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1.
Blood Cells Mol Dis ; 107: 102856, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38762921

RESUMO

COVID-19 disease progression can be accompanied by a "cytokine storm" that leads to secondary sequelae such as acute respiratory distress syndrome. Several inflammatory cytokines have been associated with COVID-19 disease progression, but have high daily intra-individual variability. In contrast, we have shown that the inflammatory biomarker γ' fibrinogen (GPF) has a 6-fold lower coefficient of variability compared to other inflammatory markers such as hs-CRP. The aims of the study were to measure GPF in serial blood samples from COVID-19 patients at a tertiary care medical center in order to investigate its association with clinical measures of disease progression. COVID-19 patients were retrospectively enrolled between 3/16/2020 and 8/1/2020. GPF was measured using a commercial ELISA. We found that COVID-19 patients can develop extraordinarily high levels of GPF. Our results showed that ten out of the eighteen patients with COVID-19 had the highest levels of GPF ever recorded. The previous highest GPF level of 80.3 mg/dL was found in a study of 10,601 participants in the ARIC study. GPF levels were significantly associated with the need for ECMO and mortality. These findings have potential implications regarding prophylactic anticoagulation of COVID-19 patients.


Assuntos
Biomarcadores , COVID-19 , Fibrinogênio , SARS-CoV-2 , Humanos , COVID-19/sangue , COVID-19/complicações , Masculino , Feminino , Pessoa de Meia-Idade , Fibrinogênio/análise , Fibrinogênio/metabolismo , Estudos Retrospectivos , Idoso , Biomarcadores/sangue , Adulto , Progressão da Doença
2.
J Immunol ; 208(6): 1500-1508, 2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-35228262

RESUMO

Oral fluids offer a noninvasive sampling method for the detection of Abs. Quantification of IgA and IgG Abs in saliva allows studies of the mucosal and systemic immune response after natural infection or vaccination. We developed and validated an enzyme immunoassay (EIA) to detect and quantify salivary IgA and IgG Abs against the prefusion-stabilized form of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein expressed in suspension-adapted HEK-293 cells. Normalization against total Ab isotype was performed to account for specimen differences, such as collection time and sample volume. Saliva samples collected from 187 SARS-CoV-2 confirmed cases enrolled in 2 cohorts and 373 prepandemic saliva samples were tested. The sensitivity of both EIAs was high (IgA, 95.5%; IgG, 89.7%) without compromising specificity (IgA, 99%; IgG, 97%). No cross-reactivity with endemic coronaviruses was observed. The limit of detection for SARS-CoV-2 salivary IgA and IgG assays were 1.98 ng/ml and 0.30 ng/ml, respectively. Salivary IgA and IgG Abs were detected earlier in patients with mild COVID-19 symptoms than in severe cases. However, severe cases showed higher salivary Ab titers than those with a mild infection. Salivary IgA titers quickly decreased after 6 wk in mild cases but remained detectable until at least week 10 in severe cases. Salivary IgG titers remained high for all patients, regardless of disease severity. In conclusion, EIAs for both IgA and IgG had high specificity and sensitivity for the confirmation of current or recent SARS-CoV-2 infections and evaluation of the IgA and IgG immune response.


Assuntos
Anticorpos Antivirais/metabolismo , COVID-19/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , Imunoglobulina A/metabolismo , Imunoglobulina G/metabolismo , SARS-CoV-2/fisiologia , Saliva/metabolismo , Adolescente , Adulto , Idoso , Doenças Assintomáticas , Criança , Pré-Escolar , Progressão da Doença , Ensaio de Imunoadsorção Enzimática/normas , Feminino , Humanos , Lactente , Masculino , Programas de Rastreamento , Pessoa de Meia-Idade , Pandemias , Padrões de Referência , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Adulto Jovem
3.
Neurobiol Learn Mem ; 205: 107821, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37666411

RESUMO

Destabilization of previously consolidated memories places them in a labile state in which they are open to modification. However, strongly encoded fear memories tend to be destabilization-resistant and the conditions required to destabilize such memories remain poorly understood. Our lab has previously shown that exposure to salient novel contextual cues during memory reactivation can destabilize strongly encoded object location memories and that activity at muscarinic cholinergic receptors is critical for this effect. In the current study, we similarly targeted destabilization-resistant fear memories, hypothesizing that exposure to salient novelty at the time of reactivation would induce destabilization of strongly encoded fear memories in a muscarinic receptor-dependent manner. First, we show that contextual fear memories induced by 3 context-shock pairings readily destabilize upon memory reactivation, and that this destabilization is blocked by systemic (ip) administration of the muscarinic receptor antagonist scopolamine (0.3 mg/kg) in male rats. Following that, we confirm that this effect is dorsal hippocampus (dHPC)-dependent by targeting M1 receptors in the CA1 region with pirenzepine. Next, we show that more strongly encoded fear memories (induced with 5 context-shock pairings) resist destabilization. Consistent with our previous work, however, we report that salient novelty (a change in floor texture) presented during the reactivation session promotes destabilization of resistant contextual fear memories in a muscarinic receptor-dependent manner. Finally, the effect of salient novelty on memory destabilization was mimicked by stimulating muscarinic receptors with the selective M1 agonist CDD-0102A (ip, 0.3 mg/kg). These findings reveal further generalizability of our previous results implicating novel cues and M1 muscarinic signaling in promoting destabilization of resistant memories and suggest possible therapeutic options for disorders characterized by persistent, maladaptive fear memories such as PTSD and phobias.


Assuntos
Memória , Receptor Muscarínico M1 , Ratos , Masculino , Animais , Memória/fisiologia , Medo/fisiologia , Antagonistas Muscarínicos/farmacologia , Escopolamina/farmacologia
4.
J Immunol ; 206(11): 2596-2604, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33972374

RESUMO

The COVID-19 pandemic is a global health emergency, and the development of a successful vaccine will ultimately be required to prevent the continued spread and seasonal recurrence of this disease within the human population. However, very little is known about either the quality of the adaptive immune response or the viral Ag targets that will be necessary to prevent the spread of the infection. In this study, we generated recombinant Vaccinia virus expressing the full-length spike protein from SARS-CoV-2 (VacV-S) to evaluate the cellular and humoral immune response mounted against this viral Ag in mice. Both CD8+ and CD4+ T cells specific to the SARS-CoV-2 spike protein underwent robust expansion, contraction, and persisted for at least 40 d following a single immunization with VacV-S. Vaccination also caused the rapid emergence of spike-specific IgG-neutralizing Abs. Interestingly, both the cellular and humoral immune responses strongly targeted the S1 domain of spike following VacV-S immunization. Notably, immunization with VacV-expressing spike conjugated to the MHC class II invariant chain, a strategy previously reported by us and others to enhance the immunogenicity of antigenic peptides, did not promote stronger spike-specific T cell or Ab responses in vivo. Overall, these findings demonstrate that an immunization approach using VacV or attenuated versions of VacV expressing the native, full-length SARS-CoV-2 spike protein could be used for further vaccine development to prevent the spread of COVID-19.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Imunidade Celular , Imunidade Humoral , Imunoglobulina G/imunologia , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Vaccinia virus , Animais , Linhagem Celular , Imunização , Camundongos , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Vaccinia virus/genética , Vaccinia virus/imunologia
5.
J Infect Dis ; 225(6): 947-956, 2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-34865053

RESUMO

The unprecedented severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has called for substantial investigations into the capacity of the human immune system to protect against reinfection and keep pace with the evolution of SARS-CoV-2. We evaluated the magnitude and durability of the SARS-CoV-2-specific antibody responses against parental WA-1 SARS-CoV-2 receptor-binding domain (RBD) and a representative variant of concern (VoC) RBD using antibodies from 2 antibody compartments: long-lived plasma cell-derived plasma antibodies and antibodies encoded by SARS-CoV-2-specific memory B cells (MBCs). Thirty-five participants naturally infected with SARS-CoV-2 were evaluated; although only 25 of 35 participants had VoC RBD-reactive plasma antibodies, 34 of 35 (97%) participants had VoC RBD-reactive MBC-derived antibodies. Our finding that 97% of previously infected individuals have MBCs specific for variant RBDs provides reason for optimism regarding the capacity of vaccination, prior infection, and/or both, to elicit immunity with the capacity to limit disease severity and transmission of VoCs as they arise and circulate.


Assuntos
COVID-19 , Células B de Memória , SARS-CoV-2/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Humanos , Índice de Gravidade de Doença , Glicoproteína da Espícula de Coronavírus
6.
Int J Neuropsychopharmacol ; 25(1): 64-74, 2022 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-34791301

RESUMO

BACKGROUND: Autism spectrum disorders (ASD) are a set of neurodevelopmental disorders marked by a lack of social interaction, restrictive interests, and repetitive behaviors. There is a paucity of pharmacological treatments to reduce core ASD symptoms. Various lines of evidence indicate that reduced brain muscarinic cholinergic receptor activity may contribute to an ASD phenotype. METHODS: The present experiments examined whether the partial M1 muscarinic receptor agonist, 5-(3-ethyl-1,2,4-oxadiazol-5-yl)-1,4,5,6-tetrahydropyrimidine hydrochloride (CDD-0102A), alleviates behavioral flexibility deficits and/or stereotyped motor behaviors in the BTBR mouse model of autism. Behavioral flexibility was tested using a reversal learning test. Stereotyped motor behaviors were measured by eliciting digging behavior after removal of nesting material in a home cage and by measuring repetitive grooming. RESULTS: CDD-0102A (0.2 and 0.6 mg/kg but not 1.2 mg/kg) injected prior to reversal learning attenuated a deficit in BTBR mice but did not affect performance in B6 mice. Acute CDD-0102A treatment (1.2 and 3 mg/kg) reduced self-grooming in BTBR mice and reduced digging behavior in B6 and BTBR mice. The M1 muscarinic receptor antagonist VU0255035 (3 mg/kg) blocked the effect of CDD-0102A on grooming behavior. Chronic treatment with CDD-0102A (1.2 mg/kg) attenuated self-grooming and digging behavior in BTBR mice. Direct CDD-0102A infusions (1 µg) into the dorsal striatum reduced elevated digging behavior in BTBR mice. In contrast, CDD-0102A injections in the frontal cortex were not effective. CONCLUSIONS: The results suggest that treatment with a partial M1 muscarinic receptor agonist may reduce repetitive behaviors and restricted interests in autism in part by stimulating striatal M1 muscarinic receptors.


Assuntos
Transtorno do Espectro Autista/tratamento farmacológico , Receptor Muscarínico M1/agonistas , Reversão de Aprendizagem/efeitos dos fármacos , Comportamento Estereotipado/efeitos dos fármacos , Animais , Colinérgicos , Modelos Animais de Doenças , Feminino , Asseio Animal/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Oxidiazóis , Pirimidinas
7.
Immunology ; 164(2): 386-397, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34056709

RESUMO

There is growing interest in understanding antibody (Ab) function beyond neutralization. The non-structural protein 1 (NS1) of Zika virus (ZIKV) is an attractive candidate for an effective vaccine as Abs against NS1, unlike the envelope or premembrane, do not carry the risk of mediating antibody-dependent enhancement. Our aim was to evaluate whether ZIKV NS1 Abs elicited following natural infection in humans can mediate antibody-dependent cellular cytotoxicity (ADCC). We evaluated the isotype specificity of ZIKV-specific Abs in immune sera and supernatants from stimulated immune PBMC and found that Abs against ZIKV NS1 and virus-like particles were predominantly of the IgG1 isotype. Using a recently developed FluoroSpot assay, we found robust frequencies of NS1-specific Ab-secreting cells in PBMC of individuals who were naturally infected with ZIKV. We developed assays to measure both natural killer cell activation by flow cytometry and target cell lysis of ZIKV NS1-expressing cells using an image cytometry assay in the presence of ZIKV NS1 Abs. Our data indicate efficient opsonization of ZIKV NS1-expressing CEM-NKR cell lines using ZIKV-immune but not ZIKV-naïve sera, a prerequisite of ADCC. Furthermore, sera from immune donors were able to induce both NK cell degranulation and lysis of ZIKV NS1 CEM-NKR cells in vitro. Our data suggest that ADCC is a possible mechanism for ZIKV NS1 Abs to eliminate virally infected target cells.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Proteínas não Estruturais Virais/imunologia , Infecção por Zika virus/imunologia , Zika virus/imunologia , Células Cultivadas , Reações Cruzadas/imunologia , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Vacinas Virais/imunologia , Infecção por Zika virus/virologia
8.
Indoor Air ; 31(6): 1826-1832, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34189769

RESUMO

Evidence continues to grow supporting the aerosol transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To assess the potential role of heating, ventilation, and air conditioning (HVAC) systems in airborne viral transmission, this study sought to determine the viral presence, if any, on air handling units in a healthcare setting where coronavirus disease 2019 (COVID-19) patients were being treated. The presence of SARS-CoV-2 RNA was detected in approximately 25% of samples taken from ten different locations in multiple air handlers. While samples were not evaluated for viral infectivity, the presence of viral RNA in air handlers raises the possibility that viral particles can enter and travel within the air handling system of a hospital, from room return air through high-efficiency MERV-15 filters and into supply air ducts. Although no known transmission events were determined to be associated with these specimens, the findings suggest the potential for HVAC systems to facilitate transfer of virions to locations remote from areas where infected persons reside. These results are important within and outside of healthcare settings and may present necessary guidance for building operators of facilities that are not equipped with high-efficiency filtration. Furthermore, the identification of SARS-CoV-2 in HVAC components indicates the potential utility as an indoor environmental surveillance location.


Assuntos
Ar Condicionado , Poluição do Ar em Ambientes Fechados , RNA Viral/isolamento & purificação , SARS-CoV-2/isolamento & purificação , Microbiologia do Ar , COVID-19 , Atenção à Saúde , Calefação , Hospitais , Humanos , Ventilação
9.
Proc Natl Acad Sci U S A ; 115(45): 11513-11518, 2018 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-30341219

RESUMO

RNA virus genomes are efficient and compact carriers of biological information, encoding information required for replication both in their primary sequences and in higher-order RNA structures. However, the ubiquity of RNA elements with higher-order folds-in which helices pack together to form complex 3D structures-and the extent to which these elements affect viral fitness are largely unknown. Here we used single-molecule correlated chemical probing to define secondary and tertiary structures across the RNA genome of dengue virus serotype 2 (DENV2). Higher-order RNA structures are pervasive and involve more than one-third of nucleotides in the DENV2 genomic RNA. These 3D structures promote a compact overall architecture and contribute to viral fitness. Disrupting RNA regions with higher-order structures leads to stable, nonreverting mutants and could guide the development of vaccines based on attenuated RNA viruses. The existence of extensive regions of functional RNA elements with tertiary folds in viral RNAs, and likely many other messenger and noncoding RNAs, means that there are significant regions with pocket-containing surfaces that may serve as novel RNA-directed drug targets.


Assuntos
Capsídeo/ultraestrutura , Vírus da Dengue/ultraestrutura , Genoma Viral , RNA Viral/ultraestrutura , Pareamento de Bases , Capsídeo/química , Capsídeo/metabolismo , Vírus da Dengue/classificação , Vírus da Dengue/genética , Vírus da Dengue/metabolismo , Aptidão Genética , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Viral/genética , RNA Viral/metabolismo , Sorogrupo , Montagem de Vírus/genética
10.
J Infect Dis ; 221(12): 2018-2025, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-31545367

RESUMO

BACKGROUND: The once-in-a-lifetime recommendation for vaccination against yellow fever virus (YFV) has been controversial, leading to increased scrutiny of the durability of immunity after 17D vaccination. METHODS: This is a cross-sectional analysis of 17D vaccinees living in nonendemic Portland, Oregon. Neutralization assays were used to determine YFV immunity. The relationships between 17D immunity and vaccination history, demographics, and travel were evaluated using nominal logistic regression. RESULTS: Seventy-one of 92 (77.2%) subjects were YFV seropositive (90 percent plaque reduction neutralization test ≥1:10) at all timepoints, and 24 of 38 (63.8%) were YFV seropositive at ≥10 years after single-dose vaccination. No relationship was found between YFV immunity and time in endemic countries, other flavivirus immunity, or demographics. Subjects were most likely to become seronegative between 3 and 12 years postvaccination (logistic regression, odds ratio [OR] = 1.75; 95% confidence interval [CI], 1.12-2.73). A comparison of our results and 4 previous studies of YFV nonendemic vaccinees found that overall, 79% (95% CI, 70%-86%) of vaccinees are likely to be seropositive ≥10 years postvaccination. CONCLUSIONS: These results suggest that 1 in 5 17D vaccinees will lack neutralizing antibodies at ~10 years postvaccination, and a booster vaccination should be considered for nonendemic vaccinees before travel to regions where there is a high risk of YFV transmission.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Imunogenicidade da Vacina , Vacina contra Febre Amarela/imunologia , Febre Amarela/prevenção & controle , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Estudos Transversais , Feminino , Humanos , Imunização Secundária , Masculino , Pessoa de Meia-Idade , Testes de Neutralização , Oregon , Fatores de Tempo , Doença Relacionada a Viagens , Febre Amarela/imunologia , Febre Amarela/transmissão , Febre Amarela/virologia , Vacina contra Febre Amarela/administração & dosagem , Vírus da Febre Amarela/imunologia , Adulto Jovem
12.
PLoS Pathog ; 13(3): e1006219, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28278237

RESUMO

Zika virus (ZIKV), an emerging flavivirus, has recently spread explosively through the Western hemisphere. In addition to symptoms including fever, rash, arthralgia, and conjunctivitis, ZIKV infection of pregnant women can cause microcephaly and other developmental abnormalities in the fetus. We report herein the results of ZIKV infection of adult rhesus macaques. Following subcutaneous infection, animals developed transient plasma viremia and viruria from 1-7 days post infection (dpi) that was accompanied by the development of a rash, fever and conjunctivitis. Animals produced a robust adaptive immune response to ZIKV, although systemic cytokine response was minimal. At 7 dpi, virus was detected in peripheral nervous tissue, multiple lymphoid tissues, joints, and the uterus of the necropsied animals. Notably, viral RNA persisted in neuronal, lymphoid and joint/muscle tissues and the male and female reproductive tissues through 28 to 35 dpi. The tropism and persistence of ZIKV in the peripheral nerves and reproductive tract may provide a mechanism of subsequent neuropathogenesis and sexual transmission.


Assuntos
Infecção por Zika virus/patologia , Infecção por Zika virus/virologia , Animais , Separação Celular , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Hibridização In Situ , Macaca mulatta , Masculino , Testes de Neutralização , Reação em Cadeia da Polimerase , Viremia/virologia , Zika virus
13.
J Virol ; 90(10): 5090-5097, 2016 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-26962223

RESUMO

UNLABELLED: The four dengue virus (DENV) serotypes, DENV1 through 4, are endemic throughout tropical and subtropical regions of the world. While first infection confers long-term protective immunity against viruses of the infecting serotype, a second infection with virus of a different serotype carries a greater risk of severe dengue disease, including dengue hemorrhagic fever and dengue shock syndrome. Recent studies demonstrate that humans exposed to DENV infections develop neutralizing antibodies that bind to quaternary epitopes formed by the viral envelope (E) protein dimers or higher-order assemblies required for the formation of the icosahedral viral envelope. Here we show that the quaternary epitope target of the human DENV3-specific neutralizing monoclonal antibody (MAb) 5J7 can be partially transplanted into a DENV1 strain by changing the core residues of the epitope contained within a single monomeric E molecule. MAb 5J7 neutralized the recombinant DENV1/3 strain in cell culture and was protective in a mouse model of infection with the DENV1/3 strain. However, the 5J7 epitope was only partially recreated by transplantation of the core residues because MAb 5J7 bound and neutralized wild-type (WT) DENV3 better than the DENV1/3 recombinant. Our studies demonstrate that it is possible to transplant a large number of discontinuous residues between DENV serotypes and partially recreate a complex antibody epitope, while retaining virus viability. Further refinement of this approach may lead to new tools for measuring epitope-specific antibody responses and new vaccine platforms. IMPORTANCE: Dengue virus is the most important mosquito-borne pathogen of humans worldwide, with approximately one-half the world's population living in regions where dengue is endemic. Dengue immunity following infection is robust and thought to be conferred by antibodies raised against the infecting virus. However, the specific viral components that these antibodies recognize and how they neutralize the virus have been incompletely described. Here we map a region on dengue virus serotype 3 recognized by the human neutralizing antibody 5J7 and then test the functional significance of this region by transplanting it into a serotype 1 virus. Our studies demonstrate a region on dengue virus necessary for 5J7 binding and neutralization. Our work also demonstrates the technical feasibility of engineering dengue viruses to display targets of protective antibodies. This technology can be used to develop new dengue vaccines and diagnostic assays.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vírus da Dengue/imunologia , Epitopos , Animais , Anticorpos Neutralizantes/genética , Anticorpos Antivirais/química , Anticorpos Antivirais/genética , Reações Cruzadas , Dengue/virologia , Vírus da Dengue/classificação , Vírus da Dengue/genética , Modelos Animais de Doenças , Epitopos/genética , Epitopos/imunologia , Engenharia Genética , Humanos , Camundongos , Testes de Neutralização , Sorogrupo
14.
Neurobiol Learn Mem ; 145: 151-164, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29030298

RESUMO

Consolidated memories can become destabilized during reactivation, resulting in a transient state of instability, a process that has been hypothesized to underlie long-term memory updating. Consistent with this notion, relatively remote memories, which are resistant to standard destabilization procedures, are reliably destabilized when novel information (i.e., the opportunity for memory updating) is present during reactivation. We have also shown that cholinergic muscarinic receptor (mAChR) activation can similarly destabilize consolidated object memories. Synaptic protein degradation via the ubiquitin proteasome system (UPS) has previously been linked to destabilization of fear and object-location memories. Given the role of calcium in regulating proteasome activity, we hypothesized that activation of cholinergic receptors, specifically M1 mAChRs, stimulates the UPS via inositol triphosphate receptor (IP3R)-mediated release of intracellular calcium stores to facilitate object memory destabilization. We present converging evidence for this hypothesis, which we tested using a modified spontaneous object recognition task for rats and microinfusions into perirhinal cortex (PRh), a brain region strongly implicated in object memory. We extend our previous findings by demonstrating that M1 mAChRs are necessary for novelty-induced object memory destabilization. We also show that proteasome inhibition or IP3R antagonism in PRh prevents object memory destabilization induced by novelty or M1 mAChR stimulation. These results establish an intracellular pathway linking M1 receptors, IP3Rs, and UPS activity to object memory destabilization and suggest a previously unacknowledged role for cholinergic signaling in long-term memory modification and storage.


Assuntos
Memória de Longo Prazo/fisiologia , Córtex Perirrinal/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptor Muscarínico M1/metabolismo , Animais , Anisomicina/administração & dosagem , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Inibidores da Síntese de Proteínas/administração & dosagem , Ratos Long-Evans , Reconhecimento Psicológico/fisiologia , Ubiquitina/metabolismo
15.
Proc Natl Acad Sci U S A ; 111(5): 1939-44, 2014 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-24385585

RESUMO

The four dengue virus (DENV) serotypes, DENV-1, -2, -3, and -4, are endemic throughout tropical and subtropical regions of the world, with an estimated 390 million acute infections annually. Infection confers long-term protective immunity against the infecting serotype, but secondary infection with a different serotype carries a greater risk of potentially fatal severe dengue disease, including dengue hemorrhagic fever and dengue shock syndrome. The single most effective measure to control this threat to global health is a tetravalent DENV vaccine. To date, attempts to develop a protective vaccine have progressed slowly, partly because the targets of type-specific human neutralizing antibodies (NAbs), which are critical for long-term protection, remain poorly defined, impeding our understanding of natural immunity and hindering effective vaccine development. Here, we show that the envelope glycoprotein domain I/II hinge of DENV-3 and DENV-4 is the primary target of the long-term type-specific NAb response in humans. Transplantation of a DENV-4 hinge into a recombinant DENV-3 virus showed that the hinge determines the serotype-specific neutralizing potency of primary human and nonhuman primate DENV immune sera and that the hinge region both induces NAbs and is targeted by protective NAbs in rhesus macaques. These results suggest that the success of live dengue vaccines may depend on their ability to stimulate NAbs that target the envelope glycoprotein domain I/II hinge region. More broadly, this study shows that complex conformational antibody epitopes can be transplanted between live viruses, opening up similar possibilities for improving the breadth and specificity of vaccines for influenza, HIV, hepatitis C virus, and other clinically important viral pathogens.


Assuntos
Vírus da Dengue/classificação , Vírus da Dengue/imunologia , Dengue/imunologia , Dengue/virologia , Imunidade/imunologia , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais/imunologia , Células HEK293 , Humanos , Células K562 , Macaca mulatta/imunologia , Macaca mulatta/virologia , Dados de Sequência Molecular , Testes de Neutralização , Multimerização Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes , Sorotipagem , Especificidade da Espécie , Relação Estrutura-Atividade , Fatores de Tempo , Proteínas do Envelope Viral/metabolismo , Viremia/imunologia
18.
Proc Natl Acad Sci U S A ; 109(19): 7439-44, 2012 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-22499787

RESUMO

Dengue is a mosquito-borne flavivirus that is spreading at an unprecedented rate and has developed into a major health and economic burden in over 50 countries. Even though infected individuals develop potent and long-lasting serotype-specific neutralizing antibodies (Abs), the epitopes engaged by human neutralizing Abs have not been identified. Here, we demonstrate that the dengue virus (DENV)-specific serum Ab response in humans consists of a large fraction of cross-reactive, poorly neutralizing Abs and a small fraction of serotype-specific, potently inhibitory Abs. Although many mouse-generated, strongly neutralizing monoclonal antibodies (mAbs) recognize epitopes that are present on recombinant DENV envelope (E) proteins, unexpectedly, the majority of neutralizing Abs in human immune sera bound to intact virions but not to the ectodomain of purified soluble E proteins. These conclusions with polyclonal Abs were confirmed with newly generated human mAbs derived from DENV-immune individuals. Two of three strongly neutralizing human mAbs bound to E protein epitopes that were preserved on the virion but not on recombinant E (rE) protein. We propose that humans produce Abs that neutralize DENV infection by binding a complex, quaternary structure epitope that is expressed only when E proteins are assembled on a virus particle. Mapping studies indicate that this epitope has a footprint that spans adjacent E protein dimers and includes residues at the hinge between domains I and II of E protein. These results have significant implications for the DENV Ab and vaccine field.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vírus da Dengue/imunologia , Dengue/imunologia , Epitopos/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Especificidade de Anticorpos/imunologia , Chlorocebus aethiops , Dengue/virologia , Vírus da Dengue/genética , Vírus da Dengue/metabolismo , Ensaio de Imunoadsorção Enzimática , Epitopos/metabolismo , Humanos , Soros Imunes/imunologia , Macaca mulatta , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Testes de Neutralização , Ligação Proteica/imunologia , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Células Vero , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo , Vírion/imunologia
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