Journal: AIDS Research and Human Retroviruses

Article Title: The Majority of HIV Type 1 DNA in Circulating CD4 + T Lymphocytes Is Present in Non-Gut-Homing Resting Memory CD4 + T Cells

doi: 10.1089/aid.2012.0351

Figure Lengend Snippet: Subject Demographics and CD4 + T Cell Subsets as Percentage of CD3 + CD4 + T Cells

Article Snippet: After enrichment with Dynabeads Untouched Human CD4 T Cell kit (Invitrogen, Oslo, Norway), 1–2×10 8 enriched CD4 + T cells were stained using the following three panels of monoclonal antibody cocktails into 14 subsets: CD3-PerCP-Cy5.5, CD4 + -PE-Cy7 (BD Biosciences, San Jose, CA), and CD45RO-ECD (Beckman Coulter, Hialeah, FL) were used in all three panels to define CD4 + T cells and to mark memory phenotype.

Techniques: Infection, Cell Counting

Journal: AIDS Research and Human Retroviruses

Article Title: The Majority of HIV Type 1 DNA in Circulating CD4 + T Lymphocytes Is Present in Non-Gut-Homing Resting Memory CD4 + T Cells

doi: 10.1089/aid.2012.0351

Figure Lengend Snippet: Gut-homing memory CD4+ T cell population in antiretroviral treatment (ART)-suppressed primary HIV-1 infection (PHI) and chronic HIV-1 infection (CHI) patients. Memory gut-homing CD4+ T cells were identified by expression of CD45RO and integrin β7. (A) The first panel shows the clearly defined β1+ and β7+ populations from the CD3+CD4+CD45RO+α4+(CD49d) cells within peripheral blood mononuclear cells (PBMCs). The second panel depicts sorting based on CD3+CD4+CD45RO+β7+ cells. These cells are clearly associated with α4, with 98% of the β7+ cells shown to be positive for α4. The presence of β7+ cells within CD45RO+CD4+ T cells is shown for one representative subject studied in the third panel. The proportions of memory gut-homing and memory non-gut-homing CD3+CD4+ T cell subsets are shown with the majority of cells demonstrated to be non-gut-homing β7−CD4+ T cells. (B) Scatter plots of medians and interquartile range (IQR) of absolute total and integrated HIV-1 DNA copies/500 ng DNA are shown for CD3+CD4+, CD3+CD4+CD45RO−, CD3+CD4+CD45RO+β7+, and CD3+CD4+CD45RO+β7− T cell subsets from CHI patients. Each individual patient is represented by a unique symbol. Both the gut-homing and non-gut-homing memory CD4+ T cell subsets contained a significantly greater number of total HIV-1 DNA copies/500 ng DNA than the naive CD4+ T cell subsets (p=0.016 for both comparisons). (C) Scatter plots of the median percentage and IQR of total and integrated HIV-1 DNA in either integrin β7+CD45RO+CD4+ T cells (16% and 19.5%, respectively) or β7−CD45RO+CD4+ T cells (84% and 80.5%, respectively) are shown for CHI patients with each individual patient represented by a unique symbol. (D) Corresponding scatter plots of medians and IQR of absolute total HIV-1 DNA copies/500 ng DNA are shown for ART-suppressed and PHI patients.

Article Snippet: After enrichment with Dynabeads Untouched Human CD4 T Cell kit (Invitrogen, Oslo, Norway), 1–2×10 8 enriched CD4 + T cells were stained using the following three panels of monoclonal antibody cocktails into 14 subsets: CD3-PerCP-Cy5.5, CD4 + -PE-Cy7 (BD Biosciences, San Jose, CA), and CD45RO-ECD (Beckman Coulter, Hialeah, FL) were used in all three panels to define CD4 + T cells and to mark memory phenotype.

Techniques: Infection, Expressing

Journal: AIDS Research and Human Retroviruses

Article Title: The Majority of HIV Type 1 DNA in Circulating CD4 + T Lymphocytes Is Present in Non-Gut-Homing Resting Memory CD4 + T Cells

doi: 10.1089/aid.2012.0351

Figure Lengend Snippet: Memory T regulatory and memory CD127high CD4+ T cell subsets in CHI. Memory Treg CD3+CD4+ cells were identified within the CD45RO+ population by high expression of CD25 and dim expression of CD127. (A) Representative histograms for one CHI subject are shown. The percentages for each population are shown. (B) Scatter plots of medians and IQR of total and integrated HIV-1 DNA copies/500 ng DNA are shown for CD3+CD4+, CD3+CD4+CD45RO−, CD3+CD4+CD45RO+CD25+CD127dim, and CD3+CD4+CD45RO+CD127high T cell subsets from CHI patients. Each individual patient is represented by a unique symbol. Total HIV-1 DNA copies/500 ng of DNA significantly resided within the Treg and CD127high memory CD4+ T cell subsets compared to the naive CD4+ T cell subset (p=0.03 for both comparisons). (C) Scatter plots of median percentage and IQR of total and integrated HIV-1 DNA in either Treg CD25+CD127dim (5% and 7%, respectively) or CD127high (95% and 93%, respectively) subsets of memory CD4+ T cells are shown for CHI patients. Each individual patient is represented by a unique symbol.

Article Snippet: After enrichment with Dynabeads Untouched Human CD4 T Cell kit (Invitrogen, Oslo, Norway), 1–2×10 8 enriched CD4 + T cells were stained using the following three panels of monoclonal antibody cocktails into 14 subsets: CD3-PerCP-Cy5.5, CD4 + -PE-Cy7 (BD Biosciences, San Jose, CA), and CD45RO-ECD (Beckman Coulter, Hialeah, FL) were used in all three panels to define CD4 + T cells and to mark memory phenotype.

Techniques: Expressing

Journal: AIDS Research and Human Retroviruses

Article Title: The Majority of HIV Type 1 DNA in Circulating CD4 + T Lymphocytes Is Present in Non-Gut-Homing Resting Memory CD4 + T Cells

doi: 10.1089/aid.2012.0351

Figure Lengend Snippet: Activated memory cells identified by their expression of CD38. (A) Representative histograms for a PHI subject and a CHI subject gated on activated memory CD4+ T cells are shown. Percentages for each population are also shown. (B) Scatter plots of medians and IQR of total and integrated HIV-1 DNA copies/500 ng DNA are shown for CD3+CD4+CD45RO−, CD3+CD4+CD45RO+CD38−, and CD3+CD4+CD45RO+CD38+ T cell subsets from CHI patients. Each individual patient is represented by a unique symbol. (C) Scatter plots of median percentage and IQR of total and integrated HIV-1 DNA in CD38+ (92% and 72%, respectively) or CD38− (8% and 28%, respectively) subsets of memory CD45RO+CD4+ T cells are shown for CHI patients. Each individual patient is represented by a unique symbol.

Article Snippet: After enrichment with Dynabeads Untouched Human CD4 T Cell kit (Invitrogen, Oslo, Norway), 1–2×10 8 enriched CD4 + T cells were stained using the following three panels of monoclonal antibody cocktails into 14 subsets: CD3-PerCP-Cy5.5, CD4 + -PE-Cy7 (BD Biosciences, San Jose, CA), and CD45RO-ECD (Beckman Coulter, Hialeah, FL) were used in all three panels to define CD4 + T cells and to mark memory phenotype.

Techniques: Expressing

Journal:

Article Title: A Chimeric Human T-Cell Lymphotropic Virus Type 1 with the Envelope Glycoprotein of Moloney Murine Leukemia Virus Is Infectious for Murine Cells

doi: 10.1128/JVI.76.15.7883-7889.2002

Figure Lengend Snippet: Cell-free viral infections of murine cells. The HTLV-1 gag gene (205 bp, positions 1616 to 1821 in Seiki ATK1 sequence [29]) was detected by PCR amplification of DNA extracted from cells infected with cell-free chimeric viruses or cell-free HTLV-1. Amplified products were detected by Southern blot hybridization with specific oligonucleotide internal probes end labeled with 32P using 3′ terminal transferase. Radioactive signals were detected and quantified using a PhosphorImager (Molecular Dynamics) and ImageQuant software. To detect plasmid contamination, PCR was also performed with primer 5′-GGCTCGTATGTTGTGTGGAA-3′ in pUC19 and primer 5′-TTAGCCATATGCGTGCCATG-3′ in the HTLV-1 LTR. (A) PCR detection of the HTLV-1 gag gene and of control plasmid in DNA extracted from different cell lines infected by chimeric (Chim, Chim.ΔR) and HTLV-1 particles. The positive control (+) corresponds to 1 copy of the HTLV-1 genome from MT2 cells. The negative control (−) is 1 μg of DNA from naïve 293T cells. (B) Standard curve for PCR detection of HTLV-1 gag gene. Dilutions of pCS-HTLV-I plasmid (top) and MT2 cell DNA (bottom) are shown. (C) Evidence for infection of mouse lymphocytes by ΔR chimeric cell particles. PCR detection of the HTLV-1 gag gene and of control plasmid in DNA. Detection by reverse transcription-PCR of the doubly spliced viral mRNA for the Tax protein (221-bp spliced fragment, primers in positions 5098 to 7438 in Seiki ATK1 sequence). The GAPDH gene was also amplified by reverse transcription-PCR for a control. The positive controls are as follows: for the Gag panel, 1 copy of the HTLV-1 genome from MT2 cells; for the pUC panel, 1 μg of pCS-HTLV-1 plasmid; and for the Tax mRNA and GAPDH panels, MT2 RNA. (D) PCR detection of HTLV-1 gag gene in mouse lymphocytes infected with ΔR chimeric particles. Lanes 1, 2, and 3 show the spread of the infection from primary infected, male CD4+ lymphocytes placed in the lower chamber of a transwell to other cells placed in the upper chamber. Lane 1, DNA from primary infected cells; lane 2, DNA from secondary infected EL4 cells; lane 3, DNA from secondary infected female 129sv CD4+ lymphocytes. Lanes 4, 5, and 6 illustrate the spread of the infection from primary infected EL4 cells placed in the lower chamber of a transwell to other cells placed in the upper chamber. Lane 4, DNA from primary infected EL4 cells; lane 5, DNA from secondary infected male 129sv CD4+ lymphocytes; lane 6, DNA from secondary infected EL4 cells. The control primers to detect any contamination in secondary infections were 5′-GACTAGACATGTCTTAACATCTGTCC-3′ and 5′-CCTATTGCATGGACAGCAGCTTATG-3′ in the Zfy gene (murine Y chromosome [Y Chr.]). The positive control for the Gag panel is 1 copy of the HTLV-1 genome from MT2 cells, while that for the Y Chr. panel is 1 μg of DNA from male mouse splenocytes. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: The target cells used were NIH 3T3, EL4 (a murine CD4 T-cell lymphoma obtained from the American Type Culture Collection), 293T, 293T4, HOS (a human osteosarcoma-derived fibroblast cell line permissive for HTLV-1 [ 19 , 35 ]), and B5 (a cell line derived from the DBS-FRhL rhesus monkey lung fibroblast line that is permissive for HTLV-1 [ 6 ]).

Techniques: Sequencing, Amplification, Infection, Southern Blot, Hybridization, Labeling, Software, Plasmid Preparation, Control, Positive Control, Negative Control, Reverse Transcription