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M T Little

Publications and source records attributed to M T Little.

17 recordsLinked to original sources
PubMed2001

Engraftment of DLA-haploidentical marrow with ex vivo expanded, retrovirally transduced cytotoxic T lymphocytes.

Genetically modified donor T cells with an inducible "suicide" gene have the potential to improve the safety and availability of allogeneic hematopoietic stem cell transplantation by enhancing engraftment and permitting control of graft-versus-host disease (GVHD). However, several clinical studies of gene-modified T cells have shown limited to no in vivo function of the ex vivo expanded T cells. Using the well-established dog model of allogeneic marrow transplantation, the question was asked if retrovirally transduced, donor derived, ex vivo expanded cytotoxic T lymphocytes (CTLs) that are recipient specific could enhance engraftment of dog leukocyte antigen (DLA)-haploidentical marrow following a single dose of 9.2 Gy total body irradiation and no postgrafting immunosuppression. In this setting, only 4 of 11 control recipients of DLA-haploidentical marrow without added CTLs engrafted. CTLs did not enhance engraftment of CD34(+) selected peripheral blood stem cells. However, recipient-specific CTLs enhanced engraftment of DLA-haploidentical marrow in 9 of 11 evaluable recipients (P =.049). All dogs that engrafted developed multiorgan GVHD. To facilitate in vivo tracking, 8 dogs received CTLs transduced with a retroviral vector encoding green fluorescent protein (GFP) and neomycin phosphotransferase (neo). Recipients that engrafted had sharp increases in the numbers of circulating GFP(+) CTLs on days +5 to +6 after transplantation. GFP(+) CTLs isolated from blood were capable of recipient-specific lysis. At necropsy, up to 7.1% of CD3(+) cells in tissues were GFP(+) and polymerase chain reaction in situ hybridization for neo showed infiltration of transduced CTLs in GVHD-affected organs. These results show that ex vivo expanded, transduced T cells maintained in vivo function and enhanced marrow engraftment.

Animals↗
PubMed2001

Hematopoietic responses to stress conditions in young dogs compared with elderly dogs.

Clinical observations show that older patients do not tolerate high-dose chemoradiotherapy as well as younger patients. It is unclear whether this is due to age-related differences in their responses to hematopoietic injury or to differential toxicities to other organs. In the present study, 6 young (0.5 years) and 6 elderly (8 years) dogs were challenged with 7 repeated nonlethal doses of 50 or 100 cGy total body irradiation (TBI) each (total 550 cGy), and 21 days of recombinant canine granulocyte-colony stimulating factor (rcG-CSF) after the last TBI dose. Recoveries of absolute neutrophil, platelet, and lymphocyte counts after each TBI dose, responses to rcG-CSF treatment, and telomere lengths in neutrophils were compared before and after the study. No differences were found in recoveries of neutrophils, platelets, or in responses to rcG-CSF among young and old dogs. In contrast, recoveries were suggestively worse in younger dogs. After rcG-CSF, platelet recoveries were poor in both groups compared with previous platelet recoveries (P <.01). Consequently, 2 old and 3 young dogs were euthanized because of persistent thrombocytopenia and bleeding. At the study's completion, marrow cellularities and peripheral blood counts of the remaining young and elderly dogs were equivalent. The telomere lengths in both groups were significantly reduced after the study versus beforehand (P =.03), but the median attritions of telomeres were not different. It was concluded that aging does not appear to affect hematopoietic cell recoveries after repeated low-dose TBI, suggesting that poor tolerance of radiochemotherapy regimens in older patients may be due to nonhematopoietic organ toxicities rather than age-related changes in hematopoietic stem cells reserves.

Aging↗
PubMed2001

Effects of extending the duration of postgrafting immunosuppression and substituting granulocyte-colony-stimulating factor-mobilized peripheral blood mononuclear cells for marrow in allogeneic engraftment in a nonmyeloablative canine transplantation model.

Stable mixed donor/host hematopoietic chimerism was uniformly achieved in dogs given 200 cGy total body irradiation (TBI) before and immunosuppression with mycophenolate mofetil (MMF) for 28 days and cyclosporine (CSP) for 35 days after transplantation of marrow from dog leukocyte antigen-identical littermates. When the TBI dose was lowered to 100 cGy, donor marrow engraftment in 6 dogs was only transient, lasting 3 to 12 weeks. In this study, we asked whether stable engraftment in this model could be achieved: (1) by substituting recombinant canine granulocyte-colony-stimulating factor-mobilized peripheral blood mononuclear cells (G-PBMCs) for marrow and (2) by extending CSP administration from 35 to 100 days. Eighteen dogs were given G-PBMC grafts and MMF for 28 days. Eight of the 18 dogs received CSP for 35 days and 10 for 100 days. We found that substituting G-PBMCs for marrow did not increase the incidence of stable allogeneic engraftment (P = .11). However, increasing the duration of posttransplantation immunosuppression with CSP from 35 to 100 days favorably influenced stable donor engraftment (P = .06).

Animals↗
PubMed2001

G-CSF-mobilized peripheral blood mononuclear cells added to marrow facilitates engraftment in nonmyeloablated canine recipients: CD3 cells are required.

Stable mixed donor/host hematopoietic chimerism can be uniformly established in dogs conditioned with 200 cGy TBI before dog leukocyte antigen (DLA)-identical marrow transplantation and immunosuppressed with a short course of mycophenolate mofetil (MMF) and cyclosporine (CSP) after the transplantation. A further decrease in the TBI dose to 100 cGy or the elimination of MMF in this model results in graft rejection. Here we asked whetherthe addition of G-CSF-mobilized peripheral blood mononuclear cells (G-PBMC) to marrow grafts would enhance donor engraftment in dogs conditioned with 100 cGy TBI and given postgrafting immunosuppression with CSP alone. Using this model, 7 of 9 dogs given only marrow cells rejected their grafts within 8 to 17 weeks after transplantation. In contrast, the addition of unmodified G-PBMC to marrow grafts resulted in stable mixed donor/host chimerism in 5 of 8 dogs studied (P = .06). However, addition of the CD3-depleted fraction of G-PBMC, which contained both CD34 cells and CD14 cells, resulted in engraftment in only 1 of 7 recipients. We conclude that adding G-PBMC to marrow grafts replaced the requirement of MMF and 100 cGy of TBI, and that CD3 cells were required to facilitate engraftment of marrow cells in DLA-identical recipients, whereas the additional CD34 cells present in G-PBMC were not sufficient for this effect.

Animals↗
PubMed2001

Nonmyeloablative hematopoietic cell transplantation. Replacing high-dose cytotoxic therapy by the graft-versus-tumor effect.

Conventional allografting produces considerable regimen-related toxicities that generally limit this treatment to patients younger than 55 years and in otherwise good medical condition. T cell-mediated graft-versus-tumor (GVT) effects are known to play an important role in the elimination of malignant disease after allotransplants. A minimally myelosuppressive regimen that relies on immunosuppression for allogeneic engraftment was developed to reduce toxicities while optimizing GVT effects. Pre-transplant total-body irradiation (200 cGy) followed by post-transplant immunosuppression with cyclosporine (CSP) and mycophenolate mofetil (MMF) permitted human leukocyte antigen (HLA)-matched sibling donor hematopoietic cell engraftment in 82% of patients (n = 55) without prior high-dose therapy. The addition of fludarabine (90 mg/m2) facilitated engraftment in all 28 subsequent patients. Overall, fatal progression of underlying disease occurred in 20% of patients after transplant. Non-relapse mortality occurred in 11% of patients. Toxicities were low. Grade 2-4 acute graft-versus-host disease (GVHD) associated with primary engraftment developed in 47% of patients, and was readily controlled in all but two patients. Donor lymphocyte infusions (DLI) were not very effective at converting a low degree of mixed donor/host chimerism to full donor chimerism; however, the addition of fludarabine reduced the need for DLI. With a median follow-up of 244 days, 68% of patients were alive, with 42% of patients in complete remission, including molecular remissions. Remissions occurred gradually over periods of weeks to a year. If long-term efficacy is demonstrated, such a strategy would expand treatment options for patients who would otherwise be excluded from conventional allografting.

Adult↗
PubMed2000

Analysis of the role of AML1-ETO in leukemogenesis, using an inducible transgenic mouse model.

As reported previously, AML1-ETO knock-in mice were generated to investigate the role of AML1-ETO in leukemogenesis and to mimic the progression of t(8;21) leukemia. These knock-in mice died in midgestation because of hemorrhaging in the central nervous system and a block of definitive hematopoiesis during embryogenesis. Therefore, they are not a good model system for the development of acute myeloid leukemia. Therefore, mice were generated in which the expression of AML1-ETO is under the control of a tetracycline-inducible system. Multiple lines of transgenic mice have been produced with the AML1-ETO complementary DNA controlled by a tetracycline-responsive element. In the absence of the antibiotic tetracycline, AML1-ETO is strongly expressed in the bone marrow of AML1-ETO and tet-controlled transcriptional activator double-positive transgenic mice. Furthermore, the addition of tetracycline reduces AML1-ETO expression in double-positive mice to nondetectable levels. Throughout the normal murine lifespan of 24 months, mice expressing AML1-ETO have not developed leukemia. In spite of this, abnormal maturation and proliferation of progenitor cells have been observed from these animals. These results demonstrate that AML1-ETO has a very restricted capacity to transform cells. Either the introduction of additional genetic changes or the expression of AML1-ETO at a particular stage of hematopoietic cell differentiation will be necessary to develop a model for studying the pathogenesis of t(8;21).

Acute Disease↗
PubMed2000

Cloning and characterization of a novel human ubiquitin-specific protease, a homologue of murine UBP43 (Usp18).

The ubiquitin-specific proteases (UBP) are a family of enzymes that cleave ubiquitin from ubiquitinated protein substrates. We have recently cloned UBP43, a novel member of this family from AML1-ETO knock-in mice. To analyze the role of UBP43 in hematopoiesis and leukemogenesis, we have cloned a full-length human UBP43 cDNA by screening a human monocytic cDNA library as well as by 5'- and 3'-rapid amplification of cDNA ends analyses. This cDNA encodes a polypeptide of 372 amino acids with all of the structural motifs of a deubiquitinating enzyme. The human UBP43 mRNA is strongly expressed in human liver and thymus. Transfection analysis has demonstrated that UBP43 is a nuclear protein. Interestingly, the gene encoding human UBP43 maps to chromosome 22q11.2. This region, known as DiGeorge syndrome critical region, contains a minimal area of 2 Mb and is consistently deleted in DiGeorge syndrome and related disorders. The syndrome is marked by thymic aplasia or hypoplasia, parathyroid hypoplasia, or congenital cardiac abnormalities. Taken together, our results broaden the understanding of a new human ubiquitin-specific protease, UBP43, and suggest that this gene may also be related to DiGeorge syndrome.

Amino Acid Sequence↗
PubMed1999

Involvement of the adapter protein CRKL in integrin-mediated adhesion.

CRKL, an SH2-SH3-SH3 adapter protein, is one of the major tyrosine phosphoproteins detected in primary leukemic neutrophils from patients with CML. CRKL binds directly to BCR/ABL through its N-terminal SH3 domain, suggesting it may be involved in BCR/ABL signal transduction. However, the biological function of CRKL in either normal or leukemic cells is still largely unknown. In this study, we have examined the effects of overexpressing full length or deletion mutants of CRKL in hematopoietic cell lines. Full length, SH2- and SH3(N)-domain deletion mutants of CRKL were transfected into an interleukin-3-dependent hematopoietic cell line, Ba/F3, and 3-5 individual sublines which stably overexpressed each transgene were obtained [Ba/F-CRKL, Ba/F-CRKL deltaSH2, and Ba/F-CRKL deltaSH3(N)]. The growth properties of these transfected cells in the presence or absence of IL-3 were not different from mock transfected or untransfected Ba/F3 cells. However, Ba/F3 cells overexpressing full length CRKL, but not deletion mutants of CRKL, were found to have an increase in their ability to bind to fibronectin-coated surfaces. Further, expression of full length, but not deltaSH2- or deltaSH3-CRKL deletion mutants, was found to alter cell morphology on fibronectin-coated plates, an effect which was further enhanced by certain kinds of stress stimuli, such as ionizing radiation. Similar results were obtained when CRKL was transiently overexpressed in Ba/F3 cells, and were also obtained in a second IL-3 dependent hematopoietic cell line, 32Dcl3. Adhesion to fibronectin was blocked by anti-beta1 integrin monoclonal antibody, but overexpression of CRKL did not affect surface expression of beta1 integrins, nor did it spontaneously induce expression of the beta1 integrin 'activation' epitope recognized by the 9EG7 monoclonal antibody. These data suggest a role for CRKL in signaling pathways which regulate adhesion to fibronectin.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Frequency of fetal cells in sorted subpopulations of nucleated erythroid and CD34+ hematopoietic progenitor cells from maternal peripheral blood.

Fetal cells that circulate in maternal peripheral blood (PB) during pregnancy offer a potential source of nucleated fetal material for noninvasive prenatal diagnosis. Fluorescence-activated cell sorting was used to target two populations of fetal cells: nucleated erythroid cells (NECs; CD71/glycophorin-A+ CD45(lo-int) CD34-) and hematopoietic progenitor cells (CD34+ cells; CD34++ CD71/glycophorin-A- CD45(int)). Fetal cells were detected by fluorescence in situ hybridization (FISH) using directly conjugated chromosome X and Y probes in 65% (13 of 20) of the maternal PBs (fetal karyotype 46,XY). The frequency of fetal cells isolated from the NEC and CD34+ fractions was, respectively, 0 to 14 and 0 to 7 cells per 2 x 10(7) previously frozen maternal cells (approximately 20 mL of blood). In nonfrozen samples, the yield and recovery of fetal cells was moderately improved. Culturing the CD34+ sorted fractions in serum-free media with cytokines improved the quality of the FISH preparations and resulted in a slight expansion in detectable fetal cells. The frequency of fetal cells isolated from cultured CD34+ fractions was 0 to 35 and 0 to 93 cells per 2 x 10(7) previously frozen and nonfrozen maternal PB cells, respectively. These results document the isolation, characterization, and enumeration of fetal cells from the maternal periphery that appear to be present in most, but not all, samples analyzed.

Antigens, CD34↗
PubMed1996

Heterogeneity in telomere length of human chromosomes.

Vertebrate chromosomes terminate in variable numbers of T2AG3 nucleotide repeats. In order to study telomere repeats at individual chromosomes, we developed novel, quantitative fluorescence in situ hybridization procedures using labeled (C3TA2)3 peptide nucleic acid and digital imaging microscopy. Telomere fluorescence intensity values from metaphase chromosomes of cultured human hematopoietic cells decreased with the replication history of the cells, varied up to six-fold within a metaphase, and were similar between sister chromatid telomeres. Surprisingly, telomere fluorescence intensity values within normal adult bone marrow metaphases did not show a normal distribution, suggesting that a minimum number of repeats at each telomere is required and/or maintained during normal hematopoiesis.

Adult↗
PubMed1995

Differential effects of the hematopoietic inhibitors MIP-1 alpha, TGF-beta, and TNF-alpha on cytokine-induced proliferation of subpopulations of CD34+ cells purified from cord blood and fetal liver.

We have previously characterized the proliferative response of primitive CD34+ cells, purified from adult bone marrow, umbilical cord blood, and fetal liver, to a mixture of hematopoietic stimulators (steel factor [SF], interleukin-3 [IL-3], IL-6, and erythropoietin [Epo]) in serum-free liquid cultures. In the present study, we assessed the effects of the hematopoietic inhibitors, macrophage inflammatory protein-1 alpha (MIP-1 alpha), transforming growth factor-beta (TGF-beta), and tumor necrosis factor-alpha (TNF-alpha), on the cytokine-induced proliferation of three different CD34+ cell subpopulations derived from cord blood and on total CD34+ cells derived from fetal liver. In cultures of cord blood cells, addition of MIP-1 alpha inhibited the numerical expansion of primitive CD34+ cells (CD34+ CD45RAlow CD71low cells) without inhibiting the proliferation of more mature subpopulations enriched for myeloid (CD34+ CD45RA+ CD71low cells) or erythroid (CD34+ CD45RAlow CD71+ cells) progenitors. TGF-beta significantly reduced the proliferation of all three subpopulations, although its effects were more pronounced on cells of the erythroid lineage, particularly immature erythroid progenitors. Similarly, TNF-alpha preferentially inhibited total nucleated and CD34+ cell production in the subpopulation enriched for erythroid cells. However, in contrast to TGF-beta, TNF-alpha preferentially inhibited the proliferation of more mature erythroid progenitors. In a separate set of experiments, MIP-1 alpha, TGF-beta, and TNF-alpha were added to cultures of total CD34+ cells purified from fetal liver. In keeping with the fact that the majority of the progenitors contained in these cells were erythroid progenitors, the inhibitory effects of the three cytokines were similar to those observed in cultures of CD34+ CD45RAlow CD71+ cord blood cells. The results of the present study demonstrate that MIP-1 alpha, TGF-beta, and TNF-alpha have the capacity to modulate cytokine-induced proliferation of cord blood and fetal liver progenitors. The differential effects of these three cytokines confirm their pleiotropic nature as regulators of hematopoiesis.

Antigens, CD↗
PubMed1994

CD45 isoform expression on human haemopoietic cells at different stages of development.

Alternate splicing and glycosylation produce multiple CD45 isoforms which are selectively expressed on the surface of cells of the haemopoietic system. The expression of CD45RA, CD45RB and CD45RO on CD34+ and CD34- haemopoietic cells from umbilical cord blood, bone marrow and fetal liver were studied by flow cytometry. CD34+ subpopulations defined by CD45 isoform expression were sorted from bone marrow and tested in long-term culture assays. By combining results of functional studies with phenotypic data and previously published information, the following pattern of CD45 isoform expression on early haemopoietic cells was established. The most primitive CD34+ cells are CD45RO+ CD45RB+ and express low or undetectable levels of CD45RA. Upon erythroid differentiation, CD34+ cells remain CD45RO+ CD45RB+, whereas commitment into the myeloid and lymphoid lineages coincides with down-regulation of CD45RO and up-regulation of CD45RA. As a result, the majority of CD34+ cells can be divided into two mutually exclusive populations of cells which express either CD45RO or CD45RA. This notion was confirmed in this study by three-colour immunofluorescence. The alternative expression of various CD45 isoforms on functionally distinct haemopoietic cells suggests an important role for these molecules in the proliferation and differentiation of haemopoietic cells.

Alternative Splicing↗
PubMed1994

Age-related decline in proliferative potential of purified stem cell candidates.

Recent studies in our laboratory have shown striking differences in the functional properties of candidate hematopoietic stem cells purified from fetal, neonatal, and adult human tissues. These differences include the ability to produce CD34+ cells, the turnover rate, and the fraction of cells that respond to a mixture of cytokines. All these parameters decrease with the age of the cell donor, and some of these observations are summarized here. Extensive qualitative changes in hematopoietic cells from various stages of development should be taken into account in the design of novel therapeutic strategies.

Adult↗
PubMed1992

Ontogeny of acyl-CoA: cholesterol acyltransferase in rat liver, intestine, and adipose tissue.

The development of acyl-coenzyme A: cholesterol acyltransferase (ACAT), was determined in the rat liver, intestine, and white (WAT) and brown adipose tissue (BAT). Animal studies have shown that dietary manipulation of cholesterol metabolism during an animal's early development can have persistent and permanent effects. Therefore it is important that the ontogeny of ACAT, one of the key enzymes in cholesterol metabolism, be clearly established. White Wistar rats were killed on day 21 of gestation, at birth, and on postnatal days 10, 14, 18, 21, 22, 25, 30, and 60. The tissues were rapidly excised, microsomes were prepared, and the activity of ACAT was measured as the rate of incorporation of [1-14C]oleoyl coenzyme A into cholesterol esters. Age-specific changes were observed in three of the four tissues investigated. Rat liver and intestine possess significant amounts of ACAT activity throughout development with marked variations in activity during this time. ACAT activity in BAT is low and variable throughout development with the exception of high activity noted in the adult animal. WAT contained little or no ACAT activity during development.

Acetyl Coenzyme A↗
PubMed1990

Diet and metabolic development.

For many years, investigators have been concerned with mechanisms that control and alter genetically regulated development. An intriguing aspect of these mechanisms is the ability of environmental factors to induce certain metabolic processes. Animal studies have shown that dietary manipulation of cholesterol and fatty acid metabolism during development can have persistent and permanent effects. In addition, there appears to be a critical period when changes in the diet can have lasting consequences. The changes in the control exerted by nutritional factors on metabolic development coincide with three phases of development: prenatal, suckling, and weaning. The effects of diet on cholesterol and fatty acid metabolism throughout these three phases of development will be addressed in this review.

Animals↗