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1.
Infect Immun ; 88(3)2020 02 20.
Artículo en Inglés | MEDLINE | ID: mdl-31907194

RESUMEN

Yersinia pestis is the causative agent of bubonic, pneumonic, and septicemic plague. We demonstrate that Toll-like receptor 2-deficient (TLR2-/-) mice are resistant to septicemic infection by the KIM5 strain of Y. pestis but not to infection by the CO92 Δpgm strain. This resistance is dependent on TLR2, the route of infection, and the isoform of YopJ. Elevated bacterial burdens were found in the spleens of CO92 Δpgm-infected animals by 24 h postinfection and in the livers by 4 days. The YopJ isoform present contributed directly to cytotoxicity and inflammatory cytokine production of bone marrow-derived macrophages from TLR2-/- mice. Immune cell trafficking is altered in CO92 Δpgm infections, with an increased neutrophil infiltration to the spleen 5 days postinfection. Immune cell infiltration to the liver was greater and earlier in KIM5-infected TLR2-/- mice. The functionality of the immune cells was assessed by the ability to develop reactive oxygen and nitrogen species. Our data suggest an inhibition of granulocytes in forming these species in CO92 Δpgm-infected TLR2-/- mice. These findings suggest that resistance to KIM5 in TLR2-/- mice is dependent on early immune cell trafficking and functionality.


Asunto(s)
Peste/inmunología , Receptor Toll-Like 2/deficiencia , Yersinia pestis/patogenicidad , Animales , Carga Bacteriana , Proteínas Bacterianas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Granulocitos/metabolismo , Hígado/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/inmunología , Neutrófilos/metabolismo , Peste/metabolismo , Peste/microbiología , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Bazo/inmunología , Bazo/microbiología , Receptor Toll-Like 2/inmunología , Virulencia/genética , Yersinia pestis/genética
2.
Am J Hematol ; 92(10): 981-988, 2017 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-28646491

RESUMEN

Sickle erythrocytes' (SSRBCs) unique physical adaptation to hypoxic conditions renders them able to home to hypoxic tumor niches in vivo, shut down tumor blood flow and induce tumoricidal responses. SSRBCs are also useful vehicles for transport of encapsulated drugs and oncolytic virus into hypoxic tumors with enhanced anti-tumor effects. In search of additional modes for arming sickle cells with cytotoxics, we turned to a lentiviral ß-globin vector with optimized Locus Control Region/ß-globin coding region/promoter/enhancers. We partially replaced the ß-globin coding region of this vector with genes encoding T cell cytolytics, perforin and granzyme or immune modulating superantigens SEG and SEI. These modified vectors efficiently transduced Sca+ ckit- Lin- hematopoietic stem cells (HSCs) from humanized sickle cell knockin mice. Irradiated mice reconstituted with these HSCs displayed robust expression of transgenic RNAs and proteins in host sickle cells that was sustained for more than 10 months. SSRBCs from reconstituted mice harboring SEG/SEI transgenes induced robust proliferation and a prototypical superantigen-induced cytokine reaction when exposed to human CD4+/CD8+ cells. The ß-globin lentiviral vector therefore produces a high level of functional, erythroid-specific immune modulators and cytotoxics that circulate without toxicity. Coupled with their unique ability to target and occlude hypoxic tumor vessels these armed SSRBCs constitute a potentially useful tool for treatment of solid tumors.


Asunto(s)
Anemia de Células Falciformes , Citotoxicidad Inmunológica , Eritrocitos Anormales/inmunología , Neoplasias Experimentales/inmunología , Neovascularización Patológica/inmunología , Globinas beta/genética , Anemia de Células Falciformes/sangre , Animales , Citotoxicidad Inmunológica/genética , Sistemas de Liberación de Medicamentos , Eritrocitos Anormales/metabolismo , Eritrocitos Anormales/trasplante , Técnicas de Sustitución del Gen , Vectores Genéticos , Trasplante de Células Madre Hematopoyéticas , Hipoxia , Lentivirus/genética , Ratones Endogámicos C57BL , Ratones Transgénicos , Neoplasias Experimentales/irrigación sanguínea , Neoplasias Experimentales/terapia , Neovascularización Patológica/patología , Neovascularización Patológica/terapia
3.
Infect Immun ; 83(4): 1507-22, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25644012

RESUMEN

The type III secretion system is employed by many pathogens, including the genera Yersinia, Shigella, Pseudomonas, and Salmonella, to deliver effector proteins into eukaryotic cells. The injectisome needle is formed by the polymerization of a single protein, e.g., YscF (Yersinia pestis), PscF (Pseudomonas aeruginosa), PrgI (Salmonella enterica SPI-1), SsaG (Salmonella enterica SPI-2), or MxiH (Shigella flexneri). In this study, we demonstrated that the N termini of some needle proteins, particularly the N terminus of YscF from Yersinia pestis, influences host immune responses. The N termini of several needle proteins were truncated and tested for the ability to induce inflammatory responses in a human monocytic cell line (THP-1 cells). Truncated needle proteins induced proinflammatory cytokines to different magnitudes than the corresponding wild-type proteins, except SsaG. Notably, N-terminally truncated YscF induced significantly higher activation of NF-κB and/or AP-1 and higher induction of proinflammatory cytokines, suggesting that a function of the N terminus of YscF is interference with host sensing of YscF, consistent with Y. pestis pathogenesis. To directly test the ability of the N terminus of YscF to suppress cytokine induction, a YscF-SsaG chimera with 15 N-terminal amino acids from YscF added to SsaG was constructed. The chimeric YscF-SsaG induced lower levels of cytokines than wild-type SsaG. However, the addition of 15 random amino acids to SsaG had no effect on NF-κB/AP-1 activation. These results suggest that the N terminus of YscF can function to decrease cytokine induction, perhaps contributing to a favorable immune environment leading to survival of Y. pestis within the eukaryotic host.


Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Citocinas/metabolismo , Proteínas de la Membrana/inmunología , Proteínas Recombinantes de Fusión/inmunología , Secuencia de Aminoácidos , Proteínas Bacterianas/farmacología , Sistemas de Secreción Bacterianos/inmunología , Línea Celular , Citocinas/biosíntesis , Células HeLa , Humanos , Evasión Inmune/genética , Inflamación/inmunología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Monocitos/inmunología , FN-kappa B/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Salmonella enterica/genética , Salmonella enterica/inmunología , Salmonella enterica/patogenicidad , Alineación de Secuencia , Eliminación de Secuencia/genética , Transducción de Señal/inmunología , Factor de Transcripción AP-1/metabolismo , Yersinia pestis/genética , Yersinia pestis/inmunología , Yersinia pestis/patogenicidad
4.
Infect Immun ; 82(6): 2300-9, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24643544

RESUMEN

Pathogens are recognized by hosts by use of various receptors, including the Toll-like receptor (TLR) and Nod-like receptor (NLR) families. Ligands for these varied receptors, including bacterial products, are identified by the immune system, resulting in development of innate immune responses. Only a couple of components from type III secretion (T3S) systems are known to be recognized by TLR or NLR family members. Known T3S components that are detected by pattern recognition receptors (PRRs) are (i) flagellin, detected by TLR5 and NLRC4 (Ipaf); and (ii) T3S rod proteins (PrgJ and homologs) and needle proteins (PrgI and homologs), detected by NAIP and the NLRC4 inflammasome. In this report, we characterize the induction of proinflammatory responses through TLRs by the Yersinia pestis T3S needle protein, YscF, the Salmonella enterica needle proteins PrgI and SsaG, and the Shigella needle protein, MxiH. More specifically, we determine that the proinflammatory responses occur through TLR2 and -4. These data support the hypothesis that T3S needles have an unrecognized role in bacterial pathogenesis by modulating immune responses.


Asunto(s)
Sistemas de Secreción Bacterianos/fisiología , Citocinas/metabolismo , Proteínas de Escherichia coli/fisiología , Proteínas de la Membrana/fisiología , Transducción de Señal/fisiología , Receptores Toll-Like/fisiología , Escherichia coli/fisiología , Células HEK293 , Humanos , Transducción de Señal/inmunología
5.
Antimicrob Agents Chemother ; 58(2): 839-50, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24247143

RESUMEN

Numerous Gram-negative pathogens rely upon type III secretion (T3S) systems to cause disease. Several small-molecule inhibitors of the type III secretion systems have been identified; however, few targets of these inhibitors have been elucidated. Here we report that 2,2'-thiobis-(4-methylphenol) (compound D), inhibits type III secretion in Yersinia pestis, Yersinia pseudotuberculosis, and Pseudomonas aeruginosa. YopD, a protein involved in the formation of the translocon and regulatory processes of the type III secretion system, appears to play a role in the inhibition of secretion by compound D. The use of compound D in T3S regulatory mutants demonstrated a difference in secretion inhibition in the presence and absence of calcium. Interestingly, compound D was effective only under conditions without calcium, indicating that a secretion-active needle structure may be necessary for compound D to inhibit secretion.


Asunto(s)
Proteínas de la Membrana Bacteriana Externa/antagonistas & inhibidores , Sistemas de Secreción Bacterianos/efectos de los fármacos , Cresoles/farmacología , Regulación Bacteriana de la Expresión Génica , Yersinia pestis/efectos de los fármacos , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Sistemas de Secreción Bacterianos/genética , Transporte Biológico/efectos de los fármacos , Calcio/metabolismo , Mutación , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Yersinia pestis/genética , Yersinia pestis/metabolismo , Yersinia pseudotuberculosis/efectos de los fármacos , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/metabolismo
6.
Artículo en Inglés | MEDLINE | ID: mdl-35690004

RESUMEN

Prostanoids are potent inflammatory mediators that play a regulatory role in the innate immune activation of the adaptive immune response to determine the duration of protection against infection. We aim to quantify the modulation of prostanoids profiles in lipopolysaccharide (LPS)-stimulated THP-1 cells treated with the novel pertussis antigen BscF. We compared the effect with pertussis antigens present in the current Tdap vaccine to understand the immunomodulatory effect that might contribute to the diminished Tdap vaccine effectiveness. The inflammatory challenge with LPS induced a robust elevation of most prostanoid family members compared to the control treatment. Treatment with BscF and Tdap significantly reduced the LPS-stimulated elevation of prostaglandins (PGs) D2, E2, and F2α, as well as thromboxane (TX) A2 levels. An opposite trend was observed for PGI2, as both antigens accelerated the LPS-stimulated upregulation. Further, we quantified cyclooxygenases (COXs) that catalyze the biosynthesis of prostanoids and found that both antigens significantly reduced LPS-stimulated COX-1 and COX-2, demonstrating that the waning of acellular pertussis vaccines' protective immunity may be due to other downstream enzymes not related to COXs. Our present study validates the potential role of BscF as an adjuvant, resulting in the next-generation pertussis vaccine discovery.


Asunto(s)
Vacunas contra Difteria, Tétanos y Tos Ferina Acelular , Tos Ferina , Anticuerpos Antibacterianos , Antígenos Bacterianos , Bordetella pertussis , Humanos , Lipopolisacáridos/farmacología , Monocitos , Prostaglandinas , Tos Ferina/prevención & control
7.
Vaccines (Basel) ; 10(2)2022 Feb 17.
Artículo en Inglés | MEDLINE | ID: mdl-35214778

RESUMEN

BscF is a type III secretion system (T3SS) needle protein from Bordetella pertussis and has previously been shown to induce a sufficient Th1 and Th17 response in human monocytes and mice as a prerequisite for long-lasting protective immunity against pertussis infection. In our current study, we aim to compare the modulation of inflammatory signaling molecules as a direct measure of the immune response to the B. pertussis antigens BscF and Tdap in the presence or absence of the adrenergic receptor agonists phenylephrine (PE) or isoproterenol (ISO) to observe differences that may contribute to the diminished protective immunity of the current acellular pertussis (aP) vaccine, Tdap. Stimulation of human monocyte THP-1 cells with LPS, BscF, and Tdap induced a robust elevation of CCL20, CXCL10, PGE2, and PGF2α among most chemokine and prostanoid members when compared with the control treatment. Treatment with the adrenergic agonist PE or ISO significantly enhanced the BscF- and Tdap-stimulated modulation of CCL20 and CXCL10 but not PGE2 and PGF2α, suggesting that adrenergic modulation of pertussis antigen responses might be a new therapeutic strategy to improve the longevity of pertussis immunity. Stimulation of THP-1 cells with BscF alone initiated significant expression of CXCL10 and PGF2α but not when Tdap was used, suggesting that BscF might be an important pertussis antigen for next-generation pertussis vaccines or when combined with the current aP vaccine. Our data offer opportunities for designing new therapeutic approaches against pertussis infection.

8.
Infect Immun ; 79(11): 4438-46, 2011 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-21859850

RESUMEN

We demonstrate that 2-month-old female B10.T(6R) mice are highly resistant to systemic infection with the KIM5 strain of Yersinia pestis and that B10.T(6R) mice become susceptible to Y. pestis infection by the age of 5 months. In this study, young (2-month-old) and middle-aged (5- to 12-month-old) B10.T(6R) mice were infected with equal CFU counts of Y. pestis. The 50% lethal dose (LD(50)) for young B10.T(6R) mice was ∼1.4 × 10(4) CFU, while middle-aged B10.T(6R) mice exhibited an LD(50) of ∼60 CFU. Elevated bacterial burdens were found in the spleens of middle-aged mice at 24 and 60 h and in the livers at 60 h postinfection. Immune cell infiltration was greater in the livers of resistant young mice than in those of middle-aged mice and mice of the susceptible C57BL/6N strain. Unlike susceptible mice, young B10.T(6R) mice did not develop necrotic lesions throughout the liver. Instead, livers from young B10.T(6R) mice contained granuloma-like structures. Immunohistochemical staining of liver sections from these mice at 60 h postinfection revealed that the majority of immune cells present in these structures were neutrophils. These findings suggest that resistance to plague in B10.T(6R) mice correlates with early formation of neutrophilic lesions in the liver.


Asunto(s)
Envejecimiento/inmunología , Susceptibilidad a Enfermedades/inmunología , Peste/inmunología , Yersinia pestis/inmunología , Animales , Quimiocinas/genética , Quimiocinas/metabolismo , Femenino , Regulación de la Expresión Génica/inmunología , Granuloma/microbiología , Granuloma/patología , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , Ratones , Ratones Endogámicos , Bazo/patología , Células Madre , Factores de Tiempo
9.
Antiviral Res ; 183: 104923, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32979401

RESUMEN

Dengue is the most prevalent arboviral disease in humans and a continually increasing global public health burden. To date, there are no approved antiviral therapies against dengue virus (DENV) and the only licensed vaccine, Dengvaxia, is exclusively indicated for individuals with prior DENV infection. Endothelial hyperpermeability and vascular leak, pathogenic hallmarks of severe dengue disease, can be directly triggered by DENV non-structural protein 1 (NS1). As such, anti-NS1 antibodies can prevent NS1-triggered endothelial dysfunction in vitro and pathogenesis in vivo. Recently, goose-derived anti-DENV immunoglobulin Y (IgY) antibodies were shown to neutralize DENV and Zika virus (ZIKV) infection without adverse effects, such as antibody-dependent enhancement (ADE). In this study, we used egg yolks from DENV-immunized geese to purify IgY antibodies specific to DENV NS1 epitopes. We determined that 2 anti-NS1 IgY antibodies, NS1-1 and NS1-8, were capable of neutralizing DENV infection in vitro. In addition, these antibodies did not cross-react with the DENV Envelope (E) protein nor enhance DENV or ZIKV infection in vitro. Intriguingly, NS1-8, but not NS1-1, partially blocked NS1-induced endothelial dysfunction in vitro while neither antibody blocked binding of soluble NS1 to cells. Finally, prophylactic treatment of mice with NS1-8 conferred significant protection against lethal DENV challenge. Although further research is needed to define the mechanism of action of these antibodies, our findings highlight the potential of anti-NS1 IgY as a promising prophylactic approach against DENV infection.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Virus del Dengue/inmunología , Dengue/inmunología , Dengue/prevención & control , Inmunización Pasiva , Inmunoglobulinas/administración & dosificación , Inmunoglobulinas/inmunología , Proteínas no Estructurales Virales/inmunología , Animales , Anticuerpos Neutralizantes/administración & dosificación , Acrecentamiento Dependiente de Anticuerpo , Chlorocebus aethiops , Dengue/terapia , Epítopos/inmunología , Femenino , Gansos/inmunología , Masculino , Ratones Endogámicos C57BL , Pruebas de Neutralización , Dengue Grave/inmunología , Dengue Grave/prevención & control , Células Vero
10.
Viruses ; 11(3)2019 03 26.
Artículo en Inglés | MEDLINE | ID: mdl-30917523

RESUMEN

The Zika virus (ZIKV) is a newly emerged pathogen in the Western hemisphere. It was declared a global health emergency by the World Health Organization in 2016. There have been 223,477 confirmed cases, including 3720 congenital syndrome cases since 2015. ZIKV infection symptoms range from asymptomatic to Gullain⁻Barré syndrome and extensive neuropathology in infected fetuses. Passive and active vaccines have been unsuccessful in the protection from or the treatment of flaviviral infections due to antibody-dependent enhancement (ADE). ADE causes an increased viral load due to an increased monocyte opsonization by non-neutralizing, low-avidity antibodies from a previous dengue virus (DENV) infection or from a previous exposure to ZIKV. We have previously demonstrated that polyclonal avian IgY generated against whole-killed DENV-2 ameliorates DENV infection in mice while not inducing ADE. This is likely due to the inability of the Fc portion of IgY to bind to mammalian Fc receptors. We have shown here that ZIKV oligoclonal IgY is able to neutralize the virus in vitro and in IFNAR-/- mice. The concentration of ZIKV-specific IgY yielding 50% neutralization (NT50) was 25 µg/mL. The exposure of the ZIKV, prior to culture with ZIKV-specific IgY or 4G2 flavivirus-enveloped IgG, demonstrated that the ZIKV-specific IgY does not induce ADE. ZIKV IgY was protective in vivo when administered following a lethal ZIKV challenge in 3-week-old IFNAR-/- mice. We propose polyclonal ZIKV-specific IgY may provide a viable passive immunotherapy for a ZIKV infection without inducing ADE.


Asunto(s)
Anticuerpos Antivirales/uso terapéutico , Inmunización Pasiva , Inmunoglobulinas/uso terapéutico , Infección por el Virus Zika/inmunología , Infección por el Virus Zika/terapia , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Acrecentamiento Dependiente de Anticuerpo , Reacciones Cruzadas/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulinas/inmunología , Ratones , Ratones Noqueados , Pruebas de Neutralización , Virus Zika
11.
Methods Mol Biol ; 1531: 165-172, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-27837490

RESUMEN

Measuring immune responses against type III secretion and related molecules has been made easier with the advent of reporter cell lines. For example, the THP-1-XBlue and HEK-Blue cells from InvivoGen provide easy detection of gene activation under NF-kB and AP-1 control. In addition, many of these cells have been engineered to express specific pattern recognition receptors (PRRs) helping to elucidate activation pathways. In addition, the THP-1 parent cell line from ATCC provides a useful tool for evaluating cytokine release upon stimulation. This chapter will outline using X-blue reporter cell lines, setting up experiments to measure cytokine secretion, and general guidelines for cytokine quantification.


Asunto(s)
Interacciones Huésped-Patógeno/inmunología , Sistemas de Secreción Tipo III/inmunología , Línea Celular , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Expresión Génica , Genes Reporteros , Humanos , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/microbiología , FN-kappa B/metabolismo
12.
Methods Mol Biol ; 1531: 193-201, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-27837493

RESUMEN

Many Gram-negative pathogens utilize a type III secretion (T3S) system to directly deliver effector molecules into host eukaryotic cells to manipulate cellular processes. These surface-exposed syringe-like structures are highly conserved, necessary for pathogenesis, and hence are therapeutic targets against a number of Gram-negative pathogens. Here we describe a protocol for using purified needle proteins to immunize mice, and subsequently, ways to characterize the immune response to immunization.


Asunto(s)
Proteínas Bacterianas/inmunología , Proteínas Recombinantes/inmunología , Sistemas de Secreción Tipo III/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Inmunización , Inmunoglobulina G/inmunología , Ratones , Proteínas Recombinantes/administración & dosificación
13.
Methods Mol Biol ; 1531: 155-164, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-27837489

RESUMEN

The type III (T3S) secretion system of many gram-negative bacteria is a surface-exposed protein secretion apparatus used to directly inject bacterial effector molecules into eukaryotic cells. These effector molecules contribute to bacterial pathogenesis in many ways, and have been shown to be crucial for infectivity. Here, we describe a protocol for using homologous recombination to generate T3S system mutants to assess the role of different T3S system proteins in bacterial pathogenesis.


Asunto(s)
Sistemas de Secreción Tipo III/genética , Sistemas de Secreción Tipo III/metabolismo , Yersinia pestis/genética , Yersinia pestis/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Western Blotting , Modelos Animales de Enfermedad , Electroforesis en Gel de Poliacrilamida , Femenino , Células HeLa , Recombinación Homóloga , Humanos , Ratones , Mutación , Peste/microbiología , Transporte de Proteínas , Yersinia pestis/patogenicidad
14.
Artículo en Inglés | MEDLINE | ID: mdl-29854047

RESUMEN

A biomedical sciences graduate program needed an introductory class that would develop skills for students interested in a wide variety of disciplines, such as microbiology or cancer biology, and a diverse array of biomedical careers. Faculty created a year-long student-centered course, Scientific Discovery, to serve this need. The course was divided into four modules with progressive skill outcomes. Each module had a focus related to each of the major research areas of the collective faculty: molecular biology, biochemistry, neuroscience, and infectious disease. First-year graduate students enter the program with relevant college-level biology and chemistry coursework but not in-depth content knowledge of any of the focus areas. Each module features a biomedical problem for the students to gain specific content knowledge while developing skills outcomes, such as the ability to conduct scholarly inquiry. In 2015, the theme of the infectious disease module was to create an effective human vaccine to prevent Lyme disease. The module required students to learn fundamental concepts of microbiology and immunology and then apply that knowledge to design their own Lyme disease vaccine. The class culminated with students communicating their creative designs in the form of a "white paper" and a pitch to "potential investors." By the end of the module, students had developed fundamental knowledge, applied that knowledge with great creativity, and met the skills learning outcomes, as evidenced by their ability to conduct scholarly inquiry and apply knowledge gained during this module to a novel problem, as part of their final exam.

15.
J Med Entomol ; 54(5): 1354-1359, 2017 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-28499036

RESUMEN

Zika virus is an emerging arbovirus of humans in the western hemisphere. With its potential spread into new geographical areas, it is important to define the vector competence of native mosquito species. We tested the vector competency of Aedes vexans (Meigen) from the Lake Agassiz Plain of northwestern Minnesota and northeastern North Dakota. Aedes aegypti (L.) was used as a positive control for comparison. Mosquitoes were fed blood containing Zika virus and 2 wk later were tested for viral infection and dissemination. Aedes vexans (n = 60) were susceptible to midgut infection (28% infection rate) but displayed a fairly restrictive midgut escape barrier (3% dissemination rate). Cofed Ae. aegypti (n = 22) displayed significantly higher rates of midgut infection (61%) and dissemination (22%). To test virus transmission, mosquitoes were inoculated with virus and 16-17 d later, tested for their ability to transmit virus into fluid-filled capillary tubes. Unexpectedly, the transmission rate was significantly higher for Ae. vexans (34%, n = 47) than for Ae. aegypti (5%, n = 22). The overall transmission potential for Ae. vexans to transmit Zika virus was 1%. Because of its wide geographic distribution, often extreme abundance, and aggressive human biting activity, Ae. vexans could serve as a potential vector for Zika virus in northern latitudes where the conventional vectors, Ae. aegypti and Ae. albopictus Skuse, cannot survive. However, Zika virus is a primate virus and humans are the only amplifying host species in northern latitudes. To serve as a vector of Zika virus, Ae. vexans must feed repeatedly on humans. Defining the propensity of Ae. vexans to feed repeatedly on humans will be key to understanding its role as a potential vector of Zika virus.


Asunto(s)
Aedes/virología , Insectos Vectores/virología , Infección por el Virus Zika/transmisión , Virus Zika/fisiología , Animales , Femenino , Humanos , Minnesota , North Dakota , Infección por el Virus Zika/virología
16.
PLoS Negl Trop Dis ; 11(7): e0005721, 2017 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-28686617

RESUMEN

Dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) are severe disease manifestations that can occur following sequential infection with different dengue virus serotypes (DENV1-4). At present, there are no licensed therapies to treat DENV-induced disease. DHF and DSS are thought to be mediated by serotype cross-reactive antibodies that facilitate antibody-dependent enhancement (ADE) by binding to viral antigens and then Fcγ receptors (FcγR) on target myeloid cells. Using genetically engineered DENV-specific antibodies, it has been shown that the interaction between the Fc portion of serotype cross-reactive antibodies and FcγR is required to induce ADE. Additionally, it was demonstrated that these antibodies were as neutralizing as their non-modified variants, were incapable of inducing ADE, and were therapeutic following a lethal, antibody-enhanced infection. Therefore, we hypothesized that avian IgY, which do not interact with mammalian FcγR, would provide a novel therapy for DENV-induced disease. We demonstrate here that goose-derived anti-DENV2 IgY neutralized DENV2 and did not induce ADE in vitro. Anti-DENV2 IgY was also protective in vivo when administered 24 hours following a lethal DENV2 infection. We were also able to demonstrate via epitope mapping that both full-length and alternatively spliced anti-DENV2 IgY recognized different epitopes, including epitopes that have not been previously identified. These observations provide evidence for the potential therapeutic applications of goose-derived anti-DENV2 IgY.


Asunto(s)
Anticuerpos Neutralizantes/administración & dosificación , Anticuerpos Antivirales/administración & dosificación , Acrecentamiento Dependiente de Anticuerpo/inmunología , Inmunoglobulinas/administración & dosificación , Dengue Grave/prevención & control , Animales , Línea Celular , Reacciones Cruzadas , Virus del Dengue , Epítopos/inmunología , Femenino , Gansos , Humanos , Ratones , Ratones Noqueados , Dengue Grave/inmunología , Vacunación , Proteínas del Envoltorio Viral/inmunología
17.
BMC Microbiol ; 5: 38, 2005 Jun 24.
Artículo en Inglés | MEDLINE | ID: mdl-15978133

RESUMEN

BACKGROUND: Yersinia pestis, the causative agent of plague, is a pathogen with a tremendous ability to cause harm and panic in populations. Due to the severity of plague and its potential for use as a bioweapon, better preventatives and therapeutics for plague are desirable. Subunit vaccines directed against the F1 capsular antigen and the V antigen (also known as LcrV) of Y. pestis are under development. However, these new vaccine formulations have some possible limitations. The F1 antigen is not required for full virulence of Y. pestis and LcrV has a demonstrated immunosuppressive effect. These limitations could damper the ability of F1/LcrV based vaccines to protect against F1-minus Y. pestis strains and could lead to a high rate of undesired side effects in vaccinated populations. For these reasons, the use of other antigens in a plague vaccine formulation may be advantageous. RESULTS: Desired features in vaccine candidates would be antigens that are conserved, essential for virulence and accessible to circulating antibody. Several of the proteins required for the construction or function of the type III secretion system (TTSS) complex could be ideal contenders to meet the desired features of a vaccine candidate. Accordingly, the TTSS needle complex protein, YscF, was selected to investigate its potential as a protective antigen. In this study we describe the overexpression, purification and use of YscF as a protective antigen. YscF immunization triggers a robust antibody response to YscF and that antibody response is able to afford significant protection to immunized mice following challenge with Y. pestis. Additionally, evidence is presented that suggests antibody to YscF is likely not protective by blocking the activity of the TTSS. CONCLUSION: In this study we investigated YscF, a surface-expressed protein of the Yersinia pestis type III secretion complex, as a protective antigen against experimental plague infection. Immunization of mice with YscF resulted in a high anti-YscF titer and provided protection against i.v. challenge with Y. pestis. This is the first report to our knowledge utilizing a conserved protein from the type III secretion complex of a gram-negative pathogen as a candidate for vaccine development.


Asunto(s)
Proteínas Bacterianas/inmunología , Peste/prevención & control , Animales , Anticuerpos Antibacterianos/sangre , Proteínas de la Membrana Bacteriana Externa/metabolismo , Vacunas Bacterianas/inmunología , Femenino , Células HeLa , Humanos , Inmunoglobulina G/sangre , Ratones , Transporte de Proteínas
18.
ASN Neuro ; 7(4)2015.
Artículo en Inglés | MEDLINE | ID: mdl-26148848

RESUMEN

In neurocysticercosis, parasite-induced immune suppressive effects are thought to play an important role in enabling site-specific inhibition of inflammatory responses to infections. It is axiomatic that microglia-mediated (M1 proinflammatory) response causes central nervous system inflammation; however, the mechanisms by which helminth parasites modulate microglia activation remain poorly understood. Here, we show that microglia display a diminished expression of M1-inflammatory mediators such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and nitric oxide synthase 2 (NOS2) in murine neurocysticercosis. Microglia also exhibited a lack of myeloid cell maturation marker major histocompatibility complex (MHC)-II in these parasite-infected brains. Treatment of microglia with helminth soluble/secreted factors (HSFs) in vitro did not induce expression of M1-inflammatory signature molecule NOS2 as well as MHC-II in primary microglia. However, HSF treatment completely inhibited lipopolysaccharide-induced increase in expression of MHC-II, NOS2 and nitric oxide production in these cells. As epigenetic modulation of chromatin states that regulates recruitment of RNA polymerase II (Pol-II) is a key regulatory step in determining gene expression and functional outcome, we next evaluated whether HSF induced modulation of these phenomenon in microglia in vitro. Indeed, HSF downregulated Pol-II recruitment to the promoter region of TNF-α, IL-6, NOS2, MHC-II, and transcription factor CIITA (a regulator of MHC-II expression), by itself. Moreover, HSF suppressed the lipopolysaccharide-induced increase in Pol-II recruitment as well. In addition, HSF exposure reduced the positive histone marks H3K4Me3 and H3K9/14Ac at the promoter of TNF-α, IL-6, NOS2, MHC-II, and CIITA. These studies provide a novel mechanistic insight into helminth-mediated immune suppression in microglia via modulation of epigenetic processes.


Asunto(s)
Sistema Nervioso Central/patología , Epigénesis Genética/inmunología , Helmintiasis/inmunología , Helmintiasis/patología , Helmintos/patogenicidad , Microglía/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Inmunoprecipitación de Cromatina , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Epigénesis Genética/efectos de los fármacos , Helmintos/metabolismo , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/metabolismo , Histonas/genética , Histonas/metabolismo , Ratones , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Polisacáridos/farmacología , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Transactivadores/genética , Transactivadores/metabolismo
19.
PLoS Negl Trop Dis ; 9(6): e0003803, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26046641

RESUMEN

Andes virus (ANDV) and ANDV-like viruses are responsible for most hantavirus pulmonary syndrome (HPS) cases in South America. Recent studies in Chile indicate that passive transfer of convalescent human plasma shows promise as a possible treatment for HPS. Unfortunately, availability of convalescent plasma from survivors of this lethal disease is very limited. We are interested in exploring the concept of using DNA vaccine technology to produce antiviral biologics, including polyclonal neutralizing antibodies for use in humans. Geese produce IgY and an alternatively spliced form, IgYΔFc, that can be purified at high concentrations from egg yolks. IgY lacks the properties of mammalian Fc that make antibodies produced in horses, sheep, and rabbits reactogenic in humans. Geese were vaccinated with an ANDV DNA vaccine encoding the virus envelope glycoproteins. All geese developed high-titer neutralizing antibodies after the second vaccination, and maintained high-levels of neutralizing antibodies as measured by a pseudovirion neutralization assay (PsVNA) for over 1 year. A booster vaccination resulted in extraordinarily high levels of neutralizing antibodies (i.e., PsVNA80 titers >100,000). Analysis of IgY and IgYΔFc by epitope mapping show these antibodies to be highly reactive to specific amino acid sequences of ANDV envelope glycoproteins. We examined the protective efficacy of the goose-derived antibody in the hamster model of lethal HPS. α-ANDV immune sera, or IgY/IgYΔFc purified from eggs, were passively transferred to hamsters subcutaneously starting 5 days after an IM challenge with ANDV (25 LD50). Both immune sera, and egg-derived purified IgY/IgYΔFc, protected 8 of 8 and 7 of 8 hamsters, respectively. In contrast, all hamsters receiving IgY/IgYΔFc purified from normal geese (n=8), or no-treatment (n=8), developed lethal HPS. These findings demonstrate that the DNA vaccine/goose platform can be used to produce a candidate antiviral biological product capable of preventing a lethal disease when administered post-exposure.


Asunto(s)
Anticuerpos Neutralizantes/uso terapéutico , Gansos/inmunología , Síndrome Pulmonar por Hantavirus/prevención & control , Inmunoglobulinas/uso terapéutico , Profilaxis Posexposición/métodos , Vacunas de ADN/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Cricetinae , Inmunoglobulinas/biosíntesis , Inmunoglobulinas/inmunología , Mesocricetus , Colorantes de Rosanilina
20.
Virus Res ; 106(1): 35-42, 2004 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-15522445

RESUMEN

Age-dependent poliomyelitis (ADPM) or murine amyotrophic lateral sclerosis (ALS) is a murine paralytic disease triggered in immunosuppressed genetically-susceptible mice by infection with the arterivirus lactate dehydrogenase-elevating virus (LDV). This disease provides an animal model for ALS, affecting anterior horn neurons and resulting in neuroparalysis 2-3 weeks after LDV infection. We have tested the hypothesis that spinal cord apoptosis is a feature of the LDV-induced murine ALS, since apoptosis is postulated to be a causal factor in human ALS. Gene microarray analyses of spinal cords from paralyzed animals revealed upregulation of several genes associated with apoptosis. Spinal cord apoptosis was investigated further by TUNEL and activated caspase-3 assays, and was observed to emerge concurrent with paralytic symptoms in both neuronal and non-neuronal cells. Caspase-3-dependent apoptosis was also triggered in cultured macrophages by neurovirulent LDV infection. Thus, virus-induced spinal cord apoptosis is a pre-mortem feature of ADPM, which affects both neuronal and support cells, and may contribute to the pathogenesis of this ALS-like disease.


Asunto(s)
Esclerosis Amiotrófica Lateral/patología , Apoptosis , Infecciones por Arterivirus/patología , Virus Elevador de Lactato Deshidrogenasa/fisiología , Macrófagos/virología , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Infecciones por Arterivirus/fisiopatología , Infecciones por Arterivirus/virología , Técnicas de Cultivo de Célula , Modelos Animales de Enfermedad , Virus Elevador de Lactato Deshidrogenasa/patogenicidad , Ratones , Ratones Endogámicos , Médula Espinal/patología
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