RESUMO
Studies investigating the cumulative incidence of and immune status against SARS-CoV-2 infection provide valuable information for shaping public health decision-making. A cross-sectional study on 935 participants, conducted in the Valencian Community (VC), measuring anti-SARS-CoV-2-receptor binding domain-RBD-total antibodies and anti-Nucleocapsid (N)-IgGs via electrochemiluminescence assays. Quantitation of neutralizing antibodies (NtAb) against ancestral and Omicron BA.1 and BA.2 variants and enumeration of SARS-CoV-2-S specific-IFNγ-producing CD4+ and CD8+ T cells was performed in 100 and 137 participants, respectively. The weighted cumulative incidence was 51.9% (95% confidence interval [CI]: 48.7-55.1) and was inversely related to age. Anti-RBD total antibodies were detected in 97% of participants; vaccinated and SARS-CoV-2-experienced (VAC-ex; n = 442) presented higher levels (p < 0.001) than vaccinated/naïve (VAC-n; n = 472) and nonvaccinated/experienced (UNVAC-ex; n = 63) subjects. Antibody levels correlated inversely with time elapsed since last vaccine dose in VAC-n (Rho, -0.52; p < 0.001) but not in VAC-ex (rho -0.02; p = 0.57). Heterologous booster shots resulted in increased anti-RBD antibody levels compared with homologous schedules in VAC-n, but not in VAC-ex. NtAbs against Omicron BA.1 were detected in 94%, 75%, and 50% of VAC-ex, VAC-n and UNVAC-ex groups, respectively. For Omicron BA.2, the figures were 97%, 84%, and 40%, respectively. SARS-CoV-2-S-reactive IFN-γ T cells were detected in 73%, 75%, and 64% of VAC-ex, VAC-n and UNVAC-ex, respectively. Median frequencies for both T-cell subsets were comparable across groups. In summary, by April 2022, around half of the VC population had been infected with SARS-CoV-2 and, due to extensive vaccination, displayed hybrid immunity.
Assuntos
COVID-19 , Humanos , COVID-19/epidemiologia , SARS-CoV-2/genética , Espanha/epidemiologia , Linfócitos T CD8-Positivos , Estudos Transversais , Incidência , Anticorpos Neutralizantes , Anticorpos AntiviraisRESUMO
A third Comirnaty vaccine dose increased severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor-binding domain antibody levels (median, 93-fold) and neutralizing antibody titers against Wuhan-Hu-1 (median, 57-fold), Beta (me 22-fold), Delta, (median, 43-fold), and Omicron (median, 8-fold) variants, but had less impact on S-reactive T-cell immunity in nursing home residents.
Assuntos
COVID-19 , Vacinas Virais , Imunidade Adaptativa , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Humanos , Testes de Neutralização , Casas de Saúde , SARS-CoV-2RESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant breakthrough infections in nursing home residents following vaccination with Comirnaty® COVID-19 vaccine were characterized. In total, 201 participants (median age, 87 years; range, 64-100; 133 female) from two nursing homes in the Valencian community (Spain) were included. SARS-CoV-2-Spike (S) antibody responses were determined by a lateral flow immunocromatography (LFIC) assay and by quantitative electrochemiluminescent assay in LFIC-negative participants. SARS-CoV-2-S-IFNγ T cells were enumerated by flow cytometry in 10 participants. Nasopharyngeal SARS-CoV-2 RNA loads were quantified by real-time polymerase chain reaction assays. Vaccine breakthrough COVID-19 due to the Delta variant occurred in 39 residents (median age, 87 years; range, 69-96; 31 female) at a median of 6.5 months after vaccination (nine requiring hospitalization). Breakthrough infections occurred at a higher rate (p < 0.0001) in residents who had not been previously infected with SARS-CoV-2 (naïve) (33/108; 18%) than in those with prior diagnosis of SARS-CoV-2 infection (experienced) (6/93; 6.4%), and were more likely (p < 0.0001) to develop in residents who tested negative by LFIC (20/49) at 3 months after vaccination as compared to their LFIC-positive counterparts (19/142). Among LFIC-negative residents, a trend towards lower plasma anti-RBD antibody levels was noticed in those developing breakthrough infection (p = 0.16). SARS-CoV-2 RNA loads in nasopharyngeal specimens were lower in SARS-CoV-2-experienced residents (p < 0.001) and in those testing positive by LFIC (p = 0.13). The frequency of SARS-CoV-2-S-reactive T cells at 3 months was similar in LFIC-negative residents with (n = 7) or without (n = 3) breakthrough infection. Prior history of SARS-CoV-2 infection and detection of S-reactive antibodies by LFIC at 3 months is associated with a lower risk of Delta-variant breakthrough infection in nursing home residents at midterm after Comirnaty® COVID-19 vaccination.
Assuntos
COVID-19 , SARS-CoV-2 , Idoso de 80 Anos ou mais , Anticorpos Antivirais , COVID-19/diagnóstico , COVID-19/epidemiologia , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Feminino , Humanos , Casas de Saúde , RNA Viral/genética , SARS-CoV-2/genética , VacinaçãoRESUMO
We investigated whether peripheral blood levels of SARS-CoV-2 Spike (S) receptor binding domain antibodies (anti-RBD), neutralizing antibodies (NtAb) targeting Omicron S, and S-reactive-interferon (IFN)-γ-producing CD4+ and CD8+ T cells measured after a homologous booster dose (3D) with the Comirnaty® vaccine was associated with the likelihood of subsequent breakthrough infections due to the Omicron variant. An observational study including 146 nursing home residents (median age, 80 years; range, 66-99; 109 female) evaluated for an immunological response after 3D (at a median of 16 days). Anti-RBD total antibodies were measured by chemiluminescent immunoassay. NtAb were quantified by an Omicron S pseudotyped virus neutralization assay. SARS-CoV-2-S specific-IFNγ-producing CD4+ and CD8+ T cells were enumerated by whole-blood flow cytometry for intracellular cytokine staining. In total, 33/146 participants contracted breakthrough Omicron infection (symptomatic in 30/33) within 4 months after 3D. Anti-RBD antibody levels were comparable in infected and uninfected participants (21 123 vs. 24 723 BAU/ml; p = 0.34). Likewise, NtAb titers (reciprocal IC50 titer, 157 vs. 95; p = 0.32) and frequency of virus-reactive CD4+ (p = 0.82) and CD8+ (p = 0.91) T cells were similar across participants in both groups. anti-RBD antibody levels and NtAb titers estimated at around the time of infection were also comparable (3445 vs. 4345 BAU/ml; p = 0.59 and 188.5 vs. 88.9; p = 0.70, respectively). Having detectable NtAb against Omicron or SARS-CoV-2-S-reactive-IFNγ-producing CD4+ or CD8+ T cells after 3D was not correlated with increased protection from breakthrough infection (OR, 1.50; p = 0.54; OR, 0.0; p = 0.99 and OR 3.70; p = 0.23, respectively). None of the immune parameters evaluated herein, including NtAb titers against the Omicron variant, may reliably predict at the individual level the risk of contracting COVID-19 due to the Omicron variant in nursing home residents.
Assuntos
Vacinas contra COVID-19 , COVID-19 , Idoso de 80 Anos ou mais , Anticorpos Neutralizantes , Anticorpos Antivirais , Linfócitos T CD8-Positivos , COVID-19/prevenção & controle , Feminino , Humanos , Casas de Saúde , SARS-CoV-2 , Proteínas do Envelope ViralRESUMO
The objective was to understand the effectiveness of the BNT162b2 and mRNA-1273 vaccines against SARS-CoV-2 in health professionals(HPs) in the Valencian Autonomous Community(Spain) who had completed a full vaccination regimen, both in terms of preventing infections and avoiding hospitalisations, according to the time elapsed since the vaccine administration. Case-controlled study with negative test results. HPs who had undergone at least one PCR or antigen(Ag) active infection diagnostic test(AIDT) to rule out SARS-CoV-2 infection between 25 January and 18 July 2021 were included. HPs with positive AIDT result were considered as cases and those with a negative result controls. Adjusted vaccine effectiveness(VEa) to prevent SARS-CoV-2 infection and its 95% confidence interval(95% CI) were calculated using the formula VEa = (1 - OR) × 100. The VEa for the prevention of SARS-CoV-2 infection 12 to 120 days after completing the full two-dose vaccine regimen was 91.6%(95%CI[89.6%,93.2%]) for the BNT162b2 vaccine and 95.2%(95%CI[88.3%,98.1%]) for the mRNA-1273 vaccine. After 120 days the VEa was 71.5%(95%CI[67.0%,75.5%]) for the BNT162b2 vaccine and 88.3%(95%CI[75.7,94.4%]) for the mRNA-1273 vaccine. The VEa for prevention of hospitalisation for COVID-19 for the complete two-dose regimen of mRNA vaccines (BNT162b2 and mRNA-1273) was 96.8%(95%CI[76.1%,99.6%]). The administration of the complete regimen of the BNT162b2 and mRNA-1273 vaccine against SARS-CoV-2 was highly effective for the prevention of COVID-19 cases in HPs when 12 to 120 days had elapsed since the second dose. However, said effectiveness decreased as time from the vaccine administration elapsed, although it was maintained for the prevention of hospitalisation of HPs.
Assuntos
COVID-19 , Vacinas Virais , Vacina de mRNA-1273 contra 2019-nCoV , Vacina BNT162 , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Hospitalização , Humanos , SARS-CoV-2 , Espanha/epidemiologia , Vacinas Sintéticas , Vacinas de mRNARESUMO
OBJECTIVE: This study aimed to evaluate the effectiveness of SARS-CoV-2 mRNA vaccines in preventing infection and hospitalization among healthcare workers (HCWs) in the Valencian Community (Spain), considering vaccination timing, dose number, and predominant variant. METHODS: A test-negative case-control design estimated vaccine effectiveness against symptomatic disease and hospitalization due to SARS-CoV-2. HCWs who underwent PCR or antigen testing for SARS-CoV-2 from January 2021 to March 2022 were included. Cases had a positive diagnostic test, while controls had negative tests. Adjusted vaccine effectiveness (aVE) was calculated using the formula: aVE = (1 - Odds ratio) × 100. RESULTS: During the Delta variant's predominance, aVE against infection within 12-120 days post-second dose was 64.8 % (BNT162b2) and 59.4 % (mRNA-1273), declining to 21.2 % and 42.2 %, respectively, after 120 days. For the Omicron variant, aVE within 12-120 days post-second dose was 61.1 % (BNT162b2) and 85.1 % (mRNA-1273), decreasing to 36.7 % and 24.9 %, respectively, after 120 days. After a booster dose of mRNA-1273, aVE was 64.0 % (BNT162b2 recipients) and 65.9 % (initial mRNA-1273 recipients). Regardless of variant, aVE for hospitalization prevention after 2 doses was 87.0 % (BNT162b2) and 89.0 % (mRNA-1273). CONCLUSION: The administration of two doses of Moderna-mRNA-1273 against SARS-CoV-2 in HCWs proved to be highly effective in preventing infections and hospitalizations in the first 120 days after the second dose during the predominance of the Omicron variant. The decline in VE after 120 days since the administration of the second dose was significantly restored by the booster dose administration. This increase in VE was greater for the Pfizer vaccine. COVID-19 hospitalization prevention remained stable with both mRNA vaccines throughout the study period.
Assuntos
Vacina BNT162 , Vacinas contra COVID-19 , COVID-19 , Pessoal de Saúde , Hospitalização , Imunização Secundária , SARS-CoV-2 , Eficácia de Vacinas , Humanos , COVID-19/prevenção & controle , COVID-19/epidemiologia , Espanha/epidemiologia , SARS-CoV-2/imunologia , SARS-CoV-2/genética , Masculino , Feminino , Hospitalização/estatística & dados numéricos , Adulto , Pessoa de Meia-Idade , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/administração & dosagem , Vacina BNT162/imunologia , Vacina BNT162/administração & dosagem , Estudos de Casos e Controles , Vacina de mRNA-1273 contra 2019-nCoV/imunologia , Vacinação/métodosRESUMO
MS-based quantitative proteomics plays an increasingly important role in biological and medical research and the development of these techniques remains one of the most important challenges in mass spectrometry. Numerous stable isotope labeling approaches have been proposed. However, and particularly in the case of (18)O-labeling, a standard protocol of general applicability is still lacking, and statistical issues associated to these methods remain to be investigated. In this work we present an improved high-throughput quantitative proteomics method based on whole proteome concentration by SDS-PAGE, optimized in-gel digestion, peptide (18)O-labeling, and separation by off-gel isoelectric focusing followed by liquid chromatography-LIT-MS. We demonstrate that the off-gel technique is fully compatible with (18)O peptide labeling in any pH range. A recently developed statistical model indicated that partial digestions and methionine oxidation do not alter protein quantification and that variances at the scan, peptide, and protein levels are stable and reproducible in a variety of proteomes of different origin. We have also analyzed the dynamic range of quantification and demonstrated the practical utility of the method by detecting expression changes in a model of activation of Jurkat T-cells. Our protocol provides a general approach to perform quantitative proteomics by (18)O-labeling in high-throughput studies, with the added value that it has a validated statistical model for the null hypothesis. To the best of our knowledge, this is the first report where a general protocol for stable isotope labeling is tested in practice using a collection of samples and analyzed at this degree of statistical detail.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Marcação por Isótopo/métodos , Proteoma/análise , Proteômica/métodos , Análise de Variância , Animais , Linhagem Celular Tumoral , Fracionamento Químico , Citoplasma/metabolismo , Eletroforese em Gel de Poliacrilamida , Humanos , Focalização Isoelétrica , Metionina/metabolismo , Proteínas de Neoplasias/metabolismo , Oxirredução , Isótopos de Oxigênio , Peptídeos/análise , RatosRESUMO
OBJECTIVES: The current study was aimed at examining SARS-CoV-2 immune responses following two doses of Comirnaty® COVID-19 vaccine among elderly people in nursing homes. METHODS: A prospective cohort study in a representative sample from nursing homes in Valencia (n = 881; males: 271, females 610; median age, 86 years) recruited residents using a random one-stage cluster sampling approach. A lateral flow immunochromatography device (LFIC) (OnSite COVID-19 IgG/IgM Rapid Test; CTK BIOTECH, Poway, CA, USA) was used as the front-line test for detecting SARS-CoV-2-Spike (S)-specific antibodies in whole blood obtained using a fingerstick. Residents returning negative LFIC results underwent venipuncture and testing for presence of SARS-CoV-2-S-reactive antibodies and T cells using the Roche Elecsys® Anti-SARS-CoV-2 S (Roche Diagnostics, Pleasanton, CA, USA), the LIAISON® SARS-CoV-2 TrimericS IgG assay (Diasorin S.p.A, Saluggia, Italy) and by flow cytometry, respectively. RESULTS: The SARS-CoV-2-S antibody detection rate in nursing home residents was 99.6% (283/284) and 98.3% (587/597) for SARS-CoV-2 recovered and naïve residents, respectively, within a median of 99 days (range 17-125 days) after full vaccination. Three out of five residents lacking SARS-CoV-2-S antibodies had detectable S-reactive CD8+ and/or CD4+ T cells. In addition, 50/50 and 40/50 participants with detectable SARS-CoV-2 antibodies also had SARS-CoV-2-S-reactive interferon-γ-producing CD4+ and CD8+ T cells, respectively. DISCUSSION: The Comirnaty® COVID-19 vaccine is highly immunogenic in nursing home residents.
Assuntos
Vacinas contra COVID-19 , COVID-19 , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antivirais , Linfócitos T CD8-Positivos , Feminino , Humanos , Masculino , Casas de Saúde , Estudos Prospectivos , SARS-CoV-2RESUMO
Burgos JS (General Directorate of Research and Healthcare Supervision, Department of Health, Valencia Government, Valencia, Spain); Meneu de Guillerna R (Vice-President Foundation Research Institute in Public Services, Valencia, Spain); Vanaclocha Luna H (General Directorate of Public Health, Department of Health, Valencia Government, Valencia, Spain); Burks DJ (The Prince Felipe Research Center-CIPF-, Valencia, Spain; Cervantes A (INCLIVA Health Research Institute, Valencia, Spain); Comas I (Biomedicine Institute of Valencia, Spanish Research Council (CSIC); Díez-Domingo J (Foundation for the promotion of health and biomedical research of the Valencian Community-FISABIO-, Valencia, Spain); Peiro S (Foundation for the promotion of health and biomedical research of the Valencian Community-FISABIO-, Valencia, Spain); González-Candelas F (CIBER in Epidemiology and Public Health, Spain; Joint Research Unit "Infection and Public Health" FISABIO-University of Valencia, Valencia, Spain; Institute for Integrative Systems Biology (I2SysBio), CSIC-University of Valencia, Valencia, Spain); Ferrer Albiach C (Fundación Hospital Provincial de Castelló); Hernández-Aguado I (University Miguel Hernández, Alicante, Spain); Oliver Ramírez N (DataPop Alliance); Sánchez-Payá J (Preventive Medicine Service, Alicante General and University Hospital, Alicante, Spain; Alicante Institute of Health and Biomedical Research (ISABIAL), Alicante, Spain; Vento Torres M (Instituto de Investigación Sanitaria La Fe); Zapater Latorre E (Fundación Hospital General Universitario de València); Navarro D (Microbiology Service, Clinic University Hospital, INCLIVA Health Research Institute, Valencia, Spain;Department of Microbiology, School of Medicine, University of Valencia, Valencia, Spain).
Assuntos
Vacinas contra COVID-19 , COVID-19 , Imunidade Adaptativa , COVID-19/prevenção & controle , Humanos , Casas de Saúde , SARS-CoV-2 , Espanha/epidemiologiaRESUMO
BACKGROUND: In 2021, four vaccines against Covid-19 (BNT162b2, mRNA-1273, ChAdOx1nCoV-19, and JNJ-78436735) were employed in the region of Valencia, Spain. We conducted a survey to identify real-world, self-reported frequency and severity of side effects during the week after vaccination. METHODS: Survey data was obtained from April 19, 2021, to October 6, 2021, at three different moments in time: day one, day three and day seven after vaccination. Answers were linked to individual-level, personal and clinical information. Respondents were stratified by the vaccine they received and reported effects were presented over time and stratified by severity. We compared our results per vaccine with the frequencies stated in each Summary of Product Characteristics (SmPC). We used binomial logistic models to identify associations between respondent characteristics and side effects. RESULTS: No symptoms were reported by 1,986 respondents (14.35 %), 6,254 informed exclusively mild symptoms (45.20 %), 3,444 up to moderate symptoms (24.89 %), and 2,153 people (15.56 %) notified also severe symptoms. Among the latter, the more frequent were extreme tiredness (7.0 %), and nausea or vomiting (7.1 %). The reported frequency of facial paralysis (0.4 %) was much higher than reflected in SmPCs. Female sex, younger age, previous positive Active Infection Diagnostic Test, chronicity, and vaccination with other than the BNT162b2 vaccine were associated to an increased risk of side effects (p < 0.001). CONCLUSIONS: Side effects after vaccination are common in the real-world. However, they are principally mild, and their frequency declines after a few days. Providing patients with dependable, beforehand information about side effects may improve outcomes and reinforce vaccination programs.
Assuntos
Vacinas contra COVID-19 , COVID-19 , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Vacina de mRNA-1273 contra 2019-nCoV , Ad26COVS1 , Vacina BNT162 , COVID-19/prevenção & controle , Vacinas contra COVID-19/efeitos adversos , ChAdOx1 nCoV-19 , Feminino , Humanos , Espanha/epidemiologia , Inquéritos e Questionários , Vacinação/efeitos adversosRESUMO
Glutamate is the major excitatory neurotransmitter of the central nervous system in vertebrates. Excitotoxicity, caused by over-stimulation of the glutamate receptors, is a major cause of neuron death in several brain diseases, including epilepsy. We describe here how behavioural seizures can be triggered in adult zebrafish by the administration of kainate and are very similar to those observed in rodent models. Kainate induced a dose-dependent sequence of behavioural changes culminating in clonus-like convulsions. Behavioural seizures were suppressed by DNQX (6,7-dinitroquinoxaline-2,3-dione) dose-dependently, whilst MK-801 (a non-competitive NMDA receptor antagonist) had a lesser effect. Kainate triggers seizures in adult zebrafish, and thus this species can be considered as a new model for studying seizures and subsequent excitotoxic brain injury.
Assuntos
Modelos Animais de Doenças , Ácido Caínico/farmacologia , Convulsões/induzido quimicamente , Peixe-Zebra/fisiologia , Animais , Maleato de Dizocilpina/uso terapêutico , Relação Dose-Resposta a Droga , Antagonistas de Aminoácidos Excitatórios/uso terapêutico , Ácido Glutâmico/metabolismo , Quinoxalinas/uso terapêutico , Ratos , Receptores de Glutamato/metabolismo , Convulsões/tratamento farmacológico , Convulsões/fisiopatologiaRESUMO
BACKGROUND: Evidence that periodontal disease is a possible risk factor for cognitive impairment may be explained by the inflammatory hypothesis. The aim of this study is to determine whether periodontitis is related to the amyloid ß (Aß) load in blood and the role of any such relationship in the association between Aß and cognitive impairment. METHODS: A case-control study was performed in elderly people diagnosed with cognitive impairment with or without dementia (cases group) and cognitively healthy elderly people (control group); data were collected on the medical and dental history of participants, and blood samples were drawn to determine Aß levels using enzyme-linked immunosorbent assay. RESULTS: The study included 166 patients and 122 control participants. Higher blood Aß1-42 levels (P = 0.01) and higher Aß42:40 ratio (P = 0.06) were observed in participants with severe attachment loss than in other participants. Periodontitis was a significant interaction variable, given that the association between Aß1-42 and Aß1-40 and cognitive impairment was only observed in patients with severe periodontitis. According to these data, periodontitis may be a modulating variable of the association between Aß and cognitive impairment. CONCLUSIONS: Plasma Aß1-42 levels are higher in individuals who have severe periodontal disease. The presence of periodontitis may modify the association between Aß and cognitive impairment.
Assuntos
Peptídeos beta-Amiloides/sangue , Disfunção Cognitiva , Periodontite/sangue , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco , EspanhaRESUMO
Traditional studies on viral neuroinvasiveness and pathogenesis have generally relied on murine models that require the sacrifice of infected animals to determine viral distributions and titers. The present paper reports the use of in vivo bioluminescence imaging to monitor the replication and tropism of KOS strain HSV-1 viruses expressing the firefly luciferase reporter protein in hematogenously infected mice. Following intraperitoneal injection, a comparison was made between real-time PCR determinations of HSV-1 DNA concentrations (requiring the sacrifice of the experimental animals) and in vivo bioluminescence emissions in living animals. For further comparison, in vitro light emission was also measured in the ovaries and adrenal glands of sacrificed mice. After infection, HSV-1 spread preferentially to the ovaries and adrenal glands (these organs showed the highest virus levels). Both the PCR and bioluminescence methods detected low viral loads in the nervous system, where the virus was restricted to the spinal cord. The concentrations of viral DNA measured correlated with the magnitude of bioluminescence in vivo, and with the photon flux determined by the in vitro luciferase enzyme assay. The results show that bioluminescence imaging can be used for non-invasive, real-time monitoring of HSV-1 hematogenous infection in living mice, but that coupling this methodology with conventional techniques aids in the characterization of the infection.
Assuntos
Bacteriemia/microbiologia , Herpes Simples/fisiopatologia , Herpesvirus Humano 1/isolamento & purificação , Luciferases de Vaga-Lume/metabolismo , Medições Luminescentes/métodos , Animais , DNA Viral/análise , Feminino , Genes Reporter , Herpes Simples/microbiologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Luciferases de Vaga-Lume/genética , Luminescência , Camundongos , Reação em Cadeia da PolimeraseRESUMO
Due to phylogenetic proximity to the human, zebrafish has been recognized as a reliable model to study Alzheimer's disease (AD) and other central nervous system disorders. Furthermore, metabotropic glutamate receptors have been previously reported to be impaired in brain from AD patients. Metabotropic glutamate 5 (mGlu5) receptors are G-protein coupled receptors proposed as potential targets for therapy of different neurodegenerative disorders. Thus, MPEP (2-methyl-6-(phenylethynyl)pyridine hydrochloride), a selective noncompetitive mGlu5 receptor antagonist, has been suggested for pharmacological treatment of AD. The aim of the present work was to quantify mGlu5 receptors in brain from zebrafish and to study the possible modulation of these receptors by MPEP treatment. To this end, radioligand binding assay and open field test were used. Results showed a slightly higher presence of mGlu5 receptors in brain from male than in that from female zebrafish. However, a significant increase of mGlu5 receptor in male without variation in female was observed after MPEP treatment. This gender specific response was also observed in locomotor behavior, being significantly decreased only in male zebrafish. These results confirm the presence of mGlu5 receptors in brain from zebrafish and their gender specific modulation by selective antagonist treatment and suggest a role of these receptors on locomotor activity, which is affected in many disorders. In addition, our data point to zebrafish as a useful model to study mGlu receptor function in both healthy and pathological conditions.
Assuntos
Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Antagonistas de Aminoácidos Excitatórios/farmacologia , Piridinas/farmacologia , Receptor de Glutamato Metabotrópico 5/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Feminino , Masculino , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Ensaio Radioligante , Caracteres Sexuais , Natação/fisiologia , Peixe-ZebraRESUMO
The aim of this study was to discuss the relevance of the Epstein-Barr virus (EBV) in the nasopharyngeal carcinoma (NPC), analyzing the variations of several molecules potentially involved in the pathogenesis of this cancer. EBV was detected in all the NPC samples by several techniques including PCR, in situ hybridization, and immunohistochemical methodologies. CD21 membrane receptor was absent after EBV infection, being a differential morphological feature of the tumoral cells. Latent membrane protein-1 (LMP1), an oncogenic viral product, was detected in a high percentage of samples, supporting a role for EBV in the pathogenesis of NPC. Bcl-2, an anti-apoptotic protein up-regulated by LMP1, was also overexpressed, coinciding with the majority of samples positive for LMP1. Finally, p53 presented abnormalities only in a low percentage of samples. These results reinforce the role of the EBV in the NPC discussing several potential mechanisms of pathogenesis in this neoplasia.
Assuntos
Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/patogenicidade , Neoplasias Nasofaríngeas/virologia , Infecções por Vírus Epstein-Barr/metabolismo , Infecções por Vírus Epstein-Barr/patologia , Humanos , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Virais/análise , Proteínas Virais/genéticaRESUMO
PURPOSE: To electrophysiologically characterize alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptors in chick retinal membrane fragments, incorporated into Xenopus oocytes by direct microinjection. METHODS: A 6-day retinal membrane suspension was injected into Xenopus oocytes by use of an electronic nanoliter injector. Fifteen to 40 hours after injection, the oocytes were assayed for kainate-elicited inward currents, under voltage-clamp conditions (membrane potential held at -70 mV). The structural incorporation of the retinal membrane fragments into the oocyte membrane was verified by specific immunofluorescent staining. RESULTS: Chick retinal membrane fragments were efficiently grafted onto Xenopus oocytes after microinjection, with 22.9% +/- 7.6% of the oocyte membrane being stained with anti-chick retina antibody. Part of the retinal material was seen as patches of relatively uniform size (292.1 +/- 72.3 microm(2)). Bath-applied kainate induced dose-dependent (EC(50): 64 +/- 7 microM), nondesensitizing inward currents (15-90 nA) in the chimeric Xenopus oocytes. Sham-injected oocytes did not respond to kainate. Kainate-driven currents were blocked by 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 1-(4-aminopropyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466), but not by gamma-D-glutamylaminomethyl sulfonic acid (GAMS) or aminophosphonoheptanoate (AP7), suggesting the involvement of AMPA receptors in the observed responses. Guanine nucleotides (GNs) also blocked kainate currents in a concentration-dependent manner. CONCLUSIONS: An alternative oocyte microinjection technique to analyze the electrophysiological properties of glutamate receptors in chick retinal membranes is described. The results show the functional activity of putative AMPA-preferring receptors from chick retina and confirm, in the chick retinal model, the antagonistic behavior of guanine nucleotides toward glutamate receptors and their potential role as neuroprotective agents under excitotoxic conditions.
Assuntos
Membrana Celular/fisiologia , Glutamina/análogos & derivados , Nucleotídeos de Guanina/farmacologia , Ácido Caínico/farmacologia , Oócitos/fisiologia , Receptores de AMPA/metabolismo , Retina/fisiologia , Retina/transplante , Animais , Membrana Celular/efeitos dos fármacos , Galinhas , Relação Dose-Resposta a Droga , Condutividade Elétrica , Eletrofisiologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Glutamina/farmacologia , Potenciais da Membrana/fisiologia , Microinjeções , Oócitos/efeitos dos fármacos , Técnicas de Patch-Clamp , Coelhos , Xenopus laevisRESUMO
Apolipoprotein E (ApoE) plays a relevant role in herpes simplex type 1 (HSV-1) infection of the CNS; after infection by the hematogenous route, the viral neuroinvasiveness directly depends on the APOE gene dose. To analyze the effect of ApoE isoforms on the HSV-1 infectivity to the brain, we have used a model of hematogenous infection of mice humanized for the ApoE3 or the ApoE4 alleles, and we have analyzed the presence of viral DNA in several organs by real time quantitative PCR. We have found that animals expressing human ApoE4 present very high levels of virus in the brain when compared to those expressing the ApoE3 allele; in contrast, there were no significant differences in the viral levels found in peripheral organs. Apolipoprotein E4 seems to facilitate the entry and/or spread of HSV-1 in the brain much more efficiently than E3, pointing to a novel potential mechanism underlying the susceptibility to neurodegenerative processes associated with the ApoE4 allele.