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1.
Front Cardiovasc Med ; 11: 1387063, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38938651

RESUMO

This case report details the identification of a novel likely pathogenic splicing variant in the TTN gene, associated with dilated cardiomyopathy (DCM), in a 42-year-old male patient presenting with early-onset heart failure and reduced ejection fraction. DCM is a nonischemic heart condition characterized by left biventricular dilation and systolic dysfunction, with approximately one-third of cases being familial and often linked to genetic mutations. The TTN gene, encoding the largest human protein essential for muscle contraction and sarcomere structure, is implicated in about 25% of DCM cases through mutations, especially truncating variants. Our investigation revealed a previously unreported G > C mutation at the splice acceptor site in intron 356 of TTN, confirmed by Sanger sequencing and not found in population databases, suggesting a novel contribution to the understanding of DCM etiology. The case emphasizes the critical role of the TTN gene in cardiac function and the genetic complexity underlying DCM. A comprehensive literature review highlighted the prevalence and significance of splice variants in the TTN gene, particularly those affecting the titin A-band, which is known for its role in muscle contraction and stability. This variant's identification underscores the importance of genetic screening in patients with DCM, offering insights into the disease's familial transmission and potential therapeutic targets. Our findings contribute to the expanding knowledge of genetic factors in DCM, demonstrating the necessity of integrating genetic diagnostics in cardiovascular medicine. This case supports the growing evidence linking splicing mutations in specific regions of the TTN gene to DCM development and underscores the importance of genetic counseling and testing in managing heart disease.

2.
Neuron ; 2024 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-38781972

RESUMO

Brain arterioles are active, multicellular complexes whose diameters oscillate at ∼ 0.1 Hz. We assess the physiological impact and spatiotemporal dynamics of vaso-oscillations in the awake mouse. First, vaso-oscillations in penetrating arterioles, which source blood from pial arterioles to the capillary bed, profoundly impact perfusion throughout neocortex. The modulation in flux during resting-state activity exceeds that of stimulus-induced activity. Second, the change in perfusion through arterioles relative to the change in their diameter is weak. This implies that the capillary bed dominates the hydrodynamic resistance of brain vasculature. Lastly, the phase of vaso-oscillations evolves slowly along arterioles, with a wavelength that exceeds the span of the cortical mantle and sufficient variability to establish functional cortical areas as parcels of uniform phase. The phase-gradient supports traveling waves in either direction along both pial and penetrating arterioles. This implies that waves along penetrating arterioles can mix, but not directionally transport, interstitial fluids.

3.
Proc Natl Acad Sci U S A ; 117(6): 2767-2769, 2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-31988118

RESUMO

While single-cell sequencing technologies have revealed tissue heterogeneity, resolving mixed cellular libraries into cellular clones is essential for many pooled screens and clonal lineage tracing. Fluorescent proteins are limited in number, while DNA barcodes can only be read after cell lysis. To overcome these limitations, we used influenza virus hemagglutinins to engineer a genetically encoded cell-surface protein barcoding system. Using antibodies paired to hemagglutinins carrying combinations of escape mutations, we developed an exponential protein barcoding system which can label 128 clones using seven antibodies. This study provides a proof of principle for a strategy to create protein-level cell barcodes that can be used in vivo in mice to track clonal populations.


Assuntos
Anticorpos Monoclonais/análise , Rastreamento de Células/métodos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/análise , Animais , Rastreamento de Células/instrumentação , Feminino , Citometria de Fluxo/métodos , Células HEK293 , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Melanoma/química , Melanoma/genética , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Orthomyxoviridae/química , Orthomyxoviridae/genética , Orthomyxoviridae/metabolismo
4.
Matern Child Nutr ; 15(2): e12717, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30315611

RESUMO

Universal salt iodization (USI) was adopted in Madagascar in 1995 within the framework of a worldwide policy to eliminate iodine deficiency disorders. Despite early USI adoption, there are no representative data on the iodine status of the Malagasy population. The aims of this study were to determine the iodine status of the Malagasy population and to assess the use of adequately iodized salt among households. We randomly sampled women of reproductive age (WRA) using a national, two-stage, stratified cross-sectional survey in 2014. Casual urine from WRA and salt samples from the household containing WRA were collected to measure urinary iodine concentration (UIC) and to assess household salt iodine content. Data from 1,721 WRA in 1,128 households were collected and analysed. The national median UIC was 46 µg L-1 (interquartile range [IQR]: 13-98 µg L-1 ), indicating a moderate iodine deficiency. The median UIC was 53 µg L-1 (IQR: 9-89 µg L-1 ) in pregnant women and 46 µg L-1 (IQR: 13-98 µg L-1 ) in non-pregnant women. The national median iodine concentration of household salt was 10 mg kg-1 (IQR: 6.3-15.8 mg kg-1 ) and 26.2% (95% CI [22.1, 31.0]) of households containing WRA used adequately iodized salt (≥15 mg kg-1 ). Women living in households with adequately iodized salt had higher median UIC (72 vs. 50 µg L-1 ). Iodine status was significantly lower among women from low socio-economic households. Madagascar's USI program needs to be revitalized. Implementing strategies to provide adequately iodized salt and enhancing iodized salt legislation to prevent severe complications resulting from iodine deficiency in the Malagasy population are essential.


Assuntos
Iodo/deficiência , Iodo/urina , Desnutrição/epidemiologia , Inquéritos Nutricionais/estatística & dados numéricos , Estado Nutricional , Adulto , Análise por Conglomerados , Estudos Transversais , Feminino , Humanos , Madagáscar/epidemiologia , Inquéritos Nutricionais/métodos , Adulto Jovem
5.
J Am Soc Nephrol ; 29(12): 2879-2889, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30385651

RESUMO

BACKGROUND: Use of enzyme replacement therapy (ERT) to treat Fabry disease, caused by deficient lysosomal α-galactosidase A activity, can lead to formation of neutralizing antidrug antibodies (ADAs). These antibodies are associated with increased accumulation of plasma globotriaosylceramide (Gb3) and disease progression. Because agalsidase ERT can saturate ADA-binding sites during infusions (achieving agalsidase/antibody equilibrium), we investigated in this open cohort study whether saturated patients (who have excess agalsidase after infusions) experience better clinical outcomes compared with not saturated patients (who have excess ADAs after infusions). METHODS: We isolated ADAs from sera of 26 men with Fabry disease receiving ERT (for a median of 94 months) and determined the amount of agalsidase necessary for antibody saturation. Clinical and biochemical outcomes included measurements of eGFR, interventricular septum thickness, and lyso-Gb3. RESULTS: ADA titers decreased significantly in all patients during infusion. Agalsidase-α and agalsidase-ß had similar ADA-binding capacity and comparable ADA saturation frequency. Fourteen patients with saturated ADAs presented with mild (but significant) loss of eGFR, stable septum thickness, and significantly decreased lyso-Gb3 levels. The 12 not saturated patients had a more pronounced and significant loss of eGFR, increased septum thickness, and a smaller, nonsignificant reduction in lyso-Gb3, over time. In three patients, dose escalation resulted in partially elevated ADA titers, but importantly, also in reduced lyso-Gb3 levels. CONCLUSIONS: A not saturated ADA status during infusion is associated with progressive loss of eGFR and ongoing cardiac hypertrophy. Dose escalation can result in saturation of ADAs and decreasing lyso-Gb3 levels, but may lead to increased ADA titers.


Assuntos
Anticorpos Neutralizantes/sangue , Terapia de Reposição de Enzimas/efeitos adversos , Doença de Fabry/tratamento farmacológico , Doença de Fabry/imunologia , Isoenzimas/administração & dosagem , Isoenzimas/efeitos adversos , alfa-Galactosidase/administração & dosagem , alfa-Galactosidase/efeitos adversos , Adulto , Idoso , Reações Antígeno-Anticorpo , Estudos de Coortes , Relação Dose-Resposta a Droga , Humanos , Isoenzimas/imunologia , Masculino , Pessoa de Meia-Idade , Modelos Imunológicos , Adulto Jovem , alfa-Galactosidase/imunologia
6.
J Vis Exp ; (132)2018 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-29553549

RESUMO

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-FcγR. Using this method, the contribution of specific Fc-FcγR interactions conferred by immunoglobulins can be determined using an in vitro assay.


Assuntos
Hemaglutininas/metabolismo , Influenza Humana/virologia , Receptores Fc/metabolismo , Receptores de IgG/metabolismo , Anticorpos Monoclonais/imunologia , Humanos
7.
Nat Commun ; 8(1): 846, 2017 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-29018261

RESUMO

The aim of candidate universal influenza vaccines is to provide broad protection against influenza A and B viruses. Studies have demonstrated that broadly reactive antibodies require Fc-Fc gamma receptor interactions for optimal protection; however, the innate effector cells responsible for mediating this protection remain largely unknown. Here, we examine the roles of alveolar macrophages, natural killer cells, and neutrophils in antibody-mediated protection. We demonstrate that alveolar macrophages play a dominant role in conferring protection provided by both broadly neutralizing and non-neutralizing antibodies in mice. Our data also reveal the potential mechanisms by which alveolar macrophages mediate protection in vivo, namely antibody-induced inflammation and antibody-dependent cellular phagocytosis. This study highlights the importance of innate effector cells in establishing a broad-spectrum antiviral state, as well as providing a better understanding of how multiple arms of the immune system cooperate to achieve an optimal antiviral response following influenza virus infection or immunization.Broadly reactive antibodies that recognize influenza A virus HA can be protective, but the mechanism is not completely understood. Here, He et al. show that the inflammatory response and phagocytosis mediated by the interaction between protective antibodies and macrophages are essential for protection.


Assuntos
Anticorpos Neutralizantes/fisiologia , Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Ativação de Macrófagos , Macrófagos Alveolares/fisiologia , Células A549 , Animais , Cães , Feminino , Células HEK293 , Hemaglutininas/imunologia , Humanos , Células Matadoras Naturais/fisiologia , Células Madin Darby de Rim Canino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/fisiologia , Infecções por Orthomyxoviridae/imunologia , Fagocitose , Receptores de IgG/metabolismo
8.
J Vis Exp ; (126)2017 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-28872136

RESUMO

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Influenza Humana/virologia , Humanos
9.
Proc Natl Acad Sci U S A ; 113(40): E5944-E5951, 2016 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-27647907

RESUMO

Influenza virus strain-specific monoclonal antibodies (mAbs) provide protection independent of Fc gamma receptor (FcγR) engagement. In contrast, optimal in vivo protection achieved by broadly reactive mAbs requires Fc-FcγR engagement. Most strain-specific mAbs target the head domain of the viral hemagglutinin (HA), whereas broadly reactive mAbs typically recognize epitopes within the HA stalk. This observation has led to questions regarding the mechanism regulating the activation of Fc-dependent effector functions by broadly reactive antibodies. To dissect the molecular mechanism responsible for this dichotomy, we inserted the FLAG epitope into discrete locations on HAs. By characterizing the interactions of several FLAG-tagged HAs with a FLAG-specific antibody, we show that in addition to Fc-FcγR engagement mediated by the FLAG-specific antibody, a second intermolecular bridge between the receptor-binding region of the HA and sialic acid on effector cells is required for optimal activation. Inhibition of this second molecular bridge, through the use of an F(ab')2 or the mutation of the sialic acid-binding site, renders the Fc-FcγR interaction unable to optimally activate effector cells. Our findings indicate that broadly reactive mAbs require two molecular contacts to possibly stabilize the immunologic synapse and potently induce antibody-dependent cell-mediated antiviral responses: (i) the interaction between the Fc of a mAb bound to HA with the FcγR of the effector cell and (ii) the interaction between the HA and its sialic acid receptor on the effector cell. This concept might be broadly applicable for protective antibody responses to viral pathogens that have suitable receptors on effector cells.


Assuntos
Anticorpos Antivirais/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Orthomyxoviridae/imunologia , Receptores Fc/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais/metabolismo , Anticorpos Antivirais/química , Citotoxicidade Celular Dependente de Anticorpos , Epitopos/química , Células HEK293 , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Humanos , Imunidade Celular , Modelos Biológicos , Modelos Moleculares , Ácido N-Acetilneuramínico/metabolismo , Receptores Fc/química
10.
Cell Host Microbe ; 19(6): 800-13, 2016 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-27281570

RESUMO

Pathogenic H7N9 avian influenza viruses continue to represent a public health concern, and several candidate vaccines are currently being developed. It is vital to assess if protective antibodies are induced following vaccination and to characterize the diversity of epitopes targeted. Here we characterized the binding and functional properties of twelve H7-reactive human antibodies induced by a candidate A/Anhui/1/2013 (H7N9) vaccine. Both neutralizing and non-neutralizing antibodies protected mice in vivo during passive transfer challenge experiments. Mapping the H7 hemagglutinin antigenic sites by generating escape mutant variants against the neutralizing antibodies identified unique epitopes on the head and stalk domains. Further, the broadly cross-reactive non-neutralizing antibodies generated in this study were protective through Fc-mediated effector cell recruitment. These findings reveal important properties of vaccine-induced antibodies and provide a better understanding of the human monoclonal antibody response to influenza in the context of vaccines.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Subtipo H7N9 do Vírus da Influenza A/imunologia , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/farmacologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Modelos Animais de Doenças , Cães , Feminino , Células HEK293 , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/virologia
11.
PLoS Pathog ; 12(4): e1005578, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27081859

RESUMO

In the early spring of 2013, Chinese health authorities reported several cases of H7N9 influenza virus infections in humans. Since then the virus has established itself at the human-animal interface in Eastern China and continues to cause several hundred infections annually. In order to characterize the antibody response to the H7N9 virus we generated several mouse monoclonal antibodies against the hemagglutinin of the A/Shanghai/1/13 (H7N9) virus. Of particular note are two monoclonal antibodies, 1B2 and 1H5, that show broad reactivity to divergent H7 hemagglutinins. Monoclonal antibody 1B2 binds to viruses of the Eurasian and North American H7 lineages and monoclonal antibody 1H5 reacts broadly to virus isolates of the Eurasian lineage. Interestingly, 1B2 shows broad hemagglutination inhibiting and neutralizing activity, while 1H5 fails to inhibit hemagglutination and demonstrates no neutralizing activity in vitro. However, both monoclonal antibodies were highly protective in an in vivo passive transfer challenge model in mice, even at low doses. Experiments using mutant antibodies that lack the ability for Fc/Fc-receptor and Fc/complement interactions suggest that the protection provided by mAb 1H5 is, at least in part, mediated by the Fc-fragment of the mAb. These findings highlight that a protective response to a pathogen may not only be due to neutralizing antibodies, but can also be the result of highly efficacious non-neutralizing antibodies not readily detected by classical in vitro neutralization or hemagglutination inhibition assays. This is of interest because H7 influenza virus vaccines induce only low hemagglutination inhibiting antibody titers while eliciting robust antibody titers as measured by ELISA. Our data suggest that these binding but non-neutralizing antibodies contribute to protection in vivo.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antígenos Virais/imunologia , Western Blotting , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Citometria de Fluxo , Humanos , Subtipo H7N9 do Vírus da Influenza A , Camundongos , Camundongos Endogâmicos BALB C
12.
Immunity ; 44(1): 46-58, 2016 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-26789921

RESUMO

Viruses are obligate parasites and thus require the machinery of the host cell to replicate. Inhibition of host factors co-opted during active infection is a strategy hosts use to suppress viral replication and a potential pan-antiviral therapy. To define the cellular proteins and processes required for a virus during infection is thus crucial to understanding the mechanisms of virally induced disease. In this report, we generated fully infectious tagged influenza viruses and used infection-based proteomics to identify pivotal arms of cellular signaling required for influenza virus growth and infectivity. Using mathematical modeling and genetic and pharmacologic approaches, we revealed that modulation of Sec61-mediated cotranslational translocation selectively impaired glycoprotein proteostasis of influenza as well as HIV and dengue viruses and led to inhibition of viral growth and infectivity. Thus, by studying virus-human protein-protein interactions in the context of active replication, we have identified targetable host factors for broad-spectrum antiviral therapies.


Assuntos
Interações Hospedeiro-Parasita/fisiologia , Vírus da Influenza A/fisiologia , Vírus da Influenza A/patogenicidade , Modelos Teóricos , Replicação Viral/fisiologia , Vírus da Dengue/patogenicidade , Vírus da Dengue/fisiologia , HIV/patogenicidade , HIV/fisiologia , Humanos , Imunoprecipitação , Espectrometria de Massas , Dobramento de Proteína , Proteômica
13.
Antimicrob Agents Chemother ; 59(7): 4162-72, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25941218

RESUMO

The emergence of influenza virus strains resistant to approved neuraminidase inhibitors and the time constrains after infection when these drugs can be effective constitute major drawbacks for this class of drugs. This highlights a critical need to discover new therapeutic agents that can be used for the treatment of influenza virus-infected patients. The use of broadly neutralizing anti-influenza monoclonal antibodies (MAbs) has been sought as an alternative immunotherapy against influenza infection. Here, we tested in mice previously characterized broadly neutralizing anti-hemagglutinin (HA) stalk MAbs prophylactically and therapeutically using different routes of administration. The efficacy of treatment against an influenza H1N1 pandemic virus challenge was compared between two systemic routes of administration, intraperitoneal (i.p.) and intravenous (i.v.), and two local routes, intranasal (i.n.) and aerosol (a.e.). The dose of MAb required for prophylactic protection was reduced by 10-fold in animals treated locally (i.n. or a.e.) compared with those treated systemically (i.p. or i.v.). Improved therapeutic protection was observed in animals treated i.n. on day 5 postinfection (60% survival) compared with those treated via the i.p. route (20% survival). An increase in therapeutic efficacy against other influenza virus subtypes (H5N1) was also observed when a local route of administration was used. Our findings demonstrate that local administration significantly decreases the amount of broadly neutralizing monoclonal antibody required for protection against influenza, which highlights the potential use of MAbs as a therapeutic agent for influenza-associated disease.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/uso terapêutico , Antivirais/administração & dosagem , Antivirais/uso terapêutico , Influenza Humana/tratamento farmacológico , Administração Intranasal , Administração Intravenosa , Aerossóis , Animais , Anticorpos Monoclonais/farmacocinética , Antivirais/farmacocinética , Disponibilidade Biológica , Feminino , Hemaglutininas/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Virus da Influenza A Subtipo H5N1/efeitos dos fármacos , Influenza Humana/patologia , Influenza Humana/virologia , Injeções Intraperitoneais , Pulmão/patologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Distribuição Tecidual
14.
J Clin Invest ; 125(3): 1255-68, 2015 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-25689254

RESUMO

The emergence and seasonal persistence of pathogenic H7N9 influenza viruses in China have raised concerns about the pandemic potential of this strain, which, if realized, would have a substantial effect on global health and economies. H7N9 viruses are able to bind to human sialic acid receptors and are also able to develop resistance to neuraminidase inhibitors without a loss in fitness. It is not clear whether prior exposure to circulating human influenza viruses or influenza vaccination confers immunity to H7N9 strains. Here, we demonstrate that 3 of 83 H3 HA-reactive monoclonal antibodies generated by individuals that had previously undergone influenza A virus vaccination were able to neutralize H7N9 viruses and protect mice against homologous challenge. The H7N9-neutralizing antibodies bound to the HA stalk domain but exhibited a difference in their breadth of reactivity to different H7 influenza subtypes. Mapping viral escape mutations suggested that these antibodies bind at least two different epitopes on the stalk region. Together, these results indicate that these broadly neutralizing antibodies may contribute to the development of therapies against H7N9 strains and may also be effective against pathogenic H7 strains that emerge in the future.


Assuntos
Anticorpos Neutralizantes/fisiologia , Anticorpos Antivirais/fisiologia , Subtipo H7N9 do Vírus da Influenza A/imunologia , Influenza Humana/prevenção & controle , Vacinação , Animais , Anticorpos Monoclonais/fisiologia , Reações Cruzadas , Cães , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Vírus da Influenza A Subtipo H3N2/imunologia , Subtipo H7N9 do Vírus da Influenza A/genética , Vacinas contra Influenza , Influenza Humana/imunologia , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Camundongos Endogâmicos BALB C , Testes de Neutralização , Mutação Puntual
15.
J Virol ; 88(23): 13580-92, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25210195

RESUMO

UNLABELLED: Due to continuous changes to its antigenic regions, influenza viruses can evade immune detection and cause a significant amount of morbidity and mortality around the world. Influenza vaccinations can protect against disease but must be annually reformulated to match the current circulating strains. In the development of a broad-spectrum influenza vaccine, the elucidation of conserved epitopes is paramount. To this end, we designed an immunization strategy in mice to boost the humoral response against conserved regions of the hemagglutinin (HA) glycoprotein. Of note, generation and identification of broadly neutralizing antibodies that target group 2 HAs are rare and thus far have yielded only a few monoclonal antibodies (MAbs). Here, we demonstrate that mouse MAb 9H10 has broad and potent in vitro neutralizing activity against H3 and H10 group 2 influenza A subtypes. In the mouse model, MAb 9H10 protects mice against two divergent mouse-adapted H3N2 strains, in both pre- and postexposure administration regimens. In vitro and cell-free assays suggest that MAb 9H10 inhibits viral replication by blocking HA-dependent fusion of the viral and endosomal membranes early in the replication cycle and by disrupting viral particle egress in the late stage of infection. Interestingly, electron microscopy reconstructions of MAb 9H10 bound to the HA reveal that it binds a similar binding footprint to MAbs CR8020 and CR8043. IMPORTANCE: The influenza hemagglutinin is the major antigenic target of the humoral immune response. However, due to continuous antigenic changes that occur on the surface of this glycoprotein, influenza viruses can escape the immune system and cause significant disease to the host. Toward the development of broad-spectrum therapeutics and vaccines against influenza virus, elucidation of conserved regions of influenza viruses is crucial. Thus, defining these types of epitopes through the generation and characterization of broadly neutralizing monoclonal antibodies (MAbs) can greatly assist others in highlighting conserved regions of hemagglutinin. Here, we demonstrate that MAb 9H10 that targets the hemagglutinin stalk has broadly neutralizing activity against group 2 influenza A viruses in vitro and in vivo.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/uso terapêutico , Linhagem Celular , Modelos Animais de Doenças , Epitopos/imunologia , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/uso terapêutico , Humanos , Imunização Passiva , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/terapia , Resultado do Tratamento , Internalização do Vírus/efeitos dos fármacos , Liberação de Vírus/efeitos dos fármacos
16.
J Grad Med Educ ; 5(1): 138-41, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24404241

RESUMO

BACKGROUND: Excessive, sometimes unnecessary consultation challenges the ophthalmology resident's ability to obtain adequate rest and personal time while taking at-home calls. Basic training of nonophthalmologists in assessing and treating common ophthalmic emergencies offers a potential solution to this problem by providing primary caregivers the knowledge base, tools, and confidence to manage them independently. OBJECTIVE: We measured ophthalmic consultation requests at a large teaching institution and the impact of an educational intervention on the number and type of requests. METHODS: During a 31-day period in 2009 all requests for urgent ophthalmic consultation were logged and characterized by reason, urgency, and source of the call. A 3-part educational intervention was targeted to residents and attending physicians in the departments of emergency medicine (half-day lecture and practice), anesthesiology (grand rounds lecture), and otolaryngology (guidelines for traumatic orbital fractures). Six months later we conducted a 26-day call log to evaluate the impact. RESULTS: Pre intervention, we received 63 total and 56 after-hours calls for urgent ophthalmic consultation as compared to 39 total and 35 after-hours calls post intervention. Ophthalmology residents reported seeing more urgent and fewer nonessential consultations in the postintervention period, and there was greater agreement between calling physicians' initial diagnosis and consulting physicians' final diagnosis. No adverse patient outcomes occurred as a result of the change in practices. CONCLUSION: Basic education of nonophthalmologists in ophthalmic diagnostic and treatment concepts, through relatively brief educational interventions, was associated with a decrease in total consultation requests and more appropriate consultations in this pilot study at a single institution.

17.
J Infect Dis ; 205 Suppl 1: S82-90, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22315391

RESUMO

BACKGROUND: Intermittent preventive treatment in infants (IPTi) is a new malaria control strategy coupled with the delivery of routine immunizations recommended by the World Health Organization since 2009 for countries with moderate to high endemicity. To evaluate its safety profile and identify potential new adverse events (AEs) following simultaneous administration of sulfadoxine-pyrimethamine (SP-IPTi) with immunizations, we measured AE incidence and evaluated spontaneous AE reporting. METHODS: A cohort event monitoring study was conducted on 24 000 infants in 2 countries after administration of SP-IPTi during routine immunizations. Additional pharmacovigilance training and supervision were conducted to stimulate AE passive reporting in 6 African countries. RESULTS: No serious AEs were found by active follow-up, representing 95% probability that the rate does not exceed 1 per 8000. No serious AEs were found by retrospective review of hospital registers. The rate of moderate AEs probably linked to immunization and/or SP-IPTi was 1.8 per 1000 doses (95% confidence interval, 1.50-2.00). Spontaneous reporting of AEs remained <1% of cases collected by active follow-up. CONCLUSIONS: Simultaneous administration of SP-IPTi and immunizations is a safe strategy for implementation with a low risk of serious AEs to infants. Strategies toward strengthening spontaneous reporting in Africa should include not only the provider but also beneficiaries or their caregivers.


Assuntos
Antimaláricos/efeitos adversos , Imunização , Vacinas Antimaláricas/efeitos adversos , Malária/prevenção & controle , Farmacovigilância , Pirimetamina/efeitos adversos , Sulfadoxina/efeitos adversos , Sistemas de Notificação de Reações Adversas a Medicamentos , África , Combinação de Medicamentos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/epidemiologia , Feminino , Humanos , Lactente , Masculino
18.
Antimicrob Agents Chemother ; 53(11): 4588-97, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19704124

RESUMO

The aim of this study was to provide the first comprehensive spatiotemporal picture of Plasmodium falciparum resistance in various geographic areas in Madagascar. Additional data about the antimalarial resistance in the neighboring islands of the Comoros archipelago were also collected. We assessed the prevalence of pfcrt, pfmdr-1, pfdhfr, and pfdhps mutations and the pfmdr-1 gene copy number in 1,596 P. falciparum isolates collected in 26 health centers (20 in Madagascar and 6 in the Comoros Islands) from 2006 to 2008. The in vitro responses to a panel of drugs by 373 of the parasite isolates were determined. The results showed (i) unusual profiles of chloroquine susceptibility in Madagascar, (ii) a rapid rise in the frequency of parasites with both the pfdhfr and the pfdhps mutations, (iii) the alarming emergence of the single pfdhfr 164L genotype, and (iv) the progressive loss of the most susceptible isolates to artemisinin derivatives. In the context of the implementation of the new national policy for the fight against malaria, continued surveillance for the detection of P. falciparum resistance in the future is required.


Assuntos
Antimaláricos/farmacologia , Artemisininas/farmacologia , Haplótipos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Plasmodium falciparum/efeitos dos fármacos , Tetra-Hidrofolato Desidrogenase/genética , Animais , Cloroquina/farmacologia , Di-Hidropteroato Sintase/genética , Combinação de Medicamentos , Resistência a Medicamentos , Madagáscar , Testes de Sensibilidade Parasitária , Pirimetamina/farmacologia , Sulfadoxina/farmacologia
19.
J Pediatr Ophthalmol Strabismus ; 46(3): 139-43; quiz 144-5, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19496494

RESUMO

PURPOSE: To describe the ocular findings in a series of children with spontaneous resolution of primary congenital glaucoma (PCG). METHODS: The medical records of 356 patients with PCG were reviewed. Nine patients with spontaneous resolution of PCG were studied. Fourteen of 18 eyes possessed evidence of early glaucoma, were found to have normal intraocular pressures, and were clinically classified as having spontaneous resolution of PCG. None of the eyes with spontaneous resolution received surgery or any glaucoma medication. RESULTS: Spontaneous resolution of PCG in all nine patients was recognized after 4 months of age. At the time of diagnosis, large corneas were observed in all affected eyes and Haab's striae in 10 of 14 eyes. Intraocular pressures were normal in 14 eyes. In all 14 eyes, the anterior chambers were deep, the lenses were clear, and the angles were abnormal with the typical appearance of PCG. The eyes with spontaneous resolution of PCG possessed angle abnormalities that were less severe compared to three fellow eyes that required glaucoma treatments. CONCLUSION: The occurrence of spontaneous resolution of PCG in these patients is supported by objective clinical evidence. The explanation for this spontaneous resolution is unknown. Its mechanism could be related to continued postnatal development of the angle structures in eyes possessing milder angle abnormalities. The anterior segment findings in patients with spontaneous resolution of PCG are similar to those of patients with treated PCG, the intraocular pressures are normal, the optic nerve damage is stable, and the filtration angles possess mild or moderate abnormalities.


Assuntos
Hidroftalmia/fisiopatologia , Adulto , Criança , Pré-Escolar , Humanos , Lactente , Pressão Intraocular , Remissão Espontânea
20.
J Microbiol Methods ; 78(2): 165-70, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19465064

RESUMO

We have developed a High-Resolution DNA Melting method to detect mutations related to Plasmodium falciparum resistance. This method is based on real-time PCR followed by High Resolution Melting ramping from 67 degrees C to 80 degrees C with fluorescence data acquisition set at 0.1 degrees C increments. The accuracy of the technique was assessed using 177 P. falciparum clinical isolates and two reference strains. Results perfectly matched those obtained by DNA sequencing for some important genetic markers of P. falciparum resistance. This technique could be of great value for epidemiological studies, especially in developing countries.


Assuntos
Antimaláricos/farmacologia , DNA de Protozoário/genética , Resistência a Medicamentos , Testes de Sensibilidade Parasitária/métodos , Plasmodium falciparum/efeitos dos fármacos , Mutação Puntual , Temperatura de Transição , Animais , Temperatura Alta , Humanos , Desnaturação de Ácido Nucleico , Plasmodium falciparum/genética , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade
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