Peripheral Nervous System Tissues Research Articles

Over 30 Years of DiI Use for Human Neuroanatomical Tract Tracing: A Scoping Review.

In the present study, we conducted a scoping review to provide an overview of the existing literature on the carbocyanine dye DiI, in human neuroanatomical tract tracing. The PubMed, Scopus, and Web of Science databases were systematically searched. We identified 61 studies published during the last three decades. While studies incorporated specimens across human life from the embryonic stage onwards, the majority of studies focused on adult human tissue. Studies that utilized peripheral nervous system (PNS) tissue were a minority, with the majority of studies focusing on the central nervous system (CNS). The most common topic of interest in previous tract tracing investigations was the connectivity of the visual pathway. DiI crystals were more commonly applied. Nevertheless, several studies utilized DiI in a paste or dissolved form. The maximum tracing distance and tracing speed achieved was, respectively, 70 mm and 1 mm/h. We identified studies that focused on optimizing tracing efficacy by varying parameters such as fixation, incubation temperature, dye re-application, or the application of electric fields. Additional studies aimed at broadening the scope of DiI use by assessing the utility of archival tissue and compatibility of tissue clearing in DiI applications. A combination of DiI tracing and immunohistochemistry in double-labeling studies have been shown to provide the means for assessing connectivity of phenotypically defined human CNS and PNS neuronal populations.

The knowns and unknowns of long COVID-19: from mechanisms to therapeutical approaches.

The coronavirus disease 2019 (COVID-19) pandemic caused by SARS-CoV-2 has been defined as the greatest global health and socioeconomic crisis of modern times. While most people recover after being infected with the virus, a significant proportion of them continue to experience health issues weeks, months and even years after acute infection with SARS-CoV-2. This persistence of clinical symptoms in infected individuals for at least three months after the onset of the disease or the emergence of new symptoms lasting more than two months, without any other explanation and alternative diagnosis have been named long COVID, long-haul COVID, post-COVID-19 conditions, chronic COVID, or post-acute sequelae of SARS-CoV-2 (PASC). Long COVID has been characterized as a constellation of symptoms and disorders that vary widely in their manifestations. Further, the mechanisms underlying long COVID are not fully understood, which hamper efficient treatment options. This review describes predictors and the most common symptoms related to long COVID's effects on the central and peripheral nervous system and other organs and tissues. Furthermore, the transcriptional markers, molecular signaling pathways and risk factors for long COVID, such as sex, age, pre-existing condition, hospitalization during acute phase of COVID-19, vaccination, and lifestyle are presented. Finally, recommendations for patient rehabilitation and disease management, as well as alternative therapeutical approaches to long COVID sequelae are discussed. Understanding the complexity of this disease, its symptoms across multiple organ systems and overlapping pathologies and its possible mechanisms are paramount in developing diagnostic tools and treatments.

Adeno-associated virus-mediated gene transfer of arginine decarboxylase to the central nervous system prevents opioid analgesic tolerance.

Agmatine, a decarboxylated form of L-arginine, prevents opioid analgesic tolerance, dependence, and self-administration when given by both central and systemic routes of administration. Endogenous agmatine has been previously detected in the central nervous system. The presence of a biochemical pathway for agmatine synthesis offers the opportunity for site-specific overexpression of the presumptive synthetic enzyme for local therapeutic effects. In the present study, we evaluated the development of opioid analgesic tolerance in ICR-CD1 mice pre-treated with either vehicle control or intrathecally delivered adeno-associated viral vectors (AAV) carrying the gene for human arginine decarboxylase (hADC). Vehicle-treated or AAV-hADC-treated mice were each further divided into two groups which received repeated delivery over three days of either saline or systemically-delivered morphine intended to induce opioid analgesic tolerance. Morphine analgesic dose-response curves were constructed in all subjects on day four using the warm water tail flick assay as the dependent measure. We observed that pre-treatment with AAV-hADC prevented the development of analgesic tolerance to morphine. Peripheral and central nervous system tissues were collected and analyzed for presence of hADC mRNA. In a similar experiment, AAV-hADC pre-treatment prevented the development of analgesic tolerance to a high dose of the opioid neuropeptide endomorphin-2. Intrathecal delivery of anti-agmatine IgG (but not normal IgG) reversed the inhibition of endomorphin-2 analgesic tolerance in AAV-hADC-treated mice. To summarize, we report here the effects of AAV-mediated gene transfer of human ADC (hADC) in models of opioid-induced analgesic tolerance. This study suggests that gene therapy may contribute to reducing opioid analgesic tolerance.

The molecular mimicry and COVID-19

A significant part of the complications of COVID-19 and manifestations of post-COVID syndrome is associated with autoimmune reactions caused by SARS-CoV-2. The key mechanism for enabling autoimmunity in COVID-19 results from molecular mimicry, which is involved in developing cytokine storm, systemic multiorgan hyperinflammation, endothelial dysfunction, also being a trigger for arising post-COVID-19 autoimmune diseases (autoimmune thrombocytopenia, autoimmune vasculitis, Guillain–Barrésyndrome, Miller–Fisher syndrome, autoimmune neuropathy, autoimmune thyroiditis, rheumatoid arthritis, etc.). Overall, there have been identified 59 common immune determinants in 80 epitopes of the SARS-CoV-2 spike protein and 53 anti-inflammatory proteins, receptors regulating cell proliferation, differentiation and apoptosis as well as immune response. It was found that among the 37 viral proteins, only 8 of them bear no immunogenic regions identical to human proteins. Cross-reactivity results in emergence of more than 15 distinct types of autoantibodies including antiphospholipid antibodies against cardiolipin and beta-2-glycoprotein I, antibodies specific to transmembrane adenosine receptor A2b, adiponectin, phosphatidylserine-prothrombin, antinuclear antigens, mitochondrial M2, type I interferons, and other cytokines, chemokines, complement components and cell membrane proteins. Autoantibodies formed during COVID-19 react to antigens of cells located in the thyroid gland, cardiac and skeletal muscles, lung, joints, liver, kidneys, brain and bone marrow, peripheral nervous system, skin and adipose tissue, gastrointestinal tract, testicles, eyes as well as mitochondrial antigens, mediating development of severe disease-related complications and post-COVID syndrome. The presence of 24 homologous pentapeptides with those found inB. pertussis, C. diphtheriae,C. tetani,H. influenzaeandN. meningitidisposes a risk of developing ineffective vaccination immune response paralleled with higher risk of autoimmune complications. It is imperative to take into account the phenomenon of molecular mimicry while proposing new approaches for rehabilitation and treatment of COVID-19 as well as in development and testing of vaccines against SARS-CoV-2.

Mayaro virus pathogenesis and immunity in rhesus macaques.

Mayaro virus (MAYV) is a mosquito-transmitted alphavirus that causes debilitating and persistent arthritogenic disease. While MAYV was previously reported to infect non-human primates (NHP), characterization of MAYV pathogenesis is currently lacking. Therefore, in this study we characterized MAYV infection and immunity in rhesus macaques. To inform the selection of a viral strain for NHP experiments, we evaluated five MAYV strains in C57BL/6 mice and showed that MAYV strain BeAr505411 induced robust tissue dissemination and disease. Three male rhesus macaques were subcutaneously challenged with 105 plaque-forming units of this strain into the arms. Peak plasma viremia occurred at 2 days post-infection (dpi). NHPs were taken to necropsy at 10 dpi to assess viral dissemination, which included the muscles and joints, lymphoid tissues, major organs, male reproductive tissues, as well as peripheral and central nervous system tissues. Histological examination demonstrated that MAYV infection was associated with appendicular joint and muscle inflammation as well as presence of perivascular inflammation in a wide variety of tissues. One animal developed a maculopapular rash and two NHP had viral RNA detected in upper torso skin samples, which was associated with the presence of perivascular and perifollicular lymphocytic aggregation. Analysis of longitudinal peripheral blood samples indicated a robust innate and adaptive immune activation, including the presence of anti-MAYV neutralizing antibodies with activity against related Una virus and chikungunya virus. Inflammatory cytokines and monocyte activation also peaked coincident with viremia, which was well supported by our transcriptomic analysis highlighting enrichment of interferon signaling and other antiviral processes at 2 days post MAYV infection. The rhesus macaque model of MAYV infection recapitulates many of the aspects of human infection and is poised to facilitate the evaluation of novel therapies and vaccines targeting this re-emerging virus.

Regenerative capacity of neural tissue scales with changes in tissue mechanics post injury

Spinal cord injuries have devastating consequences for humans, as mammalian neurons of the central nervous system (CNS) cannot regenerate. In the peripheral nervous system (PNS), however, neurons may regenerate to restore lost function following injury. While mammalian CNS tissue softens after injury, how PNS tissue mechanics changes in response to mechanical trauma is currently poorly understood. Here we characterised mechanical rat nerve tissue properties before and after in vivo crush and transection injuries using atomic force microscopy-based indentation measurements. Unlike CNS tissue, PNS tissue significantly stiffened after both types of tissue damage. This nerve tissue stiffening strongly correlated with an increase in collagen I levels. Schwann cells, which crucially support PNS regeneration, became more motile and proliferative on stiffer substrates in vitro, suggesting that changes in tissue stiffness may play a key role in facilitating or impeding nervous system regeneration.

The opioid receptor: emergence through millennia of pharmaceutical sciences.

Throughout history humanity has searched for an optimal approach to the use of opioids that maximizes analgesia while minimizing side effects. This review reflects upon the conceptualization of the opioid receptor and the critical role that the pharmaceutical sciences played in its revelation. Opium-containing formulations have been delivered by various routes of administration for analgesia and other therapeutic indications for millennia. The concept of a distinct site of opium action evolved as practitioners developed innovative delivery methods, such as intravenous administration, to improve therapeutic outcomes. The introduction of morphine and synthetic opioids engendered the prevalent assumption of a common opioid receptor. Through consideration of structure-activity relationships, spatial geometry, and pharmacological differences of known ligands, the idea of multiple opioid receptors emerged. By accessing the high-affinity property of naloxone, the opioid receptor was identified in central and peripheral nervous system tissue. The endogenous opioid neuropeptides were subsequently discovered. Application of mu-, delta-, and kappa- opioid receptor-selective ligands facilitated the pharmacological characterization and distinctions between the three receptors, which were later cloned and sequenced. Opioid receptor signal transduction pathways were described and attributed to specific physiological outcomes. The crystal structures of mu, delta, kappa, and nociceptin/orphanin FQ receptors bound to receptor-selective ligands have been elucidated. Comparison of these structures reveal locations of ligand binding and engagement of signal transduction pathways. Expanding knowledge regarding the structure and actions of the opioid receptor fuels contemporary strategies for driving the activity of opioid receptors toward maximizing therapeutic and minimizing adverse outcomes.

Mesencephalic Astrocyte-Derived Neurotrophic Factor (MANF): An Emerging Therapeutic Target for Neurodegenerative Disorders.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a member of the new family of neurotrophic factors (NTFs) with a unique structure and functions compared to other conventionally known NTFs. MANF is broadly expressed in developing and mature tissues, including the central nervous system and peripheral nervous system tissues. Growing research demonstrated that MANF protects neurons from endoplasmic reticulum (ER) stress-associated complications by restoring ER homeostasis and regulating unfolded protein response. This review discusses MANF signaling in neurodegenerative conditions with specific emphasis given to its overall effect and mechanisms of action in experimental models of Parkinson's disease, Alzheimer's disease, and stroke. Additional perspectives on its potential unexplored roles in other neurodegenerative conditions are also given.

PARIN5, a Novel Thrombin Receptor Antagonist Modulates a Streptozotocin Mice Model for Diabetic Encephalopathy.

Diabetic encephalopathy (DE) is an inflammation-associated diabetes mellitus (DM) complication. Inflammation and coagulation are linked and are both potentially modulated by inhibiting the thrombin cellular protease-activated receptor 1 (PAR1). Our aim was to study whether coagulation pathway modulation affects DE. Diabetic C57BL/6 mice were treated with PARIN5, a novel PAR1 modulator. Behavioral changes in the open field and novel object recognition tests, serum neurofilament (NfL) levels and thrombin activity in central and peripheral nervous system tissue (CNS and PNS, respectively), brain mRNA expression of tumor necrosis factor α (TNF-α), Factor X (FX), prothrombin, and PAR1 were assessed. Subtle behavioral changes were detected in diabetic mice. These were accompanied by an increase in serum NfL, an increase in central and peripheral neural tissue thrombin activity, and TNF-α, FX, and prothrombin brain intrinsic mRNA expression. Systemic treatment with PARIN5 prevented the appearance of behavioral changes, normalized serum NfL and prevented the increase in peripheral but not central thrombin activity. PARIN5 treatment prevented the elevation of both TNF-α and FX but significantly elevated prothrombin expression. PARIN5 treatment prevents behavioral and neural damage in the DE model, suggesting it for future clinical research.

Human Schwann Cells in vitro I. Nerve Tissue Processing, Pre-degeneration, Isolation, and Culturing of Primary Cells.

This paper presents versatile protocols to prepare primary human Schwann cell (hSC) cultures from mature peripheral nervous system tissues, including fascicles from long spinal nerves, nerve roots, and ganglia. This protocol starts with a description of nerve tissue procurement, handling, and dissection to obtain tissue sections suitable for hSC isolation and culturing. A description follows on how to disintegrate the nerve tissue by delayed enzymatic dissociation, plate the initial cell suspensions on a two-dimensional substrate, and culture the primary hSCs. Each section contains detailed procedures, technical notes, and background information to aid investigators in understanding and managing all steps. Some general recommendations are made to optimize the recovery, growth, and purity of the hSC cultures irrespective of the tissue source. These recommendations include: (1) pre-culturing epineurium- and perineurium-free nerve fascicles under conditions of adherence or suspension depending on the size of the explants to facilitate the release of proliferative, in vitro-activated hSCs; (2) plating the initial cell suspensions as individual droplets on a laminin-coated substrate to expedite cell adhesion and thereby increase the recovery of viable cells; and (3) culturing the fascicles (pre-degeneration step) and the cells derived therefrom in mitogen- and serum-supplemented medium to accelerate hSC dedifferentiation and promote mitogenesis before and after tissue dissociation, respectively. The hSC cultures obtained as suggested in this protocol are suitable for assorted basic and translational research applications. With the appropriate adaptations, donor-relevant hSC cultures can be prepared using fresh or postmortem tissue biospecimens of a wide range of types and sizes.

Abstract 11528: Neurotrophic Signaling Pathway Dysregulation, Cardiac Amyloidosis, and Innervation Impairment in the Tg2576 Model of Alzheimer's Disease

Alzheimer's disease (AD) is a complex and incurable neurodegenerative disorder characterized by cerebral amyloid β (Aβ) deposition and tau pathology, accompanied by a gradual loss of the two main neuromodulators, NGF and BDNF. The progressive degeneration of the cerebral neuro-signaling pathways triggers a gradual decline in neurotrophic factors with significant derangement of the peripheral nervous system. This may culminate in detrimental effects on other organs, including the heart. However, whether and how AD modulates neurotrophins, innervation, and amyloidosis in the cardiac tissue is still undefined. Here, we investigated, for the first time, cardiovascular remodeling and neuro signaling pathway dysregulation in a mouse model of Alzheimer’s disease. Hence, 12 months old Tg2576 transgenic mice, a model of cerebral amyloidosis, were compared to age-matched WT littermates. Echocardiographic analysis showed significant deterioration in LV function, evidenced by a severe decline both in ejection fraction and fraction shortening percentage in the Tg2576 group compared to WT mice. Tg2576 mice exhibited a relevant increase in interstitial fibrosis, with progressive accumulation of amyloid aggregates, resulting in severe cardiac nervous system dysfunction. The transgenic line showed a remarkable decrease in cardiac nerve fiber density, both adrenergic (stained with tyrosine hydroxylase- TH) and regenerating nerve endings (labeled with GAP-43) compared to the WT mice. This myocardial denervation was associated with a robust reduction in NGF and BDNF protein expression both at the neuronal and cardiac level, accompanied by a significant decline in GAP-43 activity in the heart and cerebral cortex lysates of Tg2576 mice. Additionally, human neuroblastoma cells (SH-SY5Y) challenged with human Aβ-40 or Aβ-42 oligomers showed a severe downregulation of both BDNF and GAP-43 protein levels. Overall, our evidence highlights a possible detrimental effect of cerebral amyloidosis on the peripheral nervous system and cardiac tissue, providing potential therapeutic targets, such as the neuro-signaling pathway, to prevent or delay some of the effects caused by AD on the cardiovascular system.

Satellite glia of the adult dorsal root ganglia harbor stem cells that yield glia under physiological conditions and neurons in response to injury.

The presence of putative stem/progenitor cells has been suggested in adult peripheral nervous system (PNS) tissue, including the dorsal root ganglion (DRG). To date, their identification and fate in pathophysiological conditions have not been addressed. Combining multiple invitro and invivo approaches, we identified the presence of stem cells in the adult DRG satellite glial population, and progenitors were present in the DRGs and sciatic nerve. Cell-specific transgenic mouse lines highlighted the proliferative potential of DRG stem cells and progenitors invitro. DRG stem cells had gliogenic and neurogenic potentials, whereas progenitors were essentially gliogenic. Lineage tracing showed that, under physiological conditions, adult DRG stem cells maintained DRG homeostasis by supplying satellite glia. Under pathological conditions, adult DRG stem cells replaced DRG neurons lost to injury in addition of renewing the satellite glial pool. These novel findings open new avenues for development of therapeutic strategies targeting DRG stem cells for PNS disorders.

P13. Transduction of the rat spinal cord using adenoassociated virus serotype 2.5

<h3>BACKGROUND CONTEXT</h3> Gene therapy is a method by which exogenous genes are introduced to treat disease or deliver drugs of interest. This is commonly done through either direct gene delivery in vivo or ex vivo through transduction of cells that are to be transplanted. There are several vectors for delivering genes, including viral vectors, naked plasmids, electroporation and lipid nanoparticles. Adeno-associated viruses (AAVs) are commonly used and considered to be well tolerated by the brain, muscles, nerves and multiple cell types. Rat Schwann cells are transduced efficiently by AAV1 whereas human Schwann cells are better transduced with AAV2 and 6. Rat nerves segments have demonstrated to be efficiently transduced by AAV1, 5, 7 and 9 whereas human nerves prefer AAV2. Many studies also agree that AAV1, 2, 4, 8 and 9 are optimal serotypes for CNS. We have utilized a novel viral vector, AAV serotype 2.5, that is a chimera of AAV2 vector modified with 5 amino acids from AAV1. AAV2.5 has shared characteristics of AAV1 and AAV2 with enhanced transduction for multiple cell types, such as in the muscle and articular tissue. Since AAV2.5 is not a naturally occurring virus, there is lower cross-reactivity to AAV2 antibodies. However, the ability of AAV2.5 to transduce tissues of the central (CNS) and peripheral nervous system (PNS) is not well understood. <h3>PURPOSE</h3> The purpose of this study is to determine the trophism and transduction efficiency of AAV2.5 for CNS and PNS tissue through in vitro transduction of dorsal root ganglion derived neurons, Schwann cells, and in vivo transduction of spinal cord. We predict that since AAV1 and AAV2 have known tropism for CNS and PNS cells to different extents depending on cell type, that the chimera AAV2.5 would be broadly effective. <h3>STUDY DESIGN/SETTING</h3> Cell culture and animal study. <h3>METHODS</h3> The cDNA encoding green fluorescent protein (GFP) was cloned into the conventional AAV packaging vector pTRUF2. For generation of self-complementing AAV vector plasmids, the cDNAs for GFP were directionally inserted into the SacII, NotI sites of pHpa-trs-SK plasmid. Transcription was driven by the cytomegalovirus promoter. Primary Schwann cells were isolated from the sciatic nerves of postnatal day 5 rat pups and dorsal root ganglion (DRG) neurons from embryonic day 15 rat pups. The cells were plated at a density of 125,000 cells per well and infected with 104 viral genomes per cell. For in vivo transduction of spinal cord tissue, 4 µL of 5 × 106– 5 × 1010vg/mL of AAV2.5-GFP was injected into 4 positions at T9. Immunostaining for markers of different spinal cord cell types colabeled with anti-GFP was done. <h3>RESULTS</h3> AAV2.5 was able to successfully infect DRG neurons and Schwann cells. AAV-2.5 vector was much more efficient than the commonly used AAV2 vector at transducing the Schwann cells. Cell survival and gene product production by AAV2.5 was greater than electroporation. When AAV2.5 -GFP was injected into the spinal cord there was a dose dependent increase in GFP expression by neurons and astrocytes up to 4 mm away from the injection site. <h3>CONCLUSIONS</h3> The study presents a novel vector for broad transduction in vivo or ex vivo of cells in the CNS and PNS. AAV2.5 was more efficient than the more commonly used AAV2 vector or electroporation. Since AAV2.5 is presently in phase 1 trials for osteoarthritis (NCT02790723), this vector type presents an interesting clinical translation opportunity for spinal cord and peripheral nerve injuries/diseases. <h3>FDA DEVICE/DRUG STATUS</h3> This abstract does not discuss or include any applicable devices or drugs.

Woman With Left Pulsatile Exophthalmos

Neurofibromatosis is rarely seen in emergency departments unless it becomes symptomatic. There are 3 types of neurofibromatosis. Neurofibromatosis type 1 (NF-1; von Recklinghausen disease) is the most common of the 3 types of neurofibromatosis disease. In the disease, there are skin, peripheral and central nervous system, bone, and soft tissue involvements, and there is an increase in the susceptibility to cancer. 1 Hirbe A.C. Gutmann D.H. Neurofibromatosis type 1: a multidisciplinary approach to care. Lancet Neurol. 2014; 13: 834-843https://doi.org/10.1016/S1474-4422(14)70063-8 Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar NF-1 is seen once in 3000 births, showing autosomal dominant inheritance and affecting many systems simultaneously. 2 Ghalayani P. Saberi Z. Sardari F. Neurofibromatosis type I (von Recklinghausen’s disease): a family case report and literature review. Dent Res J (Isfahan). 2012; 9 (PMC3491339): 483-488 Google Scholar The NF-1 gene is located on chromosome 17 and encodes a type of tumor suppressor protein called neurofibromin. As a result of incomplete or incorrect folding of this protein, benign and rarely malignant nerve tumors are seen from the early stages of life. Because the disease affects many systems at the same time, the diagnostic criteria have been shaped in this direction. Diagnostic criteria include café au lait spots, neurofibromas, axillary and/or inguinal freckles, optic glioma, Lisch nodules (iris hamartomas), typical bone lesions, and NF-1 disease in first-degree relatives. 3 Ferner R.E. Huson S.M. Thomas N. et al. Guidelines for the diagnosis and management of individuals with neurofibromatosis 1. J Med Genet. 2007; 44: 81-88https://doi.org/10.1136/jmg.2006.045906 Crossref PubMed Scopus (645) Google Scholar In uncertain cases, genetic testing may be requested to confirm the diagnosis. Metin Yadigaroglu is Assistant Professor, Faculty of Medicine, Department of Emergency Medicine, Samsun University, Samsun, Turkey. Twitter: @mtnydgrdr. Melih Imamoglu is Assistant Professor, Faculty of Medicine, Department of Emergency Medicine, Karadeniz Technical University, Trabzon, Turkey. Twitter: @melihimam. Muhammet Fatih Beser is Emergency Specialist, Faculty of Medicine, Department of Emergency Medicine, Karadeniz Technical University, Trabzon, Turkey. Twitter: @drfatih. Sinan Pasli is Assistant Professor, Faculty of Medicine, Department of Emergency Medicine, Karadeniz Technical University, Trabzon, Turkey.

Alginate-Based Hydrogels and Tubes, as Biological Macromolecule-Based Platforms for Peripheral Nerve Tissue Engineering: A Review.

Unlike the central nervous system, the peripheral nervous system (PNS) has an inherent capacity to regenerate following injury. However, in the case of large nerve defects where end-to-end cooptation of two nerve stumps is not tension-free, autologous nerve grafting is often utilized to bridge the nerve gaps. To address the challenges associated with autologous nerve grafting, neural guidance channels (NGCs) have been successfully translated into clinic. Furthermore, hydrogel-based drug delivery systems have been extensively studied for the repair of PNS injuries. There are numerous biomaterial options for the production of NGCs and hydrogels. Among different candidates, alginate has shown promising results in PNS tissue engineering. Alginate is a naturally occurring polysaccharide which is biocompatible, non-toxic, non-immunogenic, and possesses modifiable properties. In the current review, applications, challenges, and future perspectives of alginate-based NGCs and hydrogels in the repair of PNS injuries will be discussed.

Functional loss of ubiquitin-specific protease 14 may lead to a novel distal arthrogryposis phenotype.

Multiple congenital contractures (MCC) comprise a number of rare, non-progressive conditions displaying marked phenotypic and etiologic heterogeneity. A genetic cause can be established in approximately half of the affected individuals, attributed to genetic defects in the formation and functioning of the central and peripheral nervous system, neuromuscular junctions, skeletal muscles, and connective tissue. Ubiquitin-specific protease 14 (USP14) encodes a major proteasome-associated deubiquitinating enzyme with an established dual role as an inhibitor and an activator of proteolysis, maintaining protein homeostasis. Usp14-deficient mice show a phenotype similar to lethal human MCC phenotypes, with callosal anomalies, muscle wasting, and early lethality, attributed to neuromuscular junction defects due to decreased monomeric ubiquitin pool. We describe a new, autosomal recessive MCC phenotype in three fetuses from two different branches of a consanguineous family, presenting with distal arthrogryposis, underdevelopment of the corpus callosum, and dysmorphic facial features. Exome sequencing identified a biallelic 4-bp deletion (c.233_236delTTCC; p.Leu78Glnfs*11, SCV002028347) in USP14, and sequencing of family members showed segregation with the phenotype. RT-qPCR experiment in an unaffected heterozygote revealed that mutant USP14 was expressed, indicating that abnormal transcript escapes nonsense-mediated mRNA decay. We propose that herein described fetuses represent the first human phenotype of USP14 loss, with callosal anomalies and/or cortical malformations, multiple contractures, and recognizable dysmorphic facial features.

Self-renewal of peripheral nerve resident macrophage: does it represent a unique activation status?

Self-renewal of peripheral nerve resident macrophage: does it represent a unique activation status?

Abstract 2809: PET-MRI imaging of reactive macrophages in an orthotopic model of ovarian cancer

Abstract Ovarian cancer is a lethal gynecologic malignancy and the fifth leading cause of cancer-related deaths in women in the United States. Early detection is a critical unmet need, as patients have significantly improved prognosis when a tumor is discovered at an early stage, and disease staging for prognosis and treatment strategy is critical to ensure proper clinical management. Major limitations of radiological evaluation of ovarian cancer arise from the inability to consistently detect small lesions. The availability of multiplexed hybrid imaging systems, such as simultaneous PET-MRI scanners, presents unique opportunities to combine the strengths of anatomic, physiologic and molecular detection within a single exam for the purpose of early detection and staging of ovarian cancer. Tumor associated macrophages (TAMs) play a critical role in ovarian cancer and are among several promising targets in host-directed therapies in cancer. Here, we will combine the anatomic imaging capabilities of MRI with the highly sensitive molecular imaging capabilities of PET in an orthotopic syngeneic model of ovarian cancer, using [124I]iodo-DPA-713, a macrophage-specific imaging agent to define host signatures to detect and stage ovarian cancer. [124I]DPA-713 is a low molecular weight probe that is selectively trapped within reactive phagocytes in both peripheral and central nervous system tissues, including TAMs, and exhibits very low background retention. Using PET-MRI imaging, this allows for excelling visualization of ovaries and the peritoneal environment. Pieces of ID8-Defb29-VEGF tumor were orthotopically implanted onto the ovary of C57BL6 mice. Subcutaneous ID8-Defb29-VEGF tumors were also imaged for comparison. Bioluminescent imaging (BLI) was used to follow tumor progression, as these cancer cells constitutively express luciferin. Experiments were performed in mice presenting with different disease stages, from early stage when tumors are less than 3 mm with no metastases, to late stage with metastases and ascites present. BLI data were used to stratify the mice into different groups. Specific uptake of the radiotracer was observed within and proximal to the primary tumor at early stages. At later stages, uptake was observed in the peritoneal ascitic fluid and within metastases in the lungs, in addition to clear halos surrounding the primary tumor. These studies are clinically translatable and will facilitate non-invasive detection of a nursery of tumor supporting reactive macrophages. They will also provide insights into ovarian cancer progression that will complement emerging host-directed therapeutics destined for the clinic. Supported by the Hopkins Cancer Center Imaging Fund. Citation Format: Catherine Foss, Desmond Jacob, Flonné Wildes, Marie-France Penet. PET-MRI imaging of reactive macrophages in an orthotopic model of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2809.

Modeling Joint Pain on a Chip: integrating sensory neurons in the microJoint to model osteoarthritis

The development of safe and effective methods for the treatment of osteoarthritis(OA)-associated pain is a critical need. Recently, our team successfully modeled OA-like pathology in an in vitro multi-component joint-on-a-chip (microJoint), which integrates engineered osteochondral complex, synovium, adipose tissue and given their crucial role in joint pain, macrophages. In the present project, we introduce the sensory neurons into the established microJoint to generate the neu-microJoint, enabling the dynamic interplay between the peripheral nervous system and joint tissues. By recording activity in sensory neurons, joint integrity as well as therapeutic efficacy can be monitored in real time. The neu-microJoint is a hybrid PDMS/3D printed system where microchannels establish a diffusion barrier but enable sensory neuron innervation of joint tissues. Neural activity is assessed with micro electrode arrays (MEAs) and fura2-based calcium imaging. With the use of L4 and L5 dorsal root ganglion (DRG) neurons recovered from organ donors, the neu-microJoint can be fully humanized. DRG neurons cultured for 4 days were screened with increasing concentrations of KCl (10, 30, and 50mM) to detect spiking in the MEAs. There was little to no spontaneous activity under baseline conditions, but activity was detected in response to 30mM KCl. Rodent and human DRG neurons plated in the neu-microJoint cultured between 10 – 28 days showed substantial axonal growth through the microchannels as visualized through DiI or fura2 labeling. Moreover, electrical stimulation applied to the neurites evoked an increase in calcium in the corresponding soma. Conditioned media from the OA-modeled microJoint applied to the neurons caused an increase in calcium in a subpopulation of neurons. These data suggest the potential to apply an in vitro microphysiological system to study OA pain. These results also indicate that a subset of neurons, now undergoing specific characterization, is potentially responsible for OA pain modeled in the neu-microJoint. F32 1F32NS110148-01A1, UG3TR003090.

An interesting case of 3‘P’s – Primary Pericardial Primitive neuroectodermal tumor in an infant: A case report

Cardiac malignancies presenting in the infancy are rare. Primitive neuroectodermal tumor (PNET), a high-grade malignant tumor is one among them. The most common sites of occurrence of primary PNET are the peripheral nervous system, bone, and deep soft tissues. It is extremely rare in the pericardium. To the best of our knowledge, only three patients with primary PNET of the pericardium have been reported so far in the literature. We report a case of primary PNET of the pericardium in a 11-months-old female child, who was referred to our hospital with complaints of dyspnea and difficulty in feeding of 5 days duration. At presentation, she had massive pericardial effusion with tamponade and an echogenic intrapericardial mass. An emergency pericardiocentesis was done to relieve the tamponade. After stabilization, the Computer tomography (CT) imaging of the chest revealed a soft tissue mass with non-enhancing cystic areas in the pericardium with features suggestive of PNET. After two days, the child had a cardiac arrest and succumbed to death.