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David T Harris

Publications and source records attributed to David T Harris.

At least 19 recordsLinked to original sources
PubMed2006

Differential HIV-1 replication in neonatal and adult blood mononuclear cells is influenced at the level of HIV-1 gene expression.

The majority of HIV-1-infected neonates and infants have a higher level of viremia and develop AIDS more rapidly than infected adults, including differences seen in clinical manifestations. To determine the mechanisms of HIV-1 infection in neonates vs. adults, we compared the replication kinetics of HIV-1 in neonatal (cord) and adult blood T lymphocytes and monocyte-derived macrophages (MDM) from seven different donors. We found that HIV-1 replicated 3-fold better in cord blood T lymphocytes compared with adult blood T lymphocytes and 9-fold better in cord MDM than adult MDM. We also show that this differential HIV-1 replication did not depend on differences in cell proliferative capabilities, cell surface expression of CD4, CXCR4, and CCR5, or in the amount of PCR products of reverse transcription, DNA synthesis, and translocation of preintegration complex into the nucleus in cord and adult T lymphocytes and MDM. Furthermore, using a single-cycle replication competent HIV-1-NL4-3-Env(-) luciferase amphotropic virus, which measures HIV-1 transcriptional activity independent of receptor and coreceptor expression, we found there was a 3-fold increase of HIV-1 LTR-driven luciferase expression in cord T lymphocytes compared with adult T lymphocytes and 10-fold in cord MDM than in adult MDM. The HIV-1 LTR-driven luciferase expression correlated with HIV-1 LTR transcription, as measured by ribonuclease protection assay. These data suggest that the increased replication of HIV-1 in cord blood compared with adult blood mononuclear cells is regulated at the level of HIV-1 gene expression, resulting in a higher level of viremia and faster disease progression in neonates than adults.

Adult↗
PubMed2006

Heat-inducible amplifier vector for high-level expression of granulocyte-macrophage colony-stimulating factor.

PURPOSE: In cytokine immunotherapy of cancer it is critical to deliver sufficiently high local cytokine concentrations in order to reach the therapeutic threshold needed for clinical efficacy. Simultaneously, for optimal clinical safety adverse effects caused by high systemic cytokine levels must be minimized. One of the most promising anti-cancer therapeutic cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF), has elicited anti-tumour immune responses in animal studies and clinical trials. However, the clinical efficacy has been limited, with local GM-CSF levels being therapeutically insufficient and systemic toxicity being a limiting factor. METHODS: To address these problems we have developed a novel GM-CSF expression vector, pAD-HotAmp-GM-CSF, which can provide high levels of GM-CSF expression, and induction of cytokine expression to limited tissue areas. This expression system combines inducible and amplifying elements in a single multi-genic construct. The first transcriptional unit contains the inducible element, the heat shock protein 70B (HSP70B) promoter that regulates expression of the transcription-activating factor tat. RESULTS: Upon the binding of tat to the second promoter, the HIV2 long terminal repeat amplifies downstream gene expression of the therapeutic cytokine GM-CSF. Moderate hyperthermia at 42 degrees C for 30 min induced GM-CSF expression in pAD-HotAmp-GM-CSF that was over 2.5- and 2.8-fold higher than levels reached with HSP70B promoter alone and the prototypical human cytomegalovirus promoter. CONCLUSIONS: Thus, the inducible amplifier vector, pAD-HotAmp-GM-CSF, represents a novel system for regulated and enhanced GM-CSF expression, which enables both greater efficacy and safety in cytokine immunotherapy of cancer.

Animals↗
PubMed2006

Telomere length changes after umbilical cord blood transplant.

BACKGROUND: The establishment of donor-derived hematopoiesis in the recipients of hematopoietic stem cell (HSC) transplants involves extensive proliferation and differentiation of HSCs. Data from long-term survivors of HSC transplants suggest that these transplanted HSCs may experience a debilitating replicative senescence. A significant posttransplant shortening of peripheral blood mononuclear cell (PBMNC) telomeres has been observed in both marrow transplant and peripheral blood progenitor cell transplant recipients. Similar studies have not been performed for umbilical cord blood (UCB) HSC transplants, which might be expected to exhibit increased posttransplant replicative potential due to their inherently greater telomere length. STUDY DESIGN AND METHODS: Blood was obtained from donor-recipient pairs of allogeneic PBHSC transplant and UCB HSC transplant, both before transplant and at follow-up treatments (minimum 1 year after transplant) after engraftment. Telomere restriction fragment length (TRFL) analysis was performed on the blood samples. The mean TRFL and posttransplant changes in the mean TRFL were analyzed. RESULTS: Measurements of telomere lengths in the PBMNCs of transplant patients revealed a significant net decrease in telomere length in all transplant recipients compared with their respective donors. Our results also revealed that the PBMNCs of umbilical cord stem cell transplant patients retain a significantly longer posttransplant telomere length. CONCLUSION: The significantly longer telomeres observed in the allogeneic UCB HSC transplant recipients compared to the allogeneic PBHSC transplant recipients in our study may be indicative of a replicative advantage inherent in the use of UCB HSC for transplant.

Adult↗
PubMed2005

Antigen epitope-expressing cytokines for DNA immunization.

Strategies to enhance the efficacy of DNA vaccination against malignancy remain to be established. In this study, a plasmid expressing a tumor antigen incorporated into the signal peptide of human IL-2 was tested as a DNA vaccine in a murine model system. Results showed that antigen-specific CTL responses were elicited by intramuscular injection of these plasmids. Importantly, compared with a minigene vector expressing the same epitope, the OVA epitope-incorporated, IL-2 expression plasmid vaccination was more effective in protecting mice from OVA-expressing tumor challenge. The improved efficacy appears to result from enhanced antigen presentation as well as the immunostimulatory activity of IL-2. This approach may provide new perspectives in designing cytokine-adjuvant DNA vaccines for clinical applications.

Animals↗
PubMed2005

Immuno-gene therapy of melanoma by tumor antigen epitope modified IFN-gamma.

Cytokine-based vaccines play a major part in tumor immuno-gene therapy. However, down-regulated antigen expression on tumor cells may diminish the immuno-potentiating aspects of cellular vaccines. In this study, we coexpressed a tumor antigen epitope with IFN-gamma in the same gene by replacing the IFN-gamma signal peptide with an antigen epitope-expressing signal peptide. We then investigated the effect of the antigen epitope-incorporated IFN-gamma on the immunotherapy of murine melanoma B16 tumors. Results showed that TRP-2 epitope-expressing IFN-gamma decreased B16 tumorigenicity and enhanced its immunogenicity after gene transfer. Protective immunity against wild type B16 tumors was induced by vaccination with IFN-gamma transiently gene-modified tumor cells. These data suggest that cellular vaccines engineered to express an antigen epitope within an immunostimulatory cytokine could potentiate the immunization effect.

Animals↗
PubMed2005

Differential gene expression profiling of CD34+ CD133+ umbilical cord blood hematopoietic stem progenitor cells.

Umbilical cord blood (CB)-derived primitive hematopoietic stem progenitor cells (HSPC) are a promising source for stem cell-based gene therapy due to the reduced incidence and severity of graftversus- host disease (GVHD) after human leukocyte antigen (HLA)-disparate CB transplantation. Cell-surface markers such as CD34 and CD133 have been used in combination to enrich primitive HSPC for research and clinical applications. To understand the molecular characteristics of the CB HSPC, we compared the global gene expression of freshly isolated CB CD34+ CD133+ cells with their progenies using a cDNA microarray containing 22,000 human cDNA clones printed on a single chip. A total of 139 genes were differentially expressed between CB HSPC and their progenies. These transcripts included a number of known genes that might play roles in key functions of CB HSPC as well as many genes of unknown function. Among the genes showing the greatest differential expression levels in HSPC were: psoriasin 1, CRHBP, HDAC3, MLLT3, HBEX2, SPINK2, c-kit, H2BFQ, CD133, HHEX, TCF4, ALDH1A1, and FHL1. These data provide more information on the molecular phenotype of CB HSPC and may lead to the identification of new genes critical to stem cell function.

AC133 Antigen↗
PubMed2005

Substance P effects on developing thymocytes in hindlimb unloaded rats.

INTRODUCTION: The purpose of this study was to examine the effects of substance P (SP) on the immune system in a condition similar to microgravity. We analyzed immune disturbances caused by subjecting Fischer 344 rats to a 45 degrees antiorthostatic suspension technique, otherwise known as the hindlimb unloading (HU) model. METHODS: Four groups of rats were assigned to either the prone control non-substance P group (P-NSP), prone control substance P group (P-SP), hindlimb unloaded non-substance P (HU-NSP) or the hindlimb unloaded substance P group (HU-SP). SP was administered at 10 ml of a 1 micromol x L(-1) concentration for 15 min x d(-1). HU and SP exposure for all groups lasted 16 d. After 16 d, 500 microl of blood was obtained to assay for both T-cell phenotype and corticosterone (CS) levels. Thymus lobes were excised in order to examine T-cell phenotype. Thymocytes were counted and stained for lymphocyte markers (CD4, CD8, and CD3). An analysis of variance (ANOVA) test was used to determine significance between groups (p < or = 0.05). RESULTS: HU-NSP rats showed a decrease in thymic CD4+CD8 +/- cells from 85.51 +/- 1.9% to 62.06 +/- 1.9% when compared with P-NSP rats. SP reversed these effects and returned CD4+CD8+ cells to control levels (76.60 +/- 1.9%). DISCUSSION: Daily SP treatment was found to reverse the deleterious effects caused by HU and corticosterone in rat thymic immune cells. SP could prove to be an effective means for keeping the immune system functioning at normal levels in microgravity, allowing astronauts to stay in space longer and maintain a more productive immune system.

Animals↗
PubMed2005

A stem cell fusion model of carcinogenesis.

The origin of cancer remains enigmatic. Current models of carcinogenesis based on the gene mutation hypothesis have limitations in explaining many aspects of cancer. We put forward a new model of multistage carcinogenesis and propose that cancer development involves gene mutations and cell fusions. Specifically, cancer can result from a fusion between an "altered" pre-malignant cell and a bone marrow-derived stem cell (BMDSC). "Aneuploidy", which is a hallmark of malignancy, is a direct consequence of this cell fusion. The "stem cell fusion" model explains the remarkable similarities between malignant cells and BMDSC. This model also explains why non-mutagens can be carcinogens, and why non-mutagenic processes, such as wound healing and chronic inflammation, can promote malignant transformation. This model is readily testable. Cancer has been difficult to treat because of tissue heterogeneity and gene instability. However, if the malignant characteristics of cancer cells are derived from BMDSC, new conserved targets such as homing receptors for designing novel therapies may emerge.

Aging↗
PubMed2004

SING: a novel strategy for identifying tumor-specific, CTL-recognized tumor antigens.

Traditional methods for identifying T cell-recognized tumor antigens (Ags) are laborious and time-consuming. In an attempt to simplify the procedure, a novel strategy, SING (SIgnal transduction molecule-mediated, NFAT-controlled, GFP expression) was established as a direct approach for cloning T cell-recognized tumor Ags. In the SING system, a mouse T cell line (BW5147) was transduced with a chimeric H-2Kb construct containing T cell-signaling domains and a green fluorescent protein (GFP) reporter gene under the transcriptional control of nuclear factor of activated T cells (NFAT). The resultant BW5147 cells were named BS cells. This cell line could "sense" TCR stimulation through the T cell-signaling domains after coculture with Ag-specific T cells and then become fluorescent (expressing green fluorescence protein, GFP+) in the presence of Ag peptides. The interaction between BS cells and Ag-specific T cells could be enhanced by addition of costimulatory signals. Currently, BS cells have been optimized to "sense" TCR stimulation after being pulsed with the relevant peptides at concentrations as low as 10(-9) M. Endogenous Ag-expressing BS cells could also become fluorescent after coculture with Ag-specific T cells. Our results provide a proof of principle for using the SING system to directly isolate Ag-expressing BS cells from BS cell repertoires, which are retrovirally transduced with tumor-derived cDNA libraries. Once tumor Ag-marked BS cells are identified, the sequences encoding tumor Ags could be easily retrieved by PCR amplification of the genomic DNA using vector-specific primers.

3T3 Cells↗
PubMed2004

Transgenic TCR expression: comparison of single chain with full-length receptor constructs for T-cell function.

Genetic modification of T lymphocytes with T-cell receptor (TCR) genes provides a novel tool for adoptive immunotherapy. However, the efficiency of full-length TCR (flTCR)-transduced T cells could be limited by factors such as incorrect pairing between exogenous and endogenous TCR chains and downregulation of the CD3 complex. To overcome these hurdles, one promising strategy is to use three-domain single-chain TCRs (3D-scTCR), in which TCR Valpha and Vbeta chains are joined by a linker with signal transduction domains fused at the carboxyl termini as signal transducers and amplifiers. Our results showed that surface expression of scTCRs on T cells after retroviral transduction was affected by the origin of the transmembrane (TM) region and placement of signaling domains. scTCR-modified T cells were functional as shown by cytokine (IL-2 and IFN-gamma) release in response to antigen stimulation and cytolytic activity against specific target cells. CD8 and CD28, but not the complete CD3 complex, could enhance the scTCR-induced T cell activation. Compared with flTCR-modified T cells and native CTLs, scTCR-modified T cells require higher thresholds of antigen stimulation (approximately 10(-8) M peptide) to be functional. Despite the low efficiency of scTCRs, our data provide insight into further improvements in generating efficient scTCRs for in vivo applications.

Animals↗
PubMed2004

A large-scale screen for mutagen-sensitive loci in Drosophila.

In a screen for new DNA repair mutants, we tested 6275 Drosophila strains bearing homozygous mutagenized autosomes (obtained from C. Zuker) for hypersensitivity to methyl methanesulfonate (MMS) and nitrogen mustard (HN2). Testing of 2585 second-chromosome lines resulted in the recovery of 18 mutants, 8 of which were alleles of known genes. The remaining 10 second-chromosome mutants were solely sensitive to MMS and define 8 new mutagen-sensitive genes (mus212-mus219). Testing of 3690 third chromosomes led to the identification of 60 third-chromosome mutants, 44 of which were alleles of known genes. The remaining 16 mutants define 14 new mutagen-sensitive genes (mus314-mus327). We have initiated efforts to identify these genes at the molecular level and report here the first two identified. The HN2-sensitive mus322 mutant defines the Drosophila ortholog of the yeast snm1 gene, and the MMS- and HN2-sensitive mus301 mutant defines the Drosophila ortholog of the human HEL308 gene. We have also identified a second-chromosome mutant, mus215(ZIII-2059), that uniformly reduces the frequency of meiotic recombination to <3% of that observed in wild type and thus defines a function required for both DNA repair and meiotic recombination. At least one allele of each new gene identified in this study is available at the Bloomington Stock Center.

Alleles↗
PubMed2004

A novel inducible amplifier expression vector for high and controlled gene expression.

Safety and efficacy are both required for successful gene therapy. In this regard, our laboratory has created a novel expression system, pHi-Hot that combines inducible and amplifier strategies in one construct. In pHi-Hot, the first transcriptional unit contains an inducible heat shock protein (hsp70B) promoter controlling the expression of a transcriptional factor, Tat, which transactivates a second promoter, the HIV2 LTR, located downstream on the same construct. The second promoter drives the gene of interest. The magnitude of the amplified second gene expression can be regulated through manipulating the activity of the hsp promoter driving the Tat gene. Using the human interleukin-2 (IL-2) cytokine gene as the reporter gene, we demonstrated that moderate heat shock at 42 degrees C for 30 min, the pHi-Hot vector could achieve high gene expression levels while maintaining its inducibility. The induced IL-2 levels were 35- to 70-fold higher than achieved by using the hsp promoter alone, and 10- to 35-fold higher than achieved by using the CMV promoter. Using inducible and amplifier strategies, we can achieve high and controlled gene expression levels from a single construct. Finally, we discuss the advantages of using these strategies in developing new targeting and inducible vectors for genetic research and gene therapy.

Animals↗
PubMed2003

Monoclonal anti-idiotypic antibody mimicking the gastrointestinal carcinoma-associated epitope CO17-1A elicits antigen-specific humoral and cellular immune responses in colorectal cancer patients.

Monoclonal rat anti-idiotypic antibody (Ab2) BR3E4 mimicking the colorectal carcinoma (CRC)-associated epitope CO17-1A induced antigen-specific humoral and cellular immune responses in mice and rabbits. Ab2 BR3E4 was administered in a phase I trial to CRC patients either as intact IgG or as F(ab')(2) coupled to keyhole limpet hemocyanin (KLH). There was a trend for the F(ab')(2)-KLH-immunized patients to show higher immune response rates (18/21 and 5/15 patients with anti-anti-idiotypic antibodies and T cells, respectively) than the IgG-immunized patients (15/23 and 3/15 patients positive). Clinical responses were rare in these patients with liver metastases.

Antibodies, Anti-Idiotypic↗
PubMed2003

Enhanced tumor immunogenicity through coupling cytokine expression with antigen presentation.

The density of tumor antigen in conjunction with major histocompatibility complex (MHC) class I molecules on the cell surface affects cytotoxic T cell (CTL) function in an active antitumor immune response. Thus, methods to enhance antigen expression/presentation could augment the effect of cancer immune therapy. In the present study, we investigated the feasibility of modifying a cytokine signal peptide with a tumor antigenic epitope. We inserted the genes encoding the MHC class I-restricted antigenic epitope of chicken ovalbumin and tyrosinase-related protein 2 into the signal sequence of the interleukin-2 gene, replacing part of the signal sequence at different positions. Our results showed that these modified signal peptides still functioned, as indicated by cytokine secretion. The antigenic epitope within the modified signal peptide could be processed properly and presented on tumor cell surface. Tumor cells demonstrated enhanced immunogenicity as indicated by increased susceptibility to CTL lysis in vitro and decreased tumor grow in vivo after gene modification. These data provide potential perspectives in designing therapeutic or vaccine strategies in immuno-gene therapy of cancer.

Amino Acid Sequence↗
PubMed2003

Efficient transduction of murine primary T cells requires a combination of high viral titer, preferred tropism, and proper timing of transduction.

Retroviral vectors have been used exclusively for genetic modification of primary T cells. Most T cell infection protocols have been developed for human T cells, whereas systematic investigations of the optimal conditions for transduction of murine primary T cells are limited. In this study, ecotropic and 10A1-pseudotyped retroviral vectors were compared for their efficiency in infecting murine primary T cell cells, as well as T cell lines. Various factors that affect transduction efficiency were also explored, including virus titer, times of exposure, timing of infection, low-speed centrifugation, and use of fibronectin fragment. Our results showed that up to 80% of murine primary T cells could be infected after a single exposure. Successful infection required a combination of high virus titer (>10(7) CFU/ml), proper timing of infection (within 24 h after mitogen stimulation), and preferred tropism (ecotropic vectors). These optimization results may help to establish a standard protocol for infection of murine primary T cells and provide some insight into the obstacles to retroviral infection of T cells.

Animals↗
PubMed2002

JP-8 jet fuel exposure results in immediate immunotoxicity, which is cumulative over time.

The US Air Force has implemented the widespread use of JP-8 jet fuel in its operations, although a thorough understanding of its potential effects upon exposed personnel is unclear. In the present study, the immediate effects of JP-8 exposure on the immune system were analyzed. Exposure of mice once to a single 1000 mg/m3 concentration of JP-8 for one hour resulted in significant immune organ weight loss and loss of viable immune cells from the spleen within two hours post-exposure. Although a similar exposure had no effect on thymus organ weight, it did result in significant losses of viable immune cells at one hour post-exposure. It was also observed that a loss of viable bone marrow cells could be seen at four hours post-exposure, with a return to baseline levels by 24 hours post-exposure. In terms of peripheral blood immune cells, a significant loss of viable immunecells was observed within one hour post-exposure, which became more pronounced with time. Further, it was observed that a single one-hour JP-8 exposure resulted in an immediate loss of immune function at one hour post-exposure that did not recover within 24 hours. An extension of the above experiments revealed that each additional one hour/day of exposure to 1000 mg/m3 of JP-8 promulgates the significant immunotoxicity described above. That is, spleenic organ weights, as well as viable cell numbers, continued to decline with additional days of short-term exposure. Thymic organ weights were significantly reduced at three to four days of one-hour exposures, with a continuing loss of viable cell numbers. Significantly, functional immune responses continued to deteriorate with each additional day of JP-8 exposure. Thus, low concentration JP-8 jet fuel exposures have significant effects on the immune system, these effects occur rapidly and these effects are cumulative over time.

Animals↗
PubMed2002

Concentrations of estrogens and IGFs in umbilical cord blood plasma: a comparison among Caucasian, Hispanic, and Asian-American females.

It has been hypothesized that exposure to elevated levels of estrogens and IGFs before birth may increase breast cancer risk in female offspring. We examined whether the concentrations of estrone, E2, IGF-I, IGF-II, and IGF-binding protein-1 (IGFBP-1), -2, and -3 in umbilical cord blood plasma differed in female neonates of three racial/ethnic groups with contrasting breast cancer risk. The study included 57 Caucasian, 22 Hispanic, and 22 Asian-American subjects. Relative contribution of race/ethnicity to the analyte level variability was the largest for IGFBP-1 (P = 0.06). The only statistically significant (P < 0.05) mean difference was the lower IGFBP-3 levels in Asian than in Caucasian subjects. Adjusted mean levels of estrone and E2 for Asian subjects were 128% and 109% of the Caucasian means, respectively, whereas the Hispanic group showed lower means (85% and 84% of the Caucasian means). IGF-I, IGFBP-1, and IGFBP-3 showed lower adjusted means for both Hispanics and Asians compared with Caucasians. However, these differences were not statistically significant. In summary, we have shown that concentrations of estrogens, IGF-I, IGF-II, and IGFBPs are not different in cord blood samples from Caucasian, Hispanic, and Asian-American subjects. These data do not support a link between antenatal exposure to elevated levels of estrogens and IGFs and breast cancer.

Adult↗