Eroboghene E. Ubogu

Inflammatory neuropathies: pathology, molecular markers and targets for specific therapeutic intervention.

Inflammatory neuropathies encompass groups of heterogeneous disorders characterized by pathogenic immune-mediated hematogenous leukocyte infiltration of peripheral nerves, nerve roots or both, with resultant demyelination or axonal degeneration or both. Inflammatory neuropathies may be divided into three major disease categories: Guillain–Barré syndrome (particularly the acute inflammatory demyelinating polyradiculoneuropathy variant), chronic inflammatory demyelinating polyradiculoneuropathy and nonsystemic vasculitic neuropathy (or peripheral nerve vasculitis). Despite major advances in molecular biology, pathology and genetics, the pathogenesis of these disorders remains elusive. There is insufficient knowledge on the mechanisms of hematogenous leukocyte trafficking into the peripheral nervous system to guide the development of specific molecular therapies for immune-mediated inflammatory neuropathies compared to disorders such as psoriasis, inflammatory bowel disease, rheumatoid arthritis or multiple sclerosis. The recent isolation and characterization of human endoneurial endothelial cells that form the blood–nerve barrier provides an opportunity to elucidate leukocyte–endothelial cell interactions critical to the pathogenesis of inflammatory neuropathies at the interface between the systemic circulation and peripheral nerve endoneurium. This review discusses our current knowledge of the classic pathological features of inflammatory neuropathies, attempts at molecular classification and genetic determinants, the utilization of in vitro and in vivo animal models to determine pathogenic mechanisms at the interface between the systemic circulation and the peripheral nervous system relevant to these disorders and prospects for future potential molecular pathology biomarkers and targets for specific therapeutic intervention.

VIP-expressing dendritic cells protect against spontaneous autoimmune peripheral polyneuropathy.

The spontaneous autoimmune peripheral polyneuropathy (SAPP) model in B7-2 knockout nonobese diabetic mice mimics a progressive and unremitting course of chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). In this study, bone marrow-derived dendritic cells (DCs) were transduced to express vasoactive intestinal polypeptide (VIP) using a lentiviral vector (LV-VIP). These transduced DCs (LV-VIP-DCs) were then injected intravenously (i.v.) into 16-week-old (before disease onset) and 21-week-old (after disease onset) SAPP mice in order to prevent or attenuate the disease. Outcome measures included behavioral tests, clinical and histological scoring, electrophysiology, real-time PCR, flow cytometry analyses, and enzyme-linked immunosorbent assay. LV-VIP-DCs were recruited to the inflamed sciatic nerve and reduced the expression of inflammatory cytokines. A single injection of LV-VIP-DC delayed the onset of disease, stabilized, and attenuated clinical signs correlating with ameliorated behavioral functions, reduced nerve demyelination, and improved nerve conduction. This proof-of-principle study is an important step potentially leading to a clinical translational study using DCs expressing VIP in cases of CIDP refractory to standard immunosuppressive therapy.The spontaneous autoimmune peripheral polyneuropathy (SAPP) model in B7-2 knockout nonobese diabetic mice mimics a progressive and unremitting course of chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). In this study, bone marrow-derived dendritic cells (DCs) were transduced to express vasoactive intestinal polypeptide (VIP) using a lentiviral vector (LV-VIP). These transduced DCs (LV-VIP-DCs) were then injected intravenously (i.v.) into 16-week-old (before disease onset) and 21-week-old (after disease onset) SAPP mice in order to prevent or attenuate the disease. Outcome measures included behavioral tests, clinical and histological scoring, electrophysiology, real-time PCR, flow cytometry analyses, and enzyme-linked immunosorbent assay. LV-VIP-DCs were recruited to the inflamed sciatic nerve and reduced the expression of inflammatory cytokines. A single injection of LV-VIP-DC delayed the onset of disease, stabilized, and attenuated clinical signs correlating with ameliorated behavioral functions, reduced nerve demyelination, and improved nerve conduction. This proof-of-principle study is an important step potentially leading to a clinical translational study using DCs expressing VIP in cases of CIDP refractory to standard immunosuppressive therapy.

Modeling leukocyte trafficking at the human blood-nerve barrier in vitro and in vivo geared towards targeted molecular therapies for peripheral neuroinflammation

Peripheral neuroinflammation is characterized by hematogenous mononuclear leukocyte infiltration into peripheral nerves. Despite significant clinical knowledge, advancements in molecular biology and progress in developing specific drugs for inflammatory disorders such as rheumatoid arthritis, inflammatory bowel disease, and multiple sclerosis, there are currently no specific therapies that modulate pathogenic peripheral nerve inflammation. Modeling leukocyte trafficking at the blood-nerve barrier using a reliable human in vitro model and potential intravital microscopy techniques in representative animal models guided by human observational data should facilitate the targeted modulation of the complex inflammatory cascade needed to develop safe and efficacious therapeutics for immune-mediated neuropathies and chronic neuropathic pain.

Fibronectin connecting segment-1 peptide inhibits pathogenic leukocyte trafficking and inflammatory demyelination in experimental models of chronic inflammatory demyelinating polyradiculoneuropathy

ABSTRACT The molecular determinants of pathogenic leukocyte migration across the blood‐nerve barrier (BNB) in chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) are unknown. Specific disease modifying therapies for CIDP are also lacking. Fibronectin connecting segment‐1 (FNCS1), an alternatively spliced fibronectin variant expressed by microvascular endothelial cells at sites of inflammation in vitro and in situ, is a counterligand for leukocyte &agr;4 integrin (also known as CD49d) implicated in pathogenic leukocyte trafficking in multiple sclerosis and inflammatory bowel disease. We sought to determine the role of FNCS1 in CIDP patient leukocyte trafficking across the BNB in vitro and in severe chronic demyelinating neuritis in vivo using a representative spontaneous murine CIDP model. Peripheral blood mononuclear leukocytes from 7 untreated CIDP patients were independently infused into a cytokine‐treated, flow‐dependent in vitro BNB model system. Time‐lapse digital video microscopy was performed to visualize and quantify leukocyte trafficking, comparing FNCS1 peptide blockade to relevant controls. Fifty 24‐week old female B7–2 deficient non‐obese diabetic mice with spontaneous autoimmune peripheral polyneuropathy (SAPP) were treated daily with 2 mg/kg FNCS1 peptide for 5 days via intraperitoneal injection with appropriate controls. Neurobehavioral measures of disease severity, motor nerve electrophysiology assessments and histopathological quantification of inflammation and morphometric assessment of demyelination were performed to determine in vivo efficacy. The biological relevance of FNCS1 and CD49d in CIDP was evaluated by immunohistochemical detection in affected patient sural nerve biopsies. 25 &mgr;M FNCS1 peptide maximally inhibited CIDP leukocyte trafficking at the human BNB in vitro. FNCS1 peptide treatment resulted in significant improvements in disease severity, motor electrophysiological parameters of demyelination and histological measures of inflammatory demyelination. Microvessels demonstrating FNCS1 expression and CD49d + leukocytes were seen within the endoneurium of patient nerve biopsies. Taken together, these results imply a role for FNCS1 in pathogenic leukocyte trafficking in CIDP, providing a potential target for therapeutic modulation. HIGHLIGHTSFurther insights into the pathogenesis of CIDP are described.FNCS1 peptide inhibits CIDP patient leukocyte trafficking at the blood‐nerve barrier in vitro.FNCS1 peptide ameliorates severe murine chronic demyelinating neuritis in vivo.FNCS1 and its ligand, &agr;4 integrin are expressed in CIDP patient nerve biopsies.FNCS1 antagonism is a plausible targeted molecular therapy for CIDP.

The Human Blood-Nerve Barrier Transcriptome

The blood-nerve barrier (BNB), formed by tight junction-forming microvessels within peripheral nerve endoneurium, exists to regulate its internal microenvironment essential for effective axonal signal transduction. Relatively little is known about the unique human BNB molecular composition. Such knowledge is crucial to comprehend the relationships between the systemic circulation and peripheral nerves in health, adaptations to intrinsic or extrinsic perturbations and alterations that may result in disease. We performed RNA-sequencing on cultured early- and late-passage adult primary human endoneurial endothelial cells and laser-capture microdissected endoneurial microvessels from four cryopreserved normal adult human sural nerves referenced to the Genome Reference Consortium Human Reference 37 genome browser, using predefined criteria guided by known transcript or protein expression in vitro and in situ. We identified 12881 common transcripts associated by 125 independent biological networks, defined as the normal adult BNB transcriptome, including a comprehensive array of transporters and specialized intercellular junctional complex components. These identified transcripts and their interacting networks provide insights into peripheral nerve microvascular morphogenesis, restrictive barrier formation, influx and efflux transporters with relevance to understanding peripheral nerve homeostasis and pharmacology, including targeted drug delivery and the mediators of leukocyte trafficking in peripheral nerves during normal immunosurveillance.

A novel method for measuring hydraulic conductivity at the human blood-nerve barrier in vitro

Microvascular barrier permeability to water is an essential biophysical property required for the homeostatic maintenance of unique tissue microenvironments. This is of particular importance in peripheral nerves where strict control of ionic concentrations is needed for axonal signal transduction. Previous studies have associated inflammation, trauma, toxin exposure and metabolic disease with increases in water influx and hydrostatic pressure in peripheral nerves with resultant endoneurial edema that may impair axonal function. The regulation of water permeability across endoneurial microvessels that form the blood-nerve barrier (BNB) is poorly understood. Variations exist in apparatus and methods used to measure hydraulic conductivity. The objective of the study was to develop a simplified hydraulic conductivity system using commercially available components to evaluate the BNB. We determined the mean hydraulic conductivity of cultured confluent primary and immortalized human endoneurial endothelial cell layers as 2.00×10-7 and 2.17×10-7cm/s/cm H₂O respectively, consistent with restrictive microvascular endothelial cells in vitro. We also determined the mean hydraulic conductivity of immortalized human brain microvascular endothelial cell layers, a commonly used blood-brain barrier (BBB) cell line, as 0.20×10-7cm/s/cm H₂O, implying a mean 10-fold higher resistance to transendothelial water flux in the brain compared to peripheral nerves. To our knowledge, this is the first reported measurement of human BNB and BBB hydraulic conductivities. This model represents an important tool to further characterize the human BNB and deduce the molecular determinants and signaling mechanisms responsible for BNB hydraulic conductivity in normal and disease states in vitro.

Acute Myofascitis as a Manifestation of Chronic Graft Versus Host Disease

Commun 2014;2:2. 2. Musumeci O, la Marca G, Spada M, Mondello S, Danesino C, Comi GP, et al. LOPED study: looking for an early diagnosis in a late-onset Pompe disease high-risk population. J Neurol Neurosurg Psychiatry 2015 Mar 17 [Epub ahead of print]. 3. Echaniz-Laguna A, Carlier RY, Laloui K, Carlier P, Salort-Campana E, Pouget J, et al. Should patients with asymptomatic Pompe disease be treated? A nationwide study in France. Muscle Nerve 2015;51:884– 889. 4. Mercuri E, Pichiecchio A, Allsop J, Messina S, Pane M, Muntoni F. Muscle MRI in inherited neuromuscular disorders: past, present, and future. J Magn Reson Imaging 2007;25:433–440. 5. Wattjes MP, Kley RA, Fischer D. Neuromuscular imaging in inherited muscle diseases. Eur Radiol 2010;20:2447–2460. 6. Straub V, Carlier PG, Mercuri E. TREAT-NMD workshop: pattern recognition in genetic muscle diseases using muscle MRI: 25–26 February 2011, Rome, Italy. Neuromuscul Disord 2012;22(suppl 2):S42–53. 7. Hollingsworth KG, de Sousa PL, Straub V, Carlier PG. Towards harmonization of protocols for MRI outcome measures in skeletal muscle studies: consensus recommendations from two TREAT-NMD NMR workshops, 2 May 2010, Stockholm, Sweden, 1–2 October 2009, Paris, France. Neuromuscul Disord 2012;22(suppl 2):S54–67. 8. Degardin A, Morillon D, Lacour A, Cotten A, Vermersch P, Stojkovic T. Morphologic imaging in muscular dystrophies and inflammatory myopathies. Skeletal Radiol 2010;39:1219–1227. 9. Tasca G, Monforte M, Iannaccone E, Laschena F, Ottaviani P, Leoncini E, et al. Upper girdle imaging in facioscapulohumeral muscular dystrophy. PLoS One 2014;9:e100292. 10. Dlamini N, Jan W, Norwood F, Sheehan J, Spahr R, Al-Sarraj S, et al. Muscle MRI findings in siblings with juvenile-onset acid maltase deficiency (Pompe disease). Neuromuscul Disord 2008;18:408–409. 11. Carlier RY, Laforet P, Wary C, Mompoint D, Laloui K, Pellegrini N, et al. Whole-body muscle MRI in 20 patients suffering from late onset Pompe disease: involvement patterns. Neuromuscul Disord 2011;21: 791–799. 12. Pichiecchio A, Tavazzi E. Metabolic myopathies. In: Wattjes MP, Fischer D, editors. Neuromuscular imaging. Berlin: Springer; 2013. p. 127–147.

Glial-derived neurotrophic factor is essential for blood-nerve barrier functional recovery in an experimental murine model of traumatic peripheral neuropathy

ABSTRACT There is emerging evidence that glial-derived neurotrophic factor (GDNF) is a potent inducer of restrictive barrier function in tight junction-forming microvascular endothelium and epithelium, including the human blood-nerve barrier (BNB) in vitro. We sought to determine the role of GDNF in restoring BNB function in vivo by evaluating sciatic nerve horseradish peroxidase (HRP) permeability in tamoxifen-inducible GDNF conditional knockout (CKO) adult mice following non-transecting crush injury via electron microscopy, with appropriate wildtype (WT) and heterozygous (HET) littermate controls. A total of 24 age-, genotype- and sex-matched mice >12 weeks of age were injected with 30 mg/kg HRP via tail vein injection 7 or 14 days following unilateral sciatic nerve crush, and both sciatic nerves were harvested 30 minutes later for morphometric assessment by light and electron microscopy. The number and percentage of HRP-permeable endoneurial microvessels were ascertained to determine the effect of GDNF in restoring barrier function in vivo. Following sciatic nerve crush, there was significant upregulation in GDNF protein expression in WT and HET mice that was abrogated in CKO mice. GDNF significantly restored sciatic nerve BNB HRP impermeability to near normal levels by day 7, with complete restoration seen by day 14 in WT and HET mice. A significant recovery lag was observed in CKO mice. This effect was independent on VE-Cadherin or claudin-5 expression on endoneurial microvessels. These results imply an important role of GDNF in restoring restrictive BNB function in vivo, suggesting a potential strategy to re-establish the restrictive endoneurial microenvironment following traumatic peripheral neuropathies.

Immunotherapy of Guillain-Barré syndrome

ABSTRACT Guillain-Barré syndrome (GBS), the most common cause of acute neuromuscular weakness and paralysis worldwide, encompasses a group of acute immune-mediated disorders restricted to peripheral nerves and roots. Immune-mediated attack of peripheral nervous system myelin, axons or both is presumed to be triggered by molecular mimicry, with both cell- and humoral-dependent mechanisms implicated in disease pathogenesis. Good circumstantial evidence exists for a pathogenic role for molecular mimicry in GBS pathogenesis, especially with its axonal forms, providing insights that could guide future immunotherapy. Intravenous immunoglobulin (IVIg) and plasma exchange (PE) are the most commonly prescribed immunotherapies for GBS with variable efficacy dependent on GBS subtype, severity at initial presentation and other clinical and electrophysiologic prognostic factors. The mechanisms of action of IVIg and PE are not known definitely. Despite recent significant advances in molecular biology that provide insights into GBS pathogenesis, no advances in therapeutics or significant improvements in patient outcomes have occurred over the past three decades. We summarize the clinical aspects of GBS, its current pathogenesis and immunotherapy, and highlight the potential of leukocyte trafficking inhibitors as novel disease-specific immunotherapeutic drugs.