\ \ www.nhls.ac.za 및 http://www.plgcd4 : 특별 감사는 HIV 관련 흐름을 진단 서비스의 국가 조직에 남아 프리카 공화국의 국민 건강 연구소 서비스 (NHLS) (HTTP에 의한 아르 . NET). 기능 연구 Haematology과 분자 의약, Witwatersrand 대학의학과에서 실시되었습니다 (요하네스 버그, 남아 프리카, 부서 책임자, 교수 웬디 스티븐스 지원). 저자는 상업 회사 또는 자금 지원 기관이 발행물에 대한 교부금 지원을받은이 없습니다. 표현 의견은 반드시 Witwatersrand 또는 남아 프리카 NHLS 대학의 기관의 의견을 반영하지 않고 저자의 사람입니다.

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+North+American+multilaboratory+study+of+CD4+counts+using+flow+cytometric+panLeukogating+(PLG):+a+NIAID-DAIDS+Immunology+Quality+Assessment+Program+Study.">A North American multilaboratory study of CD4 counts using flow cytometric panLeukogating (PLG): a NIAID-DAIDS Immunology Quality Assessment Program Study.</a> Cytometry B Clin Cytom. 74, S52-S64 (2008).</li><li>Janossy, 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+changing+pattern+of+&#34;smart&#34;+flow+cytometry+(S-FC)+to+assist+the+cost-effective+diagnosis+of+HIV,+tuberculosis,+and+leukemias+in+resource-restricted+conditions.">The changing pattern of &#34;smart&#34; flow cytometry (S-FC) to assist the cost-effective diagnosis of HIV, tuberculosis, and leukemias in resource-restricted conditions.</a> Biotechnol J. 3, 32-42 (2008).</li></ol>

CD4 방법에 대한 국제 특허 권리는 여기에 설명된 남아 프리카 NHLS에 의해 현재 BCI, 마이애미, FL, 미국 라이센스하에 실시하고 있습니다.

최근에는 자선 중심과 보조금 - 할당된 프로그램은 높은 HIV 부담으로 지구의 자원을 제한된 영역에서 일부 우수한 임상 관행의 설립을 도왔있다. ART 프로그램은 사망률을 감소에 성공하고 지금은 높은 CD4에서 시작되고있는 것은 2,3로 계산됩니다. 이러한 새로운 조치 HIV 감염 4,5의 확산을 감소 것이라는 징후는 HIV - 특정 백신의 부족에도 불구하고, 이미있다. 우리의 영상 우리는 문서에서 예술에 환자를 모니터링 할 때, 이러한 기술은 더 초점을보다 비용 효율적인 서부 연구소 (목록 1) 수행 루틴 연습에서 관찰보다 될 수 있습니다. 이러한 아프리카 주도의 접근 방식은 잘 ART 취급 HIV 감염, 만성 질병을지고있을 때 한 번에 실험실 서비스의 업그레이 드를 지원하기 위해 필수적인 수 있습니다. 예술의 이러한 모니터링 카운팅 정확한 CD4 및 바이러스 부하 관련 기술보다 더 까다로운 작업이 단순히 ART 16 시작에 대한 결정을 지원하기 위해 임의의 그룹으로 환자를 삽입하기위한 CD4 카운트를 수행할 수 있어야합니다. 이러한 진단의 도움으로, 즉 CD8/CD38 테스트를 사용하면, 그것은 예술 16,19에있는 환자의&gt; 60 %의 VL 관련 모니터링 비용을 줄일 수 있습니다. 이 약을 복용에 VL 리바운드 및 / 또는 환자 준수의 부족의 조기 징후를 데리러 가능성이 높습니다 CD4 개수 37, CD8/CD38 시험 16,17이고, 아니라는 것을 강조하는 것도 중요하다. 조정 비용 효율적인 중앙 서비스의 필수 요소는 작업의 기술 및 의료 측면을 잘 알고있는 전문가 및 학술 고문으로 지원하는 대규모 국가 또는 지역 네트워크의 가용성이다. 이것은 작은 센터에 대한 격려, 교육 및 품질 평가를 제공하고 이들도 &#39;포인트 오브 케어&#39;서비스를 포함할 수 있습니다 남아 프리카 NHLS 네트워크의 활동과 CD4 - PLG.net 교육 제도 33, 36에 의해 증명됩니다. 중요한 것은, 중앙 및 포인트의 케어 연구소의 작업 보완하고 있습니다. 이전에 비용 효율적인 대규모 서비스의 운영뿐만 아니라 말초 연구소 33,38위한 기본 / 고급 교육을 제공의 이중 역할을 가지고 있지만, 후자를받을 수 있습니다 HIV + 환자를 모집하여 예술의 확산을 확인하는 기여 시골 진료소를 포함하여 큰 기지에서 ART. 포인트 - 오브 - 간호 진단 훨씬 더 비쌀 수 있습니다 자동으로 정확하게 38를 보장하지 않기 때문에 몇 가지주의가 필요합니다. 이것은 두 계층 시스템을 유지하고 두 팔을 줄어들 수 있도록하지 않고 두 계층 간의 협력을 촉진하는 것이 현명한 것입니다. 결론적으로, 전염성 질병의 임상 관리의 진단 모니터링 분야에 필요한 비용 절감이 크게 향상된 품질 13,39과 함께 저렴하고 더 나은 조정 현대 기술을 사용하여 APT 서비스를 통해 얻을 수 있습니다. 잘 조직된 국가 (및 지역) 진단 서비스 조항이 더 신중 사전없이는 다양한 국가에서 수입한 그대로 거의 모든 보조금 지원이 즉시 비용 및 / 또는 해보지 않은 방법에 낭비 때 시나리오를 피함으로써 6-8를 지원해야합니다 emptive 건설 감시. 마찬가지로, 지역 의사와 의료 인력의 전체 참여와 임상 치료 실험의 더 유연한 설계는 불필요하게 잦은 및 / 또는 실험실 테스트 3 야심찬 이상의 감소로 연결되어야합니다. 이 정책은 예술에 대한 환자의 조정 임상 / 실험실 평가에 사용할 수있는 정확한 진단 결과를 풀러 활용에 더 많은 기회를 줄 것입니다. 이러한 스마트 진단은 기존의 익숙한 흐름 cytometric 플랫폼 40를 사용하여 결핵의 영역과 leukaemias의 자원 빈곤 국가 8 개척 수 있습니다.

comprehensive &amp; cost effective laboratory monitoring of hiv/aids: an african role model

경제 대규모 진단 테스트 사우스 - 아프리카 역할 모델을 대표하는 - 우리는 APT 실험실 서비스에 의해 방지 retroviral 치료 (ART)을 지원에 대한 비디오를 제시한다. 저소득 국가에서 저렴 예술 HIV seropositive 환자의 생존을 위해 전망을 변화했지만 예술의 실험실 감시 및 어떤 비용이 필요가 있는지 의심 없습​​니다 - 상황에 때 병리 서비스의 전반적인 품질이 아직도 매우 낮은 수 있습니다. 적절한 대답은 더 나은 조정과 서양 국가에서 볼 수보다 엄격한 내부 품질 평가와 함께 경제적으로 소리가 서비스를 구축하는 것입니다. Witwatersrand 대학교, 요하네스 버그, 남아 프리카 공화국에서 국민 건강 연구소 서비스 (NHLS - SA)의 위치에 사진이 비디오는, 원래의 2 색 CD4 - CD45 PanLeucoGating 전략 (PLG)를 확장 이러한 조정 계획을 제공합니다. 따라서 비디오 프레 젠 테이션의 여섯 모듈은 하나의 튜브에 haematological 면역 및 바이러스학 관련 테스트를 결합하는 4 색 흐름 cytometric 분석의 단순를 공개. 이 비디오 모듈은 다음과 같습니다 (I) 설정 악기, (ii) 본 샘플 준비가, (3) 절대 계산하고 구슬 카운트 속도하여 각 샘플에 대한 모니터링의 품질을 테스트, 백혈구에 대한 (IV) heamatological CD45 테스트 계산하고 차이, (V) CD4 카운트 및 CD8의 (VI) 활성화 + CD38 표시에 의해 측정된 T 세포, 바이러스 부하와 관련된 매개 변수입니다. 잠재적인 비용 절감 뛰어난 있습니다. 이 협정 NHLS - SA 네트워크 내의 다른 실험실로 기술의 전달도 서양 실험실에 적용되는보다 엄격한 품질 관리와 함께 하루&gt; 800-1000 테스트를 수행 가능성에 대한 주요 예입니다합니다. 전문 어드바이저 실험실 관리자와 현대 의료 기술을 도입에 대한 결정의 의무를 수행 정책 입안자 자주 위치에 워크로드의 큰 부담으로 큰 지역 연구소에서 수행과 같은 최신 기술 세부 정보를 볼 수 없습니다. 따라서이 동영상이 새로운 발전의 세부 사항을 보여줍니다.

Watch this Scientific Journal Video about Comprehensive & Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model at JoVE.com

Comprehensive &amp; Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

HIV / 에이즈 치료에 방지 retroviral 요법은 대규모 남아 프리카에서 모니터합니다. 유동세포계측법는 혈액학 (CD45), 면역학 (CD4) 및 바이러스 부하 링크 CD38 분석을위한 결합입니다. NHLS - SA 실험실, 요하네스 버그에 기록이 현대적인 방법은 자원 제한 진단을위한 역할 모델 역할을, 강한 지역 내부 품질 관리와 비용 효율적인 수 있습니다.

HIV / AIDS의 종합 및 비용 효과적인 실험실 감시 : 아프리카 역할 모델

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical ...

comprehensive-cost-effective-laboratory-monitoring-hivaids-an-african

Research

JoVE Journal

Immunology and Infection

16.6K 조회수.  National Health Laboratory Services (NHLS-SA). HIV / 에이즈 치료에 방지 retroviral 요법은 대규모 남아 프리카에서 모니터합니다. 유동세포계측법는 혈액학 (CD45), 면역학 (CD4) 및 바이러스 부하 링크 CD38 분석을위한 결합입니다. NHLS - SA 실험실, 요하네스 버그에 기록이 현대적인 방법은 자원 제한 진단을위한 역할 모델 역할을, 강한 지역 내부 품질 관리와 비용 효율적인 수 있습니다.

비디오: Comprehensive & Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all gynecologic malignancies among US women 1,2,3. Serous tumors are the most widespread forms of ovarian cancer and 4,5 the Tg-MISIIR-TAg transgenic represents the only mouse model that spontaneously develops this type of tumors. Tg-MISIIR-TAg mice express SV40 transforming region under control of the Mullerian Inhibitory Substance type II Receptor (MISIIR) gene promoter 6. Additional transgenic lines have been identified that express the SV40 TAg transgene, but do not develop ovarian tumors. Non-tumor prone mice exhibit typical lifespan for C57Bl/6 mice and are fertile. These mice can be used as syngeneic allograft recipients for tumor cells isolated from Tg-MISIIR-TAg-DR26 mice.
Objective: Although tumor imaging is possible 7, early detection of deep tumors is challenging in small living animals. To enable preclinical studies in an immunologically intact animal model for serous ovarian cancer, we describe a syngeneic mouse model for this type of ovarian cancer that permits in vivo imaging, studies of the tumor microenvironment and tumor immune responses.
Methods: We first derived a TAg+ mouse cancer cell line (MOV1) from a spontaneous ovarian tumor harvested in a 26 week-old DR26 Tg-MISIIR-TAg female. Then, we stably transduced MOV1 cells with TurboFP635 Lentivirus mammalian vector that encodes Katushka, a far-red mutant of the red fluorescent protein from sea anemone Entacmaea quadricolor with excitation/emission maxima at 588/635 nm 8,9,10. We orthotopically implanted MOV1Kat in the ovary 11,12,13,14 of non-tumor prone Tg-MISIIR-TAg female mice. Tumor progression was followed by in vivo optical imaging and tumor microenvironment was analyzed by immunohistochemistry.
Results: Orthotopically implanted MOV1Kat cells developed serous ovarian tumors. MOV1Kat tumors could be visualized by in vivo imaging up to three weeks after implantation (fig. 1) and were infiltrated with leukocytes, as observed in human ovarian cancers 15 (fig. 2).
Conclusions: We describe an orthotopic model of ovarian cancer suitable for in vivo imaging of early tumors due to the high pH-stability and photostability of Katushka in deep tissues. We propose the use of this novel syngeneic model of serous ovarian cancer for in vivo imaging studies and monitoring of tumor immune responses and immunotherapies.

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

To study in vivo tumor growth and tumor microenvironment, we used a syngeneic and orthotopic mouse model of ovarian cancer in immunocompetent animals. We transduced a mouse tumor cell line (MOV1) with Katushka fluorescent protein (MOV1KAT) and here we show its orthotopic implantation in ovary and in vivo imaging.

에 대한 면역 적격 마우스에 묽은 난소암의 Orthotopic 모델 생체내에</em종양 면역 응답> 종양 이미징 및 모니터링

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

연구

면역학 및 감염병학

An Analytical Tool-box for Comprehensive Biochemical, Structural and Transcriptome Evaluation of Oral Biofilms Mediated by Mutans Streptococci

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and composition 1, 2. In general, biofilms develop from initial microbial attachment on a surface followed by formation of cell clusters (or microcolonies) and further development and stabilization of the microcolonies, which occur in a complex extracellular matrix. The majority of biofilm matrices harbor exopolysaccharides (EPS), and dental biofilms are no exception; especially those associated with caries disease, which are mostly mediated by mutans streptococci 3. The EPS are synthesized by microorganisms (S. mutans, a key contributor) by means of extracellular enzymes, such as glucosyltransferases using sucrose primarily as substrate 3.
Studies of biofilms formed on tooth surfaces are particularly challenging owing to their constant exposure to environmental challenges associated with complex diet-host-microbial interactions occurring in the oral cavity. Better understanding of the dynamic changes of the structural organization and composition of the matrix, physiology and transcriptome/proteome profile of biofilm-cells in response to these complex interactions would further advance the current knowledge of how oral biofilms modulate pathogenicity. Therefore, we have developed an analytical tool-box to facilitate biofilm analysis at structural, biochemical and molecular levels by combining commonly available and novel techniques with custom-made software for data analysis. Standard analytical (colorimetric assays, RT-qPCR and microarrays) and novel fluorescence techniques (for simultaneous labeling of bacteria and EPS) were integrated with specific software for data analysis to address the complex nature of oral biofilm research.
The tool-box is comprised of 4 distinct but interconnected steps (Figure 1): 1) Bioassays, 2) Raw Data Input, 3) Data Processing, and 4) Data Analysis. We used our in vitro biofilm model and specific experimental conditions to demonstrate the usefulness and flexibility of the tool-box. The biofilm model is simple, reproducible and multiple replicates of a single experiment can be done simultaneously 4, 5. Moreover, it allows temporal evaluation, inclusion of various microbial species 5 and assessment of the effects of distinct experimental conditions (e.g. treatments 6; comparison of knockout mutants vs. parental strain 5; carbohydrates availability 7). Here, we describe two specific components of the tool-box, including (i) new software for microarray data mining/organization (MDV) and fluorescence imaging analysis (DUOSTAT), and (ii) in situ EPS-labeling. We also provide an experimental case showing how the tool-box can assist with biofilms analysis, data organization, integration and interpretation.

Biofilms formed on tooth surfaces are highly complex and exposed to constant innate and exogenous environmental challenges, which modulate their architecture, physiology and transcriptome. We developed a toolbox to examine the composition, structural organization and gene expression of oral biofilms, which can be adapted to other areas of biofilm research.

구강 Biofilms의 종합, 생물 구조 및 Transcriptome 평가를위한 분석 도구 - 상자가 Mutans Streptococci의 중재

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and ...

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

V3의 HIV - 1 Tropism의 Genotypic 추론 사용 인구를 기반 시퀀싱

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

이식 - 대 - 호스트 질병의 유도 및<em&gt; 생체 내</emMHC 검색 손쥐 모델을 사용&gt; T 셀 모니터링

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

형광 단백질을 사용하면 아프리카 Trypanosome에서 Glycosome 역학을 모니터링하려면

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor treatment options exist. The hallmarks of these laboratories are: custom-designed airtight doors, dedicated supply and exhaust airflow systems, a negative-pressure environment, and mandatory use of positive-pressure (&#8220;space&#8221;) suits. The risk for laboratory specialists working with highly pathogenic agents is minimized through rigorous training and adherence to stringent safety protocols and standard operating procedures. Researchers perform the majority of their work in BSL-2 laboratories and switch to BSL-4 suit laboratories when work with a high-consequence pathogen is required. Collaborators and scientists considering BSL-4 projects should be aware of the challenges associated with BSL-4 research both in terms of experimental technical limitations in BSL-4 laboratory space and the increased duration of such experiments. Tasks such as entering and exiting the BSL-4 suit laboratories are considerably more complex and time-consuming compared to BSL-2 and BSL-3 laboratories. The focus of this particular article is to address basic biosafety concerns and describe the entrance and exit procedures for the BSL-4 laboratory at the NIH/NIAID Integrated Research Facility at Fort Detrick. Such procedures include checking external systems that support the BSL-4 laboratory, and inspecting and donning positive-pressure suits, entering the laboratory, moving through air pressure-resistant doors, and connecting to air-supply hoses. We will also discuss moving within and exiting the BSL-4 suit laboratories, including using the chemical shower and removing and storing positive-pressure suits.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Although researchers are generally knowledgeable about procedures and safety precautions required for biosafety level 1 or 2 (BSL-1/2) experiments, they may not be familiar with experimental procedures in BSL-4 suit laboratories. This article provides a detailed visual demonstration of BSL-4 suit laboratory systems check, laboratory entry, movement, and exit procedures.

(A)의 안전주의 사항 및 운영 절차 BSL-4 실험실 : 1. 바이오 안전성 레벨 4 정장 연구소 스위트 출입국 절차

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 3. Aerobiology

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol releases or unexpected natural aerosol transmission. Specific laboratories designed to perform these experiments require tremendous engineering controls to provide a safe and secure working environment and constant systems maintenance to sustain functionality. Class III biosafety cabinets, also referred to as gloveboxes, are gas-tight enclosures with non-opening windows. These cabinets are maintained under negative pressure by double high-efficiency-particulate-air (HEPA)-filtered exhaust systems and are the ideal primary containment for housing aerosolization equipment. A well planned workflow between staff members within high containment from, for instance, an animal biosafety level-4 (ABSL-4) suit laboratory to the ABSL-4 cabinet laboratory is a crucial component for successful experimentation. For smooth study execution, establishing a communication network, moving equipment and subjects, and setting up and placing equipment, requires staff members to meticulously plan procedures prior to study initiation. Here, we provide an overview and a visual representation of how aerobiology research is conducted at the National Institutes of Health, National Institute of Allergy and Infectious Diseases Integrated Research Facility at Fort Detrick, Maryland, USA, within an ABSL-4 environment.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 3. Aerobiology

As high-consequence pathogens can potentially infect subjects through airborne particles, aerobiology has been increasingly applied in pathogenesis research and medical countermeasure development. We present a detailed visual demonstration of aerobiology procedures during an aerosol challenge in nonhuman primates in an animal biosafety level 4 maximum containment environment.

(A)의 안전주의 사항 및 운영 절차 BSL-4 실험실 3. 공중 생물학

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol ...

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades the central nervous system (CNS). In vivo study of the late stage has been limited as traditional methodologies require the removal of the brain to determine the presence of the parasites.
Bioluminescence imaging is a non-invasive, highly sensitive form of optical imaging that enables the visualization of a luciferase-transfected pathogen in real-time. By using a transfected trypanosome strain that has the ability to produce late stage disease in mice we are able to study the kinetics of a CNS infection in a single animal throughout the course of infection, as well as observe the movement and dissemination of a systemic infection.
Here we describe a robust protocol to study CNS infections using a bioluminescence model of African trypanosomiasis, providing real time non-invasive observations which can be further analyzed with optional downstream approaches.

This manuscript describes the use of a bioluminescent strain of African trypanosomes to enable the tracking of late stage infection and demonstrates how in vivo live imaging can be used to visualize infections within the central nervous system in real-time.

생물 발광 영상은 아프리카 트리파노소마 증의 늦은 단계의 감염을 감지하는

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades ...

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and consequently poses an urgent threat to public health. Recurrence of a C. difficile infection (CDI) after successful treatment with antibiotics is high, occurring in 20-30% of patients, thus necessitating the discovery of novel therapeutics against this pathogen. Current animal models of CDI result in high mortality rates and thus do not approximate the chronic, insidious disease manifestations seen in humans with CDI. To evaluate therapeutics against C. difficile, a mouse model approximating human disease utilizing a clinically-relevant strain is needed. This protocol outlines the cefoperazone mouse model of CDI using a clinically-relevant and genetically-tractable strain, R20291. Techniques for clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol. Compared to other mouse models of CDI, this model is not uniformly lethal at the dose administered, allowing for the observation of a prolonged clinical course of infection concordant with the human disease. Therefore, this cefoperazone mouse model of CDI proves a valuable experimental platform to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile.

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

This protocol outlines the cefoperazone mouse model of Clostridium difficile infection (CDI) using a clinically-relevant and genetically-tractable strain, R20291. Emphasis on clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol.

의 Cefoperazone 처리 된 마우스 모델 임상 관련<em&gt; 클로스 트리 디움 디피</em&gt; 스트레인 R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and ...