These matters can help answer key questions in HIV/AIDS research, such as the molecular mechanism and the prognosis of HIV infection, and how and where the latency reservoir is established. The advantage of this technique is that it is non-invasive, and established a hu-NSG mice that must treat a multi-lineage development over transplanted human stem cells that are susceptible to HIV infection and are receptive to cART. Begin by filtering a digested, human, fetal, liver tissue, slurry sample through a sterile, 70 micrometer, nylon, mesh strainer to obtain a single cell suspension, and enrich for CD34+HSCs by magnetic activated cell sorting according to the manufacturer's instructions.
After counting, re-suspend the HSC at a two-times-ten-to-the-seventh viable cells per milliliter concentration in fresh DPBS on ice. Next place an entire neonatal human NOD/SCID/gamma or NSG mouse litter into a sterile, pie-shaped, irradiation cage, and treat the animals with 200 to 250 centigrades of gamma ray from a cesium-137 radiation source. Then load the cells into a custom-made, Hamilton 80508 50-microliter syringe equipped with a 30-gauge needle, and inject each neonatal animal directly into the liver with five-times-ten-to-the-fifth viable human CD34+HSCs and 25 microliters of DPBS.
Ten-to-twelve weeks post infection, spin down retro-orbital, peripheral, blood samples from each mouse by centrifugation, and transfer the plasma supernates to microcentrifuge tubes for negative 80 degrees Celsius storage as experimentally appropriate. For HSC engraftment validation, re-suspend the pellets with 200 microliters of red blood cell lysis buffer, and pull the cell suspensions in a 1.5 millimeter microcentrifuge tube for a ten-minute incubation at room temperature. Then collect the remaining white blood cells by centrifugation for two washes in 0.5 milliliters of fresh, 0.01 percent bovine serum albumin, or BSA, in DPBS per wash.
After the second wash, block the cells with 100 microliters of blocking cocktail for twenty minutes at 4 degrees Celsius followed by labeling with the appropriate anti-body cocktail of interest at a 2 microliters of anti-body per one-times-one-to-the-sixth cells concentration. After 30 minutes at 4 degrees Celsius, wash the cells two times in fresh DPBS plus BSA, and evaluate the peripheral blood samples by flow cytometry according to standard protocols to quantify the percentage of human CD45+CD3+CD14+or CD19+cells per sample. Then calculate the ratio of CD4+to-CD8+T cells, and assess the viral load with in the plasma samples according to standard, quantitative, reverse transcriptase, PCR protocols.
To assess the effects of HIV infection, deliver HIV bale virus to anesthetized, human, NSG mice with a greater than 20 percent human CD45+cell population in their peripheral blood, at a 200 nanograms of P24 per mouse concentration via intraperitoneal administration, collecting blood samples via retro-orbital bleeding every two weeks, and analyze both the CD4-to-CD8 ratio and the plasma viral load, as demonstrated. For viral suppression validation supply the infected, human NSG animals with fresh, combinatorial, anti-retroviral therapy, or cART, in sweetened water every week for 4 weeks, collecting blood samples via retro-orbital bleeding every two weeks and analyze both the CD4-to-CD8 ratio and the plasma viral load as demonstrated. To asses the viral rebound when the plasma viral loads are below the detection limit and the CD4-to-CD8 ratio is restored between 1.5 and 2.5 collect peripheral blood samples through retro-orbital bleeding every 2 weeks after cART withdrawal and evaluate the samples as demonstrated.
During the initial engraftment validation, the human CD45+cell count should range from 20 to 80 percent, and the subsets of human leukocytes should appear as discrete populations by flow cytometric analysis. The ratio of CD4+to-CD8+cells remains between 1.5 and 2.5 in healthy individuals while significant CD4+T cell depletion is typically observed upon viral infection. Notably, the healthy ratio is restored in response to cART treatment.
The detection of plasma viral loads by quantitative RT PCR throughout the course of the infection and the cART regimen can be plotted and used to evaluate the efficiency of the viral delivery and the anti-viral treatment, respectively. Though this method can provide insight into HIV/AIDS virology studies and therapeutic development, it can also be applied to other studies related to human disease, such as cancer, diabetes, and auto-immune diseases. The hu-NSG mouse model development paved the way for researchers in immunology and oncology to explore a new dysfunction and pathology in humans, particularly mechanistic and therapeutic studies targeting HIV chronic infection and the latency reservoir.
