Human Control Subjects Research Articles

Inflammatory Protein Signatures as Predictive Disease-Specific Markers for Non-Alcoholic Steatohepatitis (NASH).

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic disease worldwide, consisting of a broad spectrum of diseases such as simple steatosis (NAFL), non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma. Hepatic inflammation plays a key role in the pathophysiology of NAFLD. Inflammatory mediators such as cytokines and chemokines are considered as contributing factors to NAFLD development and progression. In the present study, we aimed to investigate the inflammatory protein signatures as predictive disease-specific markers for non-alcoholic fatty liver disease (NAFLD). This cross-sectional study included healthy control (n = 64), NAFL (n = 109), and NASH (n = 60) human subjects. Serum concentrations of various cytokines and chemokines were evaluated using sensitive multiplex assays. We used principal component analysis (PCoA) to reveal distinct differences in the levels of cytokines and chemokines between each of the study groups. Further, a random forest classification model was developed to identify the panel of markers that could predict diseases. The protein-protein network analysis was performed to determine the various signaling pathways associated with the disease-specific panel of markers. Serum concentrations of TNF-α, IL-1β, IL-1ra, G-CSF, PDGF-BB, MCP-1, MIP-1a, MIP-1b, RANTES, eotaxin, IL-8 and IP-10 were significantly increased in NASH group as compared to control group. Furthermore, serum concentrations of IL-9 and IL-13 were significantly lower in the NASH group, whereas IL-2 levels were significantly decreased in the NAFL group when compared to the control group. PCoA results demonstrated statistically significant differences in cytokines and chemokines between each of the study groups (PERMANOVA p = 0.001; R2 = 0.102). RANTES, IL-1ra, MIP-1b, IL-2, and G-CSF could differentiate the NAFL group from the controls; G-CSF, IL-1ra, TNF-α, RANTES, and IL-9 could differentiate the NASH group from the controls; and G-CSF, IL-9, IL-13, eotaxin, and TNF- α could differentiate the NASH group from the NAFL group. Our protein-protein network revealed that these markers are involved in cytokine-cytokine receptor interaction, Th1 and Th2 cell differentiation, TNF, chemokine, JAK/STAT, P13K/Akt, TLR, NOD-like receptor, NF-kB, and adipocytokine signaling pathways which might be responsible for disease pathogenesis.Our study findings revealed a set of distinct cytokine and chemokine markers and they might be considered as biomarkers in distinguishing NASH from NAFL. Future multicentre studies with larger sample size are recommended to determine the potential utility of these panels of markers.

Single particle mass photometry provides size distributions of tau oligomers in CSF samples revealing strong differences between samples from Alzheimer’s patients and healthy controls

AbstractBackgroundTau oligomers are considered toxic species in neurodegenerative disorders and their abundance correlate well with the disease onset and pathology. Assays used for detection of these toxic tau oligomer species fail to detect their low levels and to differentiate between various oligomers. This limitation reduces the potential for early diagnosis and for possible intervention. A label‐free approach that can detect these biomarkers at low concentrations while also revealing their size distribution will be ideal.MethodHere, we use a combination of tau oligomer‐specific immunoprecipitation from human CSF samples and single‐molecule mass analysis based on so‐called mass photometry to determine the size of thousands of individual oligomeric species in solution in a label‐free manner.ResultSingle‐oligomer mass analysis makes it possible to determine the number of tau monomers in thousands of individual oligomer particles in CSF samples from human patients and control subjects. The method reveals a dramatic difference in the size distribution of tau oligomer species from Alzheimer’s patients compared to control subjects. In particular, samples from AD patients contain a significant fraction of trimeric tau oligomers, which are completely absent in the control samples.ConclusionSingle‐particle mass analysis of tau protein oligomers enables discrimination of samples from patients with AD from those of age‐matched control subjects. Furthermore, single‐particle based analysis of the size distribution of oligomeric species will help to shed light on the toxicity of various oligomers species.

Boosting NAD blunts T H17 inflammation via arginine biosynthesis and redox regulatory control in healthy control and psoriasis human subjects

Abstract Although NAD +levels modulate metabolism, how this regulates immunometabolism/inflammation remains unclear. Employing Nicotinamide Riboside (NR) to boost NAD, we explored T H17-linked inflammatory in Psoriasis. Primary Psoriasis CD4 +T cells exposed to NR reduced IL-17 secretion and RNA-seq/pathway analysis implicated NR in modulating sequestosome 1 (SQSTM1/p62)-coupled oxidative stress. In parallel, NR reduced CD4 +T cell reactive oxygen species (ROS) levels and activated the NRF2 transcription factor. Depletion of NRF2 or SQSTM1 diminished NR effects on ROS and T H17 activation. Metabolomic analysis implicated NR in arginine biosynthesis and L-arginine replicated NR CD4 +T cell effects. Genetic disruption of the argininosuccinate lyase synthesis enzyme ameliorated NR antiinflammatory effects, supporting that NR, via arginine biosynthesis orchestrates NRF2 activation, CD4 +T cell antioxidant defenses and blunted T H17 responsiveness. A pilot in-vivo human study similarly showed that NR increased arginine, antioxidant enzyme gene expression and blunted T H17 regulation, implicating NAD metabolism in CD4 +T cell-linked inflammation. NHLBI Division of Intramural Research (MNS – ZIA-HL005102), NIH Bench to Bedside award (MNS and RT, HL-129510-04S1) and the NIH Office of Dietary Supplementation (JT) and the UK MRC (JLG – MR/P011705/2; UKDRI-5002; MAP UK).

IL-4-Induced Quiescence of Resting Naive B Cells Is Disrupted in Systemic Lupus Erythematosus.

Activated naive (aNAV) B cells have been shown to be the precursor of the CD11c+T-bet+ IgD-CD27- double-negative (DN)2 or atypical memory (aMEM) B cells in systemic lupus erythematosus (SLE). To determine factors that maintain resting naive (rNAV) B cells, the transcriptomic program in naive (IGHD+IGHM +) B cells in human healthy control subjects (HC) and subjects with SLE was analyzed by single-cell RNA-sequencing analysis. In HC, naive B cells expressed IL-4 pathway genes, whereas in SLE, naive B cells expressed type I IFN-stimulated genes (ISGs). In HC, aNAV B cells exhibited upregulation of the gene signature of germinal center and classical memory (cMEM) B cells. In contrast, in SLE, aNAV B cells expressed signature genes of aMEM. In vitro exposure of SLE B cells to IL-4 promoted B cell development into CD27+CD38+ plasmablasts/plasma and IgD-CD27+ cMEM B cells. The same treatment blocked the development of CD11c+Tbet+ aNAV and DN2 B cells and preserved DN B cells as CD11c-Tbet- DN1 B cells. Lower expression of IL-4R and increased intracellular IFN-β in naive B cells was correlated with the accumulation of CD21-IgD- B cells and the development of anti-Smith and anti-DNA autoantibodies in patients with SLE (n = 47). Our results show that IL-4R and type I IFN signaling in naive B cells induce the development of distinct lineages of cMEM versus aMEM B cells, respectively. Furthermore, diminished IL-4R signaling shifted activated B cell development from the DN1 to the DN2 trajectory in patients with SLE. Therapies that enhance IL-4R signaling may be beneficial for ISGhi SLE patients.

Widespread alterations in microRNA biogenesis in human Huntington’s disease putamen

Altered microRNA (miRNA) expression is a common feature of Huntington’s disease (HD) and could participate in disease onset and progression. However, little is known about the underlying causes of miRNA disruption in HD. We and others have previously shown that mutant Huntingtin binds to Ago2, a central component of miRNA biogenesis, and disrupts mature miRNA levels. In this study, we sought to determine if miRNA maturation per se was compromised in HD. Towards this end, we characterized major miRNA biogenesis pathway components and miRNA maturation products (pri-miRNA, pre-miRNA, and mature) in human HD (N = 41, Vonsattel grades HD2-4) and healthy control (N = 25) subjects. Notably, the striatum (putamen) and cortex (BA39) from the same individuals were analyzed in parallel. We show that Ago2, Drosha, and Dicer were strongly downregulated in human HD at the early stages of the disease. Using a panel of HD-related miRNAs (miR-10b, miR-196b, miR-132, miR-212, miR-127, miR-128), we uncovered various types of maturation defects in the HD brain, the most prominent occurring at the pre-miRNA to mature miRNA maturation step. Consistent with earlier findings, we provide evidence that alterations in autophagy could participate in miRNA maturation defects. Notably, most changes occurred in the striatum, which is more prone to HTT aggregation and neurodegeneration. Likewise, we observed no significant alterations in miRNA biogenesis in human HD cortex and blood, strengthening tissue-specific effects. Overall, these data provide important clues into the underlying mechanisms behind miRNA alterations in HD-susceptible tissues. Further investigations are now required to understand the biological, diagnostic, and therapeutic implications of miRNA/RNAi biogenesis defects in HD and related neurodegenerative disorders.

Omics approaches to discover pathophysiological pathways contributing to human pain.

Omics approaches to discover pathophysiological pathways contributing to human pain.

Cerebrovascular activity is a major factor in the cerebrospinal fluid flow dynamics

Cerebrospinal fluid (CSF) provides physical protection to the central nervous system as well as an essential homeostatic environment for the normal functioning of neurons. Additionally, it has been proposed that the pulsatile movement of CSF may assist in glymphatic clearance of brain metabolic waste products implicated in neurodegeneration. In awake humans, CSF flow dynamics are thought to be driven primarily by cerebral blood volume fluctuations resulting from a number of mechanisms, including a passive vascular response to blood pressure variations associated with cardiac and respiratory cycles. Recent research has shown that mechanisms that rely on the action of vascular smooth muscle cells (“cerebrovascular activity”) such as neuronal activity, changes in intravascular CO2, and autonomic activation from the brainstem, may lead to CSF pulsations as well. Nevertheless, the relative contribution of these mechanisms to CSF flow remains unclear. To investigate this further, we developed an MRI approach capable of disentangling and quantifying CSF flow components of different time scales associated with these mechanisms. This approach was evaluated on human control subjects (n = 12) performing intermittent voluntary deep inspirations, by determining peak flow velocities and displaced volumes between these mechanisms in the fourth ventricle.We found that peak flow velocities were similar between the different mechanisms, while displaced volumes per cycle were about a magnitude larger for deep inspirations. CSF flow velocity peaked at around 10.4 s (range 7.1–14.8 s, n = 12) following deep inspiration, consistent with known cerebrovascular activation delays for this autonomic challenge. These findings point to an important role of cerebrovascular activity in the genesis of CSF pulsations. Other regulatory triggers for cerebral blood flow such as autonomic arousal and orthostatic challenges may create major CSF pulsatile movement as well. Future quantitative comparison of these and possibly additional types of CSF pulsations with the proposed approach may help clarify the conditions that affect CSF flow dynamics.

Transcriptomic Profiling Reveals Discrete Poststroke Dementia Neuronal and Gliovascular Signatures

Poststroke dementia (PSD) is associated with pathology in frontal brain regions, in particular dorsolateral prefrontal cortex (DLPFC) neurons and white matter, remote from the infarct. We hypothesised that PSD results from progressive DLPFC neuronal damage, associated with frontal white matter gliovascular unit (GVU) alterations. We investigated the transcriptomic profile of the neurons and white matter GVU cells previously implicated in pathology. Laser-capture microdissected neurons, astrocytes and endothelial cells were obtained from the Cognitive Function After Stroke cohort of control, PSD and poststroke non-dementia (PSND) human subjects. Gene expression was assessed using microarrays and pathway analysis to compare changes in PSD with controls and PSND. Neuronal findings were validated using NanoString technology and compared with those in the bilateral common carotid artery stenosis (BCAS) mouse model. Comparing changes in PSD compared to controls with changes in PSND compared to controls identified transcriptomic changes associated specifically with dementia. DLPFC neurons showed defects in energy production (tricarboxylic acid (TCA) cycle, adenosine triphosphate (ATP) binding and mitochondria), signalling and communication (MAPK signalling, Toll-like receptor signalling, endocytosis). Similar changes were identified in neurons isolated from BCAS mice. Neuronal findings accompanied by altered astrocyte communication and endothelium immune changes in the frontal white matter, suggesting GVU dysfunction. We propose a pathogenic model in PSD whereby neuronal changes are associated with frontal white matter GVU dysfunction leading to astrocyte failure in supporting neuronal circuits resulting in delayed cognitive decline associated with PSD. Therefore, targeting these processes could potentially ameliorate the dementia seen in PSD.

Magnetic Resonance 3D Measurement of the Endolymphatic Space in 100 Control Human Subjects

Three Dimensional-magnetic resonance imaging of the inner ear endolymphatic space was performed in 100 control human subjects. Ear endolymphatic space was visualized using contrast-enhanced magnetic resonance imaging with intravenous gadolinium injection. The images were acquired on a 3-tesla magnetic resonance scanner using a 32-channel array head coil for all patients. The ear endolymphatic space and total fluid space volumes of the inner ear, cochlea, and vestibule were measured, and the ear endolymphatic space in the cochlea and vestibule was classified into four categories. The cochlea categories were: c-1, no ear endolymphatic space; c-2, ear endolymphatic space visualized only until the upper turn; c-3, ear endolymphatic space visualized from the upper turn to the second turn; and c-4, ear endolymphatic space visualized up to the basal turn. The vestibule categories were: v-1, no ear endolymphatic space; v-2, ear endolymphatic space visualized only in the utriculus; v-3, ear endolymphatic space visualized in the utriculus and sacculus separately; and v-4, ear endolymphatic space visualized in the utriculus and sacculus together. The mean TFS volumes of the inner ear, cochlea, and vestibule were 282.1 ± 33.2 µL, 112.9 ± 15.9 µL, and 69.1 ± 9.9 µL, respectively, and that of the ampulla of the posterior semicircular canal was 7.8 ± 1.7 µL. The mean ear endolymphatic space/total fluid space volume ratio in the cochlea was 10.3 ± 6.7% and that in the vestibule was 17.3 ± 12.2%. This ratio in the cochlea was between the results of categories c-1 and c-2 and that in the vestibule between those of categories v-2 and v-3. Two subjects had ear endolymphatic space extending from the vestibule to the posterior non-ampullated crus of the lateral semicircular canal. These findings can be applied as standard reference values for further research.

Therapeutic Targeting of PPAR Signaling in Cancer Treatment-Related Lymphedema

Lymphedema secondary to cancer treatment affects up to 1 in 5 cancer survivors. It is an irreversible late normal tissue toxicity characterized by swelling and scar formation (fibrosis) of the affected limb with physical and psychosocial health consequences for patients. Recent evidence reveals changes in peroxisome proliferator-activated receptor (PPAR) signaling as an underlying pathology of this disease. PPAR agonists have demonstrated therapeutic benefit in other fibrotic conditions; their role in lymphedema, however, remains unclear.In this study, RNA-sequencing of human and murine tissues with and without lymphedema was performed. Samples included: human lymphedema (n = 4), human normal tissue control (n = 5, matched for demographics and cancer history), murine lymphedema (n = 12), and murine control (n = 9). Pathway enrichment analysis was performed. An in vitro model of lymphedema fibrosis using cultured transforming growth factor beta (TGFβ)-stimulated fibroblasts and an in vivo model of lymphedema using a murine surgical model of tail lymphedema were utilized to evaluate the effects of PPAR agonists on gene expression and fibrotic protein expression (pro-collagen Iα1, collagen) in lymphedema compared to controls.RNA-sequencing of tissue from human and murine lymphedema and control subjects identified that suppression of PPAR signaling was a hallmark of lymphedema fibrosis on pathway enrichment analysis (FDR < 0.01). In the murine model, PPAR signaling gene expression changes co-occurred with fibrotic deposition beginning as early as two weeks after lymphedema development, and continued for at least 35 weeks. Treatment of TGFβ-stimulated fibroblasts with a PPAR agonist resulted in an increase in expression of PPAR genes and suppression of COL1α1 gene expression (n≥9 per group, q < 0.05) as well as reduction in the secretion of fibrotic proteins: pro-collagen Iα1 on ELISA (n≥8 per group, P < 0.0001) and collagen I on Western blotting. PPAR agonist treatment of the murine model of lymphedema resulted in a significant reduction in lymphedema fibrosis as measured by trichrome collagen staining (n≥4 per group, P < 0.05).These data, for the first time, demonstrated the value of upregulating PPAR signaling to treat lymphedema. For this condition, which to date, has no cure, repurposing PPAR agonists for lymphedema treatment is a highly promising therapeutic opportunity, which warrants further investigation.

Comprehensive Immunologic Evaluation of Bronchoalveolar Lavage Samples from Human Patients with Moderate and Severe Seasonal Influenza and Severe COVID-19.

Infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) or seasonal influenza may lead to respiratory failure requiring intubation and mechanical ventilation. The pathophysiology of this respiratory failure is attributed to local immune dysregulation, but how the immune response to viral infection in the lower airways of the human lung differs between individuals with respiratory failure and those without is not well understood. We used quantitative multiparameter flow cytometry and multiplex cytokine assays to evaluate matched blood and bronchoalveolar lavage (BAL) samples from control human subjects, subjects with symptomatic seasonal influenza who did not have respiratory failure, and subjects with severe seasonal influenza or SARS-CoV-2 infection with respiratory failure. We find that severe cases are associated with an influx of nonclassical monocytes, activated T cells, and plasmablast B cells into the lower airways. Cytokine concentrations were not elevated in the lower airways of moderate influenza patients compared with controls; however, 28 of 35 measured cytokines were significantly elevated in severe influenza, severe SARS-CoV-2 infection, or both. We noted the largest elevations in IL-6, IP-10, MCP-1, and IL-8. IL-1 family cytokines and RANTES were higher in severe influenza infection than severe SARS-CoV-2 infection. Interestingly, only the concentration of IP-10-correlated between blood and BAL during severe infection. Our results demonstrate inflammatory immune dysregulation in the lower airways during severe viral pneumonia that is distinct from lower airway responses seen in human patients with symptomatic, but not severe, illness and suggest that measurement of blood IP-10 concentration may predict this unique dysregulation.

Social impairments in mice lacking the voltage-gated potassium channel Kv3.1

Parvalbumin (PV)-expressing neurons have been implicated in the pathology of autism spectrum disorders (ASD). Loss of PV expression and/or reduced number of PV-expressing neurons have been reported not only in genetic and environmental rodent models of ASD, but also in post-mortem analyses of brain tissues from ASD vs. healthy control human subjects. PV-expressing neurons play a pivotal role in the maintenance of the balance between excitation and inhibition within neural circuits in part because of their fast-spiking properties. Their high firing rate is mostly regulated by the voltage-gated potassium channel Kv3.1. It is yet unknown whether disturbances in the electrophysiological properties of PV-expressing neurons per se can lead to behavioral disturbances. We assessed locomotor activity, social interaction, recognition and memory, and stereotypic behaviors in Kv3.1 wild-type (WT) and knockout (KO) mice. We then used Western Blot analyses to measure the impact of Kv3.1 deficiency on markers of GABA transmission (PV and GAD67) and neural circuit activity (Egr1). Deficiency in Kv3.1 channel is sufficient to induce social deficits, hyperactivity and stereotypic behaviors. These behavioral changes were independent of changes in GAD67 levels and associated with increased levels of PV protein in the prefrontal cortex and striatum. These findings reveal that a loss of PV expression is not a necessary factor to induce an ASD-like phenotype in mice and support the need for further investigation to fully understand the contribution of PV-expressing neurons to ASD pathology.

Characterization of Human CD4 T Cells Specific for a C-Peptide/C-Peptide Hybrid Insulin Peptide.

Hybrid Insulin Peptides (HIPs), which consist of insulin fragments fused to other peptides from β-cell secretory granule proteins, are CD4 T cell autoantigens in type 1 diabetes (T1D). We have studied HIPs and HIP-reactive CD4 T cells extensively in the context of the non-obese diabetic (NOD) mouse model of autoimmune diabetes and have shown that CD4 T cells specific for HIPs are major contributors to disease pathogenesis. Additionally, in the human context, HIP-reactive CD4 T cells can be found in the islets and peripheral blood of T1D patients. Here, we performed an in-depth characterization of the CD4 T cell response to a C-peptide/C-peptide HIP (HIP11) in human T1D. We identified the TCR expressed by the previously-reported HIP11-reactive CD4 T cell clone E2, which was isolated from the peripheral blood of a T1D patient, and determined that it recognizes HIP11 in the context of HLA-DQ2. We also identified a HIP11-specific TCR directly in the islets of a T1D donor and demonstrated that this TCR recognizes a different minimal epitope of HIP11 presented by HLA-DQ8. We generated and tested an HLA-DQ2 tetramer loaded with HIP11 that will enable direct ex vivo interrogation of CD4 T cell responses to HIP11 in human patients and control subjects. Using mass spectrometric analysis, we confirmed that HIP11 is present in human islets. This work represents an important step in characterizing the role of CD4 T cell responses to HIPs in human T1D.

ATX‐LPP3 axis mediated LPA regulation following myocardial injury

The bioactive lysophosphatidic acid (LPA) plays a well-known role in atherosclerotic disease, whereas its role in myocardial function remains virtually unexplored. Following acute myocardial infarction, serum LPA concentration rises by six-fold over control human subjects, suggesting LPA may contribute to the pathogenesis of myocardial infarction. LPA production involves hydrolysis of lysophosphatidylcholine by the secreted enzyme autotaxin, whereas lipid phosphate phosphatase-3 (LPP3) catalyzes LPA dephosphorylation to generate lipid products that are not receptor active. We present the first evidence that cardiac ischemia/reperfusion (I/R) injury enhances myocardial autotaxin levels and decreases myocardial LPP3 expression, and this is associated with increased serum LPA levels. Upon reperfusion, reactive oxygen species production arises as a burst of superoxide from mitochondria following I/R injury. The redox-sensitive transcription factor NFAT has been shown to bind to the autotaxin promoter and induce its expression. Therefore, we looked at the autotaxin and LPP3 regulation in mice following I/R injury in the myocardium. After 1h ligation followed by 3h reperfusion in the myocardium, we observed a 3 fold increase in the autotaxin protein levels, whereas LPP3 protein levels were significantly downregulated as observed through Western blot analysis in these myocardial ischemic tissues. Autotaxin and miR-92a mRNA expression levels were significantly upregulated, whereas KLF2 and LPP3 mRNA expressions were significantly downregulated following I/R injury at 24 hours. Western blot analysis showed a 3 fold increase autotaxin protein levels and immunohistochemistry of human infarct tissues at 24 hours showed disruption of the sarcomere with decreased LPP3 staining. We found that I/R injury transactivates miR-92a, and inhibit KLF2, an upstream activator of LPP3. Taken together, our in vivo data, from the myocardial I/R injury and human infarct tissues, suggest that regulation of autotaxin and LPP3 activity might cause the rise in serum LPA levels as reported with acute myocardial infarct patients.

PGI2 Inhibits Intestinal Epithelial Permeability and Apoptosis to Alleviate Colitis.

Background & AimsInflammatory bowel diseases (IBDs) that encompass both ulcerative colitis and Crohn’s disease are a major public health problem with an etiology that has not been fully elucidated. There is a need to improve disease outcomes and preventive measures by developing new effective and lasting treatments. Although polyunsaturated fatty acid metabolites play an important role in the pathogenesis of several disorders, their contribution to IBD is yet to be understood.MethodsPolyunsaturated fatty acids metabolite profiles were established from biopsy samples obtained from Crohn’s disease, ulcerative colitis, or control patients. The impact of a prostaglandin I2 (PGI2) analog on intestinal epithelial permeability was tested in vitro using Caco-2 cells and ex vivo using human or mouse explants. In addition, mice were treated with PGI2 to observe dextran sulfate sodium (DSS)-induced colitis. Tight junction protein expression, subcellular location, and apoptosis were measured in the different models by immunohistochemistry and Western blotting.ResultsA significant reduction of PGI2 in IBD patient biopsies was identified. PGI2 treatment reduced colonic inflammation, increased occludin expression, decreased caspase-3 cleavage and intestinal permeability, and prevented colitis development in DSS-induced mice. Using colonic explants from mouse and human control subjects, the staurosporine-induced increase in paracellular permeability was prevented by PGI2. PGI2 also induced the membrane location of occludin and reduced the permeability observed in colonic biopsies from IBD patients.ConclusionsThe present study identified a PGI2 defect in the intestinal mucosa of IBD patients and demonstrated its protective role during colitis.

Gender dimorphism in serotonin dopamine and norepinephrine neurons of isodendritc core in postmortem human and nonhuman primate control subjects

AbstractBackgroundComorbidity of depression in bMets, NPSs and NDDs is resource demanding clinical area considering treatment challenges and patient care. NPSs represent prodromal symptoms of dementia results of neurobiological changes in specific subcortical region. Dynamic cascade of Aβ shifts from nonamyloidogenic is implicated in early cognitive impairment and depressive symptoms. Serotonergic system is implicated in Aβ pathology and NE is important to compensatory in Aβ degradation. Although the role of DA is not investigated in AD pathology. Overall neuromodulation in IDC of midbrain is imperative and intriguing aspect of monoaminergic association in AD and other NDDs pathology.Here we present genetic dimorphism of key area of the brain (5HT: dorsal raphe, DA:substantia nigra, Noradrenaline: locus ceruleous).Method4males and 4 females midbrain of control subject were collected from Neurobiobank (NIH) and ONPRC. Brain sections were collected 20‐micron sections on cover glass plain slides. 4mm tissue punches were used to collect RNA from tissues. Enough tissues used to generate 10ug‐20 ug average quality RNA. 4ug RNA from Raphe, substantia nigra and locus ceruleous was isolated using trizol isolation and sent to UTSW microarray core. The RNA was hybridized to Clariom D Human chip and CEL chip data obtained from UTSW core. CEL files were uploaded in Transcriptome Analysis Console (TAC4.0). Statistically significant data in Excel file is uploaded in Ingenuity Pathway analysis software to find biological pathways dominate female and male brains. Microarray analysis of adult serotonin neurons in human and NHP dorsal raphe, Substantia nigra and Locus ceruleous (DR/SN/LC) Chip has expression of 500.000 known and unknown RNA species transcript encoding 135,000 RNAResultHuman DR/SNC/LC have 6643/13475/9194 number of probe set and 850/896//501 pathways, and Rhesus DR/SNC/LC have 7261/2157/1938 probe sets and 872/218/198 pathways altered respectively with one or more genes. Major dominated pathways: electron transport chain. Oxidative phosphorylation, miRNA, mitochondria dysfunction, apoptosis signaling, will be presented in the posterConclusionMajor energy metabolism pathway dominates in all three area in male DR/SN/LC area clearly state female brain having lower energy metabolism may results in reduce homeostasis in Isodendritc core midbrain area. Although compensatory mechanism may still yet investigate.

Abstract PO-034: Increased physical activity delays development of obesity-induced pancreatic ductal adenocarcinoma in mice and modulates inflammation

Abstract Background: Obesity is a risk factor for pancreatic ductal adenocarcinoma (PDAC), a deadly disease with no preventive strategies. Lifestyle interventions to decrease obesity might serve to prevent obesity-induced PDAC. We examined whether decreasing obesity via increased physical activity (PA) and/or diet-induced weight loss could prevent PDAC in mice and compared the levels of systemic inflammatory-associated cytokines in human subjects undergoing a PA intervention. Methods: Obesity-induced PDAC mouse models were exposed to various interventions before and after cancer initiation, that included increased PA, diet-induced weight loss, and/or chemotherapy. We evaluated body composition, tumor progression, growth, fibrosis, and inflammation. Circulating inflammatory-associated cytokines of overweight/obese human subjects before and after a PA intervention were also compared. Results: Genetically engineered PDAC mice on a PA intervention while on a HFD, gained less weight, displayed lower grade pancreatic intraepithelial neoplasias lesions, and fewer mice developed PDAC compared to controls. Similar results were observed in PDAC mice given a HFD to induce obesity, prior to a control diet (CD) and PA intervention, compared to mice given a CD after the HFD. PA alone or with a CD intervention decreased body weight but did not prevent tumor growth in an orthotopic mouse model of PDAC. PA combined with chemotherapy increased the percentage of splenic cluster of differentiation 8+ (CD8+) T cells in PDAC mice. Finally, PA prevented the increase of systemic inflammatory-associated cytokines in PDAC mice and in overweight/obese control human subjects. Conclusions: PA alone or with diet-induced weight loss delayed the progression of PDAC in genetically engineered mice and reduced inflammation. While these interventions did not prevent tumor growth in the orthotopic mouse model, it did modulate immune responses. Reducing obesity by increasing PA and decreasing dietary fat intake could be a way of reducing the incidence of PDAC in high-risk obese individuals. Citation Format: Valentina Pita-Grisanti, Kelly Dubay, Ali Lahooti, Niharika Badi, Olivia Ueltschi, Myrriah Chavez-Tomar, Samantha Terhorst, Sue Knoblaugh, Christopher Coss, Thomas Mace, Fouad Choueiry, Alice Hinton, Jennifer M Mitchell, Karen Basen-Engquist, Zobeida Cruz-Monserrate. Increased physical activity delays development of obesity-induced pancreatic ductal adenocarcinoma in mice and modulates inflammation [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-034.

HMGB1-Mediated Platelet Activation Is Independent of Platelet Mitochondrial Reactive Oxygen Species Generation

Background: Sickle cell disease (SCD) is a hemolytic disorder that exhibits pathologic platelet activation. Notably, hemolysis is tightly associated with platelet activation and thrombotic complications of SCD such as stroke, leg ulceration, and pulmonary hypertension. To this end, we and others have shown that free hemoglobin (Hb) released into the plasma via hemolysis directly activates healthy platelets ex vivo in a concentration-dependent manner. Treatment with Hb stimulates the production of mitochondrial reactive oxygen species (mtROS) within the platelet, resulting in thrombotic activation. Accordingly, scavenging mtROS prevents Hb-induced platelet activation. While it is established that hemolysis mediates platelet activation, the factors that modulate this response remain unknown. High mobility group box 1 (HMGB1) is an inflammatory mediator, which signals through toll-like receptor-4 (TLR4), to instigate thrombosis. Notably, we previously showed that HMGB1 is elevated in the plasma of SCD patients and augments platelet activation on its own. Importantly, complementary interactions between Hb and other pro-thrombotic plasma components such as HMGB1 have not been investigated. Preliminary data show that HMGB1, like Hb, stimulates platelet mtROS generation. Thus, we hypothesized that elevated levels of HMGB1 and Hb synergize in SCD to stimulate platelet mtROS production and downstream platelet activation. Methods: Whole blood samples were collected from healthy human control subjects or SCD subjects in steady state (n=5 per group). Plasma HMGB1 concentrations were quantified by ELISA. Platelets were isolated and treated with reagents including HMGB1, ferric Hb, or the mtROS scavenger MitoTEMPO. Platelet activation was measured by flow cytometry using PE antibody to GPIIb (CD41) to select for platelets, and two markers of platelet activation: PAC1 to detect the activation-dependent conformational change in integrin αIIbβ3 (GP IIb-IIIa), and APC to detect exposure of surface CD62P (P-selectin). Platelet mtROS were estimated using MitoSOX Red and fluorescence spectroscopy. Data was analyzed using FlowJo software and nonparametric statistical tests. Results: The baseline level of platelet activation in SCD was 7-fold higher than in controls (P=0.009). However, there was no difference in platelet activation levels between isolated SCD and control platelets in response to ex vivo treatment with agonists ADP (5 uM), collagen (50 ug/ml), or thrombin (0.1 U/ml) (P=0.38), suggesting that SCD platelets have the same agonist-induced response as control platelets when they are removed from their native plasma. We found that the concentration of HMGB1 in SCD plasma is 1.5 fold higher than controls (P=0.04), and that plasma HMGB1 concentrations positively correlate with increased platelet activation (P=0.04). Co-incubation of isolated healthy control platelets with HMGB1 (10 ug/ml) and major hemolytic byproduct Hb (10 uM, 30 uM, 50 uM) enhanced platelet activation at all doses of Hb by 3-fold compared to that of Hb alone. Mechanistically, platelets exposed to Hb alone generated mtROS which are necessary for Hb-induced platelet activation. We found that HMGB1 similarly induced platelet mtROS generation, but scavenging with MitoTEMPO did not prevent HMGB1-mediated platelet activation. However the addition of MitoTEMPO to platelets co-treated with HMGB1 + Hb attenuated platelet activation 2-fold, indicating that scavenging mtROS neutralizes the enhanced effect of Hb + HMGB1. Conclusions: We show that HMGB1 synergizes with Hb to enhance platelet activation in SCD, but unlike Hb, HMGB1-dependent platelet activation is independent of mtROS generation. Accordingly, we observed that MitoTEMPO partially attenuates the enhanced activation from co-stimulation by HMGB1 + Hb. These data characterize the synergy between HMGB1 and Hb for the first time and demonstrate differential platelet signaling between the two agonists. Disclosures No relevant conflicts of interest to declare.

Modulation of dopamine D1 receptors via histamine H3 receptors is a novel therapeutic target for Huntington's disease.

Early Huntington's disease (HD) include over-activation of dopamine D1 receptors (D1R), producing an imbalance in dopaminergic neurotransmission and cell death. To reduce D1R over-activation, we present a strategy based on targeting complexes of D1R and histamine H3 receptors (H3R). Using an HD mouse striatal cell model and HD mouse organotypic brain slices we found that D1R-induced cell death signaling and neuronal degeneration, are mitigated by an H3R antagonist. We demonstrate that the D1R-H3R heteromer is expressed in HD mice at early but not late stages of HD, correlating with HD progression. In accordance, we found this target expressed in human control subjects and low-grade HD patients. Finally, treatment of HD mice with an H3R antagonist prevented cognitive and motor learning deficits and the loss of heteromer expression. Taken together, our results indicate that D1R - H3R heteromers play a pivotal role in dopamine signaling and represent novel targets for treating HD.

Dysregulated expression of the alternatively spliced variant mRNAs of the mu opioid receptor gene, OPRM1, in the medial prefrontal cortex of male human heroin abusers and heroin self-administering male rats.

Heroin, a mu agonist, acts through the mu opioid receptor. The mu opioid receptor gene, OPRM1, undergoes extensive alternative splicing, creating an array of splice variants that are conserved from rodent to humans. Increasing evidence suggests that these OPRM1 splice variants are pharmacologically important in mediating various actions of mu opioids, including analgesia, tolerance, physical dependence, rewarding behavior, as well as addiction. In the present study, we examine expression of the OPRM1 splice variant mRNAs in the medial prefrontal cortex (mPFC), one of the major brain regions involved in decision-making and drug-seeking behaviors, of male human heroin abusers and male rats that developed stable heroin-seeking behavior using an intravenous heroin self-administration (SA) model. The results show similar expression profiles among multiple OPRM1 splice variants in both human control subjects and saline control rats, illustrating conservation of OPRM1 alternative splicing from rodent to humans. Moreover, the expressions of several OPRM1 splice variant mRNAs were dysregulated in the postmortem mPFCs from heroin abusers compared to the control subjects. Similar patterns were observed in the rat heroin SA model. These findings suggest potential roles of the OPRM1 splice variants in heroin addiction that could be mechanistically explored using the rat heroin SA model.