Showing papers by "Simon C. Watkins published in 2003"
TL;DR: Characterization of apoptotic cell/DC interaction and its outcome provides insight into the mechanisms by which apoptotic cells affect DC function without disrupting peripheral tolerance.
323 citations
TL;DR: Inhibition of poly(ADP-ribosylation) within the mitochondrial compartment preserved transmembrane potential, NAD+content, and cellular respiration, prevented release of apoptosis-inducing factor, and reduced neuronal cell death triggered by oxidative stress, and implicate intra-mitochondrial poly( ADP- ribosylations) as an important therapeutic target for central nervous system and other diseases associated with oxidative stress and energy failure.
295 citations
TL;DR: Data suggest that the anti-inflammatory and antiapoptotic properties of CO confer potent cytoprotection in a rat model of lung transplantation, which is limited by the high incidence of acute graft rejection.
Abstract: Successful lung transplantation has been limited by the high incidence of acute graft rejection. There is mounting evidence that the stress response gene heme oxygenase-1 (HO-1) and/or its catalytic by-product carbon monoxide (CO) confers cytoprotection against tissue and cellular injury. This led us to hypothesize that CO may protect against lung transplant rejection via its anti-inflammatory and antiapoptotic effects. Orthotopic left lung transplantation was performed in Lewis rat recipients from Brown-Norway rat donors. HO-1 mRNA and protein expression were markedly induced in transplanted rat lungs compared to sham-operated control lungs. Transplanted lungs developed severe intraalveolar hemorrhage, marked infiltration of inflammatory cells, and intravascular coagulation. However, in the presence of CO exposure (500 ppm), the gross anatomy and histology of transplanted lungs showed marked preservation. Furthermore, transplanted lungs displayed increased apoptotic cell death compared with the transplanted lungs of CO-exposed recipients, as assessed by TUNEL and caspase-3 immunostaining. CO exposure inhibited the induction of IL-6 mRNA and protein expression in lung and serum, respectively. Gene array analysis revealed that CO also down-regulated other proinflammatory genes, including MIP-1α and MIF, and growth factors such as platelet-derived growth factor, which were up-regulated by transplantation. These data suggest that the anti-inflammatory and antiapoptotic properties of CO confer potent cytoprotection in a rat model of lung transplantation.
229 citations
TL;DR: A novel role for SR-A is defined, and it is suggested that Ag uptake from live cells by DC may be important in the generation of immunity and in the maintenance of peripheral tolerance in vivo.
Abstract: Monocyte-derived dendritic cells (DC) possess the unique capacity to capture Ag from live cells through intimate cell contact, a process referred to as nibbling We sought to define the receptor(s) mediating DC nibbling Uptake of fluorescently labeled plasma membrane from live cells by DC was inhibited by protease treatment and by a panel of polyanionic ligands, implicating scavenger receptors (SR) in this process Differential expression of SR on DC and macrophages correlated with the capacity to acquire membrane from live cells Internalized membrane colocalized with SR ligand and entered the endosomal pathway DC very efficiently acquired and internalized gp100 tumor Ag expressed at the surface of viable adenocarcinoma cells via recombinant adenoviral infection Cross-presentation of gp100 by DC to MHC class I-restricted T cells was inhibited by polyanionic SR ligand and an Ab to type A SR (SR-A), whereas Ab to the class B SR CD36, which mediates uptake of apoptotic cells, induced no inhibition DC capture of fluorescently labeled membrane from live cells was partially blocked by SR-A-specific Ab, suggesting that other SR may also be contributing to nibbling DC maturation resulted in a switch in expression from type II SR-A (SR-AII) to the SR-AI splice variant Finally, SR-A was identified on interdigitating DC isolated from monkey lymph nodes These findings define a novel role for SR-A, and suggest that Ag uptake from live cells by DC may be important in the generation of immunity and in the maintenance of peripheral tolerance in vivo
188 citations
TL;DR: It is suggested that by inhibiting Itk, and consequently Ca2+ influx, TGF-β limits T cell differentiation along both the Th1 and Th2 lineages.
Abstract: Transforming growth factor (TGF)-β inhibits T cell proliferation and differentiation. TGF-β has been shown to inhibit the expression of transcription factors such as GATA-3 and T-bet that play important roles in T cell differentiation. Here we show that TGF-β inhibits T cell differentiation at a more proximal step. An early event during T cell activation is increased intracellular calcium levels. Calcium influx in activated T cells and the subsequent activation of transcription factors such as NFATc, events essential for T cell differentiation, are modulated by the Tec kinases that are downstream of the T cell receptor and CD28. We show that in stimulated CD4+ T cells, TGF-β inhibits phosphorylation and activation of the Tec kinase Itk, increase in intracellular Ca2+ levels, NFATc translocation, and activation of the mitogen-activated protein kinase ERK that together regulate T cell differentiation. Our studies suggest that by inhibiting Itk, and consequently Ca2+ influx, TGF-β limits T cell differentiation along both the Th1 and Th2 lineages.
152 citations
Journal Article•
TL;DR: The ability of combined cytokine gene transfer to enhance multiple effector functions mediated by intralesionally injected DCs that may concertedly promote cross-priming and the accelerated immune-mediated rejection of tumors is supported.
Abstract: Dendritic cells (DCs) were adenovirally engineered to constitutively and durably secrete the potent Th1-biasing cytokines interleukin (IL)-12 (AdIL12DC) and/or IL-18 (AdIL18DC) and evaluated for their ability to promote therapeutic antitumor immunity in murine sarcoma models. Injection of either AdIL12DC or AdIL18DC into day 7 CMS4 or MethA tumors resulted in tumor rejection or slowed tumor growth when compared with control cohorts. Importantly, intratumoral injection with DCs engineered to secrete both IL-12 and IL-18 (AdIL12/IL18DC) resulted in complete and the most acute rejection of any treatment group analyzed. This strategy was also effective in promoting the regression of contralateral, untreated tumors. Both CD4+ and CD8+ T cells were required for tumor rejection. CD8+ splenic T cells from mice treated with AdIL12/IL18DC produced the highest levels of IFN-gamma in response to tumor rechallenge in vitro and displayed the broadest repertoire of Tc1-type reactivity to acid-eluted, tumor-derived peptides among all treatment cohorts. This apparent enhancement in cross-presentation of tumor-associated epitopes in vivo may result from the increased capacity of engineered DCs to kill tumor cells, survive tumor-induced apoptosis, and present immunogenic MHC/tumor peptide complexes to T cells after intratumoral injection. In support of this hypothesis, cytokine gene-engineered DCs expressed higher levels of MHC and costimulatory molecules, as well as Fas ligand and membrane-bound tumor necrosis factor alpha, with the latter markers associated with elevated tumoricidal activity in vitro. Cytokine gene-engineered DCs appeared to have a survival advantage in situ when injected into tumor lesions, to be found in approximation with regions of tumor apoptosis, and to have the capacity to ingest apoptotic tumor bodies. These results support the ability of combined cytokine gene transfer to enhance multiple effector functions mediated by intralesionally injected DCs that may concertedly promote cross-priming and the accelerated immune-mediated rejection of tumors.
139 citations
TL;DR: This work reports the development of a stringently regulated, tetracycline-inducible, lung-specific transgenic system that allows regulated expression of KGF in the lung without causing developmental abnormalities from leaky KGF expression and protects the lung epithelium but not the endothelium from cell death.
Abstract: Oxidant-induced injury to the lung is associated with extensive damage to the lung epithelium. Instillation of keratinocyte growth factor (KGF) in the lungs of animals protects animals from oxidant-induced injury but the mechanism of protection is not well understood. An inherent problem in studying KGF function in vivo has been that constitutive overexpression of KGF in the lung causes embryonic lethality with extensive pulmonary malformation. Here we report the development of a stringently regulated, tetracycline-inducible, lung-specific transgenic system that allows regulated expression of KGF in the lung without causing developmental abnormalities from leaky KGF expression. By using this system, we show that exposure of KGF-expressing mice to hyperoxia protects the lung epithelium but not the endothelium from cell death in accordance with the selective expression of KGF receptor on epithelial and not on endothelial cells. Investigations of KGF-induced cell survival pathways revealed KGF-induced activation of the multifunctional pro-survival Akt signaling axis both in vitro and in vivo. Inhibition of KGF-induced Akt activation by a dominant-negative mutant of Akt blocked the KGF-mediated protection of epithelial cells exposed to hyperoxia. KGF-induced Akt activation may play an important role in inhibiting lung alveolar cell death thereby preserving the lung architecture and function during oxidative stress.
137 citations
TL;DR: A predominant role for the caspase-8/Bid pathway is identified in signaling associated with hyperoxic lung injury and cell death in vivo and in vitro and, for the first time, it is identified that the response to hyperoxia in vivo predominantly involved the activation of the Bid/casp enzyme pathway without apparent increases in Bax expression.
135 citations
TL;DR: The results showed that the expression profile of recombinant protein accumulation in endosperm paralleled that of the two storage proteins and that human lysozyme and storage proteins co-localized to type-II protein bodies.
Abstract: In order to understand the characteristics of recombinant protein expression and sublocalization in rice ( Oryza sativa L.) endosperm, we examined the expression level of human lysozyme protein and its subcellular location in transgenic rice seeds driven by rice glutelin and globulin promoters and signal peptides. A time course of human lysozyme expression during endosperm development was analyzed. The results showed that the expression profile of recombinant protein accumulation in endosperm paralleled that of the two storage proteins. Immunofluorescence microscopy revealed that human lysozyme and storage proteins co-localized to type-II protein bodies. Both promoter-signal peptide parings targeted recombinant protein to the protein bodies. In addition, a transgenic line with a higher lysozyme expression level exhibited morphologically different protein bodies with an unbalanced composition of lysozyme and native storage proteins. The high-level expression of recombinant protein distorted the trafficking and sorting of native storage proteins in rice endosperm and affected the expression of native storage protein.
117 citations
TL;DR: A significant prolongation of graft function suggests that the antioxidant activity of MnSOD is beneficial to transplanted islet survival and may be used in combination with other strategies aimed at islet graft protection.
Abstract: Islet transplantation is a promising cure for diabetes. However, inflammation, allorejection, and recurrent autoimmune damage all may contribute to early graft loss. Pancreatic islets express lower levels of antioxidant genes than most other tissues of the body, and β-cells in particular are sensitive to oxidative damage. Therefore, damage from oxidative stress may pose a major obstacle to islet replacement therapy in that both the islet isolation and transplantation processes generate oxygen radicals. To determine whether antioxidant gene overexpression in isolated pancreatic islets can prevent oxidative damage and prolong islet function after transplantation, we used the NOD mouse model to study oxidative stress encountered during both transplantation and autoimmune attack. We transferred an antioxidant gene, manganese superoxide dismutase (MnSOD), by adenoviral infection into isolated islets that were transplanted into streptozotocin-treated NODscid recipient mice. Functioning islet grafts were subsequently exposed to diabetogenic spleen cells and monitored until graft failure. The results show that islet grafts overexpressing MnSOD functioned ∼50% longer than control grafts. This significant prolongation of graft function suggests that the antioxidant activity of MnSOD is beneficial to transplanted islet survival and may be used in combination with other strategies aimed at islet graft protection.
116 citations
TL;DR: Like in the gut, c-kit-positive, ICC-like cells are present in the urinary tract but appear to be restricted to the proximal ureter of this murine species.
Abstract: The existence of a pacemaker system in the urinary tract capable of orchestrating the movement of filtrated urine from the ureteral pelvis to the distal ureter and lower urinary tract seems intuiti...
TL;DR: The results demonstrate that the insulin secretory granule is the major site of K(ATP) channels of the endocrine pancreas, and two different K(ir)6.2 antibodies specifically label perimeter membrane regions of the secretorygranule.
Abstract: With ATP sites on K ir 6.2 that inhibit activity and ADP sites on SUR1 that antagonize the inhibition, ATP-sensitive potassium channels (K ATP channels) are designed as exquisite sensors of adenine nucleotide levels that signal changes in glucose metabolism. If pancreatic K ATP channels localize to the insulin secretory granule, they would be well positioned to transduce changes in glucose metabolism into changes in granule transport and exocytosis. Tests for pancreatic K ATP channels localized to insulin secretory granules led to the following observations: fluorescent sulfonylureas that bind the pancreatic K ATP channel specifically label intracellular punctate structures in cells of the endocrine pancreas. The fluorescent glibenclamides colocalize with Ins-C-GFP, a live-cell fluorescent reporter of insulin granules. Expression of either SUR1-GFP or K ir 6.2-GFP fusion proteins, but not expression of GFP alone, directs GFP fluorescence to insulin secretory granules. An SUR1 antibody specifically labels insulin granules identified by anti-insulin. Two different K ir 6.2 antibodies specifically label insulin secretory granules identified by anti-insulin. Immunoelectron microscopy showed K ir 6.2 antibodies specifically label perimeter membrane regions of the secretory granule. Relatively little or no labeling of other structures, including the plasma membrane, was found. Our results demonstrate that the insulin secretory granule is the major site of K ATP channels of the endocrine pancreas.
TL;DR: In vivo studies in intact hepatocytes provide novel findings not previously disclosed by in vitro studies, and indicates the importance of several mechanisms in contributing Bid-mediated mitochondria dysfunction that could be potential cellular targets of intervention.
TL;DR: It is demonstrated, for the first time, that genetically modified DC can prevent diabetes in the context of advanced insulitis.
Abstract: Our previous studies demonstrated that adoptive transfer of dendritic cells (DC) prevents diabetes in young nonobese diabetic (NOD) mice by inducing regulatory T(H)2 cells. In this report, as a means of treating NOD mice with more advanced insulitis, we infected DC with adenoviral vectors expressing interleukin (IL)-4 (Ad.IL-4), eGFP (Ad.eGFP), or empty vector (Ad psi 5). DC infected with any of the Ad vectors expressed higher levels of CD40, CD80, and CD86 molecules than uninfected DC and Ad.IL-4 DC produced IL-4 after lipopolysaccharide (LPS) and interferon (IFN)-gamma stimulation. Ad-infected DC efficiently stimulated allogeneic T cells, and cultures of T cells with Ad.IL-4 DC produced lower levels of IFN-gamma and marginally higher levels of IL-4. In vivo studies demonstrated that the Ad.eGFP DC trafficked to the pancreatic lymph nodes within 24 hr of intravenous administration, and could be visualized in the T cell areas of the spleen. The intrapancreatic IFN-gamma:IL-4 or IFN-gamma:IL-10 cytokine ratios were lower in 10-week-old mice treated with Ad.IL-4 DC, and these mice were significantly protected from disease. These results demonstrate, for the first time, that genetically modified DC can prevent diabetes in the context of advanced insulitis.
TL;DR: Regulated modification of membrane lipid composition is required to support the cooperative interactions that enable selective transport, as it is demonstrated here for the mammalian COPII coat.
Abstract: The small GTPase Sar1p controls the assembly of the cytosolic COPII coat that mediates export from the endoplasmic reticulum (ER). Here we demonstrate that phospholipase D (PLD) activation is required to support COPII-mediated ER export. PLD activity by itself does not lead to the recruitment of COPII to the membranes or ER export. However, PLD activity is required to support Sar1p-dependent membrane tubulation, the subsequent Sar1p-dependent recruitment of Sec23/24 and Sec13/31 COPII complexes to ER export sites and ER export. Sar1p recruitment to the membrane is PLD independent, yet activation of Sar1p is required to stimulate PLD activity on ER membranes, thus PLD is temporally regulated to support ER export. Regulated modification of membrane lipid composition is required to support the cooperative interactions that enable selective transport, as we demonstrate here for the mammalian COPII coat.
TL;DR: The results suggest that proapoptotic peptides and, in particular, DP2 can be clinically useful for treatment of synovial hyperplasia, as well as inflammation.
TL;DR: Brain tissue samples removed from adult patients during the surgical management of severe intracranial hypertension after traumatic brain injury were compared with postmortem control brain tissue samples to identify additional steps in the programmed cell death cascade involving Fas death receptors and caspase‐8 after TBI in humans.
Abstract: Programmed cell death involves a complex and interrelated cascade of cysteine proteases termed caspases that are synthesized as inactive zymogens, which are proteolytically processed to active enzymes. Caspase-8 is an initiator caspase that becomes activated when Fas death receptor-Fas ligand (FasL) coupling on the cell surface leads to coalescence of a "death complex" perpetuating the programmed cell death cascade. In this study, brain tissue samples removed from adult patients during the surgical management of severe intracranial hypertension after traumatic brain injury (TBI; n=17) were compared with postmortem control brain tissue samples (n=6). Caspase-8 mRNA was measured by semiquantitative reverse transcription and polymerase chain reaction, and caspase-8 protein was examined by Western blot and immunocytochemistry. Fas and FasL were also examined using Western blot. Caspase-8 mRNA and protein were increased in TBI patients vs. controls, and caspase-8 protein was predominately expressed in neurons. Proteolysis of caspase-8 to 20-kDa fragments was seen only in TBI patients. Fas was also increased after TBI vs. control and was associated with relative levels of caspase-8, supporting formation of a death complex. These data identify additional steps in the programmed cell death cascade involving Fas death receptors and caspase-8 after TBI in humans.
TL;DR: A nontoxic biodegradable polyurethane matrix synthesized with PDI as a hard segment and sucrose as a hydroxyl group donor to obtain a microtextured spongy urethane matrix that may be useful in many tissue-engineering applications.
Abstract: Urethanes are frequently used in biomedical applications because of their excellent biocompatibility. However, their use has been limited to bioresistant polyurethanes. The aim of this study was to develop a nontoxic biodegradable polyurethane and to test its potential for tissue compatibility. A matrix was synthesized with pentane diisocyanate (PDI) as a hard segment and sucrose as a hydroxyl group donor to obtain a microtextured spongy urethane matrix. The matrix was biodegradable in an aqueous solution at 37°C in vitro as well as in vivo. The polymer was mechanically stable at body temperatures and exhibited a glass transition temperature (Tg) of 67°C. The porosity of the polymer network was between 10 and 2000 μm, with the majority of pores between 100 and 300 μm in diameter. This porosity was found to be adequate to support the adherence and proliferation of bone-marrow stromal cells (BMSC) and chondrocytes in vitro. The degradation products of the polymer were nontoxic to cells in vitro. Subdermal implants of the PDI–sucrose matrix did not exhibit toxicity in vivo and did not induce an acute inflammatory response in the host. However, some foreign-body giant cells did accumulate around the polymer and in its pores, suggesting its degradation is facilitated by hydrolysis as well as by giant cells. More important, subdermal implants of the polymer allowed marked infiltration of vascular and connective tissue, suggesting the free flow of fluids and nutrients in the implants. Because of the flexibility of the mechanical strength that can be obtained in urethanes and because of the ease with which a porous microtexture can be achieved, this matrix may be useful in many tissue-engineering applications. © 2002 Wiley Periodicals, Inc. J Biomed Mater Res 64A: 242–248, 2003
TL;DR: The results suggest that hepatic iNOS expression, associated with peroxisomal localization, is an early molecular response to HS in experimental animals and possibly in human patients with trauma with HS.
Abstract: It has been previously shown that the inducible nitric oxide (NO) synthase (iNOS; NOS-2) is elevated after hemorrhage, and that iNOS-derived NO participates in the upregulation of inflammation as well as lung and liver injury postresuscitation from shock. The purpose of this study was to elu
TL;DR: In this paper, the effects of the A-pillar and windshield effective corner radii on the local flow and its potential for noise generation using a group of idealized road vehicle models were investigated.
TL;DR: A body window is designed to allow for in vivo imaging over time of the islets transplanted under the kidney capsule and may be used to simultaneously observe beta cells and T cells that have been labeled with a fluorochrome distinguishable from Timer.
Abstract: Type 1 diabetes results from the selective destruction of insulin-producing β cells in the islets of Langerhans, and autoimmune T cells are thought to be the mediators of this destruction. T cells are also responsible for allorejection once the islets are transplanted into a patient to reduce the negative consequences of a lack of insulin. To better understand these processes, we have developed a transgenic mouse expressing proinsulin II tagged with a live-cell fluorescent reporter protein, Timer. Timer protein is unique because it changes color from green to red in the first 24 h after synthesis. With this marker, insulin synthesis can be carefully monitored through fluorescent changes over time. To complement this new biotechnological research tool, we designed a body window to allow for in vivo imaging over time of the islets transplanted under the kidney capsule. The window device, which is sutured to replace the underlying skin and body wall over the site of islet transplantation, may be used to simu...
TL;DR: It is concluded that adoptively transferred A‐NK cells are able to eliminate even well‐established metastases, which are resistant to infiltration by the transferred effector cells at time of treatment and might reduce the efficacy of cell‐based immuno‐therapeutic ventures.
Abstract: We have shown previously that i.v. injection of interleukin-2-(IL-2) activated natural killer (A-NK) cells together with IL-2 leads to a substantial localization of the A-NK cells into most, but not all, well-established B16 lung metastases in C57BL/6 mice within 12-24 hr. We demonstrate that the morphology of the lung metastases, (loose or more compact in appearance), and their location in the lungs (on the surface or deep in the lung parenchyma) are closely tied to the infiltration-permissiveness of the metastases as well as their sensitivity to treatment with A-NK cells. Although more than 1100 A-NK cells/mm(2) were found in deep metastases with a "loose" morphology (D-L), only 534, 90 and 89 cells/mm(2) were found in surface-loose (S-L), surface-compact (S-C) and deep-compact (D-C) metastases, respectively. The best infiltrated metastases responded best to the A-NK cell therapy. Thus, metastases of the D-L phenotype became reduced by 65-90% after treatment with 2 x 10(6) A-NK cells and IL-2 (120000 IU Peg-IL-2 every 12 hr for 3 days) compared to similar lesions in animals treated with PEG-IL-2 alone. In contrast, poorly infiltrated metastases, that is lesions of the compact phenotype (D-C and S-C) as well as loose metastases on the lung surface (S-L), did not react significantly to this treatment. We conclude that adoptively transferred A-NK cells are able to eliminate even well-established metastases. The existence of metastases that are resistant to infiltration by the transferred effector cells at time of treatment might reduce the efficacy of cell-based immuno-therapeutic ventures.
TL;DR: It is demonstrated that the C-terminal leucine zipper is critical to facilitate correct folding and plasma membrane trafficking of hIK1, whereas this function is not conserved in other gene family members.
TL;DR: It is concluded that Cdc42 controls cell polarity, likely in part, through its binding to active PLCgamma, which is associated with CDC42 in an EGF-dependent manner.
TL;DR: Interestingly, intradermal and intranodal injections generated a similar intensity and breadth of response, indicating that administration of Ag-expressing DC by either route may be equally effective at inducing immune responses.
Abstract: Adenoviral vectors can be used to deliver complex Ag to dendritic cells (DC), and thus may be ideal for stimulating broad T cell responses to viral pathogens and tumors. To test this hypothesis in a relevant primate model, we used recombinant adenovirus serotype 5 vectors expressing SIV Gag Ag to transduce monocyte-derived DC from rhesus macaques, and then immunized donor animals either by intradermal or intranodal injections. T cell responses were evaluated by ELISPOT assay using previously frozen PBMC pulsed with pools of 15-mer peptides representing the Gag sequence. Immunization resulted in rapid and potent induction of T cell responses to multiple regions of Gag, with frequencies approaching 1 Gag-specific T cell per 500 uncultured PBMC. Surprisingly, intradermal and intranodal injections generated a similar intensity and breadth of response, indicating that administration of Ag-expressing DC by either route may be equally effective at inducing immune responses. Detailed analysis of two monkeys revealed CD8(+) T cell responses to several peptide epitopes of Gag not previously described, at least two of which are restricted by MHC class I alleles not currently identified. Repeated vaccination did not induce T cell responses to the adenoviral vector and did not prevent Ag-expressing DC injected under the capsule of the lymph node from migrating to the paracortex and interposing between T cells. However, boost injections of adenovirus-transduced DC were generally limited in efficacy. These findings support the use of adenovirus-transduced DC in the therapy of HIV infection and cancer.
Journal Article•
TL;DR: It is shown that the interstitial diffusion of large molecules is significantly hindered in tumors with high collagen levels, and after irradiation, the decrease in K and increase in collagen type I levels could significantly hinder the convective movement and diffusion ofLarge therapeutic agents in tumors.
Abstract: Purpose: We have shown that the interstitial diffusion of large molecules is significantly hindered in tumors with high collagen levels. Because large therapeutic agents (e.g., monoclonal antibodies and viral vectors) will be combined with radiation or chemotherapy, it is significant to determine how cytotoxic therapies modify the transport and composition of the interstitial space in tumors. To test the hypothesis that radiation alters tumor interstitial transport, we measured tumor hydraulic conductivity (K) and hyaluronan and collagen type I levels after irradiation. Experimental Design: K and the quantification of interstitial matrix components were determined in sections of s.c. implants of the human colon adenocarcinoma LS174T. K was measured on days 1 and 5 after 10 Gy of irradiation or on day 5 after 30 Gy of irradiation. Results: Compared with control tumors, K decreased by approximately 12-fold after 10 or 30 Gy of irradiation on day 5. At 24 h after irradiation with 10 Gy, the decrease in K was not significant. Five days after 10 and 30 Gy of irradiation, the decrease in K was associated with significantly higher levels of collagen type I. The collagen type I content was not changed 24 h after irradiation with 10 Gy. Irradiation did not significantly increase hyaluronan levels in LS174T tumors. Conclusions: After irradiation, the decrease in K and increase in collagen type I levels could significantly hinder the convective movement and diffusion of large therapeutic agents in tumors.
TL;DR: The ability to easily create, directly from plasmid components, an oncolytic replicating VSV with a restricted host cell range is demonstrated.
TL;DR: Data implicate induction of the stress response and its modulation by the chaperone cofactor and Bcl‐2 family member BAG‐1, after TBI in humans, and suggest negative modulation of Hsp70/Hsc70 protein refolding activity in injured brain.
Abstract: The stress response in injured brain is well characterized after experimental ischemic and traumatic brain injury (TBI); however, the induction and regulation of the stress response in humans after TBI remains largely undefined. Accordingly, we examined injured brain tissue from adult patients (n = 8) that underwent emergent surgical decompression after TBI, for alterations in the inducible 72-kDa heat shock protein (Hsp70), the constitutive 73-kDa heat shock protein (Hsc70), and isoforms of the chaperone cofactor BAG-1. Control samples (n = 6) were obtained postmortem from patients dying of causes unrelated to CNS trauma. Western blot analysis showed that Hsp70, but not Hsc70, was increased in patients after TBI versus controls. Both Hsp70 and Hsc70 coimmunoprecipitated with the cofactor BAG-1. The 33 and 46, but not the 50-kDa BAG-1 isoforms were increased in patients after TBI versus controls. The ratio of the 46/33-kDa isoforms was increased in TBI versus controls, suggesting negative modulation of Hsp70/Hsc70 protein refolding activity in injured brain. These data implicate induction of the stress response and its modulation by the chaperone cofactor and Bcl-2 family member BAG-1, after TBI in humans.
TL;DR: Yeast expressing the cystic fibrosis transmembrane conductance regulator (CFTR) concentrate the protein at defined sites in the ER membrane that are not necessarily enriched for the ER molecular chaperone BiP, and it is proposed that these sites are Russell bodies, an ER subcompartment in which misfolded proteins are stored and can be targeted for degradation.
Abstract: Almost all secreted proteins pass through the endoplasmic reticulum (ER), an organelle that is equipped to tolerate and/or degrade misfolded proteins. We report here that yeast expressing the cystic fibrosis transmembrane conductance regulator (CFTR) concentrate the protein at defined sites in the ER membrane that are not necessarily enriched for the ER molecular chaperone BiP. We propose that these sites are Russell bodies, an ER subcompartment in which misfolded proteins are stored and can be targeted for degradation.
TL;DR: It is shown that MyoD, a member of the MRF family, also plays a role in fetal synapse formation and assemble a complex postsynaptic apparatus that includes muscle-specific kinase (MuSK), rapsyn, erbB, and utrophin.
Abstract: Myogenic regulatory factors (MRFs), muscle-specific transcription factors, are implicated in the activity-dependent regulation of nicotinic acetylcholine receptor (AChR) subunit genes. Here we show, with immunohistochemistry, Western blotting, and electron microscopy that MyoD, a member of the MRF family, also plays a role in fetal synapse formation. In the diaphragm of 14.5 d gestation (E14.5) wild-type and MyoD-/- mice, AChR clusters (the formation of which is under a muscle intrinsic program) are confined to a centrally located endplate zone. This distribution persists in wild-type adult muscles. However, beginning at E15.5 and extending to the adult, innervated AChR clusters are distributed all over the diaphragm of MyoD-/- mice, extending as far as the insertion of the diaphragm into the ribs. In wild-type muscle, motor axons terminate on clusters adjacent to the main intramuscular nerve; in MyoD-/- muscle, axonal bundles form extensive secondary branches that terminate on the widely distributed clusters. The number of AChR clusters on adult MyoD-/- and wild-type diaphram muscles is similar. Junctional fold density is reduced at MyoD-/- endplates, and the transition from the fetal (alpha, beta, gamma, delta) to adult-type (alpha, beta, delta, epsilon) AChRs is markedly delayed. However, MyoD-/- mice assemble a complex postsynaptic apparatus that includes muscle-specific kinase (MuSK), rapsyn, erbB, and utrophin.