Name: ex vivo infection of human lymphoid tissue and female genital mucosa with human immunodeficiency virus 1 and histoculture
Uploaded: 2018-10-12T13:00:00
Description: Watch this Scientific Journal Video about Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture at JoVE.com

This work was supported by grants from the Foundation Blanceflor Boncompagni Ludovisi nee Bildt (http://blanceflor.se/), the Foundation Swedish Physicians against AIDS (http://www.aidsfond.se/, ref. FOa2014-0006), and Fondazione Andrea e Libi Lorini (http://fondazionelorini.it/) to Andrea Introini.&#160;

The protocol for collection of human tissues may require ethical approval by the local competent authorities. In case of indicated surgeries such as tonsillectomy and hysterectomy, the specimens are not collected specifically for research purpose and the study is not considered human subjects research (National Institutes of Health. Research on Human Subjects. https://humansubjects.nih.gov/walkthrough-investigator#tabpanel11). However, obtaining tissue donors&#39; personal and medical data (e.g., sex, age, curre...

<ol>
	<li>Grivel, J. C., Margolis, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+human+tissue+explants+to+study+human+infectious+agents.">Use of human tissue explants to study human infectious agents.</a> Nature protoc. 4 (2), 256-269 (2009).</li><li>Glushakova, S., Grivel, J. C., Fitzgerald, W., Sylwester, A., Zimmerberg, J., Margolis, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+the+HIV-1+phenotype+switch+as+a+causal+factor+in+acquired+immunodeficiency.">Evidence for the HIV-1 phenotype switch as a causal factor in acquired immunodeficiency.</a> Nat Med. 4 (2), 346-349 (1998).</li><li>Dezzutti, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+and+human+mucosal+tissue+models+to+study+HIV+biomedical+interventions:+can+we+predict+success.">Animal and human mucosal tissue models to study HIV biomedical interventions: can we predict success.</a> J Int AIDS Soc. 18 (1), 20301 (2015).</li><li>Lederman, M. M., Margolis, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lymph+node+in+HIV+pathogenesis.">The lymph node in HIV pathogenesis.</a> Semin Immunol. 20 (3), 187-195 (2008).</li><li>Chun, T. W., Moir, S., Fauci, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+reservoirs+as+obstacles+and+opportunities+for+an+HIV+cure.">HIV reservoirs as obstacles and opportunities for an HIV cure.</a> Nat Immunol. 16 (6), 584-589 (2015).</li><li>Biancotto, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+induced+activation+of+CD4++T+cells+creates+new+targets+for+HIV-1+infection+in+human+lymphoid+tissue+ex+vivo.">HIV-1 induced activation of CD4+ T cells creates new targets for HIV-1 infection in human lymphoid tissue ex vivo.</a> Blood. 111 (2), 699-704 (2008).</li><li>Van Laar, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+secretion+of+immunoglobulin+by+long-lived+human+tonsil+plasma+cells.">Sustained secretion of immunoglobulin by long-lived human tonsil plasma cells.</a> Am J Pathol. 171 (3), 917-927 (2007).</li><li>Introini, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seminal+plasma+induces+inflammation+and+enhances+HIV-1+replication+in+human+cervical+tissue+explants.">Seminal plasma induces inflammation and enhances HIV-1 replication in human cervical tissue explants.</a> PLoS Pathog. 13 (5), 1006402 (2017).</li><li>Grivel, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+CCR5-+but+not+CXCR4-tropic+HIV-1+in+lymphoid+tissue+by+human+herpesvirus+6.">Suppression of CCR5- but not CXCR4-tropic HIV-1 in lymphoid tissue by human herpesvirus 6.</a> Nat Med. 7 (11), 1232-1235 (2001).</li><li>Rollenhagen, C., Lathrop, M. J., Macura, S. L., Doncel, G. F., Asin, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+type-2+stimulates+HIV-1+replication+in+cervical+tissues:+implications+for+HIV-1+transmission+and+efficacy+of+anti-HIV-1+microbicides.">Herpes simplex virus type-2 stimulates HIV-1 replication in cervical tissues: implications for HIV-1 transmission and efficacy of anti-HIV-1 microbicides.</a> Mucosal Immunol. 7 (5), 1165-1174 (2014).</li><li>Lisco, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acyclovir+is+activated+into+a+HIV-1+reverse+transcriptase+inhibitor+in+herpesvirus-infected+human+tissues.">Acyclovir is activated into a HIV-1 reverse transcriptase inhibitor in herpesvirus-infected human tissues.</a> Cell Host Microbe. 4 (3), 260-270 (2008).</li><li>Vanpouille, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+common+anti-cytomegalovirus+drug,+ganciclovir,+inhibits+HIV-1+replication+in+human+tissues+ex+vivo.">A common anti-cytomegalovirus drug, ganciclovir, inhibits HIV-1 replication in human tissues ex vivo.</a> AIDS. 31 (11), 1519-1528 (2017).</li><li>Andrei, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topical+tenofovir,+a+microbicide+effective+against+HIV,+inhibits+herpes+simplex+virus-2+replication.">Topical tenofovir, a microbicide effective against HIV, inhibits herpes simplex virus-2 replication.</a> Cell Host Microbe. 10 (4), 379-389 (2011).</li><li>Greenhead, P., Hayes, P., Watts, P. S., Laing, K. G., Griffin, G. E., Shattock, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parameters+of+human+immunodeficiency+virus+infection+of+human+cervical+tissue+and+inhibition+by+vaginal+virucides.">Parameters of human immunodeficiency virus infection of human cervical tissue and inhibition by vaginal virucides.</a> J Virol. 74 (12), 5577-5586 (2012).</li><li>Saba, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+sexual+transmission:+early+events+of+HIV-1+infection+of+human+cervico-vaginal+tissue+in+an+optimized+ex+vivo+model.">HIV-1 sexual transmission: early events of HIV-1 infection of human cervico-vaginal tissue in an optimized ex vivo model.</a> Mucosal Immunol. 3 (3), 280-290 (2010).</li><li>Introini, A., Vanpouille, C., Lisco, A., Grivel, J. C., Margolis, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-7+facilitates+HIV-1+transmission+to+cervico-vaginal+tissue+ex+vivo.">Interleukin-7 facilitates HIV-1 transmission to cervico-vaginal tissue ex vivo.</a> PLoS Pathog. 9 (2), 1003148 (2013).</li><li>Biancotto, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+sensitive+and+dynamic+immunofluorescent+cytometric+bead+assay+for+the+detection+of+HIV-1+p24.">A highly sensitive and dynamic immunofluorescent cytometric bead assay for the detection of HIV-1 p24.</a> J Virol Methods. 157 (1), 98-101 (2009).</li><li>Lisco, A., Vanpouille, C., Margolis, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=War+and+peace+between+microbes:+HIV-1+interactions+with+coinfecting+viruses.">War and peace between microbes: HIV-1 interactions with coinfecting viruses.</a> Cell Host Microbe. 6 (5), 403-408 (2009).</li><li>Keele, B. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+transmitted+and+early+founder+virus+envelopes+in+primary+HIV-1+infection.">Identification and characterization of transmitted and early founder virus envelopes in primary HIV-1 infection.</a> Proc Natl Acad Sci U S A. 105 (21), 7552-7557 (2008).</li><li>Pudney, J., Quayle, A. J., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+microenvironments+in+the+human+vagina+and+cervix:+mediators+of+cellular+immunity+are+concentrated+in+the+cervical+transformation+zone.">Immunological microenvironments in the human vagina and cervix: mediators of cellular immunity are concentrated in the cervical transformation zone.</a> Biol Reprod. 73 (6), 1253-1263 (2005).</li><li>Ganor, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Within+1h,+HIV-1+uses+viral+synapses+to+enter+efficiently+the+inner,+but+not+outer,+foreskin+mucosa+and+engages+Langerhans-T+cell+conjugates.">Within 1h, HIV-1 uses viral synapses to enter efficiently the inner, but not outer, foreskin mucosa and engages Langerhans-T cell conjugates.</a> Mucosal Immunol. 3 (5), 506-522 (2010).</li><li>Cavarelli, M., Foglieni, C., Rescigno, M., Scarlatti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=R5+HIV-1+envelope+attracts+dendritic+cells+to+cross+the+human+intestinal+epithelium+and+sample+luminal+virions+via+engagement+of+the+CCR5.">R5 HIV-1 envelope attracts dendritic cells to cross the human intestinal epithelium and sample luminal virions via engagement of the CCR5.</a> EMBO Mol Med. 5 (5), 776-794 (2013).</li><li>Cummins, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+testing+of+candidate+topical+microbicides+for+anti-human+immunodeficiency+virus+type+1+activity+and+tissue+toxicity+in+a+human+cervical+explant+culture.">Preclinical testing of candidate topical microbicides for anti-human immunodeficiency virus type 1 activity and tissue toxicity in a human cervical explant culture.</a> Antimicrob Agents Chemother. 51 (5), 1770-1779 (2007).</li><li>Abner, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+human+colorectal+explant+culture+to+evaluate+topical+microbicides+for+the+prevention+of+HIV+infection.">A human colorectal explant culture to evaluate topical microbicides for the prevention of HIV infection.</a> J Infect Dis. 192 (9), 1545-1556 (2005).</li><li>Saba, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Productive+HIV-1+infection+of+human+cervical+tissue+ex+vivo+is+associated+with+the+secretory+phase+of+the+menstrual+cycle.">Productive HIV-1 infection of human cervical tissue ex vivo is associated with the secretory phase of the menstrual cycle.</a> Mucosal Immunol. 6 (6), 1081-1090 (2013).</li></ol>

The use of human tissue explants for the study of HIV-1 infection provides advantages over traditional bi-dimensional and monotypic experimental systems, such as primary cells or cell lines, due their superior ability to reproduce intercellular interactions and cellular functions (e.g., cytokine production) as they are in vivo. Nevertheless, tonsillar and cervical tissue differ in many aspects including the number, and phenotypic and functional characteristics of HIV target cells1</sup...

Monotypic bi-dimensional cell cultures, here referred to as conventional, do not account for the spatial and functional communication between the large variety of cell types that compose tissues and organs. This aspect is of paramount importance for experimental models of disease, as interference with homeostatic intercellular interactions is the driving factor of all pathologies. Tissue explants offer major advantages for modeling health and disease in humans because they retain the cytoarchitecture and many important functional aspects of organs as they are in vivo, although for a limited amount of time1. For example, upon ex vivo challenge with recall antigens, such as diphtheria toxoid or tetanus toxoid, tonsillar tissue responds with a vigorous production of antigen-specific antibodies2. Like any other ex vivo model, histoculture has its own limitations: inter-donor variability, tissue polarization, limited tissue survival, and difficulty in monitoring cells beyond the depth of confocal microscopy1. Nevertheless, human tissue explants remain a model of choice to study homeostatic and pathogenic immunological processes in humans, including host-pathogen interactions and potential therapeutic interventions3.
The critical events of HIV-1 pathogenesis take place in tissues. Infection of the genital mucosa accounts for the majority of all HIV-1 transmission events worldwide4. Lymphoid tissue is the major site of virus replication during acute infection and harbors a significant pool of latently-infected cells5, and their persistence represents the main obstacle to achieve a cure6. The culture and ex vivo challenge of human lymphoid and mucosal tissues provide some advantages over conventional systems based on isolated cells for the study of HIV-1. For instance, tissue-resident cells can support HIV-1 infection in the absence of exogenous activation, as opposed to peripheral blood mononuclear cells1. The use of lymphoid tissue explants has allowed a better understanding of some key mechanisms underlying CD4 T cell depletion, i.e., the bystander effect7, which is the hallmark of acute infection. The preservation of structures like B cell follicles in lymphoid tissue8 and the epithelium in female genital mucosa explants9 offers the unique opportunity to integrate spatial and functional features of HIV-1 infection at these sites. Finally, histocultures were successfully used to model and study HIV-1 and herpesvirus co-infection10,11, as well as for preclinical testing of antiretrovirals and multitarget microbicides12,13,14,15.
Here, we describe a detailed protocol for the culture of human tissue explants obtained from tonsils and mucosal tissue from the lower female genital tract (cervix), covering tissue dissection to explant challenge with HIV-1 in a non-polarized way. The culture of tissue explants at the liquid-air interface, using gelatin sponges as a support, maximizes exposure to air oxygen while providing access to culture medium nutrients through the sponge capillaries, thus delaying their natural decay. The most immediate way of evaluating HIV-1 replication in our system is to measure the amount of virus released in the explant culture medium over time by immunoassay or qPCR. HIV-1 infection and pathogenesis (e.g., CD4 T cell depletion) can also be evaluated in tissue explants by bulk RNA/DNA extraction and qPCR, in situ staining, or single cell-analysis upon tissue digestion by flow cytometry.

<table border="0" cellpadding="0" cellspacing="0" style="width:941px;" width="940">
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			<td height="42" style="height:42px;width:280px;">Gelfoam 12-7 mm Absorbable gelatin sponge&#160;</td>
			<td style="width:175px;">Pfizer</td>
			<td style="width:141px;">NDC:&#160; 0009-0315-08</td>
			<td>Gelfoam and Spongostan perform equally well in histocuture for both tonsillar and cervical tissue.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:280px;">SPONGOSTAN Standard 70 &#215; 50 &#215; 10 mm Absorbable haemostatic gelatin sponge</td>
			<td>Ferrosan Medical Devices</td>
			<td>MS0002</td>
			<td>Gelfoam and Spongostan perform equally well in histocuture for both tonsillar and cervical tissue.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:280px;">RPMI 1640 with phenol red and glutamine</td>
			<td>ThermoFisher Scientific</td>
			<td>21875034</td>
			<td>To RPMI1640 add MEM non-essential aminoacids, sodium pyruvate, gentamicin, amphotericin B and FBS 15% (v/v) to make culture medium (CM).</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:280px;">Modified Eagle&#39;s medium (MEM) non-essential aminoacids (100x)</td>
			<td>ThermoFisher Scientific</td>
			<td>11140035</td>
			<td>To RPMI1640 add MEM non-essential aminoacids, sodium pyruvate, gentamicin, amphotericin B and FBS 15% (v/v) to make culture medium (CM).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Sodium pyruvate, 100 mM (100x)&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>11360070</td>
			<td>To RPMI1640 add MEM non-essential aminoacids, sodium pyruvate, gentamicin, amphotericin B and FBS 15% (v/v) to make culture medium (CM).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Gentamicin 50 mg/mL (1000x)&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>15750037</td>
			<td>To RPMI1640 add MEM non-essential aminoacids, sodium pyruvate, gentamicin, amphotericin B and FBS 15% (v/v) to make culture medium (CM).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Amphotericin B 250 &#956;g/mL (100x)&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>15290018</td>
			<td>To RPMI1640 add MEM non-essential aminoacids, sodium pyruvate, gentamicin, amphotericin B and FBS 15% (v/v) to make culture medium (CM).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Fetal bovine serum (FBS)&#160;</td>
			<td></td>
			<td></td>
			<td>To RPMI1640 add MEM non-essential aminoacids, sodium pyruvate, gentamicin, amphotericin B and FBS 15% (v/v) to make culture medium (CM).</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:49px;width:280px;">Ticarcillin disodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>T5639-1G</td>
			<td>Resuspend in sterile cell culture grade water tircacillin disodium (3g) and potassium clavulanate (100mg) and mix to obtain 100 mL of antibiotic solution (100x). Aliquote and store at -20 &#176;C. Avoid repeated freezing and thawing. Supplement CM with antibiotic solution to make CMT.</td>
		</tr>
		<tr height="82">
			<td height="82" style="height:82px;width:280px;">Potassium clavulanate</td>
			<td>Sigma-Aldrich</td>
			<td>33454-100MG</td>
			<td>Resuspend in sterile cell culture grade water tircacillin disodium (3g) and potassium clavulanate (100mg) and mix to obtain 100 mL of antibiotic solution (100x). Aliquote and store at -20 &#176;C. Avoid repeated freezing and thawing. Supplement CM with antibiotic solution to make CMT.</td>
		</tr>
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			<td height="84" style="height:84px;width:280px;">Sterile phosphate buffer saline (PBS) pH 7.4 w/o calcium, magnesium and phenol red</td>
			<td></td>
			<td></td>
			<td style="width:345px;">To use for storage and transportation of surgical specimens. PBS can be replaced with another isotonic solution with physiologic pH. Culture medium is best for overnight storage.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Sterile cell culture grade water</td>
			<td></td>
			<td></td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Sterile transportation container</td>
			<td></td>
			<td></td>
			<td style="width:345px;"></td>
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			<td height="21" style="height:21px;width:280px;">70% ethanol solution</td>
			<td></td>
			<td></td>
			<td style="width:345px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:280px;">Disinfectant solution for biological waste disposal</td>
			<td></td>
			<td></td>
			<td style="width:345px;"></td>
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			<td height="21" style="height:21px;width:280px;">Sterile metal forceps or tweezers</td>
			<td></td>
			<td></td>
			<td style="width:345px;"></td>
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			<td height="21" style="height:21px;width:280px;">Sterile metal scissors</td>
			<td></td>
			<td></td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Sterile flat weighing metal spatula</td>
			<td></td>
			<td></td>
			<td style="width:345px;"></td>
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			<td height="21" style="height:21px;width:280px;">Sterile scalpels and blades</td>
			<td></td>
			<td></td>
			<td style="width:345px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:280px;">6-well cell culture plates</td>
			<td></td>
			<td></td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">12-well cell culture plates</td>
			<td></td>
			<td></td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Sterile Petri dish, 100 mm &#215; 20 mm</td>
			<td></td>
			<td></td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:280px;">Cell-free HIV-1 viral preparation HIV-1BaL</td>
			<td>NIH AIDS Reagent Program</td>
			<td>510</td>
			<td>The virus should be propagated to generate a stock large enough to perform several experiments. Aliquote virus suspension and store at -80 &#176;C. Avoid repeated freezing and thawing.</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:280px;">Cell-free HIV-1 viral preparation HIV-1LAI.04</td>
			<td>NIH AIDS Reagent Program</td>
			<td>2522</td>
			<td>The virus should be propagated to generate a stock large enough to perform several experiments. Aliquote virus suspension and store at -80 &#176;C. Avoid repeated freezing and thawing.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Lamivudine (3TC)</td>
			<td>NIH AIDS Reagent Program</td>
			<td>8146</td>
			<td style="width:345px;">Resuspend in DMSO at 10 mg/ml, aliquote and store at -20 &#176;C. Avoid repeated freezing and thawing.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Biological safety cabinet</td>
			<td></td>
			<td></td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:280px;">Water-jacketed CO2 incubator, set at 37 &#176;C, 5% CO2, &#8805; 90% humidity</td>
			<td></td>
			<td></td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Water bath set at 37 &#176;C</td>
			<td></td>
			<td></td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Sterile 1.5- and 2-mL screw cap tubes</td>
			<td></td>
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			<td height="42" style="height:42px;width:280px;">Dry bath shaker with heating block for 1.5-mL tubes, set at 37 &#176;C, 300 rpm</td>
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ex vivo infection of human lymphoid tissue and female genital mucosa with human immunodeficiency virus 1 and histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under controlled laboratory conditions. Ex vivo infection of human tissues with human immunodeficiency virus (HIV), among other viruses, has been successfully used to investigate early disease pathogenesis, as well as a platform to test the efficacy and toxicity of antiviral drugs. In the present protocol, we explain how to process and infect with HIV-1 tissue explants from human tonsils and cervical mucosae, and maintain them in culture on top of gelatin sponges at the liquid-air interface for about two weeks. This non-polarized culture setting maximizes access to nutrients in culture medium and oxygen, although progressive loss of tissue integrity and functional architectures remains its main limitation. This method allows monitoring HIV-1 replication and pathogenesis using several techniques, including immunoassays, qPCR, and flow cytometry. Of importance, the physiologic variability between tissue donors, as well as between explants from different areas of the same specimen, may significantly affect experimental results. To ensure result reproducibility, it is critical to use an adequate number of explants, technical replicates, and donor-matched control conditions to normalize the results of the experimental treatments when compiling data from multiple experiments (i.e., conducted using tissue from different donors) for statistical analysis.

Several techniques can be used to assess HIV-1 replication in tissue explants. Our standard read-out is to measure the amount of HIV-1 p24gag released in CM over time with an immunoassay18.
Tissue explants from different donors, infected with the same inoculum of the same HIV-1 stock, may yield different amounts of virus (Table 1). This is due to several factors, such as the nu...

Watch this Scientific Journal Video about Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture at JoVE.com

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Infection of human tissues with human immunodeficiency virus (HIV) ex vivo provides a valuable 3D model of virus pathogenesis. Here, we describe a protocol to process and infect tissue specimens from human tonsils and female genital mucosae with HIV-1 and maintain them in culture at the liquid-air interface.

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under ...

This method can help answer key questions in the HIV field, such as the identification of the determinants of virus transmission and pathogenesis within two of the most effected organs by the infection. In fact, the key advanced driving disease progression take place in tissues. The features and complexity of infection are poorly reflected by cells in circulation or isolated and infected in vitro.The main advantage of the ex vivo tissue culture to study HIV pathogenesis is that it retains its original spacial and functional relationships between cells although for two to three weeks. To begin, fill a 100 millmeter by 20 millimeter Petri dish with 20 to 30 milliliters of CMT. Transfer gelatin sponges into the Petri dish using sterile forceps.Wet the sponges on both sides and allow the sponges to soak up medium for one minute. Press the sponges against the bottom of the Petri dish for about two minutes using a sterile spatula. Use sterile scissors to cut the rehydrated gelatin sponges into pieces of equal size of about one square centimeter.Then use forceps to transfer one sponge piece into each well of a culture plate. Add three to four milliliters of CMT into each well of a 6-well plate for the tonsils and one milliliter per well into a 12-well plate for the cervix. Place the plates in the incubator, set at 37 degrees Celsius, 5%CO2, and greater than or equal to 90%humidity until the tissue explants are ready to be loaded on the sponges.Transfer the tonsils from the transportation container into a 100 millimeter Petri dish containing 10 to 20 milliliters of CMT using sterile forceps. Use the lid of the Petri dish as a cutting surface to dissect the tissue. Holding the tissue gently with forceps, cut each tonsil into different, big pieces using a sterile scalpel and blade.Use forceps and a scalpel to remove cauterized, bloody, and fibroid tissue, as well as any parts containing tonsilloliths or greenish-brown color. Cut each tissue piece into slices of about two millimeters in thickness. Remove any unwanted part as in the previous step.Then cut the tissue slices into two-millimeter-thick strips. Finally, cut the tissue strips into two-millimeter-thick blocks. Transfer the explants into a new 100 millimeter Petri dish containing 10 to 20 milliliters of CMT to avoid tissue desiccation while proceeding with the dissection.At the end of the dissection, swirl the plate to randomize the explant distribution. Place nine tissue explants on top of each gelatin sponge in a 6-well plate using forceps, allowing space between them. Return the plates to the incubator.Typically, explants are cultured overnight. Inoculation with HIV is performed the next day. This is to make sure that no bacterial or fungal contamination develops in the culture.Also, cells tend to regress tissue explants immediately after dissection. Aspirate the medium in the 6-well plate with a pipette, and discard it in a container with disinfectant solution. Tilt the plate and gently push the gelatin sponges to the upper part of the well to allow the medium to gather at the bottom.Aspirate and discard the medium. Then add three to four milliliters of CMT to each well. Now thaw the HIV-1 virus preparation in the water bath at 37 degrees Celsius.Pipette the virus inoculum directly on top of each of nine explants on a gelatin sponge. Return the plate to the incubator. Use the lid of the Petri dish as a cutting surface to dissect the tissue.Holding the tissue gently with forceps, separate the mucosa of the ectocervix and/or endocervix from the underneath stromal and submucosa with a scalpel and blade to obtain strips of mucosa. Cut the mucosa into two-millimeter-wide strips. Remove and discard as much submucosa as possible, leaving only a two-millimeter-thick layer of tissue.Then cut the strips into two-millimeter-thick blocks. Transfer tissue explants into a new 100 millimeter Petri dish containing 10 to 20 milliliters of CMT to avoid tissue desiccation while proceeding with the dissection. At the end of the dissection, swirl the plate to randomize the explant distribution.With sterile forceps, transfer the explants into sterile 1.5 milliliter conical tubes. Remove any medium in the tube using a pipette. For optimum results, explants of cervix should be processed and infected as soon as possible after surgery, ideally the day of the surgery.Alternatively, tissues can be stored submerged in CMT at 4 degrees overnight and infected immediately after dissection. Thaw the HIV-1 preparation in a water bath at 37 degrees Celsius. Once thawed, transfer the virus into the tubes containing the explants.Transfer the tubes into a thermoshaker, and incubate for two hours at 37 degrees Celsius rocking at 300 rpm. Gently invert the tubes a few times every 30 to 60 minutes. Meanwhile, fill a 6-well plate with three to four milliliters per well of sterile phosphate buffered saline.At the end of the incubation with HIV-1, use a pipette to discard all virus preparation in the tubes containing the explants into a container with disinfectant solution. Then use forceps and a pipette to transfer the explants into the 6-well plate containing PBS. After allowing the explants to rest in PBS for one minute at room temperature, wash them by gently pipetting the PBS up and down into the well two to three times.Then discard the PBS into a container with disinfectant solution using a pipette. Add three to four milliliters of new PBS to each well. Repeat two more times for a total of three washes.Discard the PBS just before transferring the explants to the sponges to avoid tissue desiccation. Now use forceps to transfer five to eight explants on top of each gelatin sponge into a 12-well plate. Return the plate to the incubator.Collect a sample of culture medium with a pipette, and transfer it into sterile 1.5 or two milliliter screw cap tubes for storage at minus 80 degrees Celsius. Harvest the tissue explants with sterile forceps, and transfer the explants into sterile 1.5 or two milliliter screw cap tubes. Process or store the tissue samples as required by the experiment.Aspirate the residual culture medium in the well with a pipette, and discard it into a container with disinfectant solution. Tilt the plate and gently push the gelatin sponges to the upper part of the well to allow the medium to gather at the bottom, and then aspirate and discard it. Now add three to four milliliters of culture medium to each well.Using sterile forceps, return any tissue explants that dropped into the medium to the gelatin sponge before returning the plate to the incubator. At the end of the culture, dispose of all biological waste in apposite containers according to the institute's regulations on handling hazardous biologicals as well as the specific risk assessment. Inter-donor variability can affect HIV-1 replication as measured by the concentration of the HIV core protein p24 gag in tissue explant culture supernatant, as demonstrated for cervical tissue from three different donors infected with the same virus inoculum.For the first donor, the accumulation of p24 gag in culture medium of explants infected with HIV-1 BaL alone clearly indicates a product infection. The antiretroviral drug 3TC that completely abrogates HIV replication was used as a negative control. The amount of p24 gag measured in culture medium of 3TC-treated explants reveals the fraction of virus inoculum aspecifically absorbed and released by tissue explants during culture.In tissue explants from the second donor, HIV-1 bowel infection results in a steady decrease in the concentration of p24 gag over time, indicating an abort of infection or undetectable by p24 gag quantification. This is confirmed by 3TC-treated control explants that show a similar HIV replication kinetic. Infection of tissue explants from the third donor yields little amount of virus.In this case, the inclusion of the 3TC treatment as a negative control is necessary to ascertain the presence of infection with de novo virus production. Following this procedure, analytical methods like flow cytometry or immunostaining can be performed at different time points to answer questions about phenotype, proportion, or tissue location of the cell population of interest. When designing the experiment, it is important to estimate the impact of inter-donor variability on the experimental readout of choice.Inter-donor variability can be partially controlled by setting stringent clinical and, possibly, experimental inclusion and exclusion criteria. Ex vivo human tissue cultures can be used to study the pathogenesis of microbes other than HIV, such as vaccinia virus or herpes viruses. They have also been used as a preclinical platform for antimicrobial toxicity and ethicacy studies.Please be aware that all human specimens, even from healthy individuals, can harbor infectious agents. Appropriate education, training, and protections are required to handle human tissues to ensure the safety of you and your coworkers.

ex-vivo-infection-human-lymphoid-tissue-female-genital-mucosa-with

Research

JoVE Journal

Immunology and Infection

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals may exhibit suppressive immune microenvironment and, in contrast to activating CTL, their autologous antigen presenting cells may tend to tolerize or anergize antigen specific CTL. As a result, although still in the experimental phase, CTL-based adoptive immunotherapy has evolved to become a promising treatment for various diseases such as cancer and virus infections. In initial experiments ex vivo expanded CMV (cytomegalovirus) specific CTL have been used for treatment of CMV infection in immunocompromised allogeneic bone marrow transplant patients. While it is common to have life-threatening CMV viremia in these patients, none of the patients receiving expanded CTL develop CMV related illness, implying the anti-CMV immunity is established by the adoptively transferred CTL1. Promising results have also been observed for melanoma and may be extended to other types of cancer2. 
While there are many ways to ex vivo stimulate and expand human CTL, current approaches are restricted by the cost and technical limitations. For example, the current gold standard is based on the use of autologous DC. This requires each patient to donate a significant number of leukocytes and is also very expensive and laborious. Moreover, detailed in vitro characterization of DC expanded CTL has revealed that these have only suboptimal effector function 3. 
Here we present a highly efficient aAPC based system for ex vivo expansion of human CMV specific CTL for adoptive immunotherapy (Figure 1). The aAPC were made by coupling cell sized magnetic beads with human HLA-A2-Ig dimer and anti-CD28mAb4. Once aAPC are made, they can be loaded with various peptides of interest, and remain functional for months. In this report, aAPC were loaded with a dominant peptide from CMV, pp65 (NLVPMVATV). After culturing purified human CD8+ CTL from a healthy donor with aAPC for one week, CMV specific CTL can be increased dramatically in specificity up to 98% (Figure 2) and amplified more than 10,000 fold. If more CMV-specific CTL are required, further expansion can be easily achieved by repetitive stimulation with aAPC. Phenotypic and functional characterization shows these expanded cells have an effector-memory phenotype and make significant amounts of both TNF&alpha; and IFN&gamma; (Figure 3).

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

A new DC independent method for induction and expansion of antigen-specific T cells is described. HLA A2-Ig based artificial Antigen Presenting Cells (aAPC) are loaded with HLA-A2 restricted peptides to efficiently expand CTL of diverse antigen specificity. This technology holds great potential for CTL-based adoptive immunotherapy.

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

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

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

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

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

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes during initial entry and establishes a primary infection in the epithelium, which is followed by latent infection of neurons. After reactivation, viruses can become evident at mucocutaneous sites that appear as skin vesicles or mucosal ulcers. How HSV-1 invades skin or mucosa and reaches its receptors is poorly understood. To investigate the invasion route of HSV-1 into epidermal tissue at the cellular level, we established an ex vivo infection model of murine epidermis, which represents the site of primary and recurrent infection in skin. The assay includes the preparation of murine skin. The epidermis is separated from the dermis by dispase II treatment. After floating the epidermal sheets on virus-containing medium, the tissue is fixed and infection can be visualized at various times postinfection by staining infected cells with an antibody against the HSV-1 immediate early protein ICP0. ICP0-expressing cells can be observed in the basal keratinocyte layer already at 1.5 hr postinfection. With longer infection times, infected cells are detected in suprabasal layers, indicating that infection is not restricted to the basal keratinocytes, but the virus spreads to other layers in the tissue. Using epidermal sheets of various mouse models, the infection protocol allows determining the involvement of cellular components that contribute to HSV-1 invasion into tissue. In addition, the assay is suitable to test inhibitors in tissue that interfere with the initial entry steps, cell-to-cell spread and virus production. Here, we describe the ex vivo infection protocol in detail and present our results using nectin-1- or HVEM-deficient mice.

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To achieve this goal, the development of diagnostic techniques to recognize bacterial presence in real-time at the point-of-care is required. Optical imaging for direct identification of bacteria within the host is an attractive approach. Several attempts at chemical probe design and validation have been investigated, however none have yet been successfully translated into the clinic. Here we describe a method for ex vivo validation of bacteria-specific probes for identification of bacteria within the distal lung, imaged by fibered confocal fluorescence microscopy (FCFM). Our model used ex vivo human lung tissue and a clinically approved confocal laser endomicroscopy (CLE) platform to screen novel bacteria-specific imaging compounds, closely mimicking imaging conditions expected to be encountered with patients. Therefore, screening compounds by this technique provides confidence of potential clinical tractability.

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

This technique describes an efficient screening process for evaluating bacteria-specific optical imaging agents within ex vivo human lung tissue, by fibered confocal fluorescence microscopy for the rapid identification of small molecule chemical probe-candidates with translatable potential.

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...