HIV Infected Individuals Essay Example
HIV Infected Individuals Essay Example

HIV Infected Individuals Essay Example

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  • Published: May 6, 2022
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The study protocol was approved by the institutional review board (IRB) of East Tennessee State University (ETSU) and James H. Quillen VA Medical Center (ETSU/VA IRB, Johnson City, Tennessee State). All the individuals participated in the projects were adults and willingly agreed to sign the informed consent forms.

There were two populations participated in the study, including 150 latently HIV infected patients and 166 age-matched healthy subjects (HS). HIV infected individuals were on ART treatment, therefore they were virologically suppressed for HIV replication, as evidenced with undetectable RNA level of HIV in their blood samples. HS were supplied by Physicians Plasma Alliance (Gray, TN) and classified as healthy because they were negatively diagnosed for all HIV, hepatitis B virus (HBV) and hepatitis C virus (HCV) infections.

PBMCs are any peripheral blood ce

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lls that have a round nucleus. Human PBMCs consist of these following cell populations: T lymphocytes, B lymphocytes, natural killer cells (NKs) and dendritic cells and monocytes. The frequencies of these populations are different among individuals, however, typically the lymphocytes (including T lymphocytes, B lymphocytes and NKs) occupies around 70 to 90 percent, monocytes occupies for 10 to 20 percent and dendritic cells population is only 1 to 2 percent. In the lymphocyte population, T lymphocytes accounted for 70 percent with the ratio 2:1 for CD4+ T cells and CD8+ T cells, respectively. B lymphocytes accounted for 5 to 10 percent and NKs occupies 5 to 20 percent. Since CD4+ cells are the main interest cells in this study, therefore, they will be isolated from the mixture of cell populations in the PBMCs. The main reagent used in PBMCs isolation is Ficoll – Paque solution (G

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Healthcare, Piscataway, NJ) applied in the technique called density gradient centrifugation separation of mononuclear cells.

Technique principle. The anticoagulant-treated whole blood is diluted with RPMI 1640 medium with the ratio 1:1 and then carefully placed on top of the Ficoll-Paque solution without intermixing in the centrifugation tube. Then the tube is taken for centrifugation which after will give rise to the formation of layers containing different cell types resulted in the differential migration of cells during the centrifugation. At the most bottom is the erythrocytes which was aggregated by the Ficoll-Paque solution, therefore penetrated through the solution to reach the bottom of the tube. The immediate layer above the erythrocytes contains mostly the granulocytes. The next layer is the Ficoll-Paque solution while the top layer is the plasma. The interface layer between the Ficoll-Paque solution and the plasma contain the mononuclear cells.

Technique detail protocol. The anticoagulant-treated whole blood was placed into 50 mL centrifugation tube and centrifuged at 1500 rpm with acceleration and de-acceleration speed at 9 for 5 minutes at room temperature. The result was the formation of the two layers, the top layer contained the plasma and the bottom layers contained the mixture of cell populations. The top layer containing plasma was removed by pipette, and the remaining solution was mixed thoroughly with the RPMI 1640 media with the ratio 1:1. Then the mixture was placed carefully on top of the Ficoll-Paque solution in another 50 mL centrifugation tube without intermixing. Then the tube was centrifuged at 2000 rpm with acceleration and de-acceleration speed at 1 for 20 minutes at room temperature. After the centrifugation, the solution in the tube was separated into layers

from the bottom to the top with following orders: erythrocytes, granulocytes, Ficoll-Paque solution, mononuclear cells and remaining plasma and platelets. The mononuclear cells was collected and transferred into another sterile 50 mL centrifugation tube, then mixed with RPMI 1640 media. After that, the tube was centrifuged at 1800 rpm with acceleration and de-acceleration speed at 9 for 8 minutes to produce the two layers solution, the bottom layer contained the mononuclear cells and the upper layer contained RPMI 1640 media and remaining platelets. The upper layer was then removed, the remaining mononuclear cells could be freshly used for cell isolation or could be frozen for later used. If the cells were frozen, they then mixed with of RPMI 1640 media containing DMSO. The mixture was then transferred into cryotubes so that each of the tube containing 1 mL of media and 1 x 107 cells. Then these tubes were stored at liquid nitrogen for later use.

CD4+ T lymphocytes isolation

CD4+ T cells was isolated from PBMCs using the CD4+ T cell negative selection kit provided by Miltenyi Biotec Inn., Auburn, CA.

Technique principle. PBMCs are incubated with cocktails of biotin-conjugated monoclonal anti-human antibodies against CD8, CD14 CD15, CD16, CD19, CD36, CD56, CD123, TCRγ/δ and CD235a cells but except for CD4 cells, the non-target cells. Then the mixture of PBMCs and antibodies are incubated with Microbeads which are both conjugated to the monoclonal anti-biotin antibodies and magnetically tagged. The mixture is transferred and let to flow through the magnetic column in which the magnetically labeled cells captured inside the column while the unlabeled cells flow through.

Technique detail protocol. PBMCs were washed with RPMI 1640 media by mixing together

in 5 mL centrifugation tube, then centrifuged for at 1200 rpm for 5 minutes at room temperature. Then the supernatant was removed. The cell pellet was suspended in 40 μL isolation buffer and 10 μL of antibodies cocktail per 107 total cells. Then the mixture was incubated at 4 – 80C for 5 minutes. After incubation, 30 μL of isolation buffer and 20 μL of microbeads was added into the tube, then incubated at 4 – 80C for another 10 minutes. The magnetic column was prepared by being washed with 3 mL of isolation buffer. The incubated mixture was transferred into the column, the flow through solution was collected and placed into a new 5 mL centrifugation tube, then centrifuged at 1200 rpm for 5 minutes. The supernatant was removed, the cell pellet was suspended with RPMI 1640 media, called complete media, which had 10% fetal bovine serum (FBS) (Atlanta Biologicals, Flowery Branch, GA), 100 IU/mL penicillin and 2 mM L-glutamine (Thermo Scientific, Logan, Utah) added. Followed by that, the suspended cells were taken for cell density determination.

The isolated and suspended CD4 cells were seeded into cell culture plates and incubated in the cell culture incubator at 370C and 5% CO¬2 atmosphere. After 4 days, cultured CD4 cells were harvested and used for the necessary assays.

Flow cytometry (FCM) is a useful and rapid tool that used markedly in scientific research recently for single cellular characteristic classification in which the information obtained is both quantity and quality. FMC classified cells into distinct different populations by measuring their individual optical and fluorescence characteristics. Cell components, such as DNA, RNA can be tagged by fluorescence and analyzed by

FCM. In addition, antibodies conjugated with fluorescent dyes can be used to tag specific proteins, including plasma membrane, nuclear membrane and cytosolic proteins which present in different cell types. After being fluorescently labelled, the cells will be let to run through the flow cytometer which they pass its light source and become excited to a higher energy and give rise to the high energy emission. These emissions, or light signals, are immediately detected by photomultiplier tubes and digitized for computer analysis. All modern flow cytometer provides multiple fluorescent channels which allows the analysis of several cell properties simultaneously. In this project, FCM was used to study different characteristics of distinct cellular populations since each of cell types expressed different specific proteins, also preferred as markers. In addition, cells belong to one cell type also express disparate markers at certain states.

In order to be separated from the mixture of cellular populations in PBMCs, interested cell types were bound with specific fluorescent conjugated antibodies and being classified by FCM. In this project, CD4 cells are the main target to be studied, therefore, total CD4 and its subpopulations were sorted and their characteristics were analyzed by FCM. Extracellular markers of total CD4 was CD4+, naïve CD4 was CD4+CD45RA+ and memory CD4 was CD4+CD45RA . The memory CD4 was then further divided into several subsets, including central memory (CD4+CD45RA-CCR7+CD28+), effector memory (CD4+CD45RA-CCR7-CD28+/-) and terminally differentiated memory (CD4+CD45RA-CCR7-CD28+). There are others CD4 subset which marked by different specific proteins, such as natural killer T cell is specifically marked with CD57 and PD1 is the marker for death programmed cells. In this assay, each of the antibody was conjugated with one

specific fluorescence, including CD4-FITC, CD45RA-PerCP710, CD57-APC (Biolegend, San Diego, CA), CD28-PE (Invitrogen, Calrsbad, CA), PD1-FITC (eBioscience, San Diego, CA).

Apoptosis, also called programmed cell death, is a normal process where cells are induced to die in order to maintain an appropriate cell population during development and aging. It also serves as an immune defense when cells are under attack by pathogens, noxious agents or diseases. At certain conditions, cells are marked with apoptosis markers and the induced to go under apoptotic pathways which eventually results in self-destruction without affecting other adjacent cells. Apoptosis and necrosis, a process in which the cells are forced to die under physical or chemical pressures, are easy to be mistaken from each other since both of them induces cell deaths. Both of the processes can occur independently, sequentially or even simultaneously. The key difference between them is the way the cells die. Induced by apoptosis cell die by cell shrinkage, therefore it does not release its cytoplasmic content to the surrounding environment and later will be phagocytosed by macrophages or the adjacent cells without causing inflammation reactions. In the opposite, cell induced by necrosis die by bursting out its cytoplasmic content, which can lead to domino effect of inflammation to the whole tissue or organ. The specific morphologies of apoptotic cell are cell shrinkage, membrane bledding, chromosome condensation, nuclear fragmentation and DNA laddering. Apoptosis is considered as an irreversible process with caspase activation leading to cell suicide.

There are three pathways involved in cell apoptosis, including extrinsic or death receptor pathway, intrinsic or mitochondrial pathway and perforin/granzyme pathway. These three pathways are stimulated by different stimuli and have several distinguish proteins involved.

However, they eventually lead to the activation of a common protein kinase called caspase 3, which initiates the executive apoptotic pathway which leads to cell death. The stimuli of the extrinsic pathway are death ligands, such as Fas. When bound to its receptor, Fas can activate the adaptor protein called FADD, meanwhile, the binding of TNF ligand to its receptor activates that TRADD protein. This protein then recruits the binding of FADD and RIP proteins, then the complex comes to have an association with procaspase 8 forming death-inducing signaling complex (DISC) which in turn phosphorylates the caspase 3 protein. The perforin/granzyme pathway is stimulated by viral infections or tumors and induced under cytotoxic T cell signaling. When stimulated, T cell exerts its cytotoxic effects on targeted cells via pathway involving the secretion of transmembrane pore-forming molecule called perforin together with cytoplasmic granules such as granzyme A or granzyme B. These proteins can act as endonuclease that cleave proteins into fragments which can later activate the caspase 3 protein. There are a wide range of stimuli that can activate apoptotic intrinsic pathway, including radiation, hypoxia, viral infection or free radical. These stimulations causes the opening of mitochondria permeability transition pore results in the loss of membrane potential which lead to the leaking of the lumen content, including cytochrome c. Cytochrome c is well known for its function in cellular respiration inside the lumen of mitochondria, however, when released into the cytoplasm, it forms complex with Apaf-1 and procaspase 9 to form apoptosome which will later trigger the cascade activations of caspase 9 and caspase 3. As mention above, all three apoptosis pathways lead to the activation

of caspase 3, which can phosphorylate CAD endonuclease that cleaves proteins such as cytokeratin or PARP. These proteins can cause cellular morphological and biochemical changes, eventually lead to cell death.

Currently in scientific research, there are three assays heavily used for the detection of apoptotic markers by FCM, including Annexin/7-AAD (BD PharmingenTM PE Annexin V Apoptosis Detection Kit I, BD Biosciences, San Jose, CA), caspase 3 expression (CaspGLOWTM Fluorecein Active-Caspase-3 Staining Kit, Introvigen) and measurement of reactive oxygen species (ROS) (DCFDA-based Cellular ROS Detection Kit, Abcam, Cambridge, MA). One of the biochemical feature of early apoptosis is the externalization of phosphatidylserine, a component of plasma membrane normally inward-facing, to be expressed on the outer layers of plasma membrane. This phospholipid therefore acts as a ligand for the phagocytosis signaling of adjacent cells to digest the phosphatidylserine expressing cells. Because of its feature, phosphatidylserine is immensely used as the marker for apoptosis marker. Annexin V, a recombinant protein that strongly and specifically binds to phosphatidylserine, together with 7-ADD which is a fluorescent DNA binding dye indicating the membrane impermeability, can be used to identify the late apoptotic cells. Another method to track down the apoptotic cells is measuring the activated caspase 3 by using fluorescent conjugated antibody against it. This can be achieved by performing intracellular FCM staining. The last assay used in apoptosis identification is ROS detection. ROS is a short-lived and highly reactive molecule that play essential roles in cell development such as differentiation, proliferation, cell signaling and immune defense. However, when there is an imbalance in ROS generation and antioxidant to neutralize it, ROS can cause serious cellular damages, including protein, DNA, lipid and

organelles. Since ROS is a type of free radical, therefore its excessive level can trigger the activation of intrinsic apoptosis pathway.

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