Questa ricerca è stata sostenuta da sovvenzioni 1K01DK079019A2 (PT) dal NIH / NIDDK.

1. Preparazione per l&#39;inoculo batteri mouse Devono essere effettuata in condizioni asettiche. <ol><li> Prendere una 17 x 100 provette di polipropilene mm con tappi e aggiungere 3 ml di brodo fresco Luria (LB) media. Con punte autoclave, prendere un po &#39;congelato stock in glicerolo batteri del ceppo CP1 2 e trasferire una piccola quantità della cultura nella provetta contenente il supporto LB. Sostituire il tappo del tubo in modo che l&#39;ossigeno è ancora in grado di entrare nel tubo - la cultura ha bisogno di crescere in condizioni aerobiche. Tubo a 37 ° C in un incubatore agitazione a 220 rpm overnight. </li><li> Il giorno successivo, trasferire 5 ml di coltura durante la notte in una nuova provetta con supporti ml 3 LB e crescere in condizioni statiche in un incubatore notte a 37 ° C. </li><li> Il giorno 3 di trasferimento 40 microlitri della cultura in aa tubo da 50 ml contenente 40 ml ciascuno dei media LB. Mettere in una notte a 37 ° C statico incubatore. </li><li> Accendere e impostare centrifuga a 4 ° C.Una volta che ha raggiunto la temperatura centrifuga finale, trasferire ogni 40 ml di coltura in una provetta da centrifuga 40 ml Nalgene. </li><li> Pesare tubi per bilanciare centrifuga - regolare il volume con i media LB, se necessario. Spin tubi a 6.000 rpm per 20 min. </li><li> Rimuovere con attenzione il surnatante e delicatamente sospendere pellet batterico in 40 ml di PBS sterile. </li><li> Ripetere il punto 1.5 con PBS. </li><li> Aspirare il surnatante e sospendere off batteri in 500 microlitri di PBS sterile. Trasferire la soluzione in una provetta da microcentrifuga 1,5 ml. Prelevare 10 ml di sospensione e diluire in 990 ml di ghiacciata PBS. Usare uno spettrofotometro per leggere le OD 420 nm per determinare il volume necessario per inoculazione. Per determinare il volume appropriato di ghiacciata PBS di sospendere pellet in: Prendere l&#39;OD 420 nm numero (in ml) e sottrarre il volume stimato del pellet batterico [es. OD 420 nm = 0,545, pellet di circa 0,075 ml. 0,545-0,075 = 0,470]. </li><li> Rimuovere 10 pl di sospensione di batteri e diliuto nel 990 microlitri ghiacciata PBS. Leggi le nm 420 OD. L&#39;obiettivo è quello di raggiungere un valore di nm OD 420 per la sospensione diluita di 1.000 ± 0,010. Se il numero della diluizione è al di sopra di tale obiettivo, aggiungere più volume alla sospensione e prendere un&#39;altra lettura. Se il numero è inferiore al 0,990 poi centrifugare la sospensione e ripetere la lettura OD 420 nm fino ad ottenere lettura desiderata. </li></ol> 2. L&#39;infezione degli animali Mice (5-7 settimane di vita, dieci per gruppo) sono stati acquistati dalla Jackson Laboratory (Bar Harbor, ME). <ol><li> I topi vengono anestetizzati per inalazione del 1-4% isoflurano in una camera di plexiglas e sono controllati fino a quando reclinata. </li><li> Un mouse alla volta viene preso per instillazione di batteri tramite cateterismo con polietilene (PE10) catetere attaccato ad un ago 30 G modificata di una siringa Hamilton vetro (lunghezza di 1,5-2,0 cm). Il catetere viene inserito al mozzo dell&#39;ago. Il mouse è placed sulla sua superficie dorsale in anestesia mantenute usando un cono. </li><li> Il pene del mouse viene estrusa con una leggera pressione e la quantità di lubrificante liberali su tamponi di cotone viene utilizzato per la lubrificazione di l&#39;ingresso dell&#39;uretra peniena. </li><li> 10 pl di PBS contenente 1 x 10 8 batteri viene introdotto nell&#39;uretra di topi anestetizzati dopo cateterizzazione. </li><li> I topi vengono mantenuti in uno stato anestetizzato per 15 min nella camera plexiglas dopo introduzione batterica per consentire attacco batterico e di prevenire la minzione immediata. </li><li> I topi vengono rimesse nelle loro gabbie e monitorato per le successive 24 ore. </li></ol> 3. Comportamento Test I topi sono stati testati prima dell&#39;infezione (basale) e nei giorni 3, 7, 14, 21 e 28 dopo l&#39;infezione. Cui iperalgesia e allodinia tattile è stato testato utilizzando von Frey filamenti applicato sull&#39;addome 11,12 e il r plantareEgion della zampa posteriore 13. Il test è stato perfomed in un determinato momento della giornata, metodologia standard e test sperimentatore unico di tutti gli animali sono stati impiegati. Accecato test dei gruppi è stato utilizzato per combattere le limitazioni di comportamento basato su test del dolore in modelli animali. Cinque filamenti singoli von Frey con forze di 0,04, 0,16, 0,4, 1,0 e 4,0 g (Stoelting, USA) sono stati applicati al ventre e frequenza delle risposte ritiro è stato calcolato. <ol><li> Dopo un periodo di 30 minuti di acclimatazione, i topi sono posti in camere singole plexiglas (6 x 10 x 12 cm) realizzati in casa con un pavimento in acciaio griglia di filo di acciaio. </li><li> Riferito iperalgesia e allodinia tattile sono stati testati utilizzando i cinque filamenti di von Frey. Ogni filamento è stato applicato per 1-2 sec con un inter-stimolo intervallo di 5 secondi per un totale di 10 volte, ed i peli sono stati testati in ordine crescente di forza. </li><li> Ogni filamento è applicato alla zona addominale inferiore in prossimità generale della prostata ecura è stata presa per stimolare diverse aree all&#39;interno di questa regione per evitare di desensibilizzazione o &quot;liquidazione&quot; effetti. Filamenti sono stati applicati in ordine crescente di forza per 1-2 secondi con intervalli di almeno 5 secondi tra stimoli per un totale di 10 volte. </li><li> Tre tipi di comportamento sono considerati una risposta positiva alla stimolazione filamento: 1) retrazione acuto dell&#39;addome; 2) leccare immediata o graffiare dell&#39;area di stimolazione filamento; 3) salto. </li><li> Risposta in frequenza è stata calcolata come percentuale di risposta positiva (di 10, ad esempio 5 risposte di 10 = 50%) e di dati è stata riportata come percentuale media di risposta in frequenza ± SE </li><li> Animali con più di 25 risposte positive totale basale sono esclusi dallo studio. </li><li> Allodinia tattile è stato testato sulla regione plantare della zampa posteriore con filamenti di von Frey con le forze di 0,04, 0,16, 0,4, 1,0 e 4,0 g. La soglia media di ritiro al 50% (5) è stata valutata utilizzando il up-downmetodo in cui è stato avviato il test con 0,04 g filamento applicata perpendicolarmente alla superficie plantare della zampa posteriore finché il filamento leggermente piegate. Filamenti sono stati testati in ordine ascendente fino una risposta positiva è stata osservata. Una risposta positiva al filamento è stata definita come un brusco ritiro della zampa o leccare della zampa test. Quando una risposta positiva è stata registrata successivo filamento più debole è stato applicato, e se una risposta negativa è stata osservata, quindi il successivo filamento forte è stato applicato. </li><li> Gli esperimenti e le metodologie descritte sono stati esaminati e approvati dalla cura degli animali Northwestern University e utilizzare comitato. </li></ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+type+1+pili+and+effects+of+phase+variation+on+lower+urinary+tract+infections+produced+by+Escherichia+coli.">Role of type 1 pili and effects of phase variation on lower urinary tract infections produced by Escherichia coli.</a> Infect. Immun. 50, 370-377 (1985).</li><li>Jarrell, J., Giamberardino, M. A., Robert, M., Nasr-Esfahani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bedside+testing+for+chronic+pelvic+pain:+discriminating+visceral+from+somatic+pain.">Bedside testing for chronic pelvic pain: discriminating visceral from somatic pain.</a> Pain research and treatment. 2011, 692102 (2011).</li><li>Jarrell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demonstration+of+Cutaneous+Allodynia+in+Association+with+Chronic+Pelvic+Pain.">Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain.</a> J. Vis. Exp. (28), e1232 (2009).</li><li>Giamberardino, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viscero-visceral+hyperalgesia:+characterization+in+different+clinical+models.">Viscero-visceral hyperalgesia: characterization in different clinical models.</a> Pain. 151, 307-322 (2010).</li><li>Laird, J. M., Martinez-Caro, L., Garcia-Nicas, E., Cervero, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+of+visceral+pain+and+referred+hyperalgesia+in+the+mouse.">A new model of visceral pain and referred hyperalgesia in the mouse.</a> Pain. 92, 335-342 (2001).</li><li>Laird, J. M., Souslova, V., Wood, J. N., Cervero, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficits+in+visceral+pain+and+referred+hyperalgesia+in+Nav1.8+(SNS/PN3)-null+mice.">Deficits in visceral pain and referred hyperalgesia in Nav1.8 (SNS/PN3)-null mice.</a> J. Neurosci. 22, 8352-8356 (2002).</li><li>Chaplan, S. R., Bach, F. W., Pogrel, J. W., Chung, J. M., Yaksh, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+tactile+allodynia+in+the+rat+paw.">Quantitative assessment of tactile allodynia in the rat paw.</a> Journal of neuroscience. 53, 55-63 (1994).</li><li>Schaeffer, A. J., Schwan, W. R., Hultgren, S. J., Duncan, J. 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Nessun conflitto di interessi dichiarati.

Infezione della prostata mouse con UPEC consente la modellazione in vivo di eventi che possono essere coinvolte nella patogenesi della prostatite batterica, CP / CPPS o come un evento predisponente infiammazione cronica. I metodi descritti per la preparazione di batteri e instillazione pareggio su un vasto corpo di letteratura sui modelli UPEC nelle infezioni del tratto urinario femminile 7,14. Il modello dispone di ampia applicabilità per studiare la patogenesi, la sperimentazione vaccini candidati...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Cultura tubi </td><td> BD Falcon </td><td> 352059 </td></tr><tr><td> LB Broth Miller </td><td> EMD </td><td> EM1.10285.0500 </td></tr><tr><td> Tubi per centrifuga Nalgene </td><td> Thermo </td><td> 3118-0050 </td></tr><tr><td> Isoflurano </td><td> Butler Schein </td><td> NDC 11695-6776-1 </td></tr><tr><td> Catetere ago 30G </td><td> Hamilton </td><td> 91030 </td></tr><tr><td> PE10 tubi </td><td> BD intramedic </td><td> 427400 </td></tr><tr><td> Von Frey filamenti </td><td> Stoelting </td><td> 58025-31 </td></tr></tbody></table>

measurement of tactile allodynia in a murine model of bacterial prostatitis

Uropatogeni Escherichia coli (UPEC) sono agenti patogeni che svolgono un ruolo importante nelle infezioni delle vie urinarie e prostatite batterica 1. Abbiamo recentemente dimostrato che UPEC hanno un ruolo importante nell&#39;avvio di dolore pelvico cronico 2, una caratteristica di prostatite cronica / sindrome dolorosa pelvica cronica (CP / CPPS) 3,4. L&#39;infezione della prostata da parte UPEC clinicamente rilevante in grado di avviare e definire il dolore cronico attraverso meccanismi che possono comportare danni ai tessuti e l&#39;avvio di meccanismi di autoimmunità 5. Una sfida per comprendere la patogenesi della UPEC nella prostata è relativa inaccessibilità della prostata alla manipolazione. Abbiamo utilizzato un metodo precedentemente descritto infezione intrauretrale 6 per fornire un ceppo clinico di UPEC in topi maschi stabilendo, quindi un&#39;infezione ascendente della prostata. Qui, descriviamo i nostri protocolli per uniformare le bacterial inoculo 7 nonché la procedura per la cateterizzazione anestetizzati topi maschi per instillazione di batteri. CP / CPPS è principalmente caratterizzata dalla presenza di allodinia tattile 4. Test comportamento era basato sul concetto di iperalgesia cutanea risultante da cui 8-10 dolore viscerale. Un focus irritabile nei tessuti viscerali riduce la soglia del dolore cutaneo che consentano una risposta esagerata al normalmente non dolorosi stimoli (allodinia). Applicazione della forza normale al risultato pelle in risposte anomale che tendono ad aumentare con l&#39;intensità del dolore viscerale sottostante. Descriviamo metodologia in NOD / ShiLtJ topi che utilizzano fibre di von Frey quantificare allodinia tattile nel tempo in risposta ad una singola infezione con batteri dell&#39;UPEC.

<html> Abbiamo esaminato topi NOD / ShiLtj e maschi C57BL/6J per la presenza di dolore cronico dopo infezione batterica. Topi maschi (5 a 7 settimane di età) sono stati instillati con soluzione salina o batteri nell&#39;uretra (Figura 1). Stimolazione meccanica della zona pelvica con von Frey filamenti di soluzione salina-instillato topi C57BL/6J e UPEC topi infettati C57BL/6J portato ad una risposta in frequenza che non è cambiato durante i 28 giorni di corso dell&#39;esperimento (Figura 1).</...

Watch this Scientific Journal Video about Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis at JoVE.com

Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis

L&#39;infezione della prostata può essere un fattore che contribuisce a mediare dolore pelvico in prostatite cronica. Descriviamo la procedura per la preparazione di standard inoculo batterico, l&#39;instillazione di batteri nell&#39;uretra di topi maschi e la metodologia per la misurazione allodinia tattile nei topi nel corso del tempo.

Misurazione della tattile Allodinia in un modello murino di prostatite batterica

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis1. We have recently ...

measurement-tactile-allodynia-murine-model-bacterial

Research

JoVE Journal

Immunology and Infection

15.7K Views.  Northwestern University Feinberg School of Medicine. L'infezione della prostata può essere un fattore che contribuisce a mediare dolore pelvico in prostatite cronica. Descriviamo la procedura per la preparazione di standard inoculo batterico, l'instillazione di batteri nell'uretra di topi maschi e la metodologia per la misurazione allodinia tattile nei topi nel corso del tempo.

Video: Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

L&#39;introduzione di shear stress nello studio di adesione batterica

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Ricerca

Immunologia e infezioni

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Induzione di Graft-versus-host disease e<em&gt; In Vivo</em&gt; T monitoraggio cellulare Utilizzando un MHC-abbinato modello murino

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Murine Model of Allergen Induced Asthma

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, with the prevalence increasing.2 As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals.3 Availability of a variety of transgenic strains further increases the attractiveness of these models.4 Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma.5
The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models,5,6 and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive7 and non-invasive8 techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.

Experimental mouse models of allergic asthma offer new possibilities for studying disease pathogenesis and developing new therapeutics. These models are well suited to measuring factors governing the allergic immune response, airway inflammation, and pulmonary pathophysiology.

Modello murino di asma indotta da allergene

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

Misurazione quantitativa della risposta immunitaria e nel sonno<em&gt; Drosophila</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Generation e Multi-fenotipica Screening-Alto contenuto di<em&gt; Coxiella burnetii</em&gt; Mutants transposon

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Un modello murino di Gruppo B<em&gt; Streptococcus</em&gt; Colonizzazione vaginale

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various pathologies such as cancer, atherosclerosis and diabetes. We have previously shown that dysfunction of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/ Kelch-like erythroid cell-derived protein 1 (Keap1) signaling pathway leads to extreme ROS imbalance during cutaneous wound healing in diabetes. Since ROS levels are an important indicator of progression of wound healing, specific and accurate quantification techniques are valuable. Several in vitro assays to measure ROS in cells and tissues have been described; however, they only provide a single cumulative measurement per sample. More recently, the development of protein-based indicators and imaging modalities have allowed for unique spatiotemporal analyses. L-012 (C13H8ClN4NaO2) is a luminol derivative that can be used for both in vivo and in vitro chemiluminescent detection of ROS generated by NAPDH oxidase. L-012 emits a stronger signal than other fluorescent probes and has been shown to be both sensitive and reliable for detecting ROS. The time lapse applicability of L-012-facilitated imaging provides valuable information about inflammatory processes while reducing the need for sacrifice and overall reducing the number of study animals. Here, we describe a protocol utilizing L-012-facilitated in vivo imaging to quantify oxidative stress in a model of excisional wound healing using diabetic mice with locally dysfunctional Nrf2/Keap1.

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

We describe a non-invasive in vivo imaging protocol that is streamlined and cost-effective, utilizing L-012, a chemiluminescent luminol-analog, to visualize and quantify reactive oxygen species (ROS) generated in a mouse excisional wound model.

In Vivo Formazione immagine di specie reattive dell'ossigeno in un murino ferita modello

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various ...

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected in the urinary tract. The protocol can be broadly divided into three sections: the infection, bladder epithelial cell harvesting, and mouth micropipetting to isolate individual infected epithelial cells. The isolated epithelial cell contains viable bacterial cells and is nearly free of contaminating extracellular bacteria, making it ideal for downstream single-cell analysis. The time taken from the start of infection to obtaining a single intracellular bacterial community is about 8 h. This protocol is inexpensive to deploy and uses widely available materials, and we anticipate that it can also be utilized in other infection models to isolate single infected cells from cell mixtures even if those infected cells are rare. However, due to a potential risk in mouth micropipetting, this procedure is not recommended for highly infectious agents.

This protocol describes a simple method of isolating single, infected-bladder epithelial cells from a murine model of urinary tract infection.

Isolamento di singole comunità batteriche intracellulari generati da un modello murino di infezione delle vie urinarie per analisi unicellulare a valle

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

Un High-throughput Shigella-specifica analisi battericida

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

A Precise Pathogen Delivery and Recovery System for Murine Models of Secondary Bacterial Pneumonia

Secondary bacterial pneumonias following influenza infections consistently rank within the top ten leading causes of death in the United States. To date, murine models of co-infection have been the primary tool developed to explore the pathologies of both the primary and secondary infections. Despite the prevalence of this model, considerable discrepancies regarding instillation procedures, dose volumes, and efficacies are prevalent among studies. Furthermore, these efforts have been largely incomplete in addressing how the pathogen may be directly influencing disease progression post-infection. Herein we provide a precise method of pathogen delivery, recovery, and analysis to be used in murine models of secondary bacterial pneumonia. We demonstrate that intratracheal instillation enables an efficient and accurate delivery of controlled volumes directly and evenly into the lower respiratory tract. Lungs can be excised to recover and quantify the pathogen burden. Following excision of the infected lungs, we describe a method to extract high quality pathogen RNA for subsequent transcriptional analysis. This procedure benefits from being a non-surgical method of delivery without the use of specialized laboratory equipment and provides a reproducible strategy to investigate pathogen contributions to secondary bacterial pneumonia.

Here, we present methods to improve secondary bacterial pneumonia studies by providing a non-invasive route of instillation into the lower respiratory tract followed by pathogen recovery and transcript analysis. These procedures are reproducible and can be performed without specialized equipment such as cannulas, guide wires, or fiber optic cables.

Un sistema preciso di distribuzione e recupero di patogeni per i modelli Murine di polmonite batterica secondaria

Secondary bacterial pneumonias following influenza infections consistently rank within the top ten leading causes of death in the United States. To date, ...

Misurazione della tattile Allodinia in un modello murino di prostatite batterica

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L'introduzione di shear stress nello studio di adesione batterica

Induzione di Graft-versus-host disease e

Modello murino di asma indotta da allergene

Misurazione quantitativa della risposta immunitaria e nel sonno

Generation e Multi-fenotipica Screening-Alto contenuto di

Un modello murino di Gruppo B

In Vivo Formazione immagine di specie reattive dell'ossigeno in un murino ferita modello

Isolamento di singole comunità batteriche intracellulari generati da un modello murino di infezione delle vie urinarie per analisi unicellulare a valle

Un High-throughput Shigella-specifica analisi battericida

Un sistema preciso di distribuzione e recupero di patogeni per i modelli Murine di polmonite batterica secondaria