PP 4.4- 00059 Quantification of HIV Reservoirs in Brain: focus in bystander damage

Journal of Virus Eradication

2022 Dec 01

Hernandez, C;Eugenin, E;
| DOI: 10.1016/j.jve.2022.100212

Background: Early after primary infection, HIV reservoirs are established within multiple tissues, including the brain. As these viral reservoirs are not targeted by antiretroviral therapy (cART), we require robust methods of detection, quantification, and characterization of these viral reservoirs in human tissues. Our recent work developed a multi-component imaging methodology that characterizes and quantifies viral reservoirs within the brain. Methods: The imaging methodology demonstrated utilizes the simultaneous staining of brain tissue from HIV-infected donors using DNAscope, RNAscope, and antibodies for HIV-DNA, HIV-mRNA, and either viral or host proteins, respectively. The panel of patients included in these analyses varied in cART regimen, viral load, years living with HIV, and neurocognitive status, all contrasted to age-matched tissues from uninfected patients. Results: Our group demonstrated that cART is sufficient to reduce the size of the viral reservoirs within the brains of HIV patients. We also found that about half of the cells positive for HIV-DNA expressed HIV-mRNA, and only about one-third expressed viral proteins. HIV proteins varied in expression and bystander uptake by uninfected cells but could provide insight into bystander toxicity. Conclusions: The results found were present irrespective of cART regimen and systemic viral replication but suggested that these viral reservoirs are a major barrier to curing HIV and treating associated neurocognitive disorders.

UDP-glucuronate metabolism controls RIPK1-driven liver damage in nonalcoholic steatohepatitis

Nature communications

2023 May 11

Zhang, T;Zhang, N;Xing, J;Zhang, S;Chen, Y;Xu, D;Gu, J;
PMID: 37169760 | DOI: 10.1038/s41467-023-38371-2

Hepatocyte apoptosis plays an essential role in the progression of nonalcoholic steatohepatitis (NASH). However, the molecular mechanisms underlying hepatocyte apoptosis remain unclear. Here, we identify UDP-glucose 6-dehydrogenase (UGDH) as a suppressor of NASH-associated liver damage by inhibiting RIPK1 kinase-dependent hepatocyte apoptosis. UGDH is progressively reduced in proportion to NASH severity. UGDH absence from hepatocytes hastens the development of liver damage in male mice with NASH, which is suppressed by RIPK1 kinase-dead knockin mutation. Mechanistically, UGDH suppresses RIPK1 by converting UDP-glucose to UDP-glucuronate, the latter directly binds to the kinase domain of RIPK1 and inhibits its activation. Recovering UDP-glucuronate levels, even after the onset of NASH, improved liver damage. Our findings reveal a role for UGDH and UDP-glucuronate in NASH pathogenesis and uncover a mechanism by which UDP-glucuronate controls hepatocyte apoptosis by targeting RIPK1 kinase, and suggest UDP-glucuronate metabolism as a feasible target for more specific treatment of NASH-associated liver damage.

Characterization Of Distinct Nociceptive Populations In The Human Drg

The Journal of Pain

2023 Apr 01

Staedtler, E;Iadarola, M;Sapio, M;Maric, D;Ghetti, A;Mannes, A;
| DOI: 10.1016/j.jpain.2023.02.059

Nociceptive input to the spinal cord is transmitted by primary afferent neurons in the dorsal root ganglia (DRG). A subset of DRG neurons has the ability to attenuate nociceptive transmission through expression of the µ-opioid receptor. The relationship between algesic and analgesic properties of individual DRG neurons has not yet been evaluated in the human. By using a combination of six different 4-Plex in-situ hybridization experiments, we were able to identify three different main nociceptive populations. First, we detected a population poly-responsive, poly-modulated small-diameter nociceptors that co-express TRPV1, OPRM1, and SCN11A, and in most cases additionally SCN10A and P2RX3 and in subpopulations additionally TAC1, TRPA1, TRPM8, OPRD1, PIEZO2, and/or OPRL1. Second, we detected a medium-sized set of multimodal putative nociceptors with a very similar molecular profile, apart from a lack of expression of TRPA1. Both nociceptor types do express the µ-opioid receptor (encoded by OPRM1) and their activity and input into the spinal cord likely can be attenuated by clinically used µ-receptor agonists. The third population consists of small-diameter neurons that have a molecular expression profile characteristic for nociceptive neurons, such that they express TRPV1 together with SCN11A, SCN10A, P2RX3, PIEZO2, OPRD1, and partially TRPA1. The most distinguishable characteristic of this population is the lack of expression of the µ-opioid receptor and thus are non-susceptible to clinically used opioids. Further molecular characterization of these nociceptive populations might reveal attractive molecular candidates for targeting different types of pain indications.

Targeting translation: A review of preclinical animal models in the development of treatments for chemotherapy-induced peripheral neuropathy

Journal of the peripheral nervous system : JPNS

2023 Mar 30

White, D;Abdulla, M;Park, SB;Goldstein, D;Moalem-Taylor, G;Lees, JG;
PMID: 36995049 | DOI: 10.1111/jns.12544

The expanding use of chemotherapy in curative cancer treatment has simultaneously resulted in a substantial and growing cohort of cancer survivors with prolonged disability from chemotherapy-induced peripheral neuropathy (CIPN). CIPN is associated with several commonly prescribed chemotherapeutics, including taxanes, platinum-based drugs, vinca alkaloids, bortezomib and thalidomide. These distinct classes of chemotherapeutics, with their varied neurotoxic mechanisms, often cause patients to suffer from a broad profile of neuropathic symptoms including chronic numbness, paraesthesia, loss of proprioception or vibration sensation and neuropathic pain. Decades of investigation by numerous research groups have provided substantial insights describing this disease. Despite these advances, there is currently no effective curative or preventative treatment option for CIPN and only the dual serotonin-norepinephrine reuptake inhibitor Duloxetine is recommended by clinical guidelines for the symptomatic treatment of painful CIPN.In this review, we examine current preclinical models, with our analysis focused on translational relevance and value.Animal models have been pivotal in achieving a better understanding of the pathogenesis of CIPN. However, it has been challenging for researchers to develop appropriate preclinical models that are effective vehicles for the discovery of translatable treatment options.Further development of preclinical models targeting translational relevance will promote value for preclinical outcomes in CIPN studies.

Distribution and function of neuronal IL-1R1 in the CNS

Brain, Behavior, and Immunity

2022 Nov 01

Nemeth, D;Liu, X;Kocak, N;Niu, H;Smirnova, M;McGovern, S;Herd, A;DiSabato, D;Floyd, T;Atluri, R;Nusstein, A;Oliver, B;Witcher, K;McKim, D;Gajewski-Kurdziel, P;Godbout, J;Zhang, Q;Blakely, R;Sheridan, J;Quan, N;
| DOI: 10.1016/j.bbi.2022.07.065

Methods: Global and neuronal specific IL-1R1 reporter mice, RNA sequencing analysis, and double-immunofluorescent labeling were used to map and validate nIL-1R1 expression. NF-κB/IL-1R1 co-reporter mice were utilized to detect IL-1R1 and NF-κB expression following intracerebroventricular (i.c.v.) IL-1 injections. Basescope in situ hybridization was utilized to detect splice variants of IL-1R Accessory Protein (IL-1AcP). Unpredictable foot shock (6x shocks over 1hr for 6d) was employed as a chronic stress paradigm. Results: IL-1R1 is expressed in subsets of glutamatergic or serotonergic neurons, with highest expression in the dentate gyrus (DG) and dorsal raphe nucleus (DRN). I.c.v. IL-1β injection reveals nIL-1R1 does not signal through the canonical NF-κB pathway, whereas endothelia and ventricular IL-1R1s do. We identified that neurons of the DG and DRN express the alternatively spliced IL-1RAcP Isoform B (IL-1RAcPb). Additional results suggest that nIL-1R1 may become reactive to IL-1 when neuronal expression of IL-1RAcPb shifts to the canonical IL-1RAcP following stress. Consequently, nIL-1R1 mediates activation of microglia near nIL-1R1 neurons. Conclusions: These data suggest that regional specific nIL-1R1 may require a culmination of stress and inflammatory signals to unlock nIL-1R1 signaling. Overall, these data provide a map of nIL-1R1 and its corresponding accessory protein in the brain along with a potential output of nIL-1R1 signaling.

Protease-Activated Receptors in Health and Disease

Physiological reviews

2022 Jul 28

Peach, CJ;Edgington-Mitchell, LE;Bunnett, NW;Schmidt, BL;
PMID: 35901239 | DOI: 10.1152/physrev.00044.2021

Although generally regarded as degradatory enzymes, certain proteases are also signaling molecules that specifically control cellular functions by cleaving protease-activated receptors (PARs). The four known PARs are members of the large family of G protein-coupled receptors. These transmembrane receptors control most physiological and pathological processes and are the target of a large proportion of therapeutic drugs. Signaling proteases include enzymes from the circulation, from immune, inflammatory epithelial and cancer cells, as well as from commensal and pathogenic bacteria. Advances in our understanding of the structure and function of PARs provide insights into how diverse proteases activate these receptors to regulate physiological and pathological processes in most tissues and organ systems. The realization that proteases and PARs are key mediators of disease, coupled with advances in understanding the atomic level structure of PARs and their mechanisms of signaling in subcellular microdomains, has spurred the development of antagonists, some of which have advanced to the clinic. Herein we review the discovery, structure and function of this receptor system, highlight the contribution of PARs to homeostatic control, and discuss the potential of PAR antagonists for the treatment of major diseases.

On the use of Earth Observation to support estimates of national greenhouse gas emissions and sinks for the Global stocktake process: lessons learned from ESA-CCI RECCAP2

Carbon balance and management

2022 Oct 01

Bastos, A;Ciais, P;Sitch, S;Aragão, LEOC;Chevallier, F;Fawcett, D;Rosan, TM;Saunois, M;Günther, D;Perugini, L;Robert, C;Deng, Z;Pongratz, J;Ganzenmüller, R;Fuchs, R;Winkler, K;Zaehle, S;Albergel, C;
PMID: 36183029 | DOI: 10.1186/s13021-022-00214-w

The Global Stocktake (GST), implemented by the Paris Agreement, requires rapid developments in the capabilities to quantify annual greenhouse gas (GHG) emissions and removals consistently from the global to the national scale and improvements to national GHG inventories. In particular, new capabilities are needed for accurate attribution of sources and sinks and their trends to natural and anthropogenic processes. On the one hand, this is still a major challenge as national GHG inventories follow globally harmonized methodologies based on the guidelines established by the Intergovernmental Panel on Climate Change, but these can be implemented differently for individual countries. Moreover, in many countries the capability to systematically produce detailed and annually updated GHG inventories is still lacking. On the other hand, spatially-explicit datasets quantifying sources and sinks of carbon dioxide, methane and nitrous oxide emissions from Earth Observations (EO) are still limited by many sources of uncertainty. While national GHG inventories follow diverse methodologies depending on the availability of activity data in the different countries, the proposed comparison with EO-based estimates can help improve our understanding of the comparability of the estimates

Type 2 diabetes is associated with increased circulating levels of 3-hydroxydecanoate activating GPR84 and neutrophil migration

iScience

2022 Nov 01

Mikkelsen, R;Arora, T;Trošt, K;Dmytriyeva, O;Jensen, S;Meijnikman, A;Olofsson, L;Lappa, D;Aydin, Ö;Nielsen, J;Gerdes, V;Moritz, T;van de Laar, A;de Brauw, M;Nieuwdorp, M;Hjorth, S;Schwartz, T;Bäckhed, F;
| DOI: 10.1016/j.isci.2022.105683

Obesity and diabetes are associated with inflammation and altered plasma levels of several metabolites, which may be involved in disease progression. Some metabolites can activate G protein-coupled receptors (GPCRs) expressed on immune cells where they can modulate metabolic inflammation. Here we find that 3-hydroxydecanoate is enriched in the circulation of obese individuals with type 2 diabetes (T2D) compared with non-diabetic controls. Administration of 3-hydroxydecanoate to mice promotes immune cell recruitment to adipose tissue, which was associated with adipose inflammation and increased fasting insulin levels. Furthermore, we demonstrate that 3-hydroxydecanoate stimulates migration of primary human and mouse neutrophils, but not monocytes, through GPR84 and Gαi signaling in vitro. Our findings indicate that 3-hydroxydecanoate is a T2D-associated metabolite that increases inflammatory responses and may contribute to the chronic inflammation observed in diabetes.

Pathophysiology of Nociception and Rare Genetic Disorders with Increased Pain Threshold or Pain Insensitivity

Pathophysiology : the official journal of the International Society for Pathophysiology

2022 Aug 02

Cascella, M;Muzio, MR;Monaco, F;Nocerino, D;Ottaiano, A;Perri, F;Innamorato, MA;
PMID: 35997391 | DOI: 10.3390/pathophysiology29030035

Pain and nociception are different phenomena. Nociception is the result of complex activity in sensory pathways. On the other hand, pain is the effect of interactions between nociceptive processes, and cognition, emotions, as well as the social context of the individual. Alterations in the nociceptive route can have different genesis and affect the entire sensorial process. Genetic problems in nociception, clinically characterized by reduced or absent pain sensitivity, compose an important chapter within pain medicine. This chapter encompasses a wide range of very rare diseases. Several genes have been identified. These genes encode the Nav channels 1.7 and 1.9 (SCN9A, and SCN11A genes, respectively), NGFβ and its receptor tyrosine receptor kinase A, as well as the transcription factor PRDM12, and autophagy controllers (TECPR2). Monogenic disorders provoke hereditary sensory and autonomic neuropathies. Their clinical pictures are extremely variable, and a precise classification has yet to be established. Additionally, pain insensitivity is described in diverse numerical and structural chromosomal abnormalities, such as Angelman syndrome, Prader Willy syndrome, Chromosome 15q duplication syndrome, and Chromosome 4 interstitial deletion. Studying these conditions could be a practical strategy to better understand the mechanisms of nociception and investigate potential therapeutic targets against pain.

Selective Inhibition of Rho Kinase 2 Limits No Re-Flow in Rat Hearts Following Ischemia/Reperfusion

Circulation

2022 Jan 01

Pearce, L;He, D;Davidson, SM;Yellon, DM;
| DOI: 10.1161/circ.146.suppl_1.11926

Introduction: Myocardial infarction causes several types of injury to the myocardium including lethal cell injury and ‘no-reflow’ (NRF) /microvascular obstruction (MVO). Nonselective Rho Kinase (ROCK1/2) inhibitors such as Fasudil, ameliorate myocardial ischemia/reperfusion (I/R) injury but cause unwanted hypotension. Selective ROCK2 inhibitors (e.g.: KD025) are safe in clinical trials without causing haemodynamic compromise, however they have not been investigated in myocardial I/R. ROCK inhibitors prevent vascular smooth muscle cell (VSMC) contraction; such VSMC contraction/coronary spasm being features of ‘no-reflow’ (NRF) and microvascular obstruction (MVO). At present, there are limited therapies to improve ischemic MVO outcomes, and prognosis is poor. Hypothesis: Using a rat model, we hypothesised that, i) ROCK2 mRNA is expressed in myocardium and coronary vasculature and ii) The selective ROCK2 inhibitor KD025, would reduce infarct size (IS%) and NRF% (MVO) following I/R. Methods: RNA scope in-situ hybridisation was performed with a fluorescent, multiplex assay for ROCK1/2 & VSMC mRNA in myocardium and coronary vasculature. Male SD rats underwent in-vivo myocardial infarction with 30min ischemia, 180min reperfusion. 15min prior to reperfusion, the ROCK inhibitors Fasudil and KD025 or vehicle (DMSO) were administered i.p. For IS%, myocardium was stained with TTC, and regions not perfused with 1.5% Thioflavin S (NRF%), were visualised under UV light. Results: RNAscope confirmed the presence of ROCK2 mRNA within myocardium and VSMC of coronary arteries. Fasudil (10mg/kg) vs control significantly reduced regional IS% (30.3±4.4 vs52.9±3.8,p=0.02, n=15) and area of NRF% (12.4±2.8 vs28.6±2.2, p=0.001, n=15). However, there was significant hypotension;- Mean BP (mmHg) (72±3.9) vs control (84±2.3, p=0.007). KD025 (100mg/kg) did not reduce IS%, but significantly reduced the area of NRF% vs control (18.4±2.8 vs28.6±2.2,p=0.02, n=14) without hypotensive effect. Conclusions: Our results suggest that ROCK2 may be a prospective target in the management of coronary circulation reperfusion injury and ischemic MVO.

P.188 Early growth and metabolic abnormalities in zebrafish and cellular models of SELENON-related myopathy

Neuromuscular Disorders

2022 Oct 01

Barraza-Flores, P;Moghadaszadeh, B;Mitchell, B;Troiano, E;Mansur, A;Gupta, V;
| DOI: 10.1016/j.nmd.2022.07.331

SELENON-Related Myopathy (SELENON-RM) is a rare genetic disease caused by recessive mutations of the SELENON gene. It is characterized by the development of rigid spine, axial muscle weakness, and respiratory insufficiency. The most common histopathological feature in SELENON-RM patients is the presence of minicores in skeletal muscle biopsies, which are concentrated areas of mitochondrial depletion within fibers. Natural history data suggest that insulin-resistance as well as altered body mass index (BMI) are correlated with SELENON-RM prognosis. There is no cure or effective treatment for SELENON-RM. The SELENON gene encodes selenoprotein-N, an endoplasmic reticulum (ER) membrane glycoprotein with reducing catalytic activity. Selenoprotein-N has been shown to activate SERCA channels by reducing it at low Ca2+ concentrations in the ER. Additionally, it has also been shown to colocalize with Mitochondrial Associated Membranes (MAM), which are vital for mitochondrial function. However, the molecular mechanism(s) by which selenoprotein-N deficiency causes SELENON-RM is still unclear. A particular challenge has been the lack of cellular or animal models that exhibit readily assayable phenotypes. In this project, we aim to identify cellular and animal models suited for high throughput drug screening while elucidating the molecular mechanism of the disease. To do so, we tested selenoprotein-N deficient cells and zebrafish selenon-null models. Using different measures of metabolism, we found that Selenon-KO C2C12 cells and primary myoblasts isolated from Selenon-KO mice were metabolically impaired. We also assessed several phenotyping outcomes in zebrafish models, from embryonic development to adulthood. Our results showed muscle weakness during early development as well as reduced growth during the larval stage. Together, these data establish the potential for selenoprotein-N deficient cells and zebrafish as models for the discovery of therapeutic targets for SELENON-RM.

P.134a Phase 1/2a trial of delandistrogene moxeparvovec in patients with DMD: 4-year update

Neuromuscular Disorders

2022 Oct 01

Mendell, J;Sahenk, Z;Lehman, K;Lowes, L;Reash, N;Iammarino, M;Alfano, L;Lewis, S;Church, K;Shell, R;Potter, R;Griffin, D;Pozsgai, E;Hogan, M;Hu, L;Mason, S;Darton, E;Rodino-Klapac, L;
| DOI: 10.1016/j.nmd.2022.07.251

Delandistrogene moxeparvovec (SRP-9001) is an investigational gene transfer therapy developed for targeted skeletal and cardiac muscle expression of micro-dystrophin (a shortened, functional dystrophin protein). The objective of this phase 1/2a, single-dose, open-label clinical trial (NCT03375164) is to evaluate the safety of systemic delivery of delandistrogene moxeparvovec in patients with Duchenne muscular dystrophy (DMD). Four ambulatory patients with DMD (≥4 to ≤8 years old) were enrolled. Patients were given an intravenous infusion of delandistrogene moxeparvovec at a dose of 2.0x1014 vg/kg (supercoiled qPCR, linear plasmid standard equivalent of 1.33x1014 vg/kg) and prednisone (1 mg/kg/day) 1 day pre- to 30 days post-gene delivery. The primary outcome measure is safety. The secondary outcome measures include micro-dystrophin expression in pre- and post-muscle biopsies (Week 12 post-infusion). Key efficacy outcome measures include North Star Ambulatory Assessment (NSAA) and timed function tests. Previously, data from 3 years post-treatment were presented. Treatment-related adverse events (TRAEs) were mild to moderate, occurred mostly in the first 90 days of treatment, and all resolved. No serious adverse events (AEs), study discontinuations, or AEs associated with clinically relevant complement activation were reported. All patients demonstrated a clinically meaningful improvement on NSAA. Patients treated with delandistrogene moxeparvovec generally maintained muscle strength (Time to Rise and 4-stair Climb) and showed improvement in ambulation ability (10-metre and 100-metre Walk/Run) from baseline to Year 3. The observed safety profile and the enduring response following treatment provide proof of concept for continuation of clinical trials assessing delandistrogene moxeparvovec using single-dose gene therapy in patients with DMD. We present the latest long-term (4-year) safety and functional data from this study.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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