PubMed Health⌕ Search

Biomedical subjects

R Scollay

Publications and source records attributed to R Scollay.

At least 19 recordsLinked to original sources
PubMed2003

Enhanced systemic transgene expression after nonviral salivary gland transfection using a novel endonuclease inhibitor/DNA formulation.

Gene transfer to the major salivary glands is an attractive method for the systemic delivery of therapeutic proteins. To date, nonviral gene transfer to these glands has resulted in inadequate systemic protein concentrations. We believe that identification of the barriers responsible for this inefficient transfection will enable the development of enhanced nonviral gene transfer in salivary glands and other tissues. One potential barrier is the degradation of plasmid DNA by endonucleases. To test this hypothesis, we coadministered two endonuclease inhibitors ((zinc and aurintricarboxylic acid (ATA)) with plasmid DNA, containing the secreted alkaline phosphatase gene (SEAP), to the submandibular glands of rats. The effect of zinc and ATA on SEAP expression, tissue accumulation of plasmid DNA, and plasmid DNA stability was then characterized. We observed that mixtures containing zinc/DNA, ATA/DNA, and zinc/ATA/DNA significantly enhanced both systemic transgene expression and the amount of plasmid DNA associated with treated tissues. The relative endonuclease inhibitory activity of zinc, ATA, and zinc/ATA correlated with the observed effects on transfection efficacy. The use of zinc/ATA enhanced the efficacy of salivary gland transfection by at least 1000-fold versus DNA alone. Importantly, this improved performance resulted in robust systemic secretion of an exogenous protein (SEAP), thus demonstrating the potential this nonviral gene transfer technology has as a method to treat systemic protein deficiencies.

Animals↗
PubMed2001

Gene therapy: a brief overview of the past, present, and future.

Gene therapy has only recently begun to make serious progress, beginning with two approved gene therapy trials in the United States in late 1990. The death of an 18-year-old man participating in a gene therapy trial delivered a major setback in terms of public concerns, but the resulting improvements in scrutiny of trial design and ethical standards will benefit the field in the long run. The three main issues for the coming decade will be public perceptions, scale-up and manufacturing, and commercial considerations. Focusing on single-gene applications, which tend to be rarer diseases, will produce successful results sooner than the current focus on the commoner, yet more complex, cancer and heart disease.

Clinical Trials as Topic↗
PubMed2001

The effect of antigen stimulation on the migration of mature T cells from the peripheral lymphoid tissues to the thymus.

Although the maturation and export of T cells from the thymus has been extensively studied, the movement of cells in the opposite direction has been less well documented. In particular, the question of whether T cells which have been activated by antigen in the periphery are more likely to return to the thymus had been raised but not clearly answered. We examined this issue by activating T cells present in the periphery with their cognate antigen, and assessing migration to the thymus. TCR-transgenic cells from OT-I mice (Thy1.2+), which recognise the ovalbumin peptide OVA257-264 in the context of H-2Kb, were transferred into otherwise unmanipulated Thy1.1+ C57BL/6 mice. Recipient mice were injected i.v. with 5 microg peptide (SIINFEKL) approximately 24 hours later. The numbers of donor-derived (Thy1.2+) cells in the thymus and peripheral lymphoid tissue were determined. The results clearly show increased numbers of transgenic cells in the thymus 3 days after antigenic stimulation. However, since numbers of transgenic cells increased in the spleen and LN in about the same proportion, the data do not support the notion that there is highly increased selective migration of activated T cells to the thymus. Rather, they suggest that a sample of peripheral cells enters the thymus each day, and that the mature immigrants detected in the thymus merely reflect the contents of the peripheral T cell pool.

Animals↗
PubMed2000

Long-term multilineage expression in peripheral blood from a Moloney murine leukemia virus vector after serial transplantation of transduced bone marrow cells.

Using a mouse bone marrow transplantation model, the authors evaluated a Moloney murine leukemia virus (MMLV)-based vector encoding 2 anti-human immunodeficiency virus genes for long-term expression in blood cells. The vector also encoded the human nerve growth factor receptor (NGFR) to serve as a cell-surface marker for in vivo tracking of transduced cells. NGFR(+) cells were detected in blood leukocytes of all mice (n=16; range 16%-45%) 4 to 5 weeks after transplantation and were repeatedly detected in blood erythrocytes, platelets, monocytes, granulocytes, T cells, and B cells of all mice for up to 8 months. Transgene expression in individual mice was not blocked in the various cell lineages of the peripheral blood and spleen, in several stages of T-cell maturation in the thymus, or in the Lin(-/lo)Sca-1(+) and c-kit(+)Sca-1(+) subsets of bone marrow cells highly enriched for long-term multilineage-reconstituting activity. Serial transplantation of purified NGFR(+)c-kit(+)Sca-1(+) bone marrow cells resulted in the reconstitution of multilineage hematopoiesis by donor type NGFR(+) cells in all engrafted mice. The authors concluded that MMLV-based vectors were capable of efficient and sustained transgene expression in multiple lineages of peripheral blood cells and hematopoietic organs and in hematopoietic stem cell (HSC) populations. Differentiation of engrafting HSC to peripheral blood cells is not necessarily associated with dramatic suppression of retroviral gene expression. In light of earlier studies showing that vector elements other than the long-terminal repeat enhancer, promoter, and primer binding site can have an impact on long-term transgene expression, these findings accentuate the importance of empirically testing retroviral vectors to determine lasting in vivo expression.

Animals↗
PubMed1999

Development of T lymphocytes at extrathymic sites.

T lymphocytes expressing both CD4 and CD8 are the predominant cell type in the thymic cortex but are extremely rare outside the thymus of normal mice. In this article, we show that if precursor thymocytes (CD4-CD8-) from fetal or adult donors are injected i.v. into irradiated recipients, some of these cells will lodge in lymph nodes and develop into both CD4+CD8+ (double-positive) and CD4+ or CD8+ (single-positive) cells. This phenomenon also occurred in thymectomized recipients, strongly suggesting it is genuine extrathymic development. Prethymic precursors (e.g., fetal liver), were unable to use the lymph node for T cell development, without thymic processing. The data suggest that given unusual circumstances (irradiation or thymectomy and availability of appropriate precursors), the lymph nodes can support T cell development.

Animals↗
PubMed1999

NKT cells are phenotypically and functionally diverse.

NK1.1(+)alpha betaTCR(+) (NKT) cells have several important roles including tumor rejection and prevention of autoimmune disease. Although both CD4(+) and CD4(-)CD8(-) double-negative (DN) subsets of NKT cells have been identified, they are usually described as one population. Here, we show that NKT cells are phenotypically, functionally and developmentally heterogeneous, and that three distinct subsets (CD4(+), DN and CD8(+)) are differentially distributed in a tissue-specific fashion. CD8(+) NKT cells are present in all tissues but the thymus, and are highly enriched for CD8alpha(+)beta(-) cells. These subsets differ in their expression of a range of cell surface molecules (Vbeta8, DX5, CD69, CD45RB, Ly6C) and in their ability to produce IL-4 and IFN-gamma, with splenic NKT cell subsets producing lower levels than thymic NKT cells. Developmentally, most CD4(+) and DN NKT cells are thymus dependent, in contrast to CD8(+) NKT cells, and are also present amongst recent thymic emigrants in spleen and liver. TCR Jalpha281-deficient mice show a dramatic deficiency in thymic NKT cells, whereas a significant NKT cell population (enriched for the DN and CD8(+) subsets) is still present in the periphery. Taken together, this study reveals a far greater level of complexity within the NKT cell population than previously recognized.

Animals↗
PubMed1999

CD34+ cells from mobilized peripheral blood retain fetal bone marrow repopulating capacity within the Thy-1+ subset following cell division ex vivo.

Ex vivo cell cycling of hematopoietic stem cells (HSC), a subset of primitive hematopoietic progenitors (PHP) with engrafting capacity, is required for transduction with retroviral vectors and to increase transplantable HSC numbers. However, induction of division of HSC ex vivo also may lead to differentiation and loss of in vivo marrow repopulating potential. We evaluated mobilized peripheral blood (MPB) PHP for maintenance of stem cell function after ex vivo culture under conditions that we show can induce cycling of a majority of PHP with minimal differentiation. The following methods were combined: cell labeling with the division tracking dye carboxyfluorescein-diacetate succinimidylester (CFSE), analysis of primitive cell surface marker expression, an ex vivo PHP assay, and an in vivo marrow repopulating assay. MPB-purified CD34+ Thy-1+ cells were labeled with CFSE dye and cultured for 112 hours in serum-deprived medium in the presence of the cytokine combinations of thrombopoietin (TPO), flt3 ligand (FL), and c-kit ligand (KL), or TPO, FL, and interleukin 6 (IL-6). Both cytokine combinations supported division of greater than 95% of cells within 112 hours with an average 2.1-fold (TPO, FL, KL) or 1.3-fold (TPO, FL, IL-6) increase in total cell numbers. An average of 21.6% (TPO, FL, KL) and 27.4% (TPO, FL, IL-6) of the divided cells still expressed the Thy-1 marker after 112 hours. Functional assays were performed to compare cultured and uncultured cells. CD34+ Thy-1+ CFSElo (post division) cells showed maintenance of cobblestone area-forming cell (CAFC) frequency (a mean of 1/9.0) relative to the starting population of uncultured CD34+ Thy-1+ cells (a mean of 1/8.4). In contrast, CD34+ cells that had lost Thy-1 expression during culture (CD34+ Thy-1 CFSElo) showed a mean 5.8-fold reduction in CAFC frequency (a mean of 1/52.5). Only the Thy-1-expressing fraction of cells post culture could engraft in vivo in the SCID-hu bone assay. Because the majority of HSC functional activity post culture was found in the CD34+ Thy-1+ fraction, we focused on this fraction for subsequent analysis. CFSE labeling allows segregation and purification by flow cytometry of cells having undergone discrete numbers of divisions during culture. Very few cells that divided more than four times in culture still expressed Thy-1. Cells that retained expression of Thy-1 during culture retained CAFC activity relative to fresh CD34+ Thy-1+ cells, after undergoing at least two divisions. CAFC frequency decreased after four divisions in culture with TPO, FL, and KL or after three divisions in TPO, FL, and IL-6. We then compared populations of Thy-1+ cells that had undergone sequential numbers of divisions in culture for their ability to engraft in the SCID-hu bone assay. Engrafting ability was retained throughout four divisions in both cytokine combinations. These data demonstrate that primitive MPB CD34+ cells maintain HSC function coincident with Thy-1 expression while undergoing two to four divisions under these culture conditions. Essentially all CD34+ Thy-1+ cells divided under the conditions tested, promoting susceptibility to retroviral transduction.

Animals↗
PubMed1999

Thrombopoietin, flt3, and kit ligands together suppress apoptosis of human mobilized CD34+ cells and recruit primitive CD34+ Thy-1+ cells into rapid division.

Various combinations of cytokines have profoundly different effects on inhibition of apoptosis and stimulation of self-renewal division of hematopoietic stem cells (HSC) in short-term, ex vivo culture. Our goal was to quantitate expansion of cells with a primitive CD34+ Thy-1+ phenotype, as well as cell cycling, division history, differentiation, and apoptosis of CD34+ cells enriched from normal donor mobilized peripheral blood (MPB) cells. The balance of these parameters determines the net number of transplantable HSC produced in ex vivo cultures. Comparing several different combinations of cytokines added to 90-hour cultures of MPB CD34 cells, thrombopoietin (TPO), flt3 ligand (FL), and c-kit ligand (KL) gave the best result, with the lowest percentage of apoptotic cells and a mean 1.2-fold increase in the number of CD34+ Thy-1+ cells. A combination of interleukin 3 (IL-3), interleukin 6 (IL-6), and leukemia inhibitory factor (LIF) gave the worst outcome, including a decrease of CD34+ Thy-1+ cell number to a mean of 30% of the starting cell number. Cell division history was tracked using the dye 5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE). Division of CD34+ Thy-1+ cells was faster and more synchronous in TPO, FL, and KL than in IL-3, IL-6, and LIF, which left a significant proportion of CD34+ cells undivided. Such detailed analyses of short-term, ex vivo cultures generated "replication scores," which allowed prediction of a sixfold improvement of the efficiency of gene transduction of primitive hematopoietic progenitors from MPB, using TPO, FL, and KL to replace IL-3, IL-6, and LIF. Analysis of retroviral transduction efficiency confirmed the increase of transgene expression from MPB primitive hematopoietic progenitors assayed after stromal culture was fivefold, validating the usefulness of multiparameter analysis of short-term cultures for survival and replication of CD34+ Thy-1+ cells.

Antigens, CD34↗
PubMed1998

HIV, but not murine leukemia virus, vectors mediate high efficiency gene transfer into freshly isolated G0/G1 human hematopoietic stem cells.

Recent studies have opened the possibility that quiescent, G0/G1 hematopoietic stem cells (HSC) can be gene transduced; lentiviruses (such as HIV type 1, HIV) encode proteins that permit transport of the viral genome into the nucleus of nondividing cells. We and others have recently demonstrated efficient transduction by using an HIV-1-based vector gene delivery system into various human cell types including human CD34(+) cells or terminally differentiated neurons. Here we compare the transduction efficiency of two vectors, HIV-based and murine leukemia virus (MuLV)-based vectors, on untreated and highly purified human HSC subsets that are virtually all in G0/G1. The HIV vector, but not MuLV vector supernatants, transduced freshly isolated G0/G1 HSC from mobilized peripheral blood. Single-step transduction using replication-defective HIV resulted in HSC that expressed the green fluorescent protein (GFP) transgene while retaining their stem cell phenotype; clonal outgrowths of these GFP+ HSC on bone marrow stromal cells fully retained GFP expression for at least 5 weeks. MuLV-based vectors did not transduce resting HSC, as measured by transgene expression, but did so readily when the HSC were actively cycling after culture in vitro for 3 days in a cytokine cocktail. These results suggest that resting HSC may be transduced by lentiviral-based, but not MuLV, vectors and maintain their primitive phenotype, pluripotentiality, and at least in vitro, transgene expression.

Antigens, CD34↗
PubMed1998

Sustained gene expression in retrovirally transduced, engrafting human hematopoietic stem cells and their lympho-myeloid progeny.

Inefficient retroviral-mediated gene transfer to human hematopoietic stem cells (HSC) and insufficient gene expression in progeny cells derived from transduced HSC are two major problems associated with HSC-based gene therapy. In this study we evaluated the ability of a murine stem cell virus (MSCV)-based retroviral vector carrying the low-affinity human nerve growth factor receptor (NGFR) gene as reporter to maintain gene expression in transduced human hematopoietic cells. CD34(+) cells lacking lineage differentiation markers (CD34(+)Lin-) isolated from human bone marrow and mobilized peripheral blood were transduced using an optimized clinically applicable protocol. Under the conditions used, greater than 75% of the CD34(+) cell population retained the Lin- phenotype after 4 days in culture and at least 30% of these expressed a high level of NGFR (NGFR+) as assessed by fluorescence-activated cell sorter analysis. When these CD34(+)Lin-NGFR+ cells sorted 2 days posttransduction were assayed in vitro in clonogenic and long-term stromal cultures, sustained reporter expression was observed in differentiated erythroid and myeloid cells derived from transduced progenitors, and in differentiated B-lineage cells after 6 weeks. Moreover, when these transduced CD34(+)Lin-NGFR+ cells were used to repopulate human bone grafts implanted in severe combined immunodeficient mice, MSCV-directed NGFR expression could be detected on 37% +/- 6% (n = 5) of the donor-type human cells recovered 9 weeks postinjection. These findings suggest potential utility of the MSCV retroviral vector in the development of effective therapies involving gene-modified HSC.

Animals↗
PubMed1998

High doses of purified stem cells cause early hematopoietic recovery in syngeneic and allogeneic hosts.

In humans, autologous transplants derived from bone marrow (BM) usually engraft more slowly than transplants derived from mobilized peripheral blood. Allogeneic BM transplants show a further delay in engraftment and have an apparent requirement for donor T cells to facilitate engraftment. In mice, Thy-1.1(lo)Lin-/loSca-1+ hematopoietic stem cells (HSCs) are the principal population in BM which is responsible for engraftment in syngeneic hosts at radioprotective doses, and higher doses of HSCs can radioprotect an allogeneic host in the absence of donor T cells. Using the mouse as a preclinical model, we wished to test to what extent engraftment kinetics was a function of HSC content, and whether at high doses of c-Kit+Thy-1.1(lo)Lin-/loSca-1+ (KTLS) cells rapid allogeneic engraftment could also be achieved. Here we demonstrate that engraftment kinetics varied greatly over the range of KTLS doses tested (100-10,000 cells), with the most rapid engraftment being obtained with a dose of 5,000 or more syngeneic cells. Mobilized splenic KTLS cells and the rhodamine 123(lo) subset of KTLS cells were also able to engraft rapidly. Higher doses of allogeneic cells were needed to produce equivalent engraftment kinetics. This suggests that in mice even fully allogeneic barriers can be traversed with high doses of HSCs, and that in humans it may be possible to obtain rapid engraftment in an allogeneic context with clinically achievable doses of purified HSCs.

Animals↗
PubMed1998

Thrombopoietin, kit ligand, and flk2/flt3 ligand together induce increased numbers of primitive hematopoietic progenitors from human CD34+Thy-1+Lin- cells with preserved ability to engraft SCID-hu bone.

CD34(+)Thy-1(+)Lin- cells are enriched for primitive hematopoietic progenitor cells (PHP), as defined by the cobblestone area-forming cell (CAFC) assay, and for bone marrow (BM) repopulating hematopoietic stem cells (HSC), as defined by the in vivo SCID-hu bone assay. We evaluated the effects of different cytokine combinations on BM-derived PKH26-labeled CD34(+)Thy-1(+)Lin- cells in 6-day stroma-free cultures. Nearly all (>95%) of the CD34(+)Thy-1(+)Lin- cells divided by day 6 when cultured in thrombopoietin (TPO), c-kit ligand (KL), and flk2/flt3 ligand (FL). The resulting CD34(hi) PKHlo (postdivision) cell population retained a high CAFC frequency, a mean 3.2-fold increase of CAFC numbers, as well as a capacity for in vivo marrow repopulation similar to freshly isolated CD34(+)Thy-1(+)Lin- cells. Initial cell division of the majority of cells occurred between day 2 and day 4, with minimal loss of CD34 and Thy-1 expression. In contrast, cultures containing interleukin-3 (IL-3), IL-6, and leukemia inhibitory factor contained a mean of 75% of undivided cells at day 6. These CD34(hi) PKHhi cells retained a high frequency of CAFC, whereas the small population of CD34(hi) PKHlo postdivision cells contained a decreased frequency of CAFC. These data suggest that use of a combination of TPO, KL, and FL for short-term culture of CD34(+)Thy-1(+)Lin- cells increases the number of postdivision PHP, measured as CAFC, while preserving the capacity for in vivo engraftment.

Animals↗
PubMed1998

Emigration of mature T cells from the thymus is inhibited by the imidazole-based compound 2-acetyl-4-tetrahydroxybutylimidazole.

The primary role of the thymus is to provide mature T cells for the peripheral immune system. The mechanisms involved in the cellular export processes are as yet unknown. In this study, we examined the ability of 2-acetyl-4-tetrahydroxybutylimidazole (THI), an agent widely used as a component of ammonia caramel food colouring, to inhibit T-cell export from the thymus. BALB/c mice were maintained on drinking water containing THI for 5 days. The mice showed a twofold increase in the total number of mature medullary thymocytes (CD4+CD8- and CD4-CD8+) as well as a slight decrease in the total number of immature double-positive cells (CD4+CD8+). The mature single-positive thymocytes were found to express high levels of the homing molecule L-selectin, suggesting that these potential emigrants were prevented from leaving the thymus. To confirm this, THI-treated mice were injected intrathymically with fluorescein isothiocyanate and the number of labelled T cells appearing in the lymph nodes and spleen was determined 16 hr later. A 10-fold decrease in the number of CD4+ and CD8+ recent thymic emigrants in the lymph nodes and spleen of THI-treated mice was observed. Previous studies have shown that THI does not affect other aspects of thymocyte development, such as proliferation and differentiation. Taken together, these results suggest that the immunosuppressive effects of THI may be due, in part, to preventing of the final step of T-cell export out of the thymus.

Animals↗
PubMed1997

Recent thymic emigrants are distinct from most medullary thymocytes.

In the mouse thymus, newly formed single positive (SP) cells spend an average of 14 days in the thymic medulla. During this time, phenotypic and functional maturation occurs with down-regulation of CD69 and heat stable antigen (HSA), and up-regulation of Qa-2. Very little is known about the final steps that allow or direct these T cells to emigrate and join the recirculating peripheral T cell pool. Currently available data suggest that not all recent thymic emigrants (RTE) complete this maturational sequence in the medulla and that emigration may occur at any time during the medullary maturation stage. In this study, we have compared adhesion and activation marker expression on SP thymocytes, RTE and peripheral T cells to determine more precisely which SP medullary thymocytes are exported. Although RTE were heterogeneous for HSA and Qa-2 expression, they were quite uniform with regard to the expression of other molecules. In contrast to medullary SP thymocytes, most RTE were L-selectin(high) and CD69-. In addition, CD4+ CD8- and CD4- CD8+ RTE were phenotypically distinct from each other in that the former were beta7 integrin(-/low), CD45RB(intermediate) and CD45RC-, while the latter were beta7 integrin(high), CD45RB(high) and CD45RC(low). These phenotypes were comparable to only a minor (as little as 6%) subpopulation of medullary SP thymocytes. Overall, the data indicate that export of cells from the medullary pool of SP thymocytes is not random, but that a series of maturational events within the SP stage are necessary before export can occur.

Animals↗
PubMed1997

Thymic T cell export is not influenced by the peripheral T cell pool.

The peripheral T cell pool is maintained both by export of naive T cells from the thymus and by post-thymic expansion of activated/memory T cells. However, it is not known whether the thymus can alter its output following peripheral T cell depletion. Using intrathymic injection of fluorescein isothiocyanate to detect recent thymic emigrants (RTE), we directly tested whether the thymus is able to alter the number of RTE or the CD4:CD8 ratio of RTE emigrating to the periphery in response to in vivo depletion of total peripheral T cells or CD4 T cells, respectively. Depletion of peripheral T cells was achieved with anti-Thy-1 or anti-CD4, at doses that did not affect thymocyte numbers. Depletion of greater than 70% of peripheral T cells by treatment with anti-Thy-1 in vivo did not alter the number or cell cycle status of RTE trafficking to lymph nodes or spleen during the peripheral reconstitution phase (6, 9, 12 days). Similarly, depletion of the majority of CD4 T cells, which significantly reduced the peripheral CD4:CD8 T cell ratio, did not alter the total number or the proportion of CD4+ CD8- RTE in peripheral lymphoid organs. These data clearly indicate that thymic output is not influenced by downstream alterations in peripheral T cell pool size or CD4:CD8 ratio. Rather we contend that thymic T cell export is internally regulated by as yet undefined mechanisms.

Animals↗
PubMed1997

Specific demethylation of the CD4 gene during CD4 T lymphocyte differentiation.

The expression of CD4 during T cell development is a highly regulated process. Numerous regulatory elements have been identified including a promoter, two distinct enhancers and a silencer. Here we report a methylation site in the first intron of the CD4 gene that is specifically demethylated in cells which have previously, or are currently expressing CD4. In addition, this site becomes progressively demethylated as T lymphocytes differentiate from double-negative to double-positive to CD4 single-positive thymocytes, and finally to CD4 single-positive peripheral T lymphocytes. This specific and progressive demethylation suggests that this site represents another potential control region for the regulation of CD4.

Animals↗
PubMed1997

Stem cell antigen 2 expression in adult and developing mice.

Stem cell antigen 2 (Sca-2) expression can distinguish the most immature T-lymphocyte precursors in the thymus from the hemopoietic stem cells. Sequence analysis of the Sca-2 protein showed that Sca-2 is a glycosylphosphatidylinositol (GPI) anchored molecule that shares some characteristics with the members of the Ly-6 multigene family, and that it is the same as the thymic shared antigen-1 (TSA-1). Here we extend these studies and critically reassess the expression of the Sca-2/TSA-1 antigen in hematopoietic tissues of adult and developing mice. With more sensitive methods we show that the distribution of Sca-2/TSA-1 differs from existing reports. We find especially high expression of Sca-2/TSA1 at day 14 of fetal development.

Animals↗