Biomedical subjects

Z Qin

Publications and source records attributed to Z Qin.

At least 19 recordsLinked to original sources

PubMed2001

Decreased generation of anti-tumor immunity after intrasplenic immunization.

The localization of antigen and the nature of the host antigen-presenting cells (APC) that present it to T cells are two major determinants of antigen immunogenicity. While lymph nodes appear to be the major site for T cell priming, recently the spleen was shown to provide an optimal microenvironment for direct CD8+ cytotoxic T cell (CTL) priming by tumor cells even in the absence of known costimulatory molecules on tumor cells. We analyzed whether the splenic microenvironment would support T cell priming also when host APC are involved (cross-priming) which is probably the major pathway during the generation of anti-tumor immunity. We performed immunization/challenge experiments using different tumor cells (B7.1+, B7.1- and/or beta-gal+, beta-gal-) known to induce CTL to a variable extent either exclusively by cross-priming (B7-) or at least partially by direct priming (B7+). Our results demonstrate that tumor take in the spleen required much less cells than at a subcutaneous injection site. Additionally, intrasplenic immunization was invariably ineffective compared to subcutaneous immunization. We further showed that B cells were not responsible for the inefficient intrasplenic immunization. Therefore delivering the tumor cell antigens inside the spleen by intrasplenic immunization did not improve but rather decreased the efficacy of tumor cell vaccines.

Adenocarcinoma↗

PubMed2001

Calcium-dependent stabilization of the central sequence between Met(76) and Ser(81) in vertebrate calmodulin.

Spin-label electron paramagnetic resonance (EPR) provides optimal resolution of dynamic and conformational heterogeneity on the nanosecond time-scale and was used to assess the structure of the sequence between Met(76) and Ser(81) in vertebrate calmodulin (CaM). Previous fluorescence resonance energy transfer and anisotropy measurements indicate that the opposing domains of CaM are structurally coupled and the interconnecting central sequence adopts conformationally distinct structures in the apo-form and following calcium activation. In contrast, NMR data suggest that the opposing domains of CaM undergo independent rotational dynamics and that the sequence between Met(76) and Ser(81) in the central sequence functions as a flexible linker that connects two structurally independent domains. However, these latter measurements also resolve weak internuclear interactions that suggest the formation of transient helical structures that are stable on the nanosecond time-scale within the sequence between Met(76) and Asp(80) in apo-CaM (H. Kuboniwa, N. Tjandra, S. Grzekiek, H. Ren, C. B. Klee, and A. Bax, 1995, Nat. Struct. Biol. 2:768-776). This reported conformational heterogeneity was resolved using site-directed mutagenesis and spin-label EPR, which detects two component spectra for 1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)-methanethiosulfonate spin labels (MTSSL) bound to CaM mutants T79C and S81C that include a motionally restricted component. In comparison to MTSSL bound within stable helical regions, the fractional contribution of the immobilized component at these positions is enhanced upon the addition of small amounts of the helicogenic solvent trifluoroethanol (TFE). These results suggest that the immobilized component reflects the formation of stable secondary structures. Similar spectral changes are observed upon calcium activation, suggesting a calcium-dependent stabilization of the secondary structure. No corresponding changes are observed in either the solvent accessibility to molecular oxygen or the maximal hyperfine splitting. In contrast, more complex spectral changes in the line-shape and maximal hyperfine splitting are observed for spin labels bound to sites that undergo tertiary contact interactions. These results suggest that spin labels at solvent-exposed positions within the central sequence are primarily sensitive to backbone fluctuations and that either TFE or calcium binding stabilizes the secondary structure of the sequence between Met(76) and Ser(81) and modulates the structural coupling between the opposing domains of CaM.

Amino Acid Motifs↗

PubMed2000

A caspase-3-like protease is involved in NF-kappaB activation induced by stimulation of N-methyl-D-aspartate receptors in rat striatum.

Glutamate receptor stimulation reportedly activates NF-kappaB in vitro and in vivo, although underlying mechanisms remain to be elucidated. Here we evaluated the role of proteases in mediating N-methyl-D-aspartate (NMDA) receptor agonist-induced NF-kappaB activation and apoptosis in rat striatum. The intrastriatal infusion of quinolinic acid (QA, 60 nmol) had no effect on levels of NF-kappaB family proteins, including p65, p50, p52, c-Rel and Rel B. In contrast, QA decreased IkappaB-alpha protein levels by 60% (P<0. 05); other members of the IkappaB family, including IkappaB-beta, IkappaB-gamma, IkappaB-epsilon and Bcl-3, were not altered. The QA-stimulated degradation of IkappaB-alpha was completely blocked by the NMDA receptor antagonist MK-801. QA-induced IkappaB-alpha degradation and NF-kappaB activation were not affected by the proteasome inhibitor MG-132 (1-4 microg). On the other hand, the caspase-3 inhibitor Ac-DEVD.CHO (2-8 microgram) blocked QA-induced IkappaB-alpha degradation in a dose-dependent manner (P<0.05). Ac-DEVD.CHO (4 microgram) also substantially reduced QA-induced NF-kappaB activation (P<0.05), but had no effect on QA-induced AP-1 activation. Furthermore, Ac-DEVD.CHO, but not MG-132, dose-dependently attenuated QA-induced internucleosomal DNA fragmentation. These findings suggest that NF-kappaB activation by NMDA receptor stimulation involves IkappaB-alpha degradation by a caspase-3-like cysteine protease dependent mechanism. Caspase-3 thus appears to contribute to the excitotoxin-induced apoptosis in rat striatal neurons occurring at least partially as a consequence of NF-kappaB activation.

Animals↗

PubMed2000

Long palindromes formed in Streptomyces by nonrecombinational intra-strand annealing.

Long inverted repeats (palindromes) are ubiquitous among prokaryotic and eukaryotic genomes. Earlier work has implicated both DNA breaks and short inverted repeats (IRs) in the formation of long palindromes in yeast and Tetrahymena by a proposed mechanism of intramolecular recombination. Here we report that long-palindromic linear plasmids are formed in Streptomyces following double strand DNA breakage by a nonrecombinational intra-strand annealing process that also involves IRs. By modification of palindrome-generating linear plasmids and development of a novel procedure that enables the sequencing of palindrome junctions, we show that long-palindrome formation occurs by unimolecular intra-strand annealing of IRs followed by 3' extension of the resulting DNA fold-back. The consequent hairpin structures serve as templates for synthesis of duplex linear plasmids containing long palindromes. We suggest that this model for long-palindrome formation in Streptomyces may represent a generally applicable mechanism for generating DNA palindromes.

Base Sequence↗

PubMed2000

NMDA and non-NMDA receptor-stimulated IkappaB-alpha degradation: differential effects of the caspase-3 inhibitor DEVD.CHO, ethanol and free radical scavenger OPC-14117.

The excitotoxic response of striatal neurons to NMDA and non-NMDA receptor agonists involves the nuclear translocation of transcription factor nuclear factor-kappa B (NF-kappaB) due to IkappaB-alpha degradation. Resultant augmentation in c-Myc, p53 and cyclin D1 expression presages the apoptotic-like destruction of these cells in vivo. To differentiate molecular events triggered by intrastriatally injected quinolinic acid (QA, 60 nmol) and kainic acid (KA, 2.5 nmol), we compared the effects of a caspase-3 inhibitor (DEVD.CHO, 8 microgram intrastriatally), a free radical scavenger (OPC-14117; 600 mg/kg, orally) and ethanol (2.14-8.6 micromol, intrastriatally or 25-100 mmol/kg, orally) on changes induced by these glutamatergic agonists on NF-kappaB cascade components and the apoptotic death of rat striatal neurons in vivo. The results indicated that the QA-induced degradation of IkappaB-alpha is almost totally mediated by a caspase-3-dependent mechanism, while KA-induced IkappaB-alpha degradation is only partially dependent on caspase-3. OPC-14117 attenuated the effects of QA but not KA on IkappaB-alpha degradation, suggesting that oxidative stress contributes to the QA- but not the KA-induced degradation of IkappaB-alpha. In contrast, ethanol inhibited the KA- but not the QA-induced degradation of IkappaB-alpha and the ensuing DNA fragmentation and loss of striatal GABAergic neurons. It would now appear that NF-kappaB activation in striatal neurons induced by NMDA or KA receptor stimulation involves different biochemical mechanisms. Since excitotoxicity associated with NF-kappaB activation may contribute to neuronal degenerative disorders such as Huntington's disease, a more detailed understanding of biochemical events underlying ionotrophic glutamate receptor-stimulated cell death may assist in the discovery of alternative approaches to interdicting the deleterious consequences of excitotoxic insult.

Animals↗

PubMed2000

Relationship between ataxin-1 nuclear inclusions and Purkinje cell specific proteins in SCA-1 transgenic mice.

Spinocerebellar ataxia-1 (SCA-1), like other polyglutamine diseases, is associated with aggregation of mutant protein ataxin-1 in the nuclei of susceptible neurons. The role of ataxin-1 aggregates in the pathogenesis of susceptible neurons, especially cerebellar Purkinje cells, is unknown. The present study was initiated to determine the temporal relationship between ataxin-1 aggregation and the sequence of specific biochemical changes in Purkinje cells in SCA-1 transgenic mice (TM). Earlier, we demonstrated that SCA-1 TM with no Purkinje cell loss and no alterations in home cage behavior show decreased expression of calcium-binding proteins calbindin-D28k (CaB) and parvalbumin (PV) in Purkinje cells. To determine if increased expression of mutant ataxin-1 in TM is also associated with earlier biochemical changes in Purkinje cells, both heterozygous and homozygous (B05 line of SCA-1) TM were used. The age of onset of ataxia in SCA-1 TM was at 12 weeks in heterozygotes and 6 weeks in homozygotes. In 6 week old heterozygous TM, Western blot analysis of growth associated protein 43 (GAP-43) and synaptophysin revealed no significant alterations as compared with the age-matched nontransgenic mice (nTM), whereas CaB was significantly reduced. beta-III-Tubulin was used as a specific Purkinje cell marker protein, immunohistochemical localization showed strong beta-III-tubulin immunoreactivity (IR) in Purkinje cells in 6 week old heterozygous TM, whereas CaB and PV IR were markedly reduced in the same neurons (double immunofluorescence staining). Most Purkinje cells from heterozygous (12 weeks old) and homozygous (6 weeks old) TM contained ataxin-1 nuclear inclusions (NIs). Cells with and without visible NIs revealed reduced PV and CaB IR; however, the changes were overtly more severe in cells with visible NIs. In contrast, the same cells were strongly immunoreactive to beta-III-tubulin. CaB, which is also present in the nucleus, colocalized with ataxin-1 and ubiquitin positive NIs. Further, RT-PCR analysis of CaB mRNA in the cerebellum in 6 week old heterozygous TM demonstrated a significant decrease in mRNA in comparison with the aged-matched nTM. These data suggest that there are selective alterations in the expression of CaB and PV in Purkinje cells which possibly occur earlier than ataxin-1 aggregation. Further, we speculate that ataxin-1 aggregates may not be toxic in general; however, they may deplete specific proteins essential for Purkinje cell viability in SCA-1 TM.

Animals↗

PubMed2000

Assessment of arterial stenosis in a flow model with power Doppler angiography: accuracy and observations on blood echogenicity.

The objective of the project was to study the influence of various hemodynamic and rheologic factors on the accuracy of 3-D power Doppler angiography (PDA) for quantifying the percentage of area reduction of a stenotic artery along its longitudinal axis. The study was performed with a 3-D power Doppler ultrasound (US) imaging system and an in vitro mock flow model containing a simulated artery with a stenosis of 80% area reduction. Measurements were performed under steady and pulsatile flow conditions by circulating, at different flow rates, four types of fluid (porcine whole blood, porcine whole blood with a US contrast agent, porcine blood cell suspension and porcine blood cell suspension with a US contrast agent). A total of 120 measurements were performed. Computational simulations of the fluid dynamics in the vicinity of the axisymmetrical stenosis were performed with finite-element modeling (FEM) to locate and identify the PDA signal loss due to the wall filter of the US instrument. The performance of three segmentation algorithms used to delineate the vessel lumen on the PDA images was assessed and compared. It is shown that the type of fluid flowing in the phantom affects the echoicity of PDA images and the accuracy of the segmentation algorithms. The type of flow (steady or pulsatile) and the flow rate can also influence the PDA image accuracy, whereas the use of US contrast agent has no significant effect. For the conditions that would correspond to a US scan of a common femoral artery (whole blood flowing at a mean pulsatile flow rate of 450 mL min(-1)), the errors in the percentages of area reduction were 4.3 +/- 1.2% before the stenosis, -2.0 +/- 1.0% in the stenosis, 11.5 +/- 3.1% in the recirculation zone, and 2.8 +/- 1.7% after the stenosis, respectively. Based on the simulated blood flow patterns obtained with FEM, the lower accuracy in the recirculation zone can be attributed to the effect of the wall filter that removes low flow velocities. In conclusion, the small errors reported in vitro may support the clinical use of this technique.

Algorithms↗

PubMed2000

CD4+ T cell--mediated tumor rejection involves inhibition of angiogenesis that is dependent on IFN gamma receptor expression by nonhematopoietic cells.

Immunity against MHC class II tumors can be mediated by CD4+ T cells in the effector phase through an unknown mechanism. We show that this is IFN gamma dependent but does not require IFN gamma receptor (IFN gamma R) expression on tumor cells, T cells, or other hematopoietic cells and that IFN gamma R expression is not necessary in the priming phase. However, tumor immunity requires IFN gamma R expression on nonhematopoietic cells in the effector phase and involves inhibition of tumor-induced angiogenesis. This shows that an effective anti-tumor response involves communication between CD4+ T cells and nonhematopoietic cells, most likely within the tumor stroma, and that tumor immunity must not entirely rely on direct tumor cell killing.

Angiogenesis Inhibitors↗

PubMed2000

Bcr/abl+ autologous dendritic cells for vaccination in chronic myeloid leukemia.

In chronic myeloid leukemia (CML) ex vivo generated DC are characterized by constitutive expression of bcr/abl and possibly other yet undefined leukemia-associated antigens, since these DC share a common progeny with leukemic cells. Induction of anti-leukemic T cell responses has been described in vitro. For a phase I vaccination study, autologous bcr/abl+ DC are generated under GMP conditions mainly from monocyte precursors in chronic phase CML patients. Lin-, CD80+, CD86+, CD83+, DR+ DC could be generated in sufficient numbers for s.c. vaccination with 1 x 10(6)-5 x 10(7) DC. Using monocyte precursors, the yield of DC per seeded PBMC was in the range of 1-6%. Furthermore, we could demonstrate in vitro that the T cell stimulatory ability of CD34+-derived DC can be augmented by a factor 2-3 by retroviral transduction with a gene coding for interleukin-7. DC-based vaccination strategies are a promising clinical approach, particularly as postremission immunotherapy in the setting of autologous stem cell transplantation.

Cytotoxicity, Immunologic↗

PubMed2000

Shear rate dependence of ultrasound backscattering from blood samples characterized by different levels of erythrocyte aggregation.

The objectives were (1) to determine the effect of the erythrocyte aggregation level (wide range of aggregation) and shear rate (which also affects aggregation) on the ultrasound backscattered power, and (2) to evaluate the reproducibility of the ultrasound method. Experiments were performed under steady flow (100-1250 ml/min) in a 12.7 mm diameter vertical tube. Doppler ultrasound at 10 MHz was used to measure simultaneously the velocity and the backscattered power across the tube. For each radial position, the shear rate was computed from the derivative of the velocity profile. The backscattered power decayed exponentially as a function of the shear rate, and for a given shear rate, the power increased monotonically with the level of aggregation measured by laser reflectometry. Using blood samples simulating hypo-, normal, and hyperaggregating erythrocytes, the power of the ultrasound signal varied respectively by -7.8, -13.2, and -16.1 dB as a function of the shear rate (from 0.4 to 50 s-1). The reproducibility of the backscattered power was 5.5 dB, which is less than the variations observed as a function of the shear rate. In conclusion, ultrasound backscattering is sensitive to the level of erythrocyte aggregation. At a first glance, ultrasound seems less accurate when compared to laser reflectometry but it is suggested that this is because ultrasound backscattering may be sensitive to structural aggregate changes that are not detected by the laser method.

Animals↗

PubMed1999

Tumor necrosis factor alpha expression produces increased blood-brain barrier permeability following temporary focal cerebral ischemia in mice.

Alteration of blood-brain barrier (BBB) function occurs in both permanent and temporary cerebral ischemia. Studies in vivo and in vitro have shown that tumor necrosis factor-alpha (TNFalpha) is involved in changes of BBB permeability. However, the relationship between TNFalpha expression and BBB disruption during reperfusion is unclear. The aim of this study is to find the cell source of TNFalpha and to determine the relationship between TNFalpha expression and BBB disruption following temporary focal cerebral ischemia in mice. Adult CD-1 mice received 1 h middle cerebral artery occlusion (MCAO) followed by 2 h, 6 h, 12 h, 24 h, and 48 h of reperfusion. MCAO was achieved using an intraluminal suture technique and reperfusion was performed by the suture withdrawal. Neutralizing monoclonal anti-mouse TNFalpha antibody was administrated intraventricularly immediately after reperfusion. TNFalpha expression was determined by double labeling immunohistochemistry. BBB permeability was determined by albumin immunostaining. TNFalpha immunoreactivity (IR) was observed in the ipsilateral hemisphere from 1 h MCAO with 2 h reperfusion. TNFalpha positive cells included neurons, astrocytes, and ependymal cells. BBB disruption was detected beginning at 6 h reperfusion but was not present at 2 h of reperfusion. The areas of BBB disruption were significantly enlarged at 12 h reperfusion and plateaued at 24 h to 48 h reperfusion. BBB disruptions were significantly attenuated in the anti-TNFalpha antibody treated mice (p<0.05). Our results demonstrate that TNFalpha IR existed in neurons, astrocytes, and ependymal cells during reperfusion. TNFalpha IR following temporary focal cerebral ischemia precedes increased BBB permeability. Treatment with TNFalpha antibody reduces BBB disruption, suggesting TNFalpha may be an important mediator in altering BBB permeability during reperfusion.

Age Factors↗

PubMed1999

T helper cell type 1-associated and cytotoxic T lymphocyte-mediated tumor immunity is impaired in interleukin 4-deficient mice.

It is widely accepted that cellular immune responses are induced by CD4(+) T helper 1 (Th1) cells secreting interleukin (IL)-2 and interferon (IFN)-gamma. Tumor immunity is often mediated by cytotoxic T lymphocytes (CTLs) whose activation is supported by Th1 cytokines. Since IL-4 directs Th2 development and has been shown to inhibit Th1-dominated responses, we assumed that IL-4-deficient (IL-4(-/-)) mice would develop vigorous CTL-mediated tumor immunity compared with IL-4-competent (IL-4(+/+)) mice. Surprisingly, IL-4(-/-) mice were severely impaired to develop tumor immunity to both a mammary adenocarcinoma line and a colon carcinoma line. The lack of tumor immunity in IL-4(-/-) mice was associated with reduced IFN-gamma production, diminished levels of tumor-reactive serum IgG2a, and undetectable CTL activity, indicating a defective Th1 response in the absence of endogenous IL-4. Anti-IL-4 monoclonal antibody blocked tumor immunity in IL-4(+/+) mice when administered at the time of immunization but not at the time of challenge. Additionally, tumor immunity could be induced in IL-4(-/-) mice, if IL-4 was provided by gene-modified cells together with immunizing tumor cells. These results demonstrate that tumor immunity requires IL-4 in the priming phase for the generation of effector cells rather than for their maintenance and exclude secondary, developmental defects in the "knockout" strain. Together, our results demonstrate a novel and previously unanticipated role of IL-4 for the generation of Th1-associated, CTL-mediated tumor immunity.

Adenocarcinoma↗

PubMed1999

The effects of calbindin D-28K and parvalbumin antisense oligonucleotides on the survival of cultured Purkinje cells.

The role of calcium binding proteins, calbindin D-28k (CaB) and parvalbumin (PV) in Purkinje cell survival was investigated using oligonucleotide antisense strategy. Purkinje cell enriched cultures were prepared from the cerebella of 0-1 day old Balb/c mouse pups. Purkinje cells were identified by size, asymmetric arbors, immunoreactivity to CaB and PV, uptake of gamma-aminobutyric acid (GABA) and failure to express glial fibrillary acidic protein. The cells at different days in vitro were treated with antisense or mismatched antisense phosphorothioate oligonucleotides for CaB and PV mRNA (complexed with lipofectin). Neuronal specific [3H]-GABA uptake was used as a measure of Purkinje cell survival. The cultures treated for 24 h with antisense oligos (CaB+PV) showed a significant decrease in [3H]-GABA uptake as compared with the cultures treated with lipofectin alone or with lipofectin + mismatched antisense oligos to CaB and PV mRNA. The results of the present study suggest that the expression of calcium buffering proteins CaB and PV may have a significant involvement in Purkinje cell viability.

Animals↗

PubMed1998

Decrease in Ca-ATPase activity in aged synaptosomal membranes is not associated with changes in fatty acyl chain dynamics.

We have examined lipid peroxidation (LPO) and fatty acid acyl chain dynamics in synaptosomal membranes isolated from aged rat (Fischer 344 x Brown Norway F1 hybrids) brains, correlating these results with measurements of enzymatic activity of the synaptic plasma membrane Ca2(+)-ATPase (PMCA). Calcium-dependent ATPase activity in these membranes exhibits progressive decreases with a maximal loss of activity with age of approximately 35%. The sensitivity of this membrane-bound ion transporter to the lipid composition of the surrounding membrane, as well as the high abundance of oxidatively sensitive polyunsaturated fatty acyl chains in synaptosomal membranes, suggests that this age-related loss in catalytic turnover may result from LPO-mediated protein modification and/or changes in the physical structure of the bilayer. However, high-performance liquid chromatography analysis of 2,4-dinitrophenylhydrazone derivatives reveals no significant age-related increases in the content of reactive aldehydes (malondialdehyde, formaldehyde, acetaldehyde or acetone) which comprise breakdown products of lipid peroxidation. Electron paramagnetic resonance measurements employing 5- and 12-stearic acid spin labels with the nitroxide reporter groups at two depths in the bilayer were used to assess the fatty acyl chain dynamics (fluidity) of synaptosomal membranes. The resulting spectra demonstrate anisotropic lipid dynamics of two populations of lipids, i.e. lipids in direct association with membrane proteins (boundary lipids) and bulk lipids that do not directly associate with proteins. The nanosecond dynamics of both lipid populations is unaltered with age indicating that any compositional changes occurring with age are insufficient to result in alterations in bilayer fluidity relevant to PMCA activity. Thus, the observed age-related decline in PMCA activity may be explained by direct modification of membrane protein.

Aging↗

PubMed1998

Constitutive IL-10 production accounts for the high NK sensitivity, low MHC class I expression, and poor transporter associated with antigen processing (TAP)-1/2 function in the prototype NK target YAC-1.

Tumor cells that are treated with rIL-10 or transfected with the IL-10 gene show phenotypic changes. These include low but peptide-inducible expression of MHC class I, low sensitivity to specific CTL-mediated lysis, and increased NK sensitivity. In vitro-established mouse tumor lines were screened for IL-10 expression and production, and a large proportion of plasmocytomas or T cell lymphomas were found to produce IL-10. Since one of these lines was the prototype NK target cell YAC-1, we investigated whether the high IL-10 production of this cell line was related to its high NK sensitivity and its defects in MHC class I expression. The decrease in H-2 expression following the in vitro culture of in vivo-passaged YAC-1 cells was accompanied by a gradual increase in IL-10 production, whereas the reverse was found when passing in vitro-grown YAC-1 in vivo as an ascites tumor in syngenic mice. In addition, differences in YAC-1 MHC class I expression correlated with alterations in the functional activity of TAP-1/2 proteins. YAC-1 cells that were transduced with a retroviral IL-10 antisense construct (Y-IL-10 AS) only produced about half of the IL-10 that was produced by YAC-1 transduced with the control construct (Y-IL-10 Mock). Relative to Y-IL-10 Mock cells, the expression of H-2 on Y-IL-10 AS cells was markedly increased, and NK sensitivity was decreased. These data argue for a mechanism wherein IL-10 production is causally related to the low H-2 expression, decreased TAP function, and high NK sensitivity of YAC-1 cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

Cellular localization of tumor necrosis factor alpha following focal cerebral ischemia in mice.

Tumor necrosis factor alpha (TNFalpha) is a pleiotrophic cytokine with diverse proinflammatory actions. Focal cerebral ischemia induces rapid and dramatic increases in TNFalpha levels within and surrounding the focus of damaged brain both in striatum and cortex. The actions of TNFalpha during cerebral ischemia may be related to the cell types which deliver and/or accept TNFalpha signals. However, the cellular sources of TNFalpha following cerebral ischemia have not been fully elucidated. The present study was designed to determine the cellular localization of TNFalpha following permanent middle cerebral artery occlusion (MCAO) in mice. As judged by immunohistochemistry, TNFalpha expression in the ischemic hemisphere was increased at 3 h following MCAO, peaked at 6 to 12 h, and decreased at 24 h. Double immunostaining for TNFalpha and neuron specific enolase (NSE) or glial fibrillary acidic protein (GFAP) showed that TNFalpha positive neurons were observed in both the ischemic core and perifocal region, while TNFalpha positive astrocytes were observed in the outer cortical layer, the corpus callosum, the molecular layer of the hippocampus, and periventricular areas. The presence of TNFalpha immunoreactivity in neurons and nerve fibers following MCAO suggests that TNFalpha expressed in ischemic neurons might be delivered via axonal transport, while TNFalpha immunoreactivity in astrocyte end-feet and ependymal cells following MCAO suggests that TNFalpha may be involved in blood-brain barrier disruption and the initiation of inflammation in the brain.

Animals↗

PubMed1998

Inhibition of TNFalpha attenuates infarct volume and ICAM-1 expression in ischemic mouse brain.

The purpose of our study was to determine whether inhibiting the action of tumor necrosis factor-alpha (TNFalpha) attenuates brain injury and reduces inflammatory responses in the mouse during ischemia and reperfusion. Mice underwent middle cerebral artery occlusion (MCAO) for 1 h followed by 23 h reperfusion. Monoclonal neutralizing anti-murine TNFalpha antibody (mAb) was administrated intraventricularly in the mouse with temporary MCAO. Infarct volume in the anti-TNFalpha mAb treated mice was significantly smaller than that in the control group (p < 0.05). The number of intercellular adhesion molecule-1 (ICAM-1)-positive vessels in the ischemic area of the anti-TNFalpha mAb-treated group was significantly less than that in the control group. Our study demonstrated that blocking TNFalpha reduced brain injury and attenuated ICAM-1 expression during transient cerebral ischemia.

Animals↗

PubMed1998

Double suicide gene (cytosine deaminase and herpes simplex virus thymidine kinase) but not single gene transfer allows reliable elimination of tumor cells in vivo.

Suicide genes such as cytosine deaminase (CD) and herpes simplex virus thymidine kinase (TK) encode products that convert nontoxic substances (prodrugs) into toxic metabolites. Suicide gene transfer is currently being used in cancer therapy or can be used as a safety modality. To analyze the reliability of suicide genes as a safety modality for a vaccination study with viable cytokine/B7 gene-modified tumor cells, the individual and combined efficacy of the two suicide genes was compared for in vitro and in vivo cell killing of a murine mammary adenocarcinoma cell line (TS/A). To adapt the system to an in vivo gene delivery situation, bulk cultures cotransfected with the CD and TK gene were used instead of selected clones. In vitro, both CD and TK conferred sensitivity to the respective prodrug but the combined cytotoxic effects of both gene products were always superior. For in vivo analysis BALB/c mice were injected subcutaneously with CD- and TK-modified TS/A cells, treated with prodrugs, and tumor size was evaluated for a period of 100 days. In the in vivo situation the combination of both enzyme/prodrug systems was again most effective. The highest single concentration of 5-FC (500 mg/kg) or GCV (100 mg/kg) was not able to fully protect the animals from developing tumors, whereas a combination of 5-FC (250 mg/kg) and GCV (50 mg/kg) resulted in complete tumor eradication. In nude mice treated in the same way, most CD/TK tumors could not be eliminated. Furthermore, BALB/c mice cured of TS/A-CD/TK tumors developed a systemic tumor immunity against challenge with parental TS/A cells. These findings indicate that reliable tumor elimination by the suicide genes depends on T cells. The cooperative effect of both suicide genes was confirmed in vitro with the human renal cell carcinoma line RCC26. We conclude that TK and CD together, but neither gene alone, act as a safety mechanism for the elimination of tumor cells in a reliable fashion and suggest that a rapid and quantitative antigen release by effective TK- and CD-mediated tumor destruction is necessary for T cell immunity to develop.

Adenocarcinoma↗