HIV infection (H-S-, n = 25) provided a reference group. PBC mRNA was profiled by Affymetrix GeneChip Human Genome U133 Plus 2.0 Array. Differentially expressed genes (DEG) within outcome groups were identified by Significance Analysis of Microarrays and used for Hierarchical Clustering, Principal Component Analysis, and Ingenuity Pathways Analysis. RESULTS: HIV-1 replication resulted in > 3000 DEG involving 27 perturbed pathways. Viral suppression reduced DEG to 313, normalized all 27 pathways, and down-regulated two additional pathways, while marijuana use among virally suppressed YWH resulted in 434 DEG and no perturbed pathways. Relative to H+V-S-, multiple DEG normalized in H+V-S+[M]. In contrast, H+V-S+[M/T] had 1140 DEG and 10 dysregulated pathways, including multiple proinflammatory genes and six pathways shared by H+V+S-. CONCLUSIONS: YWH receiving ART display unique transcriptome bioprofiles based on viral replication and substance use. In the context of HIV suppression, marijuana use, alone or combined with tobacco, has opposing effects on inflammatory gene expression.

Circulating integrin α4 ß7+ CD4 T cells are enriched for proliferative transcriptional programs in HIV infection.

HIV preferentially infects α4 ß7+ CD4 T cells, forming latent reservoirs that contribute to HIV persistence during antiretroviral therapy. However, the properties of α4 ß7+ CD4 T cells in blood and mucosal compartments remain understudied. Employing two distinct models of HIV infection, HIV-infected humans and simian-human immunodeficiency virus (SHIV)-infected rhesus macaques, we show that α4 ß7+ CD4 T cells in blood are enriched for genes regulating cell cycle progression and cellular metabolism. Unlike their circulating counterparts, rectal α4 ß7+ CD4 T cells exhibited a core tissue-residency gene expression program. These features were conserved across primate species, indicating that the environment influences memory T-cell transcriptional networks. Our findings provide an important molecular foundation for understanding the role of α4 ß7 in HIV infection.

Differential expression of striatal proteins in a mouse model of DOPA-responsive dystonia reveals shared mechanisms among dystonic disorders.

Dystonia is characterized by involuntary muscle contractions that cause debilitating twisting movements and postures. Although dysfunction of the basal ganglia, a brain region that mediates movement, is implicated in many forms of dystonia, the underlying mechanisms are unclear. The inherited metabolic disorder DOPA-responsive dystonia is considered a prototype for understanding basal ganglia dysfunction in dystonia because it is caused by mutations in genes necessary for the synthesis of the neurotransmitter dopamine, which mediates the activity of the basal ganglia. Therefore, to reveal abnormal striatal cellular processes and pathways implicated in dystonia, we used an unbiased proteomic approach in a knockin mouse model of DOPA-responsive dystonia, a model in which the striatum is known to play a central role in the expression of dystonia. Fifty-seven of the 1805 proteins identified were differentially regulated in DOPA-responsive dystonia mice compared to control mice. Most differentially regulated proteins were associated with gene ontology terms that implicated either mitochondrial or synaptic dysfunction whereby proteins associated with mitochondrial function were generally over-represented and proteins associated with synaptic function were largely under-represented. Remarkably, nearly 20% of the differentially regulated striatal proteins identified in our screen are associated with pathogenic variants that cause inherited disorders with dystonia as a sign in humans suggesting shared mechanisms across many different forms of dystonia.

Induced pluripotent stem cells from subjects with Lesch-Nyhan disease.

Lesch-Nyhan disease (LND) is an inherited disorder caused by pathogenic variants in the HPRT1 gene, which encodes the purine recycling enzyme hypoxanthine-guanine phosphoribosyltransferase (HGprt). We generated 6 induced pluripotent stem cell (iPSC) lines from 3 individuals with LND, along with 6 control lines from 3 normal individuals. All 12 lines had the characteristics of pluripotent stem cells, as assessed by immunostaining for pluripotency markers, expression of pluripotency genes, and differentiation into the 3 primary germ cell layers. Gene expression profiling with RNAseq demonstrated significant heterogeneity among the lines. Despite this heterogeneity, several anticipated abnormalities were readily detectable across all LND lines, including reduced HPRT1 mRNA. Several unexpected abnormalities were also consistently detectable across the LND lines, including decreases in FAR2P1 and increases in RNF39. Shotgun proteomics also demonstrated several expected abnormalities in the LND lines, such as absence of HGprt protein. The proteomics study also revealed several unexpected abnormalities across the LND lines, including increases in GNAO1 decreases in NSE4A. There was a good but partial correlation between abnormalities revealed by the RNAseq and proteomics methods. Finally, functional studies demonstrated LND lines had no HGprt enzyme activity and resistance to the toxic pro-drug 6-thioguanine. Intracellular purines in the LND lines were normal, but they did not recycle hypoxanthine. These cells provide a novel resource to reveal insights into the relevance of heterogeneity among iPSC lines and applications for modeling LND.

Signatures of somatic mutations and gene expression from p16INK4A positive head and neck squamous cell carcinomas (HNSCC).

Human papilloma virus (HPV) causes a subset of head and neck squamous cell carcinomas (HNSCC) of the oropharynx. We combined targeted DNA- and genome-wide RNA-sequencing to identify genetic variants and gene expression signatures respectively from patients with HNSCC including oropharyngeal squamous cell carcinomas (OPSCC). DNA and RNA were purified from 35- formalin fixed and paraffin embedded (FFPE) HNSCC tumor samples. Immuno-histochemical evaluation of tumors was performed to determine the expression levels of p16INK4A and classified tumor samples either p16+ or p16-. Using ClearSeq Comprehensive Cancer panel, we examined the distribution of somatic mutations. Somatic single-nucleotide variants (SNV) were called using GATK-Mutect2 ("tumor-only" mode) approach. Using RNA-seq, we identified a catalog of 1,044 and 8 genes as significantly expressed between p16+ and p16-, respectively at FDR 0.05 (5%) and 0.1 (10%). The clinicopathological characteristics of the patients including anatomical site, smoking and survival were analyzed when comparing p16+ and p16- tumors. The majority of tumors (65%) were p16+. Population sequence variant databases, including gnomAD, ExAC, COSMIC and dbSNP, were used to identify the mutational landscape of somatic sequence variants within sequenced genes. Hierarchical clustering of The Cancer Genome Atlas (TCGA) samples based on HPV-status was observed using differentially expressed genes. Using RNA-seq in parallel with targeted DNA-seq, we identified mutational and gene expression signatures characteristic of p16+ and p16- HNSCC. Our gene signatures are consistent with previously published data including TCGA and support the need to further explore the biologic relevance of these alterations in HNSCC.

Impact of Th1 CD4 Follicular Helper T Cell Skewing on Antibody Responses to an HIV-1 Vaccine in Rhesus Macaques.

Generating durable humoral immunity through vaccination depends upon effective interactions of follicular helper T (Tfh) cells with germinal center (GC) B cells. Th1 polarization of Tfh cells is an important process shaping the success of Tfh-GC B cell interactions by influencing costimulatory and cytokine-dependent Tfh help to B cells. However, the question remains as to whether adjuvant-dependent modulation of Tfh cells enhances HIV-1 vaccine-induced antienvelope (anti-Env) antibody responses. We investigated whether an HIV-1 vaccine platform designed to increase the number of Th1-polarized Tfh cells enhances the magnitude and quality of anti-Env antibodies. Utilizing a novel interferon-induced protein 10 (IP-10)-adjuvanted HIV-1 DNA prime followed by a monophosphoryl lipid A and QS-21 (MPLA+QS-21)-adjuvanted Env protein boost (DIP-10 PALFQ) in macaques, we observed higher anti-Env serum IgG titers with greater cross-clade reactivity, specificity for V1V2, and effector functions than in macaques primed with DNA lacking IP-10 and boosted with MPLA-plus-alum-adjuvanted Env protein (DPALFA) The DIP-10 PALFQ vaccine regimen elicited higher anti-Env IgG1 and lower IgG4 antibody levels in serum, showing for the first time that adjuvants can dramatically impact the IgG subclass profile in macaques. The DIP-10 PALFQ regimen also increased vaginal and rectal IgA antibodies to a greater extent. Within lymph nodes, we observed augmented GC B cell responses and the promotion of Th1 gene expression profiles in GC Tfh cells. The frequency of GC Tfh cells correlated with both the magnitude and avidity of anti-Env serum IgG. Together, these data suggest that adjuvant-induced stimulation of Th1-Tfh cells is an effective strategy for enhancing the magnitude and quality of anti-Env antibody responses.IMPORTANCE The results of the RV144 trial demonstrated that vaccination could prevent HIV transmission in humans and that longevity of anti-Env antibodies may be key to this protection. Efforts to improve upon the prime-boost vaccine regimen used in RV144 have indicated that booster immunizations can increase serum anti-Env antibody titers but only transiently. Poor antibody durability hampers efforts to develop an effective HIV-1 vaccine. This study was designed to identify the specific elements involved in the immunological mechanism necessary to produce robust HIV-1-specific antibodies in rhesus macaques. By clearly defining immune-mediated pathways that improve the magnitude and functionality of the anti-HIV-1 antibody response, we will have the foundation necessary for the rational development of an HIV-1 vaccine.

Rectal Microbiome Composition Correlates with Humoral Immunity to HIV-1 in Vaccinated Rhesus Macaques.

The microbiome is an integral and dynamic component of the host and is emerging as a critical determinant of immune responses; however, its influence on vaccine immunogenicity is largely not well understood. Here, we examined the pivotal relationship between the mucosal microbiome and vaccine-induced immune responses by assessing longitudinal changes in vaginal and rectal microbiome profiles after intradermal immunization with a human immunodeficiency virus type 1 (HIV-1) DNA vaccine in adult rhesus macaques that received two prior DNA primes. We report that both vaginal and rectal microbiomes were dominated by Firmicutes but were composed of distinct genera, denoting microbiome specialization across mucosal tissues. Following immunization, the vaginal microbiome was resilient, except for a transient decrease in Streptococcus In contrast, the rectal microbiome was far more responsive to vaccination, exhibiting an increase in the ratio of Firmicutes to Bacteroidetes Within Bacteroidetes, multiple genera were significantly decreased, including Prevotella, Alloprevotella, Bacteroides, Acetobacteroides, Falsiporphyromonas, and Anaerocella. Decreased abundance of Prevotella correlated with induction of gut-homing α4ß7+ effector CD4 T cells. Prevotella abundance also negatively correlated with rectal HIV-1 specific IgG levels. While rectal Lactobacillus was unaltered following DNA vaccination, baseline Lactobacillus abundance showed strong associations with higher rectal HIV-1 gp140 IgA induced following a protein boost. Similarly, the abundance of Clostridium in cluster IV was associated with higher rectal HIV-1 gp140 IgG responses. Collectively, these data reveal that the temporal stability of bacterial communities following DNA immunization is site dependent and highlight the importance of host-microbiome interactions in shaping HIV-1 vaccine responses. Our findings have significant implications for microbial manipulation as a strategy to enhance HIV vaccine-induced mucosal immunity.IMPORTANCE There is considerable effort directed toward evaluating HIV-1 vaccine platforms to select the most promising candidates for enhancing mucosal HIV-1 antibody. The most successful thus far, the RV144 trial provided partial protection due to waning HIV-1 antibody titers. In order to develop an effective HIV vaccine, it may therefore be important to understand how biological factors, such as the microbiome, modulate host immune responses. Furthermore, as intestinal microbiota antigens may generate antibodies cross-reactive to the HIV-1 envelope glycoprotein, understanding the relationship between gut microbiota composition and HIV-1 envelope antibody responses after vaccination is important. Here, we demonstrate for the first time in rhesus macaques that the rectal microbiome composition can influence HIV-1 vaccine immunogenicity, and we report temporal changes in the mucosal microbiome profile following HIV-1 vaccination. Our results could inform findings from the HIV Vaccine Trials Network (HVTN) vaccine studies and contribute to an understanding of how the microbiome influences HIV-1 antibody responses.

Statistical insights into major human muscular diseases.

Muscular diseases lead to muscle fiber degeneration, impairment of mobility, and in some cases premature death. Many of these muscular diseases are largely idiopathic. The goal of this study was to identify biomarkers based on their functional role and possible mechanisms of pathogenesis, specific to individual muscular disease. We analyzed the muscle transcriptome from five major muscular diseases: acute quadriplegic myopathy (AQM), amyotrophic lateral sclerosis (ALS), mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), dermatomyositis (DM) and polymyositis (PM) using pairwise statistical comparison to identify uniquely regulated genes in each muscular disease. The genome-wide information encoded in the transcriptome provided biomarkers and functional insights into dysregulation in each muscular disease. The analysis showed that the dysregulation of genes in forward membrane pathway, responsible for transmitting action potential from neural excitation, is unique to AQM, while the dysregulation of myofibril genes, determinant of the mechanical properties of muscle, is unique to ALS, dysregulation of ER protein processing, responsible for correct protein folding, is unique to DM, and upregulation of immune response genes is unique to PM. We have identified biomarkers specific to each muscular disease which can be used for diagnostic purposes.

MicroRNA 203 Modulates Glioma Cell Migration via Robo1/ERK/MMP-9 Signaling.

Glioblastoma (GBM) is the most common and malignant primary adult brain cancer. Allelic deletion on chromosome 14q plays an important role in the pathogenesis of GBM, and this site was thought to harbor multiple tumor suppressor genes associated with GBM, a region that also encodes microRNA-203 (miR-203). In this study, we sought to identify the role of miR-203 as a tumor suppressor in the pathogenesis of GBM. We analyzed the miR-203 expression data of GBM patients in 10 normal and 495 tumor tissue samples derived from The Cancer Genome Atlas data set. Quantitative real-time PCR and in situ hybridization in 10 high-grade GBM and 10 low-grade anaplastic astrocytoma tumor samples showed decreased levels of miR-203 expression in anaplastic astrocytoma and GBM tissues and cell lines. Exogenous expression of miR-203 using a plasmid expressing miR-203 precursor (pmiR-203) suppressed glioma cell proliferation, migration, and invasion. We determined that one relevant target of miR-203 was Robo1, given that miR-203 expression decreased mRNA and protein levels as determined by RT-PCR and Western blot analysis. Moreover, cotransfection experiments using a luciferase-based transcription reporter assay have shown direct regulation of Robo1 by miR-203. We also show that Robo1 mediates miR-203 mediated antimigratory functions as up-regulation of Robo1 abrogates miR-203 mediated antimigratory effects. We also show that miR-203 expression suppressed ERK phosphorylation and MMP-9 expression in glioma cells. Furthermore, we demonstrate that miR-203 inhibits migration of the glioma cells by disrupting the Robo1/ERK/MMP-9 signaling axis. Taken together, these studies demonstrate that up-regulation of Robo1 in response to the decrease in miR-203 in glioma cells is responsible for glioma tumor cell migration and invasion.

A combined omics study on activated macrophages--enhanced role of STATs in apoptosis, immunity and lipid metabolism.

BACKGROUND: Macrophage activation by lipopolysaccharide and adenosine triphosphate (ATP) has been studied extensively because this model system mimics the physiological context of bacterial infection and subsequent inflammatory responses. Previous studies on macrophages elucidated the biological roles of caspase-1 in post-translational activation of interleukin-1ß and interleukin-18 in inflammation and apoptosis. However, the results from these studies focused only on a small number of factors. To better understand the host response, we have performed a high-throughput study of Kdo2-lipid A (KLA)-primed macrophages stimulated with ATP. RESULTS: The study suggests that treating mouse bone marrow-derived macrophages with KLA and ATP produces 'synergistic' effects that are not seen with treatment of KLA or ATP alone. The synergistic regulation of genes related to immunity, apoptosis and lipid metabolism is observed in a time-dependent manner. The synergistic effects are produced by nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and activator protein (AP)-1 through regulation of their target cytokines. The synergistically regulated cytokines then activate signal transducer and activator of transcription (STAT) factors that result in enhanced immunity, apoptosis and lipid metabolism; STAT1 enhances immunity by promoting anti-microbial factors; and STAT3 contributes to downregulation of cell cycle and upregulation of apoptosis. STAT1 and STAT3 also regulate glycerolipid and eicosanoid metabolism, respectively. Further, western blot analysis for STAT1 and STAT3 showed that the changes in transcriptomic levels were consistent with their proteomic levels. In summary, this study shows the synergistic interaction between the toll-like receptor and purinergic receptor signaling during macrophage activation on bacterial infection. AVAILABILITY: Time-course data of transcriptomics and lipidomics can be queried or downloaded from http://www.lipidmaps.org. CONTACT: shankar@ucsd.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.