RESUMO
In 2000, we reported that human cytomegalovirus (HCMV) induced specific damage on chromosome 1. The capacity of the virus to induce DNA breaks indicated potent interaction between viral proteins and these loci. We have fine mapped the 1q42 breaksite. Transcriptional analysis of genes encoded in close proximity revealed virus-induced downregulation of a single gene, nidogen 1 (NID1). Beginning between 12 and 24 hours postinfection (hpi) and continuing throughout infection, steady-state (ss) NID1 protein levels were decreased in whole-cell lysates and secreted supernatants of human foreskin fibroblasts. Addition of the proteasomal inhibitor MG132 to culture medium stabilized NID1 in virus-infected cells, implicating infection-activated proteasomal degradation of NID1. Targeting of NID1 via two separate pathways highlighted the virus' emphasis on NID1 elimination. NID1 is an important basement membrane protein secreted by many cell types, including the endothelial cells (ECs) lining the vasculature. We found that ss NID1 was also reduced in infected ECs and hypothesized that virus-induced removal of NID1 might offer HCMV a means of increased distribution throughout the host. Supporting this idea, transmigration assays of THP-1 cells seeded onto NID1-knockout (KO) EC monolayers demonstrated increased transmigration. NID1 is expressed widely in the developing fetal central and peripheral nervous systems (CNS and PNS) and is important for neuronal migration and neural network excitability and plasticity and regulates Schwann cell proliferation, migration, and myelin production. We found that NID1 expression was dramatically decreased in clinical samples of infected temporal bones. While potentially beneficial for virus dissemination, HCMV-induced elimination of NID1 may underlie negative ramifications to the infected fetus.IMPORTANCE We have found that HCMV infection promotes the elimination of the developmentally important basement membrane protein nidogen 1 (NID1) from its host. The virus both decreased transcription and induced degradation of expressed protein. Endothelial cell (EC) secretion of basement membrane proteins is critical for vascular wall integrity, and infection equivalently affected NID1 protein levels in these cells. We found that the absence of NID1 in an EC monolayer allowed increased transmigration of monocytes equivalent to that observed after infection of ECs. The importance of NID1 in development has been well documented. We found that NID1 protein was dramatically reduced in infected inner ear clinical samples. We believe that HCMV's attack on host NID1 favors viral dissemination at the cost of negative developmental ramifications in the infected fetus.
Assuntos
Membrana Basal/metabolismo , Infecções por Citomegalovirus/metabolismo , Infecções por Citomegalovirus/virologia , Citomegalovirus/fisiologia , Endotélio Vascular/metabolismo , Fibroblastos/metabolismo , Glicoproteínas de Membrana/metabolismo , Movimento Celular , Infecções por Citomegalovirus/patologia , Endotélio Vascular/virologia , Fibroblastos/virologia , Humanos , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/genética , Transdução de Sinais , Internalização do VírusRESUMO
The SARS-CoV-2 pandemic has left millions of individuals with a host of post-viral symptoms that can be debilitating and persist indefinitely. To date there are no definitive tests or treatments for the collection of symptoms known as "Long COVID" or Post-acute sequelae of COVID-19 (PASC). Following our initial case report detailing improvement of Long COVID symptoms after sequential bilateral stellate ganglion blockade (SGB), we performed a retrospective chart analysis study on individuals treated with the same protocol over the course of six months (2021-2022) in our clinic. Patients self-reported symptoms on a 10-point scale as part of optional patient follow-up using an online survey. After one month or more following treatment, patients reported striking reductions in Fatigue, Worsening of Symptoms following Mental and Physical Activity, Memory Problems, Problems Concentrating, Sleep Problems, Anxiety, and Depression. Loss of Taste and Loss of Smell in some individuals did not respond to treatment, likely indicating structural damage following infection. This study suggests that neuromodulation may provide relief of Long COVID symptoms for at least a subset of individuals, and provides support for prospective studies of this potential treatment.
Assuntos
Bloqueio Nervoso Autônomo , COVID-19 , Síndrome de COVID-19 Pós-Aguda , Gânglio Estrelado , Humanos , Estudos Retrospectivos , COVID-19/complicações , Masculino , Bloqueio Nervoso Autônomo/métodos , Feminino , Pessoa de Meia-Idade , Idoso , AdultoRESUMO
Mutations that perturb the function of photoreceptor CNG (cyclic nucleotide-gated) channels are associated with several human retinal disorders, but the molecular and cellular mechanisms leading to photoreceptor dysfunction and degeneration remain unclear. Many loss-of-function mutations result in intracellular accumulation of CNG channel subunits. Accumulation of proteins in the ER (endoplasmic reticulum) is known to cause ER stress and trigger the UPR (unfolded protein response), an evolutionarily conserved cellular programme that results in either adaptation via increased protein processing capacity or apoptotic cell death. We hypothesize that defective trafficking of cone photoreceptor CNG channels can induce UPR-mediated cell death. To test this idea, CNGA3 subunits bearing the R563H and Q655X mutations were expressed in photoreceptor-derived 661W cells with CNGB3 subunits. Compared with wild-type, R563H and Q655X subunits displayed altered degradation rates and/or were retained in the ER. ER retention was associated with increased expression of UPR-related markers of ER stress and with decreased cell viability. Chemical and pharmacological chaperones {TUDCA (tauroursodeoxycholate sodium salt), 4-PBA (sodium 4-phenylbutyrate) and the cGMP analogue CPT-cGMP [8-(4-chlorophenylthio)-cGMP]} differentially reduced degradation and/or promoted plasma-membrane localization of defective subunits. Improved subunit maturation was concordant with reduced expression of ER-stress markers and improved viability of cells expressing localization-defective channels. These results indicate that ER stress can arise from expression of localization-defective CNG channels, and may represent a contributing factor for photoreceptor degeneration.
Assuntos
Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Retículo Endoplasmático/fisiologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular , Membrana Celular/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico/metabolismo , Humanos , Camundongos , Chaperonas Moleculares/farmacologia , Transporte Proteico/efeitos dos fármacos , Fator de Transcrição CHOP/metabolismoRESUMO
After recovering from COVID-19, a significant proportion of symptomatic and asymptomatic individuals develop Long COVID. Fatigue, orthostatic intolerance, brain fog, anosmia, and ageusia/dysgeusia in Long COVID resemble "sickness behavior," the autonomic nervous system response to pro-inflammatory cytokines (Dantzer et al., 2008). Aberrant network adaptation to sympathetic/parasympathetic imbalance is expected to produce long-standing dysautonomia. Cervical sympathetic chain activity can be blocked with local anesthetic, allowing the regional autonomic nervous system to "reboot." In this case series, we successfully treated two Long COVID patients using stellate ganglion block, implicating dysautonomia in the pathophysiology of Long COVID and suggesting a novel treatment.
Assuntos
Bloqueio Nervoso Autônomo/métodos , COVID-19/complicações , Gânglio Estrelado/cirurgia , Adulto , COVID-19/cirurgia , Feminino , Humanos , SARS-CoV-2 , Síndrome de COVID-19 Pós-AgudaRESUMO
Prolactin (PRL) and vasoactive intestinal polypeptide (VIP) mRNA levels were elevated in the brainstem of neuronal nitric oxide synthase (nNOS) gene knockout (KO) mice compared to the levels in nNOS control mice. In addition, PRL mRNA levels increased in the hypothalamus and the brainstem of nNOS control mice after administration of 7-nitro-indazole (7-NI), a relatively selective nNOS inhibitor. The results suggest that NO inhibits PRL. No differences in the genes measured were observed in inducible NOS KO mice.
Assuntos
Tronco Encefálico/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Óxido Nítrico Sintase/metabolismo , Prolactina/genética , Prolactina/metabolismo , RNA Mensageiro/metabolismo , Animais , Regulação da Expressão Gênica , Hipotálamo/metabolismo , Indazóis/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo I , Peptídeo Intestinal Vasoativo/metabolismoRESUMO
Influenza viral infection induces increases in non-rapid eye movement sleep and decreases in rapid eye movement sleep in normal mice. An array of cytokines is produced during the infection, and some of them, such as IL-1beta and TNF-alpha, are well-defined somnogenic substances. It is suggested that nitric oxide (NO) may mediate the sleep-promoting effects of these cytokines. In this study, we use mice with targeted disruptions of either the neuronal NO synthase (nNOS) or the inducible NO synthase (iNOS) gene, commonly referred to as nNOS or iNOS knockouts (KOs), to investigate sleep changes after influenza viral challenge. We report that the magnitude of viral-induced non-rapid eye movement sleep responses in both nNOS KOs and iNOS KOs was less than that of their respective controls. In addition, the duration of rapid eye movement sleep in nNOS KO mice did not decrease compared with baseline values. All strains of mice had similar viral titers and cytokine gene expression profiles in the lungs. Virus was not isolated from the brains of any strain. However, gene expression in the brain stem differed between nNOS KOs and their controls: mRNA for the interferon-induced gene 2',5'-oligoadenylate synthase 1a was elevated in nNOS KOs relative to their controls at 15 h, and IL-1beta mRNA was elevated in nNOS KOs relative to their controls at 48 h. Our results suggest that NO synthesized by both nNOS and iNOS plays a role in virus-induced sleep changes and that nNOS may modulate cytokine expression in the brain.
Assuntos
Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/fisiologia , Infecções por Orthomyxoviridae/fisiopatologia , Orthomyxoviridae , Sono/genética , Sono/fisiologia , 2',5'-Oligoadenilato Sintetase/biossíntese , 2',5'-Oligoadenilato Sintetase/genética , Animais , Tronco Encefálico/enzimologia , Tronco Encefálico/metabolismo , Eletroencefalografia , Regulação da Expressão Gênica , Vírus da Influenza A/genética , Interleucina-1/biossíntese , Interleucina-1/genética , Pulmão/enzimologia , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo I , Óxido Nítrico Sintase Tipo II , Infecções por Orthomyxoviridae/enzimologia , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sono REM/genética , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genéticaRESUMO
Homer protein expression is dependent in part on neuronal activity and sleep. Homer1a differs in function from Homer1bc although both are involved in synaptic scaffolding. The effects of sleep loss, time of day and afferent activity on these molecules were investigated. Rats were sacrificed at the midpoint of either their activity or sleep phases and RNA was prepared from somatosensory cortex samples. Homer1a and 1bc mRNA levels were determined by real time PCR. There were greater amounts of both Homer1a and 1bc in night time samples. In controls, there was more Homer1bc than 1a both day and night. Sleep loss upregulated Homer1a in the morning but not at night. Homer1bc was much less affected by manipulations of sleep. Thus, Homer1a may be a state- and activity-dependent synaptic scaling factor.
Assuntos
Proteínas de Transporte/metabolismo , Ritmo Circadiano/fisiologia , Regulação da Expressão Gênica/fisiologia , Privação do Sono/metabolismo , Córtex Somatossensorial/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Arcabouço Homer , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Vibrissas/inervação , Vibrissas/fisiologiaRESUMO
Influenza virus infection up-regulates cytokines such as interleukin-1beta (IL-1beta) and activates the somatotropic axis and the hypothalamic-pituitary axis. Mice with deficits in growth hormone releasing hormone (GHRH) signaling (lit/lit mice) respond to influenza virus challenge with a progressive decrease in sleep and lower survival rates. Current experiments characterize plasma glucocorticoid responses and hypothalamic and lung mRNA expression of sleep-related genes in lit/lit mice and their heterozygous controls after influenza virus challenge. lit/lit mice had higher basal and post-infection plasma corticosterone levels compared to controls. In contrast, the heterozygous mice increased hypothalamic GHRH-receptor, CRH-type 2 receptor, IL-1beta, and tumor necrosis factor-alpha (TNF-alpha) mRNAs after virus treatment while the lit/lit mice failed to up-regulate these substances. In contrast, lung levels of IL-1beta and TNF-alpha mRNAs were greater in the lit/lit mice. These data are consistent with the hypothesis that the sleep response to influenza infection is mediated, in part, by an up-regulation of hypothalamic sleep-related transcripts and they also show that a primary deficit in GHRH signaling is associated with enhanced corticosterone secretion and attenuated hypothalamic cytokine response to infection.