الكتاب ممتنون للمجتمعات المحلية في مقاطعة يوننان، وجمهورية الصين الشعبية وأغبوفيل، وكوت ديفوار، لمشاركتهم في الدراسات المذكورة أعلاه. نحن مدينون للفرق الميدانية المحلية التي ساعدت في اختبار وتنفيذ البروتوكولات. ونحن ممتنون بشكل خاص للزملاء التالية من المعهد السويسري الصحة العامة الاستوائية و: إيفيت Endriss لتوفير الصور المجهرية، هانزبيتر مارتي وفريقه على الدعم المختبري وKrauth ستيفاني لمساعدتها أثناء تصوير الفيديو. وأخيرا، نشكر الحكام 2 مجهول لسلسلة من الملاحظات المفيدة.

1. كاتو كاتز، وتقنيات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وضع معيار كاتو كاتز-قالب مع وجود ثقب لعقد 41،7 ملليغرام (ملغم) من البراز على شريحة المجهر. </li><li style=";text-align:right;direction:rtl"> حلج القطن 2-3 غرام (ز) من عينة البراز الطازجة على قطعة من الصحيفة أو رقائق الألومنيوم، والضغط على قطعة من الأسلاك أو شبكة بلاستيكية على رأس لغربال ذلك. </li><li style=";text-align:right;direction:rtl"> باستخدام ملعقة صغيرة من البلاستيك، كشط المواد منخول قبالة شبكة وملء الحفرة تماما في قالب كاتو كاتز-معها. لإزالة المواد البرازية يزيد، ومستوى المحتوى من الحفرة مع ملعقة. </li><li style=";text-align:right;direction:rtl"> إزالة القالب عموديا من دون إزعاج المواد البرازية الانضمام الآن إلى الشريحة المجهر. يمكن تنظيفها القالب وملعقة في الماء مع مواد التنظيف، وإعادة استخدامها. </li><li style=";text-align:right;direction:rtl"> ضع قطعة من السيلوفان، قبل غارقة في الحل الخضراء الغليسيرين-المرمر ل24 ساعة على الأقل (HR)، على عينة البراز على الشريحة المجهر. </li><li style=";text-align:right;direction:rtl"> لنشر المواد البرازية إلى تشويه سميكة، اضغط بلطف الصغر نظيفةالشريحة نطاق ضد الشريحة عينة، وتوزيع المواد بشكل متساو ضمن دائرة يبلغ قطرها أصغر قليلا من اتساع الشريحة المجهر. </li><li style=";text-align:right;direction:rtl"> تسمح الشريحة لمسح ل 30-60 دقيقة (دقائق)، حيث يجب أن يبقى بعيدا عن الشرائح أشعة الشمس المباشرة. ثم، ودراسة منهجية مسحة سميكة تحت المجهر ضوء (40-100X التكبير). حساب عدد البيض STH وتطبق عليهم من قبل الأنواع (أي A.lumbricoides، T.trichiura، ودودة الأنكلستوما). من ملاحظة، ويمكن أيضا البيض الديدان الطفيلية الأخرى (مثل البلهارسيا المنسونية) يمكن الكشف عنها بواسطة هذا الأسلوب، وأن تفرز وسجلت على حدة. </li><li style=";text-align:right;direction:rtl"> للحصول على تقديرات موحدة من شدة الإصابة، وأعرب عن تقليديا عدد من البيض في كل غرام من البراز (EPG)، ضرب عدد البيض بنسبة 24 (24 × 41،7 ملغ ≈ 1 غرام). </li></ol> 2. بيرمان وتقنيات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إنشاء موقف التي يمكن أن تعقد كوب قمع وأنبوب المطاط إرفاق أغلقت الطرافةمقطع هكتار، إلى الجزء السفلي من القمع. </li><li style=";text-align:right;direction:rtl"> وضع منخل أو قطعة من سلك الشاشة على شكل مخروط، في قمع طبقة ومع قطعة من الشاش الطبي. </li><li style=";text-align:right;direction:rtl"> تملأ قمع مع ماء الصنبور والتأكد من أن كل مجموعة المتابعة لا تسرب. </li><li style=";text-align:right;direction:rtl"> حلج القطن 20-30 غرام من البراز الطازجة على منتصف الشاش الطبي وضمان مغمورة تماما في الماء. إضافة المزيد من الماء إذا لزم الأمر. طي الشاش الطبي على العينة. </li><li style=";text-align:right;direction:rtl"> من الأسفل، تألق الضوء الاصطناعي في قمع لمدة 2 ساعة. </li><li style=";text-align:right;direction:rtl"> استنزاف 50 ملليلتر (مل) من الجزء السفلي من المياه في قمع في أنبوب الطرد المركزي عن طريق الإفراج عن بلطف مقطع على أنبوب المطاط. </li><li style=";text-align:right;direction:rtl"> أجهزة الطرد المركزي لجمع المياه في 600 XG (قوة الطرد المركزي النسبية) لمدة 5 دقائق وتصب بعناية طاف دون الإخلال الرواسب. الإبقاء على قطرات القليلة الماضية في الأنبوب. </li><li style=";text-align:right;direction:rtl"> إعادة تعليق الرواسب وماصة 2 قطرات من الحل على شريحة المجهر ودراسة الشريحةتحت المجهر الخفيفة (40X التكبير للكشف والتكبير 100-400X لتأكيد الأنواع). المرحلة الأولى اليرقات (L1) من S. البرازية على قيد الحياة والتحرك بنشاط تحت المجهر. </li></ol> 3. 20-م اختبار المكوك تشغيل <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قياس 20-م مسطح ومستقيم بالطبع تشغيل وعلامة عليه مع الأقماع. </li><li style=";text-align:right;direction:rtl"> مساعدة المشاركين ارتداء اختبار رصد معدل ضربات القلب على المعصم وجهاز الإرسال المقابلة حول الصدر 18. هذا الجهاز يسمح بقياس معدل ضربات القلب من المشاركين قبل وبعد الاختبار، وبالتالي يراقب ما إذا كان المشارك بذلت جهدا القصوى. </li><li style=";text-align:right;direction:rtl"> إرشاد المشاركين إلى تشغيل ذهابا وإيابا على ملعب باتباع خطى الإشارات الصوتية المسجلة مسبقا ترسيم 20-م لفات. بدءا من سرعة تشغيل قدرها 8.5 كيلومترا في الساعة (كم / ساعة)، وتواتر الإشارات تشير إلى 20 م فترات زيادة بما يعادل 0.5 كم / ساعة كل دقيقة. </li><li style=";text-align:right;direction:rtl"> ضمان الفي المشارك يبدأ جديد 20-م الفاصل في كل مرة يتم بدا إشارة ويستكملها قبل وصول إشارة المقبل. </li><li style=";text-align:right;direction:rtl"> وقف المشارك إذا هو / هي فشل لمتابعة وتيرة فترات لمدة 20 ساعة متتالية. </li><li style=";text-align:right;direction:rtl"> تسجيل أعلى سرعة تشغيل أشار للفاصل الماضي والتي يكتمل المشارك، جنبا إلى جنب مع عمر وجنس المشارك. </li><li style=";text-align:right;direction:rtl"> تحقق من أن تحقيق معدل ضربات القلب في نهاية الاختبار هو معدل الطفل القلب القصوى 19. إذا لم يتم الوصول إلى هذا معدل ضربات القلب، قد لا الطفل بذلت جهدا القصوى، وبالتالي يجب أن يتكرر الاختبار. </li><li style=";text-align:right;direction:rtl"> تقدير السعة القصوى الهوائية (VO 2 الحد الأقصى) من المشاركين، وذلك باستخدام معادلة التي وضعتها ليجيه وزملاء العمل: VO 2 الحد الأقصى = 31.025 + 3.238 * السرعة (في كم / ساعة) - 3،248 * العمر (بالسنوات) + * 0،1536 السرعة (في كم / ساعة) * العمر (بالسنوات). </li></ol> 4. ممثل النتائج من حيثالتشخيص STH مع تقنية كاتو-كاتز، خصائص فريدة من البيض الديدان الطفيلية المختلفة تسمح لتحديد الأنواع الأكثر 20. بيض A.lumbricoides (حوالي 45-75 ميكرومتر (ميكرون) لفترة طويلة واسعة 35-50 ميكرون، الشكل 1) والجدران أكثر سمكا قذيفة من بيض T.trichiura (الشكل 2) والدودة الشصية (الشكل 3). البويضة المخصبة من A.lumbricoides (الشكل 1) لديه معطف الخارجي ألبومينية، مما يجعل الامر يبدو لونها بنيا ذهبيا، في حين أن البيض غير المخصب يفتقر هذا المعطف الخارجي وممدود قليلا وأكبر بشكل عام. بيض T.trichiura (ما يقرب من 50 ميكرومتر طولا و 22 ميكرون، الشكل 2) هي ممدود ولها سدادات القطبية متميزة. الشصية البيض (حوالي 64-76 ميكرومتر طولا و 36-40 ميكرون، الشكل 3)، من ناحية أخرى، لها جدار رقيق جدا، تليها حلقة واضحة حول كتلة كثيفة من الخلايافي وسط البيضة. S.stercoralis L 1 اليرقات (الشكل 4) ميكرومتر ما يقرب من 180-380 طويلة ولها تجويف قصيرة جدا الشدق. ويمكن ملاحظة منشم متميزة الأعضاء التناسلية (تمييز مع السهم الأزرق في الشكل 4) والذيل من اليرقات (تمييز مع سهم أحمر في الشكل 4) لها نهاية حادة. كما لا يزال الأثر الحقيقي من الإصابات STH على اللياقة البدنية للأطفال تناقش 13،14،15، لا يوجد ممثل لنتيجة تأثيرها في هذه المرحلة. لا يمكن أن يؤديها تفسير النتائج م 20-المكوك اختبار المدى بطرق مختلفة. يمكن مقارنة اللياقة البدنية للأطفال بين الموضوعات المصابة وغير المصابة، الذي تتشابه من حيث العمر والجنس، وغيرها من الظروف الصحية ذات الصلة المحتملة والحالة الاجتماعية والاقتصادية، والعادات الثقافية. ويمكن أن يتم ذلك في مسح مقطعي حيث 13،14،15يتم تقييم حالة عدوى STH واللياقة البدنية من الأطفال المصابة وغير المصابة، عند نقطة زمنية معينة. وقوة الأدلة يكون أعلى عند اتباع العشوائية التي تسيطر عليها المحاكمة التصميم حيث يتم تقييم اللياقة البدنية للطفل في سن الدراسة قبل وبعد ديدان / همي العلاج، مما يسمح لكلا تقييم القصيرة والطويلة الأجل. يمكن للمرء أن محاولة أخرى لتحديد الجرعة والاستجابة ممكن العلاقة بين شدة العدوى STH واللياقة البدنية ومقارنة مستويات مختلفة من multiparasitism 1 مع اللياقة البدنية. في العمل المقدم من قبل ياب وزملاء-15، تم العثور على اللياقة البدنية، كما هو مبين من قبل VO 2 الحد الأقصى، على أن تخفض بشكل ملحوظ في الأطفال المصابين T.trichiura مقارنة بالأطفال الذين كانوا غير مصابين T.trichiura (الشكل 5) . <img alt="الشكل 1" src="/files/ftp_upload/3966/3966fig1.jpg" /> فيقو<html> . إعادة 1 الصور المجهرية من البيض A.lumbricoides: المخصبة (A) وغير مخصبة (B) تحت التكبير 500X. شريط النطاق = 50 ميكرومتر. <img alt="الشكل 2" src="/files/ftp_upload/3966/3966fig2.jpg" /> الشكل 2. صورة مجهرية من البيض تحت التكبير 500X T.trichiura. شريط مقياس = 50 ميكرومتر. <img alt="الشكل 3" src="/files/ftp_upload/3966/3966fig3.jpg" /> الشكل 3. صورة مجهرية من البيض تحت التكبير 500X الشصية. شريط النطاق = 50 ميكرومتر. <img alt="الشكل 4" src="/files/ftp_upload/3966/3966fig4.jpg" /> الشكل 4. صورة مجهرية من يرقة S.stercoralis 1 L تحت التكبير 250x. تشير الأسهم الذيل (أحمر) والأعضاء التناسلية منشم (الأزرق). شريط النطاق = 100 ميكرومتر. d/3966/3966fig5.jpg &quot;/&gt; الشكل 5. رسم بياني يوضح مستوى اللياقة البدنية للأطفال المصابين T.trichiura بالمقارنة مع مستوى اللياقة البدنية للأطفال غير مصابين T. الشعرية.

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S., Cazorla, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secular+trends+in+the+performance+of+children+and+adolescents+(1980-2000):+an+analysis+of+55+studies+of+the+20+m+shuttle+run+test+in+11+countries.">Secular trends in the performance of children and adolescents (1980-2000): an analysis of 55 studies of the 20 m shuttle run test in 11 countries.</a> Sports Medicine. 33, 285-300 (2003).</li><li>Mahon, A. D., Marjerrison, A. D., Lee, J. D., Woodruff, M. E., Hanna, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+prediction+of+maximal+heart+rate+in+children+and+adolescents.">Evaluating the prediction of maximal heart rate in children and adolescents.</a> Research Quarterly for Exercise and Sport. 81, 466-471 (2010).</li><li>Bogitsh, B. J., Carter, C. E., Oeltmann, T. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Human Parasitology. , (2011).</li><li>Steinmann, P., Zhou, X. N., Du, Z. W., Jiang, J. Y., Wang, L. B., Wang, X. Z., Li, L. H., Marti, H., Utzinger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Occurence+of+Strongyloides+stercoralis+in+Yunnan+province,+China,+and+comparison+of+diagnostic+methods.">Occurence of Strongyloides stercoralis in Yunnan province, China, and comparison of diagnostic methods.</a> PLoS Neglected Tropical Diseases. 1, e75 (2007).</li><li>Martin, L. K., Beaver, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Kato+thick-smear+technique+for+quantitative+diagnosis+of+helminth+infections.">Evaluation of Kato thick-smear technique for quantitative diagnosis of helminth infections.</a> American Journal of Tropical Medicine and Hygiene. 17, 382-391 (1968).</li><li>Dacombe, R. J., Crampin, A. C., Floyd, S., Randall, A., Ndhlovu, R., Bickle, Q., Fine, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+delays+between+patient+and+laboratory+selectively+affect+accuracy+of+helminth+diagnosis.">Time delays between patient and laboratory selectively affect accuracy of helminth diagnosis.</a> Transactions of the Royal Society of Tropical Medicine and Hygiene. 101, 140-145 (2007).</li><li>Knopp, S., Mgeni, A. F., Khamis, I. S., Steinmann, P., Stothard, J. R., Rollinson, D., Marti, H., Utzinger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+soil-transmitted+helminths+in+the+era+of+preventive+chemotherapy:+effect+of+multiple+stool+sampling+and+use+of+different+diagnostic+techniques.">Diagnosis of soil-transmitted helminths in the era of preventive chemotherapy: effect of multiple stool sampling and use of different diagnostic techniques.</a> PloS Neglected Tropical Diseases. 2, e331 (2008).</li></ol>

الإعلان عن أي تضارب في المصالح.

وقد تم اختبار البروتوكولات الثلاثة المذكورة في هذه الورقة، وأعدم في الجزء الجنوبي الغربي من مقاطعة يوننان، وجمهورية الصين الشعبية بين أفراد الأقلية العرقية Bulang 21 و في أجزاء مختلفة من أفريقيا 6،13،14. هناك العديد من الق...

ملاحظة: مواد في هذه القائمة هي عامة ويمكن الحصول عليها من مصادر مختلفة. <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> كتالوج رقم </td><td> تعليقات </td></tr><tr><td> كاتو كاتز، طقم [400 قوالب البلاستيك مع وجود ثقب من 6 مليمتر (مم) (القطر) في قالب سميكة 1.5 ملم و 400 ملعقة من البلاستيك؛ دورا م 20 من النايلون الشاشة من حجم فتحات الشباك 80؛ دورا م 20 من السيلوفان ماء، 34 سميكة ميكرومتر.] </td><td> Vestergaard فراندسين </td><td></td><td> يرجى ملاحظة أن لا يتم تضمين حل الخضراء الجليسرين-المرمر في هذه المجموعة ولكن يمكن شراؤها من أي مورد الكيميائية. </td></tr><tr><td> قمع الزجاج [حوالي 8 سنتيمتر (سم) واسعة وعميقة 6 سم] </td><td></td><td></td><td> ينبغي للقمع زجاج لتقنية بيرمان تكون قادرة على اجراء حوالي 60 مل من الماء. ال(ه) من حجم خرطوم مطاطي والشاش يعتمد على نوع الزجاج المستخدم قمع. </td></tr><tr><td> المجهر البيولوجية </td><td> OLYMPUS </td><td> CX21LED/CX21 </td><td></td></tr><tr><td> نبذ </td><td> شنغهاي الأدوات الطبية المجموعة </td><td> 80 - 2T </td><td></td></tr><tr><td> إشارات المسجلة مسبقا </td><td> Bitworks التصميم </td><td> نسخة تجريبية فريق تنبيها 1.3.1 </td><td> اي فون تطبيق </td></tr><tr><td> رصد معدل ضربات القلب </td><td> POLAR </td><td> FT1 </td><td> رايتس ووتش والارسال </td></tr></tbody></table>

determining soil-transmitted helminth infection status and physical fitness of school-aged children

Soil-transmitted helminth (STH) infections are common. Indeed, more than 1 billion people are affected, mainly in the developing world where poverty prevails and hygiene behavior, water supply, and sanitation are often deficient1,2. Ascaris lumbricoides, Trichuris trichiura, and the two hookworm species, Ancylostoma duodenale and Necator americanus, are the most prevalent STHs3. The estimated global burden due to hookworm disease, ascariasis, and trichuriasis is 22.1, 10.5, and 6.4 million disability-adjusted life years (DALYs), respectively4. Furthermore, an estimated 30-100 million people are infected with Strongyloides stercoralis, the most neglected STH species of global significance which arguably also causes a considerable public health impact5,6. Multiple-species infections (i.e., different STHs harbored in a single individual) are common, and infections have been linked to lowered productivity and thus economic outlook of developing countries1,3.
For the diagnosis of common STHs, the World Health Organization (WHO) recommends the Kato-Katz technique7,8, which is a relatively straightforward method for determining the prevalence and intensity of such infections. It facilitates the detection of parasite eggs that infected subjects pass in their feces.
With regard to the diagnosis of S.stercoralis, there is currently no simple and accurate tool available. The Baermann technique is the most widely employed method for its diagnosis. The principle behind the Baermann technique is that active S.stercoralis larvae migrate out of an illuminated fresh fecal sample as the larvae are phototactic9. It requires less sophisticated laboratory materials and is less time consuming than culture and immunological methods5.
Morbidities associated with STH infections range from acute but common symptoms, such as abdominal pain, diarrhea, and pruritus, to chronic symptoms, such as anemia, under- and malnutrition, and cognitive impairment10. Since the symptoms are generally unspecific and subtle, they often go unnoticed, are considered a normal condition by affected individuals, or are treated as symptoms of other diseases that might be more common in a given setting. Hence, it is conceivable that the true burden of STH infections is underestimated by assessment tools relying on self-declared signs and symptoms as is usually the case in population-based surveys.
In the late 1980s and early 1990s, Stephenson and colleagues highlighted the possibility of STH infections lowering the physical fitness of boys aged 6-12 years11,12. This line of scientific inquiry gained new momentum recently13,14,15. The 20-meter (m) shuttle run test was developed and validated by L&eacute;ger et al.16 and is used worldwide to measure the aerobic fitness of children17. The test is easy to standardize and can be performed wherever a 20-m long and flat running course and an audio source are available, making its use attractive in resource-constrained settings13. To facilitate and standardize attempts at assessing whether STH infections have an effect on the physical fitness of school-aged children, we present methodologies that diagnose STH infections or measure physical fitness that are simple to execute and yet, provide accurate and reproducible outcomes. This will help to generate new evidence regarding the health impact of STH infections.

Watch this Scientific Journal Video about Determining Soil-transmitted Helminth Infection Status and Physical Fitness of School-aged Children at JoVE.com

Determining Soil-transmitted Helminth Infection Status and Physical Fitness of School-aged Children

عدوى مزمنة مع الديدان المعوية (STHs) يؤدي سوء الامتصاص، التقزم، والهزال في الطفل المتنامية. وبالتالي، فإنه من المعقول أن هذه العدوى يقلل أيضا من اللياقة البدنية للأطفال. هنا، ونحن تصور اثنين من التقنيات لتشخيص وSTHs على المدى المكوك بطول 20 مترا اختبار اللياقة البدنية لتقييم الأطفال بدنيا.

تحديد التربة المنقولة الإصابة بالديدان الحالة واللياقة البدنية من الأطفال في سن المدرسة

Soil-transmitted helminth (STH) infections are common. Indeed, more than 1 billion people are affected, mainly in the developing world where poverty ...

determining-soil-transmitted-helminth-infection-status-physical

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JoVE Journal

Immunology and Infection

23.2K Views.  Swiss Tropical and Public Health Institute, Basel, Switzerland. عدوى مزمنة مع الديدان المعوية (STHs) يؤدي سوء الامتصاص، التقزم، والهزال في الطفل المتنامية. وبالتالي، فإنه من المعقول أن هذه العدوى يقلل أيضا من اللياقة البدنية للأطفال. هنا، ونحن تصور اثنين من التقنيات لتشخيص وSTHs على المدى المكوك بطول 20 مترا اختبار اللياقة البدن?...

Video: Determining Soil-transmitted Helminth Infection Status and Physical Fitness of School-aged Children

Determining the Phagocytic Activity of Clinical Antibody Samples

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 &mu;m fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple Fc&gamma;Rs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of Fc&gamma;Rs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.

We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors&#x2014;providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.

تحديد نشاط أكلة العينات السريرية جسم

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe pathology, including liver fibrosis and splenomegaly, is caused by the immune response to the parasite eggs rather than the parasite itself 2. Parasite eggs induce a Th2 response characterized by the production of IL-4, IL-5 and IL-13, the alternative activation of macrophages and the recruitment of eosinophils. Here, we describe injection of Schistosoma mansoni eggs as a model to examine parasite-specific Th2 cytokine responses in the lung and draining lymph nodes, the formation of pulmonary granulomas surrounding the egg, and airway inflammation. 
Following intraperitoneal sensitization and intravenous challenge, S. mansoni eggs are transported to the lung via the pulmonary arteries where they are trapped within the lung parenchyma by granulomas composed of lymphocytes, eosinophils and alternatively activated macrophages 3-6. Associated with granuloma formation, inflammation in the broncho-alveolar spaces, expansion of the draining lymph nodes and CD4 T cell activation can be observed. Here we detail the protocol for isolating Schistosoma mansoni eggs from infected livers (modified from 7), sensitizing and challenging mice, and recovering the organs (broncho-alveolar lavage (BAL), lung and draining lymph nodes) for analysis. We also include representative histologic and immunologic data and suggestions for additional immunologic analysis. 
Overall, this method provides an in vivo model to investigate helminth-induced immunologic responses in the lung, which is broadly applicable to the study of Th2 inflammatory diseases including helminth infection, fibrotic diseases, allergic inflammation and asthma. Advantages of this model for the study of type 2 inflammation in the lung include the reproducibility of a potent Th2 inflammatory response in the lung and draining lymph nodes, the ease of assessment of inflammation by histologic examination of the granulomas surrounding the egg, and the potential for long-term storage of the parasite eggs.

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.

باستخدام بويضات من<em&gt; البلهارسيا المنسونية</em&gt; ك<em&gt; في الجسم الحي</em&gt; نموذج من التهاب الرئة الناجم عن الديدان الطفيلية

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe ...

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

تحليل الرئوي نضوج خلية شجيري والهجرة خلال التهاب الشعب الهوائية التحسسي

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

مضان المجهري طرق لتحديد الجدوى من البكتيريا في جمعية مع خلايا الثدييات

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

مجموع استخراج بروتين و 2-D طرق جل الكهربائي ل<em&gt; بيركولديريا</em&gt; الأنواع

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

زوجيا النمو الفحص التنافس على تحديد النسخ المتماثل للياقة البدنية من الفيروسات نقص المناعة البشرية

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

Determining Immune System Suppression versus CNS Protection for Pharmacological Interventions in Autoimmune Demyelination

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in myelin destruction, which not only slows conduction of nerve impulses, but causes axonal injury resulting in motor and cognitive decline. Current treatments for MS focus on attenuating immune cell infiltration into the central nervous system (CNS). These treatments decrease the number of relapses, improving quality of life, but do not completely eliminate relapses so long-term disability is not improved. Therefore, therapeutic agents that protect the CNS are warranted. In both animal models as well as human patients with MS, T cell entry into the CNS is generally considered the initiating inflammatory event. In order to assess if a drug protects the CNS, any potential effects on immune cell infiltration or proliferation in the periphery must be ruled out. This protocol describes how to determine whether CNS protection observed after drug intervention is a consequence of attenuating CNS-infiltrating immune cells or blocking death of CNS cells during inflammatory insults. The ability to examine MS treatments that are protective to the CNS during inflammatory insults is highly critical for the advancement of therapeutic strategies since current treatments reduce, but do not completely eliminate, relapses (i.e., immune cell infiltration), leaving the CNS vulnerable to degeneration.

This protocol describes how to determine whether pharmacological treatments for experimental autoimmune encephalomyelitis show CNS protection as a consequence of suppressing immune cell infiltration or are neuroprotective during the onslaught of immune cell infiltration.

تحديد قمع نظام المناعة مقابل حماية الجهاز العصبي المركزي للالدوائية التدخلات في إزالة الميالين المناعة الذاتية

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

تصور آثار البلغم للتنمية بيوفيلم باستخدام نموذج غرف ساترة

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, Helicoverpa armigera densovirus (HaDV2), in a lepidopteran species, the cotton bollworm, Helicoverpa armigera (Hubner). Here, we describe the protocols that are currently used to study the effect of HaDV2 on its host. First, we establish a HaDV2-free cotton bollworm colony from a single breeding pair. Then, we orally inoculate some neonate larval offspring with HaDV2-containing filtered liquid to produce two colonies with the same genetic background: one HaDV2-infected, the other uninfected. A protocol to compare life table parameters (e.g., larval, pupal, and adult periods and fecundity) between the HaDV2-infected and -uninfected individuals is also presented, as are the protocols for determining the host-tissue distribution and transmission efficiency of HaDV2. These protocols would also be suitable for investigating the effects of other orally transmitted viruses on their insect hosts, lepidopteran hosts in particular.

Here, we present a protocol to investigate the host-tissue distribution, transmission mode, and effect on the host fitness of a densovirus within a lepidopteran species, the cotton bollworm. This protocol can also be used for studying the interaction between other orally-transmitted viruses and their insect hosts.

بروتوكولات للتحقيق في توزيع الأنسجة المضيف، نقل واسطة، وتأثير ذلك على الرشاقة المضيف من Densovirus في دودة اللوز القطن

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, ...

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness during infection of Golden Syrian hamsters. Tn-Seq combines random transposon mutagenesis with the power of high-throughput sequencing technology. Animals are challenged with a pool of transposon mutants (input pool), followed by harvesting of blood and tissues a few days later to identify and quantify the number of mutants in each organ (output pools). The output pools are compared to the input pool to evaluate the in vivo fitness of each mutant. This approach enables screening of a large pool of mutants in a limited number of animals. With minor modifications, this protocol can be performed with any animal model of leptospirosis, reservoir host models such as rats and acute infection models such as hamsters, as well as in vitro studies. Tn-Seq provides a powerful tool to screen for mutants with in vivo and in vitro fitness defects.

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

We describe here a technique that combines transposon mutagenesis with high-throughput sequencing to identify and quantify transposon leptospiral mutants in tissues after a challenge of hamsters. This protocol can be used to screen mutants for survival and dissemination in animals and can also be applied to in vitro studies.

التسلسل الفائق موازية "قياس اللياقة البدنية" الإصابة إينتيروجانس Leptospira ينقول الإدراج طفرات خلال الذهبي السوري الهامستر

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness ...