Radon Inhalation Protects Against Transient Global Cerebral Ischemic Injury in Gerbils

Although brain disorders are not the main indication for radon therapy, our previous study suggested that radon inhalation therapy might mitigate brain disorders. In this study, we assessed whether radon inhalation protects against transient global cerebral ischemic injury in gerbils. Gerbils were treated with inhaled radon at a concentration of 2,000 Bq/m³ for 24 h. After radon inhalation, transient global cerebral ischemia was induced by bilateral occlusion of the common carotid artery. Results showed that transient global cerebral ischemia induced neuronal damage in hippocampal CA1, and the number of damaged neurons was significantly increased compared with control. However, radon treatment inhibited ischemic damage. Superoxide dismutase (SOD) activity in the radon-treated gerbil brain was significantly higher than that in sham-operated gerbils. These findings suggested that radon inhalation activates antioxidative function, especially SOD, thereby inhibiting transient global cerebral ischemic injury in gerbils.     

Endotoxin-Induced Hepatic Damage in BCG-Infected Mice

Systemic infection of mice with Mycobacterium BCG leads to focal liver damage by producing many granulomas. By undefined mechanisms, this infection markedly enhances the animal's susceptibility to the lethal effect of endotoxin. Small doses of endotoxin given to BCG-infected mice were found to cause acute hepatic damage, as demonstrated by elevated activities of liver enzymes in serum and by morphologic alterations documented by light and electron microscopy and by histochemical technics. The morphologic alterations caused by endotoxin included glycogen depletion, mitochondrial swelling, disruption of the continuity of sinusoidal endothelium and focal injury characterized by marked vacuolization of hepatocytes and distension and fragmentation of rough endoplasmic reticulum. Histochemical studies revealed the apparent release of acid phosphatase from granules in the central portions of granulomas, and the release of β-glucuronidase from the cytoplasm of hepatocytes.     

Antinociceptive Effects of Radon Inhalation on Formalin-Induced Inflammatory Pain in Mice

Radon therapy is clinically useful for the treatment of inflammatory diseases. The mechanisms of pain relief remain to be fully elucidated. In this study, we investigated the antinociceptive effects of radon inhalation in a mouse model of formalin-induced inflammatory pain. Immediately, after radon inhalation at a concentration of background level (ca. 19 Bq/m³), 1,000 or 2,000 Bq/m³ for 24 h, 1.35 % formalin (0.5 % formaldehyde in saline, 20 μl) was subcutaneously injected into the hind paw of mice, and we measured licking response time. Radon inhalation inhibited the second phase of response in formalin test. Formalin administration induced nociception and increased tumor necrosis factor alpha (TNF-α) and nitric oxide (NO) levels in serum and leukocyte migration in paws. Concurrently, formalin injection decreased antioxidative functions. Radon inhalation produced antinociceptive effects, i.e., lowered serum TNF-α and NO levels, and restored antioxidative functions. The results showed that radon inhalation inhibited formalin-induced inflammatory pain.     

Therapeutic Targeting of Classical and Lectin Pathways of Complement Protects from Ischemia-Reperfusion-Induced Renal Damage

Ischemia-reperfusion injury is the major cause of delayed graft function in transplanted kidneys, an early event significantly affecting long-term graft function and survival. Several studies in rodents suggest that the alternative pathway of the complement system plays a pivotal role in renal ischemia-reperfusion injury. However, limited information is currently available from humans and larger animals. Here we demonstrated that 30 minutes of ischemia resulted in the induction of C4d/C1q, C4d/MLB, and MBL/MASP-2 deposits in a swine model of ischemia-reperfusion injury. The infusion of C1-inhibitor led to a significant reduction in peritubular capillary and glomerular C4d and C5b-9 deposition. Moreover, complement-inhibiting treatment significantly reduced the numbers of infiltrating CD163⁺, SWC3a⁺, CD4a⁺, and CD8a⁺ cells. C1-inhibitor administration led to significant inhibition of tubular damage and tubular epithelial cells apoptosis. Interestingly, we report that focal C4d-deposition colocalizes with C1q and MBL at the peritubular and glomerular capillary levels also in patients with delayed graft function. In conclusion, we demonstrated the activation and a pathogenic role of classical and lectin pathways of complement in a swine model of ischemia-reperfusion−induced renal damage. Therefore, inhibition of these two pathways might represent a novel therapeutic approach in the prevention of delayed graft function in kidney transplant recipients.Delayed graft function (DGF) is the primary early post-transplant complication of kidney recipients.¹ This event, histologically characterized by the presence of acute tubular necrosis, has been reported to occur in 25% to 30% of renal transplants.¹ DGF is commonly associated with a significantly longer hospital stay and an increase in peritransplant morbidity. Moreover, this early complication seems to result in a marked reduction in long-term graft survival.² Indeed, DGF is associated with an increased rate of acute rejection and a suboptimal renal function at 1-year post-transplantation.^1,2,3Ischemia followed by reperfusion plays a pivotal role in the pathogenesis of early graft damage.¹ Ischemia-reperfusion injury is characterized by two main features at the renal level: apoptosis of tubular cells and interstitial inflammation. Although many steps in the cascade of events leading to ischemia-reperfusion injury are unclear, the most promising potential mechanisms include recruitment and activation of inflammatory cells and local priming of the complement cascade.^4,5,6The complement system is a major constituent of the innate immune system, participating in the pathogenesis of tissue damage through sequential activation of different proteases. The priming of this proteolytic cascade may occur by classical, alternative, and lectin-mediated activation pathways, and generates several pro-inflammatory mediators.^6,7 Studies in animal models have shown complement activation in the kidney after ischemia-reperfusion, leading to the generation of several mediators of inflammation, such as C3a, C5a, and C5b-9.⁴ Mice deficient in complement components such as C6 show very limited damage after renal ischemia-reperfusion injury.⁸ Moreover, the use of anti-factor B or C5a-receptor antagonists has been shown to reduce renal damage due to ischemia-reperfusion.^9,10 Therefore, prevention of complement activation is currently considered one of the best therapeutic targets to prevent or limit ischemia-reperfusion−induced renal damage.^5,6,11 C1-inhibitor (C1INH) is a potent inhibitor of proteases of the classical and lectin complement pathways (C1r, C1s, and MASP2).^6,11,12 Animal studies show that C1INH can protect liver, intestine, heart, and brain tissue from ischemia-reperfusion damage.¹³ There are no published data on the effect of C1INH on ischemia-reperfusion−induced renal damage, since most of the existing evidence suggests that in this setting complement activation is mainly induced through the alternative pathway.^5,14 However, all data reported in the literature are from murine models, and no information is currently available from larger animals and/or human subjects.The aim of the present study was to investigate the pattern of complement activation in patients with DGF and to test the efficacy of a recombinant form of human C1INH (rhC1INH) in preventing renal damage in a swine model of warm ischemia-reperfusion injury.     

Protective Effects of Radon Inhalation on Carrageenan-Induced Inflammatory Paw Edema in Mice

We assessed whether radon inhalation inhibited carrageenan-induced inflammation in mice. Carrageenan (1% v/v) was injected subcutaneously into paws of mice that had or had not inhaled approximately 2,000 Bq/m³ of radon for 24 h. Radon inhalation significantly increased superoxide dismutase (SOD) and catalase activities and significantly decreased lipid peroxide levels in mouse paws, indicating that radon inhalation activates antioxidative functions. Carrageenan administration induced paw edema and significantly increased tumor necrosis factor-alpha (TNF-α) and nitric oxide in serum. However, radon inhalation significantly reduced carrageenan-induced paw edema. Serum TNF-α levels were lower in the radon-treated mice than in sham-treated mice. In addition, SOD and catalase activities in paws were significantly higher in the radon-treated mice than in the sham-treated mice. These findings indicated that radon inhalation had anti-inflammatory effects and inhibited carrageenan-induced inflammatory paw edema.     

环孢菌素对超抗原SEB致小鼠肝损伤的保护作用

用超抗原葡萄球菌肠毒素B(SEB)、D-氨基半乳糖(D-GalN)混合腹腔注射Balb/c/小鼠及预先注射过环孢菌素(CSA)的小鼠,动态观察小鼠肝细胞变化和血清TNF,IFN-γ水平及小鼠死亡率。结果发现SEB+D-GalN注射后2h、6h时出现肝细胞凋亡,12h后出现肝坏死,小鼠24小时死亡率达 50％。小鼠血清 TNF2h升到最高,IFN-γ在 6～12h较高。而预注CSA的小鼠上述指标正常。推测SEB+D-GalN对肝细胞的作用是由T细胞介导的,可被CSA抑制。     

Renal Purge of Hemolymphatic Lipids Prevents the Accumulation of ROS-Induced Inflammatory Oxidized Lipids and Protects Drosophila from Tissue Damage

1. Download : Download high-res image (264KB)
2. Download : Download full-size image

     

Interleukin-1 Receptor Signaling Protects Mice from Lethal Intestinal Damage Caused by the Attaching and Effacing Pathogen Citrobacter rodentium

Enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and Citrobacter rodentium are classified as attaching and effacing pathogens based on their ability to adhere to the intestinal epithelium via actin-filled membranous protrusions (pedestals). Infection of mice with C. rodentium causes a breach of the intestinal epithelial barrier, leading to colitis via a vigorous inflammatory response resulting in diarrhea and a protective antibody response that clears the pathogen. Here we show that interleukin-1 receptor (IL-1R) signaling protects mice following infection with C. rodentium. Upon infection, mice lacking the type I IL-1R exhibit increased mortality together with severe colitis characterized by intramural colonic bleeding and intestinal damage including gangrenous mucosal necrosis, phenotypes also evident in MyD88-deficient mice. However, unlike MyD88^−/− mice, IL-1R^−/− mice do not exhibit increased pathogen loads in the colon, delays in the recruitment of innate immune cells such as neutrophils, or defects in the capacity to replace damaged enterocytes. Further, we demonstrate that IL-1R^−/− mice have an increased predisposition to intestinal damage caused by C. rodentium but not to that caused by chemical irritants, such as dextran sodium sulfate. Together, these data suggest that IL-1R signaling regulates the susceptibility of the intestinal epithelia to damage caused by C. rodentium.Pathogenic strains of Escherichia coli, including enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli, pose a significant public health risk, especially in developing countries, where these strains contaminate food and water supplies. EPEC causes infantile diarrhea (10), which leads to dehydration, contributing to as many as 1 million infant deaths per year (26). EPEC, enterohemorrhagic E. coli, and the murine pathogen Citrobacter rodentium are classified as attaching and effacing (A/E) pathogens based on the ability of these extracellular bacteria to intimately attach to the intestinal epithelium and flatten absorptive microvilli (effacement). Another hallmark feature of A/E pathogens is their ability to induce actin rearrangements that form membranous protrusions, called “pedestals,” beneath the attached bacteria. Pedestal formation is associated with the development of A/E lesions, breach of the epithelial barrier, and disease (25, 28).Upon infection, A/E pathogens displace the commensal flora and cause an intestinal pathology that includes damage characterized by cellular necrosis, disruption of the epithelium, and occasionally bleeding (21, 22). Damage induces a localized repair response characterized by hyperplasia (22), which reflects an increased division of stem cells at the base of crypts to replace damaged enterocytes (34). As hyperplasia develops, goblet cells become less evident because they are not replenished as readily as enterocytes. Thus, the apparent loss of goblet cells may be further evidence of repair. Infection with A/E pathogens also induces the recruitment of immune cells and causes edema within the lamina propria. Indeed, in addition to providing protection, immune cell types such as neutrophils may contribute to colitis and epithelial damage, including the formation of crypt abscesses (17). Nevertheless, an effective innate immune response allows the proper recruitment and activation of immune cell types necessary for a robust antibody response both to promote the clearance of A/E pathogens (23) and to reduce the severity of pathology upon reinfection (8).The detection of pathogens by the innate immune system is accomplished by highly conserved families of receptors, such as Toll-like receptors (TLRs), and their respective downstream signaling cascades. Several lines of evidence suggest that signaling initiated by particular TLRs is important for protective responses to C. rodentium, whereas signaling by other TLRs appears unnecessary and perhaps even deleterious. TLRs recognize and respond to conserved structural motifs associated with microbes, which include proteins (e.g., flagellin), nucleic acids (e.g., unmethylated CpG DNA), and lipids (e.g., lipid A of lipopolysaccharide [LPS]) (18). LPS is abundant on the surface of C. rodentium and is a known ligand for the TLR4 receptor complex. When LPS binds to its receptor, NF-κB becomes derepressed, and as a consequence, proinflammatory cytokines are expressed (9). Notably, infection of TLR4^−/− mice with C. rodentium results in a slower, less severe inflammatory response and reduced mortality (19), suggesting that TLR4 signaling exacerbates disease. In contrast, TLR2 signaling appears to be required for protective responses to C. rodentium. Thus, following infection TLR2^−/− mice suffer from colonic mucosal ulcerations, bleeding, increased apoptosis, and increased mortality (14). Together, these studies suggest that the activation of TLRs by C. rodentium can cause both protective and deleterious responses.A variety of innate immune receptors, including TLRs, the type I interleukin-1 receptor (IL-1R), and the IL-18 receptor, utilize the signaling adaptor myeloid differentiation factor 88 (MyD88) to activate NF-κB and produce an array of cytokines and chemokines (1, 18). Following detection of C. rodentium, MyD88 signaling in epithelial and hematopoietic cells provides protection from disseminating infection and mortality. MyD88 signaling facilitates epithelial repair responses (33), initiates recruitment of innate immune cells, limits bacterial load, and controls the amount of intestinal damage (21). We proposed that MyD88 signaling provides protection from C. rodentium by inducing the timely recruitment of neutrophils and thus stemming the growth of bacteria in the colon, which could exacerbate epithelial damage. Thus, by regulating both epithelial damage and repair, MyD88 signaling appears to prevent bacteria from escaping the colon and reduce dissemination to peripheral organs (21). However, because MyD88 signaling contributes to several aspects of the innate immune response, it is difficult to discern whether particular phenotypes result from direct or indirect consequences of TLR signaling or from other receptors that utilize MyD88. Such designations are important to understand how TLRs or other receptors mediate a balanced response to a pathogen that is sufficient for containment and clearance but limits damage due to inflammation.Here we consider whether a loss of signaling through receptors besides TLRs that also signal via MyD88 might contribute to some of the phenotypes evident in MyD88^−/− mice after C. rodentium infection. Using mice deficient in these signaling pathways, we reasoned that it might be possible to distinguish MyD88-dependent phenotypes directly controlled by TLRs or other receptors from secondary consequences of MyD88-dependent signaling. We have focused in particular on IL-1 and IL-18 and their cognate receptors. IL-18 is expressed by macrophages and was first identified as a factor that induces gamma interferon (IFN-γ) (30, 31). IL-1β and the type I IL-1R (CD121a) have been implicated in protection and control against infections caused by Staphylococcus aureus (27). Further, IL-1β and IL-18 have both been shown to be protective against Salmonella enterica serovar Typhimurium (36) and Shigella flexneri (37) infections.The type I IL-1R associates with an accessory protein and is activated by IL-1α or IL-1β (7), molecules structurally related to IL-18. The type II IL-1R is a decoy receptor, which does not lead to any cellular responses following ligand binding (7). Although IL-1α is constitutively expressed by epithelial cells, the expression of IL-1β or IL-18 precursors is induced by NF-κB in innate immune or epithelial cells, respectively (3, 15). Whereas IL-1α is produced in an active form, IL-1β and IL-18 are expressed as proforms, which, upon proteolytic cleavage, are secreted and bind to their cognate receptors. Ligand-dependent activation of IL-1R and the IL-18 receptor in turn activates NF-κB in a MyD88-dependent fashion. This positive-feedback circuit, in which IL-1β and IL-18 are both produced and signal in a MyD88-dependent fashion, has been hypothesized to amplify the NF-κB response, leading to a localized inflammatory responses (3). Although IL-1β expression is not induced by C. rodentium by 6 days postinfection (p.i.) (13), mRNA levels do increase by 2 weeks p.i. (24), raising the possibility that IL-1R signaling, perhaps activated by IL-1α or the late expression of IL-1β, provides protection.Here we demonstrate that IL-1R signaling provides protection from increased mortality and pathology upon infection with C. rodentium, whereas IL-18 signaling does not appear to participate. Notably, mice lacking the type I IL-1R provide a means to separate the complex and interrelated phenotypes associated with inflammation. Thus, the increased pathology evident in the absence of IL-1R signaling appears to result from an increased susceptibility to tissue damage by C. rodentium and not from dysregulated immune or repair responses.     

Antibody to Pneumocystis carinii Protects Rats and Mice from Developing Pneumonia

Well-proven mouse and rat models were used to show that polyclonal antisera to Pneumocystis carinii protect against P. carinii pneumonia. Antibodies were obtained from animals that were allowed to recover from severe P. carinii pneumonia after immunosuppression had been stopped and which then were given a booster injection of P. carinii from the same animal species. Mice immunosuppressed with corticosteroids or antibodies to L3T4⁺ lymphocytes (which are comparable to CD4 cells of humans) and transtracheally inoculated with mouse P. carinii did not develop P. carinii pneumonia if they were passively immunized with antiserum, while mice immunosuppressed and inoculated by identical procedures but not given antibodies developed severe infections. Rats immunosuppressed with corticosteroids and inoculated with rat P. carinii had less severe infections if they were given rat anti-P. carinii antisera. The polyclonal antisera developed in mice provided greater protection for the mice than the polyclonal rat antisera did for the rats; however, the potencies and compositions of the antisera were not quantitated and probably differed. Since both rats and mice can be protected from P. carinii infections with polyclonal antisera, it may be possible to develop vaccines that will elicit protective antibodies in humans.     

Recombinant Vaccine Displaying the Loop-Neutralizing Determinant from Protective Antigen Completely Protects Rabbits from Experimental Inhalation Anthrax

We previously showed that a multiple antigenic peptide (MAP) vaccine displaying amino acids (aa) 304 to 319 from the 2β2-2β3 loop of protective antigen was capable of protecting rabbits from an aerosolized spore challenge with Bacillus anthracis Ames strain. Antibodies to this sequence, referred to as the loop-neutralizing determinant (LND), are highly potent at neutralizing lethal toxin yet are virtually absent in rabbit and human protective antigen (PA) antiserum. While the MAP vaccine was protective against anthrax, it contains a single heterologous helper T cell epitope which may be suboptimal for stimulating an outbred human population. We therefore engineered a recombinant vaccine (Rec-LND) containing two tandemly repeated copies of the LND fused to maltose binding protein, with enhanced immunogenicity resulting from the p38/P4 helper T cell epitope from Schistosoma mansoni. Rec-LND was found to be highly immunogenic in four major histocompatibility complex (MHC)-diverse strains of mice. All (7/7) rabbits immunized with Rec-LND developed high-titer antibody, 6 out of 7 developed neutralizing antibody, and all rabbits were protected from an aerosolized spore challenge of 193 50% lethal doses (LD₅₀) of the B. anthracis Ames strain. Survivor serum from Rec-LND-immunized rabbits revealed significantly increased neutralization titers and specific activity compared to prechallenge levels yet lacked PA or lethal factor (LF) antigenemia. Control rabbits immunized with PA, which were also completely protected, appeared sterilely immune, exhibiting significant declines in neutralization titer and specific activity compared to prechallenge levels. We conclude that Rec-LND may represent a prototype anthrax vaccine for use alone or potentially combined with PA-containing vaccines.     

Inhibition of 15-PGDH Protects Mice from Immune-Mediated Bone Marrow Failure

Aplastic anemia (AA) is a human immune-mediated bone marrow failure syndrome that is treated by stem cell transplantation for patients who have a matched related donor and by immunosuppressive therapy (IST) for those who do not. Responses to IST are variable, with patients still at risk for prolonged neutropenia, transfusion dependence, immune suppression, and severe opportunistic infections. Therefore, additional therapies are needed to accelerate hematologic recovery in patients receiving front-line IST. We have shown that inhibiting 15-hydroxyprostaglandin dehydrogenase (15-PGDH) with the small molecule SW033291 (PGDHi) increases bone marrow (BM) prostaglandin E2 levels, expands hematopoietic stem cell (HSC) numbers, and accelerates hematologic reconstitution following murine BM transplantation. We now report that in a murine model of immune-mediated BM failure, PGDHi therapy mitigated cytopenias, increased BM HSC and progenitor cell numbers, and significantly extended survival compared with vehicle-treated mice. PGDHi protection was not immune-mediated, as serum IFN-γ levels and BM CD8⁺ T lymphocyte frequencies were not impacted. Moreover, dual administration of PGDHi plus low-dose IST enhanced total white blood cell, neutrophil, and platelet recovery, achieving responses similar to those seen with maximal-dose IST with lower toxicity. Taken together, these data demonstrate that PGDHi can complement IST to accelerate hematologic recovery and reduce morbidity in severe AA.     

Mucosal Administration of Flagellin Protects Mice from Streptococcus pneumoniae Lung Infection

Streptococcus pneumoniae is a major cause of pneumonia in infants and the elderly. Innate defenses are essential to the control of pneumococcal infections, and deficient responses can trigger disease in susceptible individuals. Here we showed that flagellin can locally activate innate immunity and thereby increase the resistance to acute pneumonia. Flagellin mucosal treatment improved S. pneumoniae clearance in the lungs and promoted increased survival of infection. In addition, lung architecture was fully restored after the treatment of infected mice, indicating that flagellin allows the reestablishment of steady-state conditions. Using a flagellin mutant that is unable to signal through Toll-like receptor 5 (TLR5), we established that TLR5 signaling is essential for protection. In the respiratory tract, flagellin induced neutrophil infiltration into airways and upregulated the expression of genes coding for interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), CXCL1, CXCL2, and CCL20. Using depleting antibodies, we demonstrated that neutrophils are major effectors of protection. Further, we found that B- and T-cell-deficient SCID mice clear S. pneumoniae challenge to the same extent as immunocompetent animals, suggesting that these cell populations are not required for flagellin-induced protection. In conclusion, this study emphasizes that mucosal stimulation of innate immunity by a TLR not naturally engaged by S. pneumoniae can increase the potential to cure pneumococcal pneumonia.Streptococcus pneumoniae (pneumococcus) causes respiratory infections among infants and the elderly worldwide (40, 44). Capsular polysaccharide is the main virulence factor, and its composition defines 91 serotypes of pneumococcus (42). Certain serotypes colonize the human nasopharynx asymptomatically, representing a reservoir for interindividual transmission of the bacteria. In some individuals, colonization may progress to pneumococcal pneumonia and invasive disease (19, 36). In contrast, other serotypes, such as serotype 1, are rarely associated with colonization but cause invasive infections (28).Activation of innate defenses is essential for the control of pneumococcal infection (1, 20). Toll-like receptor 2 (TLR2), TLR4, and TLR9, as well as the adaptor MyD88, participate in the early detection and clearance of pneumococcus in the lungs (reviewed in reference 42). The cytosolic receptors Nod1 (nucleotide-binding oligomerization domain 1) and Nod2 are also involved in the recognition of pneumococci (29). TLR signaling activates mucosal innate responses that culminate with the recruitment of phagocytes, such as polymorphonuclear neutrophils (PMN) and macrophages, and the production of microbicidal agents (for a review, see reference 8). This process triggers rapid eradication of the pathogen by phagocytosis as well as by extracellular killing. In MyD88-deficient animals, S. pneumoniae is unable to intrinsically trigger any PMN recruitment into the airways, and animals thus have increased susceptibility to pneumonia (1). The contribution of TLR signaling in humans has been highlighted by a recent study showing that some MyD88 polymorphisms are associated with increased susceptibility to pneumococcal infection (43).The modulation of immunity by the activity of innate receptors to elicit protective responses against infections is an emerging concept (6, 35). The rationale is to promote innate responses that greatly exceed in magnitude, quality, and dynamics the innate response triggered by the pathogen itself. The effectiveness of TLR agonists for therapeutic treatment of infectious diseases has been demonstrated in several animal models, including models of respiratory infections (6, 21, 35). TLR5 senses bacterial flagellins, which are the main constituents of flagella. Various cells of the pulmonary tract, including the epithelial cells (14, 33), express TLR5, and mucosal administration of flagellin induces MyD88-dependent signaling, characterized by the swift production of various proinflammatory cytokines and chemokines (3, 10, 15, 16, 27, 33), as well as by rapid and heavy neutrophil infiltration into the airways (2, 10, 16). Although S. pneumoniae does not have flagella, we hypothesized that activation of TLR5 signaling may promote new and appropriate protective innate defenses against ongoing acute pneumococcal infections. Here we report that local stimulation of innate immunity by flagellin from Salmonella enterica serovar Typhimurium blocks the progression of pneumococcal pneumonia in mice.     

Keratinocyte Growth Factor Protects Alveolar Epithelium and Endothelium from Oxygen-Induced Injury in Mice

Keratinocyte growth factor (KGF) has been used successfully to prevent alveolar damage induced by oxygen exposure in rodents. However, this treatment was used intratracheally and before oxygen exposure, which limited its clinical application. In the present study, mice were treated with the recombinant human KGF intravenously before (days -2 and -1) or during (days 0 and +1) oxygen exposure. In both cases, lung damage was attenuated. KGF increased the number of cells incorporating bromodeoxyuridine (BrdU) in the septa and in bronchial epithelium of air-breathing mice but not of oxygen-exposed mice, indicating that the protective effect of KGF is not necessarily associated with proliferation. Oxygen-induced damage of alveolar epithelium and, unexpectedly, of endothelium was prevented by KGF treatment as seen by electron microscopy. We investigated the effect of KGF on different mechanisms known to be involved in oxygen toxicity. The induction of p53, Bax, and Bcl-x mRNAs during hyperoxia was to a large extent prevented by KGF. Surfactant proteins A and B mRNAs were not markedly modified by KGF. The anti-fibrinolytic activity observed in the alveoli during hyperoxia was to a large extent prevented by KGF, most probably by suppressing the expression of plasminogen activator inhibitor-1 (PAI-1) mRNA and protein. As PAI-1 -/- mice are more resistant to hyperoxia, KGF might act, at least in part, by decreasing the expression of this protease inhibitor and by restoring the fibrinolytic activity into the lungs.     

REV-ERBα Agonist GSK4112 attenuates Fas-induced Acute Hepatic Damage in Mice

Fas-induced apoptosis is a central mechanism of hepatocyte damage during acute and chronic hepatic disorders. Increasing evidence suggests that circadian clock plays critical roles in the regulation of cell fates. In the present study, the potential significance of REV-ERBα, a core ingredient of circadian clock, in Fas-induced acute liver injury has been investigated. The anti-Fas antibody Jo2 was injected intraperitoneally in mice to induce acute liver injury and the REV-ERBα agonist GSK4112 was administered. The results indicated that treatment of GSK4112 decreased the level of plasma ALT and AST, attenuated the liver histological changes, and promoted the survival rate in Jo2-insulted mice. Treatment with GSK4112 also downregulated the activities of caspase-3 and caspase-8, suppressed hepatocyte apoptosis. In addition, treatment with GSK4112 decreased the level of Fas and enhanced the phosphorylation of Akt. In conclusion, treatment with GSK4112 alleviated Fas-induced apoptotic liver damage in mice, suggesting that REV-ERBα agonist might have potential value in pharmacological intervention of Fas-associated liver injury.     

Stevioside Protects LPS-Induced Acute Lung Injury in Mice

Stevioside, a diterpene glycoside component of Stevia rebaudiana, has been known to exhibit anti-inflammatory properties. To evaluate the effect and the possible mechanism of stevioside in lipopolysaccharide (LPS)-induced acute lung injury, male BALB/c mice were pretreated with stevioside or dexamethasone 1 h before intranasal instillation of LPS. Seven hours later, tumor necrosis factor-α, interleukin-1β, and interleukin-6 in bronchoalveolar lavage fluid (BALF) were measured by using enzyme-linked immunosorbent assay. The number of total cells, neutrophils, and macrophages in the BALF were also determined. The right lung was excised for histological examination and analysis of myeloperoxidase activity and nitrate/nitrite content. Cyclooxygenase 2 (COX-2), inducible NO synthase (iNOS), nuclear factor-kappa B (NF-κB), inhibitory kappa B protein were detected by western blot. The results showed that stevioside markedly attenuated the LPS-induced histological alterations in the lung. Stevioside inhibited the production of pro-inflammatory cytokines and the expression of COX-2 and iNOS induced by LPS. In addition, not only was the wet-to-dry weight ratio of lung tissue significantly decreased, the number of total cells, neutrophils, and macrophages in the BALF were also significantly reduced after treatment with stevioside. Moreover, western blotting showed that stevioside inhibited the phosphorylation of IκB-α and NF-κB caused by LPS. Taken together, our results suggest that anti-inflammatory effect of stevioside against the LPS-induced acute lung injury may be due to its ability of inhibition of the NF-κB signaling pathway. Stevioside may be a promising potential therapeutic reagent for acute lung injury treatment.     

Intestinal Glucose Uptake Protects Liver from Lipopolysaccharide and d-Galactosamine,Acetaminophen, and Alpha-Amanitin in Mice

We have recently observed that oral administration of d-glucose saves animals from lipopolysaccharide (LPS)-induced death. This effect is the likely consequence of glucose-induced activation of the sodium-dependent glucose transporter-1. In this study, we investigated possible hepatoprotective effects of glucose-induced, sodium-dependent, glucose transporter-1 activation. We show that oral administration of d-glucose, but not of either d-fructose or sucrose, prevents LPS-induced liver injury, as well as liver injury and death induced by an overdose of acetaminophen. In both of these models, physiological liver morphology is maintained and organ protection is confirmed by unchanged levels of the circulating markers of hepatotoxicity, such as alanine transaminase or lactate dehydrogenase. In addition, d-glucose was found to protect the liver from α-amanitin-induced liver injury. In this case, in contrast to the previously described models, a second signal had to be present in addition to glucose to achieve protective efficacy. Toll-like receptor 4 stimulation that was induced by low doses of LPS was identified as such a second signal. Eventually, the protective effect of orally administered glucose on liver injury induced by LPS, overdose of acetaminophen, or α-amanitin was shown to be mediated by the anti-inflammatory cytokine interleukin-10. These findings, showing glucose-induced protective effects in several animal models of liver injury, might be relevant in view of possible therapeutic interventions against different forms of acute hepatic injury.Liver failure is one of the most devastating syndromes observed in clinical practice. It is associated with high overall mortality, ranging from 30% to 80%, depending on the underlying etiology.^1,2 The most common etiologies are acute viral hepatitis, drug overdose, idiosyncratic drug reactions, and ingestion of other toxins.³ Insulting agents can cause hepatocyte death through different mechanisms of action, and when the amount of functioning cells decreases to a level at which the organ is no longer capable of fulfilling its metabolic and synthetic tasks, hepatic failure takes place.⁴Recently we demonstrated, in a murine model of septic shock, that oral administration of d-glucose saves mice from death, most likely as a result of glucose-induced activation of the intestinal sodium-dependent glucose transporter-1 (SGLT-1).⁵ Using this model, we made preliminary observations that the liver, one of the organs most severely affected by lipopolysaccharide and d-galactosamine (LPS/d-GalN) treatment, was protected by oral administration of d-glucose. The expression of SGLT-1 on the apical membrane of enterocytes and the observation that protection from LPS shock was observed only on oral administration of d-glucose, but not on i.p. administration, suggested an important role of intestinal epithelial cells in protection from LPS-induced injury to the liver and other organs.Here, we report on a more extensive investigation on the hepatoprotective effect of orally administered d-glucose. Glucose was evaluated in LPS-induced shock, as well as in two other models of acute liver failure, ie, acetaminophen (APAP)- and α-amanitin-induced hepatic toxicity. APAP overdose is a common cause of drug-induced liver injury⁶ since this drug, when administered at very elevated doses, generates the highly reactive intermediate, N-para-aminoquinonimine,⁷ which covalently binds to hepatocyte macromolecules, leading to cellular death.⁸ This can lead to a potentially fatal centrilobular hepatic necrosis.⁹ On the other hand, α-amanitin toxin, which provide the major morbidity and mortality associated with toxic mushroom ingestion, interact with RNA polymerase II and affected cells undergo apoptosis due to insufficient protein synthesis. Organs mainly affected by α-amanitin include the intestinal mucosa, hepatocytes, and proximal tubules of kidney. The most severely affected organ is the liver, and hepatic toxicity is the actual cause of α-amanitin-induced lethality.In the herein-described experiments, we show that in all three models it is possible to achieve liver protection by d-glucose-induced intestinal SGLT-1 activation. However, while in models of LPS shock and APAP overdosing, d-glucose is therapeutically active when administered alone, in the α-amanitin model the concomitant administration of a low dose of LPS was required to achieve protection.     

Intranasal Administration of Synthetic Recombinant Peptide-Based Vaccine Protects Mice from Infection by Schistosoma mansoni

Schistosomiasis is the cause of a chronic debilitating disease which accounts for significant mortality and morbidity every year, especially in tropical and subtropical areas. An epitope derived from the protective surface protein 9B-Ag of Schistosoma mansoni, designated 9B peptide-1, was previously showed to be protective in mice when conjugated to bovine serum albumin and administered subcutaneously in complete Freund's adjuvant. In this work, this protective peptide was expressed in the flagellin of a Salmonella vaccine strain, and the isolated recombinant flagella were used for immunization of mice. Since during the invasion of the parasite into the host the schistosomula migrate first to the lungs, the intranasal route of administration was employed in order to halt the parasite at an early stage of the infection. Such intranasal immunization with this peptide expressed in flagellin, without the addition of adjuvants, resulted in a significant humoral response and also led to protection against challenge infection, manifested as a reduction of the worm burden by an average of 42%.