Bu araştırma NIH / NIDDK hibe 1K01DK079019A2 (PT) tarafından desteklenmiştir.

1. Fare Aşılama için bakteri Hazırlık Aşağıdaki aseptik koşullar altında yapılmalıdır. <ol><li> Kapakları biri 17 x 100 mm polipropilen tüpler atın ve taze Luria broth (LB) medya 3 ml ekleyin. Otoklavlanmış ipuçlarını kullanarak, gerginlik CP1 2 bazı dondurulmuş bakteri gliserol stok almak ve LB ortamı içeren tüpe kültürünün küçük bir miktar transfer. Oksijen yine tüp girebilir, böylece tüp kapak değiştirin - kültür aerobik koşullar altında büyüyebilir gerekmektedir. 37 Sıra tüp ° gecede 220 rpm&#39;de bir sallayarak inkübatör C. </li><li> Ertesi gün, 3 ml LB ortamı ile yeni bir tüp vermek üzere gece boyunca kültür 5 ul aktarmak ve 37 ° C &#39;de bir gece boyunca kuluçka makinesi içinde statik koşullar altında büyüyebilir </li><li> Günde aa 50 ml tüp içinde 3 taşıma kültürün 40 ul, 40 ml LB ortamı her içeren. 37 ° C sabit inkübatör gecede yerleştirin. </li><li> Açın ve 4 ° C&#39;ye santrifüj ayarlayınBir kez santrifüj son sıcaklığa ulaştığını, 40 ml Nalgene santrifüj tüpüne her 40 ml kültürü aktarmak. </li><li> Gerektiğinde LB medya ile ses seviyesini ayarlamak - santrifüj dengelemek için tüpleri tartılır. 20 dakika boyunca 6000 rpm&#39;de tüpler Spin. </li><li> Dikkatlice süpernatant kaldırmak ve yavaşça 40 ml steril PBS bakteriyel pelet askıya. </li><li> PBS ile adım 1.5 tekrarlayın. </li><li> Süpernatantı kapalı aspire ve 500 ul steril PBS içinde bakteri askıya. 1.5 ml mikrofuge&#39;de tüpüne çözümü aktarın. Süspansiyonu 10 ul alın ve buz gibi soğuk PBS 990 ul seyreltin. Aşılama için gerekli hacim belirlemek için OD 420 nm okumak için bir spektrofotometre kullanın. Uygun miktarda belirlemek için buz gibi soğuk PBS içinde pelet askıya: OD 420 nm sayısı (ml) alın ve bakteriyel pelet [ex tahmini hacmi çıkarın. OD 420 nm = 0.545, pelet yaklaşık 0.075 ml. 0,545-0,075 = 0,470]. </li><li> 10. Bakteriyel süspansiyon ul ve di kaldırma990 ul buz gibi soğuk PBS içinde ud. OD 420 nm okuyun. Hedefi 1.000 seyreltilmiş süspansiyonu ± .010 bir OD 420 nm değeri elde etmektir. Sizin seyreltme sayısı bu hedefin üzerinde ise, süspansiyon daha fazla hacim kazandırmak ve başka bir okuma almak. Numarası altında ise 0,990 ardından istediğiniz okuma elde edilinceye kadar erteleme ve tekrar OD 420 nm okuma aşağı spin. </li></ol> 2. Hayvan Enfeksiyon Fareler (5-7 haftalık, her grupta on) Jackson Laboratory (Bar Harbor, ME) &#39;den satın alındı. <ol><li> Fare bir pleksiglas odasında izofluran% 1-4 solunması ile anestezi ve yatık kadar izlenir. </li><li> Bir seferde bir fare bir cam Hamilton şırınga (1.5-2.0 cm uzunluğunda) değiştirilmiş 30 G iğne takılı bir polietilen (PE10) kateter ile kateterizasyon ile bakterilerin instilasyonu için alınır. Kateter iğne merkeze yerleştirilir. Fare Plac olduğunued anestezi altında dorsal yüzeyi üzerinde bir burun konisi kullanılarak yapılmaktadır. </li><li> Fare penis penil üretra girişinde yağlanması için kullanılan hafif basınç ve pamuklu çubuk üzerine yağ bolca tarafından çekilmektedir. </li><li> 10 ul fosfat-tamponlu 1 x 10 8 bakteri içeren tuzlu kateterizasyon anestetize farelerin üretra içine katılmaktadır. </li><li> Bakteri bağlanma sağlamak için acil idrara çıkma ve önlemek için bakteri giriş sonra fareler pleksiglas bölme içinde 15 dakika süreyle bir anestezi altındaki bir durumda muhafaza edilir. </li><li> Fare onların tekrar kafeslerine yerleştirildi ve sonraki 24 saat boyunca izlenir. </li></ol> 3. Davranış Testi Fare öncesinde enfeksiyon (bazal) ve gün 3, 7, 14, 21 ve enfeksiyon sonrası 28 üzerinde test edildi. Referans hiperaljezi ve dokunsal allodini karın 11,12 ve plantar r uygulanan von Frey filamentleri kullanılarak test edildiarka pençe 13 egion. Test-saati günlük sabit bir de perfomed edildi, standart metodoloji ve tüm hayvanların tek deneyci testi kullanılmıştır. Grupların Kör test hayvan modellerinde davranış bazlı test ağrı ile ilgili sınırlamalar ile mücadele için kullanılmıştır. 0.04 arasında kuvvetler, 0.16, 0.4, 1.0 ve 4.0 g (Stoelting, ABD) ile beş ayrı von Frey filamentlerin çekilme yanıtların karın ve frekans uygulanmıştır hesaplanmıştır. <ol><li> Alışma sürecinden bir 30 dakika süre sonra, fareler bir paslanmaz çelik tel ızgara taban ile evde yapılan bireysel pleksiglas odacığı (6 x 10 x12 cm) yerleştirilmiştir. </li><li> Referans hiperaljezi ve dokunsal allodini beş von Frey filamentleri kullanılarak test edildi. Her filament toplam 10 kez 5 sn bir uyarıcılar arası süre ile 1-2 saniye uygulandı ve tüyleri kuvvet artan düzende test edildi. </li><li> Her bir fılaman, prostat Genel çevresinde alt karın bölgesine uygulanır vebakım duyarsızlaştırma önlemek veya etkilerini &quot;rüzgar&quot; Bu bölgedeki farklı alanlarda teşvik götürüldü. Filamentler 10 kez toplam uyarımlar arasında en az 5 saniye aralıklarla 1-2 saniye için kuvvet artan sırada uygulanmıştır. </li><li> Davranış üç tip filament stimülasyon olumlu bir yanıt olarak kabul edilir: karın 1) keskin retraksiyonu; 2) hemen yalama veya filament stimülasyon alan kazıma; 3) atlama. </li><li> Tepki frekansı olumlu yanıt yüzdesi (10 üzerinden, 10 =% 50 örneğin 5 karşılıklar) olarak hesaplandı ve data cevabı frekans ortalama yüzdesi ± SE olarak rapor edildi </li><li> 25&#39;den fazla pozitif toplam taban yanıtları Hayvanlar çalışmanın dışında tutulmuştur. </li><li> Dokunsal allodini 0.04 güçleri, 0.16, 0.4, 1.0 ve 4.0 g ile von Frey filamentleri kullanılarak arka pençenin plantar bölgeye üzerinde test edildi. Medyan% 50 geri çekme eşiği (5) yukarı-aşağı tuşlarını kullanarak değerlendirildifilaman hafif bir açı kadar test 0.04 g filaman ile başlatılan yöntem arka pençenin plantar yüzeye dik olarak uygulanır. Filament olumlu bir yanıt gözlenmiştir kadar artan sırayla test edildi. Filament pozitif tepki testi pençe pençe ya yalama keskin bir geri çekilme ya olarak tanımlandı. Olumlu bir yanıt kaydedildi zaman bir sonraki daha zayıf filaman uygulandı, ve bir negatif cevap gözlendi, sonra bir sonraki daha güçlü bir filaman uygulanmıştır. </li><li> Açıklanan deneyler ve metodoloji incelenen ve onaylanan Northwestern Üniversitesi hayvan bakımı ile ve komite kullanabilirsiniz edilmiştir. </li></ol>

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Çıkar çatışması ilan etti.

UPEC ile fare prostat enfeksiyonu bakteriyel prostatit, CP / CPPS patogenezinde veya kronik inflamasyon bir predispozan olay olarak dahil olabilir olayların in vivo modelleme için sağlar. Yöntemler kadın idrar yolu enfeksiyonu 7,14 in UPEC modellerde edebiyat büyük bir gövde üzerine bakteriyel hazırlanması ve instillasyon beraberlik için nitelendirdi. Modeli potansiyel aşı adayları ve immün modülasyon mekanizmalarının test patogenezinde çalışmak için geniş uygulanabilirliği v...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> Kültür tüpleri </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> Nalgene Santrifüj Tüpleri </td><td> Thermo </td><td> 3118-0050 </td></tr><tr><td> İzofluran </td><td> Butler Schein </td><td> NDC 11695-6776-1 </td></tr><tr><td> Kateter iğne 30G </td><td> Hamilton </td><td> 91030 </td></tr><tr><td> PE10 boru </td><td> BD intramedic </td><td> 427400 </td></tr><tr><td> Von Frey filamentler </td><td> Stoelting </td><td> 58025-31 </td></tr></tbody></table>

measurement of tactile allodynia in a murine model of bacterial prostatitis

Üropatojenik Escherichia coli (UPEC) idrar yolu enfeksiyonları ve bakteriyel prostatit 1 önemli bir rol oynamaktadır patojenlerdir. Biz son zamanlarda UPEC kronik pelvik ağrı 2, Kronik prostatit / kronik pelvik ağrı sendromu (CP / CPPS) 3,4 bir özelliği başlatılmasında önemli bir role sahip olduğunu göstermiştir. Klinik UPEC ile prostat enfeksiyonu doku hasarı ve otoimmünite 5 mekanizmalarının başlatılması içerebilir mekanizmalar yoluyla kronik ağrı başlatmak ve kurabilir. Prostat UPEC patogenezini anlamak için bir meydan okuma manipülasyon prostat bezinin nispi erişilememesi olduğunu. Böylece de prostat bir artan enfeksiyon oluşturma erkek farelere UPEC bir klinik suşu vermek üzere, daha önce anlatılan intraüretral enfeksiyon yöntemiyle 6 kullanılmıştır. Burada, biz ba standartlaştırılması için protokoller tarifgibi bakteri perfüze anestetize erkek fareler catheterizing için işlem olarak cterial inokulum 7. CP / CPPS öncelikle dokunsal allodini 4 varlığı ile karakterizedir. Davranış testleri ifade viseral ağrı 8-10 kaynaklanan kutanöz hiperaljezi kavramına dayanmaktadır. Visseral dokularda bir irritabl odak normal olmayan ağrılı uyaranlar (allodini) için abartılı bir tepki veren Kutanöz ağrı eşikleri azaltır. Yatan visseral ağrının şiddeti ile artış eğilimindedir anormal yanıtları cilt sonucu normal kuvvet uygulanması. Bu UPEC bakteri ile bir tek enfeksiyonuna yanıt olarak zaman içinde dokunsal allodini ölçmek için von Frey lifler kullanmak NOD / ShiLtJ farelerde yöntemi açıklanmaktadır.

<html> Biz bakteriyel enfeksiyon üzerine kronik ağrı varlığı için NOD / ShiLtj ve C57BL/6J erkek farelerde incelenmiştir. Erkek fareler (5-7 haftalık) üretra (Şekil 1) içine salin ya da bakteri ile damlatıldı edildi. Salin-coşturup C57BL/6J fareler ve UPEC ile enfekte C57BL/6J farelerin von Frey filamentleri ile pelvik bölgeye mekanik uyarılması deney 28 günlük kurs (Şekil 1) sırasında değişmedi bir tepki frekansı sonuçlandı. Buna karşılık, UPEC-enfekte NOD...

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

Prostat enfeksiyonu kronik prostatit pelvik ağrı arabuluculuk katkıda bulunan bir faktör olabilir. Biz standardize edilmiş bakteriyel inokulum, zamanla farelerde dokunsal allodini ölçmek için erkek farelerde ve metodoloji üretra içine bakteri instilasyon hazırlama prosedürü açıklanmaktadır.

Bakteriyel Prostatit bir fare modelinde Dokunsal allodini Ölçümü

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 Görüntüleme.  Northwestern University Feinberg School of Medicine. Prostat enfeksiyonu kronik prostatit pelvik ağrı arabuluculuk katkıda bulunan bir faktör olabilir. Biz standardize edilmiş bakteriyel inokulum, zamanla farelerde dokunsal allodini ölçmek için erkek farelerde ve metodoloji üretra içine bakteri instilasyon hazırlama prosedürü açıklanmaktadır.

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.

Bakteriyel Yapışma Çalışma Kesme Stres Tanıtımı

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

İmmünoloji ve Enfeksiyon

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.

Graft-versus-host hastalığı indüksiyonu ve<em&gt; In Vivo</emBir MHC-eşlemeli murin Model Kullanma&gt; T Hücre İzleme

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.

Alerjen Bağlı Astım murin Modeli

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.

İmmün Yanıt ve Uyku içinde kantitatif ölçümü<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.

Üretim ve Çok fenotipik Yüksek içerik Eleme<em&gt; Coxiella burnetii</em&gt; Transposon Mutantlar

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.

B Grubu bir murin modelinde<em&gt; Streptococcus</em&gt; Vajinal Kolonizasyon

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.

Vivo Reaktif oksijen türleri içinde bir fare görüntüleme modeli yara

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.

İdrar yolu enfeksiyonu aşağı akım tek hücreli analiz için bir fare modelinden oluşturulan tek hücre içi bakteriyel toplulukların yalıtım

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.

Bir yüksek-den geçerek Shigella-belirli bakterisidal tahlil

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.

Sekonder Bakteriyel Pnömoni Murine Modelleri için Hassas Patojen Dağıtım ve Kurtarma Sistemi

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

Bakteriyel Prostatit bir fare modelinde Dokunsal allodini Ölçümü

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Bakteriyel Yapışma Çalışma Kesme Stres Tanıtımı

Graft-versus-host hastalığı indüksiyonu ve

Alerjen Bağlı Astım murin Modeli

İmmün Yanıt ve Uyku içinde kantitatif ölçümü

Üretim ve Çok fenotipik Yüksek içerik Eleme

B Grubu bir murin modelinde

Vivo Reaktif oksijen türleri içinde bir fare görüntüleme modeli yara

İdrar yolu enfeksiyonu aşağı akım tek hücreli analiz için bir fare modelinden oluşturulan tek hücre içi bakteriyel toplulukların yalıtım

Bir yüksek-den geçerek Shigella-belirli bakterisidal tahlil

Sekonder Bakteriyel Pnömoni Murine Modelleri için Hassas Patojen Dağıtım ve Kurtarma Sistemi