Visualization of HIV-1 reservoir: an imaging perspective

Current opinion in HIV and AIDS

2021 Jul 01

Chapon, C;Moysi, E;Naninck, T;Mayet, C;Petrovas, C;
PMID: 34039844 | DOI: 10.1097/COH.0000000000000691

The persistence of HIV-1-infected cells, despite the introduction of the combinatorial antiretroviral therapy, is a major obstacle to HIV-1 eradication. Understanding the nature of HIV reservoir will lead to novel therapeutic approaches for the functional cure or eradication of the virus. In this review, we will update the recent development in imaging applications toward HIV-1/simian immunodeficiency virus (SIV) viral reservoirs research and highlight some of their limitations.CD4 T cells are the primary target of HIV-1/SIV and the predominant site for productive and latent reservoirs. This viral reservoir preferentially resides in lymphoid compartments that are difficult to access, which renders sampling and measurements problematical and a hurdle for understanding HIV-1 pathogenicity. Novel noninvasive technologies are needed to circumvent this and urgently help to find a cure for HIV-1. Recent technological advancements have had a significant impact on the development of imaging methodologies allowing the visualization of relevant biomarkers with high resolution and analytical capacity. Such methodologies have provided insights into our understanding of cellular and molecular interactions in health and disease.Imaging of the HIV-1 reservoir can provide significant insights for the nature (cell types), spatial distribution, and the role of the tissue microenvironment for its in vivo dynamics and potentially lead to novel targets for the virus elimination.

SALIVARY MICRORNA 155, 146a/b, AND 203: POTENTIAL NONINVASIVE DIAGNOSTIC BIOMARKERS OF PERIODONTITIS AND DIABETES

Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology

2021 Jul 01

Al-Rawi, N;Al–Marzooq, F;Hamoudi, R;
| DOI: 10.1016/j.oooo.2021.03.038

Background Dysregulated expression of microRNAs (miRNAs) plays important role in the initiation and progression of both diabetes and periodontitis. Objective The aim of the study is to identify miRNAs in saliva as potential predictive biomarkers of periodontal disease among patients with diabetes mellitus. Methods MiRNAs were extracted from the saliva of 24 adult subjects with diabetes mellitus and 27 age- and sex-matched healthy controls. Each group was subdivided into periodontally healthy or having periodontitis. In silico analysis identified 4 miRNAs (miRNA 155, 146 a/b and 203) as immune modulators. The expression of miRNAs 146a/b, 155, and 203 was tested using quantitative polymerase chain reaction. The expression levels in the study groups were compared to explore the effect of diabetes and/or periodontitis. Results In our cohort, the 4 miRNAs expressed were higher in patients with periodontitis and/or diabetes. miRNA 155 and miRNA 146 a/b were the most reliable predictors of periodontitis among subjects without diabetes with an optimum cutoff value of

Integrative analysis of the human brain mural cell transcriptome

Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism

2021 May 22

Gastfriend, BD;Foreman, KL;Katt, ME;Palecek, SP;Shusta, EV;
PMID: 34027687 | DOI: 10.1177/0271678X211013700

Brain mural cells, including pericytes and vascular smooth muscle cells, are important for vascular development, blood-brain barrier function, and neurovascular coupling, but the molecular characteristics of human brain mural cells are incompletely characterized. Single cell RNA-sequencing (scRNA-seq) is increasingly being applied to assess cellular diversity in the human brain, but the scarcity of mural cells in whole brain samples has limited their molecular profiling. Here, we leverage the combined power of multiple independent human brain scRNA-seq datasets to build a transcriptomic database of human brain mural cells. We use this combined dataset to determine human-mouse species differences in mural cell transcriptomes, culture-induced dedifferentiation of human brain pericytes, and human mural cell organotypicity, with several key findings validated by RNA fluorescence in situ hybridization. Together, this work improves knowledge regarding the molecular constituents of human brain mural cells, serves as a resource for hypothesis generation in understanding brain mural cell function, and will facilitate comparative evaluation of animal and in vitro models.

NIH HEAL Initiative: National Institute of Neurological Disorders and Stroke Preclinical Program for Non-Addictive Pain Therapeutic Development

The Journal of Pain

2021 May 01

Woller, S;Tamiz, A;Iyengar, S;
| DOI: 10.1016/j.jpain.2021.03.011

Background: The NIH Helping to End Addiction Long-term (HEAL) Initiative aims to focus efforts on advancing scientific solutions to stem the opioid crisis, improving prevention and treatment of opioid misuse/addiction, and enhancing pain management. Goal: NINDS is charged with accelerating the discovery and development of new non-addictive pharmacologic and non-pharmacologic pain therapeutics as part of the HEAL Initiative. NINDS established the Preclinical Screening Platform for Pain (PSPP) to accelerate and enhance testing of novel, non-addictive pain therapeutics. This program will evaluate new, as well as repurposed, small molecules, biologics, devices, and natural products across a range of pain conditions. PSPP is accepting assets from academic and industry sponsors, worldwide. Here we describe efforts within the PSPP program. The overall goal of the PSPP program is to provide an efficient, rigorous, one-stop in vivo screening resource to accelerate identification and efficacy profiling of non-opioid therapeutics for the treatment of pain. Under NINDS direction, preclinical testing of submitted agents is performed by contract facilities on a blinded and confidential basis at no cost to the PSPP participants. Test candidates are evaluated in a suite of in vivo pain-related endpoints and models, following in vitro receptor profiling, pharmacokinetic and safety assessment. Importantly, test candidates are also evaluated in models of abuse liability. We will describe the advances made to date towards establishing program goals of evaluating assets in a rigorous and reproducible manner.

Molecular Pathogenesis of Primary Gastrointestinal Tract Lymphomas

Seminars in Diagnostic Pathology

2021 Apr 01

Toth, L;Vasef, M;
| DOI: 10.1053/j.semdp.2021.04.003

Primary gastrointestinal lymphomas are rare though the incidence is significantly increased among adult patients in recent years. The majority of the patients present with symptoms overlapping with other gastrointestinal disorders and imaging findings are not specific. Therefore, histologic examination is necessary to establish the diagnosis. Insight into etiologies, molecular pathogenesis and critical signaling pathways in lymphomas including gastrointestinal lymphomas has significantly expanded within the last 3 decades. Given the increasing demand for incorporation of genetic data, the appropriate handling and processing of small endoscopic gastrointestinal biopsy samples of suspected lymphoma is becoming extremely crucial and at times challenging. The use of next generation sequencing with analysis of genes relevant to diagnosis, prognosis, and therapeutic targets continues to have a significant promising impact on management of patients in lymphoid malignancies. In particular, the identification of constitutively activated pathways and the emergence of novel targeted medications predict that more effective therapies will be identified for these disorders in the coming years.

ChAdOx1-vectored Lassa fever vaccine elicits a robust cellular and humoral immune response and protects guinea pigs against lethal Lassa virus challenge

NPJ vaccines

2021 Mar 02

Fischer, RJ;Purushotham, JN;van Doremalen, N;Sebastian, S;Meade-White, K;Cordova, K;Letko, M;Jeremiah Matson, M;Feldmann, F;Haddock, E;LaCasse, R;Saturday, G;Lambe, T;Gilbert, SC;Munster, VJ;
PMID: 33654106 | DOI: 10.1038/s41541-021-00291-x

Lassa virus (LASV) infects hundreds of thousands of individuals each year, highlighting the need for the accelerated development of preventive, diagnostic, and therapeutic interventions. To date, no vaccine has been licensed for LASV. ChAdOx1-Lassa-GPC is a chimpanzee adenovirus-vectored vaccine encoding the Josiah strain LASV glycoprotein precursor (GPC) gene. In the following study, we show that ChAdOx1-Lassa-GPC is immunogenic, inducing robust T-cell and antibody responses in mice. Furthermore, a single dose of ChAdOx1-Lassa-GPC fully protects Hartley guinea pigs against morbidity and mortality following lethal challenge with a guinea pig-adapted LASV (strain Josiah). By contrast, control vaccinated animals reached euthanasia criteria 10-12 days after infection. Limited amounts of LASV RNA were detected in the tissues of vaccinated animals. Viable LASV was detected in only one animal receiving a single dose of the vaccine. A prime-boost regimen of ChAdOx1-Lassa-GPC in guinea pigs significantly increased antigen-specific antibody titers and cleared viable LASV from the tissues. These data support further development of ChAdOx1-Lassa-GPC and testing in non-human primate models of infection.

P21‑activated kinase 1 mediates angiotensin II‑induced differentiation of human atrial fibroblasts via the JNK/c‑Jun pathway

Molecular medicine reports

2021 Mar 01

Zhou, Y;Xie, Y;Li, T;Zhang, P;Chen, T;Fan, Z;Tan, X;
PMID: 33495806 | DOI: 10.3892/mmr.2021.11846

Cardiac fibrosis is a common pathophysiological condition involved in numerous types of cardiovascular disease. The renin‑angiotensin system, particularly angiotensin II (AngII), serves an important role in cardiac fibrosis and remodeling. Furthermore, p21‑activated kinase 1 (PAK1) is a highly conserved serine/threonine protein kinase, which is abundantly expressed in all regions of the heart. However, the role of PAK1 in AngII‑mediated activation of cardiac fibroblasts remains unknown. Therefore, the present study aimed to investigate the role of PAK1 in cardiac fibroblasts and its underlying mechanisms. Human cardiac fibroblasts (HCFs) were cultured and treated with PAK1 inhibitor IPA‑3 or transduced with PAK1 short hairpin (sh)RNA by lentiviral particles to silence PAK1 expression levels. Subsequently, the cell proliferation and migration abilities of the HCFs were determined. Western blot analysis was used to detect the phosphorylation status of Janus kinase (JNK) and c‑Jun. A Cell Counting Kit‑8 assay showed that PAK1 inhibition following treatment of HCFs with 5 µM IPA‑3 or PAK1‑shRNA, significantly attenuated AngII‑induced proliferation of fibroblasts. In addition, wound healing and Transwell migration assays demonstrated that inhibition of PAK1 significantly inhibited AngII‑induced cell migration. Finally, decreased PAK1 expression levels downregulated AngII‑mediated upregulation of α‑smooth muscle actin (α‑SMA), collagen I, phosphorylated (p)‑JNK and p‑c‑Jun, a downstream molecule of JNK signaling. These findings indicate that PAK1 contributes to AngII‑induced proliferation, migration and transdifferentiation of HCFs via the JNK/c‑Jun pathway.

Pre-conditioning modifies the TME to enhance solid tumor CAR T cell efficacy and endogenous protective immunity

Molecular therapy : the journal of the American Society of Gene Therapy

2021 Feb 27

Murad, JP;Tilakawardane, D;Park, AK;Lopez, LS;Young, CA;Gibson, J;Yamaguchi, Y;Lee, HJ;Kennewick, KT;Gittins, BJ;Chang, WC;Tran, CP;Martinez, C;Wu, AM;Reiter, RE;Dorff, TB;Forman, SJ;Priceman, SJ;
PMID: 33647456 | DOI: 10.1016/j.ymthe.2021.02.024

Chimeric antigen receptor (CAR) T cell therapy has led to impressive clinical responses in patients with hematological malignancies; however, its effectiveness in patients with solid tumors has been limited. While CAR T cells for the treatment of advanced prostate and pancreas cancer, including those targeting prostate stem cell antigen (PSCA), are being clinically evaluated and are anticipated to show bioactivity, their safety and the impact of the immunosuppressive tumor microenvironment (TME) have not been faithfully explored preclinically. Using a novel human PSCA knockin (hPSCA-KI) immunocompetent mouse model, we evaluated the safety and therapeutic efficacy of PSCA-CAR T cells. We demonstrated that cyclophosphamide (Cy) pre-conditioning significantly modified the immunosuppressive TME and was required to uncover the efficacy of PSCA-CAR T cells in metastatic prostate and pancreas cancer models, with no observed toxicities in normal tissues with endogenous expression of PSCA. This combination dampened the immunosuppressive TME, generated pro-inflammatory myeloid and T cell signatures in tumors, and enhanced the recruitment of antigen-presenting cells, as well as endogenous and adoptively transferred T cells, resulting in long-term anti-tumor immunity.

Spinal macrophages resolve nociceptive hypersensitivity after peripheral injury

Neuron

2021 Feb 24

Niehaus, JK;Taylor-Blake, B;Loo, L;Simon, JM;Zylka, MJ;
PMID: 33667343 | DOI: 10.1016/j.neuron.2021.02.018

Peripheral nerve injury induces long-term pro-inflammatory responses in spinal cord glial cells that facilitate neuropathic pain, but the identity of endogenous cells that resolve spinal inflammation has not been determined. Guided by single-cell RNA sequencing (scRNA-seq), we found that MRC1+ spinal cord macrophages proliferated and upregulated the anti-inflammatory mediator Cd163 in mice following superficial injury (SI; nerve intact), but this response was blunted in nerve-injured animals. Depleting spinal macrophages in SI animals promoted microgliosis and caused mechanical hypersensitivity to persist. Conversely, expressing Cd163 in spinal macrophages increased Interleukin 10 expression, attenuated micro- and astrogliosis, and enduringly alleviated mechanical and thermal hypersensitivity in nerve-injured animals. Our data indicate that MRC1+ spinal macrophages actively restrain glia to limit neuroinflammation and resolve mechanical pain following a superficial injury. Moreover, we show that spinal macrophages from nerve-injured animals mount a dampened anti-inflammatory response but can be therapeutically coaxed to promote long-lasting recovery of neuropathic pain.

Activated microglia drive demyelination via CSF1R signaling

Glia

2021 Feb 23

Marzan, DE;Brügger-Verdon, V;West, BL;Liddelow, S;Samanta, J;Salzer, JL;
PMID: 33620118 | DOI: 10.1002/glia.23980

Microgliosis is a prominent pathological feature in many neurological diseases including multiple sclerosis (MS), a progressive auto-immune demyelinating disorder. The precise role of microglia, parenchymal central nervous system (CNS) macrophages, during demyelination, and the relative contributions of peripheral macrophages are incompletely understood. Classical markers used to identify microglia do not reliably discriminate between microglia and peripheral macrophages, confounding analyses. Here, we use a genetic fate mapping strategy to identify microglia as predominant responders and key effectors of demyelination in the cuprizone (CUP) model. Colony-stimulating factor 1 (CSF1), also known as macrophage colony-stimulating factor (M-CSF) - a secreted cytokine that regulates microglia development and survival-is upregulated in demyelinated white matter lesions. Depletion of microglia with the CSF1R inhibitor PLX3397 greatly abrogates the demyelination, loss of oligodendrocytes, and reactive astrocytosis that results from CUP treatment. Electron microscopy (EM) and serial block face imaging show myelin sheaths remain intact in CUP treated mice depleted of microglia. However, these CUP-damaged myelin sheaths are lost and robustly phagocytosed upon-repopulation of microglia. Direct injection of CSF1 into CNS white matter induces focal microgliosis and demyelination indicating active CSF1 signaling can promote demyelination. Finally, mice defective in adopting a toxic astrocyte phenotype that is driven by microglia nevertheless demyelinate normally upon CUP treatment implicating microglia rather than astrocytes as the primary drivers of CUP-mediated demyelination. Together, these studies indicate activated microglia are required for and can drive demyelination directly and implicate CSF1 signaling in these events.

Disruption of DNA polymerase ζ engages an innate immune response

Cell reports

2021 Feb 23

Martin, SK;Tomida, J;Wood, RD;
PMID: 33626348 | DOI: 10.1016/j.celrep.2021.108775

In mammalian cells, specialized DNA polymerase ζ (pol ζ) contributes to genomic stability during normal DNA replication. Disruption of the catalytic subunit Rev3l is toxic and results in constitutive chromosome damage, including micronuclei. As manifestations of this genomic stress are unknown, we examined the transcriptome of pol ζ-defective cells by RNA sequencing (RNA-seq). Expression of 1,117 transcripts is altered by ≥4-fold in Rev3l-disrupted cells, with a pattern consistent with an induction of an innate immune response. Increased expression of interferon-stimulated genes at the mRNA and protein levels in pol ζ-defective cells is driven by the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-signaling partner stimulator of interferon genes (STING) pathway. Expression of key interferon-stimulated chemokines is elevated in basal epithelial mouse skin cells with a disruption of Rev3l. These results indicate that the disruption of pol ζ may simultaneously increase sensitivity to genotoxins and potentially engage parts of the innate immune response, which could add an additional benefit to targeting pol ζ in cancer therapies.

FGF8 and BMP2 mediated dynamic regulation of dental mesenchyme proliferation and differentiation via Lhx8/Suv39h1 complex

Journal of cellular and molecular medicine

2021 Feb 13

Zhou, C;Chen, D;Ren, J;Huang, D;Li, R;Luo, H;Guan, C;Cao, Y;Wang, W;
PMID: 33580754 | DOI: 10.1111/jcmm.16351

The homeobox gene, LIM-homeobox 8 (Lhx8), has previously been identified as an essential transcription factor for dental mesenchymal development. However, how Lhx8 itself is regulated and regulates odontogenesis remains poorly understood. In this study, we employed an RNAscope assay to detect the co-expression pattern of Lhx8 and Suv39h1 in the dental mesenchyme, which coincided with the dynamic expression profiles of the early epithelium signal of Fibroblast Growth Factor 8 (FGF8) and the later mesenchymal signal Bone Morphogenetic Protein 2 (BMP2). Moreover, FGF8 activated Lhx8, whereas BMP2 repressed Lhx8 expression at the transcriptional level. The high expression of Lhx8 in the early dental mesenchyme maintained the cell fate in an undifferentiated status by interacting with Suv39h1, a histone-lysine N-methyltransferase constitutively expressed in the dental mesenchyme. Further in the ex vivo organ culture model, the knockdown of Suv39h1 significantly blocked the function of Lhx8 and FGF8. Mechanistically, Lhx8/Suv39h1 recognized the odontoblast differentiation-related genes and repressed gene expression via methylating H3K9 on their promoters. Taken together, our data here suggest that Lhx8/Suv39h1 complex is inversely regulated by epithelium-mesenchymal signals, balancing the differentiation and proliferation of dental mesenchyme via H3K9 methylation.

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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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