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Biomedical subjects

M Lu

Publications and source records attributed to M Lu.

At least 37 records · Page 2Linked to original sources
PubMed2003

Molecular genetic and biochemical analysis of woodchuck (Marmota monax) MHC class I polymorphism.

The woodchuck (Marmota monax) is an animal model that is used in the study of human hepatitis B virus ( HBV ) infection. A knowledge of woodchuck MHC class I (Mamo-I) genes and gene products is therefore essential for understanding the antigen-specific T-cell responses in this animal model. A number of Mamo-I genes have been identified by molecular cloning and sequencing. However, the allelic nature of these genes has not been proven by classical genetics like the segregation analysis in families. In this study, we analyzed the allelic diversity of Mamo-I in two three-generation woodchuck families including 15 members by sequencing of Mamo-I genes and immunoblotting of Mamo-I proteins after one-dimensional isoelectric focusing (1D-IEF). In addition to four published Mamo-I alleles, six new alleles that belonged to the same locus as the known Mamo-I alleles (Mamo-A) were found within the two woodchuck families. A typical Mendelian segregation of Mamo-I gene and antigens was observed in the families studied. For simple and rapid detection of allelic variability of Mamo-I gene, a typing method based on the detection of PCR products amplified by sequence specific primers (SSP) has been developed and tested in 41 unrelated animals. The most prevalent allele was Mamo-A*01 with a frequency of 21.9% followed by Mamo-A*07 (12.2%). Our study established Mamo-A as a classical MHC class I locus by the polymorphic and allelic nature of Mamo-I gene in the woodchuck.

Amino Acid Sequence↗
PubMed2003

Molecular characterization of CD28 and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) of woodchuck (Marmota monax).

Eastern woodchuck (Marmota monax) became an important animal model to study the immunological processes in hepatitis B virus infection. To facilitate further study of T-cell responses in this model, we cloned and sequenced the cDNAs of Woodchuck CD28 and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), which play important roles for the regulation of T-cell activation by delivering the costimulation signals. According to the deduced amino-acid sequences, Woodchuck CD28 showed a similarity of 70% to 80% to its mammalian homologues. Woodchuck CTLA-4 has a higher similarity of 74% to 85% to corresponding mammalian CTLA-4 molecules. The strict conservation of critical amino-acid residues like cystein and asparagine residues in Woodchuck CD28 and CTLA-4 suggests that both molecules may structurally resemble their human or mouse homologues. A hexapeptide motif, MYPPPY, which has been supposed to be essential for the interaction with CD80, is present in both Woodchuck CD28 and CTLA-4. The cloned cDNAs of Woodchuck CD28 and CTLA-4 were placed under the control of the cytomegalovirus (CMV) promoter of the mammalian expression vector pcDNA3. Both proteins were expressed and detected by respective crossreactive antibodies in transiently transfected mammalian cells. By immunohistochemical staining with these antibodies, CD28 and CTLA-4 were also detected on cultured woodchuck peripheral blood lymphocytes. The molecular characterization of Woodchuck CD28 and CTLA-4 will facilitate studies on the T-cell response to hepadnavirus in the woodchuck model.

Animals↗
PubMed2003

Pre-neutralization of C5a-mediated effects by the monoclonal antibody 137-26 reacting with the C5a moiety of native C5 without preventing C5 cleavage.

Complement C5a is aetiologically linked to inflammatory tissue damage in conditions like septicaemia, immune complex diseases and ischaemia-reperfusion injury. We here describe a monoclonal antibody (mAb), 137-26, that binds to the C5a moiety of human C5 and neutralizes the effects of C5a without interfering with C5 cleavage and the subsequent formation of lytic C5b-9 complex. Mouse anti-human C5 mAbs were generated and the reactivity with C5 and C5a was detected by ELISA and surface plasmon resonance. The inhibition of C5a binding to C5a receptor was studied using a radioligand binding assay. The effects of the antibody on C5a functions were examined using isolated neutrophils and a novel human whole blood model of inflammation. Haemolytic assays were used to study the effect on complement-mediated lysis. mAb 137-26 reacted with both solid- and solution-phase C5 and C5a in a dose-dependent manner with high affinity. The antibody competed C5a binding to C5a receptor and inhibited C5a-mediated chemotaxis of neutrophils. Furthermore, the antibody effectively abrogated complement-dependent E. coli-induced CD11b up-regulation and oxidative burst in neutrophils of human whole blood. mAb 137-26 was more potent than a C5a receptor antagonist and a previously described anti-C5a antibody. mAb 137-26 did not inhibit complement-mediated lysis, nor did it activate complement itself. Together, mAb 137-26 binds both the C5a moiety of native C5 and free C5a, thereby effectively neutralizing the biological effects of C5a. The antibody may have therapeutic potential in inflammatory diseases where C5a inhibition combined with an operative lytic pathway of C5b-9 is particularly desired.

Animals↗
PubMed2003

The infectivity of Plasmodium yoelii in different strains of mice.

We evaluated the effect of using Medium 199 alone and Medium 199 supplemented with 5% normal mouse serum, 5% fetal calf serum, 5% bovine serum albumin or 5% Albumax on Plasmodium yoelii sporozoite yield from infected mosquitoes and infectivity in BALB/c mice. The sporozoites yield, as well as their infectivity, was statistically lower (P = 0.0031) when unsupplemented Medium 199 was used to separate sporozoites from infected mosquitoes. Although Medium 199 supplemented with Albumax led to lower sporozoite yield (P < 0.0009), infectivity of the sporozoites was similar to those obtained with the other medium supplements. Because normal mouse serum supports good sporozoite infections and is also the supplement that can be used repeatedly in mice during multiple sporozoite injections without inducing anaphylaxis, we selected it to evaluate the infectivity of P. yoelii sporozoites in different strains of mice. After injecting mice with serial dilutions of sporozoites and detecting patent infections, we determined that the infective dose 50 (ID50) for BALB/c, C57Bl/6, A/J, and B10BR mice ranged between 4.9 and 10.6 sporozoites. The ID50 obtained for CD-1 mice (147 sporozoites) was significantly higher.

Animals↗
PubMed2002

Human marrow stromal cell therapy for stroke in rat: neurotrophins and functional recovery.

OBJECTIVE: To test the effect of i.v.-injected human bone marrow stromal cells (hMSC) on neurologic functional deficits after stroke in rats. METHODS: Rats were subjected to transient middle cerebral artery occlusion and IV injected with 3 x 10(6) hMSC 1 day after stroke. Functional outcome was measured before and 1, 7, and 14 days after stroke. Mixed lymphocyte reaction and the development of cytotoxic T lymphocytes measured the immune rejection of hMSC. A monoclonal antibody specific to human cellular nuclei (mAb1281) was used to identify hMSC and to measure neural phenotype. ELISA analyzed neurotrophin levels in cerebral tissue from hMSC-treated or nontreated rats. Bromodeoxyuridine injections were used to identify newly formed cells. RESULTS: Significant recovery of function was found in rats treated with hMSC at 14 days compared with control rats with ischemia. Few (1 to 5%) hMSC expressed proteins phenotypic of brain parenchymal cells. Brain-derived neurotrophic factor and nerve growth factor significantly increased, and apoptotic cells significantly decreased in the ischemic boundary zone; significantly more bromodeoxyuridine-reactive cells were detected in the subventricular zone of the ischemic hemisphere of rats treated with hMSC. hMSC induced proliferation of lymphocytes without the induction of cytotoxic T lymphocytes. CONCLUSION: Neurologic benefit resulting from hMSC treatment of stroke in rats may derive from the increase of growth factors in the ischemic tissue, the reduction of apoptosis in the penumbral zone of the lesion, and the proliferation of endogenous cells in the subventricular zone.

Animals↗
PubMed2002

Electrochemical behavior of Au colloidal electrode through layer-by-layer self-assembly.

Electrodes formed by Au colloidal nanoparticles have been obtained by layer-by-layer self-assembly using 1,6-hexanedithiol as cross-linkers. Cyclic voltammograms show that the peak-to-peak separation decreases as the number of Au colloidal layers increases. After seven layers of Au colloidal particles have been deposited, the multilayer electrodes have the electrochemical properties of metallic Au and show ideal microelectrode behavior. An equivalent circuit for the electrochemical impedance spectroscopy was established to model the working electrode. It is evident that by increasing the layer number of Au colloidals, the interfacial electron transfer is promoted, implying the electron-transfer process changes from a kinetically limited process to a diffusionally limited process.

Colloids↗
PubMed2002

Modelling the cost-effectiveness of sentinel lymph node mapping and adjuvant interferon treatment for stage II melanoma.

Clinical studies have demonstrated that high dose adjuvant interferon therapy improves disease-free and overall survival among high risk (stage IIb and III) melanoma patients. Sentinel lymph node mapping (SLM) has been shown to accurately detect micrometastasis and may be used to identify higher risk stage II patients, who might benefit most from adjuvant interferon therapy. We modelled the cost-effectiveness of first testing with SLM and then treating with adjuvant interferon (IFN) therapy for stage II melanoma. We used a decision analytical model to compare four strategies for stage II patients after surgical excision of their melanoma: (1) treat all with low dose IFN; (2) test first with SLM and then treat only those with positive micrometastasis with high dose IFN; (3) test first with SLM and treat positives with high dose IFN and negatives with low dose IFN (test and treat appropriately); and (4) surgery only. Treatment, toxicity, follow-up and relapse costs were included over a 5 year time period. The primary outcome was cost per quality-adjusted relapse-free life year saved. Our analysis shows that, compared with the current surgery-only strategy, all three treatment strategies provide incremental benefits. The test and treat appropriately strategy is the most effective, with an incremental improvement of 0.64 quality-adjusted life-years (QALY). The cost-effectiveness of test and treat some with high dose IFN compared with the surgery-only strategy is $18,700/QALY. The test and treat appropriately strategy is also cost-effective compared with test and treat some at $31,100/QALY. In conclusion appropriate dosing of IFN therapy based on the results of SLM is a cost-effective strategy for stage II melanoma patients.

Adjuvants, Immunologic↗
PubMed2002

RT-PCR-based detection of circulating calcitonin-producing cells in patients with advanced medullary thyroid cancer.

In patients with medullary thyroid carcinoma (MTC) the clinical course of disease ranges from rapid tumor progression to long-lasting stable disease. The purpose of the present study was to investigate whether circulating tumor cells can be detected in the peripheral blood of patients with MTC by RT-PCR targeted to calcitonin (CT) mRNA and whether the results of this method are correlated with disease manifestation and metastatic potential. Blood samples from 19 consecutive patients with MTC and elevated CT levels were analyzed. Four had newly diagnosed MTC, and 15 patients had undergone total thyroidectomy. Six of 19 patients had detectable CT mRNA by RT-PCR. CT levels in the CT mRNA-positive patients were significantly higher than those in CT mRNA-negative patients [2,239-265,313 pg/ml; median 80,921 pg/ml (n = 6) vs. 70-46,787 pg/ml; median, 932 pg/ml (n = 13); P = 0.006]. CT mRNA was detectable in 5 of 8 patients with distant metastases, in 1 of 6 patients with local/regional lymph node metastases, but in none of the patients with newly diagnosed, organ-confined MTC (n = 2) or surgically treated MTC without tumor manifestation by various imaging studies (n = 3). In peripheral blood from 10 healthy volunteers and 21 patients with benign thyroid nodules, no CT RNA could be detected. In conclusion, an RT-PCR-based procedure was established to detect circulating CT-producing cells in the peripheral blood of patients with MTC. RT-PCR results seem to reflect tumor spread and aggressiveness and thus may help with early identification of patients with disseminated and rapidly progressive disease.

Adult↗
PubMed2002

Efficiency and structure-activity relationship of the antioxidant action of resveratrol and its analogs.

Resveratrol and its analogs, six other polyhydroxystilbenes, were synthesized and their antioxidative activities were evaluated in vitro by determination of the levels of malondialdehyde and hydrogen peroxide. Results clearly exhibited that resveratrol and its analogs had various potencies in inhibiting lipid peroxidation in rat brain, kidney, and liver homogenates and rat erythrocyte hemolysis. Several polyhydroxystilbenes were found to be more active than resveratrol in these models, and structure-activity relationship studies on polyhydroxystilbenes are described in this paper.

Animals↗
PubMed2001

Lack of clinical significance of early ischemic changes on computed tomography in acute stroke.

CONTEXT: The prevalence and clinical significance of early ischemic changes (EICs) on baseline computed tomography (CT) scan of the head obtained within 3 hours of ischemic stroke are not established. OBJECTIVE: To determine the frequency and significance of EIC on baseline head CT scans in the National Institute of Neurological Disorders and Stroke (NINDS) rt-PA (recombinant tissue plasminogen activator) Stroke Trial. DESIGN AND SETTING: The original study, a randomized controlled trial, took place from January 1991 through October 1994 at 43 sites, during which CT images were obtained within 3 hours of symptom onset and prior to the initiation of rt-PA or placebo. For the current analysis, detailed reevaluation was undertaken after October 1994 of all baseline head CT scans with clinical data available pretreatment (blinded to treatment arm). PATIENTS: Of 624 patients enrolled in the trial, baseline CT scans were retrieved and reviewed for 616 (99%). MAIN OUTCOME MEASURES: Frequency of EICs on baseline CT scans; association of EIC with other baseline variables; effect of EICs on deterioration at 24 hours (>/=4 points increase from the baseline National Institutes of Health Stroke Scale [NIHSS] score); clinical outcome (measured by 4 clinical scales) at 3 months, CT lesion volume at 3 months, death at 90 days; and symptomatic intracranial hemorrhage (ICH) within 36 hours of treatment. RESULTS: The prevalence of EIC on baseline CT in the combined rt-PA and placebo groups was 31% (n = 194). The EIC was significantly associated with baseline NIHSS score (rho = 0.23; P<.001) and time from stroke onset to baseline CT scan (rho = 0.11; P =.007). After adjusting for baseline variables, there was no EIC x treatment interaction detected for any clinical outcome, including deterioration at 24 hours, 4 clinical scales, lesion volume, and death at 90 days (P>/=.25), implying that EIC is unlikely to affect response to rt-PA treatment. After adjusting for NIHSS score (an independent predictor of ICH), no EIC association with symptomatic ICH at 36 hours was detected in the group treated with rt-PA (P>/=.22). CONCLUSIONS: Our analysis suggests that EICs are prevalent within 3 hours of stroke onset and correlate with stroke severity. However, EICs are not independently associated with increased risk of adverse outcome after rt-PA treatment. Patients treated with rt-PA did better whether or not they had EICs, suggesting that EICs on CT scan are not critical to the decision to treat otherwise eligible patients with rt-PA within 3 hours of stroke onset.

Aged↗
PubMed2001

Thrombolysis in patients with transient neurologic deficits.

What is the risk of thrombolysis in patients with acute stroke who might recover without treatment? In the National Institute of Neurological Disorders and Stroke rt-PA for Acute Stroke Trial, 2.6% of patients taking placebo showed spontaneous 24-hour recovery, compared to 11.5% of recombinant tissue-type plasminogen activator (rt-PA)-treated patients (p < 0.001). There were no symptomatic ICH in the patients taking placebo; one hypertensive, rt-PA-treated patient hemorrhaged. Assuming the National Institute of Neurological Disorders and Stroke protocol is followed rigorously, patients with acute stroke rarely recover spontaneously and the thrombolytic risk is low.

Aged↗
PubMed2001

Postischemic (6-Hour) treatment with recombinant human tissue plasminogen activator and proteasome inhibitor PS-519 reduces infarction in a rat model of embolic focal cerebral ischemia.

BACKGROUND AND PURPOSE: The proteasome inhibitor PS-519 blocks activation of nuclear factor-kappaB, a major mediator of inflammation. We tested the hypothesis that combination treatment of recombinant human tissue plasminogen activator (rhtPA) and PS-519 extends the therapeutic window for treatment of stroke with rhtPA without increasing incidence of hemorrhagic transformation. METHODS: The middle cerebral artery (MCA) of male Wistar rats (n=56) was occluded by an embolus. After embolization, animals were randomly divided into the following groups: PS-519 treatment groups: PS-519 was given at 2, 4, or 6 hours after MCA occlusion; rhtPA treatment groups: rhtPA was given at 2 or 4 hours after MCA occlusion; combination treatment groups: PS-519 and rhtPA were given at 2, 4, or 6 hours after MCA occlusion; control group: the same volume of saline was given at 2 hours after MCA occlusion. RESULTS: Administration of PS-519 alone at 2 or 4 hours, but not 6 hours, significantly (P<0.05) reduced infarct volume and improved neurological recovery compared with the control group. Administration of rhtPA alone at 2 hours, but not 4 hours, significantly (P<0.05) reduced infarct volume and improved neurological recovery compared with the control group. Furthermore, combination treatment with rhtPA and PS-519 even at 6 hours significantly (P<0.05) reduced infarct volume, improved neurological recovery, and did not increase the incidence of hemorrhagic transformation compared with the control group or the group treated with PS-519 alone. CONCLUSIONS: Our data suggest that combination treatment with PS-519 and rhtPA extends the neuroprotective effect to at least 6 hours after embolization.

Acetylcysteine↗
PubMed2001

Exploring the function of Tyr83 in bacteriorhodopsin: features of the Y83F and Y83N mutants.

Tyrosine-83, a residue which is conserved in all halobacterial retinal proteins, is located at the extracellular side in helix C of bacteriorhodopsin. Structural studies indicate that its hydroxyl group is hydrogen bonded to Trp189 and possibly to Glu194, a residue which is part of the proton release complex (PRC) in bacteriorhodopsin. To elucidate the role of Tyr83 in proton transport, we studied the Y83F and Y83N mutants. The Y83F mutation causes an 11 nm blue shift of the absorption spectrum and decreases the size of the absorption changes seen upon dark adaptation. The light-induced fast proton release, which accompanies formation of the M intermediate, is observed only at pH above 7 in Y83F. The pK(a) of the PRC in M is elevated in Y83F to about 7.3 (compared to 5.8 in WT). The rate of the recovery of the initial state (the rate of the O --> BR transition) and light-induced proton release at pH below 7 is very slow in Y83F (ca. 30 ms at pH 6). The amount of the O intermediate is decreased in Y83F despite the longer lifetime of O. The Y83N mutant shows a similar phenotype in respect to proton release. As in Y83F, the recovery of the initial state is slowed several fold in Y83N. The O intermediate is not seen in this mutant. The data indicate that the PRC is functional in Y83F and Y83N but its pK(a) in M is increased by about 1.5 pK units compared to the WT. This suggests that Tyr83 is not the main source for the proton released upon M formation in the WT; however, Tyr83 is involved in the proton release affecting the pK(a) of the PRC in M and the rate of proton transport from Asp85 to PRC during the O --> bR transition. Both the Y83F and the Y83N mutations lead to a greatly decreased functionality of the pigment at high pH because most of the pigment is converted into the inactive P480 species, with a pK(a) 8-9.

Bacteriorhodopsins↗
PubMed2001

Ectopic expression of a Chlamydomonas mt+-specific homeodomain protein in mt- gametes initiates zygote development without gamete fusion.

The molecular mechanisms that activate expression of zygote genes after fertilization are obscure. In animals, receptor-ligand interactions during sperm-egg membrane fusion as well as delivery of putative regulatory molecules by the sperm into the egg cytoplasm are proposed to activate zygote development and subsequent transcription of zygote genes. The mechanisms of activation of zygote development in higher plants also are mysterious, in part because of the difficulty of isolating female gametes of higher plants. In the unicellular, biflagellated green alga Chlamydomonas, the early steps in zygote development are much more accessible to investigation. Within minutes after mating type plus (mt+) and mating type minus (mt-) gametes fuse, expression of several zygote-specific transcripts is induced independently of protein synthesis. Here, we show that ectopic expression in mt- gametes of an mt+ gamete-specific, homeodomain protein, GSP1, induces a zygote-like phenotype and activates expression of zygote genes. One of the genes, zsp2, expressed in these "haploid zygotes" encodes a zygote cell surface adhesion molecule that promotes formation of multicellular aggregates. In total, expression of six out of seven zygote genes examined was induced by ectopic expression of GSP1. Our experiments show that in addition to contributing their genomes to the zygote cytoplasm, gametes also deliver proteins that can activate gene transcription.

Animals↗
PubMed2001

Immunization of woodchucks (Marmota monax) with hepatitis delta virus DNA vaccine.

We investigated the DNA immunization approach in order to induce a protective immune response against hepatitis delta virus (HDV) superinfection of chronically woodchuck hepatitis virus (WHV) infected woodchucks. The animals were immunized with an expression vector encoding HDAg by gene gun. T cell and humoral immune responses induced by this protocol were determined and compared with those induced by HDAg immunization using a CpG oligonucleotide as an adjuvant. After immunization the woodchucks were challenged with 10(6) genome equivalents of HDV. The protein immunization with HDAg induced good humoral and T helper cell responses in the woodchucks, but did not protect them from HDV superinfection. The DNA immunized woodchucks were also not protected from HDV superinfection, however, the course of infection was modified: HDV viremia occurred later, the typical fluctuation of the HDV RNA titer with several peaks was absent, and antibodies to HDV were not detectable.

Animals↗
PubMed2001

Therapeutic benefit of intracerebral transplantation of bone marrow stromal cells after cerebral ischemia in rats.

We tested the hypothesis that bone marrow stromal cells (MSCs) transplanted into the ischemic boundary zone, survive, differentiate and improve functional recovery after middle cerebral artery occlusion (MCAo). MSCs were harvested from adult rats and cultured with or without nerve growth factor (NGF). For cellular identification, MSCs were prelabeled with bromodeoxyuridine (BrdU). Rats (n=24) were subjected to 2 h of MCAo, received grafts at 24 h and were euthanized at 14 days after MCAo. Test groups consisted of: (1) control-MCAo alone (n=8); (2) intracerebral transplantation of MSCs (n=8); (3) intracerebral transplantation of MSCs cultured with NGF (n=8). Immunohistochemistry was used to identify cells from MSCs. Behavioral tests (rotarod, adhesive-removal and modified neurological severity score [NSS]) were performed before and after MCAo. The data demonstrate that MSCs survive, migrate and differentiate into phenotypic neural cells. Significant recovery of somatosensory behavior (p<0.05) and NSS (p<0.05) were found in animals transplanted with MSCs compared with control animals. Animals that received MSCs cultured with NGF displayed significant recovery in motor (p<0.05), somatosensory (p<0.05) and NSS (p<0.05) behavioral tests compared with control animals. Our data suggest that intracerebral transplantation of MSCs may provide a powerful autoplastic therapy for stroke.

Animals↗
PubMed2001

Analysis of single nucleotide polymorphisms with solid phase invasive cleavage reactions.

Using microparticles as the capture surface and fluorescence resonance energy transfer as the detection technology, we have demonstrated the feasibility of performing the invasive cleavage reaction on a solid phase. An effective tool for many genomic applications, the solution phase invasive cleavage assay is a signal amplification method capable of distinguishing nucleic acids that differ by only a single base mutation. The method positions two overlapping oligonucleotides, the probe and upstream oligonucleotides, on the target nucleic acid to create a complex recognized and cleaved by a structure-specific 5'-nuclease. For microarray and other multiplex applications, however, the method must be adapted to a solid phase platform. Effective cleavage of the probe oligonucleotide occurred when either of the two required overlapping oligonucleotides was configured as the particle-bound reagent and also when both oligonucleotides were attached to the solid phase. Positioning probe oligonucleotides away from the particle surface via long tethers improved both the signal and the reaction rates. The particle-based invasive cleavage reaction was capable of distinguishing the ApoE Cys158 and Arg158 alleles at target concentrations as low as 100 amol/assay (0.5 pM).

Alleles↗