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N Blake

Publications and source records attributed to N Blake.

At least 19 recordsLinked to original sources
PubMed2006

Increased megakaryocytopoiesis in Lyn-deficient mice.

Previous studies in cell lines have shown Lyn kinase to be a negative regulator of thrombopoietin (TPO)-induced proliferation. To further investigate the role of Lyn during megakaryocytopoiesis, Lyn-deficient mice (lyn(-/-)) were analyzed. We observed that lyn(-/-) mice have more bone marrow-derived GPIIB (CD41) and Mpl(+) cells when compared to their wild-type littermates. In addition, colony-forming unit-megakaryocytes (CFU-MK) are increased and TPO-induced expansion of primary marrow cells yielded a greater number of mature megakaryocytes (MKs) with increased nuclear ploidy. Histopathology of bone marrow and spleens from lyn(-/-) mice showed an increase in the number of MKs. Mechanistic studies revealed that TPO stimulation of MKs from lyn(-/-) mice did not affect phosphorylation of Janus kinase 2 (JAK2), signal transducer and activator of transcription (STAT) 3, STAT5, or MAP kinase kinase (MEK). Lyn-deficient MKs supported greater TPO-mediated phosphorylation and kinase activity of both Erk1/2 (mitogen-activated protein kinase, MAPK) and Akt. In contrast, there was a reduction of tyrosine phosphorylation of the inositol phosphatase, SHIP. This is the first direct evidence using primary MKs from Lyn-deficient mice that confirms our prior data from cell lines that Lyn kinase is a negative regulator of TPO signaling.

Animals↗
PubMed2002

Siting of antimony dopants and gallium in Ba(8)Ga(16)Ge(30) clathrates grown from gallium flux.

A series of antimony-doped Ba(8)Ga(16)Ge(30) clathrates was grown as large crystals from gallium flux. These compounds form in the cubic space group Pm(-)3n, with the unit cell parameter varying from 10.784(5) to 10.9008(6) A as the amount of GaSb substituting for germanium atoms in the framework is increased. It was found that more antimony than extra gallium was incorporated into the material and that a specific site (the 24k Wyckoff site) was favored by this element. (71)Ga NMR was carried out to determine the siting of gallium; it fills the 6c site preferentially.

Journal Article↗
PubMed2001

Processing of a multiple membrane spanning Epstein-Barr virus protein for CD8(+) T cell recognition reveals a proteasome-dependent, transporter associated with antigen processing-independent pathway.

Epstein-Barr virus (EBV) latent membrane protein (LMP)2 is a multiple membrane spanning molecule which lacks ectodomains projecting into the lumen of the endoplasmic reticulum (ER). Human CD8(+) cytotoxic T lymphocytes (CTL)s recognize a number of epitopes within LMP2. Assays with epitope-specific CTLs in two different cell backgrounds lacking the transporter associated with antigen processing (TAP) consistently show that some, but not all, LMP2 epitopes are presented in a TAP-independent manner. However, unlike published examples of TAP-independent processing from endogenously expressed antigens, presentation of TAP-independent LMP2 epitopes was abrogated by inhibition of proteasomal activity. We found a clear correlation between hydrophobicity of the LMP2 epitope sequence and TAP independence, and experiments with vaccinia minigene constructs expressing cytosolic epitope peptides confirmed that these more hydrophobic peptides were selectively able to access the HLA class I pathway in TAP-negative cells. Furthermore, the TAP-independent phenotype of particular epitope sequences did not require membrane location of the source antigen since (i) TAP-independent LMP2 epitopes inserted into an EBV nuclear antigen and (ii) hydrophobic epitope sequences native to EBV nuclear antigens were both presented in TAP-negative cells. We infer that there is a proteasome-dependent, TAP-independent pathway of antigen presentation which hydrophobic epitopes can selectively access.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Transitional choice behavior in concurrent-chain schedules.

The choice responses of four pigeons were examined in 20 periods of transition in a concurrent-chain procedure with variable-interval schedules as initial links and fixed delays to reinforcement as terminal links. In some conditions, the delays to reinforcement were different for the two terminal links, and changes in preference were recorded after the delays for the two response keys were switched. In other conditions, the reinforcer delays were equal for the two keys, but which key delivered 80% of the reinforcers was periodically switched. Choice proportions changed more quickly after a switch in reinforcement percentages than after a switch in the delays, thereby contradicting the hypothesis that faster changes would occur when the switch in conditions was easier to discriminate. Analyses of response sequences showed that the effects of individual reinforcers were larger and lasted longer in conditions with changing reinforcement percentages than in conditions with changing terminal-link delays. Rates of change in choice behavior do not appear to be limited by the unpredictability of variable reinforcement schedules, because the changes in behavior were slow and gradual even when there was a large and sudden change in reinforcer delays.

Journal Article↗
PubMed2001

Differential immunogenicity of Epstein-Barr virus latent-cycle proteins for human CD4(+) T-helper 1 responses.

Human CD4(+) T-helper 1 cell responses to Epstein-Barr virus (EBV) infection are likely to be important in the maintenance of virus-specific CD8(+) memory and/or as antiviral effectors in their own right. The present work has used overlapping peptides as stimulators of gamma interferon release (i) to identify CD4(+) epitopes within four EBV latent-cycle proteins, i.e., the nuclear antigens EBNA1 and EBNA3C and the latent membrane proteins LMP1 and LMP2, and (ii) to determine the frequency and magnitude of memory responses to these proteins in healthy virus carriers. Responses to EBNA1 and EBNA3C epitopes were detected in the majority of donors, and in the case of EBNA1, their antigen specificity was confirmed by in vitro reactivation and cloning of CD4(+) T cells using protein-loaded dendritic cell stimulators. By contrast, responses to LMP1 and LMP2 epitopes were seen much less frequently. EBV latent-cycle proteins therefore display a marked hierarchy of immunodominance for CD4(+) T-helper 1 cells (EBNA1, EBNA3C >> LMP1, LMP2) which is different from that identified for the same proteins with respect to CD8(+)-T-cell responses (EBNA3C > EBNA1 > LMP2 >> LMP1). Furthermore, the range of CD4(+) memory T-cell frequencies in peripheral blood of healthy virus carriers was noticeably lower and narrower than the corresponding range of latent antigen-specific CD8(+)-T-cell frequencies.

Epitope Mapping↗
PubMed2000

The importance of exogenous antigen in priming the human CD8+ T cell response: lessons from the EBV nuclear antigen EBNA1.

Mouse models suggest that the processing of exogenous Ag by dendritic cells can be important for priming the CD8(+) CTL response. To study the situation in humans, we have exploited the CTL response to EBV infection. In this context EBV expresses eight latent proteins, of which EBV-encoded nuclear Ag (EBNA) 3A, 3B, and 3C appear to be immunodominant for CTL responses, whereas another nuclear Ag, EBNA1, which is completely protected from endogenous presentation via the MHC class I pathway, is thought to induce responses rarely, if ever. Here, using EBNA1 peptides and/or EBNA1 protein-loaded dendritic cells as in vitro stimuli, we have identified memory CTL responses to HLA-B*3501, -B7, and -B53-restricted EBNA1 epitopes that can be as strong as those seen in immunodominant epitopes from the "conventionally processed"" EBNA3 Ags. Furthermore, we used HLA-peptide tetramers to show that the primary response to one such EBNA1 epitope constituted up to 5% of the CD8(+) T cells in infectious mononucleosis blood, the strongest latent Ag-specific response yet detected in this setting. We conclude that exogenous protein represents a significant source of Ag for priming the human CTL response.

Adult↗
PubMed1999

Association of a syndrome resembling Wegener's granulomatosis with low surface expression of HLA class-I molecules.

BACKGROUND: Granulomatous syndromes, such as Wegener's granulomatosis, are defined according to complex criteria, but the underlying cause is rarely identified. We present evidence for a new aetiology for chronic granulomatous lesions associated with a recessive genetic defect, which is linked to the human leucocyte antigen (HLA) locus. METHODS: Five adults with necrotising granulomatous lesions in the upper respiratory tract and skin, associated with recurrent bacterial respiratory infections and skin vasculitis, were identified. A diagnosis of Wegener's granulomatosis was considered in all of them, but abandoned because of an incompatible disease course and resistance to immunosuppressive treatments. Peripheral-blood samples were taken and analysed by immunohistochemistry and fluorescent-activated-cell-sorter analysis. Since all five patients were homozygous for the HLA locus, we looked for genetic defects located within the HLA-locus with PCR and restriction fragment length polymorphism. FINDINGS: A severe decrease in cell-surface expression of HLA class-I molecule was seen in all patients. Defective expression of the transporter associated with antigen presentation (TAP) genes was responsible for the HLA class-I down-regulation, and in two patients we identified a mutation in the TAP2 gene responsible for the defective expression of the TAP complex. We showed the presence of autoreactive natural killer (NK) cells and gammadelta T lymphocytes in the peripheral blood cells of two patients. Correction of the genetic defect in vitro restored normal expression of HLA class-I molecules and prevented self-reactivity in the patients' cells. Histology of granulomatous lesions showed the presence of a large proportion of activated NK cells. INTERPRETATION: Our findings define the cause and pathogenesis of a new syndrome that affects patients with a defective surface expression of HLA class-I molecules. The syndrome resembles Wegener's granulomatosis both clinically and histologically. Patients have chronic necrotising granulomatous lesions in the upper respiratory tract and skin, recurrent infections of the respiratory tract, and skin vasculitis. A predominant NK population within the granulomatous lesions suggests that the pathophysiology of the skin lesions may relate to the inability of HLA class-I molecules to turn off NK cell responses. Accurate genetic analysis of a defined syndrome can provide a better understanding of the cause and pathogenesis of a disease.

ATP-Binding Cassette Transporters↗
PubMed1997

A case of primary immunodeficiency due to a defect of the major histocompatibility gene complex class I processing and presentation pathway.

INTRODUCTION: We report a case of primary immunodeficiency due to a defect of the TAP transporter, an heterodimeric complex which controls the expression of HLA class I molecule by delivering peptides from the cytosol into the lumen of the endoplasmic reticulum. Since childhood, the 36 year old female suffered from recurrent sinusitis/bronchitis. She later developed bronchiectasis and destructive nasal epitheloid granulomata in conjunction with a generalized vasculitic syndrome that did not improve upon immunosuppression and antibiotics. METHODS: The class I monomorphic W6/32 was used for cell surface staining and immunoprecipitation of MHC class I molecules. Peptide transport assay was carried out in semi-permeabilized cells with iodinated peptides. Antigen presentation experiments were performed using chromium 51 labelled patient B cell line and EBV specific CTL. TAP1 and TAP2 specific antibodies were used for Western blotting and immunoprecipitation of the TAP complex. RESULTS AND CONCLUSIONS: A severe reduction of MHC class I molecules at the cell surface of the B-cell lines was observed, whereas MHC class II expression was not altered. Isoelectric focusing of metabolically labelled MHC class I molecules revealed that class I heavy chains remain unsialylated, consistent with a block of TAP dependent peptide translocation. These conclusions were confirmed by further experiments showing that peptide translocation was completely abolished. We also demonstrated that presentation of viral antigens through endogenous class I molecules was severely impaired. Immunoprecipitation and Western blotting of TAP1/2 complex showed that TAP2 was not detectable. Further, experiments are in progress to identify the site of the mutation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Human CD8+ T cell responses to EBV EBNA1: HLA class I presentation of the (Gly-Ala)-containing protein requires exogenous processing.

Epstein-Barr virus (EBV)-induced cytotoxic T lymphocyte (CTL) responses have been detected against many EBV antigens but not the nuclear antigen EBNA1; this has been attributed to the presence of a glycine-alanine repeat (GAr) domain in the protein. Here we describe the isolation of human CD8+ CTL clones recognizing EBNA1-specific peptides in the context of HLA-B35.01 and HLA-A2.03. Using these clones, we show that full-length EBNA1 is not presented when expressed endogenously in target cells, whereas the GAr-deleted form is presented efficiently. However, when supplied as an exogenous antigen, the full-length protein can be presented on HLA class I molecules by a TAP-independent pathway; this may explain how EBNA1-specific CTLs are primed in vivo.

Alanine↗
PubMed1997

Endogenous IL-2 production by natural killer cells maintains cytotoxic and proliferative capacity following retroviral-mediated gene transfer.

Interleukin (IL)-2 therapy given at tolerable doses is insufficient to induce maximum activation of natural killer (NK) cells. We recently demonstrated that NK cells expanded in vivo can be maximally activated by short-term ex vivo incubation with 1000 U/mL IL-2. However, IL-2 withdrawal, which would occur with reinfusion, may lead to a rapid loss of cell viability and function. We hypothesized that retroviral transduction could provide an endogenous source of IL-2 to maintain NK function as measured by proliferation and cytotoxicity. Enriched NK cells were transduced with supernatants containing an MFG-based retrovirus designed to express murine IL-2 cDNA. Several supernatant transduction strategies were evaluated. NK cells were initially cultured in 1000 U/mL of huIL2 for 7-8 days, harvested, and replated prior to transduction (4 hours at 37degrees C); this proved insufficient to sustain NK proliferation or maintain cytotoxicity after exogenous human IL-2 (huIL-2) withdrawal. An alternative transduction procedure using phosphate-depleted medium, centrifugation, and transduction for 16 hours at 32degrees C was then evaluated. NK cells transduced under these conditions maintained significant NK proliferation in the absence of exogenous IL-2 compared with sham-transduced controls. Two consecutive daily transductions resulted in less proliferation, suggesting that several exposures to retroviral supernatant may inhibit subsequent NK proliferation. Cytotoxicity of the transduced NK cells against K562 and Raji was maintained under these conditions without exogenous IL-2. Sham-transduced NK cells produced 8.3+/-2.6 U/mL of murine IL-2 (muIL-2) by ELISA (background) after 7 days without exogenous IL-2. In contrast, 109+/-23 U/mL muIL-2 was produced by NK cells transduced with supernatant from the MFG/muIL-2 producer line. These experiments demonstrate that NK cells can be successfully transduced with retroviruses and induced to express sufficient IL-2 to maintain their proliferative and cytotoxic functions. Transduction of IL-2 genes into NK cells may offer advantages over exogenous IL-2 administration in maintaining maximum function for use in antitumor immunotherapy.

Adult↗
PubMed1996

A recombinant adenovirus expressing an Epstein-Barr virus (EBV) target antigen can selectively reactivate rare components of EBV cytotoxic T-lymphocyte memory in vitro.

While the bulk of a virus-induced cytotoxic T-lymphocyte (CTL) response may focus on a few immunodominant viral antigens, in certain tumor virus systems the detectability of clones recognizing other, subdominant antigens can assume particular importance. By using the human CTL response to Epstein-Barr virus (EBV) as a model system, here we show that even rare components of virus-specific memory can be selectively reactivated in vitro when the relevant target antigen is expressed in autologous stimulator cells from a recombinant adenovirus (RAd) vector. We generated a replication-deficient adenovirus, RAd-E3C, which in skin fibroblast cultures expressed the EBV nuclear antigen EBNA3C at a 10- to 100-fold-higher level than that naturally present in EBV-transformed lymphoblastoid cell lines (LCLs). Initial experiments with a donor whose polyclonal CTL response to LCL stimulation contained a strong EBNA3C-specific component showed that these CTLs could be efficiently reactivated by in vitro stimulation either with RAd-E3C-infected fibroblasts or with RAd-E3C-infected peripheral blood mononuclear cells. Then we studied donors whose responses to LCL stimulation contained little if any detectable EBNA3C reactivity but were dominated by clones recognizing other EBV target antigens; in vitro stimulation with RAd-E3C-infected peripheral blood mononuclear cells selectively reactivated EBNA3C-specific CTL clones from these individuals, with the epitope specificities of responses subsequently identified at the peptide level. This RAd-based approach could be applied more generally to screen for human CTL responses against any candidate target antigen expressed by tumor cells.

Adenoviridae↗