Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 20
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Proc Natl Acad Sci U S A ; 118(3)2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33384338

RESUMO

Human adenovirus species D (HAdV-D) types are currently being explored as vaccine vectors for coronavirus disease 2019 (COVID-19) and other severe infectious diseases. The efficacy of such vector-based vaccines depends on functional interactions with receptors on host cells. Adenoviruses of different species are assumed to enter host cells mainly by interactions between the knob domain of the protruding fiber capsid protein and cellular receptors. Using a cell-based receptor-screening assay, we identified CD46 as a receptor for HAdV-D56. The function of CD46 was validated in infection experiments using cells lacking and overexpressing CD46, and by competition infection experiments using soluble CD46. Remarkably, unlike HAdV-B types that engage CD46 through interactions with the knob domain of the fiber protein, HAdV-D types infect host cells through a direct interaction between CD46 and the hexon protein. Soluble hexon proteins (but not fiber knob) inhibited HAdV-D56 infection, and surface plasmon analyses demonstrated that CD46 binds to HAdV-D hexon (but not fiber knob) proteins. Cryoelectron microscopy analysis of the HAdV-D56 virion-CD46 complex confirmed the interaction and showed that CD46 binds to the central cavity of hexon trimers. Finally, soluble CD46 inhibited infection by 16 out of 17 investigated HAdV-D types, suggesting that CD46 is an important receptor for a large group of adenoviruses. In conclusion, this study identifies a noncanonical entry mechanism used by human adenoviruses, which adds to the knowledge of adenovirus biology and can also be useful for development of adenovirus-based vaccine vectors.


Assuntos
Adenovírus Humanos , Proteínas do Capsídeo , Regulação Viral da Expressão Gênica , Internalização do Vírus , Adenovírus Humanos/genética , Adenovírus Humanos/metabolismo , /metabolismo , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/genética , Linhagem Celular , Humanos
2.
ACS Chem Biol ; 15(10): 2683-2691, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-32845119

RESUMO

Coxsackievirus A24 variant (CVA24v) and human adenovirus 37 (HAdV-37) are leading causative agents of the severe and highly contagious ocular infections acute hemorrhagic conjunctivitis and epidemic keratoconjunctivitis, respectively. Currently, neither vaccines nor antiviral agents are available for treating these diseases, which affect millions of individuals worldwide. CVA24v and HAdV-37 utilize sialic acid as attachment receptors facilitating entry into host cells. Previously, we and others have shown that derivatives based on sialic acid are effective in preventing HAdV-37 binding and infection of cells. Here, we designed and synthesized novel pentavalent sialic acid conjugates and studied their inhibitory effect against CVA24v and HAdV-37 binding and infection of human corneal epithelial cells. The pentavalent conjugates are the first reported inhibitors of CVA24v infection and proved efficient in blocking HAdV-37 binding. Taken together, the pentavalent conjugates presented here form a basis for the development of general inhibitors of these highly contagious ocular pathogens.

3.
J Virol ; 94(14)2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32376620

RESUMO

Virus entry into host cells is a complex process that is largely regulated by access to specific cellular receptors. Human adenoviruses (HAdVs) and many other viruses use cell adhesion molecules such as the coxsackievirus and adenovirus receptor (CAR) for attachment to and entry into target cells. These molecules are rarely expressed on the apical side of polarized epithelial cells, which raises the question of how adenoviruses-and other viruses that engage cell adhesion molecules-enter polarized cells from the apical side to initiate infection. We have previously shown that species C HAdVs utilize lactoferrin-a common innate immune component secreted to respiratory mucosa-for infection via unknown mechanisms. Using a series of biochemical, cellular, and molecular biology approaches, we mapped this effect to the proteolytically cleavable, positively charged, N-terminal 49 residues of human lactoferrin (hLF) known as human lactoferricin (hLfcin). Lactoferricin (Lfcin) binds to the hexon protein on the viral capsid and anchors the virus to an unknown receptor structure of target cells, resulting in infection. These findings suggest that HAdVs use distinct cell entry mechanisms at different stages of infection. To initiate infection, entry is likely to occur at the apical side of polarized epithelial cells, largely by means of hLF and hLfcin bridging HAdV capsids via hexons to as-yet-unknown receptors; when infection is established, progeny virions released from the basolateral side enter neighboring cells by means of hLF/hLfcin and CAR in parallel.IMPORTANCE Many viruses enter target cells using cell adhesion molecules as receptors. Paradoxically, these molecules are abundant on the lateral and basolateral side of intact, polarized, epithelial target cells, but absent on the apical side that must be penetrated by incoming viruses to initiate infection. Our study provides a model whereby viruses use different mechanisms to infect polarized epithelial cells depending on which side of the cell-apical or lateral/basolateral-is attacked. This study may also be useful to understand the biology of other viruses that use cell adhesion molecules as receptors.


Assuntos
Infecções por Adenovirus Humanos/metabolismo , Adenovírus Humanos/metabolismo , Proteínas do Capsídeo/metabolismo , Células Epiteliais/metabolismo , Lactoferrina/metabolismo , Mucosa Respiratória/metabolismo , Células A549 , Infecções por Adenovirus Humanos/genética , Adenovírus Humanos/genética , Proteínas do Capsídeo/genética , Células Epiteliais/virologia , Humanos , Lactoferrina/genética , Mucosa Respiratória/virologia
4.
Viruses ; 11(5)2019 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-31035532

RESUMO

Human adenoviruses (HAdV) are the most common cause of ocular infections. Species B human adenovirus type 3 (HAdV-B3) causes pharyngoconjunctival fever (PCF), whereas HAdV-D8, -D37, and -D64 cause epidemic keratoconjunctivitis (EKC). Recently, HAdV-D53, -D54, and -D56 emerged as new EKC-causing agents. HAdV-E4 is associated with both PCF and EKC. We have previously demonstrated that HAdV-D37 uses sialic acid (SA)-containing glycans as cellular receptors on human corneal epithelial (HCE) cells, and the virus interaction with SA is mediated by the knob domain of the viral fiber protein. Here, by means of cell-based assays and using neuraminidase (a SA-cleaving enzyme), we investigated whether ocular HAdVs other than HAdV-D37 also use SA-containing glycans as receptors on HCE cells. We found that HAdV-E4 and -D56 infect HCE cells independent of SAs, whereas HAdV-D53 and -D64 use SAs as cellular receptors. HAdV-D8 and -D54 fiber knobs also bound to cell-surface SAs. Surprisingly, HCE cells were found resistant to HAdV-B3 infection. We also demonstrated that the SA-based molecule i.e., ME0462, designed to bind to SA-binding sites on the HAdV-D37 fiber knob, efficiently prevents binding and infection of several EKC-causing HAdVs. Surface plasmon resonance analysis confirmed a direct interaction between ME0462 and fiber knobs. Altogether, we demonstrate that SA-containing glycans serve as receptors for multiple EKC-causing HAdVs, and, that SA-based compound function as a broad-spectrum antiviral against known and emerging EKC-causing HAdVs.


Assuntos
Infecções por Adenovirus Humanos/metabolismo , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/fisiologia , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/metabolismo , Receptores Virais/metabolismo , Tropismo Viral , Células A549 , Sequência de Aminoácidos , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Humanos , Ceratoconjuntivite/metabolismo , Ceratoconjuntivite/virologia , Análise de Sequência de DNA
5.
Viruses ; 11(3)2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30870979

RESUMO

Epidemic keratoconjunctivitis (EKC) is a severe ocular disease and can lead to visual impairment. Human adenovirus type-37 (HAdV-D37) is one of the major causative agents of EKC and uses sialic acid (SA)-containing glycans as cellular receptors. Currently, there are no approved antivirals available for the treatment of EKC. Recently, we have reported that sulfated glycosaminoglycans (GAGs) bind to HAdV-D37 via the fiber knob (FK) domain of the viral fiber protein and function as decoy receptors. Based on this finding, we speculated that GAG-mimetics may act as artificial decoy receptors and inhibit HAdV-D37 infection. Repurposing of approved drugs to identify new antivirals has drawn great attention in recent years. Here, we report the antiviral effect of suramin, a WHO-approved drug and a widely known GAG-mimetic, against HAdV-D37. Commercially available suramin analogs also show antiviral effects against HAdV-D37. We demonstrate that suramin exerts its antiviral activity by inhibiting the attachment of HAdV-D37 to cells. We also reveal that the antiviral effect of suramin is HAdV species-specific. Collectively, in this proof of concept study, we demonstrate for the first time that virus binding to a decoy receptor constitutes a novel and an unexplored target for antiviral drug development.


Assuntos
Adenovírus Humanos/efeitos dos fármacos , Ceratoconjuntivite/virologia , Receptores Virais/metabolismo , Suramina/farmacologia , Ligação Viral/efeitos dos fármacos , Células A549 , Infecções por Adenovirus Humanos/tratamento farmacológico , Infecções por Adenovirus Humanos/virologia , Sítios de Ligação , DNA Viral , Reposicionamento de Medicamentos , Genoma Viral , Humanos , Ceratoconjuntivite/tratamento farmacológico , Filogenia , Estudo de Prova de Conceito , Análise de Sequência de DNA
6.
Viruses ; 11(3)2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30871026

RESUMO

Glycans on plasma membranes and in secretions play important roles in infection by many viruses. Species D human adenovirus type 37 (HAdV-D37) is a major cause of epidemic keratoconjunctivitis (EKC) and infects target cells by interacting with sialic acid (SA)-containing glycans via the fiber knob domain of the viral fiber protein. HAdV-D37 also interacts with sulfated glycosaminoglycans (GAGs), but the outcome of this interaction remains unknown. Here, we investigated the molecular requirements of HAdV-D37 fiber knob:GAG interactions using a GAG microarray and demonstrated that fiber knob interacts with a broad range of sulfated GAGs. These interactions were corroborated in cell-based assays and by surface plasmon resonance analysis. Removal of heparan sulfate (HS) and sulfate groups from human corneal epithelial (HCE) cells by heparinase III and sodium chlorate treatments, respectively, reduced HAdV-D37 binding to cells. Remarkably, removal of HS by heparinase III enhanced the virus infection. Our results suggest that interaction of HAdV-D37 with sulfated GAGs in secretions and on plasma membranes prevents/delays the virus binding to SA-containing receptors and inhibits subsequent infection. We also found abundant HS in the basement membrane of the human corneal epithelium, which may act as a barrier to sub-epithelial infection. Collectively, our findings provide novel insights into the role of GAGs as viral decoy receptors and highlight the therapeutic potential of GAGs and/or GAG-mimetics in HAdV-D37 infection.


Assuntos
Adenovírus Humanos/química , Glicosaminoglicanos/química , Heparitina Sulfato/química , Receptores Virais/química , Células A549 , Adenovírus Humanos/genética , DNA Viral/genética , Epitélio Anterior/química , Epitélio Anterior/virologia , Genoma Viral , Glicosaminoglicanos/genética , Humanos , Análise em Microsséries , Filogenia , Receptores Virais/genética , Proteínas Virais/genética , Tropismo Viral , Ligação Viral
7.
Sci Rep ; 8(1): 10019, 2018 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-29968781

RESUMO

The enteric species F human adenovirus types 40 and 41 (HAdV-40 and -41) are the third most common cause of infantile gastroenteritis in the world. Knowledge about HAdV-40 and -41 cellular infection is assumed to be fundamentally different from that of other HAdVs since HAdV-40 and -41 penton bases lack the αV-integrin-interacting RGD motif. This motif is used by other HAdVs mainly for internalization and endosomal escape. We hypothesised that the penton bases of HAdV-40 and -41 interact with integrins independently of the RGD motif. HAdV-41 transduction of a library of rodent cells expressing specific human integrin subunits pointed to the use of laminin-binding α2-, α3- and α6-containing integrins as well as other integrins as candidate co-receptors. Specific laminins prevented internalisation and infection, and recombinant, soluble HAdV-41 penton base proteins prevented infection of human intestinal HT-29 cells. Surface plasmon resonance analysis demonstrated that HAdV-40 and -41 penton base proteins bind to α6-containing integrins with an affinity similar to that of previously characterised penton base:integrin interactions. With these results, we propose that laminin-binding integrins are co-receptors for HAdV-40 and -41.


Assuntos
Adenovírus Humanos/metabolismo , Integrina alfa6/metabolismo , Integrina alfa6beta4/metabolismo , Laminina/metabolismo , Receptores Virais/metabolismo , Ligação Viral , Animais , Células CHO , Linhagem Celular , Cricetulus , Células HT29 , Humanos , Ressonância de Plasmônio de Superfície
8.
Proc Natl Acad Sci U S A ; 115(18): E4264-E4273, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29674446

RESUMO

Human adenovirus 52 (HAdV-52) is one of only three known HAdVs equipped with both a long and a short fiber protein. While the long fiber binds to the coxsackie and adenovirus receptor, the function of the short fiber in the virus life cycle is poorly understood. Here, we show, by glycan microarray analysis and cellular studies, that the short fiber knob (SFK) of HAdV-52 recognizes long chains of α-2,8-linked polysialic acid (polySia), a large posttranslational modification of selected carrier proteins, and that HAdV-52 can use polySia as a receptor on target cells. X-ray crystallography, NMR, molecular dynamics simulation, and structure-guided mutagenesis of the SFK reveal that the nonreducing, terminal sialic acid of polySia engages the protein with direct contacts, and that specificity for polySia is achieved through subtle, transient electrostatic interactions with additional sialic acid residues. In this study, we present a previously unrecognized role for polySia as a cellular receptor for a human viral pathogen. Our detailed analysis of the determinants of specificity for this interaction has general implications for protein-carbohydrate interactions, particularly concerning highly charged glycan structures, and provides interesting dimensions on the biology and evolution of members of Human mastadenovirus G.


Assuntos
Adenovírus Humanos/química , Simulação de Dinâmica Molecular , Ácidos Siálicos/química , Adenovírus Humanos/metabolismo , Linhagem Celular Tumoral , Humanos , Ácidos Siálicos/metabolismo
9.
J Virol ; 91(5)2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-27974569

RESUMO

Epidemic keratoconjunctivitis (EKC) is a severe, contagious ocular disease that affects 20 to 40 million individuals worldwide every year. EKC is mainly caused by six types of human adenovirus (HAdV): HAdV-8, -19, -37, -53, -54, and -56. Of these, HAdV-8, -19, and -37 use sialic acid-containing glycans as cellular receptors. αVß3, αVß5, and a few additional integrins facilitate entry and endosomal release of other HAdVs. With the exception of a few biochemical analyses indicating that HAdV-37 can interact physically with αVß5, little is known about the integrins used by EKC-causing HAdVs. Here, we investigated the overall integrin expression on human corneal cells and found expression of α2, α3, α6, αV, ß1, and ß4 subunits in human corneal in situ epithelium and/or in a human corneal epithelial (HCE) cell line but no or less accessible expression of α4, α5, ß3, or ß5. We also identified the integrins used by HAdV-37 through a series of binding and infection competition experiments and different biochemical approaches. Together, our data suggest that HAdV-37 uses αVß1 and α3ß1 integrins for infection of human corneal epithelial cells. Furthermore, to confirm the relevance of these integrins in the HAdV-37 life cycle, we developed a corneal multilayer tissue system and found that HAdV-37 infection correlated well with the patterns of αV, α3, and ß1 integrin expression. These results provide further insight into the tropism and pathogenesis of EKC-causing HAdVs and may be of importance for future development of new antiviral drugs.IMPORTANCE Keratitis is a hallmark of EKC, which is caused by six HAdV types (HAdV-8, -19, -37, -53, -54, and -56). HAdV-37 and some other HAdV types interact with integrin αVß5 in order to enter nonocular human cells. In this study, we found that αVß5 is not expressed on human corneal epithelial cells, thus proposing other host factors mediate corneal infection. Here, we first characterized integrin expression patterns on corneal tissue and corneal cells. Among the integrins identified, competition binding and infection experiments and biochemical assays pointed out αVß1 and α3ß1 to be of importance for HAdV-37 infection of corneal tissue. In the absence of a good animal model for EKC-causing HAdVs, we also developed an in vitro system with multilayer HCE cells and confirmed the relevance of the suggested integrins during HAdV-37 infection.


Assuntos
Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/fisiologia , Integrina alfa3beta1/fisiologia , Receptores de Vitronectina/fisiologia , Células A549 , Córnea/patologia , Córnea/virologia , Humanos , Receptores Virais , Ligação Viral , Internalização do Vírus
10.
Org Biomol Chem ; 13(35): 9194-205, 2015 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-26177934

RESUMO

Adenovirus type 37 (Ad37) is one of the principal agents responsible for epidemic keratoconjunctivitis (EKC), a severe ocular infection that remains without any available treatment. Recently, a trivalent sialic acid derivative (ME0322, Angew. Chem. Int. Ed., 2011, 50, 6519) was shown to function as a highly potent inhibitor of Ad37, efficiently preventing the attachment of the virion to the host cells and subsequent infection. Here, new trivalent sialic acid derivatives were designed, synthesized and their inhibitory properties against Ad37 infection of the human corneal epithelial cells were investigated. In comparison to ME0322, the best compound (17a) was found to be over three orders of magnitude more potent in a cell-attachment assay (IC50 = 1.4 nM) and about 140 times more potent in a cell-infection assay (IC50 = 2.9 nM). X-ray crystallographic analysis demonstrated a trivalent binding mode of all compounds to the Ad37 fiber knob. For the most potent compound ophthalmic toxicity in rabbits was investigated and it was concluded that repeated eye administration did not cause any adverse effects.


Assuntos
Adenoviridae/efeitos dos fármacos , Adenoviridae/fisiologia , Córnea/citologia , Células Epiteliais/virologia , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/farmacologia , Triazóis/química , Animais , Antivirais/síntese química , Antivirais/química , Antivirais/farmacologia , Química Click , Desenho de Fármacos , Células Epiteliais/efeitos dos fármacos , Humanos , Masculino , Modelos Moleculares , Conformação Molecular , Ácido N-Acetilneuramínico/análogos & derivados , Ácido N-Acetilneuramínico/síntese química , Coelhos
11.
PLoS Pathog ; 11(2): e1004657, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25674795

RESUMO

Most adenoviruses attach to host cells by means of the protruding fiber protein that binds to host cells via the coxsackievirus and adenovirus receptor (CAR) protein. Human adenovirus type 52 (HAdV-52) is one of only three gastroenteritis-causing HAdVs that are equipped with two different fiber proteins, one long and one short. Here we show, by means of virion-cell binding and infection experiments, that HAdV-52 can also attach to host cells via CAR, but most of the binding depends on sialylated glycoproteins. Glycan microarray, flow cytometry, surface plasmon resonance and ELISA analyses reveal that the terminal knob domain of the long fiber (52LFK) binds to CAR, and the knob domain of the short fiber (52SFK) binds to sialylated glycoproteins. X-ray crystallographic analysis of 52SFK in complex with 2-O-methylated sialic acid combined with functional studies of knob mutants revealed a new sialic acid binding site compared to other, known adenovirus:glycan interactions. Our findings shed light on adenovirus biology and may help to improve targeting of adenovirus-based vectors for gene therapy.


Assuntos
Adenovírus Humanos , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Glicoproteínas , Proteínas Virais , Tropismo Viral/fisiologia , Ligação Viral , Adenovírus Humanos/química , Adenovírus Humanos/fisiologia , Sequência de Bases , Linhagem Celular , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/química , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/metabolismo , Cristalografia por Raios X , Glicoproteínas/química , Glicoproteínas/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas Virais/química , Proteínas Virais/metabolismo
12.
J Virol ; 85(24): 13420-31, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21976659

RESUMO

Human species A adenoviruses (HAdVs) comprise three serotypes: HAdV-12, -18, and -31. These viruses are common pathogens and cause systemic infections that usually involve the airways and/or intestine. In immunocompromised individuals, species A adenoviruses in general, and HAdV-31 in particular, cause life-threatening infections. By combining binding and infection experiments, we demonstrate that coagulation factor IX (FIX) efficiently enhances binding and infection by HAdV-18 and HAdV-31, but not by HAdV-12, in epithelial cells originating from the airways or intestine. This is markedly different from the mechanism for HAdV-5 and other human adenoviruses, which utilize coagulation factor X (FX) for infection of host cells. Surface plasmon resonance experiments revealed that the affinity of the HAdV-31 hexon-FIX interaction is higher than that of the HAdV-5 hexon-FX interaction and that the half-lives of these interactions are profoundly different. Moreover, both HAdV-31-FIX and HAdV-5-FX complexes bind to heparan sulfate-containing glycosaminoglycans (GAGs) on target cells, but binding studies utilizing cells expressing specific GAGs and GAG-cleaving enzymes revealed differences in GAG dependence and specificity between these two complexes. These findings add to our understanding of the intricate infection pathways used by human adenoviruses, and they may contribute to better design of HAdV-based vectors for gene and cancer therapy. Furthermore, the interaction between the HAdV-31 hexon and FIX may also serve as a target for antiviral treatment.


Assuntos
Adenovírus Humanos/patogenicidade , Células Epiteliais/virologia , Fator IX/metabolismo , Ligação Viral , Linhagem Celular , Humanos , Mucosa Intestinal/virologia , Mucosa Respiratória/virologia , Ressonância de Plasmônio de Superfície
14.
Nat Med ; 17(1): 105-9, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21151139

RESUMO

Adenovirus type 37 (Ad37) is a leading cause of epidemic keratoconjunctivitis (EKC), a severe and highly contagious ocular disease. Whereas most other adenoviruses infect cells by engaging CD46 or the coxsackie and adenovirus receptor (CAR), Ad37 binds previously unknown sialic acid-containing cell surface molecules. By glycan array screening, we show here that the receptor-recognizing knob domain of the Ad37 fiber protein specifically binds a branched hexasaccharide that is present in the GD1a ganglioside and that features two terminal sialic acids. Soluble GD1a glycan and GD1a-binding antibodies efficiently prevented Ad37 virions from binding and infecting corneal cells. Unexpectedly, the receptor is constituted by one or more glycoproteins containing the GD1a glycan motif rather than the ganglioside itself, as shown by binding, infection and flow cytometry experiments. Molecular modeling, nuclear magnetic resonance and X-ray crystallography reveal that the two terminal sialic acids dock into two of three previously established sialic acid-binding sites in the trimeric Ad37 knob. Surface plasmon resonance analysis shows that the knob-GD1a glycan interaction has high affinity. Our findings therefore form a basis for the design and development of sialic acid-containing antiviral drugs for topical treatment of EKC.


Assuntos
Infecções por Adenoviridae/epidemiologia , Gangliosídeo G(M1)/análogos & derivados , Ceratoconjuntivite/virologia , Receptores Virais/fisiologia , Antivirais/uso terapêutico , Sítios de Ligação , Membrana Celular/virologia , Cristalografia por Raios X , Epitélio Anterior/virologia , Gangliosídeo G(M1)/química , Gangliosídeo G(M1)/imunologia , Gangliosídeo G(M1)/metabolismo , Gangliosídeo G(M1)/fisiologia , Humanos , Ceratoconjuntivite/tratamento farmacológico , Ceratoconjuntivite/epidemiologia , Ceratoconjuntivite/imunologia , Modelos Moleculares , Ligação Proteica , Ácidos Siálicos/metabolismo , Ácidos Siálicos/uso terapêutico , Ressonância de Plasmônio de Superfície
15.
J Virol ; 83(8): 3816-25, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19158249

RESUMO

Most adenoviruses bind directly to the coxsackie and adenovirus receptor (CAR) on target cells in vitro, but recent research has shown that adenoviruses can also use soluble components in body fluids for indirect binding to target cells. These mechanisms have been identified upon addressing the questions of how to de- and retarget adenovirus-based vectors for human gene and cancer therapy, but the newly identified mechanisms also suggest that the role of body fluids and their components may also be of importance for natural, primary infections. Here we demonstrate that plasma, saliva, and tear fluid promote binding and infection of adenovirus type 5 (Ad5) in respiratory and ocular epithelial cells, which corresponds to the natural tropism of most adenoviruses, and that plasma promotes infection by Ad31. By using a set of binding and infection experiments, we also found that Ad5 and Ad31 require coagulation factors IX (FIX) or X (FX) or just FIX, respectively, for efficient binding and infection. The concentrations of these factors that were required for maximum binding were 1/100th of the physiological concentrations. Preincubation of virions with heparin or pretreatment of cells with heparinase I indicated that the role of cell surface heparan sulfate during FIX- and FX-mediated adenovirus binding and infection is mechanistically serotype specific. We conclude that the use of coagulation factors by adenoviruses may be of importance not only for the liver tropism seen when administering adenovirus vectors to the circulation but also during primary infections by wild-type viruses of their natural target cell types.


Assuntos
Adenoviridae/fisiologia , Células Epiteliais/virologia , Fator IX/metabolismo , Fator X/metabolismo , Ligação Viral , Internalização do Vírus , Adenoviridae/classificação , Linhagem Celular Tumoral , Humanos , Plasma/virologia , Saliva/virologia , Lágrimas/virologia
16.
Scand J Clin Lab Invest ; 68(8): 793-800, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18766954

RESUMO

Proteomic analysis has the potential to yield vast amounts of data. The available proteomic methods have been hampered by methodological errors in quantification due to large gel-to-gel variations. The inclusion of an internal standard greatly reduces this variation, and therefore the purpose of this investigation was: 1) to develop a sample preparation protocol for human skeletal muscle for two-dimensional differentiated gel electrophoresis (DIGE) and 2) to investigate the repeatability of one particular system, the Ettan DIGE. To test repeatability, nine aliquots from the same homogenate were labelled with three different CyDye(trade mark) dyes (Cy2, Cy3, Cy5). Samples were run on 18 x 24 cm gels, scanned with a Typhoon 9410 laser scanner and analysed in the DeCyder software. When selecting spots appearing only in triplicate (n = 1314), the mean error was 1.7 % (SD: 10.5 %; 95 % CI: 1.1-2.4 %). When setting the significance level to 99 %, no false-positive changes in protein volume ratios were detected. In the protocol presented here, only 0.5 mg tissue was used and separation of >2500 distinct protein spots in the pH range 3-11 and MW 10-200 kDa. Changes in protein abundance of <20 % could be detected. The method is especially useful when comparing muscle proteins between different conditions; for example, healthy and diseased tissue, before and after treatment or different exercise protocols.


Assuntos
Eletroforese em Gel Bidimensional/métodos , Músculo Esquelético/química , Algoritmos , Western Blotting , Carbocianinas , Humanos , Concentração de Íons de Hidrogênio , Masculino , Proteínas Musculares/metabolismo , Análise de Componente Principal , Reprodutibilidade dos Testes
17.
Glycoconj J ; 24(2-3): 131-42, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17243023

RESUMO

Glycoprotein gp-340 aggregates bacteria in saliva as part of innate defence at mucosal surfaces. We have detected size- and glycoforms of gp-340 between human saliva samples (n = 7) and lung gp-340 from a proteinosis patient using antibodies and lectins in Western blots and ELISA measurements. Western blots of saliva samples, and of gp-340 purified, from the seven donors using a gp-340 specific antibody distinguished four gp-340 size variants, designated I to IV (n = 2,2,2 and 1). While saliva gp-340 variants I to III had single bands of increasing sizes, variant IV and lung gp-340 had double bands. Purified I to IV proteins all revealed a N-terminal sequence TGGWIP upon Edman degradation. Moreover, purified gp-340 from the seven donors and lung gp-340 shared N-glycans, sialylated Galbeta1-3GalNAc and (poly)lactosamine structures. However, the larger size gp-340 grouping II/III (n = 4) and smaller size grouping I/IV correlated with a secretor, Se(+), and a non secretor, Se(-), dependent glycoform of gp-340, respectively (p = 0.03). The Se(+) glycoforms contained ABH, Le(b), Le(y) and polylactosamine structures, while the Se(-) glycoforms lacked ABH antigens but expressed Le(a), Le(x) and lactosamine structures. By contrast, lung gp-340 completely lacked ABH, Le(a/b), Le(x/y) or sLe(x) structures. Gp-340 and secretor typing of saliva from additional donors (n = 29) showed gp-340 glycoforms I to IV for 6, 16, 4 and 0 donors, respectively, and 3 non-typeable donors, and verified that gp-340 glycoforms I and II/III correlate with Se(-) and Se(+) phenotypes, respectively (p < 0.0001). The glycoforms of saliva and lung gp-340 mediated differential aggregation of Le(b)- (Helicobacter pylori), sialylpolylactosamine- (Streptococcus suis) or sialic acid- (Streptococcus mutans) binding bacteria. In conclusion, variant size- and glycoforms of gp-340 are expressed by different individuals and may modulate the biological properties of gp-340 pertinent to health and disease.


Assuntos
Aderência Bacteriana/imunologia , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Receptores Imunológicos/química , Receptores Imunológicos/metabolismo , Receptores Depuradores/química , Receptores Depuradores/metabolismo , Sequência de Aminoácidos , Proteínas de Ligação ao Cálcio , Proteínas de Ligação a DNA , Glicosilação , Helicobacter pylori/imunologia , Humanos , Imunoglobulina A Secretora/química , Imunoglobulina A Secretora/metabolismo , Técnicas In Vitro , Pulmão/imunologia , Peso Molecular , Saliva/imunologia , Saliva/microbiologia , Streptococcus mutans/imunologia , Streptococcus suis/imunologia , Proteínas Supressoras de Tumor
18.
J Virol ; 81(2): 954-63, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17079302

RESUMO

Most adenoviruses bind to the coxsackie- and adenovirus receptor (CAR). Surprisingly, CAR is not expressed apically on polarized cells and is thus not easily available to viruses. Consequently, alternative mechanisms for entry of coxsackievirus and adenovirus into cells have been suggested. We have found that tear fluid promotes adenovirus infection, and we have identified human lactoferrin (HLf) as the tear fluid component responsible for this effect. HLf alone was found to promote binding of adenovirus to epithelial cells in a dose-dependent manner and also infection of epithelial cells by adenovirus. HLf was also found to promote gene delivery from an adenovirus-based vector. The mechanism takes place at the binding stage and functions independently of CAR. Thus, we have identified a novel binding mechanism whereby adenovirus hijacks HLf, a component of the innate immune system, and uses it as a bridge for attachment to host cells.


Assuntos
Adenovírus Humanos/patogenicidade , Células Epiteliais/virologia , Lactoferrina/metabolismo , Receptores Virais/metabolismo , Lágrimas/química , Adenovírus Humanos/classificação , Adenovírus Humanos/metabolismo , Linhagem Celular Tumoral , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Humanos , Especificidade da Espécie
19.
J Nutr ; 135(10): 2445-8, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16177210

RESUMO

Milk oligosaccharides have been shown to interfere with adhesion of many pathogens to host mucosal surfaces. Characterization of the adhesion mechanisms of the bacteria to host cell surface is needed to develop novel functional food, infant formulas, and anti-infective drugs. Adhesion of Neisseria meningitidis, a human specific pathogen causing meningitis and septicemia, is not completely understood but is mediated by type IV pili. Here, we developed a microtiter well pili binding assay to investigate the binding activities of N. meningitidis isolated type IV pili to different glycoproteins. Pili binding activities to bovine thyroglobulin and human salivary agglutinin but not to chicken ovalbumin were present. Inhibition of these binding activities was demonstrated by fractionated human or bovine milk oligosaccharides. The binding of neisserial pili to bovine thyroglobulin was most effective and was clearly inhibited by human milk neutral or bovine milk acidic oligosaccharides.


Assuntos
Aderência Bacteriana/efeitos dos fármacos , Leite Humano/metabolismo , Neisseria meningitidis/metabolismo , Oligossacarídeos/farmacologia , Animais , Proteínas de Ligação ao Cálcio , Bovinos , Proteínas de Ligação a DNA , Fímbrias Bacterianas/fisiologia , Humanos , Técnicas In Vitro , Leite/metabolismo , Leite/microbiologia , Leite Humano/microbiologia , Neisseria meningitidis/patogenicidade , Receptores de Superfície Celular/metabolismo , Tireoglobulina/metabolismo , Proteínas Supressoras de Tumor , Virulência
20.
Protein Expr Purif ; 27(2): 238-43, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12597882

RESUMO

The human alpha(2)-macroglobulin gene is approximately 48 kb in size and consists of 36 exons, which encode the 180 kDa subunit of this large tetrameric protein. In this investigation, a procedure of sequencing human alpha(2)-macroglobulin mRNA, using mRNA from lipopolysaccharide-stimulated peripheral blood mononuclear cells as template in RT-PCR, was developed. Incubation of peripheral blood mononuclear cell populations with lipopolysaccharide induced alpha(2)-macroglobulin mRNA expression reaching levels detectable by RT-PCR. Extracted human alpha(2)-macroglobulin mRNA was used to determine the nucleotide sequence of a 500 bp DNA segment encoding the most C-terminal, receptor-binding part of the protein, using alpha(2)-macroglobulin specific primers. The sequence obtained matched the earlier published sequence of human alpha(2)-macroglobulin, except for three point mutations, i.e., cytosine for guanine, cytosine for thymidine and thymidine for adenine substitutions at positions 4369, 4423, and 4511, respectively. None of these alterations, however, affect the amino acid sequence of the protein. In conclusion, we demonstrate a new, improved, approach to sequence human alpha(2)-macroglobulin mRNA by overexpressing the protein in peripheral blood mononuclear cells. This procedure may be useful in the search for mutations in alpha(2)-macroglobulin, examining its role in the pathogenesis of human diseases.


Assuntos
Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/metabolismo , alfa-Macroglobulinas/biossíntese , Sequência de Bases , Clonagem Molecular , Citometria de Fluxo , Humanos , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNA , Homologia de Sequência do Ácido Nucleico
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA