in-vitro-biofilm-formation-in-an-8-well-chamber-slide

<em>In vitro</em> Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

There is currently great interest worldwide in developing noninvasive methods for the delivery of vaccines for upper respiratory tract diseases, including middle ear infection (otitis media, OM). One such noninvasive approach believed to have great potential for the prevention of diseases of the airway is to deliver vaccines by the intranasal (i.n.) route. Induction of a local, mucosal immune response in the upper respiratory tract, and particularly in the nasopharynx, would be a highly efficacious approach to prevention of OM. The chinchilla is the preferred rodent host for studying OM. However, although the anatomy of the chinchilla vomeronasal organ, inner ear, middle ear and Eustachian tube have been well-studied, to date there have been no reports in the literature of a similar complete analysis of the nasopharynx and nasal cavities of the chinchilla. In order to develop a relevant animal model of i.n. delivery as a potential immunization approach for the prevention of OM and to use these models for preclinical assessments of various vaccine candidates, it was important that we better understand the anatomy of the chinchilla nasal cavities and nasopharynx. Our anatomical studies revealed that the naso- and maxilloturbinates of the chinchilla nasal cavity more closely resemble the simple turbinates found in other rodents rather than the branched or complex turbinates seen in dogs, cats, and rabbits thus facilitating the i.n. delivery of vaccine candidates. The chinchilla nasal mucosa also contains numerous lymphoid aggregates like that of other rodents. Our findings thus suggest that we will be able to deliver i.n. vaccines effectively to chinchillas and that these vaccines will likely be able to induce specific immune responses.

Anatomy of the nasal cavity in the chinchilla.

Anatomie der Nasenh&#xF6;hle in der Chinchilla.

Since Haemophilus influenzae lacks enzymes necessary for synthesis of the porphyrin ring, it has an absolute growth requirement for a porphyrin source. This requirement can be satisfied in vitro by hemoglobin and hemoglobin complexed to haptoglobin. The products of the hgp genes mediate the utilization of heme from hemoglobin-haptoglobin. These genes are also involved in the use of heme from hemoglobin, although additional gene products independently mediate the acquisition of heme from this substrate. Different strains of H. influenzae possess one to four hgp genes. A nontypeable H. influenzae mutant lacking all the hgp genes was constructed and compared to the wild-type strain in a chinchilla (Chinchilla lanigera) model of otitis media. Compared to the wild-type strain, the hgp-deficient mutant exhibited a significantly delayed onset of detectable middle ear infection and significantly reduced duration of infection as assessed by both video otoscopy and tympanometry and as evidenced by viable bacterial counts in middle ear effusions. In addition, the maximum bacterial load in the middle ears of chinchillas infected with the mutant strain was significantly reduced when compared to the parent. These data indicate that the hemoglobin/hemoglobin-haptoglobin binding proteins are required for bacterial proliferation during H. influenzae-induced otitis media in chinchillas.

Reduced severity of middle ear infection caused by nontypeable Haemophilus influenzae lacking the hemoglobin/hemoglobin-haptoglobin binding proteins (Hgp) in a chinchilla model of otitis media.

Statt Schwere der Mittelohrentz&#xFC;ndung durch Haemophilus influenzae nontypeable ohne die H&#xE4;moglobin / H&#xE4;moglobin-Haptoglobin-bindende Proteine &#x200B;&#x200B;(Hgp) in einem Chinchilla von Otitis media verursacht.

Haemophilus influenzae is considered a nonmotile organism that expresses neither flagella nor type IV pili, although H. influenzae strain Rd possesses a cryptic pilus locus. We demonstrate here that the homologous gene cluster pilABCD in an otitis media isolate of nontypeable H. influenzae strain 86-028NP encodes a surface appendage that is highly similar, structurally and functionally, to the well-characterized subgroup of bacterial pili known as type IV pili. This gene cluster includes a gene (pilA) that likely encodes the major subunit of the heretofore uncharacterized H. influenzae-expressed type IV pilus, a gene with homology to a type IV prepilin peptidase (pilD) as well as two additional uncharacterized genes (pilB and pilC). A second gene cluster (comABCDEF) was also identified by homology to other pil or type II secretion system genes. When grown in chemically defined medium at an alkaline pH, strain 86-028NP produces approximately 7-nm-diameter structures that are near polar in location. Importantly, these organisms exhibit twitching motility. A mutation in the pilA gene abolishes both expression of the pilus structure and the twitching phenotype, whereas a mutant lacking ComE, a Pseudomonas PilQ homologue, produced large appendages that appeared to be membrane bound and terminated in a slightly bulbous tip. These latter structures often showed a regular pattern of areas of constriction and expansion. The recognition that H. influenzae possesses a mechanism for twitching motility will likely profoundly influence our understanding of H. influenzae-induced diseases of the respiratory tract and their sequelae.

Demonstration of Type IV pilus expression and a twitching phenotype by Haemophilus influenzae.

Der Nachweis der Typ-IV-Pilus Ausdruck und einer zuckenden Ph&#xE4;notyp durch Haemophilus influenzae.

Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in infants and the elderly. While the primary infection is the most serious, reinfection of the upper airway throughout life is the rule. Although relatively little is known about either RSV infection of the upper respiratory tract or host mucosal immunity to RSV, recent literature suggests that RSV is the predominant viral pathogen predisposing to bacterial otitis media (OM). Herein, we describe mouse and chinchilla models of RSV infection of the nasopharynx and Eustachian tube. Both rodent hosts were susceptible to RSV infection of the upper airway following intranasal challenge; however, the chinchilla proved to be more permissive than the mouse. The chinchilla model will likely be extremely useful to test the role of RSV in bacterial OM and the efficacy of RSV vaccine candidates designed to provide mucosal and cytotoxic T-lymphocyte immunity. Ultimately, we hope to investigate the relative ability of these candidates to potentially protect against viral predisposal to bacterial OM.

Chinchilla and murine models of upper respiratory tract infections with respiratory syncytial virus.

Chinchilla-und Mausmodellen von Infektionen der oberen Atemwege mit Respiratory Syncytial Virus.

Passiven &Uuml;bertragung eines p&auml;diatrischen Humanserum-Pool generiert gegen Polysaccharid-Protein D konjugierte Impfstoffe &uuml;bertragenen rund 34 % Schutz gegen die Entwicklung von aufsteigenden NTHI induziert OM, wenn in einem Chinchilla-viral-bakteriell-Koinfektion-Modell verwendet. Diese Daten stehen im Einklang mit Ergebnissen, die mit einen &auml;hnlichen 11-Valent-Protein D-Konjugat-Impfstoff in einer p&auml;diatrischen klinischen Studie, worin eine Impfstoff Wirksamkeit von 35,6 % gegen akute OM Episoden verursacht durch NTHI gezeigt wurde. Diese Beobachtungen unterst&uuml;tzen nachdr&uuml;cklich das Chinchilla-passiven &Uuml;bertragung-Superinfektion-Modell als eine, die klinischen Ergebnisse f&uuml;r Impfstoffe verhindern NTHI induziert om Vorhersagen

Passive Immunisierung mit anti-protein D Antik&ouml;rper induziert durch Polysaccharid-Protein D-Konjugaten sch&uuml;tzt Chinchillas gegen Otitis Media nach intranasale Challenge mit Haemophilus Influenzae.

Passive transfer of a pediatric human serum pool generated against polysaccharide-protein D conjugate vaccines conferred approximately 34% protection against development of ascending NTHI-induced OM when used in a chinchilla viral-bacterial co-infection model. These data are in line with results obtained using a similar 11-valent-protein D conjugate vaccine in a pediatric clinical trial, wherein a vaccine efficacy of 35.6% was shown against acute OM episodes caused by NTHI. These observations strongly support the chinchilla passive transfer-superinfection model as one that could predict clinical trials outcomes for vaccines to prevent NTHI-induced OM.

Passive immunization with human anti-protein D antibodies induced by polysaccharide protein D conjugates protects chinchillas against otitis media after intranasal challenge with Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHI) strains are members of the normal human nasopharyngeal flora, as well as frequent opportunistic pathogens of both the upper and lower respiratory tracts. Recently, it has been shown that NTHI can form biofilms both in vitro and in vivo. NTHI strains within in vitro-formed biofilms differentially express both epitopes of lipooligosaccharide (LOS) and the outer membrane proteins P2, P5, and P6, whereas those generated either in a 96-well plate assay in vitro or in a mammalian host have been shown to incorporate a specific glycoform of sialylated LOS within the biofilm matrix. While DNA has been identified as a key component of the biofilm matrix formed in vitro by several bacterial pathogens, here we demonstrate for the first time that in addition to sialylated LOS, the biofilm formed by NTHI in vivo contains both type IV pilin protein and a significant amount of double-stranded DNA. The DNA appeared to be arranged in a dense interlaced meshwork of fine strands as well as in individual thicker "ropes" that span water channels, suggesting that DNA could be imparting structural stability to the biofilm produced by NTHI in vivo. The presence of type IV pilin protein both appearing as small aggregates within the biofilm matrix and tracking along DNA strands supports our observations which showed that type IV pili are expressed by NTHI during experimental otitis media when these bacteria form a biofilm in the middle ear space.

Biofilms formed by nontypeable Haemophilus influenzae in vivo contain both double-stranded DNA and type IV pilin protein.

Nontypeable Haemophilus Influenzae (NTHI) St&auml;mme sind Mitglieder der normalen menschlichen Nasen-Rachen-Flora, sowie als h&auml;ufige opportunistische Erreger der beiden oberen und niedrigeren Atemwege Traktate. Vor kurzem hat sich gezeigt, dass NTHI Biofilme in vitro und in-vivo bilden kann. NTHI St&auml;mme in in Vitro gebildeten Biofilme differentiell Epitopes von Lipooligosaccharide (LOS) und die &auml;u&szlig;ere Membran-Proteine, P2, P5 und P6, Ausdr&uuml;cken, w&auml;hrend erzeugt eine 96-Well-Platte-Assay in-vitro- oder in einem S&auml;ugetier-Wirt erwiesen haben, eine bestimmte Glycoform Sialylated LOS innerhalb der Biofilm-Matrix zu &uuml;bernehmen. W&auml;hrend DNA herausgestellt hat, als eine Schl&uuml;sselkomponente der Biofilm-Matrix in Vitro durch mehrere bakteriellen Krankheitserregern gebildet demonstrieren wir hier zum ersten Mal, dass neben der Sialylated LOS, der Biofilm gebildet und in-vivo NTHI enth&auml;lt beide Typ IV pilin Protein und eine erhebliche Menge an doppelstr&auml;ngige DNA. Die DNA erschien in einer dichten Zeilensprung Netzwerk der feinen Str&auml;nge ebenso wie in einzelne dickere &quot;Seile&quot;, die sich erstrecken von Wasserkan&auml;len, angeordnet werden darauf hindeutet, dass DNA strukturellen Stabilit&auml;t an den Biofilm, produziert von in-vivo NTHI Vermittlung werden k&ouml;nnte. Die Anwesenheit von Typ IV pilin Protein sowohl als kleine Aggregate innerhalb der Biofilm-Matrix und Verfolgung entlang DNA-Str&auml;nge unterst&uuml;tzt unsere Beobachtungen, die zeigte, dass die Art IV Pili w&auml;hrend experimentelle Otitis Media durch NTHI ausgedr&uuml;ckt werden, wenn diese Bakterien ein Biofilm im Mittelohr Raum bilden.

Biofilme gebildet durch Nontypeable Haemophilus Influenzae in-vivo enthalten doppelstr&auml;ngige DNA und Typ IV pilin Protein.

We recently described the expression of type IV pili (Tfp) by non-typeable Haemophilus influenzae (NTHI), a common respiratory tract pathogen. Prior to that report, Tfp were not thought to be produced by NTHI as they are not observed on NTHI when grown on chocolate agar or other commonly used growth media. To further characterize growth conditions permissive for the expression of NTHI Tfp, as well as determine their role in colonization and virulence, we transformed an NTHI otitis media isolate with a reporter plasmid containing the lux gene cluster driven by the pilA promoter. Transcription from the pilA promoter was demonstrated under a variety of in vitro growth conditions and, importantly, by ex vivo imaging of luciferase-producing NTHI in infected chinchillas. Luciferase-producing NTHI were also identified within a biofilm formed by NTHI in vivo. We further demonstrated a role for NTHI PilA in adherence to human respiratory epithelial cells, in colonization of the chinchilla respiratory tract as well as a requirement for PilA in biofilm development, both in vitro and in vivo. Collectively, our data demonstrate that NTHI express PilA in vivo, and that PilA plays an important role in the pathogenesis of an upper respiratory tract infection induced by NTHI.

The PilA protein of non-typeable Haemophilus influenzae plays a role in biofilm formation, adherence to epithelial cells and colonization of the mammalian upper respiratory tract.

Wir vor kurzem beschrieben den Ausdruck der Art IV Pili (Tfp) von nicht-darstellbares Haemophilus Influenzae (NTHI), eine verbreitete Pathogen der Atemwege. Vor diesem Bericht Tfp galten nicht als durch NTHI produziert werden, wie sie auf NTHI wenn auf Schokoladen-Agar gewachsen nicht eingehalten werden oder andere h&auml;ufig Wachstumsmedium verwendete. Um weiter f&uuml;r den Ausdruck der NTHI Tfp permissive Wachstumsbedingungen zu charakterisieren sowie ihre Rolle bei der Kolonisierung und Virulenz bestimmen, verwandelt wir NTHI Mittelohrentz&uuml;ndung isoliert mit einem Reporter-Plasmid, enth&auml;lt das Lux-gen-Cluster, angetrieben durch den Projekttr&auml;ger PilA. Gef&uuml;hrt Transkription vom Projekttr&auml;ger PilA wurde unter einer Vielzahl von in-vitro Wachstumsbedingungen und ex-Vivo Bildgebung von Luciferase erzeugenden NTHI in allem infiziert Chinchillas. Luciferase erzeugenden NTHI wurden auch innerhalb eines Biofilms durch in-vivo NTHI gebildet. In Einhaltung der menschlichen Atemwege Epithelzellen, in Besiedlung der Atemwege Chinchilla sowie eine Anforderung f&uuml;r PilA im Biofilm-Entwicklung, sowohl in vitro und in-vivo haben wir weiter eine Rolle f&uuml;r NTHI PilA gezeigt. Zusammenfassend zeigen unsere Daten, dass NTHI express PilA in-vivo und dass PilA eine wichtige Rolle in der Pathogenese von einer Infektion der oberen Atemwege spielt durch NTHI induziert.

Das PilA-Protein des nicht-darstellbares Haemophilus Influenzae spielt eine Rolle im Biofilm Formation, Einhaltung von Epithelzellen und Kolonisierung der S&auml;ugetier-Obere Atemwege.

Moraxella catarrhalis is a gram-negative mucosal pathogen of the human respiratory tract. Although little information is available regarding the initial steps of M. catarrhalis pathogenesis, this organism must be able to colonize the human mucosal surface in order to initiate an infection. Type IV pili (TFP), filamentous surface appendages primarily comprised of a single protein subunit termed pilin, play a crucial role in the initiation of disease by a wide range of bacteria. We previously identified the genes that encode the major proteins involved in the biosynthesis of M. catarrhalis TFP and determined that the TFP expressed by this organism are highly conserved and essential for natural transformation. We extended this initial study by investigating the contribution of TFP to the early stages of M. catarrhalis colonization. TFP-deficient M. catarrhalis bacteria exhibit diminished adherence to eukaryotic cells in vitro. Additionally, our studies demonstrate that M. catarrhalis cells form a mature biofilm in continuous-flow chambers and that biofilm formation is enhanced by TFP expression. The potential role of TFP in colonization by M. catarrhalis was further investigated using in vivo studies comparing the abilities of wild-type M. catarrhalis and an isogenic TFP mutant to colonize the nasopharynx of the chinchilla. These results suggest that the expression of TFP contributes to mucosal airway colonization. Furthermore, these data indicate that the chinchilla model of nasopharyngeal colonization provides an effective animal system for studying the early steps of M. catarrhalis pathogenesis.

Contribution of Moraxella catarrhalis type IV pili to nasopharyngeal colonization and biofilm formation.

Moraxella Catarrhalis ist ein Schleimhaut-Gramnegative Erreger der menschlichen Atemwege. Obwohl nur wenige Informationen &uuml;ber die ersten Schritte der M. Catarrhalis Pathogenese verf&uuml;gbar ist, muss dieser Organismus die menschliche Schleimhaut Oberfl&auml;che zu kolonisieren, um eine Infektion zu initiieren k&ouml;nnen. Typ IV Pili (TFP), filament&ouml;se Oberfl&auml;che Anh&auml;ngseln, haupts&auml;chlich bestehend aus ein einzelnes Protein-Untereinheit bezeichnet pilin, spielen eine entscheidende Rolle in der Einleitung der Krankheit durch eine Vielzahl von Bakterien. Wir haben bereits die Gene, die kodieren die wichtigsten Proteine beteiligt die Biosynthese von M. Catarrhalis TFP und festgestellt, dass die TFP, ausgedr&uuml;ckt durch dieses Organismus sind hoch konservierte und f&uuml;r nat&uuml;rliche Transformation identifiziert. Wir erweitern dieser ersten Studie durch die Untersuchung des Beitrags der TFP f&uuml;r die fr&uuml;hen Stadien der M. Catarrhalis Besiedlung. TFP-defizienten M. Catarrhalis, die Bakterien weisen verminderte Einhaltung der eukaryotischen Zellen in vitro. Dar&uuml;ber hinaus zeigen unsere Untersuchungen, dass M. Catarrhalis Zellen ein ausgereiftes Biofilms in kontinuierlichen Fluss Kammern bilden und Biofilm-Bildung von TFP Ausdruck verst&auml;rkt wird. Die potenzielle Rolle von TFP in Kolonisierung durch M. Catarrhalis wurde weiter untersucht mit in-vivo Studien zum Vergleich der F&auml;higkeiten von Wildtyp M. Catarrhalis und ein isogene TFP-Mutant, um der Nasenrachen die Chinchilla zu kolonisieren. Diese Ergebnisse legen nahe, dass der Ausdruck der TFP zur Schleimhaut Airway Kolonisation beitr&auml;gt. Diese Daten zeigen dar&uuml;ber hinaus, dass das Chinchilla-Modell der Nasen-Rachen-Kolonisation ein effektives tierisches System bietet f&uuml;r das Studium von der fr&uuml;hen Stufen des M. Catarrhalis Pathogenese.

Beitrag von Moraxella Catarrhalis geben IV Pili Nasen-Rachen-Kolonisierung und Biofilm-Bildung.

In vitro studies suggest an important role for CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1) in infection by multiple gram-negative bacteria. However, in vivo evidence supporting this role is lacking, largely because the bacterial adhesins involved in this host-microbe association do not bind to murine-derived CEACAM1. One of several adhesins expressed by nontypeable Haemophilus influenzae (NTHI), the outer membrane protein P5-homologous adhesin (or P5), is essential for colonization of the chinchilla nasopharynx and infection of the middle ear. Here we reveal that NTHI P5 binds to the chinchilla homologue of CEACAM1 and that rabbit anti-human carcinoembryonic antigen blocks NTHI colonization of the chinchilla nasopharynx, providing the first demonstration of a role for CEACAM receptor binding by any bacterial pathogen in vivo.

A carcinoembryonic antigen-related cell adhesion molecule 1 homologue plays a pivotal role in nontypeable Haemophilus influenzae colonization of the chinchilla nasopharynx via the outer membrane protein P5-homologous adhesin.

In-vitro-Studien belegen eine wichtige Rolle f&uuml;r CEACAM1 (carcinoembryonales Antigen-bezogene Zelladh&auml;sionsmolek&uuml;l 1) Infektion durch mehrere Gram-negativer Bakterien. Jedoch fehlt in-vivo Nachweise f&uuml;r diese Rolle, vor allem, weil die bakterielle Adhesins dieser Host-Mikrobe-Vereinigung beteiligt an Murine abgeleitete CEACAM1 nicht binden. Einer von mehreren Adhesins ge&auml;u&szlig;erten Nontypeable Haemophilus Influenzae (NTHI), das &auml;u&szlig;ere Membran-Protein, P5-homologe Adhesin (oder P5), ist wesentlich f&uuml;r die Kolonisation des Chinchilla-Nasopharynx und Infektion des mittleren Ohrs. Hier zeigen wir, dass NTHI P5 mit dem Chinchilla-Homolog von CEACAM1 bindet und dieses Kaninchen Anti-Human Carcinoembryonic Antigen blockiert NTHI Kolonisierung des Nasopharynx Chinchilla, bietet die erste Demonstration einer Rolle f&uuml;r CEACAM-Rezeptor-Bindung durch jede bakterielle Erreger, die in-vivo.

Ein carcinoembryonales Antigen-bezogene Zelle Adh&auml;sion Molek&uuml;l 1 Homolog spielt eine zentrale Rolle in Nontypeable Haemophilus Influenzae Kolonisation des die Chinchilla Nasopharynx &uuml;ber die &auml;u&szlig;ere Membrane Protein P5-homologe Adhesin.

Haemophilus influenzae has an absolute growth requirement for heme and the heme-binding lipoprotein (HbpA) and has been implicated in the utilization of this essential nutrient. We constructed an insertional mutation of hbpA in a type b and a nontypeable H. influenzae strain. In the type b strain, the hbpA mutant was impaired in utilization of heme complexed to either hemopexin or to albumin and in the utilization of low levels of heme but not in the utilization of heme at high levels or of hemoglobin or hemoglobin-haptoglobin complexes. In contrast, the hbpA mutant derivative of the nontypeable strain was impaired in utilization of all tested heme sources. We further examined the impact of the hbpA mutation in animal models of H. influenzae disease. The hbpA mutant of the nontypeable strain was indistinguishable from the wild-type strain in the chinchilla model of otitis media. The hbpA mutant derivative of the type b strain caused bacteremia as well as the wild-type strain in 5-day old infant rats. However, in 30-day old rats the hbpA caused significantly lower rates of bacteremia than the wild-type strain indicating a role for hbpA and heme acquisition in virulence in this model of H. influenzae disease. In conclusion, HbpA is important for heme utilization by multiple H. influenzae strains and is a virulence determinant in a model of H. influenzae invasive disease.

The heme-binding protein (HbpA) of Haemophilus influenzae as a virulence determinant.

Haemophilus Influenzae hat verwickelt wurden, bei der Nutzung von dieser wichtiger N&auml;hrstoff und hat eine absolute Wachstum Voraussetzung f&uuml;r H&auml;m und die H&auml;m-Bindung Lipoprotein (HbpA). Konstruierten wir eine muss Mutation des HbpA in einer Typ-B und eine Nontypeable H. Influenzae Belastung. Der Typ B-Stamm war der HbpA-Mutant beeintr&auml;chtigt in Nutzung des H&auml;m komplexiert, entweder Hemopexin oder Albumin und bei der Nutzung von geringen H&auml;m, aber nicht in der Nutzung von H&auml;m auf hohem Niveau oder H&auml;moglobin oder H&auml;moglobin-Haptoglobin-komplexe. Im Gegensatz dazu war die mutant HbpA-Ableitung der Nontypeable Belastung in Auslastung aller getesteten H&auml;m-Quellen beeintr&auml;chtigt. Wir weiter untersucht die Auswirkungen der HbpA Mutation in Tiermodellen f&uuml;r H. Influenzae Krankheit. Der HbpA-Mutant des Stamms Nontypeable war nicht zu unterscheiden von der Wildtyp-Stamm in der Chinchilla-Modell der Otitis Media. Die HbpA-mutant-Ableitung der Typ B-Stamm verursachte Bakteri&auml;mie sowie der Wildtyp-Stamm in 5 - Tage alten S&auml;uglings Ratten. Jedoch verursacht die HbpA 30 Tage alte Ratten deutlich niedrigere Raten von Bakteri&auml;mie als der Wildtyp-Stamm, der angibt, einer Rolle f&uuml;r HbpA und H&auml;m in Virulenz in diesem Modell der H. Influenzae Krankheit. Abschlie&szlig;end HbpA ist wichtig f&uuml;r die H&auml;m-Nutzung durch mehrere H. Influenzae St&auml;mme und Virulenz bestimmender Faktor in einem Modell von H. Influenzae invasive Erkrankung.

Die H&auml;m-verbindliches Protein (HbpA) von Haemophilus Influenzae als Virulenz bestimmender Faktor.

Otitis media (OM) is a polymicrobial disease wherein upper respiratory tract viruses compromise host airway defences, which allows bacterial flora of the nasopharynx (NP) access to the middle ear. We have shown, in vitro, that respiratory syncytial virus (RSV), a viral co-pathogen of OM, reduces transcript abundance of the antimicrobial peptide (AP), chinchilla beta-defensin-1 (cBD-1). Here, we demonstrated that chinchillas inoculated with RSV expressed approximately 40% less cBD-1 mRNA and protein than did mock-challenged animals. Further, concurrent RSV infection resulted in a 10-100-fold greater recovery of non-typeable Haemophilus influenzae (NTHI) from nasopharyngeal lavage fluids, compared with chinchillas challenged with NTHI in the absence of viral co-infection. Additionally, when either: anti-cBD-1 antibody (to bind secreted AP) or recombinant cBD-1 (to increase AP concentration at the mucosal surface) were delivered to chinchillas, we demonstrated that disruption of the availability of a single AP influenced the relative load of NTHI in the upper respiratory tract. Collectively, our data suggested that effectors of innate immunity regulate normal bacterial colonization of the NP and, further, virus-induced altered expression of APs can result in an increased load of NTHI within the NP, which likely promotes development of OM.

Respiratory syncytial virus-induced dysregulation of expression of a mucosal beta-defensin augments colonization of the upper airway by non-typeable Haemophilus influenzae.

Otitis Media (OM) ist eine polymikrobiellen Krankheit worin der oberen Atemwege Viren Host Airway S&uuml;denglands, gef&auml;hrden die Bakterienflora des Nasopharynx (NP) Zugang zum mittleren Ohr erm&ouml;glicht. Wir haben gezeigt, dass respiratorischen Synzytialvirus (RSV), eine virale Co-pathogen OM, Abschrift F&uuml;lle die antimikrobielle Peptide (AP), Chinchilla Beta-defensin-1 (cBD-1) reduziert, in Vitro. Hier wir gezeigt, dass Chinchillas mit RSV beimpft ca. 40 % weniger cBD-1 mRNA und Protein ausgedr&uuml;ckt als Mock gehandicapte Tiere haben. Weitere, gleichzeitige RSV-Infektion ergab eine 10-100-fache gr&ouml;&szlig;er Erholung des nicht-darstellbares Haemophilus Influenzae (NTHI) von Nasen-Rachen-Bronchoalveol&auml;re Lavage Fl&uuml;ssigkeiten, verglichen mit Chinchillas keine virale Koinfektion mit NTHI herausgefordert. Zus&auml;tzlich, wenn entweder: Anti-cBD-1-Antik&ouml;rper (binden sezernierte AP) oder rekombinante cBD-1 (die AP-Konzentration bei der Schleimhaut-Oberfl&auml;che erh&ouml;ht) wurden zu Chinchillas geliefert, wir gezeigt, dass die Unterbrechung der Verf&uuml;gbarkeit von einem einzigen AP die relative Belastung der NTHI in den oberen Atemwegen beeinflusst. Kollektiv, schlug unsere Daten Effektoren der angeborenen Immunit&auml;t regulieren normale bakterielle Besiedlung der NP und weiter, Virus-induzierte ver&auml;nderte Ausdruck des APs kann erh&ouml;hter Belastung des NTHI innerhalb der NP, die wahrscheinlich Entwicklung von OM f&ouml;rdert wird

Atemwege syncytial-Virus-induzierte Dysregulation des Ausdrucks von einer Schleimhaut Beta-defensin erweitert die Kolonisation der oberen Atemwege durch nicht-darstellbares Haemophilus Influenzae.

Although most viral infections of the upper respiratory tract can predispose to bacterial otitis media, human respiratory syncytial virus (HRSV) is the predominant viral copathogen of this highly prevalent pediatric polymicrobial disease. Rigorous study of the specific mechanisms by which HRSV predisposes to otitis media has been hindered by lack of a relevant animal model. We recently reported that the chinchilla, the preferred rodent host for studying otitis media, is semipermissive for upper-airway HRSV infection. In the current study, we defined the anatomy and kinetics of HRSV infection and spread in the upper airway of chinchilla hosts. Chinchillas were challenged intranasally with a fluorescent-protein-expressing HRSV. Upper-airway tissues were recovered at multiple time points after viral challenge and examined by confocal microscopy and immunohistochemistry. HRSV replication was observed from the rostral- to caudalmost regions of the nasal cavity as well as throughout the Eustachian tube in a time-dependent manner. Although fluorescence was not observed and virus was not detected in nasopharyngeal lavage fluids 14 d after infection, the latest time point examined in this study, occasional clusters of immunopositive cells were present, suggesting that the nasal cavity may serve as a reservoir for HRSV. These data provide important new information concerning the time course of HRSV infection of the uppermost airway and suggest that chinchillas may be useful for modeling the HRSV-induced changes that predispose to secondary bacterial infection.

Mapping the anatomy of respiratory syncytial virus infection of the upper airways in chinchillas (Chinchilla lanigera).

Obwohl die meisten virale Infektionen der oberen Atemwege f&uuml;r bakterielle Otitis Media pr&auml;disponieren k&ouml;nnen, ist menschliches respiratory-syncytial-Virus (HRSV) die vorherrschende virale Copathogen dieser Krankheit sehr verbreitet p&auml;diatrische polymikrobiellen. Rigorose Studie &uuml;ber die spezifischen Mechanismen, mit denen HRSV zu Otitis Media pr&auml;disponiert, worden durch Mangel an einer relevanten Tiermodell behindert. Vor kurzem berichteten wir, dass die Chinchilla, der bevorzugte Nagetier Host f&uuml;r das Studium der Otitis Media, semipermissive f&uuml;r HRSV-Infektion der oberen Atemwege ist. In der aktuellen Studie haben wir die Anatomie und die Kinetik der HRSV Infektion und Ausbreitung in die oberen Atemwege Chinchilla Hosts definiert. Chinchillas wurden intranasally mit einem Fluoreszenz-Protein-Ausdruck HRSV gestellt. Oberen Atemwege Gewebe waren erholten sich zu mehreren Zeitpunkten nach virale Herausforderung und durch konfokale Mikroskopie und Immunhistochemie untersucht. HRSV-Replikation wurde von der K&ouml;rperabschnitt - bis Caudalmost Regionen von der Nasenh&ouml;hle und in die Eustachische R&ouml;hre in einer zeitabh&auml;ngigen Weise beobachtet. Obwohl Fluoreszenz nicht beobachtet wurde und Virus wurde nicht erkannt in Nasen-Rachen-Bronchoalveol&auml;re Lavage Fl&uuml;ssigkeiten 14 d nach der Infektion, der neueste Zeitpunkt in dieser Studie untersucht, waren gelegentliche Cluster von Immunopositive Zellen anwesend, was darauf hindeutet, dass die Nasenh&ouml;hle als Reservoir f&uuml;r HRSV bedienen zu kann. Diese Daten liefern wichtige neue Informationen &uuml;ber den zeitlichen Verlauf der HRSV-Infektion der oberen Atemwege und schlagen vor, da&szlig; Chinchillas zur Modellierung der HRSV-induzierte Ver&auml;nderungen, die auf eine sekund&auml;re bakterielle Infektion pr&auml;disponieren n&uuml;tzlich sein k&ouml;nnen.

Article	Year
Anatomy of the nasal cavity in the chinchilla. Cells, tissues, organs\| PubMed ID: 12835577	2003
Reduced severity of middle ear infection caused by nontypeable Haemophilus influenzae lacking the hemoglobin/hemoglobin-haptoglobin binding proteins (Hgp) in a chinchilla model of otitis med... Microbial pathogenesis\| PubMed ID: 14643637	2004
Demonstration of Type IV pilus expression and a twitching phenotype by Haemophilus influenzae. Infection and immunity\| PubMed ID: 15731063	2005
Chinchilla and murine models of upper respiratory tract infections with respiratory syncytial virus. Journal of virology\| PubMed ID: 15857989	2005
Passive immunization with human anti-protein D antibodies induced by polysaccharide protein D conjugates protects chinchillas against otitis media after intranasal challenge with Haemophilus... Vaccine\| PubMed ID: 16616975	2006
Biofilms formed by nontypeable Haemophilus influenzae in vivo contain both double-stranded DNA and type IV pilin protein. Journal of bacteriology\| PubMed ID: 17322318	2007
The PilA protein of non-typeable Haemophilus influenzae plays a role in biofilm formation, adherence to epithelial cells and colonization of the mammalian upper respiratory tract. Molecular microbiology\| PubMed ID: 17645732	2007
Contribution of Moraxella catarrhalis type IV pili to nasopharyngeal colonization and biofilm formation. Infection and immunity\| PubMed ID: 17908808	2007
A carcinoembryonic antigen-related cell adhesion molecule 1 homologue plays a pivotal role in nontypeable Haemophilus influenzae colonization of the chinchilla nasopharynx via the outer memb... Infection and immunity\| PubMed ID: 17938212	2008
The heme-binding protein (HbpA) of Haemophilus influenzae as a virulence determinant. International journal of medical microbiology : IJMM\| PubMed ID: 19451029	2009
Respiratory syncytial virus-induced dysregulation of expression of a mucosal beta-defensin augments colonization of the upper airway by non-typeable Haemophilus influenzae. Cellular microbiology\| PubMed ID: 19500108	2009
Mapping the anatomy of respiratory syncytial virus infection of the upper airways in chinchillas (Chinchilla lanigera). Comparative medicine\| PubMed ID: 20579438	2010

Joseph A. Jurcisek

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