이 연구는 관절염과 미 국립 보건원의 Musculoskeletal와 피부 질환의 국립 연구소의 교내 연구 프로그램에 의해 지원되었다.

1. 전체 혈액 수집 <ol><li> 영향을받는 개인뿐만 아니라 나이, 성별 일치하는 건강한 컨트롤에서 말초혈 10 ML 최소를 얻습니다. 정맥 혈액이 아니라 나트륨 헤파린과 적절한 혼합을 보장하기 위해 EDTA (에틸렌 다이아 민 테트라 초산), 그리고 거꾸로 여러 번 같은 다른 anticoagulants를 사용하여보다 나트륨 헤파린의 vacutainer 혈액 수집 튜브에 수집한다. 10 ML이 특정 검정에 대한 중복의 도금 샘플에 필요한 전체 혈액의 최소 볼륨입니다. </li><li> 샘플은 최상의 결과를 위해 수집 후 가능한 빨리 처리되어야합니다. 최대 24 시간 (2) 처리 될 준비까지를 위해 그러나, heparinized 혈액이 희미한 조명 아래의 상온에서 저장할 수 있습니다. </li></ol> 2. 전체 혈액의 준비 <ol><li> 상온 10 분 3,000 RPM (브레이크가 해제된 상태로)에서 모든 혈액 샘플을 원심 분리기. 버피 코트를 방해하지 않고, 플라즈마는 위로부터 수집된 수 있으며, 다른 실험 목적을 위해 저장됩니다. 플라즈마 샘플은 일시적 ° C 장기 저장을 위해 -20 ° C 또는 -80에 저장할 수 있습니다 </li><li> 일단 플라즈마를 수집, 혈청 무료 (불완전) RPMI 1640 미디어 (주위 온도에서)와 적혈구 세포 (RBC) 펠렛과 버피 코트를 resuspending하여 샘플을 씻으십시오. 부드럽게 모음 튜브의 혼합물을 피펫과 50 ML 원뿔 관을 전송할 수 있습니다. </li><li> 최대 불완전한 RPMI 1640 문화 미디어 50 ML하고 부드럽게 동질적인 혼합을 만들 수있는 튜브를 반전 볼륨. 십분 3,000 RPM (브레이크가 해제된 상태로)에서 샘플을 원심 분리기. 확인하는 것은 명확한 분리가, 버피 코트를 방해하지 않도록주의하고, 표면에 뜨는를 대기음. 이 절차 모든 오염 물질과 바운드 크린 시토킨가 자극하기 전에 제거되는 것을 보장하기 위해 2-4 번 반복합니다. </li><li> 마지막 세척 후, 전체 볼륨이 불완전 RPMI 1640 미디어 20 ML (처음 그려 전체 혈액의 10 ML)을되도록 RBC 펠렛과 버피 코트를 resuspend. 전체 볼륨이 처음 그려진 혈액의 볼륨에 따라 조정할 수 있습니다. </li><li> Cellometer 비전을 사용하여 희석 혈액 acridine 오렌지 (AO)를 사용하여 백혈구의 숫자 옵션을 선택하여 lysing RBCs없이 계산하실 수 있습니다. 한 부분의 AO와 부하 일회용 계산 챔버에 1시 1분 혼합물 20 μL 한 부분 희석 혈액을 추가합니다. 희석 후 AO의 최종 농도는 1μg/mL해야합니다. </li><li> 2.0 X 10 6 세포 / ML의 최종 세포 농도 불완전한 RPMI 1640 미디어 따라 조정합니다. </li></ol> 3. 전체 혈액의 자극 <ol><li> 검정 네 조건 (각 중복 이루어)의 최소 필요합니다 unstimulated 혼자 LPS의 추가, ATP 혼자의 추가, 그리고 마지막으로 LPS 플러스 ATP가 함께 추가되었습니다. 24 잘 조직 문화 판의 해당 잘으로 준비 2.0 X 10 6 세포 / ML 희석 전체 혈액의 1 ML을 추가합니다. </li><li> 자극은 자극 분석을 시작하기 전에 재구성하고 실온까지 가지고 있어야합니다. 동결 건조된 LPS와 ATP는 제조 업체의 지시에 따라 재구성되어야합니다. reconstitution 후, LPS와 ATP가 더욱 불완전 RPMI 1640 미디어를 사용하여 적절한 재고 농도 희석 수 있습니다. </li><li> 물론 이렇게 LPS의 마지막 작업 농도가 1 μg / ML되는 LPS에만뿐만 아니라 LPS 플러스 ATP에 LPS를 추가합니다. 전체 자극 시간은 3 시간입니다. 그러나, 전체 혈액이 2 시간 40 분 LPS로 자극합니다. 샘플은 37 ° C와 5% CO 2 배양기에 배치해야합니다. </li><li> ATP는 3 시간 자극 기간의 나머지 이십분 동안 추가됩니다. ATP 혼자와 LPS와 인큐베이터에서 ATP의 잘과 장소 샘플로 ATP를 추가합니다. ATP의 마지막 작업 농도 2mM입니다. </li><li> 3 시간 잠복기 후, 1.5 ML의 microcentrifuge 관에 각각의 샘플을 aliquoting 및 이후 상온에서 2 분 동안 10,000 rpm으로 샘플을 centrifuging하여 supernatants를 수집합니다. 1.5 microcentrifuge 튜브 다른 설정 supernatants를 전송합니다. </li><li> 샘플은 즉시 ° C 장기 저장을 위해 -20 ° C 또는 -80에서 일시적으로 assayed하거나 저장할 수 있습니다. </li></ol> 4. 시토킨 분석 <ol><li> 냉동 샘플 분석하기 전에 실내 온도 평형해야합니다. IL - 1βcan에 대한 시토킨 농도는 다양한 방법을 사용하여 평가됩니다. 우리는 제조 업체의 지침에 따라, 바이오 플렉스 200 시스템과 함께 X - 플렉스 분석 바이오 플렉스 프로 인간 시토킨을 활용한다. 이 프로토콜에서는 IL - 1β 구체적으로 측정되지만, 우리는 동시에 작은 샘플 볼륨을 사용하는 혜택 한 분석에서 여러 크린 시토킨을 측정하기 위해이 멀티 플렉스 시토킨 분석을 활용한다. </li></ol> 5. 대표 결과 LPS와 ATP와 함께 성공적인 자극시 IL - 1β 생산 분석 할 보여줍니다SE 자극되는 세포의 숫자에 따라 반응. 우리는 2.0 X 10 6 세포 / ML은 IL - 1β의 최적 및 측정 농도뿐만 아니라 다른 크린 시토킨 (그림 2)를 생산하기 위해 충분한 농도 것으로 나타났습니다. 전체 혈액 assays는 autoinflammatory 발현과 현재 환자를 공부에 유용합니다. 이러한 환자는 혼자 LPS (그림 3)과 자극시 IL - 1β의 생산 과잉 (컨트롤에 비해)을 특징으로 할 수있는 타고난 면역 체계에 결함이 표시될 수 있습니다. <img src="/files/ftp_upload/2662/2662fig1.jpg" alt="그림 1" /> 그림 1. 희석 전체의 혈액 샘플에서 세포 농도를 결정하기 위해 사용되는 nucleated 백혈구에 대한 AO의 얼룩 대표 이미지. <img src="/files/ftp_upload/2662/2662fig2.jpg" alt="그림 2" /> 그림 2. IL - 1β 생산 증가 세포 농도에서 LPS로 자극시 건강한 기증자로부터 온 혈액 supernatants로 측정. 샘플은 세중의에 assayed되었습니다. <img src="/files/ftp_upload/2662/2662fig3.jpg" alt="그림 3" /> 그림 3. 두명의 건강한 정상 컨트롤뿐만 아니라 IL - 1β - 관련 autoinflammatory 발현과 표현이 환자에서 전체 혈액 supernatants에서 측정한 IL - 1β 분비 대표 결과입니다.

<ol>
	<li>Martinon, F., Mayor, A., Tschopp, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inflammasomes:+guardians+of+the+body.">The inflammasomes: guardians of the body.</a> Annu Rev Immunol. 27, 229-265 (2009).</li><li>Thurm, C. W., Halsey, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+cytokine+production+using+whole+blood.">Measurement of cytokine production using whole blood.</a> Curr Protoc Immunol. 66, 7-18 .</li><li>Mariathasan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopyrin+activates+the+inflammasome+in+response+to+toxins+and+ATP.">Cryopyrin activates the inflammasome in response to toxins and ATP.</a> Nature. 440, 228-232 (2006).</li><li>Gabay, C., Lamacchia, C., Palmer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-1+pathways+in+inflammation+and+human+diseases.">IL-1 pathways in inflammation and human diseases.</a> Nat Rev Rheumatol. 4, 232-241 (2010).</li><li>Franchi, L., Elgenbrod, T., Mu&#241;oz-Planillo, R., Nu&#241;ez, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inflammasome:+a+caspase-1-activation+platform+that+regulates+immune+responses+and+disease+pathogenesis.">The inflammasome: a caspase-1-activation platform that regulates immune responses and disease pathogenesis.</a> Nat Immunol. 3, 241-247 (2009).</li><li>Sims, J. E., Smith, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+IL-1+family:+regulators+of+immunity.">The IL-1 family: regulators of immunity.</a> Nat Rev Immunol. 2, 89-102 (2010).</li><li>Haug, R., Jo&#248;, G. B., Westvik, A. B., Kierulf, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokine+production+and+pattern+recognition+receptor+(PRR)+expression+in+whole+blood+stimulated+with+pathogen-associated+molecular+patterns+(PAMPs.">Chemokine production and pattern recognition receptor (PRR) expression in whole blood stimulated with pathogen-associated molecular patterns (PAMPs.</a> Cytokine. 32, 304-315 (2005).</li><li>Netea, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+requirement+for+the+activation+of+the+inflammasome+for+processing+and+release+of+IL-1bin+monocytes+and+macrophages.">Differential requirement for the activation of the inflammasome for processing and release of IL-1bin monocytes and macrophages.</a> Blood. 113, 2324-2335 (2009).</li><li>Latz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inflammasomes:+mechanisms+of+activation+and+function.">The inflammasomes: mechanisms of activation and function.</a> Curr Opin Immnol. 22, 28-33 (2010).</li><li>Masters, S. L., Simon, A., Aksentijevich, I., Kastner, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Horror+autoinflammaticus:+the+molecular+pathophysiology+of+autoinflammtory+disease.">Horror autoinflammaticus: the molecular pathophysiology of autoinflammtory disease.</a> Annu Rev Immunol. 27, 621-668 (2009).</li></ol>

관심 없음 충돌 선언하지 않습니다.

설명 전체 혈액 분석은 세포 분리 및 광범위한 시료 처리 및 조작을 필요로 다른 기술에 비해보다 밀접하게 생리 조건을 모방하는 간단한 방법입니다. 이 방법은 전체 혈액의 백혈구를 계산하기 때문에, 정확하게 셀 번호에 따라 샘플을 표준화하는 방법을 제공하기 위해 AO와 Cellometer 비전을 활용합니다. 그것은이 분석을 위해, 전체 혈액은 다른 anticoagulants가 칼슘 chelators을 ?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Sodium Heparin Blood Collection Tubes</td>
<td>&nbsp;</td>
<td>BD</td>
<td>366480</td>
<td>&nbsp;</td>
<tr>
<td>RPMI 1640 Media</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>21870-100</td>
<td>&nbsp;</td>
<tr>
<td>Acridine Orange</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A3568</td>
<td>&nbsp;</td>
<tr>
<td>Cellometer Vision</td>
<td>&nbsp;</td>
<td>Nexcelom Bioscience</td>
<td>&nbsp;</td>
<td>Contact company for instrument information</td>
</tr>
<tr>
<td>Cellometer Cell Counting Chambers</td>
<td>&nbsp;</td>
<td>Nexcelom Bioscience</td>
<td>CHT4-SD100</td>
<td>&nbsp;</td>
<tr>
<td>24-well Tissue Culture Plate</td>
<td>&nbsp;</td>
<td>Corning Costar</td>
<td>3524</td>
<td>&nbsp;</td>
<tr>
<td>Ultra Pure E. coli K12 LPS</td>
<td>&nbsp;</td>
<td>InvivoGen</td>
<td>tlrl-eklps</td>
<td>&nbsp;</td>
<tr>
<td>Adenosine 5&#180;-tripohasphate disodium salt hydrate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A26209</td>
<td>&nbsp;</td>
<tr>
<td>Bio-plex 200 system</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>&nbsp;</td>
<td>Contact company for instrument information</td>
</tr>
<tr>
<td>Bio-Plex Pro human cytokine x-plex assay</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>&nbsp;</td>
<td>x-plex assays are custom made according to specified cytokines of interest</td>
</tr>		</tbody>
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accurate and simple measurement of the pro-inflammatory cytokine il-1&beta; using a whole blood stimulation assay

면역 세포에 의해 용해 중재자의 분비로 인한 염증성 과정, 피부, 관절과 기타 조직뿐만 아니라 변경 시토킨 항상성의 다양한 발현로 이어집니다. 타고난 면역 체계는 병원균과 다른 내생 위험 자극의 인식에 중요한 역할을 담당하고 있습니다. 타고난 면역 세포에 의해 발표 주요 크린 시토킨 중 하나는 인터루킨 (IL) -1입니다. 따라서, 우리는 염증 크린 시토킨의 분비를 측정하기 위해, 특히 프로 염증 시토킨 IL - 1β 1, 2, 3의 전체 혈액 자극 분석을 활용한다. autoinflammatory syndromes를 일으키는 본래 면역 시스템의 유전자 장애 환자는 혼자 LPS로 자극시 IL - 1β 성숙의 과장 릴리스를 보여줍니다. 염증 - 관련 pathologies로 현재 환자의 타고난 면역 구성 요소를 평가하기 위해, 우리는 그람 음성 세균의 내독소 같은 병원체 - 관련 분자 패턴 (PAMPs)에 세포 면역 반응을 감지하는 특정 면역 분석법, lipopolysaccharide (LPS를 사용하여 ). 이러한 PAMPs은 본래 면역 체계 4, 5, 6, 7의 세포에서 찾을 수있는 병원체 인식 수용체 (PRRs)에 의해 인정됩니다. 보조 신호, ATP, inflammasome의 활성화, 그 성숙, bioactive 형태 4, 5, 6, 8, 9 프로 IL - 1β 처리하는 multiprotein 복합 필요한 함께 기본 신호, LPS, 10. 전체 혈액 분석은 특정 세포 집단의 노동 집약적인 고립과 culturing을 필요로 다른 방법에 비해 시토킨 생산을 평가하기 위해 최소한의 샘플 조작이 필요합니다. 이 방법은 다른 모든 혈액 자극의 assays 다릅니다; 오히려 RPMI 미디어의 비율로 샘플을 diluting보다, 우리는 백혈구가 희석 전체 혈액에서 직접 계산되므로, 문화 2 백혈구의 알려진 숫자를 자극 수행합니다. 이 특정 전체 혈액 분석의 결과는 autoinflammatory pathophysiologies와 함께 제시 환자의 동료를 elucidating에 도움이 소설 기법을 보여줍니다.

Watch this Scientific Journal Video about Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1Î² using a Whole Blood Stimulation Assay at JoVE.com

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&amp;#946; using a Whole Blood Stimulation Assay

우리는 autoinflammatory phenotypes와 함께 제시 환자에있어서 IL - 1 베타의 생산과 같은 프로 염증성 크린 시토킨의 생산, 측정하는 간단한 면역 분석법을 설명합니다. 특히 lipopolysaccharide와 병원체 - 관련된 분자 패턴과 전체 혈액 배양에서 세포를 활성화하여, 시토킨 분비가 편리 전체 혈액 supernatants에서 평가하실 수 있습니다.

전체 혈액 자극 분석을 사용하여 프로 염증성 시토킨 IL - 1β의 정확하고 간단한 측정

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues ...

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JoVE Journal

Medicine

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&beta; using a Whole Blood Stimulation Assay

26.2K 조회수.  National Institute of Arthritis and Musculoskeletal and Skin Diseases. 우리는 autoinflammatory phenotypes와 함께 제시 환자에있어서 IL - 1 베타의 생산과 같은 프로 염증성 크린 시토킨의 생산, 측정하는 간단한 면역 분석법을 설명합니다. 특히 lipopolysaccharide와 병원체 - 관련된 분자 패턴과 전체 혈액 배양에서 세포를 활성화하여, 시토킨 분비가 편리 전체 혈액 supernatants에서 평가하실 수 있습니다.

비디오: Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&#946; using a Whole Blood Stimulation Assay

Accurate and Simple Evaluation of Vascular Anastomoses in Monochorionic Placenta using Colored Dye

The presence of placental vascular anastomoses is a conditio sine qua non for the development of twin-to-twin transfusion syndrome (TTTS) and twin anemia polycythemia sequence (TAPS)1,2. Injection studies of twin placentas have shown that such anastomoses are almost invariably present in monochorionic twins and extremely rare in dichorionic twins1. Three types of anastomoses have been documented: from artery to artery, from vein to vein and from artery to vein. Arterio-venous (AV) anastomoses are unidirectional and are referred to as "deep" anastomoses since they proceed through a shared placental cotyledon, whereas arterio-arterial (AA) and veno-venous (VV) anastomoses are bi-directional and are referred to as "superficial" since they lie on the chorionic plate. Both TTTS and TAPS are caused by net imbalance of blood flow between the twins due to AV anastomoses. Blood from one twin (the donor) is pumped through an artery into the shared placental cotyledon and then drained through a vein into the circulation of the other twin (the recipient). Unless blood is pumped back from the recipient to the donor through oppositely directed deep AV anastomoses or through superficial anastomoses, an imbalance of blood volumes occurs, gradually leading to the development of TTTS or TAPS. The presence of an AA anastomosis has been shown to protect against the development of TTTS and TAPS by compensating for the circulatory imbalance caused by the uni-directional AV anastomoses1,2. 
Injection of monochorionic placentas soon after birth is a useful mean to understand the etiology of various (hematological) complications in monochorionic twins and is a required test to reach the diagnosis of TAPS2. In addition, injection of TTTS placentas treated with fetoscopic laser surgery allows identification of possible residual anastomoses3-5. This additional information is of paramount importance for all perinatologists involved in the management and care of monochorionic twins with TTTS or TAPS. Several placental injection techniques are currently being used. We provide a simple protocol to accurately evaluate the presence of (residual) vascular anastomoses using colored dye injection.

Twin-to-twin transfusion syndrome and twin anemia polycythemia sequence are two potentially devastating problems in perinatal medicine. Both disorders occur only in monochorionic twins and result from unbalanced blood flow through placental vascular anastomoses. We provide a simple protocol to accurately evaluate the presence of vascular anastomoses using colored dye injection of placental vessels after birth.

유색 염료를 사용하여 Monochorionic 태반의 혈관 Anastomoses의 정확하고 간단한 평가

The presence of placental vascular anastomoses is a conditio sine qua non for the development of twin-to-twin transfusion syndrome (TTTS) and twin anemia ...

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Epidural Intracranial Pressure Measurement in Rats Using a Fiber-optic Pressure Transducer

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and cardiac arrest. This elevation may result in further neurological injury, in the form of transtentorial herniation1,2,3,4, midbrain compression, neurological deficit or increased cerebral infarct2,4. Current therapies are often inadequate to control elevated ICP in the clinical setting5,6,7 . Thus there is a need for accurate methods of ICP measurement in animal models to further our understanding of the basic mechanisms and to develop new treatments for elevated ICP.
In both the clinical and experimental setting ICP cannot be estimated without direct measurement. Several methods of ICP catheter insertion currently exist. Of these the intraventricular catheter has become the clinical 'gold standard' of ICP measurement in humans8. This method involves the partial removal of skull and the instrumentation of the catheter through brain tissue. Consequently, intraventricular catheters have an infection rate of 6-11%9. For this reason, subdural and epidural cannulations have become the preferred methods in animal models of ischemic injury. 
Various ICP measurement techniques have been adapted for animal models, and of these, fluid-filled telemetry catheters10 and solid state catheters are the most frequently used11,12,13,14,15. The fluid-filled systems are prone to developing air bubbles in the line, resulting in false ICP readings. Solid state probes avoid this problem (Figure 1). An additional problem is fitting catheters under the skull or into the ventricles without causing any brain injury that might alter the experimental outcomes. Therefore, we have developed a method that places an ICP catheter contiguous with the epidural space, but avoids the need to insert it between skull and brain. 
An optic fibre pressure catheter (420LP, SAMBA Sensors, Sweden) was used to measure ICP at the epidural location because the location of the pressure sensor (at the very tip of the catheter) was found to produce a high fidelity ICP signal in this model. There are other manufacturers of similar optic fibre technologies13 that may be used with our methodology. Alternative solid state catheters, which have the pressure sensor located at the side of the catheter tip, would not be appropriate for this model as the signal would be dampened by the presence of the monitoring screw. 
Here, we present a relatively simple and accurate method to measure ICP. This method can be used across a wide range of ICP related animal models.

A novel technique to record the pressures within the skull is described. The minimally invasive method uses a fibre-optic pressure sensing system to accurately measure intracranial pressure (ICP) in anaesthetized rats without causing significant brain trauma. The technique may be used in a wide range of experimental models.

광섬유 압력 변환기를 사용하여 쥐에서 경막외 Intracranial 압력 측정

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and ...

Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the low-molecular-weight-heparin (LMWH), enoxaparin. There are also urgent clinical situations where it would be important if this could be determined rapidly. The present assay is designed to accomplish this. We only assayed human blood samples that were spiked with known concentrations of enoxaparin. The essential feature of the present assay is the quantification of the efficacy of enoxaparin in a patient's blood sample by degrading it to complete inactivity with heparinase. Two blood samples were drawn into Vacutainer tubes (Becton-Dickenson; Franklin Lakes, NJ) that were spiked with enoxaparin; one sample was digested with heparinase for 5 min at 37 &deg;C, the other sample represented the patient's baseline anticoagulated status. The percent shortening of clotting time in the heparinase-treated sample, as compared to the baseline state, yielded the anticoagulant contribution of enoxaparin. We used the portable, battery operated Hemochron 801 apparatus for measurements of clotting times (International Technidyne Corp., Edison, NJ). The apparatus has 2 thermostatically controlled (37 &deg;C) assay tube wells. We conducted the assays in two types of assay cartridges that are available from the manufacturer of the instrument. One cartridge was modified to increase its sensitivity. We removed the kaolin from the FTK-ACT cartridge by extensive rinsing with distilled water, leaving only the glass surface of the tube, and perhaps the detection magnet, as activators. We called this our minimally activated assay (MAA). The use of a minimally activated assay has been studied by us and others. 2-4 The second cartridge that was studied was an activated partial thromboplastin time (aPTT) assay (A104). This was used as supplied from the manufacturer. The thermostated wells of the instrument were used for both the heparinase digestion and coagulation assays. The assay can be completed within 10 min. The MAA assay showed robust changes in clotting time after heparinase digestion of enoxaparin over a typical clinical concentration range. At 0.2 anti-Xa I.U. of enoxaparin per ml of blood sample, heparinase digestion caused an average decrease of 9.8% (20.4 sec) in clotting time; at 1.0 I.U. per ml of enoxaparin there was a 41.4% decrease (148.8 sec). This report only presents the experimental application of the assay; its value in a clinical setting must still be established.

Demonstration of a rapid quantitative assay of the inhibition of blood coagulation by the low-molecular-weight-heparin, enoxaparin. The contribution of enoxaparin is assessed by removing its influence through digestion with heparinase. A fuller description of the assay is detailed in our published paper.1 The assay still requires clinical confirmation.

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the ...

The &alpha;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time and temperature restrictions, technical sophistication, and cost of reagents, in particular monoclonal antibodies to measure CD4 on blood cells, the only currently acceptable method. A commonly used cost-saving and time-saving laboratory strategy is to calculate, rather than measure certain blood values. For example, LDL levels are calculated using the measured levels of total cholesterol, HDL, and triglycerides1. Thus, identification of cell-free correlates that directly regulate the number of CD4+ T cells could provide an accurate method for calculating CD4 counts due to the physiological relevance of the correlates.
The number of stem cells that enter blood and are destined to become circulating CD4+ T cells is determined by the chemokine CXCL12 and its receptor CXCR4 due to their influence on locomotion2. The process of stem cell locomotion into blood is additionally regulated by cell surface human leukocyte elastase (HLECS) and the HLECS-reactive active &alpha;1proteinase inhibitor (&alpha;1PI, &alpha;1antitrypsin, SerpinA1)3. In HIV-1 disease, &alpha;1PI is inactivated due to disease processes 4. In the early asymptomatic categories of HIV-1 disease, active &alpha;1PI was found to be below normal in 100% of untreated HIV-1 patients (median=12 &mu;M, and to achieve normal levels during the symptomatic categories4, 5. This pattern has been attributed to immune inactivation, not to insufficient synthesis, proteolytic inactivation, or oxygenation. We observed that in HIV-1 subjects with &gt;220 CD4 cells/&mu;l, CD4 counts were correlated with serum levels of active &alpha;1PI (r2=0.93, p&lt;0.0001, n=26) and inactive &alpha;1PI (r2=0.91, p&lt;0.0001, n=26) 5. Administration of &alpha;1PI to HIV-1 infected and uninfected subjects resulted in dramatic increases in CD4 counts suggesting &alpha;1PI participates in regulating the number of CD4+ T cells in blood 3.
With stimulation, whole saliva contains sufficient serous exudate (plasma containing proteinaceous material that passes through blood vessel walls into saliva) to allow measurement of active &alpha;1PI and the correlation of this measurement is evidence that it is an accurate method for calculating CD4 counts. Briefly, sialogogues such as chewing gum or citric acid stimulate the exudation of serum into whole mouth saliva. After stimulating serum exudation, the activity of serum &alpha;1PI in saliva is measured by its capacity to inhibit elastase activity. Porcine pancreatic elastase (PPE) is a readily available inexpensive source of elastase. PPE binds to &alpha;1PI forming a one-to-one complex that prevents PPE from cleaving its specific substrates, one of which is the colorimetric peptide, succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide (SA3NA). Incubating saliva with a saturating concentration of PPE for 10 min at room temperature allows the binding of PPE to all the active &alpha;1PI in saliva. The resulting inhibition of PPE by active &alpha;1PI can be measured by adding the PPE substrate SA3NA. (Figure 1). Although CD4 counts are measured in terms of blood volume (CD4 cells/&mu;l), the concentration of &alpha;1PI in saliva is related to the concentration of serum in saliva, not to volume of saliva since volume can vary considerably during the day and person to person6. However, virtually all the protein in saliva is due to serum content, and the protein content of saliva is measurable7. Thus, active &alpha;1PI in saliva is calculated as a ratio to saliva protein content and is termed the &alpha;1PI Index. Results presented herein demonstrate that the &alpha;1PI Index provides an accurate and precise physiologic method for calculating CD4 counts.

The &amp;#945;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

A CD4 enumeration method, the &#x03B1;-test, is described which uses whole saliva to provide rapid and accurate CD4 counts. The &alpha;-test costs pennies and eliminates the need for technical training, costly reagents such as monoclonal antibodies, instrumentation, refrigeration, transport of samples, as well as collection and handling of blood.

α-테스트 : 전체 타액을 사용하여 빠른 셀 프리 CD4의 열거

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

같은 뇌 Microslices의 흥분 독성 자극<em&gt; 체외</em&gt; 스트로크의 모델

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Fast and Accurate Exhaled Breath Ammonia Measurement

This exhaled breath ammonia method uses a fast and highly sensitive spectroscopic method known as quartz enhanced photoacoustic spectroscopy (QEPAS) that uses a quantum cascade based laser. The monitor is coupled to a sampler that measures mouth pressure and carbon dioxide. The system is temperature controlled and specifically designed to address the reactivity of this compound. The sampler provides immediate feedback to the subject and the technician on the quality of the breath effort. Together with the quick response time of the monitor, this system is capable of accurately measuring exhaled breath ammonia representative of deep lung systemic levels. 
Because the system is easy to use and produces real time results, it has enabled experiments to identify factors that influence measurements. For example, mouth rinse and oral pH reproducibly and significantly affect results and therefore must be controlled. Temperature and mode of breathing are other examples. As our understanding of these factors evolves, error is reduced, and clinical studies become more meaningful. This system is very reliable and individual measurements are inexpensive. 
The sampler is relatively inexpensive and quite portable, but the monitor is neither. This limits options for some clinical studies and provides rational for future innovations.

Ammonia is an important physiologic metabolite relevant to various disease and wellness states. It is also a difficult molecule to measure in breath, which demands particular precautions be taken to obtain accurate results. Not all factors influencing ammonia are known, but progress can be difficult without accounting for these factors.

빠르고 정확한 날숨 암모니아 측정

This exhaled breath ammonia method uses a fast and highly sensitive spectroscopic method known as quartz enhanced photoacoustic spectroscopy (QEPAS) that ...

Real-time Cytotoxicity Assays in Human Whole Blood

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex&#160;vivo condition.

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

인간 전혈에서 실시간 세포 독성 분석 실험

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...

A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe the use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations of superior and uniform quality to that which can be achieved by hand. The device comprises a base that holds 4 stainless steel rods and a top, which acts a cutting guide. The rods are inserted into the lumen of the small intestine [divided into thirds] and the colon. The rods and samples are then placed over a piece of filter paper or card into the holding slots in the base of the device. The top of the device is then positioned and serves as a cutting guide. The two angled sections in the center of the top piece are used to guide a knife or scalpel and cut the intestines longitudinally on the top of the rods. Once the intestinal sections have been cut, the top is removed and the card,&#160;tissue and rods gently removed from the device and placed on the bench. The rods are then gently rolled sideways to flatten and stick the intestinal segments onto the underlying piece of filter paper or card. The final preparation can then be examined or fixed and stored for later analysis. The preparations are invaluable for the study of intestinal changes in normal or genetically modified mouse models. The preparations have been used for the study and quantification of the effects of inflammation (colitis), damage, pre-cancerous lesions (aberrant crypt foci (ACFs) and mucin depleted foci (MDFs)) and polyps or tumors.

The use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations is described.

간단한 장치는 빠르게 마우스 소장의 전체 마운트를 준비하는 방법

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

깊은 뇌 자극과 Saccadometry를 사용하여 정상 및 병적 뇌 기능을 연구하는

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

Long-term Blood Pressure Measurement in Freely Moving Mice Using Telemetry

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. Indeed, research focusing on the discovery of new potential therapeutic targets using genetically altered mice requires a reliable, long-term assessment of the systemic arterial pressure variation. Currently, the gold standard for obtaining long-term measurements of blood pressure in ambulatory mice uses implantable radio-transmitters, which require&#160;artery cannulation. This technique eliminates the need for tethering, restraining, or anesthetizing the animals which introduce stress and artifacts during data sampling. However, arterial blood pressure monitoring in mice via catheterization can be rather challenging due to the small size of the arteries. Here we present a step-by-step guide to illustrate the crucial key passages for a successful subcutaneous implantation of radio-transmitters and carotid artery cannulation in mice. We also include examples of long-term blood pressure activity taken from freely moving mice after a period of post-surgery recovery. Following this procedure will allow reliable direct blood pressure recordings from multiple animals simultaneously.

The goal of this protocol is to assess systemic blood pressure in conscious freely moving mice using implantable radio-telemetry devices.

텔레 메 트리를 사용하여 자유롭게 이동 마우스의 장기 혈압 측정

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. ...