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P Le

Publications and source records attributed to P Le.

14 recordsLinked to original sources
PubMed2007

Ex vivo expansion of umbilical cord blood stem cells for transplantation: growing knowledge from the hematopoietic niche.

Umbilical cord blood transplantation (UCBT) in adults is limited by the small number of primitive hematopoietic stem cells (HSC) in each graft, resulting in delayed engraftment post transplant, and both short- and long-term infectious complications. Initial efforts to expand UCB progenitors ex vivo have resulted in expansion of mature rather than immature HSC, confounded by the inability to accurately and reliably measure long-term reconstituting cells. Ex vivo expansion of UCB HSC has failed to improve engraftment because of resulting defects that promote apoptosis, disrupt marrow homing and initiate cell cycling. Here we discuss the future of ex vivo expansion, which we suggest will include the isolation of immature hematopoietic progenitors on the basis of function rather than surface phenotype and will employ both cytokines and stroma to maintain and expand the stem cell niche. We suggest that ex vivo expansion could be enhanced by manipulating newly discovered signaling pathways (Notch, Wnt, bone morphogenetic protein 4 and Tie2/angiopoietin-1) and intracellular mediators (phosphatase and tensin homolog and glycogen synthase kinase-3) in a manner that promotes HSC expansion with less differentiation. Improved methods for ex vivo expansion will make UCBT available to more patients, decrease engraftment times and allow more rapid immune reconstitution post transplant.

Adult↗
PubMed2001

Suppression of Ras-mediated tumorigenicity and metastasis through inhibition of the Met receptor tyrosine kinase.

Mutations in the Ras family of GTP binding proteins represent one of the most frequently observed genetic alterations in human cancers. We and others have recently demonstrated that expression of Met, the tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF/SF), is significantly up-regulated in Ras-transformed cells. Because HGF/SF-Met signaling is proposed to play a prominent role in tumor development and progression, we assessed the possible requirement for Met during Ras-mediated tumor growth and metastasis. To disrupt endogenous Met signaling, we constructed dominant-negative mutants of both human and murine Met and showed that these can inhibit HGF/SF-mediated Met signaling and cell invasion of ras-transformed cells in vitro. Moreover, ectopic expression of dominant-negative Met mutants reduced the s.c. tumor growth of ras-transformed cells and dramatically suppressed their ability to form lung metastases in vivo. Our data demonstrate that Met plays a prominent role during Ras-mediated tumor growth and metastasis, and further suggest that agents that inhibit HGF/SF-Met signaling may represent an important therapeutic avenue for the treatment of a variety of malignant tumors.

3T3 Cells↗
PubMed2000

Immunogenicity of a novel DNA vaccine cassette expressing multiple human immunodeficiency virus (HIV-1) accessory genes.

OBJECTIVE: To develop an HIV-1 accessory gene immunogen using a DNA vaccine approach. METHODS: HIV-1 accessory genes vif, vpu and nef were modified to express under the control of a single promoter with cellular proteolytic cleavage sites between the coding sequences (VVN-P). Immune responses induced by these constructs were evaluated in mice. RESULTS: DNA vaccine construct (pVVN-P) expressing Vif, Vpu and Nef was processed and the fusion protein was cleaved appropriately. Vif, Vpu and Nef as a fusion protein with proteolytic cleavage sites (VVN-P) is able to induce a significant level of cellular immune responses. We also observed that accessory genes Vif, Vpu and Nef (VVN-P) induced an effective T helper 1 proliferative response measured by cytokine production. Furthermore, expression cassette pVVN-P was able to induce cytotoxic T lymphocyte (CTL) responses against diverse HIV-1 viruses in infected target cells. CONCLUSION: We conclude that cell-mediated immune responses induced by accessory gene constructs from clade B may have a broader recognition of divergent HIV-1 viruses and should be further examined for both prophylactic and therapeutic vaccination schemes against HIV-1.

3T3 Cells↗
PubMed1998

Construction of attenuated HIV-1 accessory gene immunization cassettes.

Delivery of genetic expression cassettes into animals can effectively induce both humoral and cellular immunity to the expressed gene product. Previously, we used this strategy to immunize against HIV-1 structural and enzymatic proteins in mice, non-human primates and in humans. In contrast, the use of the accessory genes including vif, vpr, vpu and nef as immunotherapeutic vaccine targets has not been well characterized. Our goal is to design an effective genetic HIV vaccine, which includes the accessory genes as part of a multi-component immunogen. In order to develop accessory genes as genetic vaccines, we have molecularly cloned and analysed the sequence variation and immunogenic potential present in these genes derived from viral isolates obtained from HIV-1 infected patients and laboratory isolates. Prototype genetic variants were selected and their ability to induce humoral and cellular immune responses was studied in animal models. We observed that attenuated accessory genes can effectively induce both humoral and cellular responses in mice and the resulting immune response is directly correlated with DNA concentrations delivered and the number of boosts. This strategy can be used generally to develop an effective, safe DNA vaccine for any pathogen.

AIDS Vaccines↗
PubMed1997

Development of genetic vaccines for pathogenic genes: construction of attenuated vif DNA immunization cassettes.

OBJECTIVE: To develop a putative immunization cassette using HIV-1 vif accessory gene derived from HIV-1 clinical specimens as a component of a DNA vaccine for HIV-1. METHODS: vif genes were cloned from HIV-1-infected patients and the sequence variation present within the patients was analyzed. Prototypic genetic variants were selected and the ability of these clones to induce humoral and cellular immune responses was studied in animals. The selected protective genetic variants were biologically characterized through transcomplementation assays using primary cells infected with a vif-defective HIV-1 proviral clone. RESULTS: Analysis of vif variants from different patients revealed that vif is highly conserved with the open reading frame remaining intact in vivo. It was shown that attenuated vif clones from HIV-1-infected subjects can effectively induce both humoral and cellular responses against Vif protein in mice. Evaluation of the cellular responses in vitro using human cellular targets infected with a clinical HIV-1 isolate showed that vif clones could induce cellular responses capable of destroying the virus. CONCLUSIONS: The vif variants developed in this study exhibited non-productive phenotypes, yet were capable of inducing specific immune responses against HIV-1. These constructs could be used as part of a DNA vaccine strategy for HIV-1. This vaccine adaptation strategy could be used for the development of immunogens for any pathogen resulting in cross-reactive immunity and attenuated gene pathogenesis.

AIDS Vaccines↗
PubMed1996

Cloning and expression of a new HOXC6 transcript encoding a repressing protein.

Homeodomain-containing proteins are transcription factors that regulate the co-ordinated expression of multiple genes involved in development, differentiation and malignant transformation. In an attempt to characterize expressed homeobox (HOX) genes in breast cancer cells, we cloned two distinct HOXC6 transcripts from an MCF7 cDNA library, Interestingly, one of them represents a new HOXC6 mRNA encoding a homeodomain-containing protein harbouring a unique N-terminal sequence. Moreover we demonstrate that this HOXC6 transcript is less abundant in human breast cancer cells than in non-tumorigenic cell lines, is detected in breast carcinomas and adjacent tissues and is expressed in a variety of human tumours. In addition, transient co-transfection experiments illustrated that both HOXC6 transcripts encode gene products that repress transcription from a HOX binding sequence in MDA-MB231 cells and co-operate with other HOX gene products such as HOXB7 on their target genes. Taken together, our results suggest that HOXC6 proteins might contribute to the breast cell phenotype through co-operative interactions with other HOX-derived proteins and repression of their target genes.

Amino Acid Sequence↗
PubMed1993

Influence of the T cell receptor alpha-chain on T cell reactivity and tolerance to Mls-1 in T cell receptor beta-chain transgenic mice.

Previous analyses of a TCR V beta 8.1 transgenic mouse revealed multiple mechanisms of tolerance to the retroviral superantigen, Mls-1. Whereas some T cells were clonally deleted in the thymus, others became anergic in the periphery, or remained unaffected by the expression of Mls-1. In addition, a strong correlation between TCR alpha-chain usage and Mls-1 reactivity of individual transgenic V beta 8.1+ T cell hybridomas was established. Based on these observations, we speculated that the different mechanisms of tolerance were a consequence of the alpha-chain-mediated differences in Mls-1 reactivity. In the current studies, we make use of a V alpha 2-specific mAb to directly examine the role of the alpha-chain on tolerance in this transgenic model. We show, first, that V alpha 2+ CD4+ T cells, as a group, are relatively less Mls-1-reactive, and are elevated twofold in the periphery of Mls-1+ compared with Mls-1-V beta 8.1 transgenic mice. This elevated expression is also seen in the V alpha 2+ CD4+ population of mature thymocytes, but not in immature thymocytes. Second, Mls-1-induced neonatal tolerance in Mls-1-negative mice caused an increase of V alpha 2+ CD4+ T cells, comparable with the frequency of expression in transgenic mice that endogenously expressed Mls-1. Third, we have demonstrated a general correlation between the age-dependent increase in Mls-1-expression and the levels of V alpha 2+ CD4+ T cells during the first 4 wk of life. Taken together, these data suggest that the over-expression of V alpha 2+ CD4+ T cells in Mls-1+ mice is a consequence of mechanisms of tolerance, predominantly mediated by preferential lack of clonal deletion in the thymus. These data support the idea that clonal deletion is a competitive process and the influence of the TCR alpha-chain on the strength of Mls-1 reactivity of individual V beta 8.1+ transgenic T cells controls their susceptibility to clonal deletion.

Animals↗
PubMed1993

Major histocompatibility complex-specific recognition of Mls-1 is mediated by multiple elements of the T cell receptor.

We have recently shown that recognition of the mouse mammary tumor virus 9-associated superantigen (vSAG-9) by murine V beta 17+ T cells is strongly influenced by the major histocompatibility complex (MHC) class II haplotype of the presenting cells, resulting in a form of MHC-restricted recognition. This finding was unexpected, because T cell recognition of another well-characterized retroviral superantigen, minor lymphocyte-stimulating antigen 1 (Mls-1), had been shown to be independent of the MHC haplotype of the presenting cell. To determine whether recognition of vSAG-9 and Mls-1 is fundamentally different, we undertook an extensive analysis of MHC haplotype influences on vSAG-9 and Mls-1 recognition by panels of T cell hybridomas. Our results show that, although most hybridomas recognized Mls-1 regardless of the MHC haplotype of the presenting cells, as previously described by others, some hybridomas exhibited unique patterns of MHC fine specificity. Thus, T cell recognition of vSAG-9 and Mls-1 is not fundamentally different, but the apparent differences can be explained in terms of frequency. The MHC fine specificity of individual Mls-1-reactive hybridomas was influenced by both V beta and non-V beta T cell receptor (TCR) elements. First, the influence of the V beta element was apparent from the observation that V beta 8.2+ hybridomas were significantly more MHC specific in their recognition of Mls-1 than V beta 8.1 hybridomas. Second, a role for the TCR alpha chain was implicated from the distinct patterns of fine specificity of Mls-1 reactivity among a panel of transgenic hybridomas that expressed an identical beta chain (V beta 8.1D beta 2J beta 2.3C beta 2). Sequence analysis revealed that junctional residues of the TCR alpha chain and/or V alpha/J alpha combinations influenced the MHC haplotype fine specificity for Mls-1. Third, D beta J beta influences were implicated, in that the transgenic hybridomas expressed distinctive patterns of Mls-1 fine specificity not represented among V beta 8.1+ nontransgenic hybridomas. The findings that T cell recognition of endogenous superantigen is MHC specific, and that this specificity correlates with non-V beta elements of the TCR, support the hypothesis that there is a direct interaction between the TCR and either polymorphic residues of the MHC class II molecule or haplotype-specific dominant peptides presented by class II.

Animals↗
PubMed1992

T cell receptor alpha-chain influences reactivity to Mls-1 in V beta 8.1 transgenic mice.

Most, but not all, V beta 8.1+ T cells respond to M1s-1 and are clonally deleted in the thymus of M1s-1-expressing animals. To formally examine the role of the TCR alpha-chain in reactivity and tolerance to M1s-1, we have analyzed M1s-1 reactivity in a large panel of CD4+ hybridomas generated from TCR V beta 8.1 transgenic mice, that express an identical, potentially M1s-1-reactive beta-chain. The data show that the alpha-chain strongly influences the M1s-1 reactivity of the hybridomas and that the differences in reactivity had relevance for tolerance. Thus, V alpha 11+ hybridiomas were biased toward M1s-1 reactivity and V alpha 11+ T cells were correspondingly absent from the peripheral repertoire of M1s-1-expressing transgenic mice. V alpha 2+ hybridomas, on the other hand, were biased against M1s-1 reactivity, and V alpha 2+ T cells were correspondingly amplified in the M1s-1-expressing transgenic mice. Structural analysis of the alpha-chains revealed that the M1s-1 reactivity of the V alpha 11+ hybridomas segregated precisely with family member, such that V alpha 11.1+ hybridomas were M1s-1-reactive and V alpha 11.3+ hybridomas were not M1s-1-reactive. On the other hand, there was not a clear correlation between family member and M1s-1 reactivity in the V alpha 2+ hybridomas. The hybridomas also showed striking variation in their reactivity to staphylococcal enterotoxin B (SEB), and the SEB reactivity of the V alpha 11+ hybridomas correlated precisely with family member and with M1s-1 reactivity. In contrast, there was not a clear correlation with V alpha 2+ alpha-chain structure and SEB reactivity. Also, there was no correlation between M1s-1 reactivity and SEB reactivity in individual V alpha 2+ hybridomas, suggesting that the recognition of the two superantigens by the same TCR is not equivalent. Taken together, these data define a role for the TCR alpha-chain in superantigen reactivity and T cell tolerance, and provide a structural explanation for the different fates of M1s-1-reactive T cells in normal and transgenic mice.

Amino Acid Sequence↗
PubMed1984

EEG asymmetry during sleep.

The ratio of integrated EEG amplitude from left and right temporal areas was analyzed during sleep in normal subjects for two consecutive nights. The results showed that the differences between the subjects were similar in both nights and were much larger than the differences between the stages of sleep. No significant variation was found between stages, although stage 1 tended to show greater relative right activation in night 1. Also, the relative right activation was greater in wakefulness in night 2 as compared to night 1. The classification of the correlation coefficients between stages corresponded to the physiological transitions between stages, suggesting that the small source of variance due to sleep stage contains relevant information.

Adult↗
PubMed1983

Relationships between brain concentrations of desipramine and paradoxical sleep inhibition in the rat.

Desipramine (DMI), like many antidepressant drugs, inhibit the production of paradoxical sleep (PS). In the present experiment, we have investigated the relationships between brain level of DMI and PS inhibition. Groups of rats had their sleep monitored after 1, 2 or 4 mg/kg of DMI. In other animals, the brain concentration of DMI was assayed at various times after the same treatments. The results indicate that a critical threshold concentration of 300 ng/g DMI in the brain is necessary for complete PS inhibition. This stage reappears only when the DMI level falls below this value, and its production resumes at a normal rate, provided the DMI level reached initially was not largely in excess of the threshold concentration. The results are discussed with regard to the present knowledge of specific binding of tricyclics in brain and their "ex vivo" action on norepinephrine uptake resulting in enhancement of collateral inhibiton of noradrenergic cells.

Animals↗
PubMedDate not supplied

A metastatic orthotopic-transplant nude-mouse model of human patient breast cancer.

We report here the development of an orthotopic-transplant model of human patient breast cancer in nude mice. Histologically-intact patient breast tumor tissue was transplanted as intact tissue to the mammary fat pad of nude mice where the tumor tissue grew extensively and metastasized to the lung. This is the first orthotopic-transplant metastatic model of human breast cancer. The potential clinical and basic-science uses of the model are discussed.

Animals↗