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1.
Leukocyte extravasation is a prerequisite for host defense and autoimmunity alike. Detailed understanding of the tightly controlled and overlapping sequences of leukocyte extravasation might aid development of novel therapeutic strategies. Leukocyte extravasation is initiated by interaction of selectins with appropriate carbohydrate ligands. Lack of P-selectin expression leads to decreased contact hypersensitivity responses. Yet, it remains unclear if this is due to inhibition of leukocyte extravasation to the skin or due to interference with initial immune activation in lymph nodes. In line with previous data, we here report a decreased contact hypersensitivity response, induced by 2,4,-dinitrofluorobenzene (DNFB), in P-selectin-deficient mice. Eliciting an immune reaction towards DNFB in wild-type mice, followed by adoptive transfer to P-selectin-deficient mice, had no impact on inflammatory response in recipients. This was significantly reduced in wild-type recipient mice adoptively transferred with DNFB immunity generated in P-selectin-deficient mice. To investigate if platelet or endothelial P-selectin was involved, mice solely lacking platelet P-selectin expression generated by bone marrow transplantation were used. Adoptive transfer of immunity from wild-type mice reconstituted with P-selectin-deficient bone marrow led to a decrease of inflammatory response. Comparing this decrease to the one observed using P-selectin-deficient mice, no differences were observed. Our observations indicate that platelet, not endothelial, P-selectin contributes to generation of immunity in DNFB-induced contact hypersensitivity.Infiltration of leukocytes is a hallmark of inflammation. To reach the affected tissues, leukocytes must leave the bloodstream. This process of leukocyte extravasation requires several distinct steps, occurring in sequence. Leukocyte extravasation is initiated by short-lived interactions of adhesion molecules from the selectin family with appropriate carbohydrate scaffolds displayed by several glycoproteins, leading to tethering and rolling of leukocytes along the endothelial lining of the vasculature. Of the numerous adhesion molecules, endothelial P-selectin, along with E-selectin and vascular cell adhesion molecule (VCAM)-1, initiates and sustains rolling interactions of leukocytes in the skin microvasculature.In addition to a direct interaction of leukocytes with the endothelium lining the vasculature, platelets are becoming more and more recognized to initiate leukocyte rolling in several organs, including peripheral lymph nodes,1 atherosclerotic lesions2 and skin.3 In detail, platelets may influence leukocyte rolling in numerous ways: First, formation of platelet-leukocyte aggregates in the bloodstream leads to activation of bound leukocytes,2 leading to an increased avidity of leukocyte integrins,4–6 which then may allow rolling interactions through binding of leukocyte VLA-4 to endothelial VCAM-1.7 Second, rolling of activated platelets along the vasculature2,3,8,9 allows a deposition of platelet-derived pro-inflammatory substances directly to the endothelial cells, which in turn activates endothelial cells, leading to an increased expression/secretion of adhesion molecules and cytokines.10–13 Third, formation of platelet-leukocyte aggregates increases leukocyte rolling in lymph nodes9 and the skin.3 In addition, this platelet-mediated leukocyte rolling has been shown to functionally relevant in lymph nodes, lung and the skin. Impaired extravasation of naïve T lymphocytes into the lymph nodes, and thus diminished generation of immunity, in L-selectin-deficient mice,14 can be restored by infusion of activated human platelets. Interestingly, infusion of activated platelets had no impact on generation of immunity in wild-type mice.15 Likewise, pulmonary leukocyte recruitment in platelet-depleted mice was abolished in a model of ovalbumin-induced asthma.16 Furthermore, in a model of chronically induced contact dermatitis, infusion of wild-type, but not P-selectin-deficient, platelets restored the inflammatory response in mice rendered thrombocytopenic.17The latter observation is in line with previous findings, showing an impairment of cutaneous inflammation induced by application of haptens, such as oxazolone, in P-selectin-deficient mice.18 However, while the work by Tamagawa-Mineoka and colleagues17 has clearly shown a dependency of leukocyte extravasation on platelet P-selectin expression to the skin, the role of P-selectin in the generation of immunity toward cutaneous applied haptens remained to be elucidated. We therefore investigated the role of P-selectin in the generation of immunity in the model of 2,4,-dinitrofluorobenzene (DNFB)-induced cutaneous hypersensitivity.  相似文献   

2.
Nonalcoholic fatty liver disease is an increasingly prevalent spectrum of conditions characterized by excess fat deposition within hepatocytes. Affected hepatocytes are known to be highly susceptible to ischemic insults, responding to injury with increased cell death, and commensurate liver dysfunction. Numerous clinical circumstances lead to hepatic ischemia. Mechanistically, specific means of reducing hepatic vulnerability to ischemia are of increasing clinical importance. In this study, we demonstrate that the glucagon-like peptide-1 receptor agonist Exendin 4 (Ex4) protects hepatocytes from ischemia reperfusion injury by mitigating necrosis and apoptosis. Importantly, this effect is more pronounced in steatotic livers, with significantly reducing cell death and facilitating the initiation of lipolysis. Ex4 treatment leads to increased lipid droplet fission, and phosphorylation of perilipin and hormone sensitive lipase – all hallmarks of lipolysis. Importantly, the protective effects of Ex4 are seen after a short course of perioperative treatment, potentially making this clinically relevant. Thus, we conclude that Ex4 has a role in protecting lean and fatty livers from ischemic injury. The rapidity of the effect and the clinical availability of Ex4 make this an attractive new therapeutic approach for treating fatty livers at the time of an ischemic insult.The incidence of obesity and fatty liver disease is increasing worldwide. Non alcoholic fatty liver disease (NAFLD) includes a spectrum of liver abnormalities ranging from simple steatosis with preserved synthetic function to end-stage liver disease requiring transplantation.1, 2 The cause of hepatic dysfunction related to steatosis remains incompletely defined.3 However, it is known that a steatotic liver has increased susceptibility to ischemic insults, such as those induced during liver resections and liver surgery,4, 5, 6 heart failure,7 and shock.8 In addition, steatotic livers are known to weather the ischemic insult of transplantation poorly,9 resulting in increased rates of primary nonfunction and initial graft dysfunction.10, 11 As such, fatty livers are routinely turned down for transplantation and this impacts transplant wait list morbidity and mortality.12 Thus, liver steatosis contributes to the public health burden and methods to mollify the adverse effects of liver steatosis are relevant across a large spectrum of hepatic diseases.The inability of a steatotic liver to withstand ischemic insult is directly related to increased post ischemic cell death, which can occur through necrosis and apoptosis. The fundamental connection between intracellular fat and poor hepatic cell survival13 is incompletely understood. However, it has been suggested that methods that decrease intracellular fat reverse this susceptibility and the use of glucagon-like peptide-1 (GLP-1) analogues is one such approach. GLP-1 is secreted from the L cells of the small intestine and its cognate receptor (GLP-1R) is present in several organs, such as the pancreas, brain, heart, kidney, and liver. Although it is well known for its incretin action,14 it also has pleotropic effects.15, 16, 17, 18, 19 In the liver we have shown that GLP-1 or its homologue Exendin 4 (Ex4) acts directly on steatotic hepatocytes to decrease their lipid content.20, 21 In addition, a cytoprotective action of Ex4 with improvement in cell survival has also been reported.22 Thus, we hypothesize that anti-steatotic effects of Ex4 in hepatocytes and cytoprotective effects in other organs make it a rational target for investigation in steatotic livers undergoing ischemia reperfusion injury (IRI), a common clinical scenario in people with NAFLD. In this study, we explore the role of Ex4 in protecting against necrosis and apoptosis, the two forms of cell death encountered in hepatic IRI, and we provide evidence to show that Ex4 stimulates lipolysis with a short course of treatment. To our knowledge, this is the first study showing a direct and rapid action of Ex4 in acutely reversing the vulnerability of a steatotic liver to ischemic insults, supporting the investigation of Ex4 as a potential therapeutic agent for treatment of people with NAFLD undergoing ischemic injury and at the time of procurement of a fatty liver for transplantation.  相似文献   

3.
4.
Pulmonary immunity depends on the ability of leukocytes to neutralize potentially harmful and frequent insults to the lung, and appropriate regulation of leukocyte migration and adhesion is integral to this process. Arhgef1 is a hematopoietic-restricted signaling molecule that regulates leukocyte migration and integrin-mediated adhesion. To explore a possible regulatory role for Arhgef1 in pulmonary immunity we examined the lung and its leukocytes in wild-type and Arhgef1-deficient animals. Here we report that the lungs of Arhgef1−/− mice harbored significantly more leukocytes, increased expression and activity of matrix metalloproteinases (MMPs), airspace enlargement, and decreased lung elastance compared with wild-type lungs. Transfer of Arhgef1−/− lung leukocytes to wild-type mice led to airspace enlargement and impaired lung function, indicating that loss of Arhgef1 in leukocytes was sufficient to induce pulmonary pathology. Furthermore, we showed that Arhgef1-deficient peritoneal macrophages when either injected into the lungs of wild-type mice or cultured on fibronectin significantly increased expression and activity of MMPs relative to control macrophages, and the in vitro fibronectin induction was dependent on the α5β1 integrin pair. Together these data demonstrate that Arhgef1 regulates α5β1-mediated MMP expression by macrophages and that loss of Arhgef1 by leukocytes leads to pulmonary pathology.Leukocytes are resident in the lungs of healthy individuals and are necessary for the innate and adaptive immune response toward potentially harmful foreign antigens that are exposed to the lung on a constant basis. Protection provided by innate lung immunity is controlled in part through the action of sentinel alveolar macrophages (AMs)1 and alveolar epithelial cells.2 Integrin-mediated signaling is a critical component of macrophage function and has been shown to be important for pulmonary immunity.3,4 Furthermore, expression of extracellular matrix proteins are increased in several types of immune-related lung disease including chronic obstructive pulmonary disease (COPD).5Inappropriate regulation of pulmonary immune function has significant consequences and contributes to a number of lung diseases. In particular, it has been shown that the severity of COPD in humans correlates with the extent of inflammatory cell infiltrate comprising macrophages, neutrophils, CD4, CD8, and B lymphocytes.6 COPD has also been characterized by lung tissue damage, elevated production of matrix metalloproteinases (MMPs) by pulmonary leukocytes, and impaired lung function.7–9 Precisely how, if at all, the increased leukocyte presence leads to lung pathology is not yet established.Leukocytes migrate in response to chemoattractants that signal via G-protein–coupled receptors to polarize the cell.10 Cell polarization is accomplished by the localized activation within the cell of Rho GTPase family members where Cdc42 and Rac are activated at the migrating cell leading edge and Rho at the trailing edge.10 Arhgef1 (Human Genome Organization nomenclature, formerly known as Lsc in mouse and p115RhoGEF in humans) is an intracellular protein restricted in expression to hematopoietic cells that regulates signaling from select G-protein–coupled receptors as well as RhoA activation.11,12 Characterization of Arhgef1−/− mice by our lab, and similar mouse mutants by others, have demonstrated a role for Arhgef1 in regulating leukocyte migration and adhesion.13–16We have previously demonstrated that T lymphocytes depend on Arhgef1 to mount an adaptive secondary immune response to airway challenge.17 In the course of those studies, we found that naïve Arhgef1-deficient lungs reproducibly harbor an increased number of leukocytes compared with wild-type controls.17 To determine whether this increased presence of pulmonary leukocytes has functional consequences, we examined mutant pulmonary leukocytes and lung function in Arhgef1−/− mice in the absence of experimental challenge. In this report, we show that loss of Arhgef1 expression by pulmonary leukocytes leads to airspace enlargement and decreased lung elastance and that elevated integrin-mediated expression of MMPs by mutant macrophages likely contributes to lung pathology.  相似文献   

5.
Guillain-Barré syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP) are conditions that affect peripheral nerves. The mechanisms that underlie demyelination in these neuropathies are unknown. Recently, we demonstrated that the node of Ranvier is the primary site of the immune attack in patients with GBS and CIDP. In particular, GBS patients have antibodies against gliomedin and neurofascin, two adhesion molecules that play a crucial role in the formation of nodes of Ranvier. We demonstrate that immunity toward gliomedin, but not neurofascin, induced a progressive neuropathy in Lewis rats characterized by conduction defects and demyelination in spinal nerves. The clinical symptoms closely followed the titers of anti-gliomedin IgG and were associated with an important deposition of IgG at nodes. Furthermore, passive transfer of antigliomedin IgG induced a severe demyelinating condition and conduction loss. In both active and passive models, the immune attack at nodes occasioned the loss of the nodal clusters for gliomedin, neurofascin-186, and voltage-gated sodium channels. These results indicate that primary immune reaction against gliomedin, a peripheral nervous system adhesion molecule, can be responsible for the initiation or progression of the demyelinating form of GBS. Furthermore, these autoantibodies affect saltatory propagation by dismantling nodal organization and sodium channel clusters. Antibodies reactive against nodal adhesion molecules thus likely participate in the pathologic process of GBS and CIDP.Guillain-Barré syndrome (GBS) is a group of inflammatory neuropathies that affect peripheral nerves. In Europe, acute inflammatory demyelinating polyneuropathy (AIDP) is the most common form of GBS. Autopsy and biopsy studies indicated that both humoral and cellular immune reaction against Schwann cell or axonal antigens are implicated in GBS etiology.1 Early investigations have found that conduction defects closely correlate with myelin retraction and macrophage invasion in many patients.2, 3, 4, 5 Some GBS cases also involve acute demyelination without immune cell invasion and are primarily humorally mediated.6, 7 In particular, deposition of complement on the abaxonal surface of the Schwann cells has been shown during the early stage of GBS8, 9, 10 and in experimental allergic neuritis (EAN).11 In a recent study, we demonstrated that nodes of Ranvier and paranodes are the targets of the immune attack in GBS and in chronic inflammatory demyelinating polyneuropathy (CIDP).12 Notably, cell adhesion molecules (CAMs) at nodes or paranodes (gliomedin, neurofascin, and contactin) were recognized by IgG antibodies in patients with GBS or CIDP.12, 13 Autoantibodies against neurofascin and gliomedin were also detected in a rat model of AIDP and correlated with important conduction defects.14 This finding suggested that antibodies to nodal CAMs may participate to the pathogenesis of AIDP and CIDP. However, the exact mechanisms by which these humoral factors mediate demyelination and conduction defects are still elusive.Several CAMs are implicated in node formation and are responsible for the enrichment of voltage-gated sodium (Nav) channels at the nodes of Ranvier.15 At peripheral, nodes gliomedin and NrCAM are secreted into the nodal gap lumen and interact with neurofascin-186 (NF186) expressed at nodal axolemma.16, 17, 18, 19 This interaction is crucial for Nav channel aggregation at nodes.19, 20, 21 In addition, the paranodal axoglial junctions are made by the association of contactin and contactin-associated protein (Caspr) with neurofascin-155 (NF155), a variant expressed in glia.22 This adhesive junction forms a barrier to the lateral diffusion of nodal channels.19, 21, 23 In a rat model of AIDP, we found that the loss of NF186 and gliomedin at nodes preceded paranodal demyelination and the diffusion of Nav channels in demyelinated segments.14 This finding indicated that antibodies to nodal CAMs may participate to conduction defects by dismantling axoglial attachment at nodes and paranodes.We investigated whether immunity toward gliomedin and NF186 can trigger peripheral neuropathies and be responsible for demyelination in GBS patients. We found that immunization against gliomedin induced a biphasic condition associated with conduction loss and demyelination. Passive transfer of antibodies to gliomedin exacerbated the clinical signs of EAN and resulted in the disorganization of the nodes of Ranvier. Altogether, these results demonstrate that humoral immune response directed against nodal CAMs participates in conduction abnormalities in peripheral nerves and in the etiology of GBS and CIDP.  相似文献   

6.
Altered hepatic lipid homeostasis, hepatocellular injury, and inflammation are features of nonalcoholic steatohepatitis, which contributes significantly to liver-related morbidity and mortality in the Western population. A collection of inflammatory mediators have been implicated in the pathogenesis of steatohepatitis in mouse models. However, the pathways essential for coordination and amplification of hepatic inflammation and injury caused by steatosis are not completely understood. We tested the hypothesis that tissue factor (TF)-dependent thrombin generation and the thrombin receptor protease activated receptor-1 (PAR-1) contribute to liver inflammation induced by steatosis in mice. Wild-type C57Bl/6J mice fed a diet deficient in methionine and choline for 2 weeks manifested steatohepatitis characterized by increased serum alanine aminotransferase activity, macrovesicular hepatic steatosis, hepatic inflammatory gene expression, and lobular inflammation. Steatohepatitis progression was associated with thrombin generation and hepatic fibrin deposition. Coagulation cascade activation was significantly reduced in low TF mice, which express 1% of normal TF levels. Hepatic triglyceride accumulation was not affected in low TF mice or PAR-1-deficient mice. In contrast, biomarkers of hepatocellular injury, inflammatory gene induction, and hepatic accumulation of macrophages and neutrophils were greatly reduced by TF-deficiency and PAR-1-deficiency. The results suggest that TF-dependent thrombin generation and activation of PAR-1 amplify hepatic inflammation and injury during the pathogenesis of steatohepatitis.Non-alcoholic fatty liver disease (NAFLD) is increasingly appreciated as a hepatic feature of the metabolic syndrome. NAFLD may occur in 25% of the Western population and altered hepatic function increases the risk for developing diseases including diabetes and atherosclerosis.1,2 The progression of simple hepatic steatosis to the more severe nonalcoholic steatohepatitis (NASH) contributes significantly to liver-related morbidity and mortality.3 Requisite histological features of NASH include macrovesicular hepatic steatosis, evidence of hepatocellular injury, and lobular inflammation.4 In a subset of patients with chronic steatohepatitis, stellate cell activation coordinates a fibrogenic response causing fibrosis and cirrhosis.5 Of importance, the mechanisms required for the progression of hepatic inflammation during steatohepatitis are not completely understood.Animal models used to define mechanisms of steatohepatitis have used genetic and dietary modification to induce various features of the disease.2 In particular, feeding mice a diet deficient in methionine and choline (MCD diet) is an established model to study the progression of steatohepatitis and has been extensively used to study mechanisms of hepatic inflammation and fibrosis. Rodents fed an MCD diet for 2 weeks manifest a defect in hepatic β oxidation resulting in accumulation of triglyceride and the induction of steatohepatitis.2,6,7 Prolonged feeding (>4 weeks) of the MCD diet activates hepatic stellate cells and increases collagen expression and deposition in the liver. Utilization of the MCD diet model has revealed the contribution of hepatic triglyceride,8 various inflammatory mediators,9,10 nuclear receptors,11,12 and signaling pathways13 in the manifestation of steatohepatitis.An important physiological process disrupted by chronic liver disease is blood coagulation. Several studies have indicated that the progression of liver disease is associated with altered blood coagulation.14 For example, steatosis in patients with the metabolic syndrome is associated with a shift in the balance of procoagulant and antifibrinolytic factors favoring coagulation.15–17 This links the progression of NAFLD with increased risk of thrombotic complications associated with vascular disease and the metabolic syndrome. However, it is not clear whether the altered coagulation impacts progression of the liver pathology in patients with NAFLD or NASH.The coagulation cascade is initiated by tissue factor (TF), the transmembrane receptor for coagulation factor VIIa.18 TF is expressed by the normal liver,19 albeit at much lower levels compared with other tissues (eg, lung, heart).20 Of importance, potent inducers of TF expression such as bacterial lipopolysaccharide and pro-inflammatory cytokines (eg, tumor necrosis factor [TNF]α, monocyte chemoattractant protein [MCP]-1) are linked to the pathogenesis of NAFLD and NASH in humans and animal models.21–24 TF-dependent coagulation cascade activation leads to generation of the serine protease thrombin, which cleaves circulating fibrinogen to form fibrin. Thrombin also elicits intracellular signaling by activating the G-protein coupled receptor protease activated receptor-1 (PAR-1).25 This TF–PAR-1 pathway has been shown to increase inflammation in other models of tissue injury.26–29 However, the contribution of both TF and PAR-1 to coagulation and inflammation during steatohepatitis has not been determined.To this end, we characterized the procoagulant response associated with steatohepatitis induced in mice by a MCD diet. Furthermore, we used mice expressing 1% of normal TF levels (ie, low TF mice30 and PAR-1-deficient mice31 to test the hypothesis that TF-dependent thrombin generation contributes to the pathogenesis of murine steatohepatitis by activating PAR-1.  相似文献   

7.
Myocilin and optineurin are two genes linked to glaucoma, a major blinding disease characterized by progressive loss of retinal ganglion cells (RGCs) and their axons. To investigate the effects of force-expressed wild-type and mutant myocilin and optineurin on neurite outgrowth in neuronal cells, we transiently transfected cells with pEGFP-N1 (mock control) as well as myocilin and optineurin plasmids including pMYOCWT-EGFP, pMYOCP370L-EGFP, pMYOC1-367-EGFP, pOPTNWT-EGFP, and pOPTNE50K-EGFP. PC12 cells transfected with pEGFP-N1 produced, as anticipated, long and extensive neuritis on nerve growth factor induction. The neurite length in those cells transfected with myocilin constructs was shortened and the number of neurites was also reduced. A similar inhibitory effect on neurite outgrowth was also elicited by myocilin transfection in RGC5 cells. In contrast, neither transfection of the optineurin constructs pOPTNWT-EGFP and pOPTNE50K-EGFP nor the myocilin and optineurin small-interfering RNA treatments induced significant alterations in neurite outgrowth. Transfection with the wild-type optineurin construct, but not with that of the wild-type myocilin, increased the apoptotic activity in cells. These results demonstrated that the two glaucoma genes, myocilin and optineurin, exhibited differential effects on neurite outgrowth. They may contribute to the development of neurodegenerative glaucoma via distinct mechanisms.Glaucoma, one of the leading causes of irreversible blindness worldwide, is characterized by progressive loss of retinal ganglion cells (RGCs) and their axons. Primary open-angle glaucoma (POAG), the most common form of glaucoma, is frequently associated with increased intraocular pressure (IOP). The IOP is controlled by a balance between the production and outflow of the aqueous humor contained in the anterior chamber. The trabecular meshwork (TM), a specialized eye tissue neighboring the cornea, is the major site for regulation of the aqueous humor outflow.1Recent studies have revealed that POAG is genetically heterogeneous, caused by several susceptibility genes and environmental factors.2 To date, a total of 14 chromosomal loci have been mapped and designated as GLC1A to GLC1N.3,4 Three candidate genes have been identified that include myocilin as the GLC1A,5,6 optineurin as the GLC1E,7,8 and WDR36 as the GLC1G9 gene.Myocilin is the first identified gene for both juvenile- and adult-onset POAG.6 More than 70 myocilin mutations have been found in a number of families.10 Patients with glaucoma with myocilin mutation tend to have high IOP.11,12 Among the myocilin mutations, the Gln368Stop (Q368X) mutation is the most frequent10 and the Pro370Leu (P370L) mutation is responsible for one of the most severe glaucoma phenotypes.13–15The human myocilin gene encodes a protein of 504 amino acids, containing a nonmuscle myosin-like domain near the amino (N)-terminus and an olfactomedin-like domain at the carboxyl (C)-terminus.16 Myocilin interacts with itself and a number of other proteins, mainly through the leucine zipper motif and a coiled-coil region in the myosin-like domain.17–19 The wild-type myocilin is a secreted protein. Mutants with mutations in the olfatomedin-like domain, however, are not secreted. They are retained in the cells, aggregating to cause endoplasmic reticulum stress.20–22Optineurin, identified in 2002,8 is a gene that links principally to normal tension glaucoma, a subtype of POAG.23 Optineurin mutations were noted to vary with ethnic background.24,25 The Glu50Lys (E50K) mutation, found in Caucasian and Hispanic populations,25 seems to be associated with a more progressive and severe disease in patients with normal tension glaucoma.26The human optineurin gene codes for a 577-amino acid protein that contains multiple coiled-coil domains and a C-terminal zinc finger.27,28 The optineurin protein from different species has high amino acid homology.28 The amino acid 50 glutamic acid residue is conserved in mice, rats, chickens, and cows.23 Optineurin is expressed in many tissues including the brain and the retina.27,28Despite intense interest, the roles of myocilin and optineurin in cellular functions still remain largely undefined. Because glaucoma is a group of diseases known as progressive optic neuropathies, we undertook the current study to investigate the effects of myocilin and optineurin on neurite outgrowth. We demonstrated that overexpression of wild-type myocilin or P370L and Q368X mutants caused an inhibition of neurite outgrowth, whereas forced expressed wild-type or E50K optineurin did not result in any changes in neuronal rat pheochromocytoma PC1229 and RGC530 cells.  相似文献   

8.
Adhesion molecule CD44 is expressed by multiple cell types and is implicated in various cellular and immunological processes. In this study, we examined the effect of global CD44 deficiency on myelin oligodendrocyte glycoprotein peptide (MOG)-induced experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis. Compared to C57BL/6 wild-type mice, CD44-deficient mice presented with greater disease severity, increased immune cell numbers in the central nervous system, and increased anti-MOG antibody and proinflammatory cytokine production, especially those associated with T helper 17 (Th17) cells. Further, decreased numbers of peripheral CD4+CD25+FoxP3+ regulatory T cells (Tregs) were observed in CD44-knockout mice throughout the disease course. CD44-knockout CD4 T cells exhibited reduced transforming growth factor-β receptor type I (TGF-β RI) expression that did not impart a defect in Treg polarization in vitro, but did correlate with enhanced Th17 polarization in vitro. Further, EAE in bone marrow–chimeric animals suggested CD44 expression on both circulating and noncirculating cells limited disease severity. Endothelial expression of CD44 limited T-cell adhesion to and transmigration through murine endothelial monolayers in vitro. Importantly, we also identified increased permeability of the blood–brain barrier in vivo in CD44-deficient mice before and following immunization. These data suggest that CD44 has multiple protective roles in EAE, with effects on cytokine production, T-cell differentiation, T-cell–endothelial cell interactions, and blood–brain barrier integrity.Multiple sclerosis (MS) is an autoimmune, demyelinating disease resulting from chronic inflammation in the central nervous system (CNS). Experimental autoimmune encephalomyelitis (EAE), the primary and long-used animal model of MS, produces immune processes relevant to the human disease.1 The progression and pathogenesis of EAE is complex and depends on multiple cell types and processes.2–4T helper 17 (Th17) cells and their distinctive cytokine, IL-17, play pivotal roles in EAE/MS pathogenesis.5–7 Th17 cells, members of a CD4 T-cell effector subset, are generated from naive CD4 T-cell precursors in response to cytokines TGF-β and IL-6, whereas IL-23 expands this population and increases pathogenicity.8,9 In EAE, Th17 cells first infiltrate and initiate recruitment to the CNS,5,6 and Th17-produced IL-17 induces neuronal death6 and increases permeability of the blood–brain barrier (BBB), allowing continued influx of immune cells by disrupting endothelial cell (EC) junctions.6,10Regulatory T cells (Tregs), the primary suppressors of the immune system, play a pivotal role in EAE that is opposite to Th17 cells. Treg depletion exacerbates disease symptoms, whereas supplementation with additional Tregs ameliorates the disease.11,12 Identified by the expression pattern CD4+CD25+FoxP3+, Tregs are generally divided into two principal subsets: naturally occurring Tregs, which arise in the thymus during development, and induced Tregs (iTregs), which can be generated in the periphery from naive CD4 T cells in response to TGF-β.13,14Vascular EC also contribute to the complex pathogenesis of EAE. EC regulate leukocyte adhesion and extravasation, maintain vascular integrity, and limit injury and immune-mediated vascular permeability. The CNS vasculature, the primary constituent of the BBB, is especially unique and plays a critical role in protecting the CNS microenvironment. In MS/EAE, there is a characteristic breakdown of the BBB followed by accumulation of inflammatory infiltrates.15,16CD44, a ubiquitously expressed type I transmembrane glycoprotein, has been implicated in a wide variety of cellular processes within and outside of the immune system.17,18Alternative splicing and multiple posttranslational modifications generate various structural and functional versions of CD44 and are thought to be responsible for its large range of diverse and sometimes seemingly contradictory cellular functions.Although CD44 has been studied in several immunological contexts as a positive or negative regulator of inflammation, the many results are confounded by use of different mouse strains, inflammatory models, and experimental approaches. CD44 has been implicated as a proinflammatory molecule in several studies that identified an anti-inflammatory effect of a CD44 monoclonal antibody in multiple immune-mediated processes and diseases such as lymphocyte extravasation,19collagen- or proteoglycan-induced arthritis, respectively,20,21 type 1 diabetes,22 asthma,23 and EAE.24 However, most studies in CD44-knockout (KO) mice suggest an anti-inflammatory role for this molecule in various immunological processes instead. CD44-KO mice experience enhanced inflammation in several models of pulmonary inflammation that suggest various roles of CD44 in immune cell clearance, TGF-β signaling, and repression of Toll-like receptor (TLR) signaling and inflammatory gene expression.25–28 Further, CD44-KO mice show increased septic responses to lipopolysaccharide29 and enhanced inflammatory responses following myocardial infarction30 or hepatic injury.31 CD44 deficiency also led to increased collagen-induced arthritis severity with up-regulation of inflammatory genes in arthritic CD44-KO T cells.32 Clearly, antibody-mediated interference can have very different effects than genetic disruption of CD44. Hutas et al33 in 2008 reported disparate effects of CD44 monoclonal antibody treatment versus CD44 deficiency on leukocyte recruitment during proteoglycan-induced arthritis.Despite antibody-mediated interference studies, the role of CD44 in EAE/MS remains poorly understood. In active MS lesions, there is an increase in CD44 expression and accumulation of hyaluronan (HA), a major CD44 ligand.34,35 Previously, CD44 was shown to facilitate uptake of HA,36 promoting resolution of tissue-injury signals and inflammation.25,37 By contrast, a conditional mouse model of oligodendrocyte-specific overexpression of CD44 found a correlation between CD44 expression and enhanced HA accumulation, prevention of oligodendrocyte differentiation, and subsequent inflammation-independent demyelination.35Until recently, EAE had not been examined in CD44-KO mice. This report demonstrates that CD44-KO mice present with increased EAE disease severity. This was associated with loss of CD44 on circulating immune cells, and also on noncirculating cells, specifically vascular EC of the BBB. We illustrate a more proinflammatory T-cell profile in CD44-KO mice with a reduction in Treg numbers throughout the disease that is accompanied by increased permeability of the BBB. Further, we illustrate a previously unidentified role for CD44 in the baseline integrity of the BBB, which has far-reaching implications beyond MS/EAE.  相似文献   

9.
MCM7 is one of the pivotal DNA replication licensing factors in controlling DNA synthesis and cell entry into S phase. Its expression and DNA copy number are some of the most predictive factors for the growth and behavior of human malignancies. In this study, we identified that MCM7 interacts with the receptor for activated protein kinase C 1 (RACK1), a protein kinase C (PKC) adaptor, in vivo and in vitro. The RACK1 binding motif in MCM7 is located at the amino acid 221-248. Knocking down RACK1 significantly reduced MCM7 chromatin association, DNA synthesis, and cell cycle entry into S phase. Activation of PKC by 12-O-tetradecanoylphorbol-13-acetate dramatically decreased MCM7 DNA replication licensing and induced cell growth arrest. Activation of PKC induced redistribution of RACK1 from nucleus to cytoplasm and decreased RACK1-chromatin association. The MCM7 mutant that does not bind RACK1 has no DNA replication licensing or oncogenic transformation activity. As a result, this study demonstrates a novel signaling mechanism that critically controls DNA synthesis and cell cycle progression.Miniature chromosome maintenance (MCM) proteins were initially identified from autonomously replicating sequence in Saccharomyces cerevisiae. Mutations of some of these proteins, such as MCM7 or MCM3 result in loss of the large chunk of yeast chromosomes in yeast. MCM7 cDNA encodes a 543-amino acid protein and is ubiquitously expressed in all tissues. A large body of studies indicate that MCM7 is a critical component of DNA replication licensing complex in the yeast and xenopus.1–4 Some studies suggest that MCM4, MCM6, and MCM7 complex contains DNA helicase activity.5,6 DNA replication licensing complex is multimeric and phase specific. In yeast, DNA replication licensing proteins, such as MCM2-7 and several replication origin binding proteins, such as Cdc6, germinin, and Cdt1, form DNA replication licensing complex in G1 phase to enable DNA replication and to promote cell cycle entry into S phase. Initial implication of MCM7 involvement in human malignancies came from positive immunostaining of MCM7 in several human malignancies, including endometrial carcinoma,7 melanoma,8 esophageal adenocarcinoma,9 colorectal adenocarcinoma,10 oral squamous cell carcinoma,11 glioblastoma,12 and thyroid cancer.13 The first study addressing the oncogenic role of MCM7 in prostate cancer came from genome analysis of prostate cancer by performing a genome wide copy number analysis using biotin-labeled genome DNA on Affymetrix U95av2 chip.14 The DNA copy number of MCM7 was found to increase severalfold accompanied with a concomitant increase of MCM7 mRNA level. Subsequent validation analyses suggest that either copy number and/or protein level increase of MCM7 are associated with prostate cancer relapse and metastasis. Amplification of MCM7 was also found in esophageal carcinoma.9 The magnitude of MCM7 amplification correlates with the expression of MCM7, tumor grades, and the aggressiveness of esophageal cancer.9 It is presumed that amplification of MCM7 is the driving force of MCM7 overexpression in primary human malignancies. MCM7 is probably the primary target of Rb, the tumor suppressor that controls cell entry into S phase.15 There is growing evidence that other signaling pathways also regulate MCM7 activity.Receptor for activated protein kinase C 1 (RACK1), was initially identified as an adaptor of several protein kinase C (PKC) isoforms.16 The binding of RACK1 and PKC anchor PKC to its substrate to initiate second messenger signaling. It is suggested, according to recent studies that RACK1 interacts with a variety of other signaling molecules, including ras-GTPase activating protein,17 dynamin-1,18 src,19 integrins,20 PTPμ,21 phosphodiesterase,22 hypoxia induced factor-1,23 and so forth, that play an important role in several physiological processes, including, growth, hypoxia response, migration, adhesion, and cell differentiation. RACK1 only binds PKC activated by diacylglycerol or phorbol ester, but not quiescent PKC. In this study, we showed that RACK1 binds with MCM7 N-terminus. The MCM7/RACK1 interaction appears essential for DNA replication activity of MCM7.  相似文献   

10.
Molecular chaperones of the heat shock protein-90 (Hsp90) family promote cell survival, but the molecular requirements of this pathway in tumor progression are not understood. Here, we show that a mitochondria-localized Hsp90 chaperone, tumor necrosis factor receptor-associated protein-1 (TRAP-1), is abundantly and ubiquitously expressed in human high-grade prostatic intraepithelial neoplasia, Gleason grades 3 through 5 prostatic adenocarcinomas, and metastatic prostate cancer, but largely undetectable in normal prostate or benign prostatic hyperplasia in vivo. Prostate lesions formed in genetic models of the disease, including the transgenic adenocarcinoma of the mouse prostate and mice carrying prostate-specific deletion of the phosphatase tensin homolog tumor suppressor (Ptenpc−/−), also exhibit high levels of TRAP-1. Expression of TRAP-1 in nontransformed prostatic epithelial BPH-1 cells inhibited cell death, whereas silencing of TRAP-1 in androgen-independent PC3 or DU145 prostate cancer cells by small interfering RNA enhanced apoptosis. Targeting TRAP-1 with a novel class of mitochondria-directed Hsp90 inhibitors, ie, Gamitrinibs, caused rapid and complete killing of androgen-dependent or -independent prostate cancer, but not BPH-1 cells, whereas reintroduction of TRAP-1 in BPH-1 cells conferred sensitivity to Gamitrinib-induced cell death. These data identify TRAP-1 as a novel mitochondrial survival factor differentially expressed in localized and metastatic prostate cancer compared with normal prostate. Targeting this pathway with Gamitrinibs could be explored as novel molecular therapy in patients with advanced prostate cancer.Apart from skin tumors, prostate cancer is the most commonly diagnosed malignancy in men in the United States.1 Despite progress in early diagnosis,2 and prolongation of patient survival,3 the disease still carries significant morbidity and mortality, with its advanced and metastatic phase claiming over 30,000 deaths per year in the United States alone. Similar to the genetic heterogeneity of most epithelial malignancies, prostate cancer progresses through a stepwise acquisition of multiple molecular changes,4 of which insensitivity to androgen deprivation,5 emergence of an ‘osteomimetic’ phenotype responsible for metastatic tropism to the bone,6 and deregulated cell proliferation and cell survival,7 are pivotal traits.In this context, advanced prostate cancer is almost invariably associated with a heightened anti-apoptotic threshold,4 which may contribute to disease progression and resistance to therapy. This process often involves aberrant resistance to mitochondrial cell death,8 with reduced organelle permeability to solutes, and attenuated release of mitochondrial apoptogenic proteins in the cytosol.9 The regulators of such ‘mitochondrial permeability transition’ normally triggered by cell death stimuli are still largely elusive, but knockout data in mice have identified pro-apoptotic Bcl-2 family proteins and the mitochondrial matrix immunophilin, cyclophilin D, as pivotal effectors of this process, controlling the integrity of the mitochondrial outer membrane,8 and the opening a permeability transition pore,10,11 respectively.Recent data have shown that molecular chaperones of the heat shock protein-90 (Hsp90) family,12 may function as novel regulators of mitochondrial permeability transition,13 especially in tumor cells.14 Accordingly, Hsp90, and its ortholog, tumor necrosis factor receptor-associated protein-1 (TRAP-1) are abundantly localized to mitochondria of tumor, but not most normal cells, and antagonize cyclophilin D-dependent pore-forming function, potentially via a protein (re)folding mechanism.14 Consistent with a general role of Hsp90 as a drug target in prostate cancer,15 this mitochondria-compartmentalized cytoprotective pathway could provide a novel therapeutic target to enhance tumor cell apoptosis.14In the current study, we demonstrate that TRAP-1 is dramatically expressed in all lesions that comprise the entire natural history of human prostate cancer, as well as genetic disease models in rodents, but undetectable in the normal prostate. Importantly, we show that Gamitrinibs, a novel class of small molecule Hsp90 antagonists selectively engineered to target the pool of these chaperones in mitochondria,16 cause sudden prostate cancer cell death without affecting nontransformed prostatic epithelium.  相似文献   

11.
Brain hemodynamics in cerebral malaria (CM) is poorly understood, with apparently conflicting data showing microcirculatory hypoperfusion and normal or even increased blood flow in large arteries. Using intravital microscopy to assess the pial microvasculature through a closed cranial window in the murine model of CM by Plasmodium berghei ANKA, we show that murine CM is associated with marked decreases (mean: 60%) of pial arteriolar blood flow attributable to vasoconstriction and decreased blood velocity. Leukocyte sequestration further decreased perfusion by narrowing luminal diameters in the affected vessels and blocking capillaries. Remarkably, vascular collapse at various degrees was observed in 44% of mice with CM, which also presented more severe vasoconstriction. Coadministration of artemether and nimodipine, a calcium channel blocker used to treat postsubarachnoid hemorrhage vasospasm, to mice presenting CM markedly increased survival compared with artemether plus vehicle only. Administration of nimodipine induced vasodilation and increased pial blood flow. We conclude that vasoconstriction and vascular collapse play a role in murine CM pathogenesis and nimodipine holds potential as adjunctive therapy for CM.Cerebral malaria (CM) caused by Plasmodium falciparum claims the lives of nearly 1 million children every year.1 Despite antimalarial treatment, 10% to 20% of patients die, and one in every four survivors develops neurological sequelae,2,3 therefore adjunctive therapies are urgently needed. A number of clinical trials addressing potential adjunctive therapies for CM showed no proven benefits and some interventions were even deleterious,4 stressing the need for a better understanding of CM pathogenesis to develop effective therapies.An unresolved issue of CM pathogenesis regards the role of brain hemodynamic perturbations and ischemia. Sequestration of parasitized red blood cells (pRBCs) containing mature forms of the parasite in the brain microvasculature is a characteristic postmortem finding in human CM cases5 and together with rosetting6 and reduced RBC deformability7 may result in the obstruction of blood flow potentially leading to ischemia and hypoxia. In vivo studies of the microcirculation in human CM support this mechanism, with direct observation of retinal microvasculature showing impaired perfusion, retinal whitening, vascular occlusion, and ischemia.8 Accordingly, microvascular obstruction observed in the rectal mucosa of CM patients was proportional to the severity of the disease.9 In addition, hypovolemia, shock and intracranial hypertension, commonly associated with poor outcomes in CM,4 reduce tissue perfusion, and tissue hypoxia is one of the likely explanations for the acidosis frequently observed in severe malaria.7,10 Ischemic damage has also been shown in children with CM and was associated with severe neurological sequelae.11 On the other hand, transcranial Doppler sonography studies showed normal or even increased cerebral blood flow (CBF) velocities12–15 in large arteries during CM, which associated with microcirculatory obstruction has been suggested to increase cerebral blood volume leading to intracranial hypertension.16 Alternatively, collateral flow has been proposed as a mechanism to reconcile the findings of normal or increased CBF velocities and impaired perfusion,17 an interpretation supported by findings of hyperdynamic flow in capillaries adjacent to obstructed vessels.9 Interventions that improve cerebral perfusion have been proposed to be beneficial in CM.8,18The murine model of CM by Plasmodium berghei ANKA (PbA) shares many features with the human pathology,19 including the presence of multiple brain microhemorrhages and vascular obstruction, although the nature of the sequestered cell (leukocytes) differs. In murine CM, magnetic resonance imaging (MRI) and spectroscopy studies showed the presence of brain edema, decreased CBF, and ischemia.20,21 Lack of resolution in MRI, however, precludes detailed studies of the microcirculation, which is a major target and player in CM pathogenesis. A few studies have addressed the in vivo microcirculatory changes in murine models of severe malaria,22–24 however in sites other than the brain (cremaster muscle or skin). In the present work, we used for the first time brain intravital microscopy to follow the dynamic changes in the pial microcirculation during the course of PbA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow, and eventually vascular collapse, events similar to postsubarachnoid hemorrhage (SAH) vasospasm.25 We also show that nimodipine, a calcium channel blocker used to treat post-SAH vasospasm,25,26 markedly increased survival when given off-label to mice with CM as adjunctive therapy to artemether.  相似文献   

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14.
Stored tissue samples are an important resource for epidemiological genetic research. Genetic research on biological material from minors can yield valuable information on the development and genesis of early-onset genetic disorders and the early interaction of environmental and genetic factors. The use of such tissue raises some specific ethical and governance questions, which are not completely covered by the discussion on biological materials from adults. We have retrieved 29 guidelines and position papers pertaining to the storage and use of biological tissue samples for genetic research, originating from 27 different organizations. Five documents have an international scope, three have an European scope and 21 have a national scope. We discovered that 11 of these documents did not contain a section on biological materials from minors. The content of the remaining 18 documents was categorized according to four themes: consent, principles of non-therapeutic research on vulnerable populations, ethics committee approval and difference between anonymous and identifiable samples. We found out that these themes are not consistently mentioned by each document, but that documents discussing the same themes were mostly in agreement with their recommendations. However, a systematic reflection on the ethical and policy issues arising from the participation of minors in biobank research is missing.Stored tissue sample collections for genetic research exist in different forms. Some of these collections provide a resource for potentially unlimited genetic research, and gather samples and data from specific populations. An example is the ‘UK biobank''.1 Other collections are stored for research on a specific disease. Collections that were originally gathered for different purposes, for example blood spot cards for newborn screening, could be reused for genetic research.2Genetic research on biological material from minors and the associated medical records can yield valuable information on the development and genesis of early-onset genetic disorders and the early interaction of environmental and genetic factors. For example, Rasmussen3 describes the incorporation of DNA sample collections into the ‘National Birth Defects Prevention Study'' in the United States to identify the risk factors for birth defects. Studies such as the ‘Avon Longitudinal Study of Parents and Children'' in Bristol (children of the nineties) use genetic, phenotypic and environmental information of 14 000 babies from their conception onwards to study the interaction between these data.4An extensive ethical literature exists on the collection, storage and use of biological samples for genetic research. The overwhelming majority of these documents discuss issues of privacy, confidentiality, commercialization and consent.5, 6, 7, 8, 9, 10, 11, 12, 13, 14 However, research on pediatric data raises specific ethical questions with regard to consent and privacy. For example, who should give consent to the inclusion of tissue and data from children? Is the general requirement that non-therapeutic research can only be done with children if it involves no more than minimal risk, applicable to biobank research? We shall review whether and how guidelines and policy documents discuss children in the context of storing biological samples and DNA for non-therapeutic research.  相似文献   

15.
Although various guidelines and position papers have discussed, in the past, the ethical aspects of genetic testing in asymptomatic minors, the European Society of Human Genetics had not earlier endorsed any set of guidelines exclusively focused on this issue. This paper has served as a background document in preparation of the development of the policy recommendations of the Public and Professional Committee of the European Society of Human Genetics. This background paper first discusses some general considerations with regard to the provision of genetic tests to minors. It discusses the concept of best interests, participation of minors in health-care decisions, parents'' responsibilities to share genetic information, the role of clinical genetics and the health-care system in communication within the family. Second, it discusses, respectively, the presymptomatic and predictive genetic testing for adult-onset disorders, childhood-onset disorders and carrier testing.Although various guidelines and position papers have discussed, in the past, the ethical aspects of genetic testing in asymptomatic minors,1, 2 the European Society of Human Genetics had not earlier endorsed any set of guidelines exclusively focused on this issue. This background paper was preceded by an in-depth research on the topic by Eurogentest.3 Eurogentest (http://www.eurogentest.org aims to develop the necessary infrastructure, tools, resources, guidelines and procedures that will structure, harmonize and improve the overall quality of all the EU genetic services at the molecular, cytogenetic, biochemical and clinical level.4 Attention has also been paid to the provision of appropriate counselling related to genetic testing, the education of patients and professionals, as well as to the ethical, legal and social issues surrounding testing. The focus of the ethics unit of Eurogentest was oriented towards the study of the ethical issues related to genetic testing in minors. This work was the starting point for this background paper, which has been prepared and supported by different types of evidence. First, research has been performed on the existing recommendations regarding predictive genetic testing in minors1 and carrier testing,2 with the intention of identifying areas of agreement and disagreement. Second, the literature on medico–ethical and medico–legal aspects of predictive genetic testing in minors,5 carrier testing,6, 7 the position of minors8 and patient rights9 was studied. Third, a systematic literature review was performed to gather information regarding the attitudes of the different stakeholders (minors, health-care professionals, parents and relatives of the affected individuals) towards genetic testing in asymptomatic minors.10, 11 Fourth, the attitudes of European clinical geneticists regarding genetic testing in asymptomatic minors were gathered.12, 13, 14In 2007, contacts were made with the Public and Professional Policy Committee of the European Society of Human Genetics with the aim of developing policy recommendations on the issue. On the basis of a decision of the PPPC meeting during the ESHG conference in Nice (June 2007), an ad hoc committee, consisting of Pascal Borry (Eurogentest), Kris Dierickx (Eurogentest), Angus Clarke, Gerry Evers-Kiebooms (PPPC) and Martina Cornel (PPPC), was created. This ad hoc committee met on 15 November 2007 to discuss a first draft of a background paper and recommendations that were prepared by Pascal Borry under the supervision of Kris Dierickx. A revised version was discussed during a PPPC meeting in Amsterdam (April 2008) and Barcelona (June 2008). In order not to repeat issues that have been discussed elsewhere, reference will often be made to the above-referenced publications.  相似文献   

16.
Transmissible spongiform encephalopathies are fatal neurodegenerative diseases. Infection by the oral route is assumed to be important, although its pathogenesis is not understood. Using prion protein (PrP) knockout mice, we investigated the sequence of events during the invasion of orally administered PrPs through the intestinal mucosa and the spread into lymphoid tissues and the peripheral nervous system. Orally administered PrPs were incorporated by intestinal epitheliocytes in the follicle-associated epithelium and villi within 1 hour. PrP-positive cells accumulated in the subfollicle region of Peyer''s patches a few hours thereafter. PrP-positive cells spread toward the mesenteric lymph nodes and spleen after the accumulation of PrPs in the Peyer''s patches. The number of PrP molecules in the mesenteric lymph nodes and spleen peaked at 2 days and 6 days after inoculation, respectively. The epitheliocytes in the follicle-associated epithelium incorporating PrPs were annexin V-positive microfold cells and PrP-positive cells in Peyer''s patches and spleen were CD11b-positive and CD14-positive macrophages. Additionally, PrP-positive cells in Peyer''s patches and spleen were detected in the vicinity of peripheral nerve fibers in the early stages of infection. These results indicate that orally delivered PrPs were incorporated by microfold cells promptly after challenge and that macrophages might act as a transporter of incorporated PrPs from the Peyer''s patches to other lymphoid tissues and the peripheral nervous system.Transmissible spongiform encephalopathies (TSEs), or prion diseases, are fatal neurodegenerative diseases that infect humans and both wild and domestic animals. They include Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep, and bovine spongiform encephalopathy (BSE) in cattle.1 The common neuropathological features within the central nervous system (CNS) of TSEs are seen as a spongiform pathology, neuronal loss,2 glial activation,3 and the accumulation of an abnormal and protease-resistant conformer of the scrapie-associated prion proteins (PrP-res or PrPSc),4 which are closely associated with the infection.5It has been reported that variant CJD in humans is most likely to have occurred because of the transmission of BSE after the consumption of beef contaminated with the BSE agent.6 Therefore, the oral route of TSE infection is widely assumed to be important under natural conditions. Many of the infectious agents accumulate in the gut-associated lymphoid tissues (GALT) after oral infection, such as the Peyer''s patches and mesenteric lymph nodes (MLN) before spreading to the CNS.7 It is necessary for the infectious agents to cross the intestinal epithelium before they can accumulate in the GALT. In addition, there are microfold cells (M cells) within the follicle-associated epithelium (FAE) that are specialized for the transepithelial transport of macromolecules and particles.8 One in vitro study has demonstrated that M cells actively transcytose the scrapie agents into the basolateral side of the epithelium.9 However, it is still a matter of controversy as to whether M cells may be involved in the in vivo transport of the infectious agents across the intestinal epithelium. After alimentary uptake of the infectious agents, they accumulate in the GALT and the lymphoreticular systems (eg, the spleen and other peripheral lymph nodes) long before they are detected in the CNS.10 As the GALT and the lymphoreticular systems are highly innervated, they are believed to be important sites for the infectious agents to gain contact with the nervous system (ie, neuroinvasion).11 Once neuroinvasion occurs, the infectious agents reach their initial CNS target sites by spreading in a retrograde direction along efferent nerve fibers.12In the lymphoid tissues, it is believed that the macrophages, dendritic cells (DCs), and follicular dendritic cells (FDCs) are involved in the transportation and replication of the infectious agents. Macrophages are prevalent candidates for both spread13 and clearance14 of the infectious agents. DCs can capture and retain protein antigens in a nondegraded state.15,16 These characteristics suggest that the macrophages and DCs may act as a transporter of the infectious agents from the gut to lymphoid tissues. FDCs express high levels of cellular PrPs (PrPc), and therefore an early accumulation of PrPSc is seen in them.17,18 Many studies of the alimentary pathogenesis of TSEs have been conducted to elucidate how infectious agents spread from the GALT to the CNS, although this has not been clearly determined yet. Therefore, the aim of the present study was to reveal the cells involved in the early stages of the pathogenesis of oral TSE infection, such as the sites of entry, spread, and neuroinvasion.  相似文献   

17.
This paper explores the ethical implications of introducing non-invasive prenatal diagnostic tests (NIPD tests) in prenatal screening for foetal abnormalities. NIPD tests are easy and safe and can be performed early in pregnancy. Precisely because of these features, it is feared that informed consent may become more difficult, that both testing and selective abortion will become ‘normalized'', and that there will be a trend towards accepting testing for minor abnormalities and non-medical traits as well. In our view, however, the real moral challenge of NIPD testing consists in the possibility of linking up a technique with these features (easy, safe and early) with new genomic technologies that allow prenatal diagnostic testing for a much broader range of abnormalities than is the case in current procedures. An increase in uptake and more selective abortions need not in itself be taken to signal a thoughtless acceptance of these procedures. However, combining this with considerably enlarging the scope of NIPD testing will indeed make informed consent more difficult and challenge the notion of prenatal screening as serving reproductive autonomy. If broad NIPD testing includes later-onset diseases, the ‘right not to know'' of the future child will become a new issue in the debate about prenatal screening. With regard to the controversial issue of selective abortion, it may make a morally relevant difference that after NIPD testing, abortion can be done early. A lower moral status may be attributed to the foetus at that moment, given the dominant opinion that the moral status of the foetus progressively increases with its development.Since the discovery of cell-free foetal DNA/RNA (cffDNA/RNA) in maternal plasma in 1997,1 the possibility to use this cffDNA/RNA for non-invasive prenatal diagnosis (NIPD) has been investigated many times.2, 3, 4, 5, 6 cffDNA/RNA can be obtained from a maternal blood sample, as early as 4 weeks of gestation,7 but currently only reliably so from 7 weeks of gestation.4 This development holds the promise of NIPD testing early in pregnancy and without the small, but significant risk of foetal loss that the current invasive procedures of chorionic villus sampling (CVS) and amniocentesis (AP) carry. NIPD testing for the determination of a Y-signal for pregnancies at risk of X-linked disorders and for diagnosis of Rhesus factor status in RhD-negative women is now being translated into clinical practice.4 In many European countries, discussion about broader applications of NIPD testing can be expected in the coming years.8, 9 The feasibility of NIPD for trisomy 21, 13 and 18 has already been shown,2 but large-scale independent studies are still needed. Sex-chromosomal abnormalities (eg, Turner syndrome (X0) and triple X syndrome (XXX)) could in principle be diagnosed by NIPD testing as well,4 if reliable quantitative tests become available in the future and the maternal ‘background'' can be excluded from testing. Even if accurate NIPD testing for the mentioned abnormalities becomes possible, the clinical utility of the test remains to be assessed. This includes balancing the benefits to the harms also with regard to its psychological, ethical, legal, social and economic implications.10, 11 The possible ethical implications of NIPD as a new approach to prenatal testing have so far been reviewed in a few publications.4, 8, 9, 12, 13, 14, 15, 16, 17 Apart from clear benefits related to avoiding the miscarriage risk of present invasive methods, important potential drawbacks have been mentioned as well. For one thing, proper counselling and informed consent is argued to become more challenging when offering NIPD testing. Moreover, there is a concern that the ease and safety of NIPD may lead to prenatal screening being increasingly conceived as a matter of course, both by those making the offer and by the women undergoing the test. Related to this is the concern that selective abortion of foetuses with minor abnormalities, the wrong sex or unwanted paternity, will become normalized.This paper aims to expand and refine these ethical evaluations and will add some new ethical perspectives with regard to possible implications of NIPD at present and in the future.In our view, it is not so much the fact that foetal material used for prenatal testing can be obtained early and non-invasively (allowing easy and safe testing) that would lead to moral challenges. Rather, it is the fact that a technology with these features would be open to being used for testing a potentially much broader range of abnormalities than those included in the presently used method of microscopic chromosome analysis (karyotyping).Although NIPD testing can also be applied in high genetic-risk families and for the management of pregnancy, the focus of this paper will primarily be on the application of NIPD testing in the screening context. The reason for this focus on prenatal screening is that in the near future, the question if, and if so, in what way NIPD testing is to be applied within prenatal screening strategies should be considered and discussed by policy makers, health care professionals and society at large.To avoid confusion, a preliminary remark is needed on terminology. In medicine, ‘screening'' is often used as referring to a kind of test for risk assessment or disease discovery. However, after the convention in normative and regulatory discourse, we will use ‘screening'' as referring to any systematic and unsolicited offer of predictive testing (using whatever types of test) involving individuals who themselves have no reason (yet) to seek medical help for the condition in question.18 In this broader sense, screening stands in contrast to ‘diagnosis'' as testing on indication.  相似文献   

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The tumor microenvironment is crucial to the progression of various malignancies. Malignant pleural mesothelioma (MPM), which originates from the pleura, grows aggressively in the thoracic cavity. Here we describe an orthotopic implantation SCID mouse model of MPM and demonstrate that α-SMA-positive fibroblast-like cells accumulate in the tumors produced by the human MPM cell lines MSTO-211H and Y-Meso-14. We assessed the interaction between MPM cells and their microenvironments, focusing on tumor-associated fibroblasts. MSTO-211H and Y-Meso-14 cells produced fibroblast growth factor-2 (FGF-2) and/or platelet-derived growth factor-AA (PDGF-AA); they also enhanced growth, migration, and production of hepatocyte growth factor (HGF) by human lung fibroblast MRC-5 cells. MRC-5 cells stimulated HGF-mediated growth and migration of MSTO-211H and Y-Meso-14 cells in an in vitro coculture system. In the orthotopic model, tumor formation by MSTO-211H and Y-Meso-14 cells was significantly inhibited by TSU-68, an inhibitor of FGF, VEGF, and PDGF receptors; imatinib, an inhibitor of PDGF receptors; and NK4, an antagonist of HGF. Histological analyses of clinical specimens from 51 MPM patients revealed considerable tumor-associated fibroblasts infiltration and expression of HGF, together with FGF-2 or PDGF-AA, in tumors. These findings indicate that MPM instigates tumor-associated fibroblasts, promoting tumor progression via a malignant cytokine network. Regulation of this cytokine network may be therapeutically useful for controlling MPM.Malignant pleural mesothelioma (MPM) is a unique form of tumor, the development of which is highly related to asbestos exposure.1 Even after bans on asbestos were initiated in the 1970s, MPM remains a serious problem worldwide because of its long latency period (30 to 40 years) and high mortality rate. In the United States, 2000 to 3000 patients die of MPM every year. Deaths from this disease are expected to peak in 2020 to 2025, with more than 250,000 deaths expected to occur in Western Europe and Japan over the next 40 years.2 MPM grows aggressively, with dissemination in the thoracic cavity, and frequently produces a malignant pleural effusion.3 MPM is rarely diagnosed at an operable stage, and it is refractory to conventional chemotherapy and radiotherapy. Thus, the prognosis of patients with this disease is extremely poor, with median survival varying between 8 and 14 months after diagnosis, despite the recent development of a chemotherapy regimen combining cisplatin and an antifolate agent such as pemetrexed or raltitrexed.4The tumor microenvironment is crucial for the progression and chemosensitivity of various malignant diseases.5 For example, the tumor microenvironment mediates endocrine instigation of indolent metastatic tumor progression via osteopontin.6 Moreover, EGFR-TKI resistance may be induced by microenvironmental fibroblasts in epidermal growth factor receptor mutant lung cancer.7 Thus, innovative therapies may target the microenvironment. For example, antiangiogenic therapy targeting host endothelial cells and bisphosphonate targeting host osteoclasts have been successfully used to treat several malignant diseases, including colon cancer,8 non-small cell lung cancer,9,10 and metastatic bone tumors.11In MPM, angiogenesis inhibition using an anti-VEGF antibody targeting endothelial cells can successfully control the progression of MPM cells that produce high concentrations of VEGF.12 Tumor-associated fibroblasts (TAFs), also known as cancer-associated fibroblasts, are the major component of tumor microenvironments.13 TAFs regulate tumor behavior through several mediators. Although recent studies show that many populations of MPM contain TAFs,14 little is known about interactions between TAFs and MPM. We therefore investigated the molecular interaction between MPM and TAFs, using an orthotopic implantation SCID mice model and clinical specimens taken from MPM patients. We show here that MPMs produce fibroblast-growth factor 2 (FGF-2) and platelet-derived growth factor-AA (PDGF-AA), and that these growth factors stimulate TAFs to produce hepatocyte growth factor (HGF), thus promoting tumor progression through a malignant cytokine network.  相似文献   

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