Journal of The Society for Gynecologic Investigation, Apr 1, 2004
To test the hypothesis that a delay in initial fetal cell enrichment processing of maternal blood... more To test the hypothesis that a delay in initial fetal cell enrichment processing of maternal blood samples (defined as the time between blood draw and the initial density gradient centrifugation step) compromises the ability to recover fetal cells, we performed a randomized comparison of immediate (within 4 hours of draw) versus delayed (between 18-24 hours of draw) processing. Four centers participated: two centers utilized flow cytometry (FLOW), and two centers utilized magnetic-activated cell sorting (MACS) techniques. Each center collected 34 samples. The outcome was the percentage of gamma positive (gamma(+)) cells for FLOW or the number of nucleated red blood cells (NRBCs) for MACS, found in the final enriched cell population. Both outcomes reflect cell properties that are potentially fetal in origin, thus making them representative of the ability to recover fetal cells. Our results did not support our hypothesis that delay in processing compromises fetal cell recovery. Instead, in MACS processing, we observed an increase in recovered NRBCs when blood sample processing was delayed compared with immediate processing. There was no significant difference in gamma(+) cells with FLOW. Time-related changes in the density of target cells, perhaps associated with their progress towards apoptosis during the delay period, may result in increased intact fetal cells with the study methods utilized.
Optimal stimulation and prevention of anergy in T cells requires signaling through the CD28 molec... more Optimal stimulation and prevention of anergy in T cells requires signaling through the CD28 molecule. During HIV disease progression, CD28 expression is lost, particularly on CD8+ T cells. Because alterations in cytokine production patterns occur during HIV infection, we determined whether CD8+ T cell phenotype or function was affected by cytokine environment. Treatment of CD8+ T cells with IL-4 decreased levels of both CD28 surface expression and message and increased CD8 expression. Furthermore, CD8+ T cells that had down-regulated CD28 had reduced proliferative capacity. The inhibitory effects of CD28 reduction could be compensated either by increased anti-CD3 or by exogenous IL-2, suggesting that the strength of T cell signaling necessary for the production of IL-2 and subsequent proliferation is negatively regulated by IL-4. CD8+ subpopulations with differential CD28 expression produced different patterns of cytokines, particularly IL-2 and IFN-gamma. Furthermore, CD8+ T cells ...
Cyclin T1 expression in small and large memory CD4 T cells during T cell activation. Human CD4+CD... more Cyclin T1 expression in small and large memory CD4 T cells during T cell activation. Human CD4+CD45RO+ memory T cells were purified from peripheral blood and cultured without (No Costimulation) or with CD3 + CD28 mabs and IL2 (Costimulation) for 5 days, then stained for CycT1, CD69, CD25, HLA.DR, and CD38. (A) Shown are sample Isotype-FITC or CycT1-FITC dotplots gated on overall, small, or large cells, and (B) mean ± sem CycT1, CD69+CD25+, or HLA.DR+CD38+ expression (N = 5). (PPTX 595 kb)
Additional file 6. Primers used for nested PCR detection of SHIV-SF162p3 Gag and Env genes in AT-... more Additional file 6. Primers used for nested PCR detection of SHIV-SF162p3 Gag and Env genes in AT-SVF cells of acutely infected rhesus macaques.
Additional file 1. General method for isolation of stromal-vascular-fraction (AT-SVF) cells from ... more Additional file 1. General method for isolation of stromal-vascular-fraction (AT-SVF) cells from adipose tissue of rhesus macaques, and subsequent analyses. (A) 30-60Â mins collagenase digestion of solid adipose tissue samples from rhesus macaques is followed by washing and centrifugation, allowing for separation of mature adipocytes (floater fraction) from the stromal-vascular-fraction (AT-SVF) cells. AT-SVF cells were then analyzed by flow cytometry, nested PCR, and viral outgrowth assays, and floater fraction adipocytes analyzed for mRNA expression. (B) Sample flow cytometry gating schemes for examination of AT-SVF T cells, NKT cells, macrophages, and B cells.
HIV-1 is a chronic disease managed by strictly adhering to daily antiretroviral therapy (ART). Ho... more HIV-1 is a chronic disease managed by strictly adhering to daily antiretroviral therapy (ART). However, not all people living with HIV-1 have access to ART, and those with access may not adhere to treatment regimens increasing viral load and disease progression. Here, a subcutaneous nanofluidic implant was used as a long-acting (LA) drug delivery platform to address these issues. The device was loaded with tenofovir alafenamide (TAF) and implanted in treatment-naïve simian HIV (SHIV)-positive nonhuman primates (NHP) for a month. We monitored intracellular tenofovir-diphosphate (TFV-DP) concentration in the target cells, peripheral blood mononuclear cells (PBMC). The concentrations of TFV-DP were maintained at a median of 391.0 fmol/106 cells (IQR, 243.0 to 509.0 fmol/106 cells) for the duration of the study. Further, we achieved drug penetration into lymphatic tissues, known for persistent HIV-1 replication. Moreover, we observed a first-phase viral load decay of −1.14 ± 0.81 log10 ...
CD36 is a multifunctional scavenger receptor and lipid transporter implicated in metabolic and in... more CD36 is a multifunctional scavenger receptor and lipid transporter implicated in metabolic and inflammatory pathologies, as well as cancer progression. CD36 is known to be expressed by adipocytes and monocytes/macrophages, but its expression by T cells is not clearly established. We found that CD4 and CD8 T cells in adipose tissue and liver of humans, monkeys, and mice upregulated CD36 expression (ranging from~5-40% CD36+), whereas little to no CD36 was expressed by T cells in blood, spleen, and lymph nodes. CD36 was expressed predominantly by resting CD38-, HLA.DR-, and PD-1-adipose tissue T cells in monkeys, and increased during high-fat feeding in mice. Adipose tissue and liver promote a distinct phenotype in resident T cells characterized by CD36 upregulation.
Background: The regulatory cyclin, Cyclin T1 (CycT1), is a host factor essential for HIV-1 replic... more Background: The regulatory cyclin, Cyclin T1 (CycT1), is a host factor essential for HIV-1 replication in CD4 T cells and macrophages. The importance of CycT1 and the Positive Transcription Elongation Factor b (P-TEFb) complex for HIV replication is well-established, but regulation of CycT1 expression and protein levels during HIV replication and latency establishment in CD4 T cells is less characterized. Methods: To better define the regulation of CycT1 levels during HIV replication in CD4 T cells, multiparameter flow cytometry was utilized to study the interaction between HIV replication (intracellular p24) and CycT1 of human peripheral blood memory CD4 T cells infected with HIV in vitro. CycT1 was further examined in CD4 T cells of human lymph nodes. Results: In activated (CD3+CD28 costimulation) uninfected blood memory CD4 T cells, CycT1 was most significantly upregulated in maximally activated (CD69+CD25+ and HLA.DR+CD38+) cells. In memory CD4 T cells infected with HIV in vitro, two distinct infected populations of p24+CycT1+ and p24+CycT1-cells were observed during 7 days infection, suggestive of different phases of productive HIV replication and subsequent latency establishment. Intriguingly, p24+CycT1-cells were the predominant infected population in activated CD4 T cells, raising the possibility that productively infected cells may transition into latency subsequent to CycT1 downregulation. Additionally, when comparing infected p24+ cells to bystander uninfected p24-cells (after bulk HIV infections), HIV replication significantly increased T cell activation (CD69, CD25, HLA.DR, CD38, and Ki67) without concomitantly increasing CycT1 protein levels, possibly due to hijacking of P-TEFb by the viral Tat protein. Lastly, CycT1 was constitutively expressed at higher levels in lymph node CD4 T cells compared to blood T cells, potentially enhancing latency generation in lymphoid tissues. Conclusions: CycT1 is most highly upregulated in maximally activated memory CD4 T cells as expected, but may become less associated with T cell activation during HIV replication. The progression into latency may further be predicated by substantial generation of p24+CycT1-cells during HIV replication.
The Journal of allergy and clinical immunology, Jan 18, 2018
The inhibitory receptor programmed cell death protein 1 (PD-1) is upregulated on a variety of imm... more The inhibitory receptor programmed cell death protein 1 (PD-1) is upregulated on a variety of immune cells, including natural killer (NK) cells, during chronic viral infection and tumorigenesis. Blockade of PD-1 or its ligands produces durable clinical responses with tolerable side effects in patients with a broad spectrum of cancers. However, the underlying molecular mechanisms of how PD-1 regulates NK cell function remain poorly characterized. We sought to determine the effect of PD-1 signaling on NK cells. PD-1 was overexpressed in CD16-KHYG-1 (a human NK cell line with both antibody-dependent cellular cytotoxicity through CD16 and natural cytotoxicity through NKG2D) cells and stimulated by exposing the cells to NK-sensitive target cells expressing programmed death ligand 1 (PD-L1). PD-1 engagement by PD-L1 specifically blocked NK cell-mediated cytotoxicity without interfering with the conjugation between NK cells and target cells. Further examination showed that PD-1 signaling b...
HIV reservoirs pose major challenges to viral eradication. The main cellular reservoirs include C... more HIV reservoirs pose major challenges to viral eradication. The main cellular reservoirs include CD4 T cells and macrophages, whereas anatomic reservoirs are thought to be primarily lymphoid tissues. Adipose tissue represents a potentially important non-lymphoid location for HIV replication and persistence because the stromal-vascular-fraction (AT-SVF) contains activated innate and adaptive immune cells that increase in number during infections, obesity, and chronic inflammation. Adipose tissue from two groups of SHIV-SF162p3-infected (~4 weeks acute infection) or SIVmac251-infected (~38 weeks chronic infection) rhesus macaques (N = 8 for each group) were studied for immune cell content, viral infectiousness, and metabolic health. The AT-SVF cells from SHIV-infected monkeys contained abundant memory CD4 and CD8 T cells, with fewer NKT cells and macrophages, and no B cells. Proviral DNA (Gag and Env) was readily detectable by nested PCR in AT-SVF cells from multiple adipose depots (su...
Apoptosis is a process of cell death characterized by distinctive morphological changes and fragm... more Apoptosis is a process of cell death characterized by distinctive morphological changes and fragmentation of cellular DNA. Using video imaging and color thresholding techniques, we objectively quantitated the number of cultured CD4+ T-lymphoblastoid cells (HUT78 cells, RH9 subclone) displaying morphological signs of apoptosis before and after exposure to gamma-irradiation. The numbers of apoptotic cells measured by objective video imaging techniques were compared to numbers of apoptotic cells measured in the same samples by sensitive apoptotic assays that quantitate DNA fragmentation. DNA fragmentation assays gave consistently higher values compared with the video imaging assays that measured morphological changes associated with apoptosis. These results suggest that substantial DNA fragmentation can precede or occur in the absence of the morphological changes which are associated with apoptosis in gamma-irradiated RH9 cells.
Objective-To determine whether adipose tissue functions as a reservoir for HIV-1. Design-We exami... more Objective-To determine whether adipose tissue functions as a reservoir for HIV-1. Design-We examined memory CD4 T cells and HIV DNA in adipose tissue-stromal-vascularfraction (AT-SVF) of 5 patients (4 ART-treated and 1 untreated). To determine if adipocytes stimulate CD4 T cells and regulate HIV production, primary human adipose cells were co-cultured with HIV-infected CD4 T cells. Methods-AT-SVF T cells were studied by flow cytometry, and AT-SVF HIV DNA (Gag and Env) was examined by nested PCR and sequence analyses. CD4 T cell activation and HIV production were measured by flow cytometry and ELISA. Results-AT-SVF CD3 T cells were activated (>60% CD69+) memory CD4 and CD8 T cells in uninfected and HIV-infected persons, but the AT-SVF CD4/CD8 ratio was lower in HIV patients. HIV DNA (Gag and Env) was detected in AT-SVF of all 5 patients examined by nested PCR,
As part of the Houston Vanguard study, a subset of 10 patients randomized to receive IL-2 therapy... more As part of the Houston Vanguard study, a subset of 10 patients randomized to receive IL-2 therapy were compared to 4 patients randomized to not receive IL-2, for markers of T cell activation and death during the first three cycles of IL-2. All patients were treated with combination antiretroviral therapy (ART) and were virally suppressed. The purpose of the study was to examine the role of CD8(+) T cell death in responses to ART and IL-2 therapy. Lymphocytes were examined at Day 0, 5 and 30 days during three cycles of IL-2 therapy. CD25, CD38, HLA-DR expression and annexin (cell death) were examined on CD4 and CD8 subpopulations. Follow up studies examined CD4 levels and CD4:CD8 reconstitution after 6 years using both univariant and multivariate analyses. Human lymphocytes responded to IL-2 therapy by upregulation of CD25 on CD4(+) T cells, leading to an increase in CD4 cell counts. CD8(+) T cells did not increase CD25 expression, but upregulated activation antigens (CD38 and DR) an...
HIV-1 Vpr induces the cardinal metabolic defects of HIV-associated lipodystrophy and hepatosteato... more HIV-1 Vpr induces the cardinal metabolic defects of HIV-associated lipodystrophy and hepatosteatosis in vivo.
The National Institute of Child Health and Human Development Fetal Cell Isolation Study (NIFTY) i... more The National Institute of Child Health and Human Development Fetal Cell Isolation Study (NIFTY) is a prospective, multicenter clinical project to develop non-invasive methods of prenatal diagnosis. The initial objective was to assess the utility of fetal cells in the peripheral blood of pregnant women to diagnose or screen for fetal chromosome abnormalities. Results of fluorescence in situ hybridization (FISH) analysis on interphase nuclei of fetal cells recovered from maternal blood were compared to metaphase karyotypes of fetal cells obtained by amniocentesis or chorionic villus sampling (CVS). After the first 5 years of the study we performed a planned analysis of the data. We report here the data from 2744 fully processed pre-procedural blood samples; 1292 samples were from women carrying singleton male fetuses. Target cell recovery and fetal cell detection were better using magnetic-based separation systems (MACS) than with flow-sorting (FACS). Blinded FISH assessment of samples from women carrying singleton male fetuses found at least one cell with an X and Y signal in 41.4% of cases (95% CI: 37.4%, 45.5%). The false-positive rate of gender detection was 11.1% (95% CI: 6.1,16.1%). This was higher than expected due to the use of indirectly labeled FISH probes in one center. The detection rate of finding at least one aneuploid cell in cases of fetal aneuploidy was 74.4% (95% CI: 76.0%, 99.0%), with a false-positive rate estimated to be between 0.6% and 4.1%. The sensitivity of aneuploidy detection using fetal cell analysis from maternal blood is comparable to single marker prenatal serum screening, but technological advances are needed before fetal cell analysis has clinical application as part of a multiple marker method for non-invasive prenatal screening. The limitations of the present study, i.e. multiple processing protocols, are being addressed in the ongoing study.
Rare fetal cells can be recovered from maternal blood, which suggests that non-invasive prenatal ... more Rare fetal cells can be recovered from maternal blood, which suggests that non-invasive prenatal diagnosis is possible. However, recovery and analysis of fetal cells from blood is complex, and sensitivity is low because of the rarity of these cells in the maternal circulation. An alternative strategy, which suggested that intact fetal cells can be found in maternal plasma by use of simple enrichment methods, has been reported. We aimed to replicate this technique. However, five independent laboratories were unable to identify any intact male cells from the plasma of 38 women known to be carrying male fetuses. Although apoptotic intact fetal cells could contribute to the detection of fetal DNA in maternal plasma, we believe that recovery of these cells is difficult and not clinically practical.
These studies were approved by the committee for the protection of human subjects at the Universi... more These studies were approved by the committee for the protection of human subjects at the University of Texas at Houston and by the institutional review board for human subjects research at Baylor College of Medicine. All subjects provided informed consent.
Journal of The Society for Gynecologic Investigation, Apr 1, 2004
To test the hypothesis that a delay in initial fetal cell enrichment processing of maternal blood... more To test the hypothesis that a delay in initial fetal cell enrichment processing of maternal blood samples (defined as the time between blood draw and the initial density gradient centrifugation step) compromises the ability to recover fetal cells, we performed a randomized comparison of immediate (within 4 hours of draw) versus delayed (between 18-24 hours of draw) processing. Four centers participated: two centers utilized flow cytometry (FLOW), and two centers utilized magnetic-activated cell sorting (MACS) techniques. Each center collected 34 samples. The outcome was the percentage of gamma positive (gamma(+)) cells for FLOW or the number of nucleated red blood cells (NRBCs) for MACS, found in the final enriched cell population. Both outcomes reflect cell properties that are potentially fetal in origin, thus making them representative of the ability to recover fetal cells. Our results did not support our hypothesis that delay in processing compromises fetal cell recovery. Instead, in MACS processing, we observed an increase in recovered NRBCs when blood sample processing was delayed compared with immediate processing. There was no significant difference in gamma(+) cells with FLOW. Time-related changes in the density of target cells, perhaps associated with their progress towards apoptosis during the delay period, may result in increased intact fetal cells with the study methods utilized.
Optimal stimulation and prevention of anergy in T cells requires signaling through the CD28 molec... more Optimal stimulation and prevention of anergy in T cells requires signaling through the CD28 molecule. During HIV disease progression, CD28 expression is lost, particularly on CD8+ T cells. Because alterations in cytokine production patterns occur during HIV infection, we determined whether CD8+ T cell phenotype or function was affected by cytokine environment. Treatment of CD8+ T cells with IL-4 decreased levels of both CD28 surface expression and message and increased CD8 expression. Furthermore, CD8+ T cells that had down-regulated CD28 had reduced proliferative capacity. The inhibitory effects of CD28 reduction could be compensated either by increased anti-CD3 or by exogenous IL-2, suggesting that the strength of T cell signaling necessary for the production of IL-2 and subsequent proliferation is negatively regulated by IL-4. CD8+ subpopulations with differential CD28 expression produced different patterns of cytokines, particularly IL-2 and IFN-gamma. Furthermore, CD8+ T cells ...
Cyclin T1 expression in small and large memory CD4 T cells during T cell activation. Human CD4+CD... more Cyclin T1 expression in small and large memory CD4 T cells during T cell activation. Human CD4+CD45RO+ memory T cells were purified from peripheral blood and cultured without (No Costimulation) or with CD3 + CD28 mabs and IL2 (Costimulation) for 5 days, then stained for CycT1, CD69, CD25, HLA.DR, and CD38. (A) Shown are sample Isotype-FITC or CycT1-FITC dotplots gated on overall, small, or large cells, and (B) mean ± sem CycT1, CD69+CD25+, or HLA.DR+CD38+ expression (N = 5). (PPTX 595 kb)
Additional file 6. Primers used for nested PCR detection of SHIV-SF162p3 Gag and Env genes in AT-... more Additional file 6. Primers used for nested PCR detection of SHIV-SF162p3 Gag and Env genes in AT-SVF cells of acutely infected rhesus macaques.
Additional file 1. General method for isolation of stromal-vascular-fraction (AT-SVF) cells from ... more Additional file 1. General method for isolation of stromal-vascular-fraction (AT-SVF) cells from adipose tissue of rhesus macaques, and subsequent analyses. (A) 30-60Â mins collagenase digestion of solid adipose tissue samples from rhesus macaques is followed by washing and centrifugation, allowing for separation of mature adipocytes (floater fraction) from the stromal-vascular-fraction (AT-SVF) cells. AT-SVF cells were then analyzed by flow cytometry, nested PCR, and viral outgrowth assays, and floater fraction adipocytes analyzed for mRNA expression. (B) Sample flow cytometry gating schemes for examination of AT-SVF T cells, NKT cells, macrophages, and B cells.
HIV-1 is a chronic disease managed by strictly adhering to daily antiretroviral therapy (ART). Ho... more HIV-1 is a chronic disease managed by strictly adhering to daily antiretroviral therapy (ART). However, not all people living with HIV-1 have access to ART, and those with access may not adhere to treatment regimens increasing viral load and disease progression. Here, a subcutaneous nanofluidic implant was used as a long-acting (LA) drug delivery platform to address these issues. The device was loaded with tenofovir alafenamide (TAF) and implanted in treatment-naïve simian HIV (SHIV)-positive nonhuman primates (NHP) for a month. We monitored intracellular tenofovir-diphosphate (TFV-DP) concentration in the target cells, peripheral blood mononuclear cells (PBMC). The concentrations of TFV-DP were maintained at a median of 391.0 fmol/106 cells (IQR, 243.0 to 509.0 fmol/106 cells) for the duration of the study. Further, we achieved drug penetration into lymphatic tissues, known for persistent HIV-1 replication. Moreover, we observed a first-phase viral load decay of −1.14 ± 0.81 log10 ...
CD36 is a multifunctional scavenger receptor and lipid transporter implicated in metabolic and in... more CD36 is a multifunctional scavenger receptor and lipid transporter implicated in metabolic and inflammatory pathologies, as well as cancer progression. CD36 is known to be expressed by adipocytes and monocytes/macrophages, but its expression by T cells is not clearly established. We found that CD4 and CD8 T cells in adipose tissue and liver of humans, monkeys, and mice upregulated CD36 expression (ranging from~5-40% CD36+), whereas little to no CD36 was expressed by T cells in blood, spleen, and lymph nodes. CD36 was expressed predominantly by resting CD38-, HLA.DR-, and PD-1-adipose tissue T cells in monkeys, and increased during high-fat feeding in mice. Adipose tissue and liver promote a distinct phenotype in resident T cells characterized by CD36 upregulation.
Background: The regulatory cyclin, Cyclin T1 (CycT1), is a host factor essential for HIV-1 replic... more Background: The regulatory cyclin, Cyclin T1 (CycT1), is a host factor essential for HIV-1 replication in CD4 T cells and macrophages. The importance of CycT1 and the Positive Transcription Elongation Factor b (P-TEFb) complex for HIV replication is well-established, but regulation of CycT1 expression and protein levels during HIV replication and latency establishment in CD4 T cells is less characterized. Methods: To better define the regulation of CycT1 levels during HIV replication in CD4 T cells, multiparameter flow cytometry was utilized to study the interaction between HIV replication (intracellular p24) and CycT1 of human peripheral blood memory CD4 T cells infected with HIV in vitro. CycT1 was further examined in CD4 T cells of human lymph nodes. Results: In activated (CD3+CD28 costimulation) uninfected blood memory CD4 T cells, CycT1 was most significantly upregulated in maximally activated (CD69+CD25+ and HLA.DR+CD38+) cells. In memory CD4 T cells infected with HIV in vitro, two distinct infected populations of p24+CycT1+ and p24+CycT1-cells were observed during 7 days infection, suggestive of different phases of productive HIV replication and subsequent latency establishment. Intriguingly, p24+CycT1-cells were the predominant infected population in activated CD4 T cells, raising the possibility that productively infected cells may transition into latency subsequent to CycT1 downregulation. Additionally, when comparing infected p24+ cells to bystander uninfected p24-cells (after bulk HIV infections), HIV replication significantly increased T cell activation (CD69, CD25, HLA.DR, CD38, and Ki67) without concomitantly increasing CycT1 protein levels, possibly due to hijacking of P-TEFb by the viral Tat protein. Lastly, CycT1 was constitutively expressed at higher levels in lymph node CD4 T cells compared to blood T cells, potentially enhancing latency generation in lymphoid tissues. Conclusions: CycT1 is most highly upregulated in maximally activated memory CD4 T cells as expected, but may become less associated with T cell activation during HIV replication. The progression into latency may further be predicated by substantial generation of p24+CycT1-cells during HIV replication.
The Journal of allergy and clinical immunology, Jan 18, 2018
The inhibitory receptor programmed cell death protein 1 (PD-1) is upregulated on a variety of imm... more The inhibitory receptor programmed cell death protein 1 (PD-1) is upregulated on a variety of immune cells, including natural killer (NK) cells, during chronic viral infection and tumorigenesis. Blockade of PD-1 or its ligands produces durable clinical responses with tolerable side effects in patients with a broad spectrum of cancers. However, the underlying molecular mechanisms of how PD-1 regulates NK cell function remain poorly characterized. We sought to determine the effect of PD-1 signaling on NK cells. PD-1 was overexpressed in CD16-KHYG-1 (a human NK cell line with both antibody-dependent cellular cytotoxicity through CD16 and natural cytotoxicity through NKG2D) cells and stimulated by exposing the cells to NK-sensitive target cells expressing programmed death ligand 1 (PD-L1). PD-1 engagement by PD-L1 specifically blocked NK cell-mediated cytotoxicity without interfering with the conjugation between NK cells and target cells. Further examination showed that PD-1 signaling b...
HIV reservoirs pose major challenges to viral eradication. The main cellular reservoirs include C... more HIV reservoirs pose major challenges to viral eradication. The main cellular reservoirs include CD4 T cells and macrophages, whereas anatomic reservoirs are thought to be primarily lymphoid tissues. Adipose tissue represents a potentially important non-lymphoid location for HIV replication and persistence because the stromal-vascular-fraction (AT-SVF) contains activated innate and adaptive immune cells that increase in number during infections, obesity, and chronic inflammation. Adipose tissue from two groups of SHIV-SF162p3-infected (~4 weeks acute infection) or SIVmac251-infected (~38 weeks chronic infection) rhesus macaques (N = 8 for each group) were studied for immune cell content, viral infectiousness, and metabolic health. The AT-SVF cells from SHIV-infected monkeys contained abundant memory CD4 and CD8 T cells, with fewer NKT cells and macrophages, and no B cells. Proviral DNA (Gag and Env) was readily detectable by nested PCR in AT-SVF cells from multiple adipose depots (su...
Apoptosis is a process of cell death characterized by distinctive morphological changes and fragm... more Apoptosis is a process of cell death characterized by distinctive morphological changes and fragmentation of cellular DNA. Using video imaging and color thresholding techniques, we objectively quantitated the number of cultured CD4+ T-lymphoblastoid cells (HUT78 cells, RH9 subclone) displaying morphological signs of apoptosis before and after exposure to gamma-irradiation. The numbers of apoptotic cells measured by objective video imaging techniques were compared to numbers of apoptotic cells measured in the same samples by sensitive apoptotic assays that quantitate DNA fragmentation. DNA fragmentation assays gave consistently higher values compared with the video imaging assays that measured morphological changes associated with apoptosis. These results suggest that substantial DNA fragmentation can precede or occur in the absence of the morphological changes which are associated with apoptosis in gamma-irradiated RH9 cells.
Objective-To determine whether adipose tissue functions as a reservoir for HIV-1. Design-We exami... more Objective-To determine whether adipose tissue functions as a reservoir for HIV-1. Design-We examined memory CD4 T cells and HIV DNA in adipose tissue-stromal-vascularfraction (AT-SVF) of 5 patients (4 ART-treated and 1 untreated). To determine if adipocytes stimulate CD4 T cells and regulate HIV production, primary human adipose cells were co-cultured with HIV-infected CD4 T cells. Methods-AT-SVF T cells were studied by flow cytometry, and AT-SVF HIV DNA (Gag and Env) was examined by nested PCR and sequence analyses. CD4 T cell activation and HIV production were measured by flow cytometry and ELISA. Results-AT-SVF CD3 T cells were activated (>60% CD69+) memory CD4 and CD8 T cells in uninfected and HIV-infected persons, but the AT-SVF CD4/CD8 ratio was lower in HIV patients. HIV DNA (Gag and Env) was detected in AT-SVF of all 5 patients examined by nested PCR,
As part of the Houston Vanguard study, a subset of 10 patients randomized to receive IL-2 therapy... more As part of the Houston Vanguard study, a subset of 10 patients randomized to receive IL-2 therapy were compared to 4 patients randomized to not receive IL-2, for markers of T cell activation and death during the first three cycles of IL-2. All patients were treated with combination antiretroviral therapy (ART) and were virally suppressed. The purpose of the study was to examine the role of CD8(+) T cell death in responses to ART and IL-2 therapy. Lymphocytes were examined at Day 0, 5 and 30 days during three cycles of IL-2 therapy. CD25, CD38, HLA-DR expression and annexin (cell death) were examined on CD4 and CD8 subpopulations. Follow up studies examined CD4 levels and CD4:CD8 reconstitution after 6 years using both univariant and multivariate analyses. Human lymphocytes responded to IL-2 therapy by upregulation of CD25 on CD4(+) T cells, leading to an increase in CD4 cell counts. CD8(+) T cells did not increase CD25 expression, but upregulated activation antigens (CD38 and DR) an...
HIV-1 Vpr induces the cardinal metabolic defects of HIV-associated lipodystrophy and hepatosteato... more HIV-1 Vpr induces the cardinal metabolic defects of HIV-associated lipodystrophy and hepatosteatosis in vivo.
The National Institute of Child Health and Human Development Fetal Cell Isolation Study (NIFTY) i... more The National Institute of Child Health and Human Development Fetal Cell Isolation Study (NIFTY) is a prospective, multicenter clinical project to develop non-invasive methods of prenatal diagnosis. The initial objective was to assess the utility of fetal cells in the peripheral blood of pregnant women to diagnose or screen for fetal chromosome abnormalities. Results of fluorescence in situ hybridization (FISH) analysis on interphase nuclei of fetal cells recovered from maternal blood were compared to metaphase karyotypes of fetal cells obtained by amniocentesis or chorionic villus sampling (CVS). After the first 5 years of the study we performed a planned analysis of the data. We report here the data from 2744 fully processed pre-procedural blood samples; 1292 samples were from women carrying singleton male fetuses. Target cell recovery and fetal cell detection were better using magnetic-based separation systems (MACS) than with flow-sorting (FACS). Blinded FISH assessment of samples from women carrying singleton male fetuses found at least one cell with an X and Y signal in 41.4% of cases (95% CI: 37.4%, 45.5%). The false-positive rate of gender detection was 11.1% (95% CI: 6.1,16.1%). This was higher than expected due to the use of indirectly labeled FISH probes in one center. The detection rate of finding at least one aneuploid cell in cases of fetal aneuploidy was 74.4% (95% CI: 76.0%, 99.0%), with a false-positive rate estimated to be between 0.6% and 4.1%. The sensitivity of aneuploidy detection using fetal cell analysis from maternal blood is comparable to single marker prenatal serum screening, but technological advances are needed before fetal cell analysis has clinical application as part of a multiple marker method for non-invasive prenatal screening. The limitations of the present study, i.e. multiple processing protocols, are being addressed in the ongoing study.
Rare fetal cells can be recovered from maternal blood, which suggests that non-invasive prenatal ... more Rare fetal cells can be recovered from maternal blood, which suggests that non-invasive prenatal diagnosis is possible. However, recovery and analysis of fetal cells from blood is complex, and sensitivity is low because of the rarity of these cells in the maternal circulation. An alternative strategy, which suggested that intact fetal cells can be found in maternal plasma by use of simple enrichment methods, has been reported. We aimed to replicate this technique. However, five independent laboratories were unable to identify any intact male cells from the plasma of 38 women known to be carrying male fetuses. Although apoptotic intact fetal cells could contribute to the detection of fetal DNA in maternal plasma, we believe that recovery of these cells is difficult and not clinically practical.
These studies were approved by the committee for the protection of human subjects at the Universi... more These studies were approved by the committee for the protection of human subjects at the University of Texas at Houston and by the institutional review board for human subjects research at Baylor College of Medicine. All subjects provided informed consent.
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Papers by Dorothy Lewis