Chemosensory properties of murine nasal and cutaneous trigeminal neurons identified by viral tracing

BACKGROUND: Somatosensation of the mammalian head is mainly mediated by the trigeminal nerve that provides innervation of diverse tissues like the face skin, the conjunctiva of the eyes, blood vessels and the mucouse membranes of the oral and nasal cavities. Trigeminal perception encompasses thermosensation, touch, and pain. Trigeminal chemosensation from the nasal epithelia mainly evokes stinging, burning, or pungent sensations. In vitro characterization of trigeminal primary sensory neurons derives largely from analysis of complete neuronal populations prepared from sensory ganglia. Thus, functional properties of primary trigeminal afferents depending on the area of innervation remain largely unclear. RESULTS: We established a PrV based tracing technique to identify nasal and cutaneous trigeminal neurons in vitro. This approach allowed analysis and comparison of identified primary afferents by means of electrophysiological and imaging measurement techniques. Neurons were challenged with several agonists that were reported to exhibit specificity for known receptors, including TRP channels and purinergic receptors. In addition, TTX sensitivity of sodium currents and IB4 binding was investigated. Compared with cutaneous neurons, a larger fraction of nasal trigeminal neurons showed sensitivity for menthol and capsaicin. These findings pointed to TRPM8 and TRPV1 receptor protein expression largely in nasal neurons whereas for cutaneous neurons these receptors are present only in a smaller fraction. The majority of nasal neurons lacked P2X(3 )receptor-mediated currents but showed P2X(2)-mediated responses when stimulated with ATP. Interestingly, cutaneous neurons revealed largely TTX resistant sodium currents. A significantly higher fraction of nasal and cutaneous afferents showed IB4 binding when compared to randomly chosen trigeminal neurons. CONCLUSION: In conclusion, the usability of PrV mediated tracing of primary afferents was demonstrated. Using this technique it could be shown that compared with neurons innervating the skin nasal trigeminal neurons reveal pronounced chemosensitivity for TRPM8 and TRPV1 channel agonists and only partially meet properties typical for nociceptors. In contrast to P2X(3 )receptors, TRPM8 and TRPV1 receptors seem to be of pronounced physiological relevance for intranasal trigeminal sensation

A genome-wide CRISPR/Cas9 screen reveals the requirement of host sphingomyelin synthase 1 for infection with Pseudorabies virus mutant gD–Pass

Herpesviruses are large DNA viruses, which encode up to 300 different proteins including enzymes enabling efficient replication. Nevertheless, they depend on a multitude of host cell proteins for successful propagation. To uncover cellular host factors important for replication of pseudorabies virus (PrV), an alphaherpesvirus of swine, we performed an unbiased genome-wide CRISPR/Cas9 forward screen. To this end, a porcine CRISPR-knockout sgRNA library (SsCRISPRko.v1) targeting 20,598 genes was generated and used to transduce porcine kidney cells. Cells were then infected with either wildtype PrV (PrV-Ka) or a PrV mutant (PrV-gD–Pass) lacking the receptor-binding protein gD, which regained infectivity after serial passaging in cell culture. While no cells survived infection with PrV-Ka, resistant cell colonies were observed after infection with PrV-gD–Pass. In these cells, sphingomyelin synthase 1 (SMS1) was identified as the top hit candidate. Infection efficiency was reduced by up to 90% for PrV-gD–Pass in rabbit RK13-sgms1KO cells compared to wildtype cells accompanied by lower viral progeny titers. Exogenous expression of SMS1 partly reverted the entry defect of PrV-gD–Pass. In contrast, infectivity of PrV-Ka was reduced by 50% on the knockout cells, which could not be restored by exogenous expression of SMS1. These data suggest that SMS1 plays a pivotal role for PrV infection, when the gD-mediated entry pathway is blocked

T2-Weighted Dixon Turbo Spin Echo for Accelerated Simultaneous Grading of Whole-Body Skeletal Muscle Fat Infiltration and Edema in Patients With Neuromuscular Diseases

Objective The assessment of fatty infiltration and edema in the musculature of patients with neuromuscular diseases (NMDs) typically requires the separate performance of T-1-weighted and fat-suppressed T-2-weighted sequences. T-2-weighted Dixon turbo spin echo (TSE) enables the generation of T-2-weighted fat- and water-separated images, which can be used to assess both pathologies simultaneously. The present study examines the diagnostic performance of T-2-weighted Dixon TSE compared with the standard sequences in 10 patients with NMDs and 10 healthy subjects. Methods Whole-body magnetic resonance imaging was performed including T-1-weighted Dixon fast field echo, T-2-weighted short-tau inversion recovery, and T-2-weighted Dixon TSE. Fatty infiltration and intramuscular edema were rated by 2 radiologists using visual semiquantitative rating scales. To assess intermethod and interrater agreement, weighted Cohen's coefficients were calculated. Results The ratings of fatty infiltration showed high intermethod and high interrater agreement (T-1-weighted Dixon fast field echo vs T-2-weighted Dixon TSE fat image). The evaluation of edematous changes showed high intermethod and good interrater agreement (T-2-weighted short-tau inversion recovery vs T-2-weighted Dixon TSE water image). Conclusions T-2-weighted Dixon TSE imaging is an alternative for accelerated simultaneous grading of whole-body skeletal muscle fat infiltration and edema in patients with NMDs

Genetic Characterization and Clinical Features of Helicobacter pylori Negative Gastric Mucosa-Associated Lymphoid Tissue Lymphoma

Background: In Western countries, the prevalence of gastric mucosa-associated lymphoid tissue (MALT) lymphoma has declined over the last three decades. Contemporaneously, H. pylori negative gastric MALT lymphoma is increasingly encountered, and their genetic basis and clinical features remain elusive. Methods: A total of 57 cases of H. pylori negative gastric MALT lymphoma were reviewed and investigated for chromosome translocation by fluorescence in-situ hybridization and for somatic mutations by the targeted sequencing of 93 genes. Results: MALT1 translocation, most likely t(11;18)(q21;q21)/BIRC3-MALT1, was detected in 39% (22/57) cases, and IGH translocation was further seen in 12 MALT1-negative cases, together accounting for 60% of the cohort. Targeted sequencing was successful in 35 cases, and showed frequent mutations in NF-κB signaling pathways (TNFAIP3 = 23%, CARD11 = 9%, MAP3K14 = 9%), together affecting 14 cases (40%). The NF-κB pathway mutations were mutually exclusive from MALT1, albeit not IGH translocation, altogether occurring in 86% of cases. There was no significant correlation between the genetic changes and clinicopathological parameters. The patients showed a median of progression-free survival (PFS) of 66.3 months, and a significant superior PFS when treated with systemic versus antibiotic therapy (p = 0.004). Conclusion: H. pylori negative gastric MALT lymphoma is characterized by highly frequent genetic changes in the NF-κB signaling pathways

A Gammaherpesvirus Complement Regulatory Protein Promotes Initiation of Infection by Activation of Protein Kinase Akt/PKB

BACKGROUND: Viruses have evolved to evade the host's complement system. The open reading frames 4 (ORF4) of gammaherpesviruses encode homologs of regulators of complement activation (RCA) proteins, which inhibit complement activation at the level of C3 and C4 deposition. Besides complement regulation, these proteins are involved in heparan sulfate and glycosaminoglycan binding, and in case of MHV-68, also in viral DNA synthesis in macrophages. METHODOLOGY/PRINCIPAL FINDINGS: Here, we made use of MHV-68 to study the role of ORF4 during infection of fibroblasts. While attachment and penetration of virions lacking the RCA protein were not affected, we observed a delayed delivery of the viral genome to the nucleus of infected cells. Analysis of the phosphorylation status of a variety of kinases revealed a significant reduction in phosphorylation of the protein kinase Akt in cells infected with ORF4 mutant virus, when compared to cells infected with wt virus. Consistent with a role of Akt activation in initial stages of infection, inhibition of Akt signaling in wt virus infected cells resulted in a phenotype resembling the phenotype of the ORF4 mutant virus, and activation of Akt by addition of insulin partially reversed the phenotype of the ORF4 mutant virus. Importantly, the homologous ORF4 of KSHV was able to rescue the phenotype of the MHV-68 ORF4 mutant, indicating that ORF4 is functionally conserved and that ORF4 of KSHV might have a similar function in infection initiation. CONCLUSIONS/SIGNIFICANCE: In summary, our studies demonstrate that ORF4 contributes to efficient infection by activation of the protein kinase Akt and thus reveal a novel function of a gammaherpesvirus RCA protein

Entry of Herpes Simplex Virus Type 1 (HSV-1) into the Distal Axons of Trigeminal Neurons Favors the Onset of Nonproductive, Silent Infection

Following productive, lytic infection in epithelia, herpes simplex virus type 1 (HSV-1) establishes a lifelong latent infection in sensory neurons that is interrupted by episodes of reactivation. In order to better understand what triggers this lytic/latent decision in neurons, we set up an organotypic model based on chicken embryonic trigeminal ganglia explants (TGEs) in a double chamber system. Adding HSV-1 to the ganglion compartment (GC) resulted in a productive infection in the explants. By contrast, selective application of the virus to distal axons led to a largely nonproductive infection that was characterized by the poor expression of lytic genes and the presence of high levels of the 2.0-kb major latency-associated transcript (LAT) RNA. Treatment of the explants with the immediate-early (IE) gene transcriptional inducer hexamethylene bisacetamide, and simultaneous co-infection of the GC with HSV-1, herpes simplex virus type 2 (HSV-2) or pseudorabies virus (PrV) helper virus significantly enhanced the ability of HSV-1 to productively infect sensory neurons upon axonal entry. Helper-virus-induced transactivation of HSV-1 IE gene expression in axonally-infected TGEs in the absence of de novo protein synthesis was dependent on the presence of functional tegument protein VP16 in HSV-1 helper virus particles. After the establishment of a LAT-positive silent infection in TGEs, HSV-1 was refractory to transactivation by superinfection of the GC with HSV-1 but not with HSV-2 and PrV helper virus. In conclusion, the site of entry appears to be a critical determinant in the lytic/latent decision in sensory neurons. HSV-1 entry into distal axons results in an insufficient transactivation of IE gene expression and favors the establishment of a nonproductive, silent infection in trigeminal neurons

Entry of Pseudorabies Virus: an Immunogold-Labeling Study

Herpesviruses infect cells by fusion of the viral envelope with cellular membranes, primarily the plasma membrane. During this process structural components of the mature virion are lost from the invading nucleocapsid, which then travels along microtubules to the nuclear pore. We examined the penetration process by immunoelectron microscopy and analyzed which of the major tegument proteins remained associated with the incoming capsid. We show that the UL36, UL37, and US3 proteins were present at intracytoplasmic capsids after penetration, whereas the UL11, UL47, UL48, and UL49 tegument proteins were lost. Thus, the three capsid-associated tegument proteins are prime candidates for viral proteins that interact with cellular motor proteins for transport of nucleocapsids to the nucleus