Name: establishment of the dual humanized tk-nog mouse model for hiv-associated liver pathogenesis
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Description: Watch this Scientific Journal Video about Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis at JoVE.com

This work was supported by the National Institute of Health grant R24OD018546 (to L.Y.P. and S.G.). The authors would like to thank Weizhe Li, Ph.D., for the help in surgical procedures, Amanda Branch Woods, B.S., Yan Cheng for immunohistology, UNMC flow cytometry research facility members Director Phillip Hexley, Ph.D., Victoria B. Smith, B.S., and Samantha Wall, B.S., UNMC advanced microscopy core facility members Janice A. Taylor, B.S., and James R. Talaska, B.S., for the technical support. The authors acknowledge Drs. Mamoru Ito and Hiroshi Suemizu from CIEA for providing TK-NOG mice and Dr. Joachim Baumgart for providing treosulfan. The authors thank Dr. Adrian Koesters, UNMC, for her editorial contribution to the manuscript.

This protocol has been approved by the Institutional Animal Care and Use Committee (IACUC)&#160;at the University of Nebraska Medical Center.
NOTE: Obtain approval from the local IACUC before performing experiments on animals.
1. Processing of Umbilical Cord Blood and the Isolation of Human HSPCs
<ol>
	<li>Perform all steps of the protocol under sterile conditions in laminar flow cabinets.</li>
	<li>Take umbilical cord blood (CB) collected in hepa...

<ol>
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The liver is compromised and damaged in HIV-infected patients24. Experimental small animal models for studying human liver diseases in the presence of HIV-1 is extremely limited, despite the availability of a few cotransplanted animal models with CD34+ HSPCs and hepatocytes7,12,25. In in vitro experiments, hepatocytes are shown to have low-level HIV-1 infection26. Humani...

Since the advent of antiretroviral therapy, there has been a substantial decrease in deaths related to HIV-1 monoinfection. However, liver disease has emerged as a common cause of morbidity in HIV-infected patients1,2. Coinfections of hepatitis viruses with HIV-1 infection are more common, accounting for 10% - 30% of HIV-infected persons in the United States3,4,5.
The host-specificity of HIV-1 and hepatitis viruses limits the utility of small animal models to study human-specific infectious diseases or to investigate multiple aspects of HIV-1-associated liver pathogenesis. Immunodeficient mice that permit the engraftment of human cells and/or tissues (termed humanized mouse models) are acceptable animal models for preclinical studies6,7,8. Since the introduction of humanized mice in the early 2000s, multiple preclinical studies of cholestatic human liver toxicity, human-specific pathogens, including HIV-1 and HIV-associated neurocognitive disorders, Epstein Barr virus, hepatitis, and other infectious diseases, have been investigated in these mice6,9,10,11. Multiple mouse models for CD34+ HSPCs and/or human hepatocyte transplantation have long been developed and have improved over time to study the disease pathogenesis of Hepatitis B virus (HBV)-associated liver disease12,13,14. Several models for HSPC and human hepatocyte (HEP) transplantation are based on strains, known as NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac)8,13, NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ)15, Balb/C-Rag2-/- &#947;c-/- (Rag2tm1.1Flv Il2rgtm1.1Flv/J)12, and fah-/- NOD rag1-/-il2r&#947;null mouse16. However, each model has its own advantages and limitations; for example, AFC8 dual humanized mice for HEPs and human stem cells (HSCs) on a Balb/C-Rag2-/- &#947;c-/-&#160;background enables the successful engraftment of immune cells and HSCs, but there is an absence of an antigen-specific T- and B-cell response in this model12. The major concerns in reconstituting double humanized mice include suboptimal engraftment, a lack of suitable models to support different tissues, mismatched conditions, immune rejection, or graft-versus-host disease (GVHD), and technical difficulties, such as risky manipulations with newborns and high mortality rates due to metabolic abnormalities13.
Although humanized mice have been used for HIV research for many years17,18,19, the use of humanized mice to study liver damage caused by HIV-1 has been limited20. We previously reported the establishment of a dual humanized TK-NOG mouse model and its application in HIV-associated liver disease8. This model shows the robust engraftment of liver and immune cells and recapitulates HIV infection pathogenesis. This discussion presents a detailed protocol, including the most critical steps in the transplantation of human hepatocytes. A description of the HSPCs required for a successful engraftment of HEPs and the establishment of a functional immune system in TK-NOG mice is also presented. The use of these mice to study HIV-associated liver immunopathogenesis is detailed. TK-NOG male mice carrying a liver-specific herpes simplex virus type 1 thymidine kinase (HSV-TK) transgene are used. Mouse liver cells expressing this transgene can easily be ablated after a brief exposure to a nontoxic dose of GCV. Transplanted human liver cells are stably maintained within the mouse liver without exogenous drugs21. The mice are also preconditioned with nonmyeloablative doses of treosulfan to create a niche in the mouse bone marrow for human cells8. Immunodeficient TK-NOG mice are intrasplenically injected with HEPs and multipotent HSPCs. The mice are then regularly monitored for blood and liver reconstitution by blood immunophenotyping and measurements of serum human-albumin levels, respectively. Mice with a successful reconstitution of more than 15% for both human immune cells and HEPs are intraperitoneally injected with HIV-1. The effect of HIV on the liver can be assessed as early as 4 - 5 weeks postinfection. It is critical to note that, because HIV-1 is used, all necessary precautions must be taken while handling the virus and injecting it into mice.

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			<td height="84" style="height:84px;width:125px;">5 mL polystyrene&#160; round-bottom tube 12 x 75 mm style</td>
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			<td style="width:127px;">352054</td>
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			<td style="width:135px;">BD Biosciences</td>
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			<td style="width:167px;">For purity check of eluted CD34+ cells&#160;</td>
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			<td style="width:135px;">BD Biosciences</td>
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			<td style="width:167px;">Software to analysis acquired CD34+ cell on FACS array</td>
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			<td style="width:135px;">BD Biosciences</td>
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			<td style="width:167px;">Controlled substance and pain-killer</td>
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			<td style="width:167px;">For isoation of&#160;&#160; CD34+ HSPC</td>
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			<td style="width:127px;">562428</td>
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			<td style="width:135px;">Bio-rad</td>
			<td style="width:127px;">1450015</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">ChargeSwitch gDNA Mini Tissue Kit</td>
			<td style="width:135px;">Thermofisher scientific</td>
			<td style="width:127px;">CS11204</td>
			<td style="width:167px;">for extraction of genomic DNA from ear piece</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">Cobas Amplicor system v1.5&#160;</td>
			<td style="width:135px;">Roche Molecular Diagnostics</td>
			<td style="width:127px;"></td>
			<td style="width:167px;">bioanalyzer to measure viral load</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">Cotton-tipped applicators&#160;</td>
			<td style="width:135px;">&#160;McKesson</td>
			<td style="width:127px;">24-106-2S</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">Cytokeratin-18 (CK18)</td>
			<td style="width:135px;">DAKO</td>
			<td style="width:127px;">M7010</td>
			<td style="width:167px;">Specific to human</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">DMSO (Dimethyl sulfoxide)</td>
			<td style="width:135px;">Sigma-aldrich</td>
			<td style="width:127px;">D2650-5X5ML</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:125px;">Extension set Microbore Slide Clamp(s) Fixed Male Luer Lock. L: 60 in L: 152 cm PV: 0.55 mL Fluid Path Sterile</td>
			<td style="width:135px;">BD biosciences</td>
			<td style="width:127px;">30914</td>
			<td style="width:167px;">Attached to dispensing pippet and to load with HSPC and HEP suspesion</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">FACS Diva version 6</td>
			<td style="width:135px;">BD Biosciences</td>
			<td style="width:127px;"></td>
			<td style="width:167px;">flow cytometer software required for&#160; acqusition of sample</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:135px;">Gibco</td>
			<td style="width:127px;">10438026</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">FLOWJO analysis software 
			v10.2</td>
			<td style="width:135px;">FLOWJO, LLC</td>
			<td style="width:127px;"></td>
			<td style="width:167px;">flow cytometry analysis software</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">Ganciclovir</td>
			<td style="width:135px;">APP Pharmaceuticals, Inc.</td>
			<td style="width:127px;">315110</td>
			<td style="width:167px;">Prescripition drug</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">Greiner MiniCollect EDTA Tubes</td>
			<td style="width:135px;">Greiner bio-one</td>
			<td style="width:127px;">450475</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">Hepatocytes thawing medium&#160;</td>
			<td style="width:135px;">Triangle Research Labs&#160;</td>
			<td style="width:127px;">MCHT50</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">Horizon Open Ligating Clip Appliers</td>
			<td style="width:135px;">Teleflex</td>
			<td style="width:127px;">537061</td>
			<td style="width:167px;">To hold the ligating clips</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">Hospira Sterile Water for Injection</td>
			<td style="width:135px;">ACE surgical supply co. Inc.</td>
			<td style="width:127px;">001-1187</td>
			<td style="width:167px;">For dilution of Buprenorphine (pain-killer)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">Human Albumin ELISA Quantitation Set</td>
			<td style="width:135px;">Bethyl laboratories</td>
			<td style="width:127px;">E80-129</td>
			<td style="width:167px;">For assesing human albumin levels in mouse serum</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">Human hepatocyte</td>
			<td style="width:135px;">Triangle Research Labs&#160;</td>
			<td style="width:127px;">HUCP1</td>
			<td style="width:167px;">&#160;Cryopreserved human hepatocytes, induction qualified&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">Iris Scissors, Straight</td>
			<td style="width:135px;">Ted Pella, Inc.</td>
			<td style="width:127px;">13295</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:125px;">Lancet</td>
			<td style="width:135px;">MEDIpoint</td>
			<td style="width:127px;">Goldenrod 5 mm</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:125px;">LS columns&#160;</td>
			<td style="width:135px;">Miltenyi Biotec</td>
			<td style="width:127px;">130-042-401</td>
			<td style="width:167px;">Used to entrap CD34+ microbeads (positive selection)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">Lymphocyte Separation Medium (LSM)</td>
			<td style="width:135px;">MP Biomedicals</td>
			<td style="width:127px;">50494</td>
			<td style="width:167px;">For isoation of&#160;&#160; lymphocytes from peripheral blood</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">MACS MultiStand</td>
			<td style="width:135px;">Miltenyi Biotec</td>
			<td style="width:127px;">130-042-303</td>
			<td style="width:167px;">holds Qudro MACS seperator and LS columns</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:125px;">McPherson-Vannas Micro Dissecting Spring Scissors</td>
			<td style="width:135px;">Roboz Surgical Instrument Co.</td>
			<td style="width:127px;">RS-5605</td>
			<td style="width:167px;">Used to make an incision on skin to expose spleen</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">Micro Dissecting Forceps</td>
			<td style="width:135px;">Roboz Surgical Instrument Co.</td>
			<td style="width:127px;">RS-5157&#160;</td>
			<td style="width:167px;">to hold and pull out spleen from peritoneal cavity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:125px;">mouse CD45-FITC</td>
			<td style="width:135px;">BD Biosciences</td>
			<td style="width:127px;">553080</td>
			<td style="width:167px;">mouse-specific</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">PBS (Phosphate Buffered Saline)</td>
			<td style="width:135px;">Hyclone</td>
			<td style="width:127px;">SH30256.02</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">Qudro MACS separator&#160;</td>
			<td style="width:135px;">Miltenyi Biotec</td>
			<td style="width:127px;">130-090-976</td>
			<td style="width:167px;">holds four LS columns</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">RPMI 1640 medium</td>
			<td style="width:135px;">Gibco</td>
			<td style="width:127px;">11875093</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">StepOne Plus Real Time PCR&#160;</td>
			<td style="width:135px;">Applied Biosystems</td>
			<td style="width:127px;"></td>
			<td style="width:167px;">Instrument used&#160; to&#160; genotype</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:125px;">Stepper Series Repetitive Dispensing Pipette 1ml</td>
			<td style="width:135px;">DYMAX CORP</td>
			<td style="width:127px;">T15469</td>
			<td style="width:167px;">Used to&#160; dispense&#160; HSPC and HEP supension in controlled manner</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">Suturevet PGA synthetic absorbale suture</td>
			<td style="width:135px;">Henry Schein Animal Health</td>
			<td style="width:127px;">41178</td>
			<td style="width:167px;">Suturing of skin and peritoneum</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">TaqMan Gene Expression Master Mix</td>
			<td style="width:135px;">Thermofisher scientific</td>
			<td style="width:127px;">4369016</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">TC20 automated cell counter</td>
			<td style="width:135px;">Bio-rad</td>
			<td style="width:127px;">1450102</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:125px;">TK-NOG mice&#160;</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:167px;">Provided by the Central Institute for Experimental Animals (CIEA, Japan; Drs. Mamoru Ito and Hiroshi Suemizu)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">Treosulfan</td>
			<td style="width:135px;">Medac GmbH</td>
			<td style="width:127px;"></td>
			<td style="width:167px;">Provided by &#160;Dr. Joachim Baumgart (medac GmbH)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:125px;">Trypan Blue</td>
			<td style="width:135px;">Bio-rad</td>
			<td style="width:127px;">1450022</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">Vannas-type Micro Scissors, Straight, 80mm L</td>
			<td style="width:135px;">Ted Pella, Inc.</td>
			<td style="width:127px;">1346</td>
			<td style="width:167px;">Used to make an incision on skin to expose spleen</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:125px;">Weck hemoclip traditional titanium ligating clips</td>
			<td style="width:135px;">Esutures</td>
			<td style="width:127px;">523700</td>
			<td style="width:167px;">To ligate the spleen post-injection</td>
		</tr>
	</tbody>
</table>

establishment of the dual humanized tk-nog mouse model for hiv-associated liver pathogenesis

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of their morbidity. The liver immunopathology caused by HIV-1 remains elusive. Small xenograft animal models with human hepatocytes and human immune system can recapitulate the human biology of the disease&#39;s pathogenesis. Herein, a protocol is described to establish a dual humanized mouse model through human hepatocytes and CD34+ hematopoietic stem/progenitor cells (HSPCs) transplantation, to study liver immunopathology as observed in HIV-infected patients. To achieve dual reconstitution, male TK-NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(Alb-TK)7-2/ShiJic) mice are intraperitoneally injected with ganciclovir (GCV) doses to eliminate mouse transgenic liver cells, and with treosulfan for nonmyeloablative conditioning, both of which facilitate human hepatocyte (HEP) engraftment and human immune system (HIS) development. Human albumin (ALB) levels are evaluated for liver engraftment, and the presence of human immune cells in blood detected by flow cytometry confirms the establishment of human immune system. The model developed using the protocol described here resembles multiple components of liver damage from HIV-1 infection. Its establishment could prove to be essential for studies of hepatitis virus co-infection and for the evaluation of antiviral and antiretroviral drugs.

The establishment of a dual humanized mouse model with human liver and immune cells can be easily monitored at each step with very simple ELISA and flow cytometry, respectively. Flow cytometry is regularly performed to evaluate the development of a functional immune system and to see the effect of HIV infection on immune cells. In dual humanized mice, the development of functional immune cells can range from 15% to 90% of the lymphocyte gate. Representative subsets of immune cells are sho...

Watch this Scientific Journal Video about Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis at JoVE.com

Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis

This protocol provides a reliable method to establish humanized mice with both human immune system and liver cells. Dual reconstituted immunodeficient mice achieved via intrasplenic injection of human hepatocytes and CD34+ hematopoietic stem cells are susceptible to human immunodeficiency virus-1 infection and recapitulate liver damage as observed in HIV-infected patients.

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of ...

The overall aim of the protocol is to generate a humanized mouse model with functional human immune system and liver using human hematopoietic stem cells and hepato site. This method can help answer key questions in genetic liver humanized mice. This method provides a powerful tool to study immunopathology caused by HIV as observed in people.Demonstrating the process will be by Yimin Sun, a supervisor from the pathology and microbiology department, along with Weimin Wang and Edwrd Makarov, research technologist. To begin, transfer umbilical cord blood collected in heparinized tubes to a sterile laminar flow cabinet. Add PBS until the volume is increased to 35 milliliters.Layer the sample on top of lymphocyte separation medium and centrifuge it 400 times G and at four degrees celsius for 35 minutes with no breaks. Next, carefully remove the top plasma layer and use a transfer pipette to transfer the white buffy coat interface to a new tube. Re-suspend the buffy coat in 30 to 40 milliliters of ice cold buffer.Using a pipette, combine 20 microliters of the cell suspension with 20 microliters of 0.4%trypan blue. Pipette 10 microliters of this mixture into the outer opening of either of the two chambers of a counting slide, and insert the slide into an automated cell counter to count the cells. Centrifuge the cells at 300 times G and at four degrees celsius for 10 minutes.Carefully aspirate the supernatant and add 300 microliters of ice cold buffer. Add 100 microliters of human FC receptor blocking reagent and 100 microliters of monoclonal mouse anti-human CD34 antibody conjugated MicroBeads for up to 100 million cells. Incubate for 30 minutes at four degrees celsius.Add 10 milliliters of ice cold buffer to wash the cells and centrifuge it 300 times G and four degrees celsius for 10 minutes. Carefully remove the supernatant and re-suspend the pellet in 500 microliters of ice cold buffer. After this, place a positive selection LS column in the magnetic activated cell sorting field and pass it through with three milliliters of ice cold buffer.Load the sample into the LS column that can entrap MicroBeads bound to human CD34 positive cells in samples, and allow it to flow under the influence of gravity into the collection tube. Wash the column three times with ice cold buffer and collect the eluent in the same collection tube. Then, plunge the column with five milliliters of ice cold buffer to allude the CD34 positive cells into a new collection tube.Repeat the procedure to achieve a purity of over 90%Next, use trypan blue dye and a hemocytometer to count the eluded CD34 positive cells. After counting, centrifuge the cells at 300 times G for five minutes. Discard the supernatant and re-suspend the cells in 125 microliters of PBS for an injection to be used immediately in transplantation.To check the purity of the CD34 positive eluent, take 50 microliters of the suspension and incubate it with 10 microliters of PE conjugated anti-human CD34 antibody at four degrees celsius for 30 minutes. After this, wash and re-suspend the cells in PBS and proceed to perform flow cytometry. After acquisition, analyze the data as outlined in the text protocol.First, retrieve and thaw the cryopreserved hepatocytes as outlined in the text protocol. Then, remove the bio cap and pour the thawed hepatocytes into a 50 milliliter conical tube containing warmed thawing medium. Rock the tube by hand for a few seconds to suspend the cells.Pellet the cells at 100 times G and at room temperature for eight minutes. Wash the pelleted cells in PBS with 0.1%BSA and pool them with either fresh or thawed HSPCs in PBS to a final volume of 80 microliters per mouse. First, attach one end of a sterile extension tube to a 30 gauge needle and the other end to a one milliliter syringe.Fill the syringe with the suspension of pooled HEPs and HSPCs. Fit the syringe in the notch of a repetitive dispensing pipette and adjust the dispenser to dispense 10 microliters in each press. Shave each mouse as outlined in the text protocol and scrub the left side of the body of each mouse with 10%povidone iodine followed 70%isopropyl alcohol.Using Vannas type scissors, make an small incision in the skin, muscle and peritoneum at the left of the peritoneal wall to enter the peritoneal cavity approximately 5 millimeters below the lower edge of the rib cage. Locate the spleen and use forceps to pull it slightly to the operating area for easy access and insert the 30 gauge needle into the lower pole of the spleen. Next, unlock the plunger of the dispensing pipette and dispense 10 microliters of the volume at a time, with a limit of 60 to 80 microliters per spleen.Retract the needle slowly and clip the spleen with ligating clips using a ligation applier. Then, use cotton-tipped applicators wetted with sterile PBS to push the spleen back into the body cavity. Use an interrupted suture pattern to close the muscle layer of the abdominal wall.Accomplish skin closure with an interrupted suture pattern using non-absorbable sutures. Transfer all needed materials to a designated Biosafety Level Two Plus facility. Wear personal protection equipment including a disposable cover all gown, shoe covers, a face mask and double gloves, including cut resistant gloves, at all times while working with the virus.Select mice with a reconstitution of more than 15%of human CD45 positive cells and with a presence of human albumin in the serum for HIV-1 infection. Intraperitoneally inject the mice with 1, 000 to 10, 000 tissue culture infectious doses of 50 HIV-1 ADA in a volume of 100 to 200 microliters per mouse. After euthanizing the mice, excise the liver from each mouse as outlined in the text protocol.Collect and fix the livers in 4%paraformaldehyde overnight. The establishment of a dual humanized mouse model with human liver and immune cells can be easily monitored at each step with very simple ELIZA and flow cytometry, respectively. Flow cytometry is regularly performed to evaluate the development of a functional immune system and to see the effect of HIV infection on immune cells.In dual humanized mice, the development of functional immune cells can range from 15 to 90%of the lymphocyte gate. For the evaluation of the engraftment of human hepatocytes, ELIZA for human-specific albumin levels is performed monthly on mouse serum. Mice engrafted with both HSPCs and HEPs show human-specific albumin levels ranging from approximately seven micrograms per milliliter to 377 micrograms per milliliter at one month, continuing grow over the time of observation.The effect of HIV infection on human immune cells in the blood of dual humanized mice is monitored by flow cytometry and on HEPs in the liver by human-specific albumin ELIZA. By five weeks, HIV-1 causes a decrease in human albumin levels in the serum and there is a depletion of human CK18 positive hepatocytes in the liver sections of dual humanized mice. A lower of ratio of CD4 to CD8 is typically observed in the blood and liver of HIV-infected mice, compared to levels noted in the same mouse before infection.Blood should be layered carefully on top of lymphocyte separation medium for isolations of buffy coat. For the surgery, hold the spleen gently and insert the needle in lower pole of the spleen. Following the isolation of hematopoietic stem cells, it is crucial to check the purity of CD34 positive hematopoietic stem cells to avoid CD3 positive cells and acute injections of hepatocyte from different donor.As the humanized mice show physiological aspect of human immune system and liver, the developed mice could be utilized to study physiopathogenesis of HIV and physical infections and cirrhosis.

establishment-dual-humanized-tk-nog-mouse-model-for-hiv-associated

Research

JoVE Journal

Immunology and Infection

In vitro Biofilm Formation in an 8-well Chamber Slide

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

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

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

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) 1, 2 or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) 3. Both models were mainly utilized for the study of HIV infection.
One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant 4. The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency 5-8.
We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC 7, 9. This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor regression (9).
The goal of this protocol is to describe the process of generating these transgenic mice and assessing in vivo efficacy using live PET imaging. As a note, since we use human tissues and lentiviral vectors, our facilities conform to CDC NIH guidelines for Biosafety Level 2 (BSL2) with special precautions (BSL2+). In addition, the NSG mice are severely immunocompromised thus, their housing and maintenance must conform to the highest health standards (<a href="http://jaxmice.jax.org/research/immunology/005557-housing.html" target="_blank">http://jaxmice.jax.org/research/immunology/005557-housing.html</a>).

The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.

Small animal models such as mice have been extensively used to study human  disease and to develop new therapeutic interventions. Despite the wealth of  ...

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of insects as infection models provides a valuable alternative. Compared to other non-vertebrate model hosts such as nematodes, insects have a relatively advanced system of antimicrobial defenses and are thus more likely to produce information relevant to the mammalian infection process. Like mammals, insects possess a complex innate immune system1. Cells in the hemolymph are capable of phagocytosing or encapsulating microbial invaders, and humoral responses include the inducible production of lysozyme and small antibacterial peptides2,3. In addition, analogies are found between the epithelial cells of insect larval midguts and intestinal cells of mammalian digestive systems. Finally, several basic components essential for the bacterial infection process such as cell adhesion, resistance to antimicrobial peptides, tissue degradation and adaptation to oxidative stress are likely to be important in both insects and mammals1. Thus, insects are polyvalent tools for the identification and characterization of microbial virulence factors involved in mammalian infections.
Larvae of the greater wax moth Galleria mellonella have been shown to provide a useful insight into the pathogenesis of a wide range of microbial infections including mammalian fungal (Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) and bacterial pathogens, such as Staphylococcus aureus, Proteus vulgaris, Serratia marcescens Pseudomonas aeruginosa, Listeria monocytogenes or Enterococcus faecalis4-7. Regardless of the bacterial species, results obtained with Galleria larvae infected by direct injection through the cuticle consistently correlate with those of similar mammalian studies: bacterial strains that are attenuated in mammalian models demonstrate lower virulence in Galleria, and strains causing severe human infections are also highly virulent in the Galleria model8-11. Oral infection of Galleria is much less used and additional compounds, like specific toxins, are needed to reach mortality.
G. mellonella larvae present several technical advantages: they are relatively large (last instar larvae before pupation are about 2 cm long and weight 250 mg), thus enabling the injection of defined doses of bacteria; they can be reared at various temperatures (20 &deg;C to 30 &deg;C) and infection studies can be conducted between 15 &deg;C to above 37 &deg;C12,13, allowing experiments that mimic a mammalian environment. In addition, insect rearing is easy and relatively cheap. Infection of the larvae allows monitoring bacterial virulence by several means, including calculation of LD5014, measurement of bacterial survival15,16 and examination of the infection process17. Here, we describe the rearing of the insects, covering all life stages of G. mellonella. We provide a detailed protocol of infection by two routes of inoculation: oral and intra haemocoelic. The bacterial model used in this protocol is Bacillus cereus, a Gram positive pathogen implicated in gastrointestinal as well as in other severe local or systemic opportunistic infections18,19.

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

Oral and intra haemocolic infection of larvae of the greater wax moth Galleria mellonella is described. This insect can be used to study virulence factors of entomopathogenic as well as mammalian opportunistic bacteria. Rearing of the insects, methods of infection and examples of in vivo analysis are described.

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of ...

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

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

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

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

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

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

Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood vessels resulting in vascular leak, the development of purpuric rashes and eventual tissue necrosis. Studying the pathogenesis of this infection was previously limited by the human specificity of the bacteria, which makes in vivo models difficult. In this protocol, we describe a humanized model for this infection in which human skin, containing dermal microvessels, is grafted onto immunocompromised mice. These vessels anastomose with the mouse circulation while maintaining their human characteristics. Once introduced into this model, N. meningitidis adhere exclusively to the human vessels, resulting in extensive vascular damage, inflammation and in some cases the development of purpuric rash. This protocol describes the grafting, infection and evaluation steps of this model in the context of N. meningitidis infection. The technique may be applied to numerous human specific pathogens that infect the blood stream.

Neisseria meningitidis is a human specific pathogen that infects blood vessels. In this protocol human microvessels are introduced into a mouse by grafting human skin onto immunocompromised mice. Bacteria adhere extensively to the human vessels, leading to vascular damage and development of the purpuric rash typically observed in human cases.

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...

Humanized NOD/SCID/IL2r&#947;null (hu-NSG) Mouse Model for HIV Replication and Latency Studies

Ethical regulations and technical challenges for research in human pathology, immunology, and therapeutic development have placed small animal models in high demand. With a close genetic and behavioral resemblance to humans, small animals such as the mouse are good candidates for human disease models, through which human-like symptoms and responses can be recapitulated. Further, the mouse genetic background can be altered to accommodate diverse demands. The NOD/SCID/IL2r&#947;null (NSG) mouse is one of the most widely used immunocompromised mouse strains; it allows engraftment with human hematopoietic stem cells and/or human tissues and the subsequent development of a functional human immune system. This is a critical milestone in understanding the prognosis and pathophysiology of human-specific diseases such as HIV/AIDS and aiding the search for a cure. Herein, we report a detailed protocol for generating a humanized NSG mouse model (hu-NSG) by hematopoietic stem cell transplantation into a radiation-conditioned neonatal NSG mouse. The hu-NSG mouse model shows multi-lineage development of transplanted human stem cells and susceptibility to HIV-1 viral infection. It also recapitulates key biological characteristics in response to combinatorial antiretroviral therapy (cART).

This protocol provides a method to establish humanized mice (hu-NSG) via intrahepatic injection of human hematopoietic stem cells into radiation-conditioned neonatal NSG mice. The hu-NSG mouse is susceptible to HIV infection and combinatorial antiretroviral therapy (cART) and serves as a suitable pathophysiological model for HIV replication and latency investigations.

Ethical regulations and technical challenges for research in human pathology, immunology, and therapeutic development have placed small animal models in ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human diseases have been reported to be associated with four species of bacteria: Aeromonas dhakensis, Aeromonas hydrophila, Aeromonas veronii, and Aeromonas caviae. The model organism Caenorhabditis elegans is a bacterivore that provides an excellent infection model by which to study the bacterial pathogenesis of Aeromonas. Here, we introduce three different experiments to study Aeromonas infection using a C. elegans model, including survival, liquid toxicity, and muscle necrosis assays. The results of the three methods determining the virulence of Aeromonas were consistent. A. dhakensis was shown to be the most toxic among the 4 major Aeromonas species causing clinical infections. These methods are shown to be a convenient way to evaluate the toxicity among and within Aeromonas species and contribute to our understanding of the pathogenesis of Aeromonas infection.

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

Here, we introduce three different experiments to study Aeromonas infection in C. elegans. Using these convenient methods, it is easy to evaluate the toxicity among and within Aeromonas species.

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human ...