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Biomedical subjects

L Lu

Publications and source records attributed to L Lu.

At least 433 records · Page 24Linked to original sources
PubMed1995

Comparison of amplified Q beta replicase and PCR assays for detection of Mycobacterium tuberculosis.

Because of the long time required to isolate Mycobacterium tuberculosis in culture, there is an acute need for simple rapid methods for direct detection of M. tuberculosis from human sputum specimens. We have developed and characterized quantitative manual Q beta replicase and PCR assays for M. tuberculosis. The Q beta replicase assay was based on reversible target capture of M. tuberculosis 23S rRNA followed by amplification of a replicatable detector probe with Q beta replicase. For PCR assays, primers generating a 370-bp amplification product from the IS6110 insertion element were used in combination with a control plasmid containing an internal deletion in the IS6110 amplicon. Serial dilutions of M. tuberculosis were spiked into sputum and subjected to digestion and decontamination with N-acetyl-L-cysteine and NaOH. Assay conditions were optimized for hybridization and sample processing chemistries in order to maximize sample utilization. Following assay optimization, the sensitivities of the Q beta replicase and PCR assays of spiked sputum samples were 0.5 and 5.0 CFU per assay reaction, respectively. The effects of sputum matrix on each assay were examined by testing 20 patient sputum samples which had been cultured for M. tuberculosis. The culture-positive samples included smear-positive and smear-negative samples. The results of the Q beta replicase assay were not inhibited by sputum and were in 100% agreement with those of culture, including detection of 10 culture-positive specimens. However, using an internal control plasmid coamplified with each PCR as an indicator, we detected PCR inhibition in 9 of 20 samples tested. Decreasing the amount of sample assayed in the PCR 24-fold alleviated the inhibitory effects in all but two specimens, one of which was culture positive. The decreased sample utilization also resulted in a false-negative result with a third specimen which was culture positive for M. tuberculosis. Quantitative smear results and QB replicase assay estimates of the number of organisms present in these specimens were in close agreement. The QB replicase assay performed well in comparison with both culture and PCR and should offer a rapid means for detecting and controlling infection due to M. tuberculosis.

Bacteriological Techniques↗
PubMed1995

Cryopreserved cord blood myeloid progenitor cells can serve as targets for retroviral-mediated gene transduction and gene-transduced progenitors can be cryopreserved and recovered.

To determine future possibilities for gene transfer, we evaluated whether myeloid progenitor cells from human umbilical cord blood (CB) could be frozen, thawed in viable form and transduced with a Neomycin resistance (NeoR) gene using retroviral vectors, and if fresh progenitor cells transduced with a Neo gene could be cryopreserved and recovered. Fresh and thawed cryopreserved nonadherent low-density T-lymphocyte depleted (NALT-) CB cells were assayed before and after gene transduction for colony formation in the presence of multiple growth factors in the absence and presence of G418. The results demonstrate that the NeoR gene could be introduced into thawed cryopreserved myeloid progenitor cells at an efficiency similar to that of fresh cells and that fresh cells transduced with the NeoR gene could be frozen in a cryopreserved state and recovered after thawing. Proviral integration, as assessed by PCR/Southern Analysis, confirmed the G418R colony data. Proviral integration was detected not only in primary G418R-colonies, but also in replated colonies in secondary dishes derived from G418R-multipotential progenitor cells (CFU-GEMM) suggesting stable integration of the transduced gene into early subsets of replatable progenitors. This information may be of use clinically.

3T3 Cells↗
PubMed1995

Flt3 ligand stimulates/costimulates the growth of myeloid stem/progenitor cells.

The present studies evaluated effects of recombinant human (rhu) and murine (rmu) flt3 ligand (flt3-L) on colony formation by subsets of myeloid stem and progenitor cells present in low-density (LD) and cell-sorted CD34 hu cord blood (CB) and bone marrow (BM) cells and unseparated mu BM cells. By itself, flt3-L had weak colony-stimulating activity. It stimulated small dispersed CFU-GM-type colonies, but not BFU-E, CFU-GEMM, or HPP-CFC colonies, from LD and CD34 huCB and BM. However, flt3-L had additive to greater-than-additive effects on colony number and size by CFU-GM stimulated with GM-CSF or IL-3, with or without Steel factor (SLF); by CFU-G stimulated by G-CSF with or without SLF; by CFU-M stimulated by CSF-1; and by BFU-E, CFU-GEMM, and HPP-CFC stimulated by Epo with or without IL-3 or SLF. Flt3-L enhanced the effects of SLF, alone and in combination with other CSFs. Similar effects were apparent on LD and sorted CD34 cells and also at the level of single sorted and isolated CD34 cells/well. Flt3-L enhanced expansion of immature subsets of huCD34(+)-column separated CB CFU-GM stimulated by the potent combination of SLF and PIXY321 (a GM-CSF/IL-3 fusion protein). While flt3-L did not enhance the replating capacity of CFU-GEMM plated in the presence of Epo and SLF, it enhanced numbers of these CFU-GEMM colonies with the capacity to be replated. Flt3-L effects were not species-specific; rhu and rmu forms were active on huCB/BM and muBM. These results demonstrate the potent direct-acting stimulating/costimulating activities of flt3-L in vitro on myeloid stem/progenitor cells.

Adult↗
PubMed1995

Presensitization by skin grafting from major histocompatibility complex class I or major histocompatibility complex class II deficient mice identifies class I antigens as inducers of allosensitization.

Livers but not hearts are accepted spontaneously without immunosuppression when transplanted from B10 (KbAbEbDb) to C3H (KkAkEkDk) mice. Both organs however, undergo accelerated rejection in C3H recipients presensitized with B10 skin grafts. In this study, we have investigated further the role of functional cell-surface major histocompatibility complex (MHC class I or class II molecules in allosensitization. Skin from transgenic MHC class I (b2mmlUncbcr; AbEb) or class II (C2DTM, KbDb) gene 'knockout' mice was grafted onto naive recipients 2-3 weeks prior to whole organ transplantation. When C3H hosts were presensitized with skin from C2DTM (class II deficient) mice, they promptly rejected (within 4 days) subsequently transplanted B10 liver or heart allografts. In contrast, presensitization with skin from b2m (beta 2-m mutant; class I deficient) mice did not significantly affect the survival of either organ graft. Maximal sensitization was established by day 14 after skin grafting and persisted for at least 12 weeks. Splenocytes obtained from C3H mice sensitized with skin from B10, B6 (KbAbEbDb), or C2DTM but not from b2m mice exhibited an H-2b-specific cytolytic response when tested in cell-mediated lymphocytotoxicity assays. Sera from C3H mice sensitized with B10 or b2m skin contained high titres of cytotoxic activity specifically against H-2b class I. Taken together, these observations suggest that in the strain combination studied, MHC class I rather than class II molecules play an important role in allosensitization. The results indicate the potential importance of avoiding transplantation of organs into recipients of secondary grafts from donors that share human leucocyte antigen (HLA) class I antigens with the first donor.

Animals↗
PubMed1995

Influence in vitro of IL-3/Epo fusion proteins compared with the combination of IL-3 plus Epo in enhancing the proliferation of single isolated erythroid and multipotential progenitor cells from human umbilical cord blood and adult bone marrow.

Human interleukin-3/erythropoietin (IL-3/Epo) fusion protein have been constructed, expressed, and tested for biological activity. These fusion proteins were previously shown to be active on erythroid progenitors (BFU-E) from unseparated human bone marrow. We evaluated if these fusion proteins could stimulate erythroid and multipotential progenitor cells directly at the single-cell level. Two IL-3/Epo fusion proteins containing short (SL-3E, two amino acids) and long (LL-3E, 23 amino acids) linker sequences as well as a short linker Epo/IL-3 sequence (SL-E3, three amino acids) were tested. Highly enriched CD34 or BFU-E enriched CD34 CD33- cells from human umbilical cord blood or CD34 HLA-DR+CD33- cells from normal adult bone marrow were sorted as single cells into single wells. The combination of Epo plus IL-3 synergized to enhance the proliferation of BFU-E and multipotential progenitors (CFU-GEMM) in comparison to the individual effects of these cytokines. The three fusion proteins also enhanced proliferation of BFU-E and CFU-GEMM at the single-cell level and were at least as active as the combination of Epo and IL-3, demonstrating that IL-3/Epo fusion proteins directly stimulate proliferation of BFU-E and CFU-GEMM and that biological activity of IL-3 and Epo in vitro can be maintained when these proteins are fused. The activity of the combination of Epo and IL-3 or the fusion proteins was partially neutralized by preincubation with monoclonal antibodies to either Epo or IL-3 and was neutralized by greater than 90% by the combination of both antibodies, suggesting that the Epo and IL-3 components of the fusion proteins were both involved in the enhancing activity of these proteins. Additionally, use of monoclonal antibody to the human Epo receptor completely blocked the stimulating/enhancing activity of Epo alone, Epo plus IL-3, or the fusion proteins for stimulation of colony formation by BFU-E and CFU-GEMM but not for granulocyte-macrophage progenitors (CFU-GM), suggesting that the enhancing effects of the fusion proteins are most likely mediated, at least in part, by the Epo receptor.

Bone Marrow Cells↗
PubMed1995

Purification and partial amino acid sequences of a new presynaptic toxin and a cytotoxin from venom of pit veper Agkistrodon blomhoffii brevicaudus.

The technique of the reverse-phase performance liquid chromatography (RP-HPLC) was employed to separate and purify the toxic proteins from the venom of Agkistrodon blomhoffii brevicaudus collected in China. 3 toxic proteins marked as AgTx-1, AgTx-2 and AgTx-3 consisting of about 122 amino acid residues were screened. The toxicities (LD50) of the AgTx-1, AgTx-2 and AgTx-3 were 0.075, 0.51 and 6.6 mg per kg weight of mice respectively. Toxicological experiment in the chick biventer cervicis nerve-muscle preparation showed that the acetylcholine (Ach) sensitivity of the preparation was unchanged after the total failure of the indirect contraction caused by AgTx-1 and AgTx-2, suggesting that they were presynaptic blockers, namely beta-type of snake toxins. However, the amplitude of indirect contraction of the preparation was gradually reduced due to its incomplete relaxation caused by AgTx-3, indicating that it should belong to the category of cytotoxins. The partial amino acid sequences of 3 toxins have been established. It was found in ref. [1] that the sequences of the first 32 N-terminal amino acid residues of AgTx-1 and AgTx-2, as well as beta-agkistrodotoxin (beta-AgTx) reported previously were identical (the residue at the position 30 of beta-AgTx should be Trp). In view of the similarity in toxicities, and the amounts in the venom and other properties, it was concluded that AgTx-1 should be beta-AgTx and consequently was renamed beta 1-AgTx. AgTx-2 should be the isoform of beta 1-AgTx, and correspondingly named beta 2-AgTx.

Agkistrodon↗
PubMed1995

[Establishment of a human fetal thymocyte cell line and observation of its biological properties].

Thymocytes were isolated from a five month fetus after caesarean section and cultured in vitro. A cell line named human fetal thymocyte 8810 (HFT8810) was established and cultured continuously for 2 years in our lab, and grew well after recovery from the state of being frozen in liquid nitrogen. The HFT 8810 cells retained their ability to secrete thymosin after many generations in vitro culture.

Animals↗
PubMed1995

Generation of DC from mouse spleen cell cultures in response to GM-CSF: immunophenotypic and functional analyses.

In all tissues that have been studied to date, dendritic leucocytes constitute only a small proportion of total cells and are difficult both to isolate and purify. This study reports on a method for the propagation of large numbers of dendritic cells (DC) from mouse spleen using granulocyte-macrophage colony-stimulating factor (GM-CSF) and their characteristics. Within a few days of liquid culture in GM-CSF, B10 BR (H-2k, I-E+) mouse splenocytes formed loosely adherent myeloid cell clusters. Mononuclear progeny released from these clusters at and beyond 4 days exhibited distinct dendritic morphology and strongly expressed leucocyte common antigen (CD45), CD11b, heat-stable antigen, Pgp-1 (CD44) and intercellular adhesion molecule-1 (ICAM-1; CD54). The intensity of expression of the DC-restricted markers NLDC 145 and 33D1, the macrophage marker F4/80, and Fc gamma RII (CDw32) was low to moderate, whereas the cells were negative for CD3, CD45RA and NK1.1. High and moderate levels, respectively, of cell surface staining for major histocompatibility complex (MHC) class II (I-Ek) and the B7 antigens (counter-receptors of CTLA4, a structural homologue of CD28) were associated with potent stimulation of unprimed, allogeneic T cells (B10; H-2b, I-E-). DC propagated in a similar fashion from DBA/2 mouse spleen proved to be strong antigen-presenting cells (APC) for MHC-restricted, syngeneic T-helper type 2 (Th2) cell clones specifically responsive to sperm whale myoglobin. Footpad or intravenous injection of GM-CSF-stimulated B10.BR spleen-derived DC into B10 (H-2b, I-E-) recipients resulted in homing of the allogeneic cells to T-cell-dependent areas of lymph nodes and spleen, where they strongly expressed donor MHC class II antigen 1-2 days later. These findings indicate that cells can be propagated from fresh splenocyte suspensions that exhibit distinctive features of DC, namely morphology, motility, cell-surface phenotype, potent allogeneic and syngeneic APC function and in vivo homing ability. Propagation of DC in this manner from progenitors present in lymphoid tissue provides an alternative and relatively convenient source of high numbers of these otherwise difficult to isolate but functionally important APC.

Animals↗
PubMed1995

Modified vitreous surgery for subretinal neovascularization and hemorrhage.

PURPOSE: To modify vitreous surgery for subretinal neovascularization (SRNV) and to determine the effects of this operation. METHODS: Six patients with SRNV were performed with this operation. The methods of examination before and after operation included: testing the best-corrected visual acuity before operation and 1, 3 or 6 months after operation; 30 degrees and macular 10 degrees Humphrey visual field examination; FFA examination preoperatively and 1, 3 or 6 months postoperatively. Modified surgery procedure is: a preventive buckling; pars plana vitrectomy; peeling the vitreous cortical; no intraocular diathermy; small retinotomy; subretinal surgery; air-fluid exchange. RESULTS: SRNV was taken off in 4 cases. Subretinal hemorrhage was washed in 2 cases. After 4 to 7 months follow-up, the visual acuity was improved in 4 cases, unchanged in 2 cases. The visual field was improved in 4 cases, unchanged in 1 case, decraesed in 1 case. The complications included macular hole due to surgery in 1 case and subretinal hemorrage in 1 case. CONCLUSION: The surgery criteria were: 1) massive subretinal hemorrhage; 2) some patients of SRNV included: FFA evidence they showed the membrane is beneath fovea, the best V. A is 20/100 or lower, and can't be treated by laser and the patient consent. This modified subretinal operation is safe, and effective for massive hemorrhage and some SRNVS.

Adult↗
PubMed1995

Excitotoxic cell death and delayed rescue in human neurons derived from NT2 cells.

The excitotoxic response of NT2-N cells, a clonal line of human teratocarcinoma cells that are terminally differentiated into neuron-like cells, was examined using several endpoints. A 15 min exposure to glutamate produced a dose-dependent toxicity with a maximal cell loss of 80-90% in 6 week old cells. The rapidly triggered excitotoxicity induced by glutamate was blocked by NMDA selective antagonists, was calcium dependent and pH sensitive and could be mimicked by NMDA but not by non-NMDA agonists, AMPA, kainate or quisqualate. The non-NMDA agonists however caused toxicity on prolonged exposure. The NMDA receptor modulators glycine and spermidine enhanced glutamate-mediated toxicity whereas ifenprodil potently and completely inhibited toxicity suggesting that the toxic response is mediated by the NR1/NR2B combination of NMDA subunits. These cells can be rescued from death up to 1 hr after removal of glutamate by NMDA receptor blockade, removal of extracellular Ca2+ or lowering of pH. The extent of rescue is directly related to the time elapsed before intervention. Blockage of NMDA receptor activity for 1 hr immediately after removal of glutamate is both necessary and sufficient for complete rescue. Glutamate-mediated toxicity was not prevented by nitric oxide synthase inhibitors nor was nitric oxide synthase detected in NT2-N cells indicating that nitric oxide is not required for glutamate-mediated excitotoxicity. In summary, NT2-N cells exhibit a robust excitotoxic response and represent a novel model system in which to study the molecular basis of excitotoxic cell death.

2-Amino-5-phosphonovalerate↗
PubMed1995

Epidemiology of anti-human T-cell leukemia virus type I antibody and characteristics of adult T-cell leukemia in China.

We analyzed the data from seroepidemiologic survey of anti-HTLV-I serum antibody (HTLV-1 Ab) and 16 adult cases of T-cell leukemia (ATL) in China (1984-1994). The results showed that the positive rate of HTLV-I Ab of the population of China was 0.54%, the birth or residence places of HTLV-I carriers and all the patients with ATL were mainly distributed in the coastal provinces and the northeast China, and a small endemic area of HTLV-I and ATl was found in Fujian Province (HTLV-I Ab positive rate, 2.1%). The HTLV-I in China may be transmitted from Japan or originated from China. The clinical characteristics of the cases included onset in adulthood; frequent skin lesions, lymphadenopathy and hepatosplenomegaly; no mediastinal mass; the ATL cells from peripheral blood with polymorphism nuclei and mature T cell immunophenotype HTLV-IAb positive in most of the cases tested (5/8); mostly acute type; with a median survival period of 3.5 months.

Adult↗
PubMed1994

Isolation, phenotype, and allostimulatory activity of mouse liver dendritic cells.

Donor liver-derived dendritic cells (DC) have recently been identified within various lymphoid and nonlymphoid tissues of organ allograft recipients, including nonimmunosuppressed mice transplanted with and permanently accepting major histocompatibility complex (MHC)-disparate hepatic allografts. These findings have raised questions about the basis of the tolerogenicity of the liver--and, in particular, about the properties of liver-derived DC. To study further the structure, immunophenotype and allostimulatory activity of leukocytes resident in normal mouse (B10.BR;H-2k, I-Ek) liver, a procedure was developed to maximize the yield of viable, nonparenchymal cells (NPC) obtained following collagenase digestion of perfused liver fragments and density centrifugation (Percoll). These cells comprised populations expressing lymphoid and myeloid cell surface antigens. As compared with spleen cells, they proved good allostimulators of naive (B10; H-2b, I-E-) splenic T cells when tested in primary mixed leukocyte reactions (MLR). After overnight (18-hr) incubation of the NPC, enrichment for transiently adherent, low-density (LD) cells on metrizamide gradients permitted the recovery of low numbers of cells (approx. 2-5 x 10(5) per liver), many of which displayed distinct DC morphology. Flow cytometric analysis revealed that these cells were CD3-, CD4-, CD8-, and B220-, but strongly expressed CD45 (leukocyte-common antigen), and mild-to-moderate levels of CD11b, heat-stable antigen, and CD44. The cells also expressed moderate intensity of NLDC 145 but not 33D1, DC restricted markers which have been shown to be differentially expressed on mouse DC isolated from various organs. This DC-enriched population was more strongly MHC class II(I-Ek)+ than NPC, as determined by immunocytochemistry and flow cytometry and exhibited much more potent allostimulatory activity for naive T cells. These findings demonstrate that freshly isolated murine liver NPC, and perhaps their counterparts in situ, exhibit allostimulatory activity that is enhanced in the non-adherent, low-density (DC-enriched) fraction after overnight culture. They further suggest that the maturation of liver DC may play a key role in determining the immunogenicity and or tolerogenicity of hepatic allografts.

Animals↗
PubMed1994

Influence of elevated temperature on natural killer cell activity, lymphokine-activated killer cell activity and lectin-dependent cytotoxicity of human umbilical cord blood and adult blood cells.

PURPOSE: To determine whether hyperthermia is to the benefit or detriment of host immune function, the effect of hyperthermia was evaluated on various functions of T-lymphocytes from human umbilical cord blood and compared to that of adult blood. METHODS AND MATERIALS: Nonadherent mononuclear cells from cord blood or adult blood were used as the effector cells. To generate lymphokine activated killer (LAK) cells, effector cells were kept in culture for 5 days in complete medium containing recombinant human interleukin-2. To activate effector cells to become cytotoxic, cells were kept in culture in complete medium containing Con A. Cytotoxicity was determined in a standard 4-h chromium release assay using K-562 human erythroleukemic cells (in the natural killer cell activity assay) or Daudi cells (in the LAK cell activity or Lectin dependent cytotoxicity assay) as targets. For heat effects, cells in complete medium were heated at the desired temperature in a water bath for 1 h. RESULTS: Lymphokine-activated killer cell activity, lectin-dependent cytotoxicity and T-cell proliferative capacity were not deficient in human cord blood. Cytotoxic activities of T-cells from adult blood as well as from cord blood can be enhanced at febrile range (< or = 40 degrees C), and were significantly decreased by exposure to 1 h at 42 degrees C. CONCLUSION: The febrile responses (< or = 40 degrees C) to infection, in the course of malignant disease and with biological response modifiers treatment, may all be related to host defense mechanisms. Based on these observations, whole body hyperthermia (< or = 40 degrees C), in combination with the appropriate cytokines, may have therapeutic potential in the treatment of neonatal infections and malignancies under certain circumstances. Hyperthermia in febrile range may, therefore, confer an important immunoregulatory advantage to the host. In contrast, tumor killing therapeutic temperature (> 42 degrees C) which inhibits host immunocompetence should probably be used only for local hyperthermia.

Adult↗
PubMed1994

Propagation of dendritic cell progenitors from normal mouse liver using granulocyte/macrophage colony-stimulating factor and their maturational development in the presence of type-1 collagen.

Within 1 wk of liquid culture in granulocyte/macrophage colony-stimulating factor (GM-CSF), normal B10 BR (H-2k I-E+) mouse liver nonparenchymal cells (NPC) formed loosely adherent myeloid cell clusters that have been shown to contain dendritic cell (DC) progenitors in similar studies of mouse blood or bone marrow. Mononuclear cell progeny released from these clusters at and beyond 4 d exhibited distinct dendritic morphology and were actively phagocytic. After 6-10 d of culture, these cells strongly expressed CD45, CD11b, heat stable antigen, and CD44. However, the intensity of expression of the DC-restricted markers NLDC 145, 33D1, and N418, and the macrophage marker F4/80, intercellular adhesion molecule 1, and Fc gamma RII was low to moderate, whereas the cells were negative for CD3, CD45RA, and NK1.1. Splenocytes prepared in the same way also had a similar range and intensity of expression of these immunophenotypic markers. Unlike the splenic DC, however, most of the GM-CSF-propagated putative liver DC harvested at 6-10 d expressed only a low level of major histocompatibility complex (MHC) class II (I-Ek), and they failed to induce primary allogeneic responses in naive T cells, even when propagated additionally in GM-CSF and tumor necrosis alpha and/or interferon gamma-supplemented medium. However, when 7-d cultured GM-CSF-stimulated liver cells were maintained additionally for three or more days on type-1 collagen-coated plates in the continued presence of GM-CSF, they exhibited characteristics of mature DC: MHC class II expression was markedly upregulated, mixed leukocyte reaction stimulatory activity was increased, and phagocytic function was decreased. Similar observations were made when Ia+ cells were depleted from the GM-CSF-propagated cells before exposure to collagen. Further evidence that the GM-CSF-stimulated class IIdim or class II-depleted hepatic NPC were immature DC was obtained by injecting them into allogeneic B10 (H-2b I-E-) recipients. They "homed" to T cell-dependent areas of lymph nodes and spleen where they strongly expressed donor MHC class II antigen 1-5 d later. These observations provide insight into the regulation of DC maturation, and are congruent with the possibility that the migration of immature DC from normal liver and perhaps other organ allografts may help explain their inherent tolerogenicity.

Animals↗