The authors thank Dr. Lynda Partridge for the provision of advice and laboratory facilities.

세포주의 1) 준비 : <ol><li> 말 FcεRI α의 발현 RBL-2H3.1 세포주 개발 : <ol><li> 28 : 단층 세포 라인에 대한 기본 조직 배양 기술을 이용하여 말 FcεRIα 유전자 (GenBank 등록 Y18204.1)를 들고, pEE6 플라스미드를 사용하는 부모 RBL-2H3.1 세포를 형질. 1.2 X10 7 셀 ML -1의 밀도로 세포의 0.8 ml의 플라스미드 DNA의 2 μg의 μL -1을 추가합니다. 즉시 10 분 동안 얼음에서 인큐베이션 0.4 cm의 electrocuvette를 이용한 250 V 960 μF에서 세포 Electroporate. </li><li> 네티의 G418 황산 0.4 g을 포함하는 미디어를 사용하여 형질 전환 된 세포를 선택한 다음, 형광 면역 글로불린 항체로 태그를 지정하여 FACS를 통해 남아있는 살아있는 세포를 정렬 할 수 있습니다. 수사 2, 3에 대해 말 FcεRIα 세포주를 발현 RBL-얻어진 2H3.1 사용. </li></ol></li><li> 사전 분석 항체 감작 : <ol><lI&gt; 수확 합류 페트리 접시에서 말 FcεRIα 세포 발현 RBL-2H3.1. 5 × 5 셀 ML의 -1의 세포 밀도로 배양 배지에 세포를 다시 중단 후 씻는다. </li><li> 1 NG ml의 -1의 최종 농도로 일시 중단 된 세포에 관심 IgE에 추가 한 후 1-6 열에서 96 웰 플레이트에 세포를 100 μl를 판 37 ° C ~ + 5 % CO 2 + 90 % 부화 16 시간 동안 상대 습도. 배양 시간 후 및 박리 시험을 수행하기 전에, 잘 자랄 및 세포 부착에 대한 현미경 웰을 확인. </li></ol></li></ol> 2) 자료 분석 : <ol><li> 세포를 세척 : <ol><li> 따뜻한 방출 완충액 37 ° C에서 (25 밀리미터 PIPES, 120 mM 염화나트륨, 5 mM 염화칼륨, 0.04 mM의 염화 마그네슘, 및 1mM의 염화칼슘)은 부드러운 셀 세정을 허용한다. </li><li> 세포 용지를 제거하기 위해 판을 튕기 따뜻한 100 μl를 추가하여 3 세포를 씻으7 ° C를 방출 버퍼입니다. 두 번 반복합니다. </li></ol></li><li> 항원 과제 : <ol><li> 항원의 용액을 희석 0 NG ml의 -1, 0.1 NG ml의 -1, 1 NG ml의 -1, 10 NG ml의 -1, 100 NG ml의 -1 (NIP-HSA 또는 DNP-HSA), 1000 NG ml의 준비 -1, 10,000 NG 릴리스 버퍼 ml의 -1, 37 ° C에서 따뜻한. </li><li> 두 번째 셀을 세척 한 후, 미디어를 무시하고 항원 용액 100 ㎕로 대체. 같은 행의 우물 (예를 들어 A1-6)이 그들에 추가 된 동일한 항원 농도가 있는지 확인하십시오. </li><li> 0 NG ml의 -1 항원을 추가하여 행에서 음성 대조군을 설정합니다. 트리톤 X-완충액 (5 % 트리톤 X-100), 행 H 세포는 세포를 용해하는 다음 행 (BG) 아래로 증가하는 항원의 농도를 추가 양성 대조군으로 사용한다. 20 분 세포는 매개체를 해제 할 수 있도록, 37 ° C에서 배양하라. </li></ol></li><li> 개별 잘 컨트롤을 설정 : <ol><li> 배양 후, 플레이트의 나머지 절반 (A1-6 웰 웰 A7-12 행, 등)에 세포 상청액 50 μl를 옮긴다. 상청액의 나머지 50 μl를 버리고 컬럼 1-6 셀 내부 전체 매개체의 비율로 잘 컬럼 7-12의 각 매개체 방출의 양의 측정을 허용하도록 트리톤 X-완충액 50㎕의 교체 . </li></ol></li><li> 효소 기판 : </li><li> β-소사 미나 기판 (50 mM의 4- 니트로 N 아세틸 β-D-글루코 시트르산 완충액, 0.2 M 시트르산 및 0.2 M 아세트산 나트륨을 첨가하여 2 mM의 아래로 희석 된 DMSO에서 제조하여 pH 4.5) 50 μl를 추가 모든 웰에 β-소사 미나 제 효소에 의해 4- 니트로 페놀에 대한 기판의 전환을 촉진한다. 2 시간 동안 37 ° C에서 번호판을 품어. </li></ol><img alt="그림 2.4.1" fo:content-width="4in" src="/files/ftp_upload/52222/52222fig241.jpg" widtH = &quot;400&quot;/&gt; <ol start="5"><li> 반응을 종료 : <ol><li> 각각 150 ㎕를 첨가 트리스 완충액 (1 M 트리스 -HCl, pH가 9)뿐만 아니라 버퍼의 높은 pH로 반응을 정지하여, 반응을 정지하고 황색로 -4- 니트로 페놀 변. </li></ol></li><li> 결과를 읽고 분석 : <ol><li> 노란 색의 흡광도를 측정하기 위해 405 nm에서 플레이트 분광 광도계를 사용하여 판을 읽어보십시오. 다음 식을 이용하여 풀어 β 헥 소사 미나 백분율 계산 값 : </li></ol></li></ol><img alt="그림 2.6.1" fo:content-width="1in" src="/files/ftp_upload/52222/52222fig261.jpg" width="100" /><ol start="2"><li> 평균이 각 행에 대해 수행 된 후 각 웰에이 수식을 적용합니다. A1 및 A7은 96 웰 플레이트에서 웰의 위치를​​ 나타낸다. 전압 강하 (총 중재자 릴리스에 해당) β-소사 미나 릴리스의 비율의 그래프를 그린다NST 항원 농도 (2, 3). </li></ol>

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The authors declare that they have no competing financial interests in this paper.

요약하면,이 조사 결과는 말 FcεRIα 발현 RBL-2H3.1 세포는 항원에 의해 말의 IgE로 감작 및 도전 될 때, 그들은 내부 매개체의 총량 4.88 % ± 36.68 %의 피크 매개체 방출을주는 것으로 나타났다 세포, RBL-2H3.1에 ​​비해 부모의 세포가 말 FcεRIα을 표현하지. 따라서이 분석은 조사하고 체외에서 말 알레르기 반응을 연구하기위한 유용한 도구를 제공합니다. 그는 비만 세포 / ?...

알레르기는 천년 알려져있다. 천식 치료는 Ebers 파피루스 (~ 1550 BCE)로 알려진 고대 이집트 의료 텍스트에 설명하고 7을 치료하는 한약을 논의했다. 현재 알레르기는 T 헬퍼 세포 유형 2 (TH (2)) 면역계의 아암 알레르겐이라고 환경 항원에 반응하여 면역 글로불린 E (IgE의) 항체의 생산을 조향 타입 I의 과민 반응으로 분류된다. 이들은 일반적으로 면역계 세포와 상호 작용 된 IgE 합성을 담배 연기 또는 디젤 배기 미립자 입자 향상 않은로서 인터루킨 -4 및 인터루킨 13 (8, 9)을 포함하여 합성 및 염증성 사이토 카인의 분비를 자극하는 다양한 물질이다 10. 지난 50 년 동안 선진국에서 알레르기 증상의 상승의 효과의 조합에 기인 된&#39;위생 가설&#39;(11)에 의해 제안 된 TH 사이토 카인이 지배적 프로파일 향해 면역 반응을 전환하기 위해 결합 환경 오염 물질 및보다 위생적 환경 동향. 전술 한 바와 같이, 인간 알레르기 시달리다 만 포유류 아니다. 특히 말과 개는 클래식 알레르기 반응을 개발할 수 있고, 말 알레르기가 유전 적, 환경 적 요인에 기인 인간에로 (12)의 연구는 것을 보여 주었다. 결과적으로,이 동물은 한 번 임상 증상이에서 설정 한 알레르기의 유전 적, 환경 적 원인, 질병에 감작에서의 진행, 가능한 한 개입 전략 사이의 상호 작용 연구를위한 좋은 모델을 제시 1887 년, Stömmer는 말 심혈관 시스템 히스타민의 효과는 인간과 말, 천식 (13) 사이의 유사성을 설명하는 첫 번째 사람이었다인간 (14)의 그것과 매우 유사합니다. 말은 15 매년 115,000,000,000달러 미국의 도박 매출과 가치가 미국 720 억 달러 인 경마 산업의 초석이다. 대부분의 현대 경주마는 이후 1878에서 레이디 앤 블런트가 자란 아라비아 말의 적은 수의 자손입니다. 현대 경주마는 일반적으로 성능 능력에 대한 선택 근친된다. 이들은 알레르기 반응을 탑재 감수성 중 하나는 유전 질환,되기 쉽다. 또한 심지어 가장 심각한 알레르기 인간 (16)보다 1000 배 더 높은 혈청 IgE의 수준을 가지고있다. 말 알레르기 반응은 일반적으로 곤충 물린 과민 반응 (IBH) 17, 18로 나타나있다. 때문에 물기에 피부염에 IBH 결과가 속 Culicoides 곤충을 형성한다. 말 알레르기 질환의 또 다른 형태는 재발 성기도에 장애물 (RAO)이이 폐와기도에 명시되어 있습니다. 그것은 천명 및 실험실 특징OR 연산 호흡. RAO 일반적 포자를 성형하는 응답하여 발생하고, 다른 조사이 20를 확인되지 않았지만 높은 알레르겐 - 특이 IgE 수준 한 연구 19 RAO 앓고 말에 기록되었다. 말 알레르기에 대한 연구가 시도 모니터링에서 안티 말 IgE의 단클론 항체 (21, 22)을 개발하여 말 IgE의 중화 주위에 회귀. 또한 23 의한 연구 말 고친 화성 Fc 수용체의 α의 세포 외 도메인의 생성을 논의 말 혈청의 IgE를 감지하고 정량화하려는 시도에 체인 (FcεRIα) 수용체. Ledin (24)에 의해 관련 연구는 자기 / 비 자기 면역원을 사용하여 면역 체계를 프라이밍에 의해 혈청의 IgE를 중화하기위한 새로운 접근 방법에 대해 설명합니다. 이러한 모든 연구는, 그러나, 이들 프로토콜의 안전성과 효능을 테스트하기 위해 효과적인 분석 결여. 이 글에서, 우리는 지금 이러한 분석 SYS을 제시β-소사 미나을 출시, 세포 매개 탈과립의 지표로, 말 FcεRIα을 표현 RBL-2H3.1 세포에 평가 된 말 시스템, 관련 진단 및 치료 전략의 연구에 적용 TEM. 이 프로토콜은 다른 종으로부터 IgE에 대한 고친 화성 수용체의 IgE 결합 도메인을 코딩하는 유전자로 형질 RBL 세포 공학 기술하는 이전의 간행물 25, 4, 5, 2, 3에 기초한다. 이 프로토콜의 결과는 세중의 실험 표준 편차 평균 ±로 표시되는 β-소사 미나 자료 분석을 수행하는 방법에 대해 설명합니다. 자료 분석은 처음 Siraganian에 의해 개발과 인간의 알레르기를 연구하는 25 후크했다. 닥터 루벤 Siraganian 이끄는 실험 그룹은 RBL 세포주를 개발했다. 이러한 RBL 세포는 인간 FcεRIα을 표현하기 위해 개발되었고, 프로토콜은 (4)에 의해 출판되었다. 마지막 조각분석은 4- 히드 록시 3 합텐을 대상 IgE의 가변 영역에 대한 마우스 유전자의 하류 중쇄 유전자를 클로닝하여 IgE 항체의 생산을 설명 NEUBERGER (26)에 의해 종이 PSV 플라스미드의 개발과 함께의 니트로 - phenacetyl은 (NP), 결과 키메라 항체는 완벽하게 작동했다. 또한 호 염기성 세포의 탈과립을 측정하는 데 유용하게 분석 프로토콜의 표준화 결과 RBL 세포 표면 수용체를 클로닝하는 동안 능력은 동일한 합텐 타겟팅 임의의 IgE를 개발. 분석은 장점과 단점을 가지고있다. 분석의 장점은 우리 실험실 따라서 인간, 송곳니와 말 시스템의 탈과립을 테스트하는 데 사용되는 한 모든 포유류 시스템에서 사용되는 적응성이며,이 단순히 유기체의 IgE의 합성과 이의 수용체를 복제하여 달성 RBL 세포의 표면 상. 한편,분석의 단점 RBL 세포들이 동일한 분석 내에서 탈과립 레벨의 변화를 만들어주고, 열적, 기계적, PH 변화에 매우 민감하다는 것이다. 따라서 그것은 강하게 분석은 항상 3 회 반복 반복 한 후 평균 그들로부터 촬영하는 것이 좋습니다. 나아가, RBL 세포는 그들의 유지 관리를 복잡하게 연장 된 시간 (&gt; 10 주) 27 조직 배양에 남아 있다면 비 방출 표현형을 향해 이동하는 경향이있다. 또한 광학 현미경에서 볼 수없는 셀의 형태를 변경하지 않는 마이코 플라스마 균에 의한 감염,하는 경향이 있지만, 크게 자신의 탈과립 수준을 변경합니다. 따라서 정기적 인 마이코 플라스마 시험이 필요하다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>RBL-2H3.1 Expressing Equine Fc&#949;RI&#945;</td>
			<td>-</td>
			<td>-</td>
			<td>Produced in the lab</td>
		</tr>
		<tr>
			<td>Equine IgE anti NIP-HSA</td>
			<td>-</td>
			<td>-</td>
			<td>Produced in the lab</td>
		</tr>
		<tr>
			<td>96 Well Plate</td>
			<td>Sigma</td>
			<td>CLS3595</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Multi Channel Pipette</td>
			<td>Anachem</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Galaxy R</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>4Hydroxy-5-iodo-3-nitrophenylacetic acid</td>
			<td>Cambridge Research Biochemicals</td>
			<td>N-1070-1</td>
			<td>NIP-OH was conjugated with Human Serum Albumin to make NIP-HSA in the lab</td>
		</tr>
		<tr>
			<td>Dinitrophenyl Conjugated to Human Serum Albumin</td>
			<td>Sigma</td>
			<td>A6661</td>
			<td>Abbreviated DNP-HSA</td>
		</tr>
		<tr>
			<td>Plate Spectrophotometer</td>
			<td>Anthos Labtec HT2</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Pipes</td>
			<td>Sigma</td>
			<td>P1851</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Sigma</td>
			<td>M2670</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma</td>
			<td>C1016</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Triton x100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td>-</td>
		</tr>
		<tr>
			<td>4-nitrophenyl N-acetyl-&#946;-D-glucosaminide</td>
			<td>Sigma</td>
			<td>N9376</td>
			<td>Stock solution called &#946;-hexosaminidase substrate was 50mM prepared in DMSO</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Citric Acid</td>
			<td>Sigma</td>
			<td>251275</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sodium Acetate</td>
			<td>Sigma</td>
			<td>S7670</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sigma</td>
			<td>T5941</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

development of an in vitro model system for studying the interaction of equus caballus ige with its high-affinity receptor fc&#949;ri

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and Fc&#949;RI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine Fc&#949;RI&#945; was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine &#945;-chain formed a functional receptor complex with the endogenous rat &#946;- and &#947;-chains 1. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine Fc&#949;RI&#945; transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using &#946;-hexosaminidase release as a readout 2, 3. Mediator release peaked at 36.68% &#177; 4.88% at 100 ng ml-1 of antigen. This assay was modified from previous assays used to study human and canine allergic responses 4, 5. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease 6, 2, 3.

부모 RBL-2H3.1 세포 및 말 FcεRIα 수용체 유전자로 형질들은 먼저 마우스의 IgE 항 DNP-HSA 감작과 DNP-HSA 항원으로 감염시켰다. 마우스 IgE의 양쪽 세포주에서 내인성 쥐 수용체에 결합하므로 매개체 (그림 1 A)를 풀어 두 세포주의 분리 가능성을 테스트하기 위해 대조군으로서 작용한다. 이 중요한 검사이며, 세포 배양에 확장 (&gt; 10 주) 통과에 정기적으로 이후 수행되어야한다, RBL-2H3.1 세포...

Watch this Scientific Journal Video about Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcÎµRI at JoVE.com

Development of an &lt;em&gt;in vitro&lt;/em&gt; model system for studying the interaction of &lt;em&gt;Equus&lt;/em&gt; &lt;em&gt;caballus&lt;/em&gt; IgE with its high-affinity receptor Fc&amp;#949;RI

The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine Fc&#949;RI&#945; gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen.

개발<em&gt; 체외</em의 상호 작용을 연구&gt; 모델 시스템<em&gt; 에쿠스</em&gt;<em&gt; caballus</em높은 친 화성 수용체 FcεRI에&gt;의 IgE

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated ...

development-an-vitro-model-system-for-studying-interaction-equus

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Immunology and Infection

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

12.3K 조회수.  King Abdulaziz University. The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine FcεRIα gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen.

비디오: Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

에 대한 부정적인 선택 마커의 개발 Entamoeba histolytica</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

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In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

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8 잘 회의소 슬라이드에서 체외의 Biofilm 형성에

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

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

설립<em&gt; 체외</em&gt; 시스템의 세포 내 행동을 연구하는<em&gt; 칸디다 글라 브라</em&gt; 인간 THP-1 식세포에

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.
We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNF&#945;) and found the presence of CD16+ cells to be significantly higher on TNF&#945; stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

소설<em&gt; 시험관</em&gt; 인간의 전체 혈액과 내피 세포 사이의 상호 작용 유학을위한 모델

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

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 ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

<em&gt; 체외</em&gt; HIV 공동 감염의 맥락에서 말라리아에 면역 반응을 측정하기위한 모델

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

미생물 커뮤니티를 공부<em&gt; 생체</em&gt; 사이에 호스트 매개 상호 작용의 모델<em&gt; 칸디다 알비 칸스</em&gt; 및<em&gt; 녹농균</em&gt; 항공에

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

<em&gt; 체외</em&gt; 임피던스 분광학을 사용하여 혈액 - 뇌 장벽 모델 : T 세포 - 내피 세포 상호 작용에 초점

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

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.

챔버의 coverglass 모델을 사용하여 바이오 필름 개발에 객담의 효과를 시각화

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 of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a fascinating approach to investigate immune modulation of cutaneous inflammation in mouse models of human diseases. Here we present a cost-effective protocol that delivered naked DNA in mouse skin and leads to transgene expression. The method is coined &#34;acufection&#34;, denoting acupuncture-mediated DNA transfection. To perform acufection, mouse skin was first infused with DNA in phosphate-buffered saline (PBS) and then pricked lightly with a bundle of acupuncture needles to facilitate the absorption of DNA and transfection into cells. The plasmid DNA is presumably taken up by the keratinocyte and dendritic cells (DCs) in the skin and expressed into protein. Mechanical prick with the needles per se did not cause skin damage or induce keratinocyte activation. The expression of the transfected genes was detected in the skin at both transcriptional and translational levels following acufection for 2 days and maintained up to 7 days. The primary goal for the development of this acufection method was to investigate a previously undefined isoform of IL-15. Using this method, an alternatively spliced IL-15 isoform with partially deleted exon 7 (IL-15&#916;E7) was expressed in the skin and subsequently treated with a Toll-like receptor 7 (TLR7) agonist, imiquimod (IMQ), to induce inflammation. Acufection-delivered IL-15&#916;E7 in skin suppressed keratinocyte proliferation, epidermal thickness and neutrophil recruitment in IMQ-induced cutaneous inflammation. With increasing interest in identifying the regulatory mechanisms of cutaneous inflammation, the protocol described here provides a cost effective and versatile alternative to the gene gun system or microseeding for DNA delivery in vivo. It may potentially allow discovery of the function of a novel gene in the skin or for investigating new treatment for cutaneous diseases.

Development of an Economical DNA Delivery System by &quot;Acufection&quot; and its Application to Skin Research

This protocol is a cost-effective alternative for expressing naked plasmid DNA in mouse skin. The overall goal of the protocol is to deliver immune-related genes into skin tissue to delineate the functional role of a specific gene in cutaneous inflammation.

&quot;Acufection&quot;와 피부 연구에의 응용에 의한 경제적 인 DNA 전달 시스템의 개발

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a ...