저자는 Therwa 함자, 존 E. Tidwell, 니나 클로비스, 그리고 실험 지원 및 교정을위한 수잔 스미스 수잔 스미스 감사드립니다. 저자는 자신의 지원과 미생물학, 면역학 및 웨스트 버지니아 대학에서 세포 생물학의 부에있는 생물 안전 실험실의 사용에 대한 세균의 임상 격리하고 존 B. 바넷, 박사 학위를 제공하는 존 토마스, 박사 학위를 감사합니다. 저자 Osteosynthesis 및 외상 관리 재단과 국립 과학 재단 (National Science Foundation) (# 1003907)에서 재정 지원을 인정합니다. 현미경 실험 및 이미지 분석도 메리 Babb 랜돌프 암 센터와 NIH 보조금 P20 RR016440에 의해 부분적으로 지원되는 웨스트 버지니아 대학 이미징 시설에서 수행되었다. 피를 동물 사용은 웨스트 버지니아 대학 기관 동물 케어 및 사용위원회에 의해 승인되었습니다립니다. 모든 실험은 모든 관련 guidelin을 준수 실행 된어, 규정 및 규제 기관.

1. 준비와 PRP의 활성화 1.1 혈액 무승부 <ol><li> isoflurane의 흡입 (유도를위한 O 2 2 % 유지 보수 1 %)에서 토끼를 마취. </li><li> 20 ML의 주사기로 2 ML 0.129 M 트라이 - 나트륨 구연산을 (anticoagulent 솔루션)립니다. 트라이 나트륨 구연산 솔루션은 50 ML 증류수 H 2 O에 1.897 g 트라이 - 나트륨 구연산을 용해하고 0.22 μm 살균 필터로 필터링하여 준비가되어 있습니다. </li><li> 70 % 에탄올을 사용하여 토끼 귀를 살균. </li><li> 나비 바늘 (25 G) 주사기에 연결을 통해 토끼 귀 정맥에서 (예를 들면 5 ML) 혈액을 그립니다. </li><li> 부드러운 교반하여 트라이 나트륨 구연산 솔루션으로 피를 섞는다. 혈액 및 트라이 - 나트륨 구연산 솔루션의 볼륨 비율이 9시 1분입니다. </li></ol> 1.2 PRP 준비 (그림 1) <ol><li> 50 ML 플라스틱 원심 분리기 튜브에 anticoagulated 혈액을 전송합니다. 10 μl의 나누어지는 해hemocytometry를 사용하여 기준 혈소판 수를 확인하기 위해 혈액. </li><li> 스윙 아웃 로터와 원심 분리기에 실온에서 10 분 (RT) 300 XG에서 혈액을 원심 분리기. 낮은 가속 및 브레이크 속도 (그림 1)을 설정합니다. </li><li> 원심 분리 후, 피가 3 층으로 분리된다. 아래 층은 주로 적혈구이며, 중간 층 (일반적으로 &#39;버피 코트 &quot;이라한다) 농축 혈소판과 백혈구로 구성하고, 상단 층은 주로 혈액의 액체 성분이 플라즈마, 그리고 혈소판 (입니다 그림 1). 세포 배양 후드에 원심 분리기 튜브를 조심스럽게 수송, 레이어를 방해하지 않습니다. 1 ML 플라스틱 pipettor를 사용하여 15 ML 플라스틱 튜브에 플라즈마의 모든 버피 코트, 그리고 2~3밀리미터 두께 적혈구 레이어를 전송합니다. </li><li> RT에서 15 분 3,000 XG에서 이전 샘플 두 번째 시간을 원심 분리기. 상단 층 (표면에 뜨는)는 혈소판 가난한 플라즈마 (PPP)로 간주됩니다D는 새로운 튜브로 전송됩니다. </li><li> PPP 2.0 X 10 6 혈소판 / μl를 (hemocytometry에 의해 결정)을 획득하는 데 사용 나머지 혈액 샘플에서 혈소판 농도를 조정하여 PRP를 구합니다. </li></ol> 1.3 PRP 활성화 <ol><li> 1000 IU / ML의 작업 농도에 5 ML 10 % 염화칼슘과 5,000 IU 소 트롬빈을 용해하여 PRP 활성화 솔루션을 준비합니다. </li><li> PRP와 PPP에 활성화 솔루션을 추가하고 반복적으로 PRP와 PPP-젤을 형성하기 위해 pipetting하여 솔루션을 섞는다. PRP 또는 PPP를 활성화 솔루션의 볼륨 비율이 1시 4분입니다. </li></ol> 2. 죽 곡선 분석을 사용하여 PRP의 체외 항균 시험 (그림 2)에서 <ol><li> 멸균 inoculating 루프를 사용 S. 여러 식민지를 추가 플라스틱 관 뮐러 힌튼 국물 (MHB)의 5 ML에 자사의 하룻밤 판 문화의 구균. 소용돌이가 잠시 후 37 2 시간 동안 샘플을 품다 ° C. 다음, 박테리아 미디어의 광학 밀도 분광 광도계를 사용하여 결정하고 ~ 1 이상으로 광학 밀도로 조정 된 X 10 8 CFU / 미리 정해진 표준 곡선에 따라 ML. </li><li> 1 개 10 6 CFU / ML을 취득과 얼음에 inoculums을 배치 PBS를 사용하여 100x 희석하십시오. </li><li> 설정 및 살균, 일회용 5 ML 왕복 아래 폴리스티렌 튜브를 라벨 및 각 관 2 ML의 최종 볼륨에 대해 표 1에 표시된 다음 예제 그룹을 준비합니다. </li><li> 활성화 (겔 형성)에 대한 트롬빈 솔루션 다음 폴리스티렌 튜브 먼저 PRP, PPP, 또는 PBS를 추가합니다. 다음 MHB를 추가 한 후 S. 구균의 inoculums (1 X 10 6 CFU / ML) 1 × 10 5 CFU / ML의 최종 농도를 얻을 수 있습니다. </li><li> ° C 150 rpm으로 궤도 동요와 함께 37 튜브를 품다. </li><li> 미리 정해진 시간 포인트 (예 : 0, 1, 2 시간)에서 반복 pipetting (이 통해 각 튜브의 솔루션을 혼합박테리아는 PRP 젤 안에 갇혀 될 수 있기 때문에 단계) 중요합니다. , 샘플의 10 μl를 멸균 0.9 %의 식염수에 순차적으로 희석하고, Tryptic 간장 한천 (TSA, 5 %의 양 피) CFU 계산을위한 플레이트로 각 희석의 100 μl 나누어지는을 피펫. </li><li> 37 번 밤 문화 한천 플레이트 ° C 한 다음 계산과 판 식민지를 기록합니다. 플롯 X-축과 CFU / y 축에 ML에서 시간 (시간)와 logarithimic 규모 데이터입니다. </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=A+technique+for+improving+the+handling+of+particulate+cancellous+bone+and+marrow+grafts+using+platelet+gel.">A technique for improving the handling of particulate cancellous bone and marrow grafts using platelet gel.</a> J. Oral. Maxillofac. Surg. 56, 1217-1218 (1998).</li><li>Currie, L. J., Sharpe, J. R., Martin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+fibrin+glue+in+skin+grafts+and+tissue-engineered+skin+replacements:+a+review.">The use of fibrin glue in skin grafts and tissue-engineered skin replacements: a review.</a> Plast. Reconstr. Surg. 108, 1713-1726 (2001).</li><li>Nikulin, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+calcium,+thrombin+and+nucleotides+(ADP,+cAMP,+cGMP)+on+blood+platelet+glycolysis+and+energy+metabolism.">Effect of calcium, thrombin and nucleotides (ADP, cAMP, cGMP) on blood platelet glycolysis and energy metabolism.</a> Farmakol. Toksikol. 43, 585-590 (1980).</li><li>Hantgan, R. R., Taylor, R. G., Lewis, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelets+interact+with+fibrin+only+after+activation.">Platelets interact with fibrin only after activation.</a> Blood. 65, 1299-1311 (1985).</li><li>Hantgan, R., Fowler, W., Erickson, H., Hermans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+assembly:+a+comparison+of+electron+microscopic+and+light+scattering+results.">Fibrin assembly: a comparison of electron microscopic and light scattering results.</a> Thromb. Haemost. 44, 119-124 (1980).</li><li>Li, B., Jiang, B., Boyce, B. M., Lindsey, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilayer+polypeptide+nanoscale+coatings+incorporating+IL-12+for+the+prevention+of+biomedical+device-associated+infections.">Multilayer polypeptide nanoscale coatings incorporating IL-12 for the prevention of biomedical device-associated infections.</a> Biomaterials. 30, 2552-2558 (2009).</li><li>Li, B., Jiang, B., Dietz, M. J., Smith, E. S., Clovis, N. B., Rao, K. M. K. <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+local+MCP-1+and+IL-12+nanocoatings+for+infection+prevention+in+open+fractures.">Evaluation of local MCP-1 and IL-12 nanocoatings for infection prevention in open fractures.</a> J. Orthop. Res. 28, 48-54 (2010).</li><li>Boyce, B. M., Lindsey, B. A., Clovis, N. B., Smith, E. S., Hobbs, G. R., Hubbard, D. F., Emery, S. E., Barnett, J. B., Li, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Additive+effects+of+exogenous+IL-12+supplementation+and+antibiotic+treatment+in+infection+prophylaxis.">Additive effects of exogenous IL-12 supplementation and antibiotic treatment in infection prophylaxis.</a> J. Orthop. Res. 30 (2), 196-202 (2012).</li></ol>

제작자들은 더 경쟁 금융 이익이 없다는 점 선언합니다.

혈소판이 풍부한 혈장은 점점 그 치유 - 홍보 등록 15-17으로 인해 임상 응용에 사용되었습니다. 본 연구에서는 PRP는 감염 예방을위한 새로운 접근 방법으로 제시되었다. PRP는 MRSA, MSSA, 그룹 연쇄상 구균 및 Neisseria gonorrhoeae에 대한 강력한 항균 특성을 가지고 발견되었습니다. 기존의 항생제 치료에 비해 PRP의 주요 장점은, 감염 예방을 위해 포함 : (1) 현재 항생제 치료는 점점 ?...

임플란트 - 관련 감염은 상당한 임상 합병증입니다. 포도상 구균 (S. 구균)이 임플란트 - 관련 감염으로부터 격리 가장 흔한 미생물 중 하나입니다. 그것은 임플란트의 표면을 커버하고 항생제 내성 감염 1,2으로 이어질 수있는 biofilm을 생산 할 수 있습니다. 임플란트 - 관련 감염의 치료가 자주 반복 ​​debridements과 장기간 비경 항생제 치료 장기 입원이 필요합니다. 항생제 내성의 경우, 보형물의 제거가 필요할 수 있습니다. 항생제에 대한 박테리아의 증가 저항도 등의 질병 통제 및 예방 (CDC)의 센터에 의해 참조되었습니다 &quot;세계에서 가장 급박 한 건강 문제 중 하나입니다.&quot; 시간이, 새로운과 효과적인 항균 요법의 개발없이, 그것은 다중 약물 내성 병원균은 기존의 항생제와 untreatable 될 가능성이 있습니다. 임플란트 - 관련 예방감염 것이 중요하며, 새로운 예방 대리인 또는 접근 방법은 이러한 감염을 예방하기 위해 필요합니다. 혈소판이 풍부한 혈장 (PRP)은 뼈와 뼈 이식 치료 3-5으로 도움을 줄 수 30 성장 요인을 포함 autologous 혈액의 농도이다. 뼈 재생과 부드러운 조직 성숙을 향상시키는 PRP의 응용 프로그램이 점점 때문에 혈소판에 의해 발표 다양한 성장 요인의 높은 농도의 병원에서보고되었습니다. PRP의 여러 특성은 PRP는 항균 특성에게 6-9있을 수 있습니다 것으로 나타났습니다. PRP는 혈소판의 큰 번호, 백혈구의 높은 농도 (세균 및 곰팡이에 대한 호스트 방어 동작을 가지고 할 수있는), 그리고 여러 항균 펩티드 7,8,10가 포함되어 있습니다. 심장 수술 환자의 큰 일대의 최근 연구에서,이 드러났다 그 상처 폐쇄 signif 동안 PRP-젤의 intraoperative 사용icantly 표면과 깊은 가슴 감염 (11)의 발생률을 감소. 이러한 이유와 관찰의 경우, 우리는 PRP는 그 잘 공부 치료 - 추진 특성 외에, 항균 특성을 가지고 가상. 감염을 방지하기 위해 PRP를 사용하는 잠재적 인 장점이 포함될 수 있습니다 :은 (i) PRP는 기존의 항생제 치료에 비해 저항을 유도 할 가능성이 적습니다. PRP의 치료 - 홍보 특성은 병원균과 인간의 세포가 이식 표면 12 질주를하고 같이함으로써 감염 가능성을 줄여 세균 첨부 파일을 방지하기 위해 인감을 제공 할 수 경우, (ii) PRP는 감염 예방에 시너지 효과가있을 수 있습니다 치유를 촉진 속성을 가지고 있습니다 13. (III) PRP는 본질적으로 biocompatible하고, 안전하고 전도 할 수있는 질병의 위험에서 무료입니다. 우리의 장기 목표는 임플란트 - 관련 infecti을 방지하기 위해 새로운 접근 방법으로 PRP를 사용하는 것입니다기능. 본 연구의 목적은 체외 항균 속성에 PRP의를 검사하기 위해, 두 번 원심 분리 방식을 사용하여 PRP를 준비하고, 이러한 항균 속성을 평가하기위한 프로토콜을 설명하는 것이 었습니다.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Bovine thrombin</td>
 <td>King Pharmaceuticals, Inc </td>
 <td>60793-215-05</td>
 <td>Thrombin (bovine origin)</td>
 </tr>
 <tr>
 <td>Calcium chloride</td>
 <td>King Pharmaceuticals, Inc</td>
 <td>60793-215-05</td>
 <td>10% calcium chloride</td>
 </tr>
 <tr>
 <td>Ethanol</td>
 <td>Sigma-Aldrich</td>
 <td>E7023</td>
 <td></td>
 </tr>
 <tr>
 <td>Isoflurane</td>
 <td>Baxter</td>
 <td>1001936060</td>
 <td></td>
 </tr>
 <tr>
 <td>Mueller Hinton broth</td>
 <td>Becton, Dickinson and Company</td>
 <td>275710</td>
 <td></td>
 </tr>
 <tr>
 <td>Phosphate-buffered saline</td>
 <td>Sigma-Aldrich</td>
 <td>D8662</td>
 <td></td>
 </tr>
 <tr>
 <td>Tri-sodium citrate</td>
 <td>Sigma-Aldrich</td>
 <td>W302600</td>
 <td></td>
 </tr>
 <tr>
 <td>Tryptic soy agar</td>
 <td>Fisher Scientific</td>
 <td>R01202</td>
 <td></td>
 </tr>
 <tr>
 <td>Centrifuge</td>
 <td>Kendro Laboratory Products</td>
 <td>750043077</td>
 <td></td>
 </tr>
 <tr>
 <td>Syringe filter</td>
 <td>Millipore</td>
 <td>SLGP033RS</td>
 <td></td>
 </tr>
 </tbody>
</table>

prp as a new approach to prevent infection: preparation and in vitro antimicrobial properties of prp

임플란트 - 관련 감염 항생제 저항, 동물과 인간 사이의 항생제 내성 박테리아의 전송과 감염을 치료의 높은 비용을 증가로 인해 점점 더 의료 산업에 더 도전 세계적되고 있습니다. 본 연구에서는, 우리는 혈소판이 풍부한 혈장 (PRP)의 잠재적 인 항균 특성을 바탕으로 임플란트 - 관련 감염을 예방에 효과가있을 수 있습니다 새로운 전략을 공개. 치유를 촉진위한 잘 연구 특성으로 인해, PRP는 (생물 제품) 점점 정형 외과 수술, 치주 및 구강 수술, 악안면 수술, 플라스틱 수술, 스포츠 의학 등 임상 응용에 사용되었습니다 PRP는 임플란트 - 관련 감염을 예방 기존의 항생제 치료에 대한 고급 대안이 될 수 있습니다. PRP의 사용은 기존의 항생제 치료시에 비해 유리할 수있다PRP는 항생제 저항을 유도 할 수 있고 PRP의 항균 및 치료 - 홍보 속성이 감염 예방에 시너지 효과가있을 수 있습니다 적은 가능성이 nce. 그것은 잘 병원균과 인간의 세포가 이식 표면에 경주하는 알려져 있으며, 홍보 치유의 PRP의 속성함으로써 감염 가능성을 줄여 인간의 세포 첨부 파일을 향상 수 있습니다. 또한, PRP는 본질적으로 biocompatible하고, 안전하고 전도 할 수있는 질병의 위험에서 무료입니다. 우리의 연구를 들어, 우리는 일반적으로 정형 외과 감염에서 발견하고 PRP이 박테리아에 대한 체외 항균 속성에 있는지 여부를 검사 몇 가지 임상 박테리아 변종을 선택했습니다. 우리는 같은 혈소판 농도가 모든 샘플에 대해 얻을 수 할 수있는 두 번 원심 분리 방식을 사용하여 PRP를 준비했습니다. 우리는 일관성있는 항균 결과를 달성하고 PRP는 methi 같은 세균에 대한 시험 관내 항균 속성에 강한 것으로 나타났습니다cillin에 민감한와 methicillin - 내성 황색 포도상 구균, 그룹 연쇄상 구균과 Neisseria gonorrhoeae. 따라서, PRP의 사용은 감염을 예방하고 임플란트 - 관련 감염의 비용이 많이 드는 수술 치료의 필요성을 줄일 수있는 가능성이있을 수 있습니다.

PRP는 reproducibly 두 번 원심 분리 방식 (그림 1)를 사용하여 준비가되어 있습니다. PRP는 methicillin 내성 S.에 대한 체외 항균 속성에 (CFUs에서 최대 100 배 감소) 강한 제시 찾을 수 있습니다 구균 (MRSA) (그림 3)은, 그는 일반적으로 전세계 병원 14에서 발견됩니다. 마찬가지로, PRP는 methicillin에 민감한 S.에 대한 강력한 항균 특성을 가지고...

Watch this Scientific Journal Video about PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP at JoVE.com

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

임플란트 - 관련 감염은 중요한 임상 합병증입니다. 이 연구는 임플란트 - 관련 감염을 방지하기 위해 혈소판이 풍부한 혈장 (PRP)를 사용하여 접근 방식을 설명 지속적인 혈소판 농도와 PRP를 준비 프로토콜을 제시하고, PRP의 새로 확인 된 항균 특성 및 항균 특성을 조사에 대한 관련 프로토콜을보고<em&gt; 체외 인치</em

준비를 한 다음 감염을 방지하기 위해 새로운 접근 방식으로 PRP<em&gt; 체외에서</emPRP의&gt; 항균 속성

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, ...

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JoVE Journal

Immunology and Infection

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

21.2K 조회수.  West Virginia University. 임플란트 - 관련 감염은 중요한 임상 합병증입니다. 이 연구는 임플란트 - 관련 감염을 방지하기 위해 혈소판이 풍부한 혈장 (PRP)를 사용하여 접근 방식을 설명 지속적인 혈소판 농도와 PRP를 준비 프로토콜을 제시하고, PRP의 새로 확인 된 항균 특성 및 항균 특성을 조사에 대한 관련 프로토콜을보고 체외 인치

비디오: PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

마우스 T - 세포에서 T - 세포 수용체의 Retroviral 전달

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

연구

면역학 및 감염병학

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.

세균성 접착 연구에 전단 응력을 소개합니다

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

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal phase during establishment of infection in a given host. Consequently, study of encapsidation deciphers the information regarding the know-how of the mechanism regulating virus assembly to form infectious virions. Such information is vital in formulating novel methods of curbing virus spread and disease control. Virus encapsidation can be studied in vivo and in vitro. Genome encapsidation in vivo is a highly regulated selective process involving macromolecular interactions and subcellular compartmentalization. Therefore, study leading to dissect events encompassing virus encapsidation in vivo would provide basic knowledge to understand how viruses proliferate and assemble. Recently in vitro encapsidation has been exploited for the research in the area of biomedical imaging and therapeutic applications. Non-enveloped plant viruses stand far ahead in the venture of in vitro encapsidation of the negatively charged foreign material. Brome mosaic virus (BMV), a non-enveloped multicomponent RNA virus pathogenic to plants, has been used as a model system for studying genome packaging in vivo and in vitro. For encapsidation assays in Nicotiana benthamiana plants, Agrobacterium -mediated transient expression, refer to as agroinfiltration, is an efficient and robust technique for the synchronized delivery and expression of multiple components to the same cell. In this approach, a suspension of Agrobacterium tumefaciens cells carrying binary plasmid vectors carrying cDNAs of desiredviral mRNAs is infiltrated into the intercellular space withina leaf using nothing more sophisticated than a 1 ml disposable syringe (without needle). This process results in the transfer of DNA insert into plant cells; the T-DNA insert remains transiently in the nucleus and is then transcribed by the host polymerase II, leading to the transient expression. The resulting mRNA transcript (capped and polyadenylated) is then exported to the cytoplasm for translation. After approximately 24 to 48 hours of incubation,sections of infiltrated leaves can be sampled for microscopyor biochemical analyses. Agroinfiltration permits large numbers (hundreds to thousands) of cells to be transfected simultaneously. For in vitro encapsidation, purified virions of BMV are dissociated into capsid protein by dialyzing against dissociation buffer containing calcium chloride followed by removal of RNA and un-dissociated virions by centrifugation. Genome depleted capsid protein subunits are then reassembled with desired viral genome components or non-viral components such as indocyanine dye.

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

A simple, efficient and robust way to synchronize the delivery of multiple viral components to plant cells via Agrobacterium-mediated transient expression is described. This approach is amenable for studying replication, encapsidation followed by in vitro reassembly of non-viral components into genome depleted optical viral ghosts suitable for biomedical applications.

간편하고 강력한 생체내에서과 체외에서</em바이러스 조립 유학을위한> 접근법

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

위반 항생제 효능을 테스트하는 인공 가래 매체의 사용<em&gt; 녹농균</em&gt; 낭성 섬유증의 폐 관련성이 높은 조건에서

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

사용<em&gt; 갤러리아 mellonella</em&gt; 모델 생물로 공부하기<em&gt; 레지오넬라 뉴모 필라</em&gt; 감염

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

막 극성, 멤브레인 무결성 및 세포 내 이온 농도의 모니터링 변경<em&gt; 폐렴 구균</em&gt; 형광 염료를 사용

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

웨스트 나일 바이러스 돌연변이 균주의 감염에에서 MyD88 매개 세포 면역 반응의 생체 외 분석

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

간단하고 효율적인 접근 방법은 돌연변이 백시 니아 바이러스 벡터를 구축하기

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

공부하는 유전 적 접근으로 헬퍼 T 세포의 레트로 바이러스 형질 도입은 메커니즘 자신의 차별화 및 기능 제어

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...