Showing papers by "Simon C. Watkins published in 2000"
TL;DR: Analysis of the expression and constitutive activation of Stat3 in tumors and normal mucosa from patients with head and neck cancer provides evidence that constitutively activated Stat3 is an early event in head and head carcinogenesis that contributes to the loss of growth control by an anti-apoptotic mechanism.
Abstract: Field cancerization predisposes the upper aerodigestive tract mucosa to the formation of multiple primary tumors, when exposed to environmental carcinogens. Up-regulation of epidermal growth factor receptor occurs early in squamous cell carcinogenesis and is critical for the loss of growth control in a variety of human cancers, including head and neck squamous cell carcinomas. In these tumor cells in culture, epidermal growth factor receptor stimulation initiates signaling via persistent activation of selective STAT proteins. To determine the timing of Stat3 activation in head and neck carcinogenesis, we studied the expression and constitutive activation of Stat3 in tumors and normal mucosa from patients with head and neck cancer compared with mucosa from controls without cancer. Stat3 was up-regulated and constitutively activated in both primary human head and neck tumors as well as in normal mucosa from these cancer patients compared with control normal mucosa from patients without cancer. In vivo liposome-mediated gene therapy with a Stat3 antisense plasmid efficiently inhibited Stat3 activation, increased tumor cell apoptosis, and decreased Bcl-x(L) expression in a head and neck xenograft model. These findings provide evidence that constitutively activated Stat3 is an early event in head and neck carcinogenesis that contributes to the loss of growth control by an anti-apoptotic mechanism.
611 citations
TL;DR: It is shown that delayed and persistent activation of ERKs is associated with glutamate-induced oxidative toxicity in HT22 cells and immature primary cortical neuron cultures, and that U0126, a specific inhibitor of the ERK-activating kinase, MEK-1/2, protects both HT22cells and immaturePrimary cortex neuron cultures from glutamate toxicity.
535 citations
TL;DR: Lipid accumulation within muscle fibers is significantly increased in obesity and is reduced by weight loss, which provides important information regarding the accumulation and distribution of skeletal muscle triglyceride in type 2 DM and obesity.
Abstract: The triglyceride content of skeletal muscle samples determined by lipid extraction correlates with the severity of insulin-resistant glucose metabolism in muscle. To determine whether this reflects increased triglyceride within muscle fibers and to test the-hypothesis that the lipid content in muscle fibers is increased in obesity, the present study was undertaken using quantitative histochemistry of Oil Red 0 staining of vastus lateralis muscle. A percutaneous muscle biopsy was performed in 9 lean subjects, 15 obese subjects without type 2 diabetes mellitus (DM), and 10 obese subjects with type 2 DM (body mass index [BMI], 23 .4 ± 1 .0, 33 .6 ± 0 .6, and 36.0 ± 1.1 kg. M −2 for lean, obese, and DM, respectively). Eight obese and 7 DM subjects had a weight loss and reassessment of muscle lipid content. Transverse muscle cryosections were examined by light microscopy with quantitative image analysis (grayscale images obtained by analog to digital conversion) to determine a lipid accumulation index (LAI) based on the percentage of cross-sectional fiber area occupied by lipid droplets. Muscle fiber lipid content was greater in obese individuals with DM than in lean individuals (3 .62% ± 0.65% v 1 .42%: 0.28%, P
496 citations
TL;DR: The results suggest a critical role of TNF-alpha and MMPs in myocardial matrix remodeling and functional regulation and support the hypothesis that both TNFs and M MPs may serve as potential therapeutic targets in the treatment of heart failure.
Abstract: Myocardial fibrosis caused by maladaptive extracellular matrix (ECM) remodeling is implicated in the dysfunction of the failing heart. Matrix metalloproteinases (MMPs) regulate ECM remodeling, and are regulated by cytokines. Transgenic mice with cardiac-specific overexpression of tumor necrosis factor alpha (TNF-alpha) (TNF1.6) develop heart failure. We hypothesized that modulation of TNF-alpha and/or MMP activity might alter the myocardial ECM remodeling process and the development of heart failure. To test this hypothesis, we took advantage of the TNF1.6 mice and studied soluble and total collagens and collagen type profiling by using hydroxyproline quantification, Sircol collagen assay, Northern blot analysis, and immunohistochemistry and studied myocardial function by using echocardiography. Progressive ventricular hypertrophy and dilation in the TNF1.6 mice were accompanied by a significant increase in MMP-2 and MMP-9 activity, an increase in collagen synthesis, deposition, and denaturation, and a decrease in undenatured collagens. In young TNF1.6 mice, these changes in the ECM were associated with marked diastolic dysfunction as demonstrated by significantly reduced transmitral Doppler echocardiographic E/A wave ratio. Anti-TNF-alpha treatment with adenoviral vector expressing soluble TNF-alpha receptor type I attenuated both MMP-2 and MMP-9 activity, prevented further collagen synthesis, deposition and denaturation, and preserved myocardial diastolic function in young, but not old, TNF1.6 mice. The results suggest a critical role of TNF-alpha and MMPs in myocardial matrix remodeling and functional regulation and support the hypothesis that both TNF-alpha and MMPs may serve as potential therapeutic targets in the treatment of heart failure.
323 citations
TL;DR: It is reported that insulin stimulation of HIRcB fibroblasts leads to accumulation of Ras, Raf-1, phosphorylated MEK,osphorylated MAPK, and PA on endosomal membranes and results indicate that agonist-dependent Raf- 1 translocation is primarily mediated by a direct interaction with PA and is independent of association with Ras.
321 citations
TL;DR: Using the yeast two-hybrid method, a skeletal muscle-specific form of filamin is identified, which is term filamin 2 (FLN2), as a γ- and δ-sarcoglycan interacting protein which introduces new implications for the pathogenesis of muscular dystrophy.
Abstract: Mutations in genes encoding for the sarcoglycans, a subset of proteins within the dystrophin–glycoprotein complex, produce a limb-girdle muscular dystrophy phenotype; however, the precise role of this group of proteins in the skeletal muscle is not known. To understand the role of the sarcoglycan complex, we looked for sarcoglycan interacting proteins with the hope of finding novel members of the dystrophin–glycoprotein complex. Using the yeast two-hybrid method, we have identified a skeletal muscle-specific form of filamin, which we term filamin 2 (FLN2), as a γ- and δ-sarcoglycan interacting protein. In addition, we demonstrate that FLN2 protein localization in limb-girdle muscular dystrophy and Duchenne muscular dystrophy patients and mice is altered when compared with unaffected individuals. Previous studies of filamin family members have determined that these proteins are involved in actin reorganization and signal transduction cascades associated with cell migration, adhesion, differentiation, force transduction, and survival. Specifically, filamin proteins have been found essential in maintaining membrane integrity during force application. The finding that FLN2 interacts with the sarcoglycans introduces new implications for the pathogenesis of muscular dystrophy.
296 citations
TL;DR: Formula-fed neonatal rats, with or without hypoxia, exhibit morphological changes in the intestinal epithelium similar to those seen in patients with acute NEC, and this model may offer a simple reproducible method for inducing experimental NEC.
224 citations
TL;DR: This work has identified a new mechanism by which potentially immunogenic glycoprotein tumor Ags, such as MUC1, fail to stimulate strong immune responses, and shows that a 140-aa-long synthetic peptide is very efficiently processed by dendritic cells.
Abstract: Immunity to tumor Ags in patients is typically weak and not therapeutic. We have identified a new mechanism by which potentially immunogenic glycoprotein tumor Ags, such as MUC1, fail to stimulate strong immune responses. MUC1 is a heavily glycosylated membrane protein that is also present in soluble form in sera and ascites of cancer patients. We show that this soluble protein is readily taken up by dendritic cells (DC), but is not transported to late endosomes or MHC class II compartments for processing and binding to class II MHC. MUC1 uptake is mediated by the mannose receptor, and the protein is then retained long term in early endosomes without degradation. Long-term retention of MUC1 does not interfere with the ability of DC to process and present other Ags. We also demonstrate inhibited processing of another important glycoprotein tumor Ag, HER-2/neu. This may, therefore, be a frequent obstacle to presentation of tumor Ags and an important consideration in the design of cancer vaccines. It should be possible to overcome this obstacle by providing DC with a form of tumor Ag that can be better processed. For MUC1 we show that a 140-aa-long synthetic peptide is very efficiently processed by DC.
164 citations
TL;DR: In vitro maturation is not a requirement for effective migration of DC in vivo and it is suggested that administration of Ag-loaded immature DC that undergo natural maturation following injection may be preferred for DC-based immunotherapy.
Abstract: Human dendritic cells (DC) have polarized responses to chemokines as a function of maturation state, but the effect of maturation on DC trafficking in vivo is not known. We have addressed this question in a highly relevant rhesus macaque model. We demonstrate that immature and CD40 ligand-matured monocyte-derived DC have characteristic phenotypic and functional differences in vitro. In particular, immature DC express CC chemokine receptor 5 (CCR5) and migrate in response to macrophage inflammatory protein-1alpha (MIP-1alpha), whereas mature DC switch expression to CCR7 and respond exclusively to MIP-3beta and 6Ckine. Mature DC transduced to express a marker gene localized to lymph nodes after intradermal injection, constituting 1.5% of lymph node DC. In contrast, cutaneous DC transfected in situ via gene gun were detected in the draining lymph node at a 20-fold lower frequency. Unexpectedly, the state of maturation at the time of injection had no influence on the proportion of DC that localized to draining lymph nodes, as labeled immature and mature DC were detected in equal numbers. Immature DC that trafficked to lymph nodes underwent a significant up-regulation of CD86 expression indicative of spontaneous maturation. Moreover, immature DC exited completely from the dermis within 36 h of injection, whereas mature DC persisted in large numbers associated with a marked inflammatory infiltrate. We conclude that in vitro maturation is not a requirement for effective migration of DC in vivo and suggest that administration of Ag-loaded immature DC that undergo natural maturation following injection may be preferred for DC-based immunotherapy.
161 citations
Journal Article•
TL;DR: The results suggest that the CCR7 expression of DCs is enhanced with direct contact with apoptotic tumor cells and may have a critical role for DC migrating to regional lymph nodes.
Abstract: Dendritic cells (DCs) are very potent antigen-presenting cells and play critical roles in regulating immune responses in cancer. The migrating of DCs from the tumor site to the lymphoid organs is believed to be one of the critical events. To examine this important DC function in tumor situations, bone marrow-derived DCs, cultured for 6 days with granulocyte macrophage colony-stimulating factor and interleukin 4, were inoculated at the tumor site. We have shown (Y. Nishioka et al., Cancer Res., 59: 40354041, 1999) that DCs can migrate from tumor site to the draining lymph nodes within 24 h (approximately 0.1% of administrated DCs). The DCs then form clusters with adjacent lymphoid cells, which produce IFN-gamma (1500-3200pg/10(6) cells/48 h) in response to tumor stimulation. The number of the DCs migrating into lymph nodes were greater when they were inoculated into the tumor rather than the skin. Coculture of DCs and apoptotic tumor cells resulted in decreased expression of CC chemokine receptor (CCR) 1 and increased CCR7 expression at mRNA level without alteration in other phenotypical markers on DCs. Chemotaxis assay showed that CCR7 ligands, macrophage inflammatory protein 3beta and secondary lymphoid-tissue chemokine significantly (P < 0.05) induced the migration of DCs when cocultured with apoptotic tumor cells. To directly examine the involvement of CCR7 expression in DC migration, we investigated the functions of DCs genetically modified to express high levels of CCR7. CCR7 transduction promotes DC migration in response to relevant ligands in vitro and in vivo. These results suggest that the CCR7 expression of DCs is enhanced with direct contact with apoptotic tumor cells and may have a critical role for DC migrating to regional lymph nodes. The means to promote DC delivery to tumor and to nodal sites represent novel targets for the biological therapy of cancer.
148 citations
TL;DR: Cell death regulatory genes of the bcl-2 and caspase families may play a role in ongoing neuropathologic processes in human epilepsy, and offer novel targets as an adjunct to anticonvulsant therapy.
Abstract: Objective: To address the role of cell death regulatory genes of the bcl-2 and caspase families in the neuropathology of human epilepsy using tissue extracted from patients undergoing temporal lobectomy for intractable seizures Methods: Using Western blotting and immunohistochemistry, the authors investigated the expression of bcl-2, bcl-x L , bax, caspase-1, and caspase-3 in temporal cortex samples from patients who had undergone temporal lobectomy surgery for intractable epilepsy (n = 19) Nonepileptic postmortem tissue from a brain bank served as control (n = 6) Results: Western blot analysis demonstrated significant increases in levels of bcl-2 and bcl-x L protein in seizure brain compared to control Cleavage of caspase-1 was evidenced by a reduction in levels of the 45 kDa proenzyme form and an increase in levels of the p10 fragment Levels of the 32 kDa proenzyme form of caspase-3 were elevated in seizure patients, as were levels of the 12 kDa cleaved fragment Bcl-2, bax, and caspase-3 immunoreactivity was increased predominantly in cells with the morphologic appearance of neurons, whereas bcl-x L immunoreactivity was increased in cells with the appearance of glia DNA fragmentation was detected in some but not all sections from epileptic brain samples Conclusions: Cell death regulatory genes of the bcl-2 and caspase families may play a role in ongoing neuropathologic processes in human epilepsy, and offer novel targets as an adjunct to anticonvulsant therapy
TL;DR: Lyophilization of LPD, a novel cationic lipid-based gene delivery system, was studied and both particle size and transfection efficiency could be preserved in the presence of sufficient amount of appropriate lyoprotectant.
TL;DR: Nitric oxide inhibits VSMC proliferation in association with the upregulation of p21; both occur independent of p53 and cGMP while being partially mediated through the p42/44 MAPK signaling cascade.
TL;DR: It is shown that isolated brain mitochondria are more resistant to induction of permeability transition than mitochondria from liver, which may have important implications for the study of the mechanisms involved in neuronal cell death.
TL;DR: These transgenic studies represent the first in vivo demonstration that the HNF-3β transcriptional network regulates expression of hepatocyte-specific genes required for bile acid and glucose homeostasis, as well as postnatal growth.
Abstract: The winged helix transcription factor, hepatocyte nuclear factor-3β (HNF-3β), mediates the hepatocyte-specific transcription of numerous genes important for liver function. However, the in vivo role of HNF-3β in regulating these genes remains unknown because homozygous null HNF3β mouse embryos die in utero prior to liver formation. In order to examine the regulatory function of HNF-3β, we created transgenic mice in which the −3-kb transthyretin promoter functions to increase hepatocyte expression of the rat HNF-3β protein. Postnatal transgenic mice exhibit growth retardation, depletion of hepatocyte glycogen storage, and elevated levels of bile acids in serum. The retarded growth phenotype is likely due to a 20-fold increase in hepatic expression of insulin-like growth factor binding protein 1 (IGFBP-1), which results in elevated levels in serum of IGFBP-1 and limits the biological availability of IGFs required for postnatal growth. The defects in glycogen storage and serum bile acids coincide with diminished postnatal expression of hepatocyte genes involved in gluconeogenesis (phosphoenolpyruvate carboxykinase and glycogen synthase) and sinusoidal bile acid uptake (Ntcp), respectively. These changes in gene transcription may result from the disruptive effect of HNF-3β on the hepatic expression of the endogenous mouse HNF-3α,-3β, -3γ, and -6 transcription factors. Furthermore, adult transgenic livers lack expression of the canalicular phospholipid transporter, mdr2, which is consistent with ultrastructure evidence of damage to transgenic hepatocytes and bile canaliculi. These transgenic studies represent the first in vivo demonstration that the HNF-3β transcriptional network regulates expression of hepatocyte-specific genes required for bile acid and glucose homeostasis, as well as postnatal growth.
TL;DR: Saturation of the actin-scavenging system by addition of exogenous G-actin to plasma produces direct pulmonary endothelial cell injury and plasma from patients with ARDS secondary to bacterial pneumonia is toxic to SPAEC, and a small but significant contributory role of actin is apparent in these studies.
Abstract: Actin released from damaged cells after a variety of tissue injuries appears to be involved in multiple organ dysfunction syndrome. Under experimental conditions, when the quantity of actin present in plasma is made to exceed the protective capacity of the actin-scavenging mechanism, microembolism and pulmonary vascular angiopathy have been noted in rats. It remains to be determined whether this injury is a result of a direct toxic effect or occurs indirectly via platelet activation or fibrin interactions. We examined the effect of sera from patients with adult respiratory distress syndrome (ARDS), as well as G-actin added to normal serum, on the viability, morphology, and function of cultured sheep pulmonary artery endothelial cells (SPAEC). Both patient sera and normal sera to which actin was added were toxic in the cell culture model; this toxicity could be abrogated, at least partially, by preincubation with gelsolin, which is known to complex with actin. A significant portion of the toxicity of sera ...
TL;DR: It is suggested that nitrative/oxidative stress to the transplanted multilineage hematopoietic cells due to exposure to nitric oxide released by host bone marrow stromal cells may contribute to the genotoxic events associated with malignant alterations in bone marrow tissue of transplant recipients who are prepared for engraftment by total-body irradiation.
Abstract: Gorbunov, N. V., Pogue-Geile, K. L., Epperly, M. W., Bigbee, W. L., Draviam, R., Day, B. W., Wald, N., Watkins, S. C. and Greenberger, J. S. Activation of the Nitric Oxide Synthase 2 Pathway in the Response of Bone Marrow Stromal Cells to High Doses of Ionizing Radiation. Reverse transcription-polymerase chain reaction and immunofluorescence analysis of D2XRII murine bone marrow stromal cells showed that γ irradiation with doses of 2–50 Gy from 137Cs stimulated expression of nitric oxide synthase 2 (Nos2, also known as iNos). The activation of Nos2 was accompanied by an increase in the fluorescence of 4,5-diaminofluorescein diacetate, a nitric oxide trap, and accumulation of 3-nitrotyrosine within cellular proteins in a dose-dependent manner. These effects were inhibited by actinomycin D and by N-[3-(aminomethyl)benzyl]acetamidine dihydrochloride, a specific inhibitor of Nos2. The induction of Nos2 expression and Nos2-dependent release of nitric oxide in D2XRII cells was observed within 24 h afte...
TL;DR: The reproductive tract can be an inductive site for mucosally induced tolerance, however, unlike other mucosal sites such as the lung and gastrointestinal tract, reproductive tract tolerance induction is hormonally regulated.
Abstract: These data demonstrate that tolerance can be induced by vaginal Ag exposure. In these experiments, mice were given vaginal agarose gel suppositories containing either 5 mg OVA or saline for 6 h. Mice were given suppositories either during the estrous (estrogen dominant) or diestrous (progesterone dominant) stage of the estrous cycle. Mice were restrained during the inoculation period to prevent orovaginal transmission of the Ag. After 1 wk, mice were immunized s.c. with OVA in CFA. After 3 wk, mice were tested for delayed-type hypersensitivity responses by measuring footpad swelling and measuring in vitro proliferation of lymphocytes to Ag. Using ELISA, the magnitude of the serum Ab response was also measured. In some mice, FITC conjugated to OVA was used to track the dissemination of the protein into the systemic tissues. The magnitude of footpad swelling was significantly reduced in mice receiving OVA-containing suppositories during estrus compared with mice receiving saline suppositories. Concomitant decreases in the Ag-specific proliferative response were also observed in lymph node lymphocytes and splenocytes. Conversely, mice inoculated during diestrus did not show a decreased response to Ag by either footpad response or in vitro proliferation. Serum Ab titers in the estrus-inoculated mice did not decrease significantly. These data demonstrate that the reproductive tract can be an inductive site for mucosally induced tolerance. However, unlike other mucosal sites such as the lung and gastrointestinal tract, reproductive tract tolerance induction is hormonally regulated.
TL;DR: The results suggested that endogenously expressed bcl-2 promoted survival in ischemic neurons and was not simply an epiphenomenon in neurons already destined to live or die.
Abstract: Previous studies have shown that overexpression of bcl-2 in transgenic mice or by viral vectors protects the brain against cerebral ischemia However, it is not known whether bcl-2, which is endogenously expressed in response to ischemia, exerts a protective effect To address this question, the authors blocked the endogenous expression of bcl-2 after ischemia using antisense oligodeoxynucleotides (ODN) Antisense, sense, scrambled ODN, or vehicles were infused in the lateral ventricle of the rat for 24 hours after 30 minutes of temporary middle cerebral artery occlusion Twenty-four hours later the brains were removed and bcl-2 protein expression was assayed by Western blot Antisense ODN, but not sense or scrambled ODN treatment, significantly inhibited bcl-2 protein expression after ischemia Bcl-2 protein expression was also studied 24 hours after 60 minutes of temporary middle cerebral artery occlusion in vehicle and antisense ODN-treated rats After 60 minutes of ischemia and vehicle treatment, bcl-2 was expressed in many neurons in the ventral cortical mantle and the medial striatum After antisense ODN treatment there were few neurons in this region expressing bcl-2, instead most neurons TUNEL labeled Treatment with the antisense ODN, but not sense ODN, increased infarction volume as determined by cresyl violet staining 72 hours after ischemia compared with vehicle controls These results suggested that endogenously expressed bcl-2 promoted survival in ischemic neurons and was not simply an epiphenomenon in neurons already destined to live or die
TL;DR: Developing replication-defective recombinant adenoviruses that express M2-901/CFTR and using them to probe cell surface CFTR in forskolin (FSK)-stimulated polarized Madin-Darby canine kidney (MDCK) cells may reconcile previous conflicting reports regarding the effect of cAMP stimulation on CFTR trafficking.
Abstract: Channel gating of the cystic fibrosis transmembrane conductance regulator (CFTR) is activated in response to cAMP stimulation. In addition, CFTR activation may also involve rapid insertion of a sub...
TL;DR: In vivo and in vitro angiogenesis assays using NO donor S-nitroso-N-acetylpenicillamine (SNAP) and NO synthase inhibitor N-iminoethyl- l -ornithine (L-NIO) showed significant changes in capillary ingrowth into subcutaneously implanted Matrigel plugs in mice.
TL;DR: It is found that eN OS plays a protective role in allografts, and that in eNOS-deficient allografteds, early overexpression of iNOS is capable of preventing the development of allogRAFT arteriosclerosis.
Abstract: Background Inducible nitric oxide synthase (iNOS) is up-regulated in rejecting allografts and is protective against allograft arteriosclerosis; it suppresses neointimal smooth muscle cell accumulation and inhibits adhesion of platelets and leukocytes to the endothelium. However, the functional importance of endothelial NOS (eNOS) in the rejecting allografts remains unclear. Methods We examined the effects of selective eNOS deficiency in aortic allografts in a murine chronic rejection model using grafts from eNOS knockout (KO) mice (C57BL/6 background; H2b) and normal C3H (H2K) as recipients. Grafts from wild-type C57BL/6 mice served as controls. Grafts from iNOS KO mice served as a second group of controls where the contribution from iNOS was eliminated but eNOS was preserved. Aortic grafts were harvested and analyzed at days 10-14, 18-22, and 26-30 after transplantation. Results Endothelial NOS-deficient grafts showed significantly increased intima/media ratios at days 26-30 compared to controls. Immunostaining demonstrated that in eNOS KO grafts, eNOS was not detectable whereas iNOS was expressed prominently in infiltrating recipient mononuclear cells. In control grafts, eNOS expression was preserved in the endothelium even by day 30, and associated with a decrease in intimal thickening. We further demonstrated that early overexpression of iNOS by ex vivo gene transfer completely prevented the development of arteriosclerosis associated with eNOS deficiency. Conclusions We found that eNOS plays a protective role in allografts, and that in eNOS-deficient allografts, early overexpression of iNOS is capable of preventing the development of allograft arteriosclerosis. In allografts with dysfunctional vascular endothelium and impaired eNOS activity as a result of ischemia or native arteriosclerotic disease, iNOS gene therapy may serve to improve their long-term survival and function.
Journal Article•
TL;DR: These findings demonstrate that apoptotic cascades induced in Jurkat T lymphocytes by anti-Fas Ab or tumor cells are differentially susceptible to a panel of inhibitors of mitochondrial apoptotic events.
Abstract: We have recently reported that apoptosis of T cells induced by squamous cell carcinoma of the head and neck (SCCHN) is partly Fas dependent. This tumor-induced T-cell death is mediated by the activities of caspase-8 and caspase-3 and is partially inhibited by antibodies to either Fas or Fas ligand. We report here that in contrast to apoptosis induced by agonistic anti-Fas antibody (Ab), the tumor-induced apoptotic cascade in Jurkat cells is significantly amplified by a mitochondrial loop. The involvement of mitochondria in tumor-induced apoptosis of T cells was demonstrated by changes in mitochondrial permeability transition as assessed by 3,3'-dihexiloxadicarbocyanine staining, by cleavage of cytosolic BID and its translocation to the mitochondria, by release of cytochrome c to the cytosol, and by the presence of active subunits of caspase-9 in Jurkat T cells cocultured with tumor cells. To further elucidate the significance of mitochondria in tumor-induced T-cell death, we investigated the effects of various inhibitors of the mitochondrial pathway. Specific antioxidants, as well as two inhibitors of mitochondria permeability transition, bongkrekic acid and cyclosporin A, significantly blocked the DNA degradation induced in Jurkat T cells by SCCHN cells. However, these inhibitors had no effect on cells triggered by anti-Fas Ab. Furthermore, a cell-permeable inhibitor of caspase-9, Ac-LEHD.CHO, which did not inhibit T-cell apoptosis induced by anti-Fas Ab, markedly inhibited apoptosis induced by etoposide or by coculture of Jurkat with SCCHN cells. These findings demonstrate that apoptotic cascades induced in Jurkat T lymphocytes by anti-Fas Ab or tumor cells are differentially susceptible to a panel of inhibitors of mitochondrial apoptotic events. It appears that besides the Fas-mediated pathway, additional mitochondria-dependent cascades are involved in apoptosis of tumor-associated lymphocytes. Inhibition of mitochondria-dependent cascades of caspase activation should be considered to enhance the success of immunotherapy or vaccination protocols in cancer.
TL;DR: Gene transfer by donor pretreatment with AdEGFP induces early and efficient gene transduction to liver grafts compared with back-table delivery with CT, and provides early transgene expression in liver grafting that potentially could be used to deliver genes to decrease preservation injury or rejection.
TL;DR: It is clear that the dystrophin cytoskeletal complex is not completely understood and component molecules continue to be discovered, but currently defined molecules (such as alpha‐actinin or neuronal nitric oxide synthase) are being recognized as being specifically associated with the complex.
Abstract: The discovery of dystrophin and its definition as the causative molecule in Duchenne Muscular Dystrophy has led to a renewed interest in the molecular structure of the muscle fiber plasma membrane and its association with the extracellular basal lamina. The original identification of dystrophin gave credence to the possibility that the plasma membrane of the muscle fiber may be highly organized and involved in maintaining appropriate homeostasis in this actively contracting cellular system. In this review, we examine the currently known members of the muscle fiber plasma membrane cytoskeleton and the interactions that occur between the different members of this complex using histological, electron microscopic, and confocal methods. From our studies and others cited in this review, it is clear that the dystrophin cytoskeletal complex is not completely understood and component molecules continue to be discovered. Perhaps equally importantly, currently defined molecules (such as alpha-actinin or neuronal nitric oxide synthase) are being recognized as being specifically associated with the complex. What is striking from all of the studies, to date, is that while we are able to identify members of the dystrophin cytoskeletal complex and while we are able to associate mutations of individual molecules with disease(s), we are still unable to truly define the roles of each of the molecules in maintaining the normal physiology of the muscle fiber.
TL;DR: A possible role for the addition of exogenous, primary myoblasts in the treatment of traumatized and/or diseased muscles that are characterized by myofiber loss is suggested.
TL;DR: The observations suggest that the cryptal plexus is perfused preferentially during hemorrhagic shock, as a consequence of its peculiar microvascular organization, which may explain the relative resistance of the crypts, compared to villi, to hypoxic injuries in order to sustain endocrine function and the regenerative capability of the mucosa after prolonged hypoperfusion conditions.
Abstract: While injuries due to a hypoxic state commonly appear later in both intestinal crypts and basal portion of the villi than in the apical portion, a nonhomogeneous distribution of blood flow in the intestinal mucosa may be supposed. The presence of two different microvascular plexuses inside the mucosa, corresponding to the cryptal plexus and the villous plexus, supports the above hypothesis. This work studies the intestinal microvasculature in shocked versus normal rats. Forty-five rats were divided into four groups to study the histological damage and the microvascular bed by ink injection, fluorescent microsphere infusion, and resin injection for scanning electron microscopy (SEM) of vascular corrosion cast (VCC) observations. An infusion pressure of 100 +/- 5 mm Hg was used in control animals, while 30 +/- 5 mm Hg infusion pressure was adopted for controls as well as for shocked animals to simulate physiological or shock conditions. Hemorrhagic shock was induced by removing blood and maintaining a mean arterial pressure of 30 +/- 5 mm Hg for 45-120 mins. A close connection among the patterns of microvasculature obtained with VCC and ink injection technique can be appreciated. In normal rats the whole microvasculature was visualized, but in both normal and shocked animals injected at low pressure different patterns could be found, generally showing a highly incomplete visualization of the vascular network. A significant decrease of visualization of both the entire microvasculature and the villous plexus is present in shocked animals when compared to unshocked controls, while no difference in the cryptal plexus visualization was observed. These observations suggest that the cryptal plexus is perfused preferentially during hemorrhagic shock, as a consequence of its peculiar microvascular organization. This may explain the relative resistance of the crypts, compared to villi, to hypoxic injuries in order to sustain endocrine function and the regenerative capability of the mucosa after prolonged hypoperfusion conditions that can lead to villous damage and temporary loss of the intestinal barrier function.
TL;DR: In this paper, a theoretical model for the calculation of Specific Dissipation (SD) was developed based on themodel, and the effect of ambient and coolant radiator inlet temperature variation on SD has been predicted.
Abstract: In this paper, a theoretical model for the calculation ofSpecific Dissipation (SD) was developed. Based on themodel, the effect of ambient and coolant radiator inlettemperatures on SD has been predicted.Results indicate that the effect of ambient and coolantinlet temperature variation on SD is small (less than 2%)when ambient temperature varies between 10 and 50°Cand coolant radiator inlet temperature between 60 and120°C. The effect of coolant flowrate on SD is larger if there is alarger flowrate variation. Experimental results indicatethat a 1 % variation at 1.0 L/s will cause about ±0.6% SDvariation. Therefore the flowrate should be carefully con-trolled. INTRODUCTION Aerodynamic drag reduction is desirable to minimizevehicle fuel usage, yet a practical requirement of vehicledesign is adequate airflow through the radiator to ensureadequate engine cooling under all operating conditions.Therefore, it is important to optimize the cooling air flowthrough a vehicle’s radiator without detriment to aerody-namic drag.In the design and development stage of a vehicle enginecooling system, the final evaluation of the system is usu-ally made by either on-road testing or placing the vehiclein an environmental wind tunnel (EWT) and simulatingsome of the climatic properties that exist on the road.However, environmental wind tunnels have tended toevolve in a rather pragmatic way. Initially dynamometerswere installed to test the car operation. An air supplywas needed to cool the engine, so a jet of air wasdirected at the radiator. Development then progressedinto the demands of solar radiator, cooling, etc.The cost associated with cooling and heating an EWT isconsiderable, so EWTs were not made any bigger thanessential. Consequently, the air supply and cross-sec-tion of the wind tunnel was kept to a minimum.However, for the precision needed in reproducing on-roadflow in the wind tunnel, the pressure distribution over thecar should be more representative than that delivered bya jet of air exiting from a typically 2-4 square meter noz-zle in front of the car. Thus the larger types of wind tun-nels used for the measurement of aerodynamiccoefficients should be used.Over the last 15 years of commercial cooling tests atRMIT, the demand for resolving the front-end cooling hasincreased from a SD resolution of about 3% to the needto resolve under 1%, often to 0.5% to determine whetherone configuration is better than another. This is difficult toachieve in an EWT. On-road, the effect of atmosphericwinds combined with ambient temperature variationsmake repeatability and thus resolution even more diffi-cult.Besides improving precision, it is also desirable to beable to test the effects of tail-winds [14] and cross-winds[10]; and thus larger aerodynamic wind tunnels at RMITand Monash have been used. In addition, many aerodynamic wind tunnels do not havedynamometers or extensive cooling capability. Thustechniques were developed to tune the geometry of carfront ends without using a dynamometer and where thetemperature of the wind tunnel could not be indepen-dently varied.
TL;DR: Recombinant human IL-11 prevents mucosal atrophy and shortening of defunctionalized intestinal loops and may help reduce the incidence of short gut syndrome in infants with extensive NEC.