NK recognition and lysis of targets are mediated by activation receptor(s) whose effects may be over-ridden by inhibitory receptors recognizing class I MHC on the target. Incubation of normal ...lymphoblasts with a peptide that can bind to their class I MHC renders them sensitive to lysis by syngeneic NK cells. By binding to class I MHC, the peptide alters or masks the target structure recognized by an inhibitory NK receptor(s). This target structure is most likely an "empty" dimer of class I heavy chain and beta2m as opposed to a "full" class I trimer formed by binding of specific peptide that is recognized by CTL.
Natural killer (NK) cells are lymphocytes that provide an important line of defense against viruses and tumors. Technical hurdles in genetic modifications of primary NK cell ex vivo had limited our ...studies of protein function(s) in NK cell differentiation, acquisition of self-tolerance, and induction of anti-tumor responses. We used VSV-G-pseudotyped, EGFP-expressing lentiviral vectors to develop an efficient gene transfer system to modify gene expression in primary murine NK cells with or without prior IL-2 activation. Lentiviral vector transduction did not impair NK cellular viability, phenotype, or functions. We also demonstrated the use of this system in modifying differentiating NK cells derived from lentiviral-transduced murine hematopoietic progenitor cells. Furthermore, the same transduction protocol is amendable to delivery of short-hairpin RNA (shRNA) for specific gene silencing. Collectively, our approach in genetic engineering of primary murine NK cells will prove useful in studying basic NK cell biology and in exploring therapeutic potentials of NK cells in inbred and transgenic mouse models.
We constructed human immunodeficiency virus type 1 (HIV-1) vectors that will allow higher levels of gene expression in T cells. Gene expression under the control of an internal cytomegalovirus (CMV) ...immediate-early promoter in a self-inactivating lentiviral vector (CSCG) is 4- to 15-fold lower in T-cell lines (SUPT1 and CEMX174) than in non-lymphoid-cell lines (HeLa and 293T). This is in contrast to a Moloney murine leukemia virus (MoMLV)-based retrovirus vector (SRalphaLEGFP). We therefore replaced the internal CMV promoter of CSCG with three different murine oncoretroviral long terminal repeat (LTR) promoters-murine sarcoma virus (MSV), MoMLV (MLV), and the LTR (termed Rh-MLV) that is derived from the ampho-mink cell focus-forming (AMP/MCF) retrovirus in the serum of one rhesus macaque monkey that developed T-cell lymphoma following autologous transplantation of enriched bone marrow stem cells transduced with a retrovirus vector preparation containing replication-competent viruses (E. F. Vanin, M. Kaloss, C. Broscius, and A. W. Nienhuis, J. Virol. 68:4241-4250, 1994). We found that the combination of Rh-MLV LTR and a partial gag sequence of MoMLV (Deltagag(871-1612)) in CS-Rh-MLV-E gave the highest level of enhanced green fluorescent protein (EGFP) gene expression compared with MLV, MSV LTR, phosphoglycerate kinase, and CMV promoters in T-cell lines, as well as activated primary T cells. Interestingly, there was a further two- to threefold increase in EGFP expression (thus, 10-fold-higher expression than with CMV) when the Rh-MLV promoter and Deltagag(871-1612) were used in a self-inactivating-vector setting that has a further deletion in the U3 region of the HIV-1 LTR. These hybrid vectors should prove useful in gene therapy applications for T cells.
Modeling human hematopoietic progenitor cell gene therapy in nonhuman primates allows long-term evaluation of safety, maintenance of gene expression, and potential immune response against transgene ...products. We transplanted autologous G-CSF/SCF-mobilized CD34+ cells transduced with lentiviral vectors expressing EGFP into myeloablated rhesus macaques. To date, more than 4 years posttransplantation, 0.5–8% EGFP expression is maintained in multiple cell lineages. The animals remain healthy with no evidence of hematopoietic abnormalities or malignancies. To assess immune functions, we actively immunized two of our transplanted animals with purified rEGFP proteins and CpG adjuvant and demonstrated stable levels of EGFP+ cell populations maintained for over 29 months despite four active immunizations. We did not detect a persistent anti-EGFP antibody response or anti-EGFP T cell response in these immunized animals. Immune response to an irrelevant antigen was normal. Taken together, our data provide formal support that transplantation of lentivirally transduced CD34+ progenitor cells in myeloablated rhesus macaques induces specific immunological tolerance toward a foreign transgene.
To identify concepts including misconceptions among the community members regarding family doctors, and determine factors affecting decisions on which doctor to consult in different clinical ...scenarios.
Household telephone survey conducted between 4 and 13 September 2006.
Hong Kong community.
Cantonese-speaking Hong Kong residents aged 18 years or more were targeted. Randomly selected participants were asked to complete a questionnaire, which was designed based on a literature search and subsequent focus group discussions.
Among the 1811 households with eligible subjects to survey, 1204 completed the questionnaire (response rate, 67%). More than 85% considered a family doctor to be the first doctor they wanted to see even if it was inconvenient. "Clearly knowing my physical conditions", "fast-acting and effective treatment", and "doctor with friendly and sincere attitude" were the three most important factors influencing the choice of a family doctor. When affected by flu-like symptoms, 65% would go to a private clinic, 20% to a general out-patient clinic, 6% to a designated clinic with staff approved by their respective medical insurance/medical benefit scheme, and 5% to a private hospital outpatient clinic. Among the latter two groups, 65% consulted the same doctor every time when they felt sick. More than 50% of those willing to have regular follow-up by a family doctor for hypertension and diabetes paid more than HK$300 per month. Approximately 64% might consider having regular follow-up at a general out-patient clinic by a nurse specialist.
Hong Kong inhabitants already have their own ideas regarding how to care for their own health, and what kind of family doctors they prefer. This survey should help both doctors and health care policy makers to realign their current thinking, and thus provide a platform for the development of a primary care model unique to Hong Kong.
We have investigated the capacity of human MHC class I HLA-B gene products, HLA-B27, -B7 (fully human), and -B7kb (human-mouse hybrid consisting of the alpha1 and alpha2 domains of HLA-B7, and the ...alpha3 and cytoplasmic domains of mouse H-2Kb), expressed on mouse NK cells during ontogeny to influence NK recognition of otherwise syngeneic mouse target cells. Despite a high level of surface expression of the transgene (comparable to that of endogeneous H-2DbKb molecules), the direct killing of YAC-1 targets, and the killing of P815 targets in a redirected lysis assay, the NK effectors of these transgenic mice could not mediate hybrid resistance-like killing of nontransgenic C57BL/6 target cells either in vitro or in vivo. Splenocytes from B6-B27 mice could be used to generate CTL lines against a B27-binding peptide, implying that T cells restricted by HLA-B27 developed during ontogeny. NK cells from B6-B27 could lyse B6-B27 Con A lymphoblasts pulsed with Db-binding peptide but not B27-binding peptides. Taken together, our results show that these human HLA-B transgene products cannot function as class I MHC "self" elements for mouse NK cells, even when present throughout ontogeny.
Although studies indicate LIGHT (lymphotoxin (LT)-like, exhibits inducible expression and competes with HSV glycoprotein D for herpes virus entry mediator (HVEM), a receptor expressed by T ...lymphocytes) enhances inflammation and T cell-mediated immunity, the mechanisms involved in this process remain obscure. In this study, we assessed the role of LIGHT in IL-12 production and development of CD4 super(+) Th cells type one (Th1) in vivo. Bone marrow-derived dendritic cells from LIGHT super(-/-) mice were severely impaired in IL-12p40 production following IFN- gamma and LPS stimulation in vitro. Furthermore, blockade of LIGHT in vitro and in vivo with HVEM-Ig and LT beta receptor (LT beta R)-Ig leads to impaired IL-12 production and defective polyclonal and Ag-specific IFN- gamma production in vivo. In an infection model, injection of HVEM-Ig or LT beta R-Ig into the usually resistant C57BL/6 mice results in defective IL-12 and IFN- gamma production and severe susceptibility to Leishmania major that was reversed by rIL-12 treatment. This striking susceptibility to L. major in mice injected with HVEM-Ig or LT beta R-Ig was also reproduced in LIGHT super(-/-) arrow right RAG1 super(-/-) chimeric mice. In contrast, L. major-infected LT beta super(-/-) mice do not develop acute disease, suggesting that the effect of LT beta R-Ig is not due to blockade of membrane LT (LT alpha 1 beta 2) signaling. Collectively, our data show that LIGHT plays a critical role for optimal IL-12 production by DC and the development of IFN- gamma -producing CD4 super(+) Th1 cells and its blockade results in severe susceptibility to Leishmania major.
The prevalence of abnormalities of fibrinolysis in patients with venous thromboembolism is as yet unknown. Defined abnormalities include congenital dysfunction and deficiency of plasminogen, and ...probably impaired plasminogen activation secondary to elevated levels of plasminogen activator inhibitor type 1 (PAI-1) or to impaired release of tissue plasminogen activator (tPA). In this preliminary study, we analyzed plasma samples from 21 patients for whom an investigation for possible thrombophilia was requested. Twenty of the patients had venous thromboembolism, and one had arterial thrombosis at an early age. Two patients had deficiency of protein C or protein S, but no other recognized biochemical disturbances related to thrombophilia were identified. Patient samples and plasma from 25 normal controls were assayed for tPA activity, PAI-1 activity, and urokinase (uPA) activity and antigen. tPA activity and antigen were not significantly different in patients than in controls. PAI-1 activity was significantly greater in patients (P < 0.0001). uPA activity was not different in the two groups. However, uPA antigen was significantly reduced in patients compared to controls (P = 0.001). These data suggest that hypofibrinolysis leading to a risk of thrombosis may be caused not only by elevated PAI-1 activity but also by reduced total uPA concentration.
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