RESUMEN
A large body of evidence indicates that nitric oxide (NO)/cGMP signaling essentially contributes to the processing of chronic pain. In general, NO-induced cGMP formation is catalyzed by 2 isoforms of guanylyl cyclase, NO-sensitive guanylyl cyclase 1 (NO-GC1) and 2 (NO-GC2). However, the specific functions of the 2 isoforms in pain processing remain elusive. Here, we investigated the distribution of NO-GC1 and NO-GC2 in the spinal cord and dorsal root ganglia, and we characterized the behavior of mice lacking either isoform in animal models of pain. Using immunohistochemistry and in situ hybridization, we demonstrate that both isoforms are localized to interneurons in the spinal dorsal horn with NO-GC1 being enriched in inhibitory interneurons. In dorsal root ganglia, the distribution of NO-GC1 and NO-GC2 is restricted to non-neuronal cells with NO-GC2 being the major isoform in satellite glial cells. Mice lacking NO-GC1 demonstrated reduced hypersensitivity in models of neuropathic pain, whereas their behavior in models of inflammatory pain was normal. By contrast, mice lacking NO-GC2 exhibited increased hypersensitivity in models of inflammatory pain, but their neuropathic pain behavior was unaltered. Cre-mediated deletion of NO-GC1 or NO-GC2 in spinal dorsal horn neurons recapitulated the behavioral phenotypes observed in the global knockout. Together, these results indicate that cGMP produced by NO-GC1 or NO-GC2 in spinal dorsal horn neurons exert distinct, and partly opposing, functions in chronic pain processing.
Asunto(s)
Inflamación/enzimología , Neuralgia/enzimología , Isoformas de Proteínas/metabolismo , Guanilil Ciclasa Soluble/metabolismo , Animales , Modelos Animales de Enfermedad , Adyuvante de Freund/toxicidad , Ganglios Espinales/enzimología , Inflamación/inducido químicamente , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Neuralgia/etiología , Dimensión del Dolor , Isoformas de Proteínas/genética , ARN Mensajero/metabolismo , Guanilil Ciclasa Soluble/genética , Médula Espinal/enzimología , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/metabolismoRESUMEN
Nucleic acid polymers (NAPs) block the release of subviral particles from hepatocytes, a mechanism consistent with their antiviral activity against hepatitis B virus (HBV) in patients. Analysis of immunostimulatory properties of NAPs were conducted with several NAP species: REP 2006, the prototypic degenerate NAP [dN]40, containing TLR9-stimulatory CpG; REP 2055 a clinically active NAP with a sequence [dAdC]20 devoid of CpG content; REP 2139 (also clinically active) and REP 2165 (REP 2055 analogues further rendered immunologically inactive by replacing cytidine with 5-methylcytidine and incorporating 2'-O methylation of riboses). These analyses revealed pro-inflammatory responses in human peripheral blood mononuclear cells with REP 2006 and with REP 2139 and REP 2165 only at high dose but displayed no significant antiviral activity. In primary isolated human hepatocytes and liver sinusoidal endothelial cells no significant inflammatory or antiviral responses were detected for any NAPs. In human Kupffer cells pro-inflammatory activity was observed with REP 2006 and REP 2055, whereas a weak but significant induction of interferon genes was only observed with REP 2006 at the highest concentration. We therefore hypothesize that the antiviral activity of NAPs optimized to treat HBV infection in patients cannot be explained by direct induction of innate antiviral responses.
Asunto(s)
Virus de la Hepatitis B/efectos de los fármacos , Hepatitis B/prevención & control , Hepatocitos/efectos de los fármacos , Ácidos Nucleicos/química , Polímeros/farmacología , Antivirales/farmacología , Células Cultivadas , Hepatitis B/virología , Virus de la Hepatitis B/fisiología , Hepatocitos/metabolismo , Humanos , Macrófagos del Hígado/efectos de los fármacos , Macrófagos del Hígado/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Ácidos Nucleicos/farmacología , Oligodesoxirribonucleótidos/farmacología , Polímeros/químicaRESUMEN
The hepatitis B virus (HBV) has been described as stealth virus subverting immune responses initially upon infection. Impaired toll-like receptor signaling by the HBV surface antigen (HBsAg) attenuates immune responses to facilitate chronic infection. This implies that HBV replication may trigger host innate immune responses in the absence of HBsAg. Here we tested this hypothesis, using highly replicative transgenic mouse models. An HBV replication-dependent expression of antiviral genes was exclusively induced in HBsAg-deficient mice. These interferon responses attributed to toll-like receptor 3 (TLR3)-activated Kupffer and liver sinusoidal endothelial cells and further controlled the HBV genome replication. However, activation of TLR3 with exogenous ligands indicated additional HBs-independent immune evasion events. Our data demonstrate that in the absence of HBsAg, hepatic HBV replication leads to Tlr3-dependent interferon responses in non-parenchymal liver cells. We hypothesize that HBsAg is a major HBV-mediated evasion mechanism controlling endogenous antiviral responses in the liver. Eradication of HBsAg as a therapeutic goal might facilitate the induction of endogenous antiviral immune responses in patients chronically infected with HBV.
Asunto(s)
Antígenos de Superficie de la Hepatitis B/metabolismo , Virus de la Hepatitis B/inmunología , Virus de la Hepatitis B/fisiología , Evasión Inmune , Interferones/antagonistas & inhibidores , Receptor Toll-Like 3/antagonistas & inhibidores , Replicación Viral , Animales , Antígenos de Superficie de la Hepatitis B/genética , Humanos , Ratones , Ratones TransgénicosRESUMEN
Novel shuttle vectors named pEBP were constructed to allow the gene expression in different bacterial hosts including Escherichia coli, Bacillus subtilis and Pseudomonas putida. These vectors share the inducible promoters P(T7) and P(Xyl) and a cos site to enable packaging of plasmid DNA into phage, and carry different multiple cloning sites and antibiotic resistance genes. Vector pEBP41 generally replicates episomally while pEBP18 replicates episomally in Gram-negative bacteria only, but integrates into the chromosome of B. subtilis. Plasmid copy numbers determined for E. coli and P. putida were in the range of 5-50 per cell. The functionality of pEBP18 and pEBP41 was confirmed by expression of two lipolytic enzymes, namely lipase A from B. subtilis and cutinase from the eukaryotic fungus Fusarium solani pisi in three different host strains. Additionally, we report here the construction of a T7 RNA polymerase-based expression strain of P. putida.