Sample records for adjuvant-free murine experimental

  1. Isotype specific immune responses in murine experimental toxocariasis

    Cuéllar C


    Full Text Available In this work, a murine experimental model of toxocariasis has been developed in BALB/c, C57BL/10 and C3H murine strains orally inoculated with 4,000 Toxocara canis embryonated eggs, in order to investigate the isotype-specific immune responses against excretory-secretory antigens from larvae. T. canis specific IgG+M, IgM, IgG, IgA, IgG1, IgG2a and IgG3 were tested by ELISA. The dynamics of the specific immunoglobulins (IgG+IgM production showed a contrasting profile regarding the murine strain. Conversely to the results obtained with the IgM isotype, the IgG antibody class showed similar patterns to those obtained with IgG+IgM antibodies, only in the case of the BALB/c strain, being different and much higher than the obtained with IgG+IgM antibodies, when the C3H murine strain was used. The antibodies IgG+IgM tested in BALB/c and C57BL/10 were both of the IgM and IgG isotypes. Conversely, in the C3H strain only IgG specific antibody levels were detected. The IgG1 subclass responses showed a similar profile in the three murine strains studied, with high values in BALB/c, as in the case of the IgG responses.

  2. Successful adjuvant-free vaccination of BALB/c mice with mutated amyloid β peptides

    Wahi Monika M


    Full Text Available Abstract Background A recent human clinical trial of an Alzheimer's disease (AD vaccine using amyloid beta (Aβ 1–42 plus QS-21 adjuvant produced some positive results, but was halted due to meningoencephalitis in some participants. The development of a vaccine with mutant Aβ peptides that avoids the use of an adjuvant may result in an effective and safer human vaccine. Results All peptides tested showed high antibody responses, were long-lasting, and demonstrated good memory response. Epitope mapping indicated that peptide mutation did not lead to epitope switching. Mutant peptides induced different inflammation responses as evidenced by cytokine profiles. Ig isotyping indicated that adjuvant-free vaccination with peptides drove an adequate Th2 response. All anti-sera from vaccinated mice cross-reacted with human Aβ in APP/PS1 transgenic mouse brain tissue. Conclusion Our study demonstrated that an adjuvant-free vaccine with different Aβ peptides can be an effective and safe vaccination approach against AD. This study represents the first report of adjuvant-free vaccines utilizing Aβ peptides carrying diverse mutations in the T-cell epitope. These largely positive results provide encouragement for the future of the development of human vaccinations for AD.

  3. Cyclosporine Inhibits Apoptosis in Experimental Murine Xerophthalamia Conjunctival Epithelium

    SUN Jinghua; WANG Jingxin


    apoptosis and protect goblet cell against the loss in experimental murine xerophathala-mia. Inhibition of apoptosis appears to be a key mechanism responsible for the therapeutic effect of CsA on xerophthalamia.

  4. An adjuvant-free mouse model to evaluate the allergenicity of milk whey protein.

    Gonipeta, B; Parvataneni, S; Tempelman, R J; Gangur, V


    Milk allergy is the most common type of food allergy in humans with the potential for fatality. An adjuvant-free mouse model would be highly desirable as a preclinical research tool to develop novel hypoallergenic or nonallergenic milk products. Here we describe an adjuvant-free mouse model of milk allergy that uses transdermal sensitization followed by oral challenge with milk protein. Groups of BALB/c mice were exposed to milk whey protein via a transdermal route, without adjuvant. Systemic IgG1 and IgE antibody responses to transdermal exposure as well as systemic anaphylaxis and hypothermia response to oral protein challenge were studied. Transdermal exposure resulted in a time- and dose-dependent induction of significant IgE and IgG1 antibody responses. Furthermore, oral challenge of sensitized mice resulted in significant clinical symptoms of systemic anaphylaxis within 1 h and significant hypothermia at 30 min postchallenge. To study the underlying mechanism, we examined allergen-driven spleen cell T-helper 2 cytokine (IL-4) responses. There was a robust dose- and time-dependent activation of memory IL-4 responses in allergic mice but not in healthy control mice. These data demonstrate for the first time a novel transdermal sensitization followed by oral challenge mouse model of milk allergy that does not use adjuvant. It is expected that this model may be used not only to study mechanisms of milk allergy, but also to evaluate novel milk products for allergenic potential and aid in the production of hypo- or nonallergenic milk products.

  5. Low concentrations of human neutrophil peptide ameliorate experimental murine colitis.

    Maeda, Takuro; Sakiyama, Toshio; Kanmura, Shuji; Hashimoto, Shinichi; Ibusuki, Kazunari; Tanoue, Shiroh; Komaki, Yuga; Arima, Shiho; Nasu, Yuichiro; Sasaki, Fumisato; Taguchi, Hiroki; Numata, Masatsugu; Uto, Hirofumi; Tsubouchi, Hirohito; Ido, Akio


    Human neutrophil peptides (HNPs) not only have antimicrobial properties, but also exert multiple immunomodulatory effects depending on the concentration used. We have previously demonstrated that the intraperitoneal administration of high-dose HNP-1 (100 µg/day) aggravates murine dextran sulfate sodium (DSS)-induced colitis, suggesting a potential pro-inflammatory role for HNPs at high concentrations. However, the role of low physiological concentrations of HNPs in the intestinal tract remains largely unknown. The aim of this study was to examine the effects of low concentrations of HNPs on intestinal inflammation. We first examined the effects of the mild transgenic overexpression of HNP-1 in DSS-induced colitis. HNP-1 transgenic mice have plasma HNP-1 levels similar to the physiological concentrations in human plasma. Compared to wild-type mice treated with DSS, HNP-1 transgenic mice treated with DSS had significantly lower clinical and histological scores, and lower colonic mRNA levels of pro-inflammatory cytokines, including interleukin (IL)-1β and tumor necrosis factor (TNF)-α. We then injected low-dose HNP-1 (5 µg/day) or phosphate-buffered saline (PBS) intraperitoneally into C57BL/6N and BALB/c mice administered DSS. The HNP-1-treated mice exhibited significantly milder colitis with reduced expression levels of pro-inflammatory cytokines compared with the PBS-treated mice. Finally, we examined the in vitro effects of HNP-1 on the expression of cytokines associated with macrophage activation. Low physiological concentrations of HNP-1 did not significantly affect the expression levels of IL-1β, TNF-α, IL-6 or IL-10 in colonic lamina propria mononuclear cells activated with heat-killed Escherichia coli, suggesting that the anti-inflammatory effects of HNP-1 on murine colitis may not be exerted by direct action on intestinal macrophages. Collectively, our data demonstrated a biphasic dose-dependent effect of HNP-1 on DSS-induced colitis: an

  6. Costimulatory signal blockade in murine relapsing experimental autoimmune encephalomyelitis

    Schaub, M; Issazadeh-Navikas, Shohreh; Stadlbauer, T H;


    Blockade of the CD28-B7 or CD40L-CD40 T cell costimulatory signals prevents induction of experimental autoimmune encephalomyelitis (EAE). However, the effect of simultaneous blockade of these signals in EAE is unknown. We show that administration of either MR1 (to block CD40L) or CTLA4Ig (to block...... cells in the periphery. Selective B7-1 blockade did not protect from EAE. These observations have implications for therapy of autoimmune diseases....

  7. Study on The Role of Interleukin-4 in Experimental Murine Systemic Candidiasis


    In order to investigate the role of interleukin-4 (IL-4) in experimental murine systemic Candidiasis, we created the intact and dexamethasone-induced immunosuppressed murine systemic Candidiasis models. In these models, two-site ELISA and RT-PCR were applied to determine the level of IL-4 protein and mRNA expression in spleens respectively, clone forming units (CFUs) of infected kidneys were determined with the plating dilution method, and mean survival time (MST) of the mice was recorded. The results showed that, when compared with the controls, protein level of IL-4 increased in both intact mice infected with lethal doses of yeast (day 3, P0.05; day 7, P<0.05). Furthermore, the level of IL-4 was higher on day 7 than on day 3 after infection (P<0.001 and P<0.05 respectively in two groups). The tendency of IL-4mRNA expression was similar with that of IL-4 protein. As for fungal loads in kidneys, CFUs were significantly higher on day 7 than on day 3 after infection (P<0.001 in both groups). Mice in both groups succumbed to infection within several days. It was suggested that IL-4 might play a promoting role in the development of murine systemic Candidiasis.

  8. A comprehensive collection of experimentally validated primers for Polymerase Chain Reaction quantitation of murine transcript abundance

    Wang Xiaowei


    Full Text Available Abstract Background Quantitative polymerase chain reaction (QPCR is a widely applied analytical method for the accurate determination of transcript abundance. Primers for QPCR have been designed on a genomic scale but non-specific amplification of non-target genes has frequently been a problem. Although several online databases have been created for the storage and retrieval of experimentally validated primers, only a few thousand primer pairs are currently present in existing databases and the primers are not designed for use under a common PCR thermal profile. Results We previously reported the implementation of an algorithm to predict PCR primers for most known human and mouse genes. We now report the use of that resource to identify 17483 pairs of primers that have been experimentally verified to amplify unique sequences corresponding to distinct murine transcripts. The primer pairs have been validated by gel electrophoresis, DNA sequence analysis and thermal denaturation profile. In addition to the validation studies, we have determined the uniformity of amplification using the primers and the technical reproducibility of the QPCR reaction using the popular and inexpensive SYBR Green I detection method. Conclusion We have identified an experimentally validated collection of murine primer pairs for PCR and QPCR which can be used under a common PCR thermal profile, allowing the evaluation of transcript abundance of a large number of genes in parallel. This feature is increasingly attractive for confirming and/or making more precise data trends observed from experiments performed with DNA microarrays.

  9. Ceftobiprole medocaril (BAL5788) treatment of experimental Haemophilus influenzae, Enterobacter cloacae, and Klebsiella pneumoniae murine pneumonia.

    Rouse, Mark S; Hein, Melanie M; Anguita-Alonso, Paloma; Steckelberg, James M; Patel, Robin


    Ceftobiprole (BAL9141) is an investigational cephalosporin active against methicillin- and vancomycin-resistant staphylococci administered as a water-soluble prodrug, ceftobiprole medocaril (BAL5788). Using an immunocompetent murine pneumonia model of Haemophilus influenzae, Enterobacter cloacae, or extended-spectrum beta-lactamase (ESBL) nonproducing or producing Klebsiella pneumoniae pneumonia, we compared results of treatment with ceftobiprole medocaril (71 mg/kg, sc, qid), ceftriaxone (50 mg/kg, im, bid), or cefepime (50 mg/kg, ip, q.i.d.). Results were expressed as median and 25th to 75th percentile log10 colony forming units per gram of lung tissue. Ceftobiprole, ceftriaxone, and cefepime were each more active than was no treatment and were equally active for treatment of experimental H. influenzae, E. cloacae, or ESBL-nonproducing K. pneumoniae pneumonia. For ESBL-producing K. pneumoniae, no differences were detected between no treatment and treatment with ceftobiprole, ceftriaxone, or cefepime. Ceftobiprole is active against H. influenzae, E. cloacae, and ESBL-nonproducing K. pneumoniae in an immunocompetent experimental murine pneumonia model.

  10. Strain-related effects of fenbendazole treatment on murine experimental autoimmune encephalomyelitis.

    Ramp, A A; Hall, C; Orian, J M


    Parasitic infections are a concern in animal facilities, in view of their influence on physiological processes and the immune status of animals. Pinworms are effectively controlled with the anthelminthic fenbendazole (FBZ, [5-(phenylthio)-1H-benzamidazol-2-yl]carbamic acid methyl ester; C(15)H(13)N(3)O(2)S); however, questions remain as to whether prolonged FBZ exposure alters the disease course in specific experimental models, such as those pertaining to the immune system. We report that a three-month regimen of FBZ-medicated feed severely affected the onset and disease severity of murine experimental autoimmune encephalomyelitis (EAE), a disease that mimics multiple sclerosis. Differences were recorded between mouse strains used. Our data suggest that where the use of FBZ is mandatory, its full effect should be verified on the particular EAE variant adopted by the laboratory.

  11. Comparison of histopathology and PCR based assay for detection of experimentally induced toxoplasmosis in murine model

    Vikrant Sudan; A K Tewari; R Singh; Harkirat Singh


    Objective:To compare histopathology and PCR based detection in diagnosis of experimentally induced toxoplasmosis of RH human strain of the parasite in murine models. Methods:A comparison of histopathology and PCR based detection was done to diagnose experimentally induced toxoplasmosis in ten inbred swiss albino mice after intraperitoneal inoculation of 100 tachyzoites of laboratory mantained human RH strain of the parasite. Tissue samples from lung, liver, spleen, brain, heart and kidney were taken and processed for histopathological examination while all the samples also were subjected to PCR, using primers directed to the multicopy of SAG 3 gene, in dublicates. Results: Histopathology revealed presence of tachyzoites only in liver while along with lung, liver, spleen and brain tissue yielded desired positive PCR amplicons. Conclusions:The SAG 3 based PCR is able to diagnose toxoplasmosis in those tissues which are declared negative by histopathological assay.

  12. Oral treatment with Bifidobacterium longum 51A reduced inflammation in a murine experimental model of gout.

    Vieira, A T; Galvão, I; Amaral, F A; Teixeira, M M; Nicoli, J R; Martins, F S


    Gout is an acute inflammatory disease characterised by the presence of uric acid crystals in the joint. This event promotes neutrophil infiltration and activation that leads to tissue damage. We investigated here whether the oral administration of the probiotic strain Bifidobacterium longum 5(1A) (BL) could ameliorate monosodium urate crystal (MSU)-induced inflammation in a murine model of gout. Mice received oral administration of BL or saline daily for 7 days and then were injected with MSU in the knee cavity. Treatment with BL significantly alleviated the inflammatory parameters, as seen by reduced hypernociception, reduced neutrophil accumulation in the joint and myeloperoxidase activity in periarticular tissue. There was inhibition of the production of CXCL1 and interleukin(IL)-1β in joints. Levels of the anti-inflammatory cytokine IL-10 were significantly higher in the knee tissue of mice treated with than control mice injected with MSU. In conclusion, oral BL treatment reduced the inflammatory response in an experimental murine model of gout, suggesting it may be useful as an adjuvant treatment in patients with gout.

  13. Kinetics of expression of costimulatory molecules and their ligands in murine relapsing experimental autoimmune encephalomyelitis in vivo

    Issazadeh-Navikas, Shohreh; Navikas, V; Schaub, M;


    We studied the kinetics of expression of costimulatory molecules and cytokines in the central nervous system (CNS) in murine relapsing experimental autoimmune encephalomyelitis (EAE). During the natural course of EAE, B7-2 expression in the CNS correlated with clinical signs, while B7-1 was exclu...

  14. Flucytosine + fluconazole association in the treatment of a murine experimental model of cryptococcosis

    A. J. Bava


    Full Text Available The efficacy of flucytosine (5-FC and fluconazole (FLU association in the treatment of a murine experimental model of cryptococcosis, was evaluated. Seven groups of 10 Balb C mice each, were intraperitoneally inoculated with 10(7 cells of Cryptococcus neoformans. Six groups were allocated to receive 5-FC (300 mg/kg and FLU (16 mg/ kg, either combined and individually, by daily gavage beginning 5 days after the infection, for 2 and 4 weeks. One group received distilled water and was used as control. The evaluation of treatments was based on: survival time; macroscopic examination of brain, lungs, liver and spleen at autopsy; presence of capsulated yeasts in microscopic examination of wet preparations of these organs and cultures of brain homogenate. 5-FC and FLU, individually or combined, significantly prolonged the survival time of the treated animals with respect to the control group (p<0.01. Animals treated for 4 weeks survived significantly longer than those treated for 2 weeks (p<0.01. No significant differences between the animals treated with 5-FC and FLU combined or separately were observed in the survival time and morphological parameters. The association of 5-FC and FLU does not seem to be more effective than 5-FC or FLU alone, in the treatment of this experimental model of cryptococcosis.

  15. A novel adjuvant-free H fusion system for the production of recombinant immunogens in Escherichia coli

    Costa, Sofia J; Silva, Pedro; Almeida, André; Conceição, Antónia; Domingues, Lucília; Castro, António


    The production of recombinant antigens in Escherichia coli and specific polyclonal antibodies for diagnosis and therapy is still a challenge for world-wide researchers. Several different strategies have been explored to improve both antigen and antibody production, all of them depending on a successful expression and immunogenicity of the antigen. Gene fusion technology attempted to address these challenges: fusion partners have been applied to optimize recombinant antigen production in E. coli, and to increase protein immunogenicity. Taking a 12-kDa surface adhesion antigen from Cryptosporidium parvum (CP12) by example, the novel H fusion partner was presented in this work as an attractive option for the development of recombinant immunogens and its adjuvant-free immunization. The H tag (of only 1 kDa) efficiently triggered a CP12-specific immune response, and it also improved the immunization procedure without requiring co-administration of adjuvants. Moreover, polyclonal antibodies raised against the HCP12 fusion antigen detected native antigen structures displayed on the surface of C. parvum oocysts. The H tag proved to be an advanced strategy and promising technology for the diagnosis and therapy of C. parvum infections in animals and humans, allowing a rapid and simple recombinant production of the CP12 antigen. PMID:23941978

  16. Detection of antibodies against Theiler's murine encephalomyelitis virus GDVII strain in experimental guinea pigs.

    Häger, C; Glage, S; Held, N; Bleich, E M; Burghard, A; Mähler, M; Bleich, André


    A disease affecting guinea pigs called 'guinea pig lameness' characterized by clinical signs of depression, lameness of limbs, flaccid paralysis, weight loss and death within a few weeks was first described by Römer in 1911. After a research group in our facility kept laboratory guinea pigs from two different origins together in one room, lameness was observed in two animals. Further investigations revealed a serological immune response against Theiler's murine encephalomyelitis virus (TMEV; GDVII strain) in these animals. Histopathology of the lumbar spinal cord of these animals showed mononuclear cell infiltration and necrotic neurons in the anterior horn. Therefore, all guinea pigs from this contaminated animal unit, from other units in our facility, as well as from different European institutions and breeding centres were screened for antibodies directed against GDVII. Our investigations showed that approximately 80% of all guinea pigs from the contaminated animal unit were seropositive for GDVII, whereas animals from other separate units were completely negative. In addition, 43% of tested sera from the different European institutions and breeding centres contained antibodies against GDVII. The present data confirm that an unknown viral infection causes an immune response in experimental guinea pigs leading to seroconversion against GDVII and that guinea pigs from a commercial breeder are the source of the infection.

  17. Vinegar Treatment Prevents the Development of Murine Experimental Colitis via Inhibition of Inflammation and Apoptosis.

    Shen, Fengge; Feng, Jiaxuan; Wang, Xinhui; Qi, Zhimin; Shi, Xiaochen; An, Yanan; Zhang, Qiaoli; Wang, Chao; Liu, Mingyuan; Liu, Bo; Yu, Lu


    This study investigated the preventive effects of vinegar and acetic acid (the active component of vinegar) on ulcerative colitis (UC) in mice. Vinegar (5% v/v) or acetic acid (0.3% w/v) treatment significantly reduced the disease activity index and histopathological scores, attenuated body weight loss, and shortened the colon length in a murine experimental colitis model induced by dextran sulfate sodium (DSS). Further mechanistic analysis showed that vinegar inhibited inflammation through suppressing Th1 and Th17 responses, the NLRP3 inflammasome, and MAPK signaling activation. Vinegar also inhibited endoplasmic reticulum (ER) stress-mediated apoptosis in the colitis mouse model. Surprisingly, pretreatment with vinegar for 28 days before DSS induction increased levels of the commensal lactic acid-producing or acetic acid-producing bacteria, including Lactobacillus, Bifidobacteria, and Enterococcus faecalis, whereas decreased Escherichia coli levels were found in the feces of mice. These results suggest that vinegar supplementation might provide a new dietary strategy for the prevention of UC.

  18. Experimental murine chromoblastomycosis obtained from Fonsecaea pedrosoi isolate cultured for a long periodt

    AP Machado


    Full Text Available The present study aimed to describe F. pedrosoi propagules capable of causing chronic murine disease. Several changes in F. pedrosoi hyphae were identified in fungal cells cultured for a long period. Optical microscopy found many rounded cells with double-rigid melanin-rich walls. Terminal and intercalary chlamydoconidia were also frequently observed. Analyses of images from transmission electron microscopy (TEM revealed several cells with walls composed of at least three layers and an outer layer enriched with melanin. Two groups of twenty BALB/c mice were subcutaneously infected in their footpads with F. pedrosoi cells at an inoculum concentration of approximately 1 x 10(4 cells/mL. In one group, long-term cultured F. pedrosoi cells were inoculated in one footpad, whereas in the other group, both footpads were infected. Active lesions were observed up to seven months post-infection, particularly in mice inoculated at two sites. After this period, animals were killed. Histological sections revealed characteristics bearing a strong resemblance to the human form of the disease such as tissue hyperplasia, granulomas with microabscesses and sclerotic cells. Based on this study, we identified fungal cells from old cultures capable of provoking chronic chromoblastomycosis under experimental conditions, especially when more than one site is infected.

  19. Pharmacokinetics of indium-111-labeled antimyosin monoclonal antibody in murine experimental viral myocarditis

    Yamada, T.; Matsumori, A.; Watanabe, Y.; Tamaki, N.; Yonekura, Y.; Endo, K.; Konishi, J.; Kawai, C. (Kyoto Univ. (Japan))


    The pharmacokinetics of indium-111-labeled antimyosin monoclonal antibody Fab were investigated with use of murine experimental viral myocarditis as a model. The biodistribution of indium-111-labeled antimyosin antibody Fab on days 3, 5, 7, 14, 21 and 28 after encephalomyocarditis virus inoculation demonstrated that myocardial uptake increased significantly on days 5, 7 and 14 (maximum on day 7) in infected versus uninfected mice (p less than 0.001). In vivo kinetics in infected mice on day 7 demonstrated that the heart to blood ratio reached a maximum 48 h after the intravenous administration of indium-111-labeled antimyosin Fab, which was considered to be the optimal time for scintigraphy. The scintigraphic images obtained with indium-111-labeled antimyosin Fab demonstrated positive uptake in the cardiac lesion in infected mice. The pathologic study demonstrated that myocardial uptake correlated well with pathologic grades of myocardial necrosis. High performance liquid chromatography revealed the presence of an antigen-antibody complex in the circulation of infected mice after the injection of indium-111-labeled antimyosin Fab. This antigen bound to indium-111-labeled antimyosin Fab in the circulation might be whole myosin and this complex may decrease myocardial uptake and increase liver uptake. It is concluded that indium-111-labeled antimyosin monoclonal antibody Fab accumulates selectively in damaged heart tissue in mice with acute myocarditis and that indium-111-labeled antimyosin Fab scintigraphy may be a useful method for the visualization of acute myocarditis.

  20. Lipid alterations in experimental murine colitis: role of ceramide and imipramine for matrix metalloproteinase-1 expression.

    Jessica Bauer

    Full Text Available BACKGROUND: Dietary lipids or pharmacologic modulation of lipid metabolism are potential therapeutic strategies in inflammatory bowel disease (IBD. Therefore, we analysed alterations of bioactive lipids in experimental models of colitis and examined the functional consequence of the second messenger ceramide in inflammatory pathways leading to tissue destruction. METHODOLOGY/PRINCIPAL FINDINGS: Chronic colitis was induced by dextran-sulphate-sodium (DSS or transfer of CD4(+CD62L(+ cells into RAG1(-/--mice. Lipid content of isolated murine intestinal epithelial cells (IEC was analysed by tandem mass spectrometry. Concentrations of MMP-1 in supernatants of Caco-2-IEC and human intestinal fibroblasts from patients with ulcerative colitis were determined by ELISA. Imipramine was used for pharmacologic inhibition of acid sphingomyelinase (ASM. Ceramide increased by 71% in chronic DSS-induced colitis and by 159% in the transfer model of colitis. Lysophosphatidylcholine (LPC decreased by 22% in both models. No changes were detected for phosphatidylcholine. Generation of ceramide by exogenous SMase increased MMP-1-protein production of Caco-2-IEC up to 7-fold. Inhibition of ASM completely abolished the induction of MMP-1 by TNF or IL-1beta in Caco-2-IEC and human intestinal fibroblasts. CONCLUSIONS/SIGNIFICANCE: Mucosal inflammation leads to accumulation of ceramide and decrease of LPC in the intestinal epithelium. One aspect of ceramide generation is an increase of MMP-1. Induction of MMP-1 by TNF or IL-1beta is completely blocked by inhibition of ASM with imipramine. Therefore, inhibition of ASM may offer a treatment strategy to reduce MMP-1 expression and tissue destruction in inflammatory conditions.

  1. Macrophage activation associated with chronic murine cytomegalovirus infection results in more severe experimental choroidal neovascularization.

    Scott W Cousins

    Full Text Available The neovascular (wet form of age-related macular degeneration (AMD leads to vision loss due to choroidal neovascularization (CNV. Since macrophages are important in CNV development, and cytomegalovirus (CMV-specific IgG serum titers in patients with wet AMD are elevated, we hypothesized that chronic CMV infection contributes to wet AMD, possibly by pro-angiogenic macrophage activation. This hypothesis was tested using an established mouse model of experimental CNV. At 6 days, 6 weeks, or 12 weeks after infection with murine CMV (MCMV, laser-induced CNV was performed, and CNV severity was determined 4 weeks later by analysis of choroidal flatmounts. Although all MCMV-infected mice exhibited more severe CNV when compared with control mice, the most severe CNV developed in mice with chronic infection, a time when MCMV-specific gene sequences could not be detected within choroidal tissues. Splenic macrophages collected from mice with chronic MCMV infection, however, expressed significantly greater levels of TNF-α, COX-2, MMP-9, and, most significantly, VEGF transcripts by quantitative RT-PCR assay when compared to splenic macrophages from control mice. Direct MCMV infection of monolayers of IC-21 mouse macrophages confirmed significant stimulation of VEGF mRNA and VEGF protein as determined by quantitative RT-PCR assay, ELISA, and immunostaining. Stimulation of VEGF production in vivo and in vitro was sensitive to the antiviral ganciclovir. These studies suggest that chronic CMV infection may serve as a heretofore unrecognized risk factor in the pathogenesis of wet AMD. One mechanism by which chronic CMV infection might promote increased CNV severity is via stimulation of macrophages to make pro-angiogenic factors (VEGF, an outcome that requires active virus replication.

  2. The murine gammaherpesvirus-68 chemokine-binding protein M3 inhibits experimental autoimmune encephalomyelitis

    Millward, Jason M; Holst, Peter J; Høgh-Petersen, Mette


    actions, in order to evade host immune responses. The murine gammaherpesvirus-68 encodes a chemokine-binding protein called M3, which has unique biochemical features that enable it to bind to and inhibit an unusually broad range of chemokines. We applied a replication-defective adenoviral vector encoding...

  3. Therapeutic effects of garenoxacin in murine experimental secondary pneumonia by Streptococcus pneumoniae after influenza virus infection.

    Fukuda, Yoshiko; Furuya, Yuri; Nozaki, Yusuke; Takahata, Masahiro; Nomura, Nobuhiko; Mitsuyama, Junichi


    In a pneumococcal pneumonia murine model following influenza virus infection, garenoxacin was more effective than other fluoroquinolones and demonstrated high levels of bacterial eradication in the lung, low mortality, and potent histopathological improvements. Garenoxacin could potentially be used for the treatment of secondary pneumococcal pneumonia following influenza.

  4. Assessment and in vivo scoring of murine experimental autoimmune uveoretinitis using optical coherence tomography.

    Chu, Colin J; Herrmann, Philipp; Carvalho, Livia S; Liyanage, Sidath E; Bainbridge, James W B; Ali, Robin R; Dick, Andrew D; Luhmann, Ulrich F O


    Despite advances in clinical imaging and grading our understanding of retinal immune responses and their morphological correlates in experimental autoimmune uveoretinitis (EAU), has been hindered by the requirement for post-mortem histology. To date, monitoring changes occurring during EAU disease progression and evaluating the effect of therapeutic intervention in real time has not been possible. We wanted to establish whether optical coherence tomography (OCT) could detect intraretinal changes during inflammation and to determine its utility as a tool for accurate scoring of EAU. EAU was induced in C57BL/6J mice and animals evaluated after 15, 26, 36 and 60 days. At each time-point, contemporaneous Spectralis-OCT scanning, topical endoscopic fundal imaging (TEFI), fundus fluorescein angiography (FFA) and CD45-immunolabelled histology were performed. OCT features were further characterised on retinal flat-mounts using immunohistochemistry and 3D reconstruction. Optic disc swelling and vitreous opacities detected by OCT corresponded to CD45+ cell infiltration on histology. Vasculitis identified by FFA and OCT matched perivascular myeloid and T-cell infiltrates and could be differentiated from unaffected vessels. Evolution of these changes could be followed over time in the same eye. Retinal folds were visible and found to encapsulate mixed populations of activated myeloid cells, T-cells and microglia. Using these features, an OCT-based EAU scoring system was developed, with significant correlation to validated histological (Pearson r(2) = 0.6392, P<0.0001, n = 31 eyes) and TEFI based scoring systems (r(2) = 0.6784, P<0.0001). OCT distinguishes the fundamental features of murine EAU in vivo, permits dynamic assessment of intraretinal changes and can be used to score disease severity. As a result, it allows tissue synchronisation with subsequent cellular and functional assessment and greater efficiency of animal usage. By relating OCT signals with

  5. Protein tyrosine phosphatase 1B deficiency ameliorates murine experimental colitis via the expansion of myeloid-derived suppressor cells.

    Jing Zhang

    Full Text Available Protein tyrosine phosphatase 1B (PTP1B is a key molecule in modulating low-degree inflammatory conditions such as diabetes. The role of PTP1B in other chronic inflammations, however, remains unknown. Here, we report that PTP1B deficiency ameliorates Dextran Sulfate Sodium (DSS-induced murine experimental colitis via expanding CD11b(+Gr-1(+ myeloid-derived suppressor cells (MDSCs. Employing DSS-induced murine experimental colitis as inflammatory animal model, we found that, compared with wild-type littermates, PTP1B-null mice demonstrated greater resistance to DSS-induced colitis, as reflected by slower weight-loss, greater survival rates and decreased PMN and macrophage infiltration into the colon. The evidence collectively also demonstrated that the resistance of PTP1B-null mice to DSS-induced colitis is based on the expansion of MDSCs. First, PTP1B-null mice exhibited a greater frequency of MDSCs in the bone marrow (BM, peripheral blood and spleen when compared with wild-type littermates. Second, PTP1B levels in BM leukocytes were significantly decreased after cells were induced into MDSCs by IL-6 and GM-CSF, and the MDSC induction occurred more rapidly in PTP1B-null mice than in wild-type littermates, suggesting PTP1B as a negative regulator of MDSCs. Third, the adoptive transfer of MDSCs into mice with DSS-colitis significantly attenuated colitis, which accompanies with a decreased serum IL-17 level. Finally, PTP1B deficiency increased the frequency of MDSCs from BM cells likely through enhancing the activities of signal transducer and activator of transcription 3 (STAT3 and Janus kinase 2 (JAK2. In conclusion, our study provides the first evidences that PTP1B deficiency ameliorates murine experimental colitis via expanding MDSCs.

  6. Characterization of murine hepatitis virus (JHM) RNA from rats with experimental encephalomyelitis.

    Jackson, D P; Percy, D H; Morris, V L


    When Wistar Furth rats are inoculated intracerebrally with the murine hepatitis virus JHM they often develop a demyelinating disease with resulting hind leg paralysis. Using an RNA transfer procedure and hybridization kinetic analysis, the virus-specific RNA in these rats was characterized. The pattern of JHM-specific RNA varied with individual infections of Wistar Furth rats. However, two species of JHM-specific RNA, the nucleocapsid and a 2.1-2.4 X 10(6)-Da RNA species were generally present. A general decrease in JHM-specific RNA in brains and spinal cord samples taken later than 20 days postinoculation was observed; however, JHM-specific RNA persisted in the spinal cord longer than in the brain of these rats.

  7. IL-25, IL-33 and TSLP receptor are not critical for development of experimental murine malaria

    Akiko Shibui


    Full Text Available IL-25, IL-33 and TSLP, which are produced predominantly by epithelial cells, can induce production of Th2-type cytokines such as IL-4, IL-5 and/or IL-13 by various types of cells, suggesting their involvement in induction of Th2-type cytokine-associated immune responses. It is known that Th2-type cytokines contribute to host defense against malaria parasite infection in mice. However, the roles of IL-25, IL-33 and TSLP in malaria parasite infection remain unclear. Thus, to elucidate this, we infected wild-type, IL-25−/−, IL-33−/− and TSLP receptor (TSLPR−/− mice with Plasmodium berghei (P. berghei ANKA, a murine malaria strain. The expression levels of IL-25, IL-33 and TSLP mRNA were changed in the brain, liver, lung and spleen of wild-type mice after infection, suggesting that these cytokines are involved in host defense against P. berghei ANKA. However, the incidence of parasitemia and survival in the mutant mice were comparable to in the wild-type mice. These findings indicate that IL-25, IL-33 and TSLP are not critical for host defense against P. berghei ANKA.

  8. Ultrastructural study on experimental infection of rotavirus in a murine heterologous model

    Selma Majerowicz


    Full Text Available Viral replication, histopathological and ultrastructural changes were observed for a period of nine days in the small intestine of suckling mice infected with a simian rotavirus (SA11. Samples taken from duodenum, jejunun and ileum were prepared for light microscopy, transmission and scanning electron microscopy analysis. Histopathologic effect could be detected within 8 hr post-infection, when only a few altered cells were observed. Damage was extensive after 16 hr post-infection, showing swollen enterocytes and reduced and irregularly oriented microvilli at intestinal villi tips. Virus particles were detected at 16 and 48 hr post-infection, budding from the viroplasm into the rough endoplasmic reticulum cisternae in ileum enterocytes. Clear evidence of viral replication, observed by electron microscopy was not described before in heterologous murine models. Regeneration of the intestinal villi began at the third day post-infection. Despite some differences observed in clinical symptoms and microscopic analysis of homologous and heterologous rotavirus infections, we concluded that mechanisms of heterologous rotavirus infection in mice follow similar patterns to those observed in the homologous models.

  9. Vaccine potential of plasma bead-based dual antigen delivery system against experimental murine candidiasis.

    Ahmad, Ejaj; Zia, Qamar; Fatima, Munazza Tamkeen; Owais, Mohammad; Saleemuddin, Mohammed


    The development of prophylactic anti-candidal vaccine comprising the Candida albicans cytosolic proteins (Cp) as antigen and plasma beads (PB) prepared from plasma as sustained delivery system, is described. The immune-prophylactic potential of various PBs-based dual antigen delivery systems, co-entrapping Cp pre-entrapped in PLGA microspheres were tested in the murine model. Induction of cell mediated immunity was measured by assaying DTH and NO production as well as in vitro proliferation of lymphocytes derived from the immunized animals. Expression of surface markers on APCs (CD80, CD86) and T-cells (CD4+, CD8+) was also evaluated. Humoral immune response was studied by measuring circulating anti-Cp antibodies and their subclasses. When the prophylactic efficacy of the vaccines was tested in mice challenged with virulent C. albicans, the PB-based formulation (PB-PLGA-Cp vaccine) was found to be most effective in the generation of desirable immune response, in terms of suppression of fungal load and facilitating the survival of the immunized animals.

  10. Mitogen-activated protein kinase pathways contribute to hypercontractility and increased Ca2+ sensitization in murine experimental colitis.

    Ihara, Eikichi; Beck, Paul L; Chappellaz, Mona; Wong, Josee; Medlicott, Shaun A; MacDonald, Justin A


    Inflammatory bowel disease (IBD) is associated with intestinal smooth muscle dysfunction. Many smooth muscle contractile events are associated with alterations in Ca(2+)-sensitizing pathways. The aim of the present study was to assess the effect of colitis on Ca(2+) sensitization and the signaling pathways responsible for contractile dysfunction in murine experimental colitis. Colitis was induced in BALB/c mice by providing 5% dextran sulfate sodium (DSS) in drinking water for 7 days. Contractile responses of colonic circular smooth muscle strips to 118 mM K(+) and carbachol (CCh) were assessed. DSS induced a T(H)2 colitis [increased interleukin (IL)-4 and IL-6] with no changes in T(H)1 cytokines. Animals exposed to DSS had increased CCh-induced contraction (3.5-fold) and CCh-induced Ca(2+)-sensitization (2.2-fold) responses in intact and alpha-toxin permeabilized colonic smooth muscle, respectively. The contributions of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) to CCh-induced contractions were significantly increased during colitis. Ca(2+)-independent contraction induced by microcystin was potentiated (1.5-fold) in mice with colitis. ERK and p38MAPK (but not Rho-associated kinase) contributed to this potentiation. ERK1/2 and p38MAPK expression were increased in the muscularis propria of colonic tissue from both DSS-treated mice and patients with IBD (ulcerative colitis > Crohn's disease). Murine T(H)2 colitis resulted in colonic smooth muscle hypercontractility with increased Ca(2+) sensitization. Both ERK and p38MAPK pathways contributed to this contractile dysfunction, and expression of these molecules was altered in patients with IBD.

  11. Comparative study of the biological properties of Trypanosoma cruzi I genotypes in a murine experimental model.

    Cruz, Lissa; Vivas, Angie; Montilla, Marleny; Hernández, Carolina; Flórez, Carolina; Parra, Edgar; Ramírez, Juan David


    Chagas disease is an endemic zoonosis in Latin America and caused by the parasite Trypanosoma cruzi. This kinetoplastid displays remarkable genetic variability, allowing its classification into six Discrete Typing Units (DTUs) from TcI to TcVI. T. cruzi I presents the broadest geographical distribution in the continent and has been associated to severe forms of cardiomyopathies. Recently, a particular genotype associated to human infections has been reported and named as TcIDOM (previously named TcIa-b). This genotype shows to be clonal and adapted to the domestic cycle but so far no studies have determined the biological properties of domestic (TcIDOM) and sylvatic TcI strains (previously named TcIc-e). Hence, the aim of this study was to untangle the biological features of these genotypes in murine models. We infected ICR-CD1 mice with five TcI strains (two domestic, two sylvatic and one natural mixture) and determined the course of infection during 91 days (acute and chronic phase of the disease) in terms of parasitemia, tissue tropism, immune response (IgG titers) and tissue invasion by means of histopathology studies. Statistically significant differences were observed in terms of parasitemia curves and prepatent period between domestic (TcIDOM) and sylvatic strains. There were no differences in terms of IgG antibodies response across the mice infected with the five strains. Regarding the histopathology, our results indicate that domestic strains present higher parasitemias and low levels of histopathological damage. In contrast, sylvatic strains showed lower parasitemias and high levels of histopathological damage. These results highlight the sympatric and behavioral differences of domestic and sylvatic TcI strains; the clinical and epidemiological implications are herein discussed.

  12. Dynamic ocular surface and lacrimal gland changes induced in experimental murine dry eye.

    Bing Xiao

    Full Text Available Dry eye disease can be a consequence of lacrimal gland insufficiency in Sjögren's Syndrome or increased tear film evaporation despite normal lacrimal gland function. To determine if there is a correlation between severity effects in these models and underlying pathophysiological responses, we compared the time dependent changes in each of these parameters that occur during a 6 week period. Dry eye was induced in 6-week-old female C57BL/6 mice by exposing them to an Intelligently Controlled Environmental System (ICES. Sixty mice were housed in ICES for 1, 2, 4 and 6 weeks respectively. Twelve were raised in normal environment and received subcutaneous injections of scopolamine hydrobromide (SCOP 3 times daily for 5 days. Another sixty mice were housed in a normal environment and received no treatment. Corneal fluorescein staining along with corneal MMP-9 and caspase-3 level measurements were performed in parallel with the TUNEL assay. Interleukin-17(IL-17, IL-23, IL-6, IL-1, TNF-α, IFN-γ and TGF-β2 levels were estimated by real-time PCR measurements of conjunctival and lacrimal gland samples (LGs. Immunohistochemistry of excised LGs along with flow cytometry in cervical lymph nodes evaluated immune cell infiltration. Light and transmission electron microscopy studies evaluated LGs cytoarchitectural changes. ICES induced corneal epithelial destruction and apoptosis peaked at 2 weeks and kept stable in the following 4 weeks. In the ICES group, lacrimal gland proinflammatory cytokine level increases were much lower than those in the SCOP group. In accord with the lower proinflammatory cytokine levels, in the ICES group, lacrimal gland cytosolic vesicular density and size exceeded that in the SCOP group. ICES and SCOP induced murine dry eye effects became progressively more severe over a two week period. Subsequently, the disease process stabilized for the next four weeks. ICES induced local effects in the ocular surface, but failed to elicit

  13. Continuous oral chloroquine as a novel route for Plasmodium prophylaxis and cure in experimental murine models

    Pfeil Johannes


    Full Text Available Abstract Background Chloroquine (CQ is utilized as both cure and prophylaxis to Plasmodium infection. In animal studies, CQ administration to experimental animals is via intraperitoneal (i.p. injection of a single dose that varies from daily to several times per week. Such daily administration can be distressing to the animals and provoke aggressive behaviors that may affect the immune responses of the animal and interfere with data read-outs. Findings We describe a novel, viable and efficacious prophylactic and curative administration route whereby chloroquine is continuously supplied in the drinking water to experimental animals. The prophylactic effect is robust and the curative effect against patent blood stage infection comparable to the traditional route of i.p. administration. Continuous drinking water administration may decrease animal stress responses and thus improve the reliability of experimental data.

  14. Prevention of murine experimental autoimmune orchitis by recombinant human interleukin-6

    Li, Lu; Itoh, Masahiro; Ablake, Maila;


    We studied the effect of exogenously administered recombinant human interleukin (IL)-6 on the development of experimental autoimmune orchitis (EAO) in C3H/Hej mice. IL-6 significantly reduced histological signs of EAO and appearance of delayed type hypersensitivity against the immunizing testicul...

  15. Sepsis otopathy: experimental sepsis leads to significant hearing impairment due to apoptosis and glutamate excitotoxicity in murine cochlea

    Joachim Schmutzhard


    Hearing loss is frequent in intensive care patients and can be due to several causes. However, sepsis has not been examined as a possible cause. The aim of this study is to assess the influence of experimental sepsis on hearing thresholds and to evaluate pathological changes in the cochlea. The cecal ligation puncture technique was used to induce sepsis in 18 mice. Results were compared with those from 13 sham-operated and 13 untreated control mice. The hearing thresholds of the animals were evaluated with auditory evoked brainstem responses prior to the induction of sepsis and again at the peak of the disease. Immediately after the second measurement, the mice were sacrificed and the inner ears harvested and prepared for further evaluation. The cochleae were examined with light microscopy, electron microscopy and immunohistochemistry for Bax, cleaved caspase-3 and Bcl-2. The mice with sepsis showed a significant hearing loss but not the control groups. Induction of apoptosis could be shown in the supporting cells of the organ of Corti. Furthermore, excitotoxicity could be shown at the basal pole of the inner hair cells. In this murine model, sepsis leads to significant hearing impairment. The physiological alteration could be linked to apoptosis in the supporting cells of the organ of Corti and to a disturbance of the synapses of the inner hair cells.

  16. Effects of ionizing radiation on bone cell differentiation in an experimental murine bone cell model

    Baumstark-Khan, Christa; Lau, Patrick; Hellweg, Christine; Reitz, Guenther

    During long-term space travel astronauts are exposed to a complex mixture of different radiation types under conditions of dramatically reduced weight-bearing activity. It has been validated that astronauts loose a considerable amount of bone mass at a rate up to one to two percent each month in space. Therapeutic doses of ionizing radiation cause bone damage and increase fracture risks after treatment for head-and-neck cancer and in pelvic irradiation. For low radiation doses, the possibility of a disturbed healing potential of bone was described. Radiation induced damage has been discussed to inflict mainly on immature and healing bone. Little is known about radiation effects on bone remodelling and even less on the combined action of microgravity and radiation. Bone remodelling is a life-long process performed by balanced action of cells from the osteoblast and osteoclast lineages. While osteoblasts differentiate either into bone-lining cells or into osteocytes and play a crucial role in bone matrix synthesis, osteoclasts are responsible for bone resorption. We hypothesize that the balance between bone matrix assembly by osteocytes and bone degradation by osteoclasts is modulated by microgravity as well as by ionizing radiation. To address this, a cell model consisting of murine cell lines with the potential to differentiate into bone-forming osteoblasts (OCT-1, MC3T3-E1 S24, and MC3T3-E1 S4) was used for studying radiation response after exposure to simulated components of cosmic radiation. Cells were exposed to graded doses of 150 kV X-rays, α particles (0.525 MeV/u, 160 keV/µm; PTB, Braunschweig, Germany) and accelerated heavy ions (75 MeV/u carbon, 29 keV/µm; 95 MeV/u argon, 230 keV/µm; GANIL, Caen, France). Cell survival was measured as colony forming ability; cell cycle progression was analyzed via fluorescence-activated cell scanning (FACS) by measurement of the content of propidium iodide-stained DNA, DNA damage was visualized by γH2AX

  17. Vaccinia virus Transmission through Experimentally Contaminated Milk Using a Murine Model.

    Rehfeld, Izabelle Silva; Guedes, Maria Isabel Maldonado Coelho; Fraiha, Ana Luiza Soares; Costa, Aristóteles Gomes; Matos, Ana Carolina Diniz; Fiúza, Aparecida Tatiane Lino; Lobato, Zélia Inês Portela


    Bovine vaccinia (BV) is a zoonosis caused by Vaccinia virus (VACV), which affects dairy cattle and humans. Previous studies have detected the presence of viable virus particles in bovine milk samples naturally and experimentally contaminated with VACV. However, it is not known whether milk contaminated with VACV could be a route of viral transmission. However, anti-Orthopoxvirus antibodies were detected in humans from BV endemic areas, whom had no contact with affected cows, which suggest that other VACV transmission routes are possible, such as consumption of contaminated milk and dairy products. Therefore, it is important to study the possibility of VACV transmission by contaminated milk. This study aimed to examine VACV transmission, pathogenesis and shedding in mice orally inoculated with experimentally contaminated milk. Thirty mice were orally inoculated with milk containing 107 PFU/ml of VACV, and ten mice were orally inoculated with uncontaminated milk. Clinical examinations were performed for 30 consecutive days, and fecal samples and oral swabs (OSs) were collected every other day. Mice were euthanized on predetermined days, and tissue and blood samples were collected. Nested-PCR, plaque reduction neutralization test (PRNT), viral isolation, histopathology, and immunohistochemistry (IHC) methods were performed on the collected samples. No clinical changes were observed in the animals. Viral DNA was detected in feces, blood, OSs and tissues, at least in one of the times tested. The lungs displayed moderate to severe interstitial lymphohistiocytic infiltrates, and only the heart, tonsils, tongue, and stomach did not show immunostaining at the IHC analysis. Neutralizing antibodies were detected at the 20th and 30th days post infection in 50% of infected mice. The results revealed that VACV contaminated milk could be a route of viral transmission in mice experimentally infected, showing systemic distribution and shedding through feces and oral mucosa, albeit

  18. Role of CD8^+ T Cells in Murine Experimental Allergic Encephalomyelitis

    Jiang, Hong; Zhang, Sheng-Le; Pernis, Benvenuto


    The course of experimental allergic encephalomyelitis (EAE), an animal model for multiple sclerosis, is affected by immunoregulatory T lymphocytes. When animals are immunized with encephalitogenic peptide of myelin basic protein and recover from the first episode of EAE, they become resistant to a second induction of this disease. Animals depleted of CD8^+ T cells by antibody-mediated clearance were used to examine the role of CD8^+ T cells in EAE. These cells were found to be major participants in the resistance to a second induction of EAE but were not essential for spontaneous recovery from the first episode of the disease.

  19. Effect of piroxicam, metamizol, and S-adenosylmethionine in a murine model of experimental trichomoniasis

    Nogal-Ruiz J.J.


    Full Text Available Biological effects of piroxicam, metamizol, and S-adenosylmethionine (S-AMET have been tested in NMRI mice infected intraperitoneally with Trichomonas vaginalis. An intraperitoneal treatment during ten preinfection days with piroxicam (10 mg/Kg/day, or metamizol (275 mg/Kg/day, but not with S-AMET (17 mg/Kg/day induced a significant decrease of abdominal lesions and mortality, assessed by means of a pathogenicity index. The trichomonicidal activity of piroxicam, metamizol, and S-AMET was tested in vitro at the concentration of 300 μM, but found ineffective. These assays have shown the usefulness of the experimental trichomoniasis model for the study of the immunomodulating activity of synthetic drugs.

  20. Pomegranate (Punica granatum) peel is effective in a murine model of experimental Cryptosporidium parvum.

    Al-Mathal, Ebtisam M; Alsalem, Afaf M


    Cryptosporidiosis, a major health issue for neonatal calves, is caused by the parasite Cryptosporidium parvum, which is highly resistant to drug treatments. To date, many anti-parasitic drugs have been tested, but only a few have been shown to be partially effective in treating cryptosporidiosis. Previous studies have indicated that pomegranate (Punica granatum) possesses anti-plasmodium, anti-cestode, and anti-nematode activities. Therefore, the aim of this study was to evaluate the effect of P. granatum peel on suckling mice infected with experimental C. parvum. At 4days of age, 72 neonatal albino mice were randomly divided into five groups: G1: healthy controls, G2: infected/untreated controls, G3: uninfected/distilled water-treated, G4: uninfected/P. granatum peel-treated, and G5: infected/P. granatum peel-treated. Mice were experimentally-infected by oral administration of 1×10(3)C. parvum oocysts per animal. On day 7 post-inoculation (pi), treated mice received an aqueous suspension of P. granatum peel orally (3g/kg body weight). The presence of diarrhea, oocyst shedding, and weight gain/loss, and the histopathology of ileal sections were examined. Infected mice treated with the P. granatum peel suspension showed improvement in all parameters examined. Additionally, these mice did not exhibit any clinical symptoms and no deaths occurred. Oocyst shedding was very significantly reduced in the P. granatum-treated mice by day 14 pi (Parchitecture of villi from the P. granatum-treated mice on day 14 pi showed visible improvement in comparison with the infected/untreated controls, including renewed brush borders, reduced numbers of C. parvum trophozoites, and reduced lymphatic infiltration. On day 28 pi, tissues of the P. granatum-treated mice were very similar to those of healthy control mice. These results suggest that P. granatum peel is a promising anti-coccidial therapeutic treatment that lacks negative side effects.

  1. Genetic deletion of dectin-1 does not affect the course of murine experimental colitis

    Heinsbroek Sigrid EM


    Full Text Available Abstract Background It is believed that inflammatory bowel diseases (IBD result from an imbalance in the intestinal immune response towards the luminal microbiome. Dectin-1 is a widely expressed pattern recognition receptor that recognizes fungi and upon recognition it mediates cytokine responses and skewing of the adaptive immune system. Hence, dectin-1 may be involved in the pathogenesis of IBD. Methods We assessed the responses of dectin-1 deficient macrophages to the intestinal microbiota and determined the course of acute DSS and chronic Helicobacter hepaticus induced colitis in dectin-1 deficient mice. Results We show that the mouse intestinal microbiota contains fungi and the cytokine responses towards this microbiota were significantly reduced in dectin-1 deficient macrophages. However, in two different colitis models no significant differences in the course of inflammation were found in dectin-1 deficient mice compared to wild type mice. Conclusions Together our data suggest that, although at the immune cell level there is a difference in response towards the intestinal flora in dectin-1 deficient macrophages, during intestinal inflammation this response seems to be redundant since dectin-1 deficiency in mice does not affect intestinal inflammation in experimental colitis.

  2. Murine pattern recognition receptor dectin-1 is essential in the development of experimental autoimmune uveoretinitis.

    Stoppelkamp, Sandra; Reid, Delyth M; Yeoh, Joyce; Taylor, Julie; McKenzie, Emma J; Brown, Gordon D; Gordon, Siamon; Forrester, John V; Wong, Simon Y C


    Mycobacteria in complete Freund's adjuvant (CFA) are an essential component of immunization protocols in a number of autoimmune disease animal models including experimental autoimmune encephalomyelitis and uveoretinitis (EAE and EAU, respectively). We determined the role in EAU of two C-type lectin receptors on myeloid cells that recognize and respond to mycobacteria. Using receptor-specific antibodies and knockout mice, we demonstrated for the first time that the macrophage mannose receptor delays disease development but does not affect severity. In contrast, dectin-1 is critically involved in the development of CFA-mediated EAU. Disease severity is reduced in dectin-1 knockout mice and antibody blockade of dectin-1 during the induction, but not the effector phase, prevents EAU development. Significantly, similar blockade of dectin-1 in vivo has no effect in non-CFA-mediated, spontaneously induced or adoptive transfer models of EAU. Thus dectin-1 plays a critical role in the ability of complete Freund's adjuvant to induce EAU in mice.

  3. Notch signalling suppresses regulatory T-cell function in murine experimental autoimmune uveitis.

    Rong, Hua; Shen, Hongjie; Xu, Yueli; Yang, Hai


    Autoimmune uveitis is an intraocular inflammatory disorder in developed countries. Understanding the mechanisms underlying the development and modulation of immune reaction in uveitic eyes is critical for designing therapeutic interventions. Here we investigated the role of Notch signalling in regulatory T-cell (Treg cell) function during experimental autoimmune uveitis (EAU). Using the Foxp3-GFP reporter mouse strain, the significance of Notch signalling for the function of infiltrating Treg cells was characterized in an EAU model. We found that infiltrating Treg cells substantially expressed Notch-1, Notch-2, JAG1 and DLL1 in uveitic eyes. Activation of Notch signalling, represented by expression of HES1 and HES5, was enhanced in infiltrating Treg cells. Treatment with JAG1 and DLL1 down-regulated Foxp3 expression and immunosuppressive activity of isolated infiltrating Treg cells in vitro, whereas neutralizing antibodies against JAG1 and DLL1 diminished Notch ligand-mediated negative effects on Treg cells. To investigate the significance of Notch signalling for Treg cell function in vivo, lentivirus-derived Notch short hairpin RNAs were transduced into in vitro expanded Treg cells before adoptive transfer of Treg cells into EAU mice. Transfer of Notch-1-deficient Treg cells remarkably reduced pro-inflammatory cytokine production and inflammatory cell infiltration in uveitic eyes. Taken together, Notch signalling negatively modulates the immunosuppressive function of infiltrating Treg cells in mouse EAU.

  4. Efficacy of Lychnopholide Polymeric Nanocapsules after Oral and Intravenous Administration in Murine Experimental Chagas Disease.

    de Mello, Carlos Geraldo Campos; Branquinho, Renata Tupinambá; Oliveira, Maykon Tavares; Milagre, Matheus Marques; Saúde-Guimarães, Dênia Antunes; Mosqueira, Vanessa Carla Furtado; Lana, Marta de


    The etiological treatment of Chagas disease remains neglected. The compounds available show several limitations, mainly during the chronic phase. Lychnopholide encapsulated in polymeric nanocapsules (LYC-NC) was efficacious in mice infected with Trypanosoma cruzi and treated by intravenous administration during the acute phase (AP). As the oral route is preferred for treatment of chronic infections, such as Chagas disease, this study evaluated the use of oral LYC-NC in the AP and also compared it with LYC-NC administered to mice by the oral and intravenous routes during the chronic phase (CP). The therapeutic efficacy was evaluated by fresh blood examination, hemoculture, PCR, and enzyme-linked immunosorbent assay (ELISA). The cure rates in the AP and CP were 62.5% and 55.6%, respectively, upon oral administration of LYC-poly(d,l-lactide)-polyethylene glycol nanocapsules (LYC-PLA-PEG-NC) and 57.0% and 30.0%, respectively, with LYC-poly-ε-caprolactone nanocapsules (LYC-PCL-NC). These cure rates were significantly higher than that of free LYC, which did not cure any animals. LYC-NC formulations administered orally during the AP showed cure rates similar to that of benznidazole, but only LYC-NC cured mice in the CP. Similar results were achieved with intravenous treatment during the CP. The higher cure rates obtained with LYC loaded in PLA-PEG-NC may be due to the smaller particle size of these NC and the presence of PEG, which influence tissue diffusion and the controlled release of LYC. Furthermore, PLA-PEG-NC may improve the stability of the drug in the gastrointestinal tract. This work is the first report of cure of experimental Chagas disease via oral administration during the CP. These findings represent a new and important perspective for oral treatment of Chagas disease.

  5. An experimental study of artificial murine bladder reflex arc established by abdominal reflex

    WANG Jin-wu; ZHAO Yu-wu; HOU Chun-lin; NI Wei-feng; RUI Bi-yu; GUO Shang-chun; ZHENG Xian-you; DAI Ke-rong


    Background The neurogenic bladder dysfunction caused by spinal cord injury is difficult to treat clinically. The aim of this research was to establish an artificial bladder reflex arc in rats through abdominal reflex pathway above the level of spinal cord injury, reinnervate the neurogenic bladder and restore bladder micturition.Methods The outcome was achieved by intradural microanastomosis of the right T13 ventral root to S2 ventral root with autogenous nerve grafting, leaving the right T13 dorsal root intact. Long-term function of the reflex arc was assessed from nerve electrophysiological data and intravesical pressure tests during 8 months postoperation. Horseradish peroxidase (HRP) tracing was performed to observe the effectiveness of the artificial reflex.Results Single stimulus (3 mA, 0.3 ms pulses, 20 Hz, 5-second duration) on the right T13 dorsal root resulted in evoked action potentials, raised intravesical pressures and bladder smooth muscle, compound action potential recorded from the right vesical plexus before and after the spinal cord transaction injury between L5 and S4 segmental in 12 Sprague-Dawley rats. There were HRP labelled cells in T13 ventral horn on the experimental side and in the intermediolateral nucleus on both sides of the L6-S4 segments after HRP injection. There was no HRP labelled cell in T13 ventral horn on the control side.Conclusion Using the surviving somatic reflex above the level of spinal cord injury to reconstruct the bladder autonomous reflex arc by intradural microanastomosis of ventral root with a segment of autologous nerve grafting is practical in rats and may have clinical applications for humans.

  6. Systemic Inflammatory Effects of Traumatic Brain Injury, Femur Fracture, and Shock: An Experimental Murine Polytrauma Model

    C. Probst


    Full Text Available Objective. Despite broad research in neurotrauma and shock, little is known on systemic inflammatory effects of the clinically most relevant combined polytrauma. Experimental investigation in an animal model may provide relevant insight for therapeutic strategies. We describe the effects of a combined injury with respect to lymphocyte population and cytokine activation. Methods. 45 male C57BL/6J mice (mean weight 27 g were anesthetized with ketamine/xylazine. Animals were subjected to a weight drop closed traumatic brain injury (WD-TBI, a femoral fracture and hemorrhagic shock (FX-SH. Animals were subdivided into WD-TBI, FX-SH and combined trauma (CO-TX groups. Subjects were sacrificed at 96 h. Blood was analysed for cytokines and by flow cytometry for lymphocyte populations. Results. Mortality was 8%, 13% and 47% for FX-SH, WD-TBI and CO-TX groups (P<0.05. TNFα (11/13/139 for FX-SH/WD-TBI/CO-TX; P<0.05, CCL2 (78/96/227; P<0.05 and IL-6 (16/48/281; P=0.05 showed significant increases in the CO-TX group. Lymphocyte populations results for FX-SH, WD-TBI and CO-TX were: CD-4 (31/21/22; P= n.s., CD-8 (7/28/34, P<0.05, CD-4-CD-8 (11/12/18; P= n.s., CD-56 (36/7/8; P<0.05. Conclusion. This study shows that a combination of closed TBI and femur-fracture/ shock results in an increase of the humoral inflammation. More attention to combined injury models in inflammation research is indicated.

  7. Experimental parameters differentially affect the humoral response of the cholera-toxin-based murine model of food allergy

    Kroghsbo, S.; Christensen, Hanne Risager; Frøkiær, Hanne


    Background: Recent studies have developed a murine model of IgE-mediated food allergy based on oral coadministration of antigen and cholera toxin (CT) to establish a maximal response for studying immunopathogenic mechanisms and immunotherapeutic strategies. However, for studying subtle immunomodu......Background: Recent studies have developed a murine model of IgE-mediated food allergy based on oral coadministration of antigen and cholera toxin (CT) to establish a maximal response for studying immunopathogenic mechanisms and immunotherapeutic strategies. However, for studying subtle...

  8. Immunogenic multistage recombinant protein vaccine confers partial protection against experimental toxoplasmosis mimicking natural infection in murine model

    Yaprak Gedik


    To generate a protective vaccine against toxoplasmosis, multistage vaccines and usage of challenging models mimicking natural route of infection are critical cornerstones. In this study, we generated a BAG1 and GRA1 multistage vaccine that induced strong immune response in which the protection was not at anticipated level. In addition, the murine model was orally challenged with tissue cysts to mimic natural route of infection.

  9. Expression and Significance of NF-κB, IL-1β and COX-2 in the Murine Model of Estrogen-dependent Experimental Vulvovaginal Candidiasis

    Xue-rong CHEN; Ya-li LIU; Dun-zhen XIAO; Jun GAO


    Objective To investigate the possible role of estrogen in the pathogenesis of vulvovaginal candidiasis(VVC).Methods Estrogen-dependent experimental murine model of C. albicans vaginal infection was established by injecting subcutaneously with estradiol benzoate and then 5 × 106 stationary-phase C. albicans blastoconidia was inoculated intravaginally to mice (group EI),and other 3 groups were set up: estrogen-treated but not infected (group E) ;estrogen-untreated but infected (group Ⅰ);normal control (group C).The dynamic change of colony-forming unit (CFU) of cervivovaginal lavage fluid was observed. Vaginal tissues at different time points (d 2,d 4,d 7 and d 14) after inoculation of C.albicans were obtained.In situ hybridization staining was used to detect expression of on d 4 and d 7 (P<0.01).Conclusions In the murine model of estrogen-dependent experimental VVC,estrogen promotes the infection establishment by up-regulating expression of CO X-2 via activating NF-κB signal pathway,and the high expression of COX-2 promoted by the interaction of IL-1β and NF-κB after infection formation was involved in persistence of infection.

  10. NLRP3 inflammasome as a target of berberine in experimental murine liver injury: interference with P2X7 signalling.

    Vivoli, Elisa; Cappon, Andrea; Milani, Stefano; Piombanti, Benedetta; Provenzano, Angela; Novo, Erica; Masi, Alessio; Navari, Nadia; Narducci, Roberto; Mannaioni, Guido; Moneti, Gloriano; Oliveira, Claudia P; Parola, Maurizio; Marra, Fabio


    Berberine (BRB) is commonly used in herbal medicine, but its mechanisms of action are poorly understood. In the present study, we tested BRB in steatohepatitis induced by a methionine- and choline-deficient (MCD) diet, in acute acetaminophen intoxication and in cultured murine macrophages. BRB markedly improved parameters of liver injury and necroinflammation induced by the MCD diet, although increased mortality was observed by mechanisms independent of bacterial infections or plasma levels of BRB. The MCD diet induced up-regulation of all components of the NLRP3 (NACHT, LRR and PYD domain-containing protein 3) inflammasome, and increased hepatic levels of mature IL-1β (interleukin 1β). All of these parameters were significantly reduced in mice treated with BRB. In mice administered an acetaminophen overdose, a model dependent on inflammasome activation, BRB reduced mortality and ALT (alanine aminotransferase) elevation, and limited the expression of inflammasome components. In vitro, LPS (lipopolysaccharide)-induced activation of NLRP3 inflammasome in RAW264.7 murine macrophages was markedly decreased by pre-incubation with BRB. BRB significantly limited the activation of the purinergic receptor P2X7, involved in the late phases of inflammasome activation. Upon P2X7 knockdown, the ability of BRB to block LPS-induced secretion of IL-1β was lost. These data indicate that administration of BRB ameliorates inflammation and injury in two unrelated murine models of liver damage. We demonstrate for the first time that BRB interferes with activation of the NLRP3 inflammasome pathway in vivo and in vitro, through a mechanism based on interference with activation of P2X7, a purinergic receptor involved in inflammasome activation.

  11. Murine experimental autoimmune encephalomyelitis is diminished by treatment with the angiogenesis inhibitors B20-4.1.1 and angiostatin (K1-3.

    Carolyn J MacMillan

    Full Text Available Angiogenesis is the formation of new blood vessels form pre-existing vasculature whose contribution to inflammatory conditions of the Central Nervous System is being studied in order to generate novel therapeutic targets. This study is the first to investigate the impact of two particular angiogenesis inhibitors on murine Experimental Autoimmune Encephalomyelitis (EAE, an inflammatory disease that mimics aspects of the human disease Multiple Sclerosis. The inhibitors were chosen to reduce angiogenesis by complimentary means. Extrinsic factors were targeted with B20-4.1.1 through its ability to bind to murine Vascular Endothelial Growth Factor (VEGF. Vascular processes connected to angiogenesis were targeted directly with K(1-3, the first three kringle domains of angiostatin. Mice treated with B20-4.1.1 and K(1-3 from onset of signs had reduced clinical scores 18-21 days after EAE induction. Both agents suppressed spinal cord angiogenesis without effect on local VEGF expression. B20-4.1.1 reduced spinal cord vascular permeability while K(1-3 had no effect. T cell infiltration into the spinal cord at day 21 was unaffected by either treatment. B20-4.1.1 reduced peripheral T cell proliferation while K(1-3 had no effect. Lymphoid cells from treated mice produced reduced levels of the T helper-17 (Th-17 cell cytokine interleukin (IL-17 with no effect on the Th-1 cytokine interferon (IFN-γ or Th-2 cytokine IL-4. However, when both drugs were added in vitro to naive T cells or to antigen stimulated T cells from mice with untreated EAE they had no effect on proliferation or levels of IL-17 or IFN-γ. We conclude that these angiogenesis inhibitors mitigate EAE by both suppressing spinal cord angiogenesis and reducing peripheral T cell activation.

  12. Plecanatide and dolcanatide, novel guanylate cyclase-C agonists, ameliorate gastrointestinal inflammation in experimental models of murine colitis

    Kunwar; Shailubhai; Vaseem; Palejwala; Krishna; Priya; Arjunan; Sayali; Saykhedkar; Bradley; Nefsky; John; A; Foss; Stephen; Comiskey; Gary; S; Jacob; Scott; E; Plevy


    AIM: To evaluate the effect of orally administeredplecanatide or dolcanatide, analogs of uroguanylin, on amelioration of colitis in murine models.METHODS: The cyclic guanosine monophosphate(cG MP) stimulatory potency of plecanatide and dolcanatide was measured using a human colon carcinoma T84 cellbased assay. For animal studies all test agents were formulated in phosphate buffered saline. Sulfasalazine or 5-amino salicylic acid(5-ASA) served as positive controls. Effect of oral treatment with test agents on amelioration of acute colitis induced either by dextran sulfate sodium(DSS) in drinking water or by rectal instillation of trinitrobenzene sulfonic(TNBS) acid, was examined in BALB/c and/or BDF1 mice. Additionally, the effect of orally administered plecanatide on the spontaneous colitis in T-cell receptor alpha knockout(TCRα-/-) mice was also examined. Amelioration of colitis was assessed by monitoring severity of colitis, disease activity index and by histopathology. Frozen colon tissues were used to measure myeloperoxidase activity.RESULTS: Plecanatide and dolcanatide are structurally related analogs of uroguanylin, which is an endogenous ligand of guanylate cyclase-C(GC-C). As expected from the agonists of GC-C, both plecanatide and dolcanatide exhibited potent cG MP-stimulatory activity in T84 cells. Once-daily treatment by oral gavage with either of these analogs(0.05-0.5 mg/kg) ameliorated colitis in both DSS and TNBS-induced models of acute colitis, as assessed by body weight, reduction in colitis severity(P < 0.05) and disease activity index(P < 0.05). Amelioration of colitis by either of the drug candidates was comparable to that achieved by orally administered sulfasalazine or 5-ASA. Plecanatide also effectively ameliorated colitis in TCRα-/- mice, a model of spontaneous colitis. As dolcanatide exhibited higher resistance to proteolysis in simulated gastric and intestinal juices, it was selected for further studies. CONCLUSION: This is the first

  13. Does Carica papaya leaf-extract increase the platelet count? An experimental study in a murine model

    Susiji Wickramasinghe; Roshitha Nilmini Waduge


    Objective:To investigate the potential role of fresh Carica papaya (C. papaya) leaf extract on haematological and biochemical parameters and toxicological changes in a murine model. Methods: In total 36 mice were used for the trial. Fresh C. papaya leaf extract [0.2 mL (2 g)/mouse] was given only to the test group (18 mice). General behavior, clinical signs and feeding patterns were recorded. Blood and tissue samples were collected at intervals. Haematological parameters including platelet, red blood cell (RBC), white blood cell (WBC), packed cell volume (PCV), serum biochemistry including serum creatinine, serum glutamic-oxaloacetic transaminase (SGOT) and serum glutamic-pyruvic transaminase (SGPT) were determined. Organs for possible histopathological changes were examined. Results: Neither group exhibited alteration of behavior or reduction in food and water intake. Similarly, no significant changes in SGOT, SGPT and serum creatinine levels were detected in the test group. Histopathological organ changes were not observed in either group of mice except in three liver samples of the test group which had a mild focal necrosis. The platelet count (11.33±0.35)í105/µL (P=0.000 04) and the RBC count (7.97±0.61)í106/µL (P=0.000 03) were significantly increased in the test group compared to that of the controls. However, WBC count and PCV (%) values were not changed significantly in the test group. The platelet count in the test group started to increase significantly from Day 3 (3.4±0.18í105/µL), reaching almost a fourfold higher at Day 21 (11.3í105/µL), while it was 3.8í105/µL and 5.5í105/µL at Day 3 and Day 21 respectively in the control. Likewise, the RBC count in the test group increased from 6í106/µL to 9í106/ µL at Day 21 while it remained near constant in the control group (6í106/µL). Conclusions: Fresh C. papaya leaf extract significantly increased the platelet and RBC counts in the test group as compared to controls. Therefore, it is very

  14. Folate-targeted paclitaxel-conjugated polymeric micelles inhibits pulmonary metastatic hepatoma in experimental murine H22 metastasis models

    Zhang Y


    Full Text Available Yan Zhang,1 Hui Zhang,2 Wenbin Wu,2 Fuhong Zhang,3,4 Shi Liu,3 Rui Wang,3 Yingchun Sun,1 Ti Tong,1 Xiabin Jing3 1Department of Thoracic Surgery, The Second Hospital of Jilin University, Changchun, Jilin, People's Republic of China; 2Department of Thoracic Surgery, Xuzhou Central Hospital, Xuzhou, Jiangsu, People's Republic of China; 3State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, People's Republic of China; 4Department of Otolaryngology, The First Hospital of Lanzhou University, Lanzhou, Gansu, People's Republic of China Abstract: Hepatocellular carcinoma shows low response to most conventional chemotherapies; additionally, extrahepatic metastasis from hepatoma is considered refractory to conventional systemic chemotherapy. Target therapy is a promising strategy for advanced hepatoma; however, targeted accumulation and controlled release of therapeutic agents into the metastatic site is still a great challenge. Folic acid (FA and paclitaxel (PTX containing composite micelles (FA-M[PTX] were prepared by coassembling the FA polymer conjugate and PTX polymer conjugate. The main purpose of this study is to investigate the inhibitory efficacy of FA-M(PTX on the pulmonary metastasis of intravenously injected murine hepatoma 22 (H22 on BALB/c mice models. The lung metastatic burden of H22 were measured and tissues were analyzed by immunohistochemistry and histology (hematoxylin and eosin stain, followed by survival analysis. The results indicated that FA-M(PTX prevented pulmonary metastasis of H22, and the efficacy was stronger than pure PTX and simple PTX-conjugated micelles. In particular, the formation of lung metastasis colonies in mice was evidently inhibited, which was paralleled with the downregulated expression of matrix metalloproteinase-2 and matrix metalloproteinase-9. Furthermore, the mice bearing pulmonary metastatic hepatoma in the FA

  15. The Effect of Phosphatidylcholine and Deoxycholate Compound Injections to the Localized Adipose Tissue: An Experimental Study with a Murine Model

    Yongjoon Noh


    Full Text Available Background Phosphatidylcholine (PPC and deoxycholate (DCA compound has been recentlyused for the purpose of partial lipolysis and is valued for its efficacy and lower invasivenesscompared to liposuction and dermolipectomy used previously. In this article, the authors discussthe efficacy of the PPC dissolved in DCA via an experimental rat study model, along with suggestinga useful animal experimental model for the study of adipose tissue and lipolysis.Methods Bilateral inguinal fat pads of an experimental rat were elevated with the deep inferiorepigastric vessel as the sole vascular pedicle. Normal saline was injected on one side as acontrol group and a PPC and DCA compound was injected on the other side. After 4 days, therats were euthanized for microscopic tissue examination. The pathology was scored by a semiquantitativesystem in 4 categories: normal fat amount, fat necrosis, inflammatory activity,and stage of fibrosis. A Wilcoxon signed-rank test powered by SPSS packet program was usedfor statistical analysis and to determine significance.Results Microscopic examination was performed on the obtained samples, and theexperimental data of all four categories showed significant histologic differences compared tothe control group. All of the data also showed statistical significance by the Wilcoxon signedranktest (P<0.01.Conclusions In the inguinal fat pad rat model, the control group and the experimental grouphad a differed significantly in the amount of normal fat tissue, inflammation, necrosis, andfibrosis. We recommend the rat inguinal fat pad model used in this study, as it is likely to beuseful in related research.

  16. Murine Typhus

    Dzul-Rosado, Karla R; Zavala Velázquez, Jorge Ernesto; Zavala-Castro, Jorge


    Rickettsia typhi: is an intracellular bacteria who causes murine typhus. His importance is reflected in the high frequency founding specific antibodies against Rickettsia typhi in several worldwide seroepidemiological studies, the seroprevalence ranging between 3-36%. Natural reservoirs of R. typhi are rats (some species belonging the Rattus Genus) and fleas (Xenopsylla cheopis) are his vector. This infection is associated with overcrowding, pollution and poor hygiene. Typically presents fever, headache, rash on trunk and extremities, in some cases may occur organ-specific complications, affecting liver, kidney, lung or brain. Initially the disease is very similar to other diseases, is very common to confuse the murine typhus with Dengue fever, therefore, ignorance of the disease is a factor related to complications or non-specific treatments for the resolution of this infection. This paper presents the most relevant information to consider about the rickettsiosis caused by Rickettsia typhi. PMID:24893060

  17. Effect of Topical Administration of Fractions and Isolated Molecules from Plant Extracts on Skin Wound Healing: A Systematic Review of Murine Experimental Models

    Mariáurea Matias Sarandy


    Full Text Available Background and Purpose. Skin wound healing is a dynamic process driven by molecular events responsible for the morphofunctional repair of the injured tissue. In a systematic review, we analyzed the relevance of plant fractions and isolates on skin wound healing. By revising preclinical investigations with murine models, we investigated if the current evidence could support clinical trials. Methods. Studies were selected in the MEDLINE/PubMed and Scopus databases according to the PRISMA statement. All 32 identified studies were submitted to data extraction and the methodological bias was investigated according to ARRIVE strategy. Results. The studies demonstrated that plant fractions and isolates are able to modulate the inflammatory process during skin wound healing, being also effective in attenuating the oxidative tissue damage in the scar tissue and stimulating cell proliferation, neoangiogenesis, collagen synthesis, granulation tissue expansion, reepithelialization, and the wound closure rate. However, we identified serious methodological flaws in all studies, such as the high level of reporting bias and absence of standardized experimental designs, analytical methods, and outcome measures. Conclusion. Considering these limitations, the current evidence generated from flawed methodological animal studies makes it difficult to determine the relevance of herbal medicines to treat skin wounds and derails conducting clinical studies.

  18. An Insight into the Behavior, Course and Kinetics of Acute Infection of Toxoplasma gondii Human RH Strain in Experimentally Infected Murine Model.

    Vikrant Sudan


    Full Text Available Toxoplasma gondii, an apicomplexan parasite, is capable of infecting a broad range of intermediate warm-blooded hosts including humans. The parasite seems to be capable of altering the natural behavior of the host to favor its transmission in the environment. The aim of this study was to evaluate the course, alterations in behavior along with normal kinetics of the abnormally induced experimental acute toxoplasmosis in murine models.Ten Swiss albino mice were intraperitoneally inoculated with 100 virulent RH strain tachyzoites and finally, the alterations in behavior were described and compared with other known alterations in humans and animals.The behavior and the other symptoms of the acute toxoplasmosis were recorded. Such mice showed typical symptoms like normal coat, severe ascites with pendulous abdomen and tachypnoea exhibited by resting fore legs either on walls of the cage, or nozzle of water bottle or other resting mice and yielded a creamy colored cloudy natured peritoneal fluid on aspiration.Finally the alterations in behavior were described and compared with other known alterations in humans and animals. The study has generated some important data related to possible causes of behavioral alterations and generation of suitable strategies for control of these alterations in behavior vis-à-vis better understanding of the effect of acute infection of parasite on normal behavior of infected intermediate host.

  19. Effect of Topical Administration of Fractions and Isolated Molecules from Plant Extracts on Skin Wound Healing: A Systematic Review of Murine Experimental Models

    Lopes, Fernanda Barbosa; Pinto, Marcus Vinicius Mello; Sartori, Sirlene Souza Rodrigues


    Background and Purpose. Skin wound healing is a dynamic process driven by molecular events responsible for the morphofunctional repair of the injured tissue. In a systematic review, we analyzed the relevance of plant fractions and isolates on skin wound healing. By revising preclinical investigations with murine models, we investigated if the current evidence could support clinical trials. Methods. Studies were selected in the MEDLINE/PubMed and Scopus databases according to the PRISMA statement. All 32 identified studies were submitted to data extraction and the methodological bias was investigated according to ARRIVE strategy. Results. The studies demonstrated that plant fractions and isolates are able to modulate the inflammatory process during skin wound healing, being also effective in attenuating the oxidative tissue damage in the scar tissue and stimulating cell proliferation, neoangiogenesis, collagen synthesis, granulation tissue expansion, reepithelialization, and the wound closure rate. However, we identified serious methodological flaws in all studies, such as the high level of reporting bias and absence of standardized experimental designs, analytical methods, and outcome measures. Conclusion. Considering these limitations, the current evidence generated from flawed methodological animal studies makes it difficult to determine the relevance of herbal medicines to treat skin wounds and derails conducting clinical studies. PMID:27829707

  20. Atorvastatin treatment is effective when used in combination with mefloquine in an experimental cerebral malaria murine model

    Souraud Jean-Baptiste


    Full Text Available Abstract Background One of the major complications of Plasmodium falciparum infection is cerebral malaria (CM, which causes one million deaths worldwide each year, results in long-term neurological sequelae and the treatment for which is only partially effective. Statins are recognized to have an immunomodulatory action, attenuate sepsis and have a neuroprotective effect. Atorvastatin (AVA has shown in vitro anti-malarial activity and has improved the activity of mefloquine (MQ and quinine. Methods The efficiency of 40 mg/kg intraperitoneal AVA, alone or in association with MQ, was assessed in an experimental Plasmodium berghei ANKA rodent parasite model of CM and performed according to different therapeutic schemes. The effects on experimental CM were assessed through the evaluation of brain histopathological changes and neuronal apoptosis by TUNEL staining. Results AVA alone in the therapeutic scheme show no effect on survival, but the prophylactic scheme employing AVA associated with MQ, rather than MQ alone, led to a significant delay in mouse death and had an effect on the onset of CM symptoms and on the level of parasitaemia. Histopathological findings show a correlation between brain lesions and CM onset. A neuronal anti-apoptotic effect of AVA in the AVA + MQ combination was not shown. Conclusions The combination of AVA and MQ therapy led to a significant delay in mouse mortality. There were differences in the incidence, time to cerebral malaria and the level of parasitaemia when the drug combination was administered to mice. When used in combination with MQ, AVA had a relevant effect on the in vivo growth inhibition and clinical outcome of P. berghei ANKA-infected mice.

  1. The efficacy of hydro alcoholic extract of Seidlitzia rosmarinus on experimental zoonotic cutaneous leishmaniasis lesions in murine model

    Maryam Ahmadi


    Full Text Available Objective: Leishmaniasis is one of the most important parasitic infectious diseases in the world. Since last century, many efforts have been made to control and treat the disease, but appropriate vaccines, pesticides and medicines are not available or even eligible. The purpose of this study was to evaluate the effect of hydro-alcoholic extract of Seidlitzia rosmarinus on the lesions of experimental Cutaneous Leishmaniasis (CL in Balb/c mice. Materials and Methods: The population study was 60 Ballb/c mice which divided to 6 groups, all infected with Leishmania major [MRHO/75/IR]. Soon after the ulcer started to appear in the early stage, a dose of provided herbal extract with 5, 10 and 15% concentration applied on each lesion. The surface area of the lesions measured during an interval of 10 days. Direct Giemsa stained smears prepared two and four weeks after treatment. Results: Increasing the mean size of the lesions was statistically significant compared to those in control group (p>0.001. Visceral Leishmaniasis (VL developed in all of the mice including the control group that received Eucerine alone. Survival rate in group receiving 15% S. rosmarinus extracts showed significantly higher  compared to mice in control group (p

  2. The role of lipoprotein-associated phospholipase A2 in a murine model of experimental autoimmune uveoretinitis.

    G L Crawford

    Full Text Available Macrophage activation is, in part, regulated via hydrolysis of oxidised low density lipoproteins by Lipoprotein-Associated phospholipase A2 (Lp-PLA2, resulting in increased macrophage migration, pro-inflammatory cytokine release and chemokine expression. In uveitis, tissue damage is mediated as a result of macrophage activation; hence inhibition of Lp-PLA2 may limit macrophage activation and protect the tissue. Utilising Lp-PLA2 gene-deficient (KO mice and a pharmacological inhibitor of Lp-PLA2 (SB-435495 we aimed to determine the effect of Lp-PLA2 suppression in mediating retinal protection in a model of autoimmune retinal inflammation, experimental autoimmune uveoretinitis (EAU. Following immunisation with RBP-3 (IRBP 1-20 or 161-180 peptides, clinical disease was monitored and severity assessed, infiltrating leukocytes were enumerated by flow cytometry and tissue destruction quantified by histology. Despite ablation of Lp-PLA2 enzyme activity in Lp-PLA2 KO mice or wild-type mice treated with SB-435495, the number of infiltrating CD45+ cells in the retina was equivalent to control EAU animals, and there was no reduction in disease severity. Thus, despite the reported beneficial effects of therapeutic Lp-PLA2 depletion in a variety of vascular inflammatory conditions, we were unable to attenuate disease, show delayed disease onset or prevent progression of EAU in Lp-PLA2 KO mice. Although EAU exhibits inflammatory vasculopathy there is no overt defect in lipid metabolism and given the lack of effect following Lp-PLA2 suppression, these data support the hypothesis that sub-acute autoimmune inflammatory disease progresses independently of Lp-PLA2 activity.

  3. Cáncer experimental e inflamación sistémica en un modelo murino Systemic inflammation and experimental cancer in a murine model

    Juan Bruzzo


    both animals and human beings. In contrast, the relationship between cancer and systemic inflammation has been less studied. In this work, we demonstrated that the growth of the murine fibrosarcoma MC-C, was accompanied by manifestations of systemic inflammation, as demonstrated by an increase in both the number of circulating polymorphonuclear neutrophils (PMN and the serum concentration of the proinflammatory cytokines interleukin-1β (IL-1β, interleukin-6 (IL-6 and tumor necrosis factor-α (TNF-α and the acute phase proteins C reactive (CRP and serum A amyloid (SAA. Two temporally separate peaks of systemic inflammation were detected during tumor development. The first was displayed during the first week after tumor inoculation. The second peak began around day 14 and its intensity was proportional to tumor size. In mice bearing a large MC-C tumor, a high number of circulating PMN and myeloid precursors were evident. Most of these cells exhibited activation evidenced by an increased reactive oxygen species generation and high expression of the Gr1+/Mac1+ markers. Inoculation of thioglycolate -which generates a transient systemic inflammation- accelerated the growth of MC-C tumor and reciprocally, inhibition of such systemic inflammation by using indomethacin, prevented that enhancing effect. This suggests that the systemic inflammation that the tumor generates on its own, could be part of its growth strategy.

  4. Therapeutic immunization with radio-attenuated Leishmania parasites through i.m. route revealed protection against the experimental murine visceral leishmaniasis.

    Datta, Sanchita; Manna, Madhumita; Khanra, Supriya; Ghosh, Moumita; Bhar, Radhaballav; Chakraborty, Anindita; Roy, Syamal


    After our promising results from prophylactic and therapeutic study (i.p. route) with the radio-attenuated Leishmania donovani parasites against experimental murine visceral leishmaniasis, we prompted to check their therapeutic efficacy through i.m route. BALB/c mice were infected with highly virulent L. donovani parasites. After 75 days, mice were treated with gamma (γ)-irradiated parasites. A second therapeutic immunization was given after 15 days of first immunization. The protection against kala-azar was estimated with the reduction of Leishman-Donovan unit from spleen and liver that scored up to 80% and 93%, respectively, while a twofold increase in nitric oxide (NO) and reactive oxygen species (ROS) productions has been observed in the immunized groups of animals. These groups of mice also showed disease regression by skewing Th2 cytokines (IL-10) towards Th1 cytokine (IFN-γ) bias along with the increased generation of NO and ROS, while the infected control group of mice without such treatment surrendered to the disease. Establishment of Th1 ambience in the treated groups has also been supported from the measured antileishmanial antibody IgG subsets (IgG2a and IgG1) with higher anti-soluble Leishmania antigen-specific IgG2a titer. As seen in our previous studies, doses of attenuation by γ-radiation should be taken into serious consideration. Attenuation of parasites at 50 Gy of absorbed dose of gamma rays has not worked well. Thus, therapeutic use of L. donovani parasites radio-attenuated at particular doses can be exploited as a promising vaccine agent. Absence of any adjuvant may increase its acceptability as vaccine candidate further.

  5. Efficacy of amoxycillin-clavulanate in an experimental model of murine pneumonia caused by AmpC-non-hyperproducing clinical isolates of Escherichia coli resistant to cefoxitin.

    Docobo-Pérez, F; Fernández-Cuenca, F; Pachón-Ibáñez, M E; Pascual, A; Pichardo, C; Martínez-Martínez, L; Pachón, J


    The algorithms included in most automated systems used for antimicrobial susceptibility testing (e.g., Vitek 2) consider that Escherichia coli isolates resistant to cefoxitin are AmpC-hyperproducers and, consequently, resistant also to amoxycillin-clavulanate. However, a recent study revealed that 30% of E. coli clinical isolates resistant to cefoxitin remained susceptible in vitro to amoxycillin-clavulanate. The aim of the present study was to evaluate the in-vivo efficacy of amoxycillin-clavulanate in the treatment of an experimental model of pneumonia, using two clonally related isolates (with identical repetitive extragenic palindromic sequence (REP)-PCR patterns) of AmpC-non-hyperproducing and OmpF-lacking E. coli (Ec985 and Ec571) that were resistant to cefoxitin and susceptible to cefotaxime and amoxycillin-clavulanate. MICs were determined using a microdilution technique, and in-vitro bactericidal activity was tested using time-kill assays. The in-vivo efficacy of amoxycillin, amoxycillin-clavulanate and cefotaxime against both isolates was tested in a murine pneumonia model using immunocompetent C57BL/6 mice. Ec571 (a TEM-1/2 producer) was resistant to amoxycillin, whereas Ec985 (a TEM-1/2 non-producer) was susceptible. Amoxycillin, amoxycillin-clavulanate and cefotaxime were bactericidal for Ec985, and amoxycillin-clavulanate and cefotaxime were bactericidal for Ec571 at different concentrations and time-points, as determined using time-kill assays. Treatment with amoxycillin, amoxycillin-clavulanate and cefotaxime reduced the bacterial lung concentration of Ec985 compared with non-treated controls (p AmpC-non-hyperproducing strains of E. coli resistant to cefoxitin.

  6. Efficacy of High-Dose Meropenem (Six Grams per Day) in Treatment of Experimental Murine Pneumonia Induced by Meropenem-Resistant Pseudomonas aeruginosa.

    Oshima, Kazuhiro; Nakamura, Shigeki; Iwanaga, Naoki; Takemoto, Koji; Miyazaki, Taiga; Yanagihara, Kastunori; Miyazaki, Yoshitsugu; Mukae, Hiroshi; Kohno, Shigeru; Izumikawa, Koichi


    High-dose meropenem (MEPM; 6 g/day) has been approved as a treatment for purulent meningitis; however, little is known regarding its in vivo efficacy in refractory lower respiratory tract infections. The purpose of this study was to evaluate the efficacy of MEPM at 6 g/day in a murine model of severe pneumonia caused by MEPM-resistant Pseudomonas aeruginosa Experimental pneumonia induced by MEPM-resistant P. aeruginosa was treated with normal-dose MEPM (150 mg/kg of body weight, simulating a 3-g/day regimen in humans) or high-dose MEPM (500 mg/kg, simulating a 6-g/day regimen in humans). Mice treated with high-dose MEPM showed significantly restored survival relative to that of untreated mice and tended to show a survival rate higher than that of mice treated with normal-dose MEPM. The viable bacterial counts (of two clinical isolates) in the lungs decreased significantly in mice treated with high-dose MEPM from those for untreated mice (P high-dose MEPM than in untreated mice. The free MEPM concentration in the epithelial lining fluid (ELF) exceeded 16 μg/ml for 85 min in mice treated with high-dose MEPM, but not for mice treated with normal-dose MEPM. Our results demonstrate that high-dose MEPM (6 g/day) might provide better protection against pneumonia caused by MEPM-resistant strains of P. aeruginosa than the dose normally administered (less than 3 g/day).

  7. Cholestasis in a murine experimental model: lesions include hepatocyte ischemic necrosis Colestase em modelo experimental em murinos: lesões incluem necrose isquêmica dos hepatócitos

    Ivete Bedin Prado


    Full Text Available OBJECTIVE: To establish a murine experimental model of bile duct obstruction that would enable controlled observations of the acute and subacute phases of cholestasis. METHODOLOGY: Adult male isogenic BALB/c mice underwent a bile duct ligation (22 animals or a sham operation (10 animals. Fifteen days after surgery, or immediately after the animal's death, macroscopic findings were noted and histological study of the liver, biliary tree, and pancreas was performed (hematoxylin-eosin and Masson trichromic staining. RESULTS: Beginning 24 hours after surgery, all animals from the bile duct ligation group presented progressive generalized malaise. All animals presented jaundice in the parietal and visceral peritoneum, turgid and enlarged liver, and accentuated dilatation of gallbladder and common bile duct. Microscopic findings included marked dilatation and proliferation of bile ducts with accentuated collagen deposits, frequent areas of ischemic necrosis, hepatic microabscesses, and purulent cholangitis. Animals from the sham operation group presented no alterations. CONCLUSION: We established a murine experimental model of induced cholestasis, which made it possible to study acute and subacute tissue lesions. Our data suggests that in cholestasis, hepatic functional ischemia plays an important role in inducing hepatic lesions, and it also suggests that the infectious process is an important factor in morbidity and mortality.OBJETIVO: Realizar um modelo experimental de obstrução do ducto biliar que permita uma observação controlada das fases aguda e subaguda da colestase. MÉTODOS: Submeteram-se camundongos BALB/c, adultos, machos, a ligadura do ducto biliar (22 animais ou a cirurgia-controle (10 animais. Quinze dias após a cirurgia, ou imediatamente após a morte do animal, foram observados os achados macroscópicos e realizado o estudo histológico do fígado, árvore biliar e pâncreas (haematoxylina-eosina e tricrômico de Masson




    The aim of this study was to determine how stem cell factor (SCF) modifies hemopoietic cell production. First we determined the effects of a prolonged SCP administration on murine hemopoiesis and analyzed the results by a mathematical simulation model of hemopoiesis in order to explain the data. Sub

  9. 沙利度胺治疗肝癌的实验研究%Experimental study thalidomide for treatment of murine hepatocellular carcinoma

    杨义明; 杜钢军; 林海红


    Objective To study the therapeutic effect of thalidomide(Tha) on murine hepatocellular carcinoma. Methods In murine transplanted hepatoma model, thalidomide was administered intragastrically alone (200 mg/kg daily for 10 days) or in combination with doxorubicin. The antitumor activity of Tha was observed in solid and ascitic tumor models. Results Tha induced significant growth inhibition of solid hepatoma without obvious toxicity on peripheral blood cells and lymphocyte proliferation. Although Tha alone had no effect on the survival of mice with ascitic tumor, it showed a synergistic antitumor activity in combination with doxorubicin (Dox) in both solid and ascitic tumor models. Moreover, Tha reduced Dox-induced cytopenia and immunosuppression. Histological analysis of Tha-treated tumors revealed remarkably enhanced tumor necrosis and lymphocyte infiltration on the edge of tumor tissues. Conclusion Tha has definite therapeutic effect on murine hepatoma,and the combination with Dox shows an enhanced therapeutic potential.%目的研究沙利度胺对肝癌的治疗作用.方法采用小鼠肝癌移植性模型,观察沙利度胺对实体型和腹水型肿瘤的治疗作用.结果沙利度胺按每日200mg/kg连续给药10 d,能明显抑制肝癌实体型肿瘤的生长,不降低小鼠血细胞数及淋巴细胞增殖;对腹水型肿瘤小鼠虽无明显生命延长作用,但沙利度胺与阿霉素联合用药对肝癌实体型及腹水型均有协调抗肿瘤作用,且可阻止阿霉素造成的小鼠血细胞减少、免疫功能降低.沙利度胺日剂量200mg/kg能明显增加肿瘤组织坏死,促进肿瘤组织边缘淋巴细胞侵润.结论沙利度胺对小鼠肝癌有确切治疗作用,与阿霉素联合用药效果更好.

  10. [Protective activity of S-PT84, a heat-killed preparation of Lactobacillus pentosus, against oral and gastric candidiasis in an experimental murine model].

    Hayama, Kazumi; Ishijima, Sanae; Ono, Yoshiko; Izumo, Takayuki; Ida, Masayuki; Shibata, Hiroshi; Abe, Shigeru


    The effect of S-PT84, a heat-killed preparation of Lactobacillus pentosus on growth of Candida albicans was examined in vitro and in vivo. The mycelial growth was effectively inhibited by S-PT84 and seemed to bind to the hyphae. We assessed the potential of S-PT84 for treatment of oral and gastric candidiasis using a murine model. When 2 mg of S-PT84 was administered three times into the oral cavity of orally Candida infected mice, the score of lesions on the tongue was improved on day 2. When 50 μl and 200 μl of S-PT84 (10 mg/ml) were administered three times into the oral cavity (0.5 mg × 3) and the stomach (2 mg × 3) of the same mouse model, the number of viable Candida cells in the stomach was reduced significantly on day 2. These findings suggest the possibility that S-PT84 has potential as a food ingredient supporting anti-Candida treatment, especially for Candida infection in the gastrointestinal tract.

  11. Experimental study on therapeutic effect of in vivo expression of Cell I-Hep II recombinant polypeptide of fibronectin on murine H22 hepatocellular carcinoma

    Gui-Mei Zhang; Yan Yang; Bo Huang; Hui Xiao; Dong Li; Zuo-Hua Feng


    AIM: To investigate the inhibitory effect of in vivo expression of expressing plasmid pCH510 of recombinant fibronectin polypeptide (CH50) on hepatocellular carcinoma and the improved therapeutic effect of pCH510 in combination with chemotherapeutic agents and Hsp70-H22 hepatocarcinoma antigen peptide on tumor.METHODS: Mice were inoculated with H22 hepatocarcinoma ceils. The chemotactic effect of the expression of plasmid pCH510 on immunocytes was observed after in vivo transfection, tissue slicing and HE staining. Inhibitory effect of transfection with pCH510 on murine tumor originated from different inoculative doses was observed. The inhibitory effect of immediate transfection with pCH510after chemotherapy on tumor was compared with that of transfection 5 days after chemotherapy. The change of function and amount of mouse peritoneal macrophages and the peripheral blood immunocytes resulted from administration of chemotherapeutic agents were detected. The peptides mixture was prepared from H22hepatocarcinoma cells. pCH510 + Hsp70-H22 antigen peptides were injected into tumor-bearing mice with or without chemotherapy, to observe the inhibitory effects on tumor.RESULTS: At the tumor tissue site injected with pCH510,there were a great number of immunocytes which mainly were macrophages, lymphocytes and neutrophils.Transfection of plasmid pCH510 inhibited significantly the murine tumor induced by different inoculative doses. The inhibitory effect was negatively correlated with the inoculative dose. The therapeutic effect was not improved by immediate transfection with pCH510 after chemotherapy, but was significantly improved by transfection with pCH510 5 days after chemotherapy. Chemotherapeutic agent decreased the number of immunocytes and suppressed their activation in vivo. After injection of drug, the amount of immunocytes was the lowest from d 1 to d 3 and returned to normal level on the 10th day. Transfection with plasmid pCH510 alone could inhibit tumor

  12. Lung inflammation and epithelial changes in a murine model of atopic asthma.

    Blyth, D I; Pedrick, M S; Savage, T J; Hessel, E M; Fattah, D


    A murine model of allergen-induced airway inflammation and epithelial phenotypic change, and the time-courses of these events, are described. Mice were sensitized to ovalbumin using an adjuvant-free protocol, and challenged by multiple intratracheal instillations of ovalbumin by a non-surgical technique. Many of the characteristic features of human atopic asthma were seen in the mice. A marked eosinophilic infiltration of lung tissue and airways followed allergen challenge, and its severity increased with each challenge, as did the number of eosinophils in the blood. Lymphocytes, neutrophils, and monocytes also invaded the lungs. Airway macrophages showed signs of activation, their appearance resembling those recovered from antigen-challenged human asthmatic airways. The airway epithelium was thickened and displayed a marked goblet cell hyperplasia in terminal bronchioles and larger airways. After repeated challenges, the reticular layer beneath the basement membrane of the airway epithelium showed fibrosis, reproducing a commonly observed histologic feature of human asthma. Goblet cell hyperplasia began to appear before eosinophils or lymphocytes had migrated across the airway epithelium, and persisted for at least 11 days after the third intratracheal challenge with ovalbumin, despite the number of inflammatory cells in the lungs and airways having decreased to near-normal levels by 4 days. Plugs of mucus occluded some of the airways. These results indicate that some of the phenotypic changes in airway epithelium that follow an allergic response in the lung can be initiated before the migration of eosinophils or lymphocytes across the epithelial layer.

  13. Suppression of NF-κB signaling and NLRP3 inflammasome activation in macrophages is responsible for the amelioration of experimental murine colitis by the natural compound fraxinellone

    Wu, Xue-Feng; Ouyang, Zi-Jun; Feng, Li-Li; Chen, Gong; Guo, Wen-Jie; Shen, Yan; Wu, Xu-Dong; Sun, Yang, E-mail:; Xu, Qiang, E-mail:


    Inflammatory bowel disease (IBD) affects millions of people worldwide. Although the etiology of this disease is uncertain, accumulating evidence indicates a key role for the activated mucosal immune system. In the present study, we examined the effects of the natural compound fraxinellone on dextran sulfate sodium (DSS)-induced colitis in mice, an animal model that mimics IBD. Treatment with fraxinellone significantly reduced weight loss and diarrhea in mice and alleviated the macroscopic and microscopic signs of the disease. In addition, the activities of myeloperoxidase and alkaline phosphatase were markedly suppressed, while the levels of glutathione were increased in colitis tissues following fraxinellone treatment. This compound also decreased the colonic levels of interleukin (IL)-1β, IL-6, IL-18 and tumor necrosis factor (TNF)-α in a concentration-dependent manner. These effects of fraxinellone in mice with experimental colitis were attributed to its inhibition of CD11b{sup +} macrophage infiltration. The mRNA levels of macrophage-related molecules in the colon, including intercellular adhesion molecule 1 (ICAM1), vascular cell adhesion molecule 1 (VCAM1), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX2), were also markedly inhibited following fraxinellone treatment. The results from in vitro assays showed that fraxinellone significantly reduced lipopolysaccharide (LPS)-induced production of nitric oxide (NO), IL-1β and IL-18 as well as the activity of iNOS in both THP-1 cells and mouse primary peritoneal macrophages. The mechanisms responsible for these effects were attributed to the inhibitory role of fraxinellone in NF-κB signaling and NLRP3 inflammasome activation. Overall, our results support fraxinellone as a novel drug candidate in the treatment of colonic inflammation. - Highlights: • Fraxinellone, a lactone compound, alleviated DSS induced colitis. • The effects of fraxinellone were attributed to its inhibition on

  14. Evaluation of three recombinant multi-antigenic vaccines composed of surface and secretory antigens of Toxoplasma gondii in murine models of experimental toxoplasmosis.

    Dziadek, Bozena; Gatkowska, Justyna; Brzostek, Anna; Dziadek, Jaroslaw; Dzitko, Katarzyna; Grzybowski, Marcin; Dlugonska, Henryka


    The great clinical and economical impact of Toxoplasma gondii infections makes the development of an effective vaccine for controlling toxoplasmosis an extremely important aim. In the presented study, we evaluate the protective and immunogenic properties of three recombinant subunit vaccines composed of rROP2+rGRA4+rSAG1, rROP2+rROP4+rGRA4 and rROP2+rROP4+rSAG1 proteins of T. gondii in an experimental toxoplasmosis model in the C3H/HeJ and C57BL/6 mouse strains. All three recombinant vaccines induced partial protection as measured by the reduction of brain cyst burden following challenge with five tissue cysts of the low virulence DX T. gondii strain. The level of protection was dependent on the antigen composition of the vaccine and the genetic background of the laboratory animals. The strongest protection against chronic toxoplasmosis was induced in both C3H/HeJ and C57BL/6 mice by the mixture of rhoptry proteins rROP2 and rROP4 combined with tachyzoite major protein rSAG1. The average parasite burden in these groups of mice was reduced by 71% and 90%, respectively, compared to non-vaccinated mice. The observed protective effect was related to the vaccine-induced cellular and humoral immune responses, as measured by the antigen-induced release of the Th1 cytokines IFN-γ and IL-2, the antigen-stimulated proliferation of spleen cells of vaccinated animals in comparison to control animals and the development of systemic antigen-specific IgG1 and IgG2a (C3H/HeJ) or IgG2c (C57BL/6) antibodies. Our studies show that recombinant rROP2, rROP4, rGRA4 and rSAG1 antigens may be promising candidates for a subunit vaccine against toxoplasmosis. Additionally, we demonstrate that the ideal composition of vaccine antigens can be equally effective in mice with different genetic backgrounds and variable levels of innate resistance to toxoplasmosis, resulting in strong protection against T. gondii invasion.

  15. Local immunotherapy in experimental murine lung inflammation



    Authors: Caroline Uebel, Sonja Koch, Anja Maier, Nina Sopel, Anna Graser, Stephanie Mousset & Susetta Finotto ### Abstract Innovative local immunotherapy for severe lung diseases such as asthma, chronic obstructive pulmonary disease or lung cancer requires a successful delivery to access the desired cellular target in the lung. An important route is the direct instillation into the airways in contrast to delivery through the digestive tract. This protocol details a method to deliv...

  16. Effects of FVIII immunity on hepatocyte and hematopoietic stem cell-directed gene therapy of murine hemophilia A.

    Lytle, Allison M; Brown, Harrison C; Paik, Na Yoon; Knight, Kristopher A; Wright, J Fraser; Spencer, H Trent; Doering, Christopher B


    Immune responses to coagulation factors VIII (FVIII) and IX (FIX) represent primary obstacles to hemophilia treatment. Previously, we showed that hematopoietic stem cell (HSC) retroviral gene therapy induces immune nonresponsiveness to FVIII in both naive and preimmunized murine hemophilia A settings. Liver-directed adeno-associated viral (AAV)-FIX vector gene transfer achieved similar results in preclinical hemophilia B models. However, as clinical immune responses to FVIII and FIX differ, we investigated the ability of liver-directed AAV-FVIII gene therapy to affect FVIII immunity in hemophilia A mice. Both FVIII naive and preimmunized mice were administered recombinant AAV8 encoding a liver-directed bioengineered FVIII expression cassette. Naive animals receiving high or mid-doses subsequently achieved near normal FVIII activity levels. However, challenge with adjuvant-free recombinant FVIII induced loss of FVIII activity and anti-FVIII antibodies in mid-dose, but not high-dose AAV or HSC lentiviral (LV) vector gene therapy cohorts. Furthermore, unlike what was shown previously for FIX gene transfer, AAV-FVIII administration to hemophilia A inhibitor mice conferred no effect on anti-FVIII antibody or inhibitory titers. These data suggest that functional differences exist in the immune modulation achieved to FVIII or FIX in hemophilia mice by gene therapy approaches incorporating liver-directed AAV vectors or HSC-directed LV.

  17. Experimental study on regulating the influence of quantum dots on the survival of murine embryo stem cells%调控量子点对小鼠胚胎干细胞活性影响的研究

    阮静; 沈洁; 汪铮; 崔大祥


    Objective: To regulate the influence of quantum dots on the survival of murine embryo stem cells. Methods: We prepared the quantum dots by using microwave synthesis method; labeled the murine embryo stem cells in vitro with quantum dots; analyzed the influence of quantum dots on the murine embryo stem cells by using confocal microscopy and flow eytometer, such as labeling rate, viability and proliferation capability; regulated the viability and proliferation capability of murine embryo stem cells by adding ROCK inhibitor Y-27632. Results: Murine embryo stem cells had 92.5% labeled rate and 40.7% survival rate after cultured with 20 nmol· L-1 quantum dots for 24 hours, murine embryo stem cells had 20% survival rate of growth on the same culture condition after regulated by 10 μmol· L-1 Y-27632. Conclusion: The more concentration of quantum dots treated, the higher rate of murine embryo stem cells being labeled and the lower rate of murine embryo stem cells surviving. ROCK inhibitor Y-27632 could prevent murine embryo stem cells from apoptosis and increase murine embryo stem cells' bioactivity.%目的:探讨调控量子点对小鼠胚胎干细胞活性的影响.方法:通过微波合成法制备荧光性能优越的量子点,并在体外对小鼠胚胎干细胞进行标记,采用激光共聚焦显微镜和流式细胞仪观察分析量子点对小鼠胚胎干细胞标记率、活力、增殖等方面的影响,并通过ROCK抑制剂Y-27632调控小鼠胚胎干细胞的活力和增殖能力.结果:用20 nmol·L-1的量子点标记小鼠胚胎干细胞培养24 h后,小鼠胚胎干细胞的标记率达到92.5%,存活率只有40.7%;使用10 μmol·L-1 Y-27632调控量子点标记的小鼠胚胎干细胞培养24 h后,小鼠胚胎干细胞的存活率提高20%.结论:量子点的浓度越大,小鼠胚胎干细胞的标记率越高,成活率越低,添加Y-27632可以阻止量子点标记的胚胎干细胞的凋亡,明显增加胚胎干细胞的生物活性.

  18. Movimentação dentária experimental em murinos: período de observação e plano dos cortes microscópicos Experimental tooth movement in murines: study period and direction of microscopic sections

    Ana Carolina Cuzzuol Fracalossi


    Full Text Available OBJETIVO: este trabalho tem por finalidade explicitar aspectos microscópicos relevantes da movimentação dentária induzida em murinos quanto aos: (1 diferentes períodos de observação e (2 planos dos cortes microscópicos transversais e longitudinais. Os estudos experimentais sobre a movimentação dentária induzida em murinos variam quanto aos períodos e planos de cortes microscópicos, mesmo os trabalhos que utilizam especificamente o modelo de Heller e Nanda, de 1979. Para contribuir (1 na escolha do melhor design experimental de movimentação dentária induzida em murinos em futuros trabalhos, e (2 no aperfeiçoamento dos critérios de análise por outros pesquisadores, propusemo-nos a publicar este artigo. MÉTODOS: Empregaram-se 50 ratos machos Wistar, com 90 dias de vida, submetidos à movimentação dentária induzida por períodos de 3, 5, 7 e 9 dias. Utilizou-se movimento de inclinação mesial no primeiro molar superior esquerdo, com uma força equivalente a 75cN. A análise microscópica qualitativa avaliou os fenômenos teciduais e celulares decorrentes da movimentação dentária induzida, nos diferentes períodos de observação e entre os cortes microscópicos transversais e longitudinais. RESULTADOS: dos fenômenos observados, as áreas hialinas tiveram expressão máxima no período de 5 dias e as reabsorções radiculares apresentaram-se exuberantes e bem demarcadas no período de 9 dias. Nos dois fenômenos, as raízes mais afetadas foram as distais, especialmente a raiz distovestibular. CONCLUSÃO: mediante o delineamento proposto, pode-se sugerir, para futuros trabalhos nesta linha de pesquisa, períodos de análise de 5 a 9 dias e cortes microscópicos transversais.AIM: This study aims to elucidate the relevant microscopic aspects of induced tooth movement in murines with regard to: (1 different study periods; and (2 transverse and longitudinal directions of microscopic sections. Experimental studies on induced

  19. "肤敏"膏抑制小鼠变应性接触性皮炎机制的研究%Experimental Study on "Fu Min" Gao Inhibiting Allergic Contact Dermatitis in Murine Model

    李静平; 李庆生; 李杰


    目的:研究"肤敏"膏滋剂对小鼠变应性接触性皮炎(ACD)的抑制作用,探讨其作用机理.方法:以DNFB和FITC分别建立小鼠ACD模型.采用ELISA法检测ACD小鼠产生干扰素-γ (IFN-γ)、肿瘤坏死因子-α(TNF-α)、白介素4和6(IL-4、IL-6)的水平及"肤敏"膏对4种细胞因子的作用.结果:口服不同剂量"肤敏"膏对4种细胞因子均有显著或不同程度的抑制作用(P<0.05或P<0.01).结论:"肤敏"膏对小鼠ACD疗效可能通过抑制Th1/Th2细胞因子而发挥作用.%Objective: To investigate the mechanism of "Fu Min" Gao inhibiting allergic contact dermatitis (ACD) in murine model. Methods:The murine model of allergic contact dermatitis by topical DNCB and FITC were used. After oral administration of "Fu Min" Gao in different doses, The levels of IFN - γ,TNF - α, IL - 4 and IL - 6 in the serum of these mice were detected by enzyme- linked immunosorbent assay (ELISA). Result: Different doses of "Fu Min" Gao inhibited the expression of these four cytokines to different degrees. ( P <0.05 or P < 0.01 ). Conclusion: The therapeutic effect of "Fu Min" Gao on murine ACD may be played by inhibiting the expression of some cytokines of helper T lymphocyte.

  20. Análisis de la resistencia inmune en un modelo murino experimental alimentado con dietas lipídicas e infectado con Listeria monocytogenes Analysis of the immune resistance in an experimental murine model fed dietary lipids and infected with Listeria monocytogenes

    M.ª A. Puertollano


    infectious diseases. The purpose of the present study was to determinate the immune status of mice fed dietary lipids and experimentally infected with a virulent strain of Listeria monocytogenes. Balb/c mice were divided into four groups and were fed with their respective diet: low fat diet (LF, 20%, olive oil diet (OO, 20%, fish oil diet (FO, 20% and hydrogenated coconut oil (HCO, 20%. Mice were fed for four weeks and infected with L. monocytogenes by endovenous route. Results have shown a survival reduction in mice fed a diet containing FO, as well as a significant increase in the number of viable bacteria from spleen. In addition, we have observed an increase in the bactericidal activity in peritoneal cells from OO group, although the invasion of L. monocytogenes in cells from this group was larger. Finally, a significant reduction of lymphocyte proliferation was observed in the group fed an FO diet, whereas natural killer (NK cell activity was not modified. These results indicate that dietary lipids constituted by polyunsaturated n-3 fatty acids reduce the murine immune resistance, whereas a diet constituted by OO-does not exert an immunosuppressor effect as relevant as FO diet, and it does not reduce the immune resistance leading to an efficient L. monocytogenes elimination.

  1. Murine gamma interferon fails to inhibit Toxoplasma gondii growth in murine fibroblasts.

    Schwartzman, J D; Gonias, S L; Pfefferkorn, E R


    Although treatment of human macrophages or fibroblasts with human gamma interferon results in the inhibition of intracellular Toxoplasma gondii, murine gamma interferon stimulated only murine macrophages, not murine fibroblasts, to inhibit T. gondii. This species difference may be important in understanding the control of acute and chronic toxoplasmosis. PMID:2106497

  2. Direct demonstration of the infiltration of murine central nervous system by Pgp-1/CD44high CD45RB(low) CD4+ T cells that induce experimental allergic encephalomyelitis

    Zeine, R; Owens, T


    In experimental allergic encephalomyelitis (EAE), autoimmune T cells infiltrate the central nervous system (CNS) and initiate demyelinating pathology. We have used flow cytometry to directly analyse the migration to the CNS of MBP-reactive CD4+ T cells labelled with a lipophilic fluorescent dye (...

  3. Patogenesis of pipe-stem fibrosis of the liver (experimental observation on murine Schistosomiasis Patogenia da fibrose "pipe-stem" do fígado (observações experimentais na esquistossomose murina

    Zilton A. Andrade


    Full Text Available Mice infected with 30 cercariae of Schistosoma mansoni developed portal and septal fibrosis due to the massive and concentrated deposition of eggs in the periportal areas which occurred following the 16th week after infection. The lesion resembled pipe-stem fibrosis seen in human hepatosplenic schistosomiasis in the following characters: portal fibrosis interconnecting portal spaces as well as portal spaces and central canals; portal inflammation; periovular granulomas; vascular obstruction and telangiectasia. The liver parenchyma maintained its normal architecture. Vascular injection techniques with Indian ink and vinylite revealed that the portal system developed numerous dilated collateral venules coming from the large and medium-sized portal branches, about 10 weeks after schistosome infection. The lodging of schistosome eggs into these collaterals resulted in granulomatous inflammation and fibrosis along all the portal tracts, thus forming the pipe-stem lesion. Although not readily demonstrable grossly, the pipe-stem fibrosis of murine schistosomiasis has many similarities with the human lesion and can be considered to have the same basic pathogenesis.Camundongos infectados com 30 cercárias do Schistosoma mansoni desenvolveram fibrose porta em virtude de um depósito progressivo e concentrado de ovos na região periportal, o que aconteceu a partir da 16ª semana da infecção. Esta fibrose certas características da chamada fibrose "pipe-stem" do homem vista na forma hepatoesplênica da esquistossomose, tais como obstrução das radiculas porta, telangiectasia, conexão fibrosa entre espaços porta e entre estes e veias centrais, além de certo grau de fibrose septal, presença dos granulomas em várias fases evolutivas e reação inflamatória crônica difusa, enquanto o parênquima hepático mantinha a sua estrutura lobular normal. As técnicas de injeção vascular com tinta da China e com vinilite feitas no sistema porta permitiram a

  4. Lactobacillus rhamnosus GG as an Effective Probiotic for Murine Giardiasis

    Nisha Goyal


    Full Text Available The gut microflora is an important constituent in the intestinal mucosal barrier and has been introduced as the concept of probiotic therapy that beneficially affects the host by improving its intestinal microbial balance. Therefore, the main objective of the study was to explore the protective potential of various lactobacilli strains for murine giardiasis. By experimentation, it was found that the probiotic supplementation of either Lactobacillus casei, L. acidophilus, L. plantarum, or L. rhamnosus GG, 7 days prior to inoculation with G. lamblia trophozoites, reduced the rate of cyst excretion compared with Giardia-infected mice. Interestingly, L. GG was found to be the most effective probiotic in reducing the duration of giardia cycle and acts as an effective prophylactic probiotic for murine giardiasis but needs to be clinically correlated due to entirely different human microflora.

  5. The B Cell-Stimulatory Cytokines BLyS and APRIL Are Elevated in Human Periodontitis and Are Required for B Cell-Dependent Bone Loss in Experimental Murine Periodontitis.

    Abe, Toshiharu; AlSarhan, Mohammed; Benakanakere, Manjunatha R; Maekawa, Tomoki; Kinane, Denis F; Cancro, Michael P; Korostoff, Jonathan M; Hajishengallis, George


    B-lineage cells (B lymphocytes and plasma cells) predominate in the inflammatory infiltrate of human chronic periodontitis. However, their role in disease pathogenesis and the factors responsible for their persistence in chronic lesions are poorly understood. In this regard, two cytokines of the TNF ligand superfamily, a proliferation-inducing ligand (APRIL) and B-lymphocyte stimulator (BLyS), are important for the survival, proliferation, and maturation of B cells. Thus, we hypothesized that APRIL and/or BLyS are upregulated in periodontitis and contribute to induction of periodontal bone loss. This hypothesis was addressed in both human and mouse experimental systems. We show that, relative to healthy controls, the expression of APRIL and BLyS mRNA and protein was upregulated in natural and experimental periodontitis in humans and mice, respectively. The elevated expression of these cytokines correlated with increased numbers of B cells/plasma cells in both species. Moreover, APRIL and BLyS partially colocalized with κ L chain-expressing B-lineage cells at the epithelial-connective tissue interface. Ligature-induced periodontitis resulted in significantly less bone loss in B cell-deficient mice compared with wild-type controls. Ab-mediated neutralization of APRIL or BLyS diminished the number of B cells in the gingival tissue and inhibited bone loss in wild-type, but not in B cell-deficient, mice. In conclusion, B cells and specific cytokines involved in their growth and differentiation contribute to periodontal bone loss. Moreover, APRIL and BLyS have been identified as potential therapeutic targets in periodontitis.

  6. Diagnostic imaging advances in murine models of colitis.

    Brückner, Markus; Lenz, Philipp; Mücke, Marcus M; Gohar, Faekah; Willeke, Peter; Domagk, Dirk; Bettenworth, Dominik


    Inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis are chronic-remittent inflammatory disorders of the gastrointestinal tract still evoking challenging clinical diagnostic and therapeutic situations. Murine models of experimental colitis are a vital component of research into human IBD concerning questions of its complex pathogenesis or the evaluation of potential new drugs. To monitor the course of colitis, to the present day, classical parameters like histological tissue alterations or analysis of mucosal cytokine/chemokine expression often require euthanasia of animals. Recent advances mean revolutionary non-invasive imaging techniques for in vivo murine colitis diagnostics are increasingly available. These novel and emerging imaging techniques not only allow direct visualization of intestinal inflammation, but also enable molecular imaging and targeting of specific alterations of the inflamed murine mucosa. For the first time, in vivo imaging techniques allow for longitudinal examinations and evaluation of intra-individual therapeutic response. This review discusses the latest developments in the different fields of ultrasound, molecularly targeted contrast agent ultrasound, fluorescence endoscopy, confocal laser endomicroscopy as well as tomographic imaging with magnetic resonance imaging, computed tomography and fluorescence-mediated tomography, discussing their individual limitations and potential future diagnostic applications in the management of human patients with IBD.

  7. Osteopontin Is Upregulated in Human and Murine Acute Schistosomiasis Mansoni

    Pereira, Thiago Almeida; Syn, Wing-Kin; Amâncio, Frederico Figueiredo; Cunha, Pedro Henrique Diniz; Caporali, Julia Fonseca Morais; Trindade, Guilherme Vaz de Melo; Santos, Elisângela Trindade; Souza, Márcia Maria; Andrade, Zilton Araújo; Witek, Rafal P; Secor, William Evan; Pereira, Fausto Edmundo Lima; Lambertucci, José Roberto; Diehl, Anna Mae


    Background Symptomatic acute schistosomiasis mansoni is a systemic hypersensitivity reaction against the migrating schistosomula and mature eggs after a primary infection. The mechanisms involved in the pathogenesis of acute schistosomiasis are not fully elucidated. Osteopontin has been implicated in granulomatous reactions and in acute hepatic injury. Our aims were to evaluate if osteopontin plays a role in acute Schistosoma mansoni infection in both human and experimentally infected mice and if circulating OPN levels could be a novel biomarker of this infection. Methodology/Principal Findings Serum/plasma osteopontin levels were measured by ELISA in patients with acute (n = 28), hepatointestinal (n = 26), hepatosplenic (n = 39) schistosomiasis and in uninfected controls (n = 21). Liver osteopontin was assessed by immunohistochemistry in needle biopsies of 5 patients. Sera and hepatic osteopontin were quantified in the murine model of schistosomiasis mansoni during acute (7 and 8 weeks post infection, n = 10) and chronic (30 weeks post infection, n = 8) phase. Circulating osteopontin levels are increased in patients with acute schistosomiasis (p = 0.0001). The highest levels of OPN were observed during the peak of clinical symptoms (7–11 weeks post infection), returning to baseline level once the granulomas were modulated (>12 weeks post infection). The plasma levels in acute schistosomiasis were even higher than in hepatosplenic patients. The murine model mirrored the human disease. Macrophages were the major source of OPN in human and murine acute schistosomiasis, while the ductular reaction maintains OPN production in hepatosplenic disease. Soluble egg antigens from S. mansoni induced OPN expression in primary human kupffer cells. Conclusions/Significance S. mansoni egg antigens induce the production of OPN by macrophages in the necrotic-exudative granulomas characteristic of acute schistosomiasis mansoni. Circulating OPN levels are upregulated in human and

  8. Sexual transmission of Trypanosoma cruzi in murine model.

    Ribeiro, Marcelle; Nitz, Nadjar; Santana, Camilla; Moraes, Aline; Hagström, Luciana; Andrade, Rafael; Rios, Adriano; Sousa, Alessandro; Dallago, Bruno; Gurgel-Gonçalves, Rodrigo; Hecht, Mariana


    Trypanosoma cruzi is mainly transmitted by blood-sucking triatomines, but other routes also have epidemiological importance, such as blood transfusion and congenital transmission. Although the possibility of sexual transmission of T. cruzi has been suggested since its discovery, few studies have been published on this subject. We investigated acquisition of T. cruzi by sexual intercourse in an experimental murine model. Male and female mice in the chronic phase of Chagas disease were mated with naive partners. Parasitological, serological and molecular tests demonstrated the parasites in tissues and blood of partners. These results confirm the sexual transmission of T. cruzi in mice.

  9. Flow cytometry of murine spermatocytes.

    Gaysinskaya, Valeriya; Bortvin, Alex


    Protocols for purification of murine male germ cells by FACS based on Hoechst 33342 (Ho342) dye staining have been reported and optimized. However, the protocols are often challenging to follow, partly due to difficulties related to sample preparation, instrument parameters, data display, and selection strategies. In addition, troubleshooting of flow cytometry experiments usually requires some fluency in technical principles and instrument specifications and settings. This unit describes setup and procedures for analysis and sorting of male meiotic prophase I (MPI) cells and other germ cells. Included are procedures that guide data acquisition, display, gating, and back-gating critical for optimal data visualization and cell sorting. Additionally, a flow cytometry analysis of spermatogenesis-defective testis is provided to illustrate the applicability of the technique to the characterization and purification of cells from mutant testis.

  10. Two Epitopes Shared by Taenia crassiceps and Taenia solium Confer Protection against Murine T. crassiceps Cysticercosis along with a Prominent T1 Response

    Toledo, Andrea; Fragoso, Gladis; Rosas, Gabriela; Hernández, Marisela; Gevorkian, Goar; López-Casillas, Fernando; Hernández, Beatriz; Acero, Gonzalo; Huerta, Mirna; Larralde, Carlos; Sciutto, Edda


    Taenia crassiceps recombinant antigens KETc1 and KETc12 have been shown to induce high level of protection against experimental murine T. crassiceps cysticercosis, an experimental model successfully used to test candidate antigens for use in vaccination against porcine Taenia solium cysticercosis. Based on the deduced amino acid sequence, KETc1 and KETc12 were chemically synthesized in linear form. Immunization with KETc1 induced 66.7 to 100% protection against murine cysticercosis, and immun...

  11. Treatment of experimental murine arthritis with transdermal photodynamic therapy

    Ratkay, Leslie G.; Chowdhary, R. K.; Neyndorff, Herma C.; Levy, Julia G.; Waterfield, J. D.


    Photodynamic therapy (PDT) using benzoporphyrin derivative, monoacid ring A (BPD), and transdermal light was able to significantly treat symptoms of adjuvant-enhanced arthritis in MRL-lpr mice. Clinical and histological evaluation showed that PDT was able to modify the progression of adjuvant-enhanced arthritis up to 10 days after induction. When PDT was used on arthritic joints displaying swelling, it prevented further deterioration of clinical symptoms (76%, 16/21). However, it did not significantly effect the histopathologic parameters. As we have previously reported that mitogen activated MRL-lpr splenocytes were shown to be more susceptible to in vitro PDT we postulate that our findings reflect a selective destruction of adjuvant activated lymphocytes in the circulation and/or joints. The application of PDT to eliminate activated cells responsible for the inflammatory reaction at the arthritic site may have significant clinical implications for the treatment of rheumatoid arthritis.

  12. Apoptosis in irradiated murine tumors.

    Stephens, L C; Ang, K K; Schultheiss, T E; Milas, L; Meyn, R E


    Early radiation responses of transplantable murine ovarian (OCaI) and hepatocellular (HCaI) carcinomas were examined at 6, 24, 48, 96, and 144 h after single photon doses of 25, 35, or 45 Gy. Previous studies using tumor growth delay and tumor radiocurability assays had shown OCaI tumors to be relatively radiosensitive and HCaI tumors to be radioresistant. At 6 h, approximately 20% of nuclei in OCaI tumors showed aberrations characteristic of cell death by apoptosis. This contrasted to an incidence of 3% in HCaI tumors. Mitotic activity was eliminated in OCaI tumors but was only transiently suppressed in HCaI tumors. At 24-96 h, OCaI tumors continued to display apoptosis and progressive necrosis, whereas HCaI tumors responded by exhibiting marked pleomorphism. Factors other than mitotic activity may influence tumor radiosensitivity, and one of these may be susceptibility to induction of apoptosis (programmed cell death), because this was a prominent early radiation response by the radiosensitive OCaI tumors.

  13. Cloning and expression of murine immune interferon cDNA.


    The murine immune interferon (IFN-gamma) gene was cloned and expressed under control of the simian virus 40 early promoter in the monkey COS-1 cell line. A protein is secreted from these cells having the biological, antigenic, and biochemical characteristics of natural murine IFN-gamma. Cloned murine IFN-gamma cDNAs were obtained by using RNA from both mitogen-induced murine spleens and the transfected COS cells, and both code for identical proteins. The mature murine IFN-gamma encoded is 136...

  14. Structure of the murine Thy-1 gene

    V. Giguere; K-I. Isobe; F.G. Grosveld (Frank)


    textabstractWe have cloned the murine Thy-1.1 (AKR) and Thy-1.2 (Balb/c) genes. The complete exon/intron structure and the nucleotide sequence of the Thy-1.2 gene was determined. The gene contains four exons and three intervening sequences. The complete transcriptional unit gives rise to a tissue an

  15. Reemergence of Murine Typhus in the US


    Dr. Lucas Blanton discusses the Reemergence of Murine Typhus in Galveston Texas in 2013.  Created: 4/21/2015 by National Center for Emerging and Zoonotic Infectious Diseases (NCEZID).   Date Released: 4/27/2015.

  16. Angelica acutiloba Kitagawa Extract Attenuates DSS-Induced Murine Colitis.

    Jang, Jong-Chan; Lee, Kang Min; Ko, Seong-Gyu


    We examined the protective effects of Angelica acutiloba Kitagawa (AAK) extract on a murine model of acute experimental colitis. Colitis was induced by 4% dextran sulfate sodium (DSS) in the drinking water of male C57BL/6 mice, for 7 consecutive days. Oral administration of AAK extract (500 mg/kg/day) significantly alleviated DSS-induced symptoms such as anorexia, weight loss, events of diarrhea or bloody stools, and colon shortening. Histological damage was also ameliorated, as evidenced by the architectural preservation and suppression of inflammatory cell infiltration in colonic samples. Treatment improved the colonic mRNA expression of different inflammatory markers: cytokines, inducible enzymes, matrix metalloproteinases, and tight junction-related proteins. In the isolated serum, IgE levels were downregulated. Collectively, these findings indicate the therapeutic potentials of AAK as an effective complementary or alternative modality for the treatment of ulcerative colitis.

  17. Limited Role of Murine ATM in Oncogene-Induced Senescence and p53-Dependent Tumor Suppression

    Martinez-Pastor, Barbara; Ortega-Molina, Ana; Soria, Rebeca; Collado, Manuel; Fernandez-Capetillo, Oscar; Serrano, Manuel


    Recent studies in human fibroblasts have provided a new general paradigm of tumor suppression according to which oncogenic signaling produces DNA damage and this, in turn, results in ATM/p53-dependent cellular senescence. Here, we have tested this model in a variety of murine experimental systems. Overexpression of oncogenic Ras in murine fibroblasts efficiently induced senescence but this occurred in the absence of detectable DNA damage signaling, thus suggesting a fundamental difference between human and murine cells. Moreover, lung adenomas initiated by endogenous levels of oncogenic K-Ras presented abundant senescent cells, but undetectable DNA damage signaling. Accordingly, K-Ras-driven adenomas were also senescent in Atm-null mice, and the tumorigenic progression of these lesions was only modestly accelerated by Atm-deficiency. Finally, we have examined chemically-induced fibrosarcomas, which possess a persistently activated DNA damage response and are highly sensitive to the activity of p53. We found that the absence of Atm favored genomic instability in the resulting tumors, but did not affect the persistent DNA damage response and did not impair p53-dependent tumor suppression. All together, we conclude that oncogene-induced senescence in mice may occur in the absence of a detectable DNA damage response. Regarding murine Atm, our data suggest that it plays a minor role in oncogene-induced senescence or in p53-dependent tumor suppression, being its tumor suppressive activity probably limited to the maintenance of genomic stability. PMID:19421407

  18. Limited role of murine ATM in oncogene-induced senescence and p53-dependent tumor suppression.

    Alejo Efeyan

    Full Text Available Recent studies in human fibroblasts have provided a new general paradigm of tumor suppression according to which oncogenic signaling produces DNA damage and this, in turn, results in ATM/p53-dependent cellular senescence. Here, we have tested this model in a variety of murine experimental systems. Overexpression of oncogenic Ras in murine fibroblasts efficiently induced senescence but this occurred in the absence of detectable DNA damage signaling, thus suggesting a fundamental difference between human and murine cells. Moreover, lung adenomas initiated by endogenous levels of oncogenic K-Ras presented abundant senescent cells, but undetectable DNA damage signaling. Accordingly, K-Ras-driven adenomas were also senescent in Atm-null mice, and the tumorigenic progression of these lesions was only modestly accelerated by Atm-deficiency. Finally, we have examined chemically-induced fibrosarcomas, which possess a persistently activated DNA damage response and are highly sensitive to the activity of p53. We found that the absence of Atm favored genomic instability in the resulting tumors, but did not affect the persistent DNA damage response and did not impair p53-dependent tumor suppression. All together, we conclude that oncogene-induced senescence in mice may occur in the absence of a detectable DNA damage response. Regarding murine Atm, our data suggest that it plays a minor role in oncogene-induced senescence or in p53-dependent tumor suppression, being its tumor suppressive activity probably limited to the maintenance of genomic stability.

  19. Local IL-23 Expression in Murine Vaginal Candidiasis and Its Relationship with Infection and Immune Status

    WU Yan; TAN Zhijian; LIU Zhixiang; XIA Dechao; LI Jiawen


    To investigate the expression of vaginal IL-23 and its role in experimental murine vaginal candidiasis and its relationship with infection and immune status, immuno-competent (group A) and immuno-suppressed (group B) murine models of vaginal candidiasis were established in estrogentreated mice. Non-estrogen-treated mice were used as controls (group C). The level of IL-23 p19 mRNA in murine vaginal tissue was determined by RT-PCR. Significantly increased levels of IL23p19mRNA were observed on the 4th, the 7th and 14th day after inoculation in immuno-competent group when compared with that in control group (P<0.01, P<0.05), However, significant increase of IL-23 p19mRNA were only observed on the 7th day and the 14th day after inoculatuon in immuno-suppressed groups (P<0.05). On the 4th and 7th day, the levels of IL-23 p19mRNA were significantly increased in immuno-competent group than those in immuno-suppressed group (P <0.05). Local IL-23 may play a role in the pathogenesis of murine vaginal candidiasis and has a protective function during infection. Low vaginal IL-23 level may correlate with the increased susceptibility to Candida albicans in immuno-suppressed group.

  20. Hyperlipidemia affects multiscale structure and strength of murine femur.

    Ascenzi, Maria-Grazia; Lutz, Andre; Du, Xia; Klimecky, Laureen; Kawas, Neal; Hourany, Talia; Jahng, Joelle; Chin, Jesse; Tintut, Yin; Nackenhors, Udo; Keyak, Joyce


    To improve bone strength prediction beyond limitations of assessment founded solely on the bone mineral component, we investigated the effect of hyperlipidemia, present in more than 40% of osteoporotic patients, on multiscale structure of murine bone. Our overarching purpose is to estimate bone strength accurately, to facilitate mitigating fracture morbidity and mortality in patients. Because (i) orientation of collagen type I affects, independently of degree of mineralization, cortical bone׳s micro-structural strength; and, (ii) hyperlipidemia affects collagen orientation and μCT volumetric tissue mineral density (vTMD) in murine cortical bone, we have constructed the first multiscale finite element (mFE), mouse-specific femoral model to study the effect of collagen orientation and vTMD on strength in Ldlr(-/-), a mouse model of hyperlipidemia, and its control wild type, on either high fat diet or normal diet. Each µCT scan-based mFE model included either element-specific elastic orthotropic properties calculated from collagen orientation and vTMD (collagen-density model) by experimentally validated formulation, or usual element-specific elastic isotropic material properties dependent on vTMD-only (density-only model). We found that collagen orientation, assessed by circularly polarized light and confocal microscopies, and vTMD, differed among groups and that microindentation results strongly correlate with elastic modulus of collagen-density models (r(2)=0.85, p=10(-5)). Collagen-density models yielded (1) larger strains, and therefore lower strength, in simulations of 3-point bending and physiological loading; and (2) higher correlation between mFE-predicted strength and 3-point bending experimental strength, than density-only models. This novel method supports ongoing translational research to achieve the as yet elusive goal of accurate bone strength prediction.

  1. Murine Typhus: Clinical and epidemiological aspects

    Gaspar Peniche Lara


    Full Text Available Rickettsia typhi is an intracellular bacteria who causes murine typhus. His importance is reflected in the high frequency founding specific antibodies against R. typhi in several worldwide seroepidemiological studies, the seroprevalence ranging between 3-36%. Natural reservoirs of Rickettsia typhi are rats (some species belonging the Rattus Genus and fleas (Xenopsylla cheopis are his vector. This infection is associated with overcrowding, pollution and poor hygiene. Typically presents fever, headache, rash on trunk and extremities, in some cases may occur organ-specific complications, affecting liver, kidney, lung or brain. Initially the disease is very similar to other diseases, is very common to confuse the murine typhus with Dengue fever, therefore, ignorance of the disease is a factor related to complications or non-specific treatments for the resolution of this infection. This paper presents the most relevant information to consider about the rickettsiosis caused by Rickettsia typhi.

  2. Advances in Murine Models of Diabetic Nephropathy

    Li-li Kong


    Full Text Available Diabetic nephropathy (DN is one of the microvascular complications of both type 1 and type 2 diabetes, which is also associated with a poor life expectancy of diabetic patients. However, the pathogenesis of DN is still unclear. Thus, it is of great use to establish appropriate animal models of DN for doing research on pathogenesis and developing novel therapeutic strategies. Although a large number of murine models of DN including artificially induced, spontaneous, and genetically engineered (knockout and transgenic animal models have been developed, none of them develops renal changes sufficiently reflecting those seen in humans. Here we review the identified murine models of DN from the aspects of genetic background, type of diabetes, method of induction, gene deficiency, animal age and gender, kidney histopathology, and phenotypic alterations in the hope of enhancing our comprehension of genetic susceptibility and molecular mechanisms responsible for this disease and providing new clues as to how to choose appropriate animal models of DN.

  3. Immunodetection of Murine Lymphotoxins in Eukaryotic Cells.

    Boitchenko, Veronika E.; Korobko, Vyacheslav G.; Prassolov, Vladimir S.; Kravchenko, Vladimir V.; Kuimov, Alexander N.; Turetskaya, Regina L.; Kuprash, Dmitry V.; Nedospasov, Sergei A.


    Lymphotoxins alpha and beta (LTalpha and LTbeta) are members of tumor necrosis factor superfamily. LT heterotrimers exist on the surface of lymphocytes and signal through LTbeta receptor while soluble LTalpha homotrimer can signal through TNF receptors p55 and p75. LT-, as well as TNF-mediated signaling are important for the organogenesis and maintenance of microarchitecture of secondary lymphoid organs in mice and has been implicated in the mechanism of certain inflammatory syndromes in humans. In this study we describe the generation of eukaryotic expression plasmids encoding murine LTalpha and LTbeta genes and a prokaryotic expression construct for murine LTalpha. Using recombinant proteins expressed by these vectors as tools for antisera selection, we produced and characterized several polyclonal antibodies capable of detecting LT proteins in eukaryotic cells.

  4. Murine models of human wound healing.

    Chen, Jerry S; Longaker, Michael T; Gurtner, Geoffrey C


    In vivo wound healing experiments remain the most predictive models for studying human wound healing, allowing an accurate representation of the complete wound healing environment including various cell types, environmental cues, and paracrine interactions. Small animals are economical, easy to maintain, and allow researchers to take advantage of the numerous transgenic strains that have been developed to investigate the specific mechanisms involved in wound healing and regeneration. Here we describe three reproducible murine wound healing models that recapitulate the human wound healing process.

  5. Preparation and characterization of polysulfone affinity membranes bearing a synthetic peptide ligand for the separation of murine immunoglobulins.

    Boi, Cristiana; Algeri, Cristian; Sarti, Giulio C


    Affinity membranes have been prepared by immobilizing D-PAM, a synthetic ligand that exhibits affinity for the Fc portion of antibodies, onto poliethersulfone microporous membranes. The ligand density has been measured and the ligand utilization was evaluated and compared with literature data available for chromatographic beads. The resulting new affinity membranes have been experimentally characterized and tested by using pure murine IgG solutions and mouse serum. Equilibrium and kinetic parameters have been obtained in batch experiments using pure protein solutions. The highest binding capacity measured for murine IgG was 45 microg/cm(2) obtained at 1.2 mg/mL protein concentration at equilibrium, while the maximum static binding capacity calculated with the Langmuir model was 81 microg/cm(2). The adsorption of murine IgG on the affinity membranes was described using different isotherms: Freundlich and Temkin models have been considered and critically compared with the Langmuir adsorption model. A dynamic binding capacity of 21 microg/cm(2) was obtained by feeding a solution of 0.3 mg/mL of murine IgG, confirming the results obtained in batch experiments at the same concentration. The affinity membranes considered are endowed with good binding capacity for murine IgG and good selectivity for immunoglobulins and can be considered for the capturing step of an antibody production process.

  6. Nanoliposomal artemisinin for the treatment of murine visceral leishmaniasis

    Want MY


    Full Text Available Muzamil Y Want,1 Mohammad Islammudin,1 Garima Chouhan,1 Hani A Ozbak,2 Hassan A Hemeg,2 Asoke P Chattopadhyay,3 Farhat Afrin2 1Parasite Immunology Laboratory, Department of Biotechnology, Jamia Hamdard, Hamdard University, New Delhi, India; 2Department of Clinical Laboratory Sciences, Faculty of Applied Medical Sciences, Taibah University, Medina, Saudi Arabia; 3Department of Chemistry, University of Kalyani, Kalyani, India Abstract: Visceral leishmaniasis (VL is a fatal, vector-borne disease caused by the intracellular protozoa of the genus Leishmania. Most of the therapeutics for VL are toxic, expensive, or ineffective. Sesquiterpenes are a new class of drugs with proven antimicrobial and antiviral activities. Artemisinin is a sesquiterpene lactone with potent antileishmanial activity, but with limited access to infected cells, being a highly lipophilic molecule. Association of artemisinin with liposome is a desirable strategy to circumvent the problem of poor accessibility, thereby improving its efficacy, as demonstrated in a murine model of experimental VL. Nanoliposomal artemisinin (NLA was prepared by thin-film hydration method and optimized using Box–Behnken design with a mean particle diameter of 83±16 nm, polydispersity index of 0.2±0.03, zeta potential of -27.4±5.7 mV, and drug loading of 33.2%±2.1%. Morphological study of these nanoliposomes by microscopy showed a smooth and spherical surface. The mechanism of release of artemisinin from the liposomes followed the Higuchi model in vitro. NLA was free from concomitant signs of toxicity, both ex vivo in murine macrophages and in vivo in healthy BALB/c mice. NLA significantly denigrated the intracellular infection of Leishmania donovani amastigotes and the number of infected macrophages ex vivo with an IC50 of 6.0±1.4 µg/mL and 5.1±0.9 µg/mL, respectively. Following treatment in a murine model of VL, NLA demonstrated superior efficacy compared to artemisinin with a

  7. Murine fundus fluorescein angiography: An alternative approach using a handheld camera.

    Ehrenberg, Moshe; Ehrenberg, Scott; Schwob, Ouri; Benny, Ofra


    In today's modern pharmacologic approach to treating sight-threatening retinal vascular disorders, there is an increasing demand for a compact, mobile, lightweight and cost-effective fluorescein fundus camera to document the effects of antiangiogenic drugs on laser-induced choroidal neovascularization (CNV) in mice and other experimental animals. We have adapted the use of the Kowa Genesis Df Camera to perform Fundus Fluorescein Angiography (FFA) in mice. The 1 kg, 28 cm high camera has built-in barrier and exciter filters to allow digital FFA recording to a Compact Flash memory card. Furthermore, this handheld unit has a steady Indirect Lens Holder that firmly attaches to the main unit, that securely holds a 90 diopter lens in position, in order to facilitate appropriate focus and stability, for photographing the delicate central murine fundus. This easily portable fundus fluorescein camera can effectively record exceptional central retinal vascular detail in murine laser-induced CNV, while readily allowing the investigator to adjust the camera's position according to the variable head and eye movements that can randomly occur while the mouse is optimally anesthetized. This movable image recording device, with efficiencies of space, time, cost, energy and personnel, has enabled us to accurately document the alterations in the central choroidal and retinal vasculature following induction of CNV, implemented by argon-green laser photocoagulation and disruption of Bruch's Membrane, in the experimental murine model of exudative macular degeneration.

  8. DMPD: The actions of bacterial DNA on murine macrophages. [Dynamic Macrophage Pathway CSML Database

    Full Text Available 10534106 The actions of bacterial DNA on murine macrophages. Sester DP, Stacey KJ, ... Show The actions of bacterial DNA on murine macrophages. PubmedID 10534106 Title The actions of bacterial DNA on murine macrophage

  9. Handling stress may confound murine gut microbiota studies

    Allen-Blevins, Cary R.; You, Xiaomeng; Hinde, Katie


    . Results Our results demonstrate a significant difference between the amount of Firmicutes in pups receiving water passively and those receiving FOS actively (p-value = 0.009). Additionally, we found significant differences between the fecal microbiota from handled and non-handled mouse pups. Discussion From our results, we conclude even handling pups for experimental purposes, without gavage, may induce enough stress to alter the murine gut microbiota profile. We suggest further studies to examine potential stress effects on gut microbiota caused by experimental techniques. Stress from experimental techniques may need to be accounted for in future gut microbiota studies. PMID:28097073

  10. Handling stress may confound murine gut microbiota studies

    Cary R. Allen-Blevins


    Firmicutes. Results Our results demonstrate a significant difference between the amount of Firmicutes in pups receiving water passively and those receiving FOS actively (p-value = 0.009. Additionally, we found significant differences between the fecal microbiota from handled and non-handled mouse pups. Discussion From our results, we conclude even handling pups for experimental purposes, without gavage, may induce enough stress to alter the murine gut microbiota profile. We suggest further studies to examine potential stress effects on gut microbiota caused by experimental techniques. Stress from experimental techniques may need to be accounted for in future gut microbiota studies.

  11. Effects of Murine Cytomegalovirus Infection on Sperm Viability in Mice


    In order to explore the effects of testicular infection of murine cytomegalovirus (MCMV) on mature sperm viability at different periods following MCMV inoculation in mice, 91 BALB/c mice without MCMV infection were randomly divided into two groups: an experimental group (n=56) and a control group (n= 35). The mice in the experimental group were treated by inoculating MCMV intratesticularly, while those in the controlled group were directly inoculated with DMEM without MCMV. The mice in both groups were sacrificed separately on the day 1,1.5, 2, 4, 6, 9 and 14 post-inoculation (D1, 1.5,2, 4, 6, 9 and 14 PI). The MCMV M83 mRNA gene was detected in the testis by in situ hybridization (ISH) with MCMV late-mRNA probe labeled with digoxin.Sperm viability of mature sperm in the epididymis cauda was measured. The results demonstrated the positive signal of ISH of MCMV was found mainly in the cytoplasm of the testicular interstitial cells and spermatogenic cells in the experimental group. Compared with that in the controlled group, the sperm viability in the experimental group was decreased significantly on D1 PI and D1.5PI (P< 0.05). No statistically significant difference in the sperm viability was found after D2 PI between two groups (P>0.05). This suggested that sperm viability in mice might be descended significantly shortly after MCMV infection and might return to normal with time, indicating that MCMV acute infection might temporarily degrade sperm quality and influence procreation transiently.

  12. Murine erythrocytes contain high levels of lysophospholipase activity

    Kamp, J.A.F. op den; Roelofsen, B.; Sanderink, G.; Middelkoop, E.; Hamer, R.


    Murine erythrocytes were found to be unique in the high levels of lysophospholipase activity in the cytosol of these cells. The specific activity of the enzyme in the cytosol of the murine cells is 10-times higher than in the cytosol of rabbit erythrocytes and approximately three orders of magnitude

  13. Exploring the translational disconnect between the murine and human inflammatory response: analysis of LPS dose–response relationship in murine versus human cell lines and implications for translation into murine models of sepsis

    McCarron EP


    Full Text Available Eamon P McCarron,1 Dominic P Williams,1 Daniel J Antoine,1 Anja Kipar,2 Jana Lemm,3 Sebastian Stehr,3 Ingeborg D Welters,4 1Department of Clinical and Molecular Pharmacology, Centre for Drug Safety Science, Institute of Translational Medicine, 2Department of Veterinary Pathology, University of Liverpool, Liverpool, UK; 3Department of Anaesthesiology and Intensive Care Medicine, Jena University Hospital, Jena, Germany; 4Department of Obesity and Endocrinology, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, UK Background: Inflammation forms an important part of the human innate immune system and is largely dependent on the activation of the "classical" NF-κB pathway through Toll-like receptors (TLRs. Understanding this has allowed researchers to explore roles of therapeutic targets in managing conditions such as sepsis. Recapitulating an inflammatory response using lipopolysaccharide (LPS, a "sterile" technique, can provide information that is dissimilar to the clinical condition. By examining NF-κB activation (through immunoblotting of the p65 subunit in two separate cell lines (murine and human and analyzing two murine models of sepsis (intraperitoneal [IP] LPS and IP stool inoculation, an evaluation of the translational disconnect between experimental and clinical sepsis can be made. Methods: THP-1 (human cells and RAW 264.7 (murine cells were dosed with concentrations of LPS (human, 1 pg/mL to 100 ng/mL; murine, 30 pg/mL to 1,000 ng/mL and nuclear actin and p65 were immunoblotted to measure changes in nuclear density. In vivo, C57BL/6 mice received either IP injection of stool suspension (5 µL/g or LPS (25 mg/kg or saline (1 mL/kg. Animals were culled at 6 hours and tissues were analyzed. Results: An increase in basal p65:actin density in THP-1 cells (mean 0.214, standard error of the mean 0.024 was seen at doses as small as 0.1 ng/mL (0.519±0.064. In contrast to RAW 264.7 cells, basal increases (0.170±0

  14. Posttraumatic Chondrocyte Apoptosis in the Murine Xiphoid

    Davis, Christopher G.; Eisner, Eric; McGlynn, Margaret; Shelton, John M.; Richardson, James


    Objective. To demonstrate posttraumatic chondrocyte apoptosis in the murine xiphoid after a crush-type injury and to ultimately determine the pathway (i.e., intrinsic or extrinsic) by which chondrocytes undergo apoptosis in response to mechanical injury. Design. The xiphoids of adult female wild-type mice were injured with the use of a modified Kelly clamp. Postinjury xiphoid cartilage was analyzed via 3 well-described independent means of assessing apoptosis in chondrocytes: hematoxylin and eosin staining, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, and activated caspase-3 staining. Results. Injured specimens contained many chondrocytes with evidence of apoptosis, which is characterized by cell shrinkage, chromatin condensation, nuclear fragmentation, and the liberation of apoptotic bodies. There was a statistically significant increase in the number of chondrocytes undergoing apoptosis in the injured specimens as compared with the uninjured specimens. Conclusions. Chondrocytes can be stimulated to undergo apoptosis as a result of mechanical injury. These experiments involving predominantly cartilaginous murine xiphoid in vivo establish a baseline for future investigations that employ the genetic and therapeutic modulation of chondrocyte apoptosis in response to mechanical injury. PMID:26069679

  15. Thermal resistance in a spontaneous murine tumour.

    Maher, J; Urano, M; Rice, L; Suit, H D


    Resistance to subsequent hyperthermia as a result of prior heating was investigated using a spontaneous murine tumour implanted into the feet of C3H/Sed mice. Tumours were treated by immersing the tumour-bearing foot into a constant-temperature hot water bath set at 45.5 degrees C and were given single and split doses of heat. Response was assessed using a tumour-growth time assay. Three aspects of thermally-induced resistance were particularly considered: the time course of development and decay; the importance of the magnitude of the priming dose and the influence of the size of the tumour at the time of treatment. Substantial resistance was induced in this tumour by short priming doses at 45.5 degrees C, rising rapidly 1-2 days after the first treatment and then starting to decay. There was no significant difference in the kinetics of thermal resistance induced in tumours treated at 4mm and those treated at 8 mm in size, although the large tumours were more sensitive to single doses of heat. Increasing the magnitude of the priming dose of heat resulted in an increase in the magnitude of resistance to the second dose. The results of this study are compared with results of similar studies in this and other laboratories using murine normal tissues and cells in culture. Possible clinical implications are considered.

  16. Splenectomy normalizes hematocrit in murine polycythemia vera.

    Jan-Rung Mo

    Full Text Available Splenic enlargement (splenomegaly develops in numerous disease states, although a specific pathogenic role for the spleen has rarely been described. In polycythemia vera (PV, an activating mutation in Janus kinase 2 (JAK2(V617 induces splenomegaly and an increase in hematocrit. Splenectomy is sparingly performed in patients with PV, however, due to surgical complications. Thus, the role of the spleen in the pathogenesis of human PV remains unknown. We specifically tested the role of the spleen in the pathogenesis of PV by performing either sham (SH or splenectomy (SPL surgeries in a murine model of JAK2(V617F-driven PV. Compared to SH-operated mice, which rapidly develop high hematocrits after JAK2(V617F transplantation, SPL mice completely fail to develop this phenotype. Disease burden (JAK2(V617 is equivalent in the bone marrow of SH and SPL mice, however, and both groups develop fibrosis and osteosclerosis. If SPL is performed after PV is established, hematocrit rapidly declines to normal even though myelofibrosis and osteosclerosis again develop independently in the bone marrow. In contrast, SPL only blunts hematocrit elevation in secondary, erythropoietin-induced polycythemia. We conclude that the spleen is required for an elevated hematocrit in murine, JAK2(V617F-driven PV, and propose that this phenotype of PV may require a specific interaction between mutant cells and the spleen.

  17. Murine Typhus: Clinical and epidemiological aspects

    Gaspar Peniche Lara


    Full Text Available 14.00 Normal 0 21 false false false ES-CO X-NONE X-NONE Rickettsia typhi is an intracellular bacteria who causes murine typhus. His importance is reflected in the high frequency founding specific antibodies against R. typhi in several worldwide seroepidemiological studies, the seroprevalence ranging between 3-36%. Natural reservoirs of Rickettsia typhi are rats (some species belonging the Rattus Genus and fleas (Xenopsylla cheopis are his vector. This infection is associated with overcrowding, pollution and poor hygiene. Typically presents fever, headache, rash on trunk and extremities, in some cases may occur organ-specific complications, affecting liver, kidney, lung or brain. Initially the disease is very similar to other diseases, is very common to confuse the murine typhus with Dengue fever, therefore, ignorance of the disease is a factor related to complications or non-specific treatments for the resolution of this infection. This paper presents the most relevant information to consider about the rickettsiosis caused by Rickettsia typhi.

  18. Benzaldehyde suppresses murine allergic asthma and rhinitis.

    Jang, Tae Young; Park, Chang-Shin; Kim, Kyu-Sung; Heo, Min-Jeong; Kim, Young Hyo


    To evaluate the antiallergic effects of oral benzaldehyde in a murine model of allergic asthma and rhinitis, we divided 20 female BALB/c mice aged 8-10 weeks into nonallergic (intraperitoneally sensitized and intranasally challenged to normal saline), allergic (intraperitoneally sensitized and intranasally challenged to ovalbumin), and 200- and 400-mg/kg benzaldehyde (allergic but treated) groups. The number of nose-scratching events in 10 min, levels of total and ovalbumin-specific IgE in serum, differential counts of inflammatory cells in bronchoalveolar lavage (BAL) fluid, titers of Th2 cytokines (IL-4, IL-5, IL-13) in BAL fluid, histopathologic findings of lung and nasal tissues, and expressions of proteins involved in apoptosis (Bcl-2, Bax, caspase-3), inflammation (COX-2), antioxidation (extracellular SOD, HO-1), and hypoxia (HIF-1α, VEGF) in lung tissue were evaluated. The treated mice had significantly fewer nose-scratching events, less inflammatory cell infiltration in lung and nasal tissues, and lower HIF-1α and VEGF expressions in lung tissue than the allergic group. The number of eosinophils and neutrophils and Th2 cytokine titers in BAL fluid significantly decreased after the treatment (Pbenzaldehyde exerts antiallergic effects in murine allergic asthma and rhinitis, possibly through inhibition of HIF-1α and VEGF.

  19. Efficacy of ceftobiprole Medocaril against Enterococcus faecalis in a murine urinary tract infection model.

    Singh, Kavindra V; Murray, Barbara E


    We evaluated ceftobiprole against the well-characterized Enterococcus faecalis strain OG1RF (with and without the β-lactamase [Bla] plasmid pBEM10) in a murine urinary tract infection (UTI) model. Ceftobiprole was equally effective for Bla(+) and Bla(-) OG1 strains, while ampicillin was moderately to markedly (depending on the inoculum) less effective against Bla(+) than Bla(-) OG1 strains. These data illustrate an in vivo effect on ampicillin of Bla production by E. faecalis and the stability and efficacy of ceftobiprole in experimental UTI.

  20. In vitro development of murine embryos in presence of Campylobacter fetus

    Catena, María; Teruel, Miriam; Morán, P.; Chiapparrone, María Laura; Echeverria, Hernan Eduardo; Soto, Pedro de (O.F.M.)


    Bovine campylobacteriosis caused by Campylobacter fetus is associated with reproductive losses. The knowledge about the mechanisms of bacterial pathogenesis is limited, then a murine experimental model is proposed. BALB/c females and males were used. Two-cell embryos were cultured in Ham-F10 as control group (CG). Treatment groups were constituted by the addition of Cfv 1 and 3, or Cff 2 and 5. Morulae were placed in Ham-F10 (CG); treatment groups were constituted by the addition of Cfv27, CF...

  1. Isolation and culture of murine macrophages.

    Davies, John Q; Gordon, Siamon


    The two most convenient sources of primary murine macrophages are the bone marrow and the peritoneal cavity. Resident peritoneal macrophages can readily be harvested from mice and purified by adherence to tissue culture plastic. The injection of Bio-Gel polyacrylamide beads or thioglycollate broth into the peritoneal cavity produces an inflammatory response allowing the purification of large numbers of elicited macrophages. The production of an activated macrophage population can be achieved by using Bacillus-Calmette-Guerin as the inflammatory stimulus. Resident bone marrow macrophages can be isolated following enzymatic separation of cells from bone marrow plugs and enrichment on 30% fetal calf serum containing medium or Ficoll-Hypaque gradients. Bone marrow-derived macrophages can be produced by differentiating nonadherent macrophage precursors with medium containing macrophage colony-stimulating factor.

  2. Extracellular proteolysis in the adult murine brain.

    Sappino, A P; Madani, R; Huarte, J; Belin, D; Kiss, J Z; Wohlwend, A; Vassalli, J D


    Plasminogen activators are important mediators of extracellular metabolism. In the nervous system, plasminogen activators are thought to be involved in the remodeling events required for cell migration during development and regeneration. We have now explored the expression of the plasminogen activator/plasmin system in the adult murine central nervous system. Tissue-type plasminogen activator is synthesized by neurons of most brain regions, while prominent tissue-type plasminogen activator-catalyzed proteolysis is restricted to discrete areas, in particular within the hippocampus and hypothalamus. Our observations indicate that tissue-type plasminogen activator-catalyzed proteolysis in neural tissues is not limited to ontogeny, but may also contribute to adult central nervous system physiology, for instance by influencing neuronal plasticity and synaptic reorganization. The identification of an extracellular proteolytic system active in the adult central nervous system may also help gain insights into the pathogeny of neurodegenerative disorders associated with extracellular protein deposition.

  3. Molecular cloning and chromosome assignment of murine N-ras.

    Ryan, J.; Hart, C P; Ruddle, F H


    The murine N-ras gene was cloned by screening an EMBL-3 recombinant phage library with a human N-ras specific probe. Hybridization of two separate unique sequence N-ras probes, isolated from the 5' and 3' flanking sequences of the murine gene, to a mouse-Chinese hamster hybrid mapping panel assigns the N-ras locus to mouse chromosome three.

  4. microRNA-222 modulates liver fibrosis in a murine model of biliary atresia

    Shen, Wen-jun; Dong, Rui; Chen, Gong, E-mail:; Zheng, Shan


    Highlights: • The RRV infected group showed cholestasis, retardation and extrahepatic biliary atresia. • miR-222 was highly expressed, and PPP2R2A was inhibited in the murine biliary atresia model. • miR-222 profoundly modulated the process of fibrosis in the murine biliary atresia model. • miR-222 might represent a potential target for improving biliary atresia prognosis. - Abstract: microRNA-222 (miR-222) has been shown to initiate the activation of hepatic stellate cells, which plays an important role in the pathogenesis of liver fibrosis. The aim of our study was to evaluate the role of miR-22 in a mouse model of biliary atresia (BA) induced by Rhesus Rotavirus (RRV) infection. New-born Balb/c mice were randomized into control and RRV infected groups. The extrahepatic bile ducts were evaluated. The experimental group was divided into BA group and negative group based on histology. The expression of miR-222, protein phosphatase 2 regulatory subunit B alpha (PPP2R2A), proliferating cell nuclear antigen (PCNA) and phospho-Akt were detected. We found that the experimental group showed signs of cholestasis, retardation and extrahepatic biliary atresia. No abnormalities were found in the control group. In the BA group, miR-222, PCNA and Akt were highly expressed, and PPP2R2A expression was significantly inhibited. Our findings suggest that miR-222 profoundly modulated the process of fibrosis in the murine BA model, which might represent a potential target for improving BA prognosis.

  5. Stability of murine scrapie strain 87V after passage in sheep and comparison with the CH1641 ovine strain.

    González, Lorenzo; Chianini, Francesca; Hunter, Nora; Hamilton, Scott; Gibbard, Louise; Martin, Stuart; Dagleish, Mark P; Sisó, Sílvia; Eaton, Samantha L; Chong, Angela; Algar, Lynne; Jeffrey, Martin


    Breed- and prion protein (PRNP) genotype-related disease phenotype variability has been observed in sheep infected with the 87V murine scrapie strain. Therefore, the stability of this strain was tested by inoculating sheep-derived 87V brain material back into VM mice. As some sheep-adapted 87V disease phenotypes were reminiscent of CH1641 scrapie, transgenic mice (Tg338) expressing ovine prion protein (PrP) were inoculated with the same sheep-derived 87V sources and with CH1641. Although at first passage in VM mice the sheep-derived 87V sources showed some divergence from the murine 87V control, all the characteristics of murine 87V infection were recovered at second passage from all sheep sources. These included 100 % attack rates and indistinguishable survival times, lesion profiles, immunohistochemical features of disease-associated PrP accumulation in the brain and PrP biochemical properties. All sheep-derived 87V sources, as well as CH1641, were transmitted to Tg338 mice with identical clinical, pathological, immunohistochemical and biochemical features. While this might potentially indicate that sheep-adapted 87V and CH1641 are the same strain, profound divergences were evident, as murine 87V was unable to infect Tg338 mice but was lethal for VM mice, while the reverse was true for CH1641. These combined data suggest that: (i) murine 87V is stable and retains its properties after passage in sheep; (ii) it can be isolated from sheep showing a CH1641-like or a more conventional scrapie phenotype; and (iii) sheep-adapted 87V scrapie, with conventional or CH1641-like phenotype, is biologically distinct from experimental CH1641 scrapie, despite the fact that they behave identically in a single transgenic mouse line.

  6. Murine cerebrovascular cells as a cell culture model for cerebral amyloid angiopathy: isolation of smooth muscle and endothelial cells from mouse brain.

    Gauthier, Sebastien A; Sahoo, Susmita; Jung, Sonia S; Levy, Efrat


    The use of murine cerebrovascular endothelial and smooth muscle cells has not been widely employed as a cell culture model for the investigation of cellular mechanisms involved in cerebral amyloid angiopathy (CAA). Difficulties in isolation and propagation of murine cerebrovascular cells and insufficient yields for molecular and cell culture studies have deterred investigators from using mice as a source for cerebrovascular cells in culture. Instead, cerebrovascular cells from larger mammals are preferred and several methods describing the isolation of endothelial and smooth muscle cells from human, canine, rat, and guinea pig have been published. In recent years, several transgenic mouse lines showing CAA pathology have been established; consequently murine cerebrovascular cells derived from these animals can serve as a key cellular model to study CAA. Here, we describe a procedure for isolating murine microvessels that yields healthy smooth muscle and endothelial cell populations and produce sufficient material for experimental purposes. Murine smooth muscle cells isolated using this protocol exhibit the classic "hill and valley" morphology and are immunoreactive for the smooth muscle cell marker α-actin. Endothelial cells display a "cobblestone" pattern phenotype and show the characteristic immunostaining for the von Willebrand factor and the factor VIII-related antigen. In addition, we describe methods designed to preserve these cells by storage in liquid nitrogen and reestablishing viable cell cultures. Finally, we compare our methods with protocols designed to isolate and maintain human cerebrovascular cell cultures.

  7. Research on Growth Behavior of Embryos for Bovine and Murine on Primary Murine Embryos Fibroblast Cell Feeder Layer

    AN Li-long; XIAO Mei; FENG Xiu-Liang; DOU Zhong-ying; QIU Huai; YANG Qi; LEI An-min; YANG Chun-rong; GAO Zhi-min


    The difference in growth behavior between bovine embryos and murine embryos was studied on PMEF(primary murine embryos fibroblast)feeder layer. The results showed as follows: With embryos having attached, bovine embryonic trophoblast formed a transparent membranous structure covering on inner cell mass (ICM), however, murine embryonic trophoblast formed disc structure. Bovine embryos formed four kinds of ICM colonies with different morphology including the mass-like, the net-like, the stream-like and the mixture-like colonies. Compared with Murine ICM, the bovine ICM grew more fast. So, the bovine ICM was passaged at first after a culture of approximately 5 - 6 days in vitro, but murine ICM was passaged at first after an attachment of 3 - 4 days on PMEF feeder layer. The mixture colonies of bovine ICM differentiated very early, while the others differentiated very late. Most ICM-like mass of Bovine grew in a defined spot, but bovine ICMs like stream and ICMs like net proliferated fast and dispersed quickly. We found that the single blastomeres derived from late bovine morula and late murine morula formed sub-blastophere; moreover, the bovine ICM cell would differentiate rapidly if the trophoblast was removed.

  8. Metabolic syndrome components in murine models.

    Lawson, Heather A; Cheverud, James M


    Animal models have enriched understanding of the physiological basis of metabolic disorders and advanced identification of genetic risk factors underlying the metabolic syndrome (MetS). Murine models are especially appropriate for this type of research, and are an excellent resource not only for identifying candidate genomic regions, but also for illuminating the possible molecular mechanisms or pathways affected in individual components of MetS. In this review, we briefly discuss findings from mouse models of metabolic disorders, particularly in light of issues raised by the recent flood of human genome-wide association studies (GWAS) results. We describe how mouse models are revealing that genotype interacts with environment in important ways, indicating that the underlying genetics of MetS is highly context dependant. Further we show that epistasis, imprinting and maternal effects each contribute to the genetic architecture underlying variation in metabolic traits, and mouse models provide an opportunity to dissect these aspects of the genetic architecture that are difficult if not impossible to ascertain in humans. Finally we discuss how knowledge gained from mouse models can be used in conjunction with comparative genomic methods and bioinformatic resources to inform human MetS research.

  9. Proteomic analysis of normal murine brain parts.

    Taraslia, Vasiliki K; Kouskoukis, Alexandros; Anagnostopoulos, Athanasios K; Stravopodis, Dimitrios J; Margaritis, Lukas H; Tsangaris, George Th


    Murine brain is an excellent tool for studying protein expression and brain function in mammals. Although mice are an extensively used model to recapitulate various pathological conditions, the proteome of the normal mouse brain has not been yet reported. In the present study, we identified the total proteins of different parts of the brain of CB7BL/6 mice, a widely used strain, by applying proteomic methodologies. The adult mouse brain was dissected anatomically into the following regions: frontal cortex, olfactory bulb, hippocampus, midbrain, cerebellum, hypothalamus and medulla. Total protein extracts of these regions were separated by two-dimensional gel electrophoresis and analyzed by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry, following in-gel digestion with trypsin. Protein identification was carried out by peptide mass fingerprint. Thus, 515 different single-gene products were identified in total, 54 expressed specifically in the olfactory bulb, 62 in the hippocampus, 36 in the frontal cortex, five in the cerebellum, nine in the midbrain, eight in the hypothamamus and 10 in the medulla. The majority of the proteins were enzymes, structural proteins and transporters. Moreover, the distribution of these molecules appears to exhibit direct correlation with the function of the brain regions where they were expressed. This study leads to the complete characterization of the normal mouse brain proteome as well as the protein expression profile of the different brain regions. These results will aid in addressing unmet scientific needs regarding physiological and pathological brain functions.

  10. Amphotropic murine leukemia viruses induce spongiform encephalomyelopathy.

    Münk, C; Löhler, J; Prassolov, V; Just, U; Stockschläder, M; Stocking, C


    Recombinants of amphotropic murine leukemia virus (A-MuLV) have found widespread use in retroviral vector systems due to their ability to efficiently and stably infect cells of several different species, including human. Previous work has shown that replication-competent recombinants containing the amphotropic env gene, encoding the major SU envelope glycoprotein that determines host tropism, induce lymphomas in vivo. We show here that these viruses also induce a spongiform encephalomyelopathy in mice inoculated perinatally. This fatal central nervous system disease is characterized by noninflammatory spongiform lesions of nerve and glial cells and their processes, and is associated with moderate astro- and microgliosis. The first clinical symptoms are ataxia, tremor, and spasticity, progressing to complete tetraparesis and incontinence, and finally death of the animal. Sequences within the amphotropic env gene are necessary for disease induction. Coinfection of A-MuLV recombinants with nonneuropathogenic ecotropic or polytropic MuLV drastically increases the incidence, degree, and distribution of the neurodegenerative disorder. The consequence of these results in view of the use of A-MuLV recombinants in the clinic is discussed.

  11. Glycosaminoglycan interactions in murine gammaherpesvirus-68 infection.

    Laurent Gillet

    Full Text Available Glycosaminoglycans (GAGs commonly participate in herpesvirus entry. They are thought to provide a reversible attachment to cells that promotes subsequent receptor binding. Murine gamma-herpesvirus-68 (MHV-68 infection of fibroblasts and epithelial cells is highly GAG-dependent. This is a function of the viral gp150, in that gp150-deficient mutants are much less GAG-dependent than wild-type. Here we show that the major MHV-68 GAG-binding protein is not gp150 but gp70, a product of ORF4. Surprisingly, ORF4-deficient MHV-68 showed normal cell binding and was more sensitive than wild-type to inhibition by soluble heparin rather than less. Thus, the most obvious viral GAG interaction made little direct contribution to infection. Indeed, a large fraction of the virion gp70 had its GAG-binding domain removed by post-translational cleavage. ORF4 may therefore act mainly to absorb soluble GAGs and prevent them from engaging gp150 prematurely. In contrast to gp70, gp150 bound poorly to GAGs, implying that it provides little in the way of adhesion. We hypothesize that it acts instead as a GAG-sensitive switch that selectively activates MHV-68 entry at cell surfaces.

  12. B-cell directed therapies in antiphospholipid antibody syndrome--new directions based on murine and human data.

    Khattri, Saakshi; Zandman-Goddard, Gisele; Peeva, Elena


    The increased awareness of the role of humoral immunophysiology in antiphospholipid syndrome (APS) has aroused interest in B cells as therapeutic targets in this disease. This paper reviews the literature on B cell directed therapies in human and experimental APS. The clinical data is limited to B cell depletion with rituximab and comprises case reports and case series. Murine studies include use of modulators of B cell function such as belimumab and abatacept. In both human and murine studies, B cell directed therapies appeared to have clinical and serologic beneficial effects including a decrease in the antiphospholipid antibody titers after treatment. Randomized controlled clinical trials are needed to determine whether B cell depletors and/or B cell modulators can be effective agents for treating patients with APS.

  13. Structural and biochemical characterization of the inhibitor complexes of xenotropic murine leukemia virus-related virus protease

    Li, Mi; Gustchina, Alla; Matúz, Krisztina; Tözsér, Jozsef; Namwong, Sirilak; Goldfarb, Nathan E.; Dunn, Ben M.; Wlodawer, Alexander (Debrecen); (NCI); (Florida); (Suan Sunandha)


    Interactions between the protease (PR) encoded by the xenotropic murine leukemia virus-related virus and a number of potential inhibitors have been investigated by biochemical and structural techniques. It was observed that several inhibitors used clinically against HIV PR exhibit nanomolar or even subnanomolar values of K{sub i}, depending on the exact experimental conditions. Both TL-3, a universal inhibitor of retroviral PRs, and some inhibitors originally shown to inhibit plasmepsins were also quite potent, whereas inhibition by pepstatin A was considerably weaker. Crystal structures of the complexes of xenotropic murine leukemia virus-related virus PR with TL-3, amprenavir and pepstatin A were solved at high resolution and compared with the structures of complexes of these inhibitors with other retropepsins. Whereas TL-3 and amprenavir bound in a predictable manner, spanning the substrate-binding site of the enzyme, two molecules of pepstatin A bound simultaneously in an unprecedented manner, leaving the catalytic water molecule in place.

  14. A murine model of muscle training by neuromuscular electrical stimulation.

    Ambrosio, Fabrisia; Fitzgerald, G Kelley; Ferrari, Ricardo; Distefano, Giovanna; Carvell, George


    Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore (1), maintain (2) or enhance (3-5) muscle functional capacity. Transcutaneous surface stimulation of skeletal muscle involves a current flow between a cathode and an anode, thereby inducing excitement of the motor unit and the surrounding muscle fibers. NMES is an attractive modality to evaluate skeletal muscle adaptive responses for several reasons. First, it provides a reproducible experimental model in which physiological adaptations, such as myofiber hypertophy and muscle strengthening (6), angiogenesis (7-9), growth factor secretion (9-11), and muscle precursor cell activation (12) are well documented. Such physiological responses may be carefully titrated using different parameters of stimulation (for Cochrane review, see (13)). In addition, NMES recruits motor units non-selectively, and in a spatially fixed and temporally synchronous manner (14), offering the advantage of exerting a treatment effect on all fibers, regardless of fiber type. Although there are specified contraindications to NMES in clinical populations, including peripheral venous disorders or malignancy, for example, NMES is safe and feasible, even for those who are ill and/or bedridden and for populations in which rigorous exercise may be challenging. Here, we demonstrate the protocol for adapting commercially available electrodes and performing a NMES protocol using a murine model. This animal model has the advantage of utilizing a clinically available device and providing instant feedback regarding positioning of the electrode to elicit the desired muscle contractile effect. For the purpose of this manuscript, we will describe the protocol for muscle stimulation of the anterior compartment muscles of a mouse hindlimb.

  15. Murine and human CFTR exhibit different sensitivities to CFTR potentiators.

    Cui, Guiying; McCarty, Nael A


    Development of therapeutic molecules with clinical efficacy as modulators of defective CFTR includes efforts to identify potentiators that can overcome or repair the gating defect in mutant CFTR channels. This has taken a great leap forward with the identification of the potentiator VX-770, now available to patients as "Kalydeco." Other small molecules with different chemical structure also are capable of potentiating the activity of either wild-type or mutant CFTR, suggesting that there are features of the protein that may be targeted to achieve stimulation of channel activity by structurally diverse compounds. However, neither the mechanisms by which these compounds potentiate mutant CFTR nor the site(s) where these compounds bind have been identified. This knowledge gap partly reflects the lack of appropriate experimental models to provide clues toward the identification of binding sites. Here, we have compared the channel behavior and response to novel and known potentiators of human CFTR (hCFTR) and murine (mCFTR) expressed in Xenopus oocytes. Both hCFTR and mCFTR were blocked by GlyH-101 from the extracellular side, but mCFTR activity was increased with GlyH-101 applied directly to the cytoplasmic side. Similarly, glibenclamide only exhibited a blocking effect on hCFTR but both blocked and potentiated mCFTR in excised membrane patches and in intact oocytes. The clinically used CFTR potentiator VX-770 transiently increased hCFTR by ∼13% but potentiated mCFTR significantly more strongly. Our results suggest that mCFTR pharmacological sensitivities differ from hCFTR, which will provide a useful tool for identifying the binding sites and mechanism for these potentiators.

  16. Antitumor Activity and Immune Enhancement of Murine Interleukin-23 Expressed in Murine Colon Carcinoma Cells

    Baoen Shan; Jingsheng Hao; Qiaoxia Li; Masatoshi Tagawa


    Interleukin (IL)-23, a cytokine composed of p19 and the p40 subunit of IL-12, can enhance the proliferation of memory T cells and production of IFN-γ from activated T cells. It can also induce antitumor effects in murine model. To further evaluate the antitumor activity and immune enhancement of IL-23 in vivo, murine colon carcinoma cells retrovirally transduced with mIL-23 gene were injected subcutaneously (s.c.) into BALB/c mice.Survival time and tumor volume were observed. LDH release assay, [3H]-TdR incorporation assay and ELISA were used to determine CTL activity, proliferation of splenocytes and level of cytokines, respectively. Number of dendritic cells (DCs) was analyzed by flow cytometry (FCM). IL-23 secreted by Colon26/IL-23 cells suppressed the growth of tumor and prolonged the survival time of mice, enhanced proliferation of splenocytes, CTL activity, and number of DCs. IL-23 also promoted the production of Th1 cytokines such as IFN-γ, IL-12 and TNF-o. However,the level of IL-4 was not enhanced significantly. These data suggested that IL-23 secreted by tumor cells can induce antitumor activity by enhancing immune response.

  17. Effect of Red Wine Polyphenols on the Expression of Transthyretin in Murine Choroid Plexus.

    Tenore, Gian C; Morisco, Filomena; Lembo, Vincenzo; Ritieni, Alberto

    Plasmatic transthyretin may be regarded as a suitable candidate biomarker for the onset, severity, and progression of Alzheimer disease. The aim of the present experimental work was to evaluate the effect of red wine polyphenols (RWPs) on the expression of transthyretin in murine choroid plexus. In contrast to what generally reported in literature for polyphenols, our experimental results indicated a correlation between RWPs assumption and a decrease of transthyretin expression, with a non-dose dependent trend. The present study would point out the attention on the possible pro-oxidant effects of red wine polyphenols at certain doses, although further in vitro, in vivo, and clinical experiments must be performed in order to clarify the mechanisms of action at the base of observed results.

  18. Murine Typhus: An Important Consideration for the Nonspecific Febrile Illness

    Gurjot Basra


    Full Text Available Murine typhus is a widely distributed flea-borne infection caused by Rickettsia typhi. Symptoms of murine typhus are nonspecific and mimic a variety of other infectious diseases. We herein report a case of murine typhus in an area where the broad use of DDT in the mid-20th century has now made it a rare disease. The patient described presented with headache, fever, and a faint macular rash. Initial laboratory studies revealed a slight transaminase elevation. Further questioning revealed exposure to opossums, prompting the consideration of murine typhus as a diagnosis. Although typhus group antibodies were not present during the patient’s acute illness, empiric therapy with doxycycline was initiated, and the patient defervesced. One month after convalescence, the patient returned to clinic with serum that contained typhus group antibodies with an IgG titer of 1 : 1024. Murine typhus is an important consideration during the workup of a patient with a nonspecific febrile illness. Exposure to reservoir hosts and the flea vector place humans at risk for this disease. Clinician recognition of this entity is required for diagnosis and effective therapy.

  19. Analysis of cardiomyocyte movement in the developing murine heart

    Hashimoto, Hisayuki [Department of Cardiology, Keio University School of Medicine, Tokyo (Japan); Yuasa, Shinsuke, E-mail: [Department of Cardiology, Keio University School of Medicine, Tokyo (Japan); Tabata, Hidenori [Department of Anatomy, Keio University School of Medicine, Tokyo (Japan); Tohyama, Shugo; Seki, Tomohisa; Egashira, Toru; Hayashiji, Nozomi; Hattori, Fumiyuki; Kusumoto, Dai; Kunitomi, Akira; Takei, Makoto; Kashimura, Shin; Yozu, Gakuto; Shimojima, Masaya; Motoda, Chikaaki; Muraoka, Naoto [Department of Cardiology, Keio University School of Medicine, Tokyo (Japan); Nakajima, Kazunori [Department of Anatomy, Keio University School of Medicine, Tokyo (Japan); Sakaue-Sawano, Asako; Miyawaki, Atsushi [Life Function and Dynamics, ERATO, JST, 2-1 Hirosawa, Wako-city, Saitama 351-0198 (Japan); Laboratory for Cell Function and Dynamics, Advanced Technology Development Group, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama 351-0198 (Japan); Fukuda, Keiichi [Department of Cardiology, Keio University School of Medicine, Tokyo (Japan)


    The precise assemblage of several types of cardiac precursors controls heart organogenesis. The cardiac precursors show dynamic movement during early development and then form the complicated heart structure. However, cardiomyocyte movements inside the newly organized mammalian heart remain unclear. We previously established the method of ex vivo time-lapse imaging of the murine heart to study cardiomyocyte behavior by using the Fucci (fluorescent ubiquitination-based cell cycle indicator) system, which can effectively label individual G1, S/G2/M, and G1/S-transition phase nuclei in living cardiomyocytes as red, green, and yellow, respectively. Global analysis of gene expression in Fucci green positive ventricular cardiomyocytes confirmed that cell cycle regulatory genes expressed in G1/S, S, G2/M, and M phase transitions were upregulated. Interestingly, pathway analysis revealed that many genes related to the cell cycle were significantly upregulated in the Fucci green positive ventricular cardiomyocytes, while only a small number of genes related to cell motility were upregulated. Time-lapse imaging showed that murine proliferating cardiomyocytes did not exhibit dynamic movement inside the heart, but stayed on site after entering the cell cycle. - Highlights: • We directly visualized cardiomyocyte movement inside the developing murine heart. • Cell cycle related genes were upregulated in the proliferating cardiomyocytes. • Time-lapse imaging revealed that proliferating murine cardiomyocytes stayed in place. • Murine ventricular cardiomyocytes proliferate on site during development.

  20. Apoptosis and the thymic microenvironment in murine lupus.

    Takeoka, Y; Taguchi, N; Shultz, L; Boyd, R L; Naiki, M; Ansari, A A; Gershwin, M E


    The thymus of New Zealand black (NZB) mice undergoes premature involution. In addition, cultured thymic epithelial cells from NZB mice undergo accelerated preprogrammed degeneration. NZB mice also have distinctive and well-defined abnormalities of thymic architecture involving stromal cells, defined by staining with monoclonal antibodies specific for the thymic microenvironment. We took advantage of these findings, as well as our large panel of monoclonal antibodies which recognize thymic stroma, to study the induction of apoptosis in the thymus of murine lupus and including changes of epithelial architecture. We studied NZB, MRL/lpr, BXSB/Yaa, C3H/gld mice and BALB/c and C57BL/6 as control mice. Apoptosis was studied both at basal levels and following induction with either dexamethasone or lipopolysaccharide (LPS). The apoptotic cells were primarily found in the thymic cortex, and the frequency of apoptosis in murine lupus was less than 20% of controls. Moreover, all strains of murine lupus had severe abnormalities of the cortical network. These changes were not accentuated by dexamethasone treatment in cultured thymocytes. However, the thymus in murine lupus was less susceptible to LPS-induced apoptosis than control mice. Finally we note that the number of thymic nurse cells (TNC) was lowest in NZB mice. Our findings demonstrate significant abnormalities in the induction of apoptosis and the formation of TNC-like epithelial cells in SLE mice, and suggest that the abnormalities of the thymic microenvironment have an important role in the pathogenesis of murine lupus.

  1. Aminoglycoside ototoxicity in three murine strains and effects on NKCC1 of stria vascularis

    CHU Han-qi; XIONG Hao; Zhou Xiao-qin; HAN Fang; WU Zhen-gong; ZHANG Ping; HUANG Xiao-wen; CUI Yong-hua


    Background After establishing a murine model of aminoglycoside antibiotic (AmAn) induced ototoxicity, the sensitivity of AmAn induced ototoxicity in three murine strains and the effect of kanamycin on the expression of Na-K-2C1 cotransporter-1 (NKCC 1) in stria vascularis were investigated.Methods C57BL/6J, CBA/CaJ, NKCC1+/- mice (24 of each strain) were randomly divided into four experimental groups: A: kanamycin alone; B: kanamycin plus 2,3-dihydroxybenzoate; C: 2,3-dihydroxybenzoate alone; and D: control group. Mice were injected with kanamycin or/and 2,3-dihydroxybenzoate twice daily for 14 days. Auditory brainstem response (ABR) was measured and morphology of cochlea delineated with succinate dehydrogenase staining. Expression of NKCC1 in stria vascularis was detected immunohistochemically.Results All three strains in groups A and B developed significant ABR threshold shifts (P<0.01), which were accompanied by outer hair cell loss. NKCC 1 expression in stria vascularis was the weakest in group A (A cf D,P<0.01) and the strongest in groups C and D (P<0.05). CBA/CaJ mice had the highest sensitivity to AmAn.Conclusions Administration of kanamycin established AmAn induced ototoxicity. Kanamycin inhibited the expression of NKCC1 in stria vascularis. 2, 3-dihydroxybenzoate attenuated AmAn induced ototoxicitypossibly by enhancing the expression of NKCC1. Age related hearing loss did not show additional sensitivity to AmAn induced ototoxicity in murine model.

  2. Integrating murine gene expression studies to understand obstructive lung disease due to chronic inhaled endotoxin.

    Peggy S Lai

    Full Text Available RATIONALE: Endotoxin is a near ubiquitous environmental exposure that that has been associated with both asthma and chronic obstructive pulmonary disease (COPD. These obstructive lung diseases have a complex pathophysiology, making them difficult to study comprehensively in the context of endotoxin. Genome-wide gene expression studies have been used to identify a molecular snapshot of the response to environmental exposures. Identification of differentially expressed genes shared across all published murine models of chronic inhaled endotoxin will provide insight into the biology underlying endotoxin-associated lung disease. METHODS: We identified three published murine models with gene expression profiling after repeated low-dose inhaled endotoxin. All array data from these experiments were re-analyzed, annotated consistently, and tested for shared genes found to be differentially expressed. Additional functional comparison was conducted by testing for significant enrichment of differentially expressed genes in known pathways. The importance of this gene signature in smoking-related lung disease was assessed using hierarchical clustering in an independent experiment where mice were exposed to endotoxin, smoke, and endotoxin plus smoke. RESULTS: A 101-gene signature was detected in three murine models, more than expected by chance. The three model systems exhibit additional similarity beyond shared genes when compared at the pathway level, with increasing enrichment of inflammatory pathways associated with longer duration of endotoxin exposure. Genes and pathways important in both asthma and COPD were shared across all endotoxin models. Mice exposed to endotoxin, smoke, and smoke plus endotoxin were accurately classified with the endotoxin gene signature. CONCLUSIONS: Despite the differences in laboratory, duration of exposure, and strain of mouse used in three experimental models of chronic inhaled endotoxin, surprising similarities in gene

  3. A murine herpesvirus closely related to ubiquitous human herpesviruses causes T-cell depletion.

    Patel, Swapneel J; Zhao, Guoyan; Penna, Vinay R; Park, Eugene; Lauron, Elvin J; Harvey, Ian B; Beatty, Wandy L; Plougastel-Douglas, Beatrice; Poursine-Laurent, Jennifer; Fremont, Daved H; Wang, David; Yokoyama, Wayne M


    Mouse models of human herpesvirus infections The human roseoloviruses HHV6A, HHV6B, and HHV7 comprise the Roseolovirus genus of the human Betaherpesvirinae subfamily. Infections with these viruses have been implicated in many diseases; however, it has been challenging to establish infections with Roseoloviruses as direct drivers of pathology because they are nearly ubiquitous and display species-specific tropism. Furthermore, controlled study of infection has been hampered by the lack of experimental models, and until now, a mouse roseolovirus has not been identified. Herein we describe a virus that causes severe thymic necrosis in neonatal mice, characterized by a loss of CD4(+) T-cells. These phenotypes resemble those caused by the previously described mouse thymic virus (MTV), a putative herpesvirus that has not been molecularly characterized. By Next Generation sequencing of infected tissue homogenates, we assembled a contiguous 174Kb genome sequence encoding 128 unique predicted open reading frames (ORFs), many of which were most closely related to herpesvirus genes. Moreover, the structure of the virus genome and phylogenetic analysis of multiple genes strongly suggested that this virus is a betaherpesvirus more closely related to the roseoloviruses, HHV6A, HHV6B, and HHV7, than another murine betaherpesvirus, mouse cytomegalovirus (MCMV). As such, we have named this virus murine roseolovirus (MRV) because these data strongly suggest that MRV is a mouse homolog of HHV6A/HHV6B/HHV7.Importance: Herein we describe the complete genome sequence of a novel murine herpesvirus. By sequence and phylogenetic analyses, we show that it is a betaherpesvirus most closely related to the roseoloviruses, human herpesvirus 6A, 6B, and 7. These data combined with physiological similarities with human roseoloviruses collectively suggest that this virus is a murine roseolovirus (MRV), the first definitively described rodent roseolovirus, to our knowledge. Many biological and

  4. A rapid murine coma and behavior scale for quantitative assessment of murine cerebral malaria.

    Ryan W Carroll

    Full Text Available BACKGROUND: Cerebral malaria (CM is a neurological syndrome that includes coma and seizures following malaria parasite infection. The pathophysiology is not fully understood and cannot be accounted for by infection alone: patients still succumb to CM, even if the underlying parasite infection has resolved. To that effect, there is no known adjuvant therapy for CM. Current murine CM (MCM models do not allow for rapid clinical identification of affected animals following infection. An animal model that more closely mimics the clinical features of human CM would be helpful in elucidating potential mechanisms of disease pathogenesis and evaluating new adjuvant therapies. METHODOLOGY/PRINCIPAL FINDINGS: A quantitative, rapid murine coma and behavior scale (RMCBS comprised of 10 parameters was developed to assess MCM manifested in C57BL/6 mice infected with Plasmodium berghei ANKA (PbA. Using this method a single mouse can be completely assessed within 3 minutes. The RMCBS enables the operator to follow the evolution of the clinical syndrome, validated here by correlations with intracerebral hemorrhages. It provides a tool by which subjects can be identified as symptomatic prior to the initiation of trial treatment. CONCLUSIONS/SIGNIFICANCE: Since the RMCBS enables an operator to rapidly follow the course of disease, label a subject as affected or not, and correlate the level of illness with neuropathologic injury, it can ultimately be used to guide the initiation of treatment after the onset of cerebral disease (thus emulating the situation in the field. The RMCBS is a tool by which an adjuvant therapy can be objectively assessed.

  5. Effects of murine and human bone marrow-derived mesenchymal stem cells on cuprizone induced demyelination.

    Jasmin Nessler

    Full Text Available For the treatment of patients with multiple sclerosis there are no regenerative approaches to enhance remyelination. Mesenchymal stem cells (MSC have been proposed to exert such regenerative functions. Intravenous administration of human MSC reduced the clinical severity of experimental autoimmune encephalomyelitis (EAE, an animal model mimicking some aspects of multiple sclerosis. However, it is not clear if this effect was achieved by systemic immunomodulation or if there is an active neuroregeneration in the central nervous system (CNS. In order to investigate remyelination and regeneration in the CNS we analysed the effects of intravenously and intranasally applied murine and human bone marrow-derived MSC on cuprizone induced demyelination, a toxic animal model which allows analysis of remyelination without the influence of the peripheral immune system. In contrast to EAE no effects of MSC on de- and remyelination and glial cell reactions were found. In addition, neither murine nor human MSC entered the lesions in the CNS in this toxic model. In conclusion, MSC are not directed into CNS lesions in the cuprizone model where the blood-brain-barrier is intact and thus cannot provide support for regenerative processes.

  6. Dendritic cell-based vaccination in cancer: therapeutic implications emerging from murine models

    Soledad eMac Keon


    Full Text Available Dendritic cells (DCs play a pivotal role in the orchestration of immune responses, and are thus key targets in cancer vaccine design. Since the 2010 FDA approval of the first cancer DC-based vaccine (Sipuleucel T there has been a surge of interest in exploiting these cells as a therapeutic option for the treatment of tumors of diverse origin. In spite of the encouraging results obtained in the clinic, many elements of DC-based vaccination strategies need to be optimized. In this context, the use of experimental cancer models can help direct efforts towards an effective vaccine design. This paper reviews recent findings in murine models regarding the antitumoral mechanisms of DC-based vaccination, covering issues related to antigen sources, the use of adjuvants and maturing agents, and the role of DC subsets and their interaction in the initiation of antitumoral immune responses. The summary of such diverse aspects will highlight advantages and drawbacks in the use of murine models, and contribute to the design of successful DC-based translational approaches for cancer treatment.

  7. R-phycoerythrin-conjugated antibodies are inappropriate for intracellular staining of murine plasma cells.

    Kim, Myun Soo; Kim, Tae Sung


    Phycoerythrin (PE) is a type of phycobiliproteins found in cyanobacteria and red algae. PE-conjugated antibodies are broadly used for flow cytometry and immunofluorescence microscopy. Because nonspecific binding of antibodies results in decreased analytic accuracy, numerous efforts have been made to unveil cases and mechanisms of nonspecific bindings. However, nonspecific binding of specific cell types by a fluorescent dye-conjugated form of antibody has been rarely reported. In the present study, we discovered that PE-conjugated antibodies, but not FITC- or APC-antibodies, selectively stained lamina propria plasma cells (LP-PCs) from the murine small intestine after membrane permeabilization. We demonstrated that LP-PC-selective staining with PE-antibodies was not due to interactions of antibody-epitope or antibody-Fc receptor. This unexpected staining by PE-antibody was not dependent on the mouse strain of LP-PCs, experimental methods, or origin species of the antibody, but dependent on PE itself. This phenomenon was also observed in plasma cells isolated from bone marrow, spleen, and mesenteric lymph nodes. Furthermore, in vitro activated B cells and in vivo generated LP-PCs were also selectively stained by PE-conjugated antibodies. Taken together, these results show that PE-conjugated antibodies are inappropriate for intracellular staining of murine plasma cells.

  8. Dendritic Cell-Based Vaccination in Cancer: Therapeutic Implications Emerging from Murine Models

    Mac Keon, Soledad; Ruiz, María Sol; Gazzaniga, Silvina; Wainstok, Rosa


    Dendritic cells (DCs) play a pivotal role in the orchestration of immune responses, and are thus key targets in cancer vaccine design. Since the 2010 FDA approval of the first cancer DC-based vaccine (Sipuleucel-T), there has been a surge of interest in exploiting these cells as a therapeutic option for the treatment of tumors of diverse origin. In spite of the encouraging results obtained in the clinic, many elements of DC-based vaccination strategies need to be optimized. In this context, the use of experimental cancer models can help direct efforts toward an effective vaccine design. This paper reviews recent findings in murine models regarding the antitumoral mechanisms of DC-based vaccination, covering issues related to antigen sources, the use of adjuvants and maturing agents, and the role of DC subsets and their interaction in the initiation of antitumoral immune responses. The summary of such diverse aspects will highlight advantages and drawbacks in the use of murine models, and contribute to the design of successful DC-based translational approaches for cancer treatment. PMID:26042126

  9. Standardization, Evaluation, and Area-Under-Curve Analysis of Human and Murine Treg Suppressive Function.

    Akimova, Tatiana; Levine, Matthew H; Beier, Ulf H; Hancock, Wayne W


    FOXP3+ T-regulatory (Treg) cells have important roles in immune homeostasis, and alterations in their number and function can predispose to diseases ranging from autoimmunity to allograft rejection and tumor growth. Reliable identification of human Tregs remains a persistent problem due to a lack of specific markers. The most definitive Treg characterization currently involves combined assessment of phenotypic, epigenetic and functional parameters, with the latter typically involving in vitro Treg suppression assays. Unfortunately, suppression assays are frequently performed using differing methods and readouts, limiting comparisons between studies. We provide a perspective on our experience with human and murine Treg suppression assay conditions, including Treg data obtained in clinical transplant studies, Tregs isolated from healthy donors and treated with epigenetically active compounds, and Tregs from standard murine strains (C57BL/6 and BALB/c). We provide detailed descriptions and illustrations of typical problems, shortcomings and troubleshooting; describe new modifications and approaches; and present a new method for calculation of suppressive assay data using a modified area-under-curve (AUC) method. This method allows us to directly compare Treg suppressive function between multiple patients (such as in clinical transplant studies), to reliably track changes in Treg function from the same person over time, or compare effects of Treg-modulating compounds tested with different healthy donors Tregs in separate or combined experimental settings.

  10. Comparison of LAIR-1 genetic pathways in murine vs human internal organs.

    Sun, Shuqiu; Jiao, Yan; Wei, Wei; Postlethwaite, Arnold E; Gu, Weikuan; Sun, Dianjun


    Growing evidence suggests that defective expression or dysfunction of LAIR-1, a novel immunoinhibitory receptor for collagen, is closely associated with some autoimmune diseases, cancers, as well as viral infections. We analyzed the variation of LAIR-1 genetic pathways in murine versus human internal organs, including the lung and brain. The results showed that, under physiological conditions, LAIR-1 links more closely to the common genes in mouse than in human, which poses tissue specificity. It means that mice experimental data in relation to the role of LAIR-1 immune regulation may be overestimated when applied to assess human conditions. Moreover, we found that the in vivo interaction of LAIR-1 with LAIR-2 rarely occurs, implying that the species difference in LAIR-1 genetic pathways could not be primarily attributed to the existence of human LAIR-2. In summary, this study opens the door for insight into LAIR-1 functions inside the human body, and raises concern as to extrapolative credibility of the murine model in biomedical research.

  11. Activity of an Intralipid formulation of nystatin in murine systemic candidiasis.

    Semis, R; Mendlovic, S; Polacheck, I; Segal, E


    Since nystatin (NYT) is used only topically owing to its toxicity upon systemic administration, a study was initiated aiming to develop a formulation of NYT that could be used systemically against invasive mycoses. The present research is a continuation of previous in vitro investigation of the antifungal effect of nystatin-Intralipid (NYT-IL) against Candida, exploring its in vivo activity. NYT-IL was tested in murine systemic candidiasis induced in naïve as well as cyclophosphamide-immunosuppressed female ICR mice. The infection was assessed by survival rate (SR), mean survival time (MST) and qualitative and quantitative fungal organ colonisation. Mice were treated by intravenous administration of various doses of NYT-IL for 5 consecutive days starting either 24h or 48 h after the initiation of infection. The experiments showed that NYT-IL is therapeutically effective in the murine candidiasis model. NYT-IL was found to be less toxic in vivo than NYT and therefore higher doses of NYT-IL could be used. The efficacy of NYT-IL was expressed in treated naïve and immunosuppressed mice by increased SR, prolonged MST and reduced fungal organ colonisation. Early initiation of treatment increased efficacy. In summary, the Intralipid formulation of NYT can be administered parenterally and is effective against systemic experimental Candida infection.

  12. Anatomy of the Murine Hepatobiliary System: A Whole-Organ-Level Analysis Using a Transparency Method.

    Higashiyama, Hiroki; Sumitomo, Hiroyuki; Ozawa, Aisa; Igarashi, Hitomi; Tsunekawa, Naoki; Kurohmaru, Masamichi; Kanai, Yoshiakira


    The biliary tract is a well-branched ductal structure that exhibits great variation in morphology among vertebrates. Its function is maintained by complex constructions of blood vessels, nerves, and smooth muscles, the so-called hepatobiliary system. Although the mouse (Mus musculus) has been used as a model organism for humans, the morphology of its hepatobiliary system has not been well documented at the topographical level, mostly because of its small size and complexity. To reconcile this, we conducted whole-mount anatomical descriptions of the murine extrahepatic biliary tracts with related blood vessels, nerves, and smooth muscles using a recently developed transparentizing method, CUBIC. Several major differences from humans were found in mice: (1) among the biliary arteries, the arteria gastrica sinistra accessoria was commonly found, which rarely appears in humans; (2) the sphincter muscle in the choledochoduodenal junction is unseparated from the duodenal muscle; (3) the pancreatic duct opens to the bile duct without any sphincter muscles because of its distance from the duodenum. This state is identical to a human congenital malformation, an anomalous arrangement of pancreaticobiliary ducts. However, other parts of the murine hepatobiliary system (such as the branching patterns of the biliary tract, blood vessels, and nerves) presented the same patterns as humans and other mammals topologically. Thus, the mouse is useful as an experimental model for studying the human hepatobiliary system.

  13. Murine model of disseminated fusariosis: evaluation of the fungal burden by traditional CFU and quantitative PCR.

    González, Gloria M; Márquez, Jazmín; Treviño-Rangel, Rogelio de J; Palma-Nicolás, José P; Garza-González, Elvira; Ceceñas, Luis A; Gerardo González, J


    Systemic disease is the most severe clinical form of fusariosis, and the treatment involves a challenge due to the refractory response to antifungals. Treatment for murine Fusarium solani infection has been described in models that employ CFU quantitation in organs as a parameter of therapeutic efficacy. However, CFU counts do not precisely reproduce the amount of cells for filamentous fungi such as F. solani. In this study, we developed a murine model of disseminated fusariosis and compared the fungal burden with two methods: CFU and quantitative PCR. ICR and BALB/c mice received an intravenous injection of 1 × 10(7) conidia of F. solani per mouse. On days 2, 5, 7, and 9, mice from each mice strain were killed. The spleen and kidneys of each animal were removed and evaluated by qPCR and CFU determinations. Results from CFU assay indicated that the spleen and kidneys had almost the same fungal burden in both BALB/c and ICR mice during the days of the evaluation. In the qPCR assay, the spleen and kidney of each mouse strain had increased fungal burden in each determination throughout the entire experiment. The fungal load determined by the qPCR assay was significantly greater than that determined from CFU measurements of tissue. qPCR could be considered as a tool for quantitative evaluation of fungal burden in experimental disseminated F. solani infection.

  14. Rapamycin Inhibits ALDH Activity, Resistance to Oxidative Stress, and Metastatic Potential in Murine Osteosarcoma Cells

    Xiaodong Mu


    Full Text Available Osteosarcoma (OS is the most common primary malignancy of bone. Mortality is determined by the presence of metastatic disease, but little is known regarding the biochemical events that drive metastases. Two murine OS cell lines, K7M2 and K12, are related but differ significantly in their metastatic potentials: K7M2 is highly metastatic whereas K12 displays much less metastatic potential. Using this experimental system, the mammalian target of rapamycin (mTOR pathway has been implicated in OS metastasis. We also discovered that aldehyde dehydrogenase (ALDH, a stem cell marker activity is higher in K7M2 cells than K12 cells. Rapamycin treatment reduces the expression and enzymatic activity of ALDH in K7M2 cells. ALDH inhibition renders these cells more susceptible to apoptotic death when exposed to oxidative stress. Furthermore, rapamycin treatment reduces bone morphogenetic protein-2 (BMP2 and vascular endothelial growth factor (VEGF gene expression and inhibits K7M2 proliferation, migration, and invasion in vitro. Inhibition of ALDH with disulfiram correlated with decreased mTOR expression and activity. In conclusion, we provide evidence for interaction between mTOR activity, ALDH activity, and metastatic potential in murine OS cells. Our work suggests that mTOR and ALDH are therapeutic targets for the treatment and prevention of OS metastasis.

  15. Spontaneous murine lupus-like syndromes. Clinical and immunopathological manifestations in several strains.

    Andrews, B S; Eisenberg, R A; Theofilopoulos, A N; Izui, S; Wilson, C B; McConahey, P J; Murphy, E D; Roths, J B; Dixon, F J


    MRL/1 and BXSB male mice have a systemic lupus erythematosus (SLE)-like disease similar to but more acute than that occurring in NZB X W mice. The common elements of lymphoid hyperplasia, B-cell hyperactivity, autoantibodies, circulating immune complex (IC), complement consumption, IC glomerulonephritis with gp70 deposition, and thymic atrophy were found in all three kinds of SLE mice. On the basis of these common elements, SLE seen in these mice can be considered a single disease in the same sense that human SLE is one disease. The differences in the SLE expressed in the different mice are no greater than those found in an unselected series of humans with SLE. However, the significant quantitative and qualitative variations in abnormal immunologic expression suggest that different constellations of factors, genetic and/or pathophysiologic, may operate in the three murine strains and that each constellation is capable of leading, via its particular abnormal immunologic consequences, to the activation of common immunopathologic effector mechanisms that cause quite similar SLE-like syndromes. From an experimental point of view, the availability of several inbred murine strains of commonplace histocompatibility types that express an SLE-like syndrome makes possible innumerable manipulations which should help to elucidate the nature and cause(s) of this disorder.

  16. Mutational dynamics of murine angiogenin duplicates

    Fares Mario A


    Full Text Available Abstract Background Angiogenin (Ang is a protein involved in angiogenesis by inducing the formation of blood vessels. The biomedical importance of this protein has come from findings linking mutations in Ang to cancer progression and neurodegenerative diseases. These findings highlight the evolutionary constrain on Ang amino acid sequence. However, previous studies comparing human Angiogenin with homologs from other phylogenetically related organisms have led to the conclusion that Ang presents a striking variability. Whether this variability has an adaptive value per se remains elusive. Understanding why many functional Ang paralogs have been preserved in mouse and rat and identifying functional divergence mutations at these copies may explain the relationship between mutations and function. In spite of the importance of testing this hypothesis from the evolutionarily and biomedical perspectives, this remains yet unaccomplished. Here we test the main mutational dynamics driving the evolution and function of Ang paralogs in mammals. Results We analysed the phylogenetic asymmetries between the different Ang gene copies in mouse and rat in the context of vertebrate Ang phylogeny. This analysis shows strong evidence in support of accelerated evolution in some Ang murine copies (mAng. This acceleration is not due to non-functionalisation because constraints on amino acid replacements remain strong. We identify many of the amino acid sites involved in signal localization and nucleotide binding by Ang to have evolved under diversifying selection. Compensatory effects of many of the mutations at these paralogs and their key structural location in or nearby important functional regions support a possible functional shift (functional divergence in many Ang copies. Similarities between 3D-structural models for mAng copies suggest that their divergence is mainly functional. Conclusions We identify the main evolutionary dynamics shaping the variability of

  17. Chemokine (C-C motif ligand 2 mediates direct and indirect fibrotic responses in human and murine cultured fibrocytes

    Ekert Jason E


    Full Text Available Abstract Background Fibrocytes are a population of circulating bone-marrow-derived cells that express surface markers for leukocytes and mesenchymal cells, and are capable of differentiating into myofibroblasts. They have been observed at sites of active fibrosis and increased circulating numbers correlate with mortality in idiopathic pulmonary fibrosis (IPF. Inhibition of chemokine (C-C motif receptor 2 (CCR2 during experimental models of lung fibrosis reduces lung collagen deposition, as well as reducing lung fibrocyte accumulation. The aim of the present study was to determine whether human and mouse fibrocytes express functional CCR2. Results Following optimized and identical human and murine fibrocyte isolation, both cell sources were shown to be positive for CCR2 by flow cytometry and this expression colocalized with collagen I and CD45. Human blood fibrocytes stimulated with the CCR2 ligand chemokine (C-C motif ligand 2 (CCL2, demonstrated increased proliferation (P P P Conclusions This study directly compares the functional responses of human and murine fibrocytes to CCR2 ligands, and following comparable isolation techniques. We have shown comparable biological effects, strengthening the translatability of the murine models to human disease with respect to targeting the CCR2 axis to ameliorate disease in IPF patients.

  18. Human Mesenchymal Stromal Cells Transplantation May Enhance or Inhibit 4T1 Murine Breast Adenocarcinoma through Different Approaches

    T. Jazedje


    Full Text Available The use of Mesenchymal Stromal Cells (MSCs aiming to treat cancer has shown very contradictory results. In an attempt to clarify the contradictory results reported in the literature and the possible role of human fallopian tube Mesenchymal Stromal Cells (htMSCs against breast cancer, the aim of this study was to evaluate the clinical effect of htMSCs in murine mammary adenocarcinoma using two different approaches: (1 coinjections of htMSCs and 4T1 murine tumor cell lineage and (2 injections of htMSCs in mice at the initial stage of mammary adenocarcinoma development. Coinjected animals had a more severe course of the disease and a reduced survival, while tumor-bearing animals treated with 2 intraperitoneal injections of 106 htMSCs showed significantly reduced tumor growth and increased lifespan as compared with control animals. Coculture of htMSCs and 4T1 tumor cells revealed an increase in IL-8 and MCP-1 and decreased VEGF production. For the first time, we show that MSCs isolated from a single source and donor when injected in the same animal model and tumor can lead to opposite results depending on the experimental protocol. Also, our results demonstrated that htMSCs can have an inhibitory effect on the development of murine mammary adenocarcinoma.

  19. Local Expression of Vaginal Th1 and Th2 Cytokines in Murine Vaginal Candidiasis under Different Immunity Conditions

    Shanjuan CHEN; Shaohua LI; Yan WU; Zhixiang LIU; Jiawen LI


    To investigate the expression of vaginal Th1 and Th2 cytokines in rats with experimental vaginal candidiasis under different immune conditions, ICR murine vaginal candidiasis model was established and immno-suppressed murine models of vaginal cadidiasis were established in estrogen-treated mice. Non-estrogen-treated mice were used as controls. The mRNA level of Th1(IL-2)/Th2 (IL-4, IL-10, TGF-β1) cytokines in murine vaginal tissues was determined by RT-PCR.The cykotine in local tissues was increased to different extent under normal immune condition. IL-2mRNA was increased during early stage of infection, while IL-10 was increased transiently during late stage of infection. TGF-β1 production was found to be increased persistently. At same time, the expression of IL-2 mRNA was suppressed in immno-suppressed group, and the level of IL-4, IL-10,and TGF-β1 were higher than the normal immunity group to different degree during infection. The high level of IL-2 mRNA during early stage of infection was associated with clearance of mucosal Candidia albicans (C. albicans), and its expression suppressed leading to decreased clearance of mucosal C. albican in immuno-suppression. The over-expression of IL-4 and IL-10 could significantly enhance the susceptibility to C. albicans infection in mice.

  20. Directed evolution and targeted mutagenesis to murinize listeria monocytogenes internalin A for enhanced infectivity in the murine oral infection model

    Hill Colin


    Full Text Available Abstract Background Internalin A (InlA is a critical virulence factor which mediates the initiation of Listeria monocytogenes infection by the oral route in permissive hosts. The interaction of InlA with the host cell ligand E-cadherin efficiently stimulates L. monocytogenes entry into human enterocytes, but has only a limited interaction with murine cells. Results We have created a surface display library of randomly mutated InlA in a non-invasive heterologous host Lactococcus lactis in order to create and screen novel variants of this invasion factor. After sequential passage through a murine cell line (CT-26, multiple clones with enhanced invasion characteristics were identified. Competitive index experiments were conducted in mice using selected mutations introduced into L. monocytogenes EGD-e background. A novel single amino acid change was identified which enhanced virulence by the oral route in the murine model and will form the basis of further engineering approaches. As a control a previously described EGD-InlAm murinized strain was also re-created as part of this study with minor modifications and designated EGD-e InlAm*. The strain was created using a procedure that minimizes the likelihood of secondary mutations and incorporates Listeria-optimized codons encoding the altered amino acids. L. monocytogenes EGD-e InlAm* yielded consistently higher level murine infections by the oral route when compared to EGD-e, but did not display the two-fold increased invasion into a human cell line that was previously described for the EGD-InlAm strain. Conclusions We have used both site-directed mutagenesis and directed evolution to create variants of InlA which may inform future structure-function analyses of this protein. During the course of the study we engineered a murinized strain of L. monocytogenes EGD-e which shows reproducibly higher infectivity in the intragastric murine infection model than the wild type, but does not display enhanced

  1. Directed evolution and targeted mutagenesis to murinize Listeria monocytogenes Internalin A for enhanced infectivity in the murine oral infection model

    Monk, Ian R


    Abstract Background Internalin A (InlA) is a critical virulence factor which mediates the initiation of Listeria monocytogenes infection by the oral route in permissive hosts. The interaction of InlA with the host cell ligand E-cadherin efficiently stimulates L. monocytogenes entry into human enterocytes, but has only a limited interaction with murine cells. Results We have created a surface display library of randomly mutated InlA in a non-invasive heterologous host Lactococcus lactis in order to create and screen novel variants of this invasion factor. After sequential passage through a murine cell line (CT-26), multiple clones with enhanced invasion characteristics were identified. Competitive index experiments were conducted in mice using selected mutations introduced into L. monocytogenes EGD-e background. A novel single amino acid change was identified which enhanced virulence by the oral route in the murine model and will form the basis of further engineering approaches. As a control a previously described EGD-InlAm murinized strain was also re-created as part of this study with minor modifications and designated EGD-e InlA m*. The strain was created using a procedure that minimizes the likelihood of secondary mutations and incorporates Listeria-optimized codons encoding the altered amino acids. L. monocytogenes EGD-e InlA m* yielded consistently higher level murine infections by the oral route when compared to EGD-e, but did not display the two-fold increased invasion into a human cell line that was previously described for the EGD-InlAm strain. Conclusions We have used both site-directed mutagenesis and directed evolution to create variants of InlA which may inform future structure-function analyses of this protein. During the course of the study we engineered a murinized strain of L. monocytogenes EGD-e which shows reproducibly higher infectivity in the intragastric murine infection model than the wild type, but does not display enhanced entry into human

  2. Pharmacodynamics of Doxycycline in a Murine Malaria Model▿

    Batty, Kevin T.; Law, Angela S. F.; Stirling, Verity; Moore, Brioni R.


    Doxycycline is reported to impair second-generation parasite schizogony. The effects of doxycycline alone and combined with dihydroartemisinin were investigated in a murine malaria model. Doxycycline lowered the rate of parasite growth within 2 days, with maximum effect in 6 days. Addition of dihydroartemisinin led to an additive antimalarial effect.

  3. Immunotherapy of hepatoma with a monoclonal antibody against murine endoglin

    Guang-Hong Tan; Feng-Ying Huang; Hua Wang; Yong-Hao Huang; Ying-Ying Lin; Yue-Nan Li


    AIM: To explore the capability of a monoclonal antibody(mAb) against murine endoglin to inhibit tumor angiogenesis and suppression of hepatoma growth in murine models.METHODS: A monoclonal antibody against murine endoglin was purified by affinity chromatography and passively transfused through tail veins in two murine hepatoma models. Tumor volume and survival time were observed at three-day intervals for 48 d. Microvessels in tumor tissues were detected by immunohistochemistry against CD31, and angiogenesis in vivo was determined by alginate encapsulated assay. In addition, tumor cell apoptosis was detected by TUNEL assay.RESULTS: Passive immunotherapy with anti-endoglin mAb could effectively suppress tumor growth, and prolonged the survival time of hepatoma-bearing mice.Angiogenesis was apparently inhibited within the tumor tissues, and the vascularization of alginate beads was also reduced in the mice passively transfused with antiendoglin mAb. In addition, increased apoptotic cells were observed within the tumor tissues from the mice passively transfused with anti-endoglin mAb.CONCLUSION: Passive immunotherapy with antiendoglin mAb effectively inhibits tumor growth via inhibiting tumor angiogenesis and increasing tumor cell apoptosis, which may be highly correlated with the blockage of endoglin-related signal pathway induced by anti-endoglin mAb.

  4. Murine Sirt3 protein isoforms have variable half-lives

    Sirt3 is a NAD+-dependent protein deacetylase mainly localized in mitochondria. Recent studies indicate that the murine Sirt3 gene expresses different transcript variants resulting in three possible Sirt3 protein isoforms with variable lengths at the N-terminus: M1 (aa 1-334), M2 (aa 15-334), and M3...

  5. Increased rejection of murine allogeneic bone marrow in presensitized recipients

    vanOs, R; deWitte, T; Dillingh, JH; Mauch, PM; Down, JD


    The role of presensitizing murine recipients with donor spleen cells prior to T cell-depleted or -repleted H-2 compatible allogeneic bone marrow transplantation (BMT) was investigated at two different doses of total body irradiation (TBI). Recipients that were presensitized with 2 x 10(7) irradiated

  6. Macropinocytosis is the Entry Mechanism of Amphotropic Murine Leukemia Virus

    Rasmussen, Izabela; Vilhardt, Frederik


    The entry mechanism of murine amphotropic retrovirus (A-MLV) has not been unambiguously determined. We show here that A-MLV does not internalize by caveolae or other pinocytic mechanism, but by macropinocytosis. Thus A-MLV infection of mouse embryonic fibroblasts deficient for caveolin or dynamin...

  7. Immunohistochemical diagnosis of systemic bovine zygomycosis by murine monoclonal antibodies

    Jensen, H.E.; Aalbaek, B.; Lind, Peter


    Murine monoclonal antibodies (Mabs) against water-soluble somatic antigens (WSSA) and the wall fraction (WF) from Rhizopus arrhizus (Rhizopus oryzae) were produced in vitro by fusion of splenocytes from immunized BALB/c mice with mouse myeloma X63-Ag 8.653 cells. Supernatants reacting only with h...... for the in situ diagnosis of systemic bovine zygomycosis....

  8. Expression of biologically active murine interleukin-18 in Lactococcus lactis.

    Feizollahzadeh, Sadegh; Khanahmad, Hossein; Rahimmanesh, Ilnaz; Ganjalikhani-Hakemi, Mazdak; Andalib, Alireza; Sanei, Mohammad Hossein; Rezaei, Abbas


    The food-grade bacterium Lactococcus lactis is increasingly used for heterologous protein expression in therapeutic and industrial applications. The ability of L. lactis to secrete biologically active cytokines may be used for the generation of therapeutic cytokines. Interleukin (IL)-18 enhances the immune response, especially on mucosal surfaces, emphasizing its therapeutic potential. However, it is produced as an inactive precursor and has to be enzymatically cleaved for maturation. We genetically manipulated L. lactis to secrete murine IL-18. The mature murine IL-18 gene was inserted downstream of a nisin promoter in pNZ8149 plasmid and the construct was used to transform L. lactis NZ3900. The transformants were selected on Elliker agar and confirmed by restriction enzyme digestion and sequencing. The expression and secretion of IL-18 protein was verified by SDS-PAGE, western blotting and ELISA. The biological activity of recombinant IL-18 was determined by its ability to induce interferon (IFN)-γ production in L. lactis co-cultured with murine splenic T cells. The amounts of IL-18 in bacterial lysates and supernatants were 3-4 μg mL(-1) and 0.6-0.7 ng mL(-1), respectively. The successfully generated L. lactis strain that expressed biologically active murine IL-18 can be used to evaluate the possible therapeutic effects of IL-18 on mucosal surfaces.

  9. Herpesvirus delivery to the murine respiratory tract.

    Tan, Cindy S E; Frederico, Bruno; Stevenson, Philip G


    Herpesvirus transmission is sporadic, and infection may be asymptomatic or present only with secondary lesions after dissemination. Consequently host entry remains ill-understood. Experimental infections can be informative, but depend on inoculations that are inherently artificial and so need validation. Mice are a widely used experimental host. Alert mice inhale readily small (5 μl) liquid volumes, and Indian ink, luciferase or radiolabel delivered thus distributed to the nasopharynx and oropharynx. Murid Herpesvirus-4 or Herpes simplex virus type 1 delivered thus infected only the nose, arguing that host entry is nasal rather than oral. Marker or virus delivery to the lung depended on general anesthesia and a large inoculum volume (30 μl), and so needs further validation of physiological relevance. While lungs could be infected at lower doses than the upper respiratory tract, tracking experiments showed that nasal inocula pass mostly into the oropharynx, even when restricted to 1 μl. Thus, the relative inefficiency of experimental upper respiratory tract infection was attributable to limited liquid retention in this site. Nonetheless low volume intranasal delivery to alert mice provides a convenient way to model experimentally an apparently natural mode of herpesvirus host entry.

  10. [Anti-Candida activity of aroma candy and its protective activity against murine oral candidiasis].

    Hayama, Kazumi; Takahashi, Miki; Suzuki, Motofumi; Ezawa, Kunio; Yamazaki, Masatoshi; Matsukawa, Taiji; Kishi, Akinobu; Sato, Nobuya; Abe, Shigeru


    A daily eatable candy that has possible protective activity against oral candidiasis was experimentally produced. The candy was made from reduced-maltose as main constituent and from several natural products, such as oligonol (depolymerized polyphenols derived from lychee), cinnamon (cassia), citral, and capric acid, which are known to have anti-Candida activity in vitro and in vivo. The candy effectively inhibited the mycelial growth of C. albicans, even when it was diluted 1,000 times with culture media. We assessed the protective activity of the candy against murine candidiasis. When 50μl of candy dissolved and diluted 4 times with water was administered 3 times into the oral cavity of Candida infected mice, the score of lesions on the Candida-infected tongues improved on day 2. These findings suggest that this candy has potential as food that provides protective activity against oral candidiasis.

  11. Overexpression of mclca3 in airway epithelium of asthmatic murine models with airway inflammation

    ZHANG Hui-lan; HE Li


    Asthma is a worldwide prevalent disease that is a considerable health burden in many countries.1 In recent years, the airway epithelium is increasingly recognized as a central contributor to the pathogenesis of asthma.2 One of the most highly induced genes in epithelial cells in experimental allergic airway disease is the third murine calcium-activated chloride channel homologue (mclca3, alias gob-5). Its human homology protein is hCLCA1,3,4 which has been identified as clinically relevant molecules in diseases with secretory dysfunctions including asthma and cystic fibrosis. In initial studies, mclca3 was thought to be a member of calcium-activated chloride channel (CaCCs) family,whereas some new interesting reports suggest that the two mclca3 cleavage products cannot form an anion channel on their own but may instead act as extracellular signaling molecules with as yet unknown functions and interacting partners.5

  12. Angiogenesis gene expression in murine endothelial cells during post-pneumonectomy lung growth

    Konerding Moritz A


    Full Text Available Abstract Although blood vessel growth occurs readily in the systemic bronchial circulation, angiogenesis in the pulmonary circulation is rare. Compensatory lung growth after pneumonectomy is an experimental model with presumed alveolar capillary angiogenesis. To investigate the genes participating in murine neoalveolarization, we studied the expression of angiogenesis genes in lung endothelial cells. After left pneumonectomy, the remaining right lung was examined on days 3, 6, 14 and 21days after surgery and compared to both no surgery and sham thoracotomy controls. The lungs were enzymatically digested and CD31+ endothelial cells were isolated using flow cytometry cell sorting. The transcriptional profile of the CD31+ endothelial cells was assessed using quantitative real-time polymerase chain reaction (PCR arrays. Focusing on 84 angiogenesis-associated genes, we identified 22 genes with greater than 4-fold regulation and significantly enhanced transcription (p

  13. Baicalin Inhibits IL-17-Mediated Joint Inflammation in Murine Adjuvant-Induced Arthritis

    Xue Yang


    Full Text Available T-helper-17 (Th17 cells are implicated in a number of inflammatory disorders including rheumatoid arthritis. Antagonism of Th17 cells is a treatment option for arthritis. Here, we report that Baicalin, a compound isolated from the Chinese herb Huangqin (Scutellaria baicalensis Georgi, relieved ankle swelling and protected the joint against inflammatory destruction in a murine adjuvant-induced arthritis model. Baicalin inhibited splenic Th17 cell population expansion in vivo. Baicalin prevented interleukin- (IL- 17-mediated lymphocyte adhesion to cultured synoviocytes. Baicalin also blocked IL-17-induced intercellular adhesion molecule 1, vascular cell adhesion molecule 1, IL-6, and tumor necrosis factor-alpha mRNA expression in cultured synoviocytes. Collectively, these findings suggest that Baicalin downregulates the joint inflammation caused by IL-17, which is likely produced by an expanded population of splenic Th17 cells in experimental arthritis. Baicalin might be a promising novel therapeutic agent for treating rheumatoid arthritis in humans.

  14. Directed evolution and targeted mutagenesis to murinize Listeria monocytogenes internalin A for enhanced infectivity in the murine oral infection model.

    Monk, Ian R


    Internalin A (InlA) is a critical virulence factor which mediates the initiation of Listeria monocytogenes infection by the oral route in permissive hosts. The interaction of InlA with the host cell ligand E-cadherin efficiently stimulates L. monocytogenes entry into human enterocytes, but has only a limited interaction with murine cells.

  15. Increased Cerebral Tff1 Expression in Two Murine Models of Neuroinflammation

    Eva B Znalesniak


    Full Text Available Background/Aims: The trefoil factor family (TFF peptide TFF1 is a typical secretory product of the gastric mucosa and a very low level of expression occurs in nearly all regions of the murine brain. TFF1 possesses a lectin activity and binding to a plethora of transmembrane glycoproteins could explain the diverse biological effects of TFF1 (e.g., anti-apoptotic effect. It was the aim to test whether TFF expression is changed during neuroinflammation. Methods: Expression profiling was performed using semi-quantitative RT-PCR analyses in two murine models of neuroinflammation, i.e. Toxoplasma gondii-induced encephalitis and experimental autoimmune encephalomyelitis (EAE, the latter being the most common animal model of multiple sclerosis. Tff1 expression was also localized using RNA in situ hybridization histochemistry. Results: We report for the first time on a significant transcriptional induction in cerebral Tff1 expression in both T. gondii-induced encephalitis and EAE. In contrast, Tff2 and Tff3 expression were not altered. Tff1 transcripts were predominantly localized in the internal granular layer of the cerebellum indicating neuronal expression. Furthermore, also glial cells are expected to express Tff1. Characterization of both experimental models by expression profiling (e.g., inflammasome sensors, inflammatory cytokines, microglial marker Iba1, ependymin related protein 1 revealed differences concerning the expression of the inflammasome sensor Nlrp1 and interleukin 17a. Conclusion: The up-regulated expression of Tff1 is probably the result of a complex inflammatory process as its expression is induced by tumor necrosis factor α as well as interleukins 1β and 17. However on the transcript level, Tff1KO mice did not show any significant signs of an altered immune response after infection with T. gondii in comparison with the wild type animals.

  16. Cytokines and Immune Responses in Murine Atherosclerosis.

    Kusters, Pascal J H; Lutgens, Esther


    Atherosclerosis is an inflammatory disease of the vessel wall characterized by activation of the innate immune system, with macrophages as the main players, as well as the adaptive immune system, characterized by a Th1-dominant immune response. Cytokines play a major role in the initiation and regulation of inflammation. In recent years, many studies have investigated the role of these molecules in experimental models of atherosclerosis. While some cytokines such as TNF or IFNγ clearly had atherogenic effects, others such as IL-10 were found to be atheroprotective. However, studies investigating the different cytokines in experimental atherosclerosis revealed that the cytokine system is complex with both disease stage-dependent and site-specific effects. In this review, we strive to provide an overview of the main cytokines involved in atherosclerosis and to shed light on their individual role during atherogenesis.

  17. Evaluation of the antitumor activity of interleukin-12 in an experimental murine model of colorectal cancer induced by 1,2 dimethylhydrazine (DMH Estudio de la respuesta antitumoral de la interleucina-12 en cáncer de colon inducido mediante 1,2-dimetilhidracina (DMH

    S. Coca


    I (p=0.028 and group III (p = 0.019. Other parameters measured, such as biggest tumor size and total tumoral volume were found to be lower in group II, although no statistical differences were found between groups. Only 10% of rats in group I showed moderated/extensive NK cell infiltration, vs. 60% of rats in group II (p = 0.077 and 70% in group III (p = 0.02. Conclusion: The administration of DMH to rodents provides a reliable and consistent means of inducing CRC that may be suitable for the evaluation of anti-cancer therapies. Our findings suggest that IL-12 is effective against the development of experimental CRC. Its antineoplastic effect could be attributed to the stimulus of this cytokine on the intratumoral infiltration of NK cells.Objetivo: la interleucina (IL-12 es una citocina que estimula la proliferación y la actividad citotóxica de los linfocito T y las células natural killer (NK. En trabajos previos se ha observado una relación entre la infiltración intratumoral de células NK y una mayor supervivencia en carcinomas colorrectales (CCR. Existen evidencias de un efecto aditivo en la actividad inmunomoduladora de la asociación de IL-12 con IL-2. Así, nos hemos propuesto el estudio de la capacidad de respuesta inmune antitumoral, tras la administración sistémica de IL-12 sola o combinada con IL-2, en un modelo experimental de CCR inducidos mediante la administración de 1,2-dimetilhidracina (DMH. Método: sesenta y cinco ratas Wistar de 6 semanas a las que se administró en inyección subcutánea una dosis semanal de DMH a razón de 20 mg/kg de peso durante 26 semanas. Finalizado el periodo de inducción, los animales se distribuyeron aleatoriamente en tres grupos. I: grupo control. Grupo II, se administró IL-12 recombinante murina. Grupo III: se administró IL-12, combinada con IL-2. Las ratas se sacrificaron en la semana 30, estudiándose los siguientes parámetros: número y localización de tumores, tamaño y carga tumoral. Se realiz

  18. Broadband acoustic properties of a murine skull.

    Estrada, Héctor; Rebling, Johannes; Turner, Jake; Razansky, Daniel


    It has been well recognized that the presence of a skull imposes harsh restrictions on the use of ultrasound and optoacoustic techniques in the study, treatment and modulation of the brain function. We propose a rigorous modeling and experimental methodology for estimating the insertion loss and the elastic constants of the skull over a wide range of frequencies and incidence angles. A point-source-like excitation of ultrawideband acoustic radiation was induced via the absorption of nanosecond duration laser pulses by a 20 μm diameter microsphere. The acoustic waves transmitted through the skull are recorded by a broadband, spherically focused ultrasound transducer. A coregistered pulse-echo ultrasound scan is subsequently performed to provide accurate skull geometry to be fed into an acoustic transmission model represented in an angular spectrum domain. The modeling predictions were validated by measurements taken from a glass cover-slip and ex vivo adult mouse skulls. The flexible semi-analytical formulation of the model allows for seamless extension to other transducer geometries and diverse experimental scenarios involving broadband acoustic transmission through locally flat solid structures. It is anticipated that accurate quantification and modeling of the skull transmission effects would ultimately allow for skull aberration correction in a broad variety of applications employing transcranial detection or transmission of high frequency ultrasound.

  19. Effects of the murine skull in optoacoustic brain microscopy.

    Kneipp, Moritz; Turner, Jake; Estrada, Héctor; Rebling, Johannes; Shoham, Shy; Razansky, Daniel


    Despite the great promise behind the recent introduction of optoacoustic technology into the arsenal of small-animal neuroimaging methods, a variety of acoustic and light-related effects introduced by adult murine skull severely compromise the performance of optoacoustics in transcranial imaging. As a result, high-resolution noninvasive optoacoustic microscopy studies are still limited to a thin layer of pial microvasculature, which can be effectively resolved by tight focusing of the excitation light. We examined a range of distortions introduced by an adult murine skull in transcranial optoacoustic imaging under both acoustically- and optically-determined resolution scenarios. It is shown that strong low-pass filtering characteristics of the skull may significantly deteriorate the achievable spatial resolution in deep brain imaging where no light focusing is possible. While only brain vasculature with a diameter larger than 60 µm was effectively resolved via transcranial measurements with acoustic resolution, significant improvements are seen through cranial windows and thinned skull experiments.

  20. T Cell Integrin Overexpression as a Model of Murine Autoimmunity

    Yung Raymond L.


    Full Text Available Integrin adhesion molecules have important adhesion and signaling functions. They also play a central role in the pathogenesis of many autoimmune diseases. Over the past few years we have described a T cell adoptive transfer model to investigate the role of T cell integrin adhesion molecules in the development of autoimmunity. This report summarizes the methods we used in establishing this murine model. By treating murine CD4+ T cells with DNA hypomethylating agents and by transfection we were able to test the in vitro effects of integrin overexpression on T cell autoreactive proliferation, cytotoxicity, adhesion and trafficking. Furthermore, we showed that the ability to induce in vivo autoimmunity may be unique to the integrin lymphocyte function associated antigen-1 (LFA-1.

  1. Antibody responses against xenotropic murine leukemia virus-related virus envelope in a murine model.

    Natalia Makarova

    Full Text Available BACKGROUND: Xenotropic murine leukemia virus-related virus (XMRV was recently discovered to be the first human gammaretrovirus that is associated with chronic fatigue syndrome and prostate cancer (PC. Although a mechanism for XMRV carcinogenesis is yet to be established, this virus belongs to the family of gammaretroviruses well known for their ability to induce cancer in the infected hosts. Since its original identification XMRV has been detected in several independent investigations; however, at this time significant controversy remains regarding reports of XMRV detection/prevalence in other cohorts and cell type/tissue distribution. The potential risk of human infection, coupled with the lack of knowledge about the basic biology of XMRV, warrants further research, including investigation of adaptive immune responses. To study immunogenicity in vivo, we vaccinated mice with a combination of recombinant vectors expressing codon-optimized sequences of XMRV gag and env genes and virus-like particles (VLP that had the size and morphology of live infectious XMRV. RESULTS: Immunization elicited Env-specific binding and neutralizing antibodies (NAb against XMRV in mice. The peak titers for ELISA-binding antibodies and NAb were 1:1024 and 1:464, respectively; however, high ELISA-binding and NAb titers were not sustained and persisted for less than three weeks after immunizations. CONCLUSIONS: Vaccine-induced XMRV Env antibody titers were transiently high, but their duration was short. The relatively rapid diminution in antibody levels may in part explain the differing prevalences reported for XMRV in various prostate cancer and chronic fatigue syndrome cohorts. The low level of immunogenicity observed in the present study may be characteristic of a natural XMRV infection in humans.

  2. Integrin Based Isolation Enables Purification of Murine Lineage Committed Cardiomyocytes.

    Laura Tarnawski

    Full Text Available In contrast to mature cardiomyocytes which have limited regenerative capacity, pluripotent stem cells represent a promising source for the generation of new cardiomyocytes. The tendency of pluripotent stem cells to form teratomas and the heterogeneity from various differentiation stages and cardiomyocyte cell sub-types, however, are major obstacles to overcome before this type of therapy could be applied in a clinical setting. Thus, the identification of extracellular markers for specific cardiomyocyte progenitors and mature subpopulations is of particular importance. The delineation of cardiomyocyte surface marker patterns not only serves as a means to derive homogeneous cell populations by FACS, but is also an essential tool to understand cardiac development. By using single-cell expression profiling in early mouse embryonic hearts, we found that a combination of integrin alpha-1, alpha-5, alpha-6 and N-cadherin enables isolation of lineage committed murine cardiomyocytes. Additionally, we were able to separate trabecular cardiomyocytes from solid ventricular myocardium and atrial murine cells. These cells exhibit expected subtype specific phenotype confirmed by electrophysiological analysis. We show that integrin expression can be used for the isolation of living, functional and lineage-specific murine cardiomyocytes.

  3. A GPR18-based signalling system regulates IOP in murine eye

    Caldwell, Meggie D; Hu, Sherry Shu-Jung; Viswanathan, Suresh; Bradshaw, Heather; Kelly, Melanie EM; Straiker, Alex


    Background and Purpose GPR18 is a recently deorphaned lipid receptor that is activated by the endogenous lipid N-arachidonoyl glycine (NAGly) as well the behaviourally inactive atypical cannabinoid, abnormal cannabidiol (Abn-CBD). The presence and/or function of any GPR18-based ocular signalling system remain essentially unstudied. The objectives of this research are: (i) to determine the disposition of GPR18 receptors and ligands in anterior murine eye, (ii) examine the effect of GPR18 activation on intraocular pressure (IOP) in a murine model, including knockout mice for CB1, CB2 and GPR55. Experimental Approach IOP was measured in mice following topical application of Abn-CBD, NAGly or the GPR55/GPR18 agonist O-1602, alone or with injection of the GPR18 antagonist, O-1918. GPR18 protein localization was assessed with immunohistochemistry. Endocannabinoids were measured using LC/MS-MS. Key Results GPR18 protein was expressed most prominently in the ciliary epithelium and the corneal epithelium and, interestingly, in the trabecular meshwork. The GPR18 ligand, NAGly, was also detected in mouse eye at a level comparable to that seen in the brain. Abn-CBD and NAGly, but not O-1602, significantly reduced IOP in all mice tested. The antagonist, O-1918, blocked the effects of Abn-CBD and NAGly. Conclusions and Implications We present evidence for a functional GPR18-based signalling system in the murine anterior eye, including receptors and ligands. GPR18 agonists, Abn-CBD and NAGly, reduce IOP independently of CB1, CB2 or GPR55. These findings suggest that GPR18 may serve as a desirable target for the development of novel ocular hypotensive medications. Linked Articles This article is part of a themed section on Cannabinoids. To view the other articles in this section visit & PMID:23461720

  4. Optimal in vitro culture conditions for murine predominant immature CD8a+ dendritic cells

    NA Ning; XU Lin; CAO Kai-yuan; LUO Yun; YUAN Guang-qing; XIANG Peng; HONG Liang-qing; LI Shu-nong


    Background The prospects of using immature CD8a+ dendritic cells (DC2) to establish transplant immunologic tolerance and treatments for autoimmune diseases in the future are promising. However, the methods for inducing DC2 are still being explored. The present study was aimed to investigate the optimal in vitro conditions for preparing large numbers f predominant DC2 from murine bone marrow cells.Methods Three groups of bone marrow cells cultured under different conditions were examined, namely a cytokine-induced experimental group (cytokine group), a control group with a low concentration of granulocyte-macrophage colony stimulating factor (GM-CSF, low GM-CSF group) and a control group without ndogenous cytokines. The cytokine group was cultured with 5 ng/ml GM-CSF, 25 ng/ml Fit3 ligand (Flt3L), 20 ng/ml interleukin 4 (IL-4) and 100 ng/ml stem cell factor (SCF). The low GM-CSF control group was cultured with 0.4 ng/ml GM-CSF, 25 ng/ml FIt3L and 100 ng/ml SCF, without IL-4. The control group without exogenous cytokines was cultured without dditional cytokines. All cells were cultured at 37℃ under 5% CO2. On days 3, 7 and 16, 4-color flow cytometry was carried out to analyze the cell phenotypes, and the total cell numbers were counted to analyze the cell yields. Phase-contrast microscopy was used to observe the cell morphologies.Results The cytokine group exhibited higher proportions f typical immature CD8a+ DC, especially on day 3, but the total cell number and DC2 proportion decreased during prolonged culture. The low GM-CSF control group showed the same tendencies as the cytokine group on days 16 and 22, but produced higher total cell numbers (P <0.05) with lower DC2 proportions and cell numbers. The control group without exogenous cytokines spontaneously generated a certain proportion of DC2, but with low total cell and DC2 numbers that decreased rapidly, especially during prolonged culture (days 7 and 16, P <0.05).Conclusions Culture in the presence of 5 ng

  5. Intradermal Infection Model for Pathogenesis and Vaccine Studies of Murine Visceral Leishmaniasis


    The levels of protection found in vaccine studies of murine visceral leishmaniasis are significantly lower than for cutaneous leishmaniasis; whether this is due to the high-challenge murine model employed and/or is a consequence of differences required in tissue-specific local immune responses is not understood. Consequently, an intradermal murine model of visceral leishmaniasis has been explored. Intradermal inoculation established a chronic infection in susceptible mice which was associated...

  6. Monopoly experimentation


    This paper considers a firm facing an uncertain demand curve. The firm can experimentally adjust its output in order to gain information that willincrease expected future profits. We examine two basic questions. Under whatconditions is it worthwhile for the firm to experiment? How does the firmadjust its output away from the myopic optimism to exploit its ability to experiment? Two necessary conditions are established for experimentation tooccur, involving requirements that experimentation be...

  7. Microbiome and Asthma: What Have Experimental Models Already Taught Us?

    R. Bonamichi-Santos


    Full Text Available Asthma is a chronic inflammatory disease that imposes a substantial burden on patients, their families, and the community. Although many aspects of the pathogenesis of classical allergic asthma are well known by the scientific community, other points are not yet understood. Experimental asthma models, particularly murine models, have been used for over 100 years in order to better understand the immunopathology of asthma. It has been shown that human microbiome is an important component in the development of the immune system. Furthermore, the occurrence of many inflammatory diseases is influenced by the presence of microbes. Again, experimental models of asthma have helped researchers to understand the relationship between the microbiome and respiratory inflammation. In this review, we discuss the evolution of murine models of asthma and approach the major studies involving the microbiome and asthma.

  8. Combination therapy with metformin and coenzyme Q10 in murine experimental autoimmune arthritis.

    Jhun, JooYeon; Lee, SeungHoon; Kim, Se-Young; Na, Hyun Sik; Kim, Eun-Kyung; Kim, Jae-Kyung; Jeong, Jeong-Hee; Park, Sung Hwan; Cho, Mi-La


    Metformin (Met) and coenzyme Q10 (CoQ10) are reported to have therapeutic functions in several inflammatory diseases. These drugs have shown anti-inflammatory effects and have been utilized in mouse models of rheumatoid arthritis (RA). However, there is no evidence of the additive effect of Met and CoQ10 in RA. Although Met and CoQ10 may be involved in the improvement of mitochondrial dysfunction, limited information is available regarding whether this effect can improve mitochondrial dysfunction in RA in particular. In this study, we sought to determine whether Met and CoQ10 attenuate the severity of collagen-induced arthritis (CIA) and show an additive effect in a mouse model. The combination of Met and CoQ10 improved CIA, reducing joint inflammation, Th17 differentiation and IgG production. In contrast, the combination of Met and CoQ10 induced Treg differentiation. Osteoclastogenesis was reduced by the combination of Met and CoQ10. The protein expression of interleukin-1β, interleukin-6 and tumor necrosis factor-alpha in mice splenocytes exposed to lipopolysaccharide decreased after drug combination therapy. We also found that the expression of JC-1 and COX IV were enhanced by treatment with the combination of Met and CoQ10. Moreover, the combination of Met and CoQ10 promoted mitochondrial O2 consumption. These findings suggest that the combination of Met and CoQ10 reduced CIA severity, improving mitochondrial dysfunction compared to Met or CoQ10 alone. These results present a novel, significant preventive targets in RA and may enhance our understanding of its pathogenesis.

  9. The Roles of Streptozotocin Neurotoxicity and Neutral Endopeptidase in Murine Experimental Diabetic Neuropathy

    Eric Davidson


    Full Text Available We demonstrated that inhibition of neutral endopeptidase (NEP, a protease that degrades vaso- and neuroactive peptides, improves vascular and neural function in diabetic animal models. In this study we explored the role of NEP in neuropathy related to either insulin-deficient diabetes or diet-induced obesity using NEP deficient (−/− mice. Initial studies showed that streptozotocin, in the absence of subsequent hyperglycemia, did not induce nerve conduction slowing or paw thermal hypoalgesia. Glucose disposal was impaired in both C57Bl/6 and NEP −/− mice fed a high fat diet. Thermal hypoalgesia and nerve conduction slowing were present in both streptozotocin-diabetic and high fat fed C57Bl/6 mice but not in NEP −/− mice exposed to either streptozotocin-induced diabetes or a high fat diet. These studies suggest that streptozotocin does not induce neurotoxicity in mice and that NEP plays a role in regulating nerve function in insulin-deficient diabetes and diet-induced obesity.

  10. The Roles of Streptozotocin Neurotoxicity and Neutral Endopeptidase in Murine Experimental Diabetic Neuropathy

    Davidson, Eric; Coppey, Lawrence; Lu, Bao; Arballo, Victor; Calcutt, Nigel A.; Gerard, Craig; Yorek, Mark


    We demonstrated that inhibition of neutral endopeptidase (NEP), a protease that degrades vaso- and neuroactive peptides, improves vascular and neural function in diabetic animal models. In this study we explored the role of NEP in neuropathy related to either insulin-deficient diabetes or diet-induced obesity using NEP deficient (−/−) mice. Initial studies showed that streptozotocin, in the absence of subsequent hyperglycemia, did not induce nerve conduction slowing or paw thermal hypoalgesia. Glucose disposal was impaired in both C57Bl/6 and NEP −/− mice fed a high fat diet. Thermal hypoalgesia and nerve conduction slowing were present in both streptozotocin-diabetic and high fat fed C57Bl/6 mice but not in NEP −/− mice exposed to either streptozotocin-induced diabetes or a high fat diet. These studies suggest that streptozotocin does not induce neurotoxicity in mice and that NEP plays a role in regulating nerve function in insulin-deficient diabetes and diet-induced obesity. PMID:20148083

  11. Local IL-17 Production Exerts a Protective Role in Murine Experimental Glomerulonephritis.

    Sally Hamour

    Full Text Available IL-17 is a pro-inflammatory cytokine implicated in the pathogenesis of glomerulonephritis and IL-17 deficient mice are protected from nephrotoxic nephritis. However, a regulatory role for IL-17 has recently emerged. We describe a novel protective function for IL-17 in the kidney. Bone marrow chimeras were created using wild-type and IL-17 deficient mice and nephrotoxic nephritis was induced. IL-17 deficient hosts transplanted with wild-type bone marrow had worse disease by all indices compared to wild-type to wild-type bone marrow transplants (serum urea p<0.05; glomerular thrombosis p<0.05; tubular damage p<0.01, suggesting that in wild-type mice, IL-17 production by renal cells resistant to radiation is protective. IL-17 deficient mice transplanted with wild-type bone marrow also had a comparatively altered renal phenotype, with significant differences in renal cytokines (IL-10 p<0.01; IL-1β p<0.001; IL-23 p<0.01, and macrophage phenotype (expression of mannose receptor p<0.05; inducible nitric oxide synthase p<0.001. Finally we show that renal mast cells are resistant to radiation and produce IL-17, suggesting they are potential local mediators of disease protection. This is a novel role for intrinsic cells in the kidney that are radio-resistant and produce IL-17 to mediate protection in nephrotoxic nephritis. This has clinical significance as IL-17 blockade is being trialled as a therapeutic strategy in some autoimmune diseases.

  12. Glibenclamide, a Blocker of K+ATP Channels, Shows Antileishmanial Activity in Experimental Murine Cutaneous Leishmaniasis▿

    Serrano-Martín, Xenón; Payares, Gilberto; Mendoza-León, Alexis


    Glibenclamide reduced the rate of lesion growth in BALB/c mice infected with Leishmania (Leishmania) mexicana, the effect was dose dependent, and the highest dose proved more effective than glucantime. Cross-resistance to glucantime was found in animals infected with a glibenclamide-resistant line, but combined therapy reduced lesion progression even in the glibenclamide-resistant strain. PMID:17015627

  13. Examination of Neisseria Gonorrhoeae Opacity Protein Expression During Experimental Murine Genital Tract Infection


    E., M. Virji, K. Zak and J. N. Fletcher (1987). "Immunobiology of gonococcal outer membrane protein I." Antonie Van Leeuwenhoek 53(6): 461-4. 88...pathogenic Neisseriae." Antonie Van Leeuwenhoek 53(6): 435-40. 146. Murphy, G. L., T. D. Connell, D. S. Barritt, M. Koomey and J. G. Cannon (1989

  14. Morphometric evaluation of murine pulmonary mast cells in experimental hemorrhagic shock

    I Kasacka


    Full Text Available Respiratory failure resulting frequently in death is one of the complications in the course of post-hemorrhagic changes. A systemic inflammatory reaction plays a significant role in the pathogenesis of this syndrome. Mast cells also contribute to this effect. To broaden our knowledge of the pathogenesis of respiratory insufficiency, we evaluated morphometrically lung mast cells in hemorrhagically shocked rats. Lung sections were stained with alcian blue and safranin, and four separate locations were distinguished: under the lung pleura, around the bronchi and the large vessels, and in the interalveolar septa. A decrease in the area and volume of mast cells and an increase in their circularity index in interalveolar septa and around the bronchi was observed. An enlargement of mast cells around lung vessels was also found. There were no changes in the morphometric parameters of mast cells under pleura. The results suggest an activation and degranulation of mast cells and a role in the inflammatory process causing acute lung injury in hemorrhagic shock.

  15. Role of nociceptin/orphanin FQ (Noc/oFQ) in murine experimental colitis.

    Kato, Shingo; Tsuzuki, Yoshikazu; Hokari, Ryota; Okada, Yoshikiyo; Miyazaki, Junichi; Matsuzaki, Koji; Iwai, Atsuhiro; Kawaguchi, Atsushi; Nagao, Shigeaki; Itoh, Kazuro; Suzuki, Hidekazu; Nabeshima, Toshitaka; Miura, Soichiro


    Nociceptin/orphanin (Noc/oFQ), endogenous agonist for nociceptin receptor (NOR), is thought to be a stimulator of neurogenic inflammation. We investigated the possible role of Noc/oFQ in the development of colitis using NOR-deficient mice treated with dextran sulfate sodium (DSS). Colitis was significantly improved in NOR-deficient mice against wild-type mice. Expression level of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) and infiltrating cells also significantly decreased in NOR-deficient mice against wild-type mice. Nociceptin expression increased in wild-type mice after DSS treatment. These results suggest stimulation by Noc/oFQ deteriorates colonic inflammation via up-regulation of adhesion molecule.

  16. Local Th1/Th2 Cytokine Expression in Experimental Murine Vaginal Candidiasis

    Weixiang OUYANG; Shanjuan CHEN; Zhixiang LIU; Yan WU; Jiawen LI


    In order to investigate the expression of Th1 and Th2 cytokines in the vaginal candidiasis caused by Candida, the fungal vaginitis model was established in female ICR mice by intravaginal inoculation of suspension of C. albicans after the animals were pretreated with estradiol. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the expression of IL-2, IL-4, IL-10 and TGF-β1 in the vagina in the mice of different groups at different time points after the beginning of the experiment. The average expression level of IL-2 mRNA in group D (estrogen-treated mice) was significantly higher than that in groups H (estrogen-untreated mice) and I (control group) on the day 2. The average expression level of IL-4 mRNA in group D was significantly higher than that in groups I and H on the day 5. The average expression level of IL-10 mRNA in group D was significantly higher than that in groups H and I from day 7 to 11. The average expression level of TGF-β1 mRNA in group D was significantly higher than that in groups H and I at all time points. It was concludes that the high-level expression of IL-2 mRNA during early infection was associated with clearance of mucosal C. albicans, and the high-level expression of IL-10 mRNA during late stage of the infection was related to susceptibility to infection. TGF-β1 may play a predominant role when the virtual absence of changes in other Th-type cytokines during infection.

  17. Evaluation of potential antidepressant-like activity of chalcone-1203 in various murine experimental depressant models.

    Guan, Li-Ping; Tang, Li-Ming; Pan, Cheng-Yan; Zhao, Shui-Lian; Wang, Si-Hong


    Two classic animal behavior despair tests-the forced swimming test (FST) and the tail suspension test (TST) were used to evaluate antidepressant-like activity of a new chalcone compound, chalcone-1203 in mice. It was observed that chalcone-1203 at dose of 1, 5, and 10 mg/kg significantly reduced the immobility time in the FST and TST in mice 30 min after treatment. In addition, chalcone-1203 was found to exhibit significant oral activity in the FST in mice. It also produced a reduction in the ambulation in the open-field test in mice not previously habituated to the arena, but no effect in the locomotor activity in mice previously habituated to the open-field. The main monoamine neurotransmitters and their metabolites in mouse brain regions were also simultaneously determined by HPLC-ECD. It was found that chalcone-1203 significantly increased the concentrations of the main neurotransmitters 5-HT and NE in the hippocampus, hypothalamus and cortex. Chalcone-1203 also significantly reduced the ratio of 5-HIAA/5-HT in the hippocampus and cortex, shown down 5-HT metabolism compared with mice treated with stress vehicle. In conclusion, chalcone-1203 produced significant antidepressant-like activity, and the mechanism of action may be due to increased 5-HT and NE in the mouse hippocampus and cortex.

  18. Anti-Lipid IgG Antibodies Are Produced via Germinal Centers in a Murine Model Resembling Human Lupus

    Wong-Baeza, Carlos; Reséndiz-Mora, Albany; Donis-Maturano, Luis; Wong-Baeza, Isabel; Zárate-Neira, Luz; Yam-Puc, Juan Carlos; Calderón-Amador, Juana; Medina, Yolanda; Wong, Carlos; Baeza, Isabel; Flores-Romo, Leopoldo


    Anti-lipid IgG antibodies are produced in some mycobacterial infections and in certain autoimmune diseases [such as anti-phospholipid syndrome, systemic lupus erythematosus (SLE)]. However, few studies have addressed the B cell responses underlying the production of these immunoglobulins. Anti-lipid IgG antibodies are consistently found in a murine model resembling human lupus induced by chlorpromazine-stabilized non-bilayer phospholipid arrangements (NPA). NPA are transitory lipid associations found in the membranes of most cells; when NPA are stabilized they can become immunogenic and induce specific IgG antibodies, which appear to be involved in the development of the mouse model of lupus. Of note, anti-NPA antibodies are also detected in patients with SLE and leprosy. We used this model of lupus to investigate in vivo the cellular mechanisms that lead to the production of anti-lipid, class-switched IgG antibodies. In this murine lupus model, we found plasma cells (Gr1−, CD19−, CD138+) producing NPA-specific IgGs in the draining lymph nodes, the spleen, and the bone marrow. We also found a significant number of germinal center B cells (IgD−, CD19+, PNA+) specific for NPA in the draining lymph nodes and the spleen, and we identified in situ the presence of NPA in these germinal centers. By contrast, very few NPA-specific, extrafollicular reaction B cells (B220+, Blimp1+) were found. Moreover, when assessing the anti-NPA IgG antibodies produced during the experimental protocol, we found that the affinity of these antibodies progressively increased over time. Altogether, our data indicate that, in this murine model resembling human lupus, B cells produce anti-NPA IgG antibodies mainly via germinal centers. PMID:27746783

  19. A Murine Model of Candida glabrata Vaginitis Shows No Evidence of an Inflammatory Immunopathogenic Response

    Nash, Evelyn E.; Peters, Brian M.; Lilly, Elizabeth A.; Noverr, Mairi C.; Fidel, Paul L.


    Candida glabrata is the second most common organism isolated from women with vulvovaginal candidiasis (VVC), particularly in women with uncontrolled diabetes mellitus. However, mechanisms involved in the pathogenesis of C. glabrata-associated VVC are unknown and have not been studied at any depth in animal models. The objective of this study was to evaluate host responses to infection following efforts to optimize a murine model of C. glabrata VVC. For this, various designs were evaluated for consistent experimental vaginal colonization (i.e., type 1 and type 2 diabetic mice, exogenous estrogen, varying inocula, and co-infection with C. albicans). Upon model optimization, vaginal fungal burden and polymorphonuclear neutrophil (PMN) recruitment were assessed longitudinally over 21 days post-inoculation, together with vaginal concentrations of IL-1β, S100A8 alarmin, lactate dehydrogenase (LDH), and in vivo biofilm formation. Consistent and sustained vaginal colonization with C. glabrata was achieved in estrogenized streptozotocin-induced type 1 diabetic mice. Vaginal PMN infiltration was consistently low, with IL-1β, S100A8, and LDH concentrations similar to uninoculated mice. Biofilm formation was not detected in vivo, and co-infection with C. albicans did not induce synergistic immunopathogenic effects. This data suggests that experimental vaginal colonization of C. glabrata is not associated with an inflammatory immunopathogenic response or biofilm formation. PMID:26807975

  20. Vitamin D Deficiency in Human and Murine Sepsis*

    Parekh, Dhruv; Patel, Jaimin M.; Scott, Aaron; Lax, Sian; Dancer, Rachel C. A.; D’Souza, Vijay; Greenwood, Hannah; Fraser, William D.; Gao, Fang; Sapey, Elizabeth; Perkins, Gavin D.


    Objectives: Vitamin D deficiency has been implicated as a pathogenic factor in sepsis and ICU mortality but causality of these associations has not been demonstrated. To determine whether sepsis and severe sepsis are associated with vitamin D deficiency and to determine whether vitamin D deficiency influences the severity of sepsis. Design, Setting, and Patients: Sixty-one patients with sepsis and severe sepsis from two large U.K. hospitals and 20 healthy controls were recruited. Murine models of cecal ligation and puncture and intratracheal lipopolysaccharide were undertaken in normal and vitamin D deficient mice to address the issue of causality. Measurements and Main Results: Patients with severe sepsis had significantly lower concentrations of 25-hydroxyvitamin D3 than patients with either mild sepsis or age-matched healthy controls (15.7 vs 49.5 vs 66.5 nmol/L; p = 0.0001). 25-hydroxyvitamin D3 concentrations were significantly lower in patients who had positive microbiologic culture than those who were culture negative (p = 0.0023) as well as those who died within 30 days of hospital admission (p = 0.025). Vitamin D deficiency in murine sepsis was associated with increased peritoneal (p = 0.037), systemic (p = 0.019), and bronchoalveolar lavage (p = 0.011) quantitative bacterial culture. This was associated with reduced local expression of the cathelicidin-related antimicrobial peptide as well as evidence of defective macrophage phagocytosis (p = 0.029). In the intratracheal lipopolysaccharide model, 1,500 IU of intraperitoneal cholecalciferol treatment 6 hours postinjury reduced alveolar inflammation, cellular damage, and hypoxia. Conclusions: Vitamin D deficiency is common in severe sepsis. This appears to contribute to the development of the condition in clinically relevant murine models and approaches to correct vitamin D deficiency in patients with sepsis should be developed. PMID:27632669

  1. Characterization of a Novel Murine Model to Study Zika Virus

    Rossi, Shannan L.; Tesh, Robert B.; Azar, Sasha R.; Muruato, Antonio E.; Hanley, Kathryn A.; Auguste, Albert J.; Langsjoen, Rose M.; Paessler, Slobodan; Vasilakis, Nikos; Weaver, Scott C.


    The mosquito-borne Zika virus (ZIKV) is responsible for an explosive ongoing outbreak of febrile illness across the Americas. ZIKV was previously thought to cause only a mild, flu-like illness, but during the current outbreak, an association with Guillain–Barré syndrome and microcephaly in neonates has been detected. A previous study showed that ZIKV requires murine adaptation to generate reproducible murine disease. In our study, a low-passage Cambodian isolate caused disease and mortality in mice lacking the interferon (IFN) alpha receptor (A129 mice) in an age-dependent manner, but not in similarly aged immunocompetent mice. In A129 mice, viremia peaked at ∼107 plaque-forming units/mL by day 2 postinfection (PI) and reached high titers in the spleen by day 1. ZIKV was detected in the brain on day 3 PI and caused signs of neurologic disease, including tremors, by day 6. Robust replication was also noted in the testis. In this model, all mice infected at the youngest age (3 weeks) succumbed to illness by day 7 PI. Older mice (11 weeks) showed signs of illness, viremia, and weight loss but recovered starting on day 8. In addition, AG129 mice, which lack both type I and II IFN responses, supported similar infection kinetics to A129 mice, but with exaggerated disease signs. This characterization of an Asian lineage ZIKV strain in a murine model, and one of the few studies reporting a model of Zika disease and demonstrating age-dependent morbidity and mortality, could provide a platform for testing the efficacy of antivirals and vaccines. PMID:27022155

  2. Forced recombination of psi-modified murine leukaemia virus-based vectors with murine leukaemia-like and VL30 murine endogenous retroviruses

    Mikkelsen, J G; Lund, Anders Henrik; Duch, M;


    -impaired Akv-MLV-derived vectors, we here examine putative genetic interactions between vector RNAs and copackaged endogenous retroviral RNAs of the murine leukaemia virus (MLV) and VL30 retroelement families. We show (i) that MLV recombination is not blocked by nonhomology within the 5' untranslated region...... harbouring the supposed RNA dimer-forming cis -elements and (ii) that copackaged retroviral RNAs can recombine despite pronounced sequence dissimilarity at the cross-over site(s) and within parts of the genome involved in RNA dimerization, encapsidation and strand transferring during reverse transcription....... We note that recombination-based rescue of primer binding site knock-out retroviral vectors may constitute a sensitive assay to register putative genetic interactions involving endogenous retroviral RNAs present in cells of various species....

  3. Adrenaline influences the release of interleukin-6 from murine pituicytes

    Christensen, J D; Hansen, E W; Frederiksen, C


    In this study, we examined the effect of adrenaline and interleukin-1beta on interleukin-6 secretion from cultured murine neurohypophyseal cells. Cells were cultured from neurohypophyses of 3- to 5-week-old mice and experiments were performed after 13 days in culture. Interleukin-6 was measured...... in 24-h samples using a sandwich fluoroimmunoassay. Unstimulated cells released 19+/-3 fmol interleukin-6/neurohypophysis/24 h (mean +/- S.E.M., n = 42). Adrenaline and interleukin-1beta increased the release of interleukin-6 from the cells in a concentration-dependent manner. Incubation with adrenaline...

  4. Drosophila homolog of the murine Int-1 protooncogene.


    We have isolated phage clones from Drosophila melanogaster genomic and cDNA libraries containing a sequence homologous to the murine Int-1 protooncogene. The Drosophila gene is represented by a single locus at position 28A1-2 on chromosome 2. The gene is expressed as a 2.9-kilobase-long polyadenylylated mRNA in embryo, larval, and pupal stages. It is hardly detectable in adult flies. The longest open reading frame of the cDNA clone corresponds to a protein 469 amino acids long. Alignment of t...

  5. Investigation of Murine Models for Sleep, Wakefulness and Target Discovery


    subjective day, 7pm-7am w R NR 0 50 100 150 200 250 state c o u n t s F2 Females F2 Males Different from humans, mice exhibit polyphasic sleep with many...early trends, e.g., in the 1p.m. (6 hrs of subjective night) group, became non-significant with additional mice tested • The polyphasic sleep ...REPORT Final report - Investigation of Murine Models for Sleep , Wakefulness and Target Discovery 14. ABSTRACT 16. SECURITY CLASSIFICATION OF

  6. Purification of Murine Monoclonal IgM Antibody


    This paper presents the purification of a monoclonal IgM antibody against human tumor associated antigen Lewis-Y by ion exchange chromatography and gel filtration.Enzyme-linked immunosorbent assay (ELISA) and SDS-polyacrylamide gel electrophoresis (PAGE) were used to identify purified IgM antibody.In flow cytometry analysis, the purified IgM antibody recognizes human breast tumor cell line MCF-7 which expresses Lewis-Y antigen.This work presents a new way for the purification of murine monoclonal IgM antibody.

  7. Contribution of Candida albicans ALS1 to the Pathogenesis of Experimental Oropharyngeal Candidiasis


    We investigated the contribution of Candida albicans ALS1, which encodes a candidal adhesin, to the pathogenesis of experimental murine oropharyngeal candidiasis. Our results indicate that the ALS1 gene product is important for the adherence of the organism to the oral mucosa during the early stage of the infection.

  8. Proinflammatory effects of exogenously administered IL-10 in experimental autoimmune orchitis

    Kaneko, Tetsushi; Itoh, Masahiro; Nakamura, Yoichi;


    We studied the effects of exogenously administered recombinant murine interleukin (IL)-10 on the development of experimental autoimmune orchitis (EAO) in C3H/He mice. IL-10 significantly augments histological signs of EAO when administered for 6 consecutive days from days 15 to 20 after primary...

  9. Analysis of the capacity to produce IL-3 in murine AIDS

    Neuenschwander, A U; Marker, O; Thomsen, Allan Randrup


    Adult C57BL/6 mice infected with LP-BM5 murine leukaemia virus represent a model of murine AIDS (MAIDS). In this study we have analysed the capacity of CD4+ T cells from infected mice to produce IL-3 following stimulation with ConA for 24-72 h. In contrast to the position with IL-2, the production...

  10. Species difference of CD137 ligand signaling in human and murine monocytes.

    Qianqiao Tang

    Full Text Available BACKGROUND: Stimulation of CD137 ligand on human monocytes has been shown to induce DC differentiation, and these CD137L-DCs are more potent than classical DCs, in stimulating T cell responses in vitro. To allow an in vivo evaluation of the potency of CD137L-DCs in murine models we aimed at generating murine CD137L-DCs. METHODOLOGY/PRINCIPAL FINDINGS: When stimulated through CD137 ligand murine monocytes responded just as human monocytes with an increased adherence, morphological changes, proliferation and an increase in viable cell numbers. But CD137 ligand signaling did not induce expression of inflammatory cytokines and costimulatory molecules in murine monocytes and these cells had no T cell stimulatory activity. Murine monocytes did not differentiate to inflammatory DCs upon CD137 ligand signaling. Furthermore, while CD137 ligand signaling induces maturation of human immature classical DCs it failed to do so with murine immature classical DCs. CONCLUSIONS/SIGNIFICANCE: These data demonstrate that both human and murine monocytes become activated by CD137 ligand signaling but only human and not murine monocytes differentiate to inflammatory DCs.

  11. Multi-walled carbon nanotubes: biodegradation by gastric agents in vitro and effect on murine intestinal system

    Masyutin, A.; Erokhina, M.; Sychevskaya, K.; Gusev, A.; Vasyukova, I.; Smirnova, E.; Onishchenko, G.


    One of the main questions limiting application of fibrous carbon nanomaterials (CNM) in medicine and food industry concerns presumptive degradation of CNM in living organisms. In this study, we have investigated biodegradation of multi-walled carbon nanotubes (MWCNTs) by gastric agents in vitro and influence of ingested MWCNTs on murine intestine. Using scanning, conventional transmission and analytical electron microscopy, we demonstrated that industrial MWCNTs treated in vitro by 0.1 M hydrochloric acid (pH=1) and gastric juice (pH=2-3) isolated from murine stomach, are subjected to incomplete degradation. After 30 days of oral administration to experimental mice, we did find MWCNTs in the cells of small intestine, and it may indicate that agglomerates of MWCNTs do not penetrate into colon epithelia and do not accumulate in enterocytes. However, we observed local areas of necrotic damages of intestinal villi. It seems likely, therefore, that MWCNTs end up leaving gastrointestinal tract by excretion with the feces. Our results suggest that MWCNTs do not undergo complete degradation in gastrointestinal tract of mice, and passing through non-degraded particles may negatively affect intestinal system.

  12. Great efficacy of sulfachloropyrazine-sodium against acute murine toxoplasmosis

    Yan-Bo Zeng; Bing Huang; Shun-Hai Zhu; Hui Dong; Hong-Yu Han; Lian-Lian Jiang; Quan Wang; Jun Cheng; Qi-Ping Zhao; Wei-Jiao Ma


    Objective: To identify more effective and less toxic drugs to treat animal toxoplasmosis.Methods:Efficacy of seven kinds of sulfonamides against Toxoplasma gondii (T. gondii) in an acute murine model was evaluated. The mice used throughout the study were randomly assigned to many groups (10 mice each), which either remained uninfected or were infected intraperitoneally with tachyzoites of T. gondii (strains RH and CN). All groups were then treated with different sulfonamides and the optimal treatment protocol was determined candidates. Sulfadiazine-sodium (SD) was used for comparison. Results: The optimal therapy involved gavaging mice twice per day with 250 mg/kg bw of sulfachloropyrazine-sodium (SPZ) for five days. Using this protocol, the average survival time and the time-point of 50% fatalities were prolonged significantly compared with SD treatment. Treatment with SPZ protected 40% of mice from death, and the heart and kidney tissue of these animals was parasite-free, as determined by nested-PCR. SPZ showed excellent therapeutic effects in the treatment of T. gondii in an acute murine model and is therefore a promising drug candidate for the treatment and prevention of T. gondii in animals. Conclusions: It can be concluded that the effective drug sulfachloropyrazine may be the new therapeutic options against animal toxoplasmosis.

  13. Isoforms of murine and human serum amyloid P component

    Nybo, Mads; Hackler, R; Kold, B;


    Isoelectric focusing (IEF) and immunofixation of murine serum amyloid P component (SAP), purified and in serum, showed a distinct and strain-dependent isoform pattern with up to seven bands (pI 5.1-5.7). Neuraminidase treatment caused a shift of the isoforms to more basic pI values, but did not a...... treatment caused a shift of the isoforms, but no reduction in isoform number. Two-dimensional gel electrophoresis confirmed the existence of multiple isoforms of human SAP monomers.......Isoelectric focusing (IEF) and immunofixation of murine serum amyloid P component (SAP), purified and in serum, showed a distinct and strain-dependent isoform pattern with up to seven bands (pI 5.1-5.7). Neuraminidase treatment caused a shift of the isoforms to more basic pI values, but did...... not affect their number. When the acute-phase response was analysed in three mouse strains, CBA/J and C3H/HeN initially showed seven SAP isoforms in serum and C57BL/6 J three or four. The responses in all three strains peaked at day 2 and were normalized within 14 days. On days 2 and 4, CBA/J and C3H...

  14. Rapamycin improves lymphoproliferative disease in murine autoimmune lymphoproliferative syndrome (ALPS).

    Teachey, David T; Obzut, Dana A; Axsom, Kelly; Choi, John K; Goldsmith, Kelly C; Hall, Junior; Hulitt, Jessica; Manno, Catherine S; Maris, John M; Rhodin, Nicholas; Sullivan, Kathleen E; Brown, Valerie I; Grupp, Stephan A


    Autoimmune lymphoproliferative syndrome (ALPS) is a disorder of abnormal lymphocyte survival caused by defective Fas-mediated apoptosis, leading to lymphadenopathy, hepatosplenomegaly, and an increased number of double-negative T cells (DNTs). Treatment options for patients with ALPS are limited. Rapamycin has been shown to induce apoptosis in normal and malignant lymphocytes. Since ALPS is caused by defective lymphocyte apoptosis, we hypothesized that rapamycin would be effective in treating ALPS. We tested this hypothesis using rapamycin in murine models of ALPS. We followed treatment response with serial assessment of DNTs by flow cytometry in blood and lymphoid tissue, by serial monitoring of lymph node and spleen size with ultrasonography, and by enzyme-linked immunosorbent assay (ELISA) for anti-double-stranded DNA (dsDNA) antibodies. Three-dimensional ultrasound measurements in the mice correlated to actual tissue measurements at death (r = .9648). We found a dramatic and statistically significant decrease in DNTs, lymphadenopathy, splenomegaly, and autoantibodies after only 4 weeks when comparing rapamycin-treated mice with controls. Rapamycin induced apoptosis through the intrinsic mitochondrial pathway. We compared rapamycin to mycophenolate mofetil, a second-line agent used to treat ALPS, and found rapamycin's control of lymphoproliferation was superior. We conclude that rapamycin is an effective treatment for murine ALPS and should be explored as treatment for affected humans.

  15. Scanning electron microscopy of the neuropathology of murine cerebral malaria

    Brenneis Christian


    Full Text Available Abstract Background The mechanisms leading to death and functional impairments due to cerebral malaria (CM are yet not fully understood. Most of the knowledge about the pathomechanisms of CM originates from studies in animal models. Though extensive histopathological studies of the murine brain during CM are existing, alterations have not been visualized by scanning electron microscopy (SEM so far. The present study investigates the neuropathological features of murine CM by applying SEM. Methods C57BL/6J mice were infected with Plasmodium berghei ANKA blood stages. When typical symptoms of CM developed perfused brains were processed for SEM or light microscopy, respectively. Results Ultrastructural hallmarks were disruption of vessel walls, parenchymal haemorrhage, leukocyte sequestration to the endothelium, and diapedesis of macrophages and lymphocytes into the Virchow-Robin space. Villous appearance of observed lymphocytes were indicative of activated state. Cerebral oedema was evidenced by enlargement of perivascular spaces. Conclusion The results of the present study corroborate the current understanding of CM pathophysiology, further support the prominent role of the local immune system in the neuropathology of CM and might expose new perspectives for further interventional studies.

  16. Filaria-induced IL-10 suppresses murine cerebral malaria.

    Specht, Sabine; Ruiz, Daniel Fernández; Dubben, Bettina; Deininger, Susanne; Hoerauf, Achim


    Filarial nematodes achieve long survival in their hosts due to their capacity to modulate immune responses. Therefore, immunomodulation by filarial nematodes may alter responses to concomitant infections such as malaria. Cerebral malaria (CM), a severe complication of Plasmodium falciparum infections, is triggered as a consequence of the immune response developed against malaria parasites. The question arises whether prior infection with helminth parasites is beneficial against CM. In the present work a murine model for subsequent has been used to assess this hypothesis. C57BL/6 mice were infected with the rodent filarial parasite Litomosoides sigmodontis and the murine model parasite for CM, Plasmodium berghei ANKA. Previously filaria-infected C57BL/6 mice showed significantly reduced CM rates. CD8(+) T cell recruitment to the brain, a hallmark for CM development, was reduced in protected mice. Furthermore, in contrast to P. berghei single-infected animals, filaria-infected mice had significantly higher levels of circulating IL-10. The requirement for IL-10 in CM protection was demonstrated by the lack of protection in IL-10 KO mice. This suggests that the anti-inflammatory IL-10 elicited by filarial nematodes is able to suppress the overwhelming inflammatory reaction otherwise triggered against malaria parasites in C57BL/6 mice, preventing full progress to CM.

  17. Neurological Disorders in a Murine Model of Chronic Renal Failure

    Jean-Marc Chillon


    Full Text Available Cardiovascular disease is highly prevalent in patients with chronic renal failure (CRF. However, data on the impact of CRF on the cerebral circulatory system are scarce—despite the fact that stroke is the third most common cause of cardiovascular death in people with CRF. In the present study, we examined the impact of CRF on behavior (anxiety, recognition and ischemic stroke severity in a well-defined murine model of CRF. We did not observe any significant increases between CRF mice and non-CRF mice in terms of anxiety. In contrast, CRF mice showed lower levels of anxiety in some tests. Recognition was not impaired (vs. controls after 6 weeks of CRF but was impaired after 10 weeks of CRF. Chronic renal failure enhances the severity of ischemic stroke, as evaluated by the infarct volume size in CRF mice after 34 weeks of CRF. Furthermore, neurological test results in non-CRF mice tended to improve in the days following ischemic stroke, whereas the results in CRF mice tended to worsen. In conclusion, we showed that a murine model of CRF is suitable for evaluating uremic toxicity and the associated neurological disorders. Our data confirm the role of uremic toxicity in the genesis of neurological abnormalities (other than anxiety.

  18. Immunological impact of magnetic nanoparticles (Ferucarbotran) on murine peritoneal macrophages

    Yeh, Chen-Hao [National Taiwan University, Department of Horticulture (China); Hsiao, Jong-Kai [National Taiwan University Hospital and College of Medicine, Department of Medical Imaging (China); Wang, Jaw-Lin [National Taiwan University, Institute of Biomedical Engineering (China); Sheu, Fuu, E-mail: [National Taiwan University, Department of Horticulture (China)


    Ferucarbotran, a clinically used superparamagnetic iron oxide, is widely developed as a magnetic resonance imaging (MRI) contrast agent and has the potential to improve the monitoring of macrophage recirculation in vivo. However, the biological effect of Ferucarbotran or magnetic nanoparticles (MNPs) on macrophage is not clearly understood yet. This study is aimed to examine the immunological impact of Ferucarbotran toward murine peritoneal macrophages. Cells treated with Ferucarbotran demonstrated a dose-responsive increase of granularity in the cytoplasm. After 24 h of incubation, viability and cytotoxicity in macrophages treated with 200 {mu}g Fe/mL of Ferucarbotran were not affected. Macrophages loaded with Ferucarbotran above 100 {mu}g Fe/mL showed a significant (p < 0.01) increase in cytokine (TNF-{alpha}, IL-1{beta}, IL-6) secretion and mRNA expression, followed by nitric oxide (NO) secretion and iNOS mRNA expression. Chemotactic responses of Ferucarbotran-preloaded macrophages toward CX3CL1 were significantly (p < 0.05) lower than those of untreated macrophages. Taking together, Ferucarbotran at high dose (100 {mu}g Fe/mL) could induce murine peritoneal macrophages activation in pro-inflammatory cytokine secretion and NO production.

  19. Effect of blockage of costimulatory signal on murine abortion-prone model

    ZHAO Fu-xi; ZHANG Yuan-yuan; LIU Run-hua; LI Shuan-ming


    Background Inhibition of the key costimulatory signals results in T cell anergy, indicating the alloantigen-specific immunologic unresponsiveness. In this study, the effect of blockage of costimulatory signal CD86 on murine abortion-prone model was studied.Methods Thirty CBA/J female mice cohabitated with DBA/2 male or BALB/c male mice were investigated. CBA/J ×DBA/2 matings were used as the abortion-prone model, and CBA/J × BALB/c matings were used as the normal pregnant model. The abortion-prone models were divided into experimental and control groups, and the normal pregnant models were set as a normal group (10 mice in each group). The mice in the experimental group were treated with anti-mouse CD86 monoclonal antibody (mAb) (100 μg) on day 4.5 of gestation, while the controls received irrelevant-isotype matched rat IgG2b. As for the normal group, nothing was given to the mice. The mice were killed on day 13.5 of gestation, embryo resorption rate and the expression of transforming growth factor β1 (TGF-β1), plasminogen activator inhibitor 1 (PAI-1), and matrix metalloproteinase 9 (MMP-9) were detected. Then the data were analyzed by Chi-square test and Fisher's exact test.Results The embryo resorption rate in the experimental (8.2%) and normal groups (7.7%) was significantly lower than that of the control (23.5%, P<0.05). No significant difference was detected between the experimental and normal groups (P>0.05). The positive expression rates of TGF-β1 and PAI-1 proteins in the experimental and normal groups were significantly higher than those in the control group (P<0.05). The positive expression rate of MMP-9 protein in the experimental and normal groups was significantly lower than that in the control group (P<0.05). No significant difference in the positive expression rates of the three proteins was detected between the experimental and normal groups (P>0.05).Conclusions Blockage of costimulatory signal CD86 at early pregnancy can treat

  20. Research progress of murine lupus nephritis models%狼疮样肾炎小鼠模型研究进展

    何艺磊; 杨林承; 李卫东


    Systemic lupus erythematosus ( SLE ) is characterized by the presence of anti-nuclear antibodies (ANA) directed against naked DNA and entire nucleosomes. Systemic lupus erythematosus is a multifactorial autoimmune disease affected by genetic, environmental, hormonal and infection factors. Murine model of lupus nephritis and its SLE pathogenesis and pathological changes have similar characteristics to that of SLE in humans. Therefore , murine SLE model is an ideal experimental pathological model for the study of SLE and screening of anti-inflammatory and anti-immune drugs. In this article, we will review the preparation methods and evaluations of murine models of lupus nephritis.%系统性红斑狼疮(systemic lupus erythematosus,SLE)是一种以体内产生抗核抗体为特征的自身免疫性疾病,其病因目前未明,一般认为是遗传、环境、内分泌、感染等因素相互作用的结果.狼疮样肾炎小鼠模型与人SLE发病机制和病理变化等有着相似特点,是研究SLE、筛选抗炎免疫药物较理想的实验性病理模型.本文对近年来狼疮样肾炎小鼠模型制备的方法及评价进行了综述.

  1. Convenience experimentation.

    Krohs, Ulrich


    Systems biology aims at explaining life processes by means of detailed models of molecular networks, mainly on the whole-cell scale. The whole cell perspective distinguishes the new field of systems biology from earlier approaches within molecular cell biology. The shift was made possible by the high throughput methods that were developed for gathering 'omic' (genomic, proteomic, etc.) data. These new techniques are made commercially available as semi-automatic analytic equipment, ready-made analytic kits and probe arrays. There is a whole industry of supplies for what may be called convenience experimentation. My paper inquires some epistemic consequences of strong reliance on convenience experimentation in systems biology. In times when experimentation was automated to a lesser degree, modeling and in part even experimentation could be understood fairly well as either being driven by hypotheses, and thus proceed by the testing of hypothesis, or as being performed in an exploratory mode, intended to sharpen concepts or initially vague phenomena. In systems biology, the situation is dramatically different. Data collection became so easy (though not cheap) that experimentation is, to a high degree, driven by convenience equipment, and model building is driven by the vast amount of data that is produced by convenience experimentation. This results in a shift in the mode of science. The paper shows that convenience driven science is not primarily hypothesis-testing, nor is it in an exploratory mode. It rather proceeds in a gathering mode. This shift demands another shift in the mode of evaluation, which now becomes an exploratory endeavor, in response to the superabundance of gathered data.

  2. Experimental philosophy.

    Knobe, Joshua; Buckwalter, Wesley; Nichols, Shaun; Robbins, Philip; Sarkissian, Hagop; Sommers, Tamler


    Experimental philosophy is a new interdisciplinary field that uses methods normally associated with psychology to investigate questions normally associated with philosophy. The present review focuses on research in experimental philosophy on four central questions. First, why is it that people's moral judgments appear to influence their intuitions about seemingly nonmoral questions? Second, do people think that moral questions have objective answers, or do they see morality as fundamentally relative? Third, do people believe in free will, and do they see free will as compatible with determinism? Fourth, how do people determine whether an entity is conscious?

  3. Experimental Techniques

    Engelfried, J


    In this course we will give examples for experimental techniques used in particle physics experiments. After a short introduction, we will discuss applications in silicon microstrip detectors, wire chambers, and single photon detection in Ring Imaging Cherenkov (RICH) counters. A short discussion of the relevant physics processes, mainly different forms of energy loss in matter, is enclosed.

  4. Effect of Cold Plasma on Cell Viability and Collagen Synthesis in Cultured Murine Fibroblasts

    Shi, Xingmin; Cai, Jingfen; Xu, Guimin; Ren, Hongbin; Chen, Sile; Chang, Zhengshi; Liu, Jinren; Huang, Chongya; Zhang, Guanjun; Wu, Xili


    An argon atmospheric pressure plasma jet was employed to treat L929 murine fibroblasts cultured in vitro. Experimental results showed that, compared with the control cells, the treatment of fibroblasts with 15 s of plasma led to a significant increase of cell viability and collagen synthesis, while the treatment of 25 s plasma resulted in a remarkable decrease. Exploration of related mechanisms suggested that cold plasma could up-regulate CyclinD1 gene expression and down-regulate p27 gene expression at a low dose, while it could down-regulate CyclinD1 expression and up-regulate p27 expression at a higher dose, thus altering the cell cycle progression, and then affecting cell viability and collagen synthesis of fibroblasts. supported partly by National Natural Science Foundation of China (Nos. 81372076, 51307133 and 51221005), China National Funds for Distinguished Young Scientists (No. 51125029), the Sci-Tech Project of Shaanxi Province of China (No. 2010K16-04), and the Fundamental Research Funds for the Central Universities of China (No. xkjc2013004)

  5. Transcutaneous photodynamic therapy delays the onset of paralysis in a murine multiple sclerosis model

    Hunt, David W. C.; Leong, Simon; Levy, Julia G.; Chan, Agnes H.


    Photodynamic therapy (PDT) using benzoporphyrin derivative (BPD, Verteporfin) and whole body irradiation, can affect the course of adoptively transferred experimental allergic (autoimmune) encephalomyelitis (EAE) in PL mice. Murine EAE is a T cell-mediated autoimmune disease which serves as a model for human multiple sclerosis. Using a novel disease induction protocol, we found that mice characteristically developed EAE within 3 weeks of receipt of myelin basic protein (MBP)-sensitized, in vitro-cultured spleen or lymph node cells. However, if animals were treated with PDT (1 mg BPD/kg bodyweight and exposed to whole body 15 Joules cm2 of LED light) 24 hours after receiving these cells, disease onset time was significantly delayed. PDT-treated mice developed disease symptoms 45 +/- 3 days following cell administration whereas untreated controls were affected within 23 +/- 2 days. In contrast, application of PDT 48 or 120 hours following injection of the pathogenic cells had no significant effect upon the development of EAE. Experiments are in progress to account for the protective effect of PDT in this animal model. These studies should provide evidence on the feasibility of PDT as a treatment for human autoimmune disease.

  6. The iron chelator deferasirox enhances liposomal amphotericin B efficacy in treating murine invasive pulmonary aspergillosis

    Ibrahim, Ashraf S.; Gebremariam, Teclegiorgis; French, Samuel W.; Edwards, John E.; Spellberg, Brad


    Objectives Increased bone marrow iron levels in patients with haematological malignancies is an independent risk factor for developing invasive pulmonary aspergillosis (IPA), suggesting an important role for iron uptake in the pathogenesis of IPA. We sought to determine the potential for combination therapy with the iron chelator deferasirox + liposomal amphotericin B (LAmB) to improve the outcome of murine IPA compared with LAmB monotherapy. Methods In vitro MIC and minimum fungicidal concentration (MFC) values of the iron chelator, deferasirox, for Aspergillus fumigatus were determined by microdilution assay. In addition, we studied the efficacy of deferasirox alone or combined with LAmB in treating immunocompromised mice infected with A. fumigatus via inhalation. Results Deferasirox was cidal in vitro against A. fumigatus, with an MIC and MFC of 25 and 50 mg/L, respectively. Deferasirox monotherapy modestly prolonged survival of mice with IPA. Combination deferasirox + LAmB therapy synergistically improved survival and reduced lung fungal burden compared with either monotherapy alone. Conclusions Iron chelation therapy with deferasirox alone or in combination with LAmB is effective in treating experimental IPA. Further study of deferasirox is warranted as adjunctive therapy for IPA infections. PMID:19942619

  7. Differential Nucleosome Occupancies across Oct4-Sox2 Binding Sites in Murine Embryonic Stem Cells.

    Sebeson, Amy; Xi, Liqun; Zhang, Quanwei; Sigmund, Audrey; Wang, Ji-Ping; Widom, Jonathan; Wang, Xiaozhong


    The binding sequence for any transcription factor can be found millions of times within a genome, yet only a small fraction of these sequences encode functional transcription factor binding sites. One of the reasons for this dichotomy is that many other factors, such as nucleosomes, compete for binding. To study how the competition between nucleosomes and transcription factors helps determine a functional transcription factor site from a predicted transcription factor site, we compared experimentally-generated in vitro nucleosome occupancy with in vivo nucleosome occupancy and transcription factor binding in murine embryonic stem cells. Using a solution hybridization enrichment technique, we generated a high-resolution nucleosome map from targeted regions of the genome containing predicted sites and functional sites of Oct4/Sox2 regulation. We found that at Pax6 and Nes, which are bivalently poised in stem cells, functional Oct4 and Sox2 sites show high amounts of in vivo nucleosome displacement compared to in vitro. Oct4 and Sox2, which are active, show no significant displacement of in vivo nucleosomes at functional sites, similar to nonfunctional Oct4/Sox2 binding. This study highlights a complex interplay between Oct4 and Sox2 transcription factors and nucleosomes among different target genes, which may result in distinct patterns of stem cell gene regulation.

  8. Differential Nucleosome Occupancies across Oct4-Sox2 Binding Sites in Murine Embryonic Stem Cells.

    Amy Sebeson

    Full Text Available The binding sequence for any transcription factor can be found millions of times within a genome, yet only a small fraction of these sequences encode functional transcription factor binding sites. One of the reasons for this dichotomy is that many other factors, such as nucleosomes, compete for binding. To study how the competition between nucleosomes and transcription factors helps determine a functional transcription factor site from a predicted transcription factor site, we compared experimentally-generated in vitro nucleosome occupancy with in vivo nucleosome occupancy and transcription factor binding in murine embryonic stem cells. Using a solution hybridization enrichment technique, we generated a high-resolution nucleosome map from targeted regions of the genome containing predicted sites and functional sites of Oct4/Sox2 regulation. We found that at Pax6 and Nes, which are bivalently poised in stem cells, functional Oct4 and Sox2 sites show high amounts of in vivo nucleosome displacement compared to in vitro. Oct4 and Sox2, which are active, show no significant displacement of in vivo nucleosomes at functional sites, similar to nonfunctional Oct4/Sox2 binding. This study highlights a complex interplay between Oct4 and Sox2 transcription factors and nucleosomes among different target genes, which may result in distinct patterns of stem cell gene regulation.

  9. Simultaneous administration of vitamin A and DTP vaccine modulates the immune response in a murine cerebral malaria model

    Hein-Kristensen, L; Jørgensen, M J; Ravn, H


    The World Health Organisation recommends vitamin A supplementation (VAS) to children aged 6 months to 5 years in low-income countries, and for logistic reasons, this has been linked to routine childhood immunizations. Observational studies suggest that VAS given with diphtheria-tetanus-pertussis ......The World Health Organisation recommends vitamin A supplementation (VAS) to children aged 6 months to 5 years in low-income countries, and for logistic reasons, this has been linked to routine childhood immunizations. Observational studies suggest that VAS given with diphtheria......-tetanus-pertussis (DTP) vaccine may increase mortality from non-targeted diseases. We investigated the non-targeted effect of pretreatment with VAS and DTP vaccine in a murine model of experimental cerebral malaria. Our a priori hypothesis was that VAS/DTP would aggravate the infection. We found that the effect of VAS...

  10. In vitro proteolytic cleavage of Gazdar murine sarcoma virus p65gag.

    Maxwell, S.; Arlinghaus, R B


    Moloney murine leukemia virus, disrupted in concentrations of 0.1 to 0.5% Nonidet P-40, catalyzed the cleavage of p65, the gag gene polyprotein of the Gazdar strain of murine sarcoma virus, into polypeptides with sizes and antigenic determinants of murine leukemia virus-specified p30, p15, pp12, and p10. Cleavage performed in the presence of 0.15% Nonidet P-40 in water yielded polypeptides of approximately 40,000 (P40) and 25,000 (P25) Mr. In vitro cleavage performed in a buffered solution co...

  11. Proteolytically modified human beta 2-microglobulin augments the specific cytotoxic activity in murine mixed lymphocyte culture

    Nissen, Mogens Holst; Claësson, M H


    (M-beta 2-m) bind to murine lymphocytes expressing H-2 class I antigens; M-beta 2-m, when added at day 0 and 1 of culture in nanomolar concentrations to a one-way murine allogeneic mixed lymphocyte culture (MLC) augments the generation of specific cytotoxic T lymphocytes; M-beta 2-m increases...... the endogenous production of interleukin 2 in the MLC culture; monoclonal antibody which reacts with both the native beta 2-m and M-beta 2-m molecule blocks the augmentation of cytotoxic T lymphocyte production induced by M-beta 2-m; murine as well as human MLC responder cells can proteolytically modify native...

  12. Haemopedia: An Expression Atlas of Murine Hematopoietic Cells

    Carolyn A. de Graaf


    Full Text Available Hematopoiesis is a multistage process involving the differentiation of stem and progenitor cells into distinct mature cell lineages. Here we present Haemopedia, an atlas of murine gene-expression data containing 54 hematopoietic cell types, covering all the mature lineages in hematopoiesis. We include rare cell populations such as eosinophils, mast cells, basophils, and megakaryocytes, and a broad collection of progenitor and stem cells. We show that lineage branching and maturation during hematopoiesis can be reconstructed using the expression patterns of small sets of genes. We also have identified genes with enriched expression in each of the mature blood cell lineages, many of which show conserved lineage-enriched expression in human hematopoiesis. We have created an online web portal called Haemosphere to make analyses of Haemopedia and other blood cell transcriptional datasets easier. This resource provides simple tools to interrogate gene-expression-based relationships between hematopoietic cell types and genes of interest.

  13. A novel inexpensive murine model of oral chronic digitalization.

    Helber, Izo; Kanashiro, Rosemeire M; Alarcon, Ernesto A; Antonio, Ednei L; Tucci, Paulo J F


    A novel inexpensive murine model of oral administration of digitoxin (100 micro g/kg per day) added to routine chow is described. Serum digitoxin levels achieved after oral (n = 5; 116 +/- 14 ng/mL) and subcutaneous (n = 5; 124 +/- 11 ng/mL) administration were similar. A significant increase in the maximal left ventricular pressure rise of treated (n = 9) compared with control (n = 6) rats (dP/dt: 8956 +/- 233 vs 7980 +/- 234 mmHg/s, respectively; P = 0.01) characterized the positive inotropic action of digitoxin. In addition, no differences were observed in treated compared with control rats with regard to the electrocardiogram and systolic and diastolic left ventricular pressures.

  14. In vitro stimulation of murine peritoneal monocytes induced by alginates.

    Pasquali, Paolo; Zalcman, Amy; Murtas, Susanna; Adone, Rosanna; Brambilla, Gianfranco; Marianelli, Cinzia; Cagiola, Monica; Ciuchini, Franco


    In this trial we assessed the effect of soluble alginates on murine cells. Mouse peritoneal monocytes were stimulated in vitro with a solution of alginate. The production of TNF-alpha and nitric oxide (NO), the expression of surface molecules CD80 and CD86, and the ability of monocytes to phagocyte bacteria were assessed, in order to evaluate the effect of alginate on cell functionality. We showed that mouse peritoneal monocytes stimulated with alginate produce NO and TNF-alpha. In addition, alginate is able also to increase their phagocytic activity and to a lesser extent also to increase the expression of CD80. Even with different degrees, it implies that alginates per se act directly on immune response, being able to effectively stimulate proinflammatory activity. These findings corroborate the idea that alginates can represent interesting adjuvants to use to increase the efficacy of antigenic stimulation.

  15. The immunological relationship between filtrable agent, Salmonella and murine leukosis



    Full Text Available Salmonella typhimurium was invariably isolated from our J strain murine leukosis. Immunization of D103 mice with either inactivated Salmonella typhimurium or the cell-free extract of leukosis inhibited the transplantation of leukosis. The adoptive immunization of D103 mice with spleen cells of Strong A mice immunized with either Salmonella or the cell-free extract of leukosis inhibited the transplantation of leukosis. The addition of either Salmonella or the cell-free extract of leukosis inhibited the migration of macrophages of leukosis spleen in tissue culture. Strong A mice is non-susceptible to J strain leukosis. However, inoculation of neonatal Strong A mice with the cell-free extract of leukosis produced a susceptibility to the transplantation of leukosis. These results suggest that both a filtrable agent and Salmonella typhimurium are present in cells of this leukosis and might be etiologically related to the leukosis.

  16. Expression of human adenosine deaminase in murine hematopoietic cells.

    Belmont, J W; MacGregor, G R; Wager-Smith, K; Fletcher, F A; Moore, K A; Hawkins, D; Villalon, D; Chang, S M; Caskey, C T


    Multiple replication-defective retrovirus vectors were tested for their ability to transfer and express human adenosine deaminase in vitro and in vivo in a mouse bone marrow transplantation model. High-titer virus production was obtained from vectors by using both a retrovirus long terminal repeat promoter and internal transcriptional units with human c-fos and herpes virus thymidine kinase promoters. After infection of primary murine bone marrow with one of these vectors, human adenosine deaminase was detected in 60 to 85% of spleen colony-forming units and in the blood of 14 of 14 syngeneic marrow transplant recipients. This system offers the opportunity to assess methods for increasing efficiency of gene transfer, for regulation of expression of foreign genes in hematopoietic progenitors, and for long-term measurement of the stability of expression in these cells. Images PMID:3072474

  17. The kin17 Protein in Murine Melanoma Cells

    Anelise C. Ramos


    Full Text Available kin17 has been described as a protein involved in the processes of DNA replication initiation, DNA recombination, and DNA repair. kin17 has been studied as a potential molecular marker of breast cancer. This work reports the detection and localization of this protein in the murine melanoma cell line B16F10-Nex2 and in two derived subclones with different metastatic potential, B16-8HR and B16-10CR. Nuclear and chromatin-associated protein fractions were analyzed, and kin17 was detected in all fractions, with an elevated concentration observed in the chromatin-associated fraction of the clone with low metastatic potential, suggesting that the kin17 expression level could be a marker of melanoma.

  18. Dye-mediated photosensitization of murine neuroblastoma cells

    Sieber, F.; Sieber-Blum, M.


    The purpose of this study was to determine if photosensitization mediated by the fluorescent dye, merocyanine 540, could be used to preferentially kill murine neuroblastoma cells in simulated autologous remission marrow grafts. Simultaneous exposure of Neuro 2a or NB41A3 neuroblastoma cells to merocyanine 540 and white light reduced the concentration of in vitro-clonogenic tumor cells 50,000-fold. By contrast, the same treatment had little effect on the graft's ability to rescue lethally irradiated syngeneic hosts. Lethally irradiated C57BL/6J X A/J F1 mice transplanted with photosensitized mixtures of neuroblastoma cells and normal marrow cells (1:100 or 1:10) survived without developing neuroblastomas. It is conceivable that merocyanine 540-mediated photosensitization will prove useful for the extracorporeal purging of residual neuroblastoma cells from human autologous remission marrow grafts.

  19. Separation and sequencing of familiar and novel murine proteins using preparative two-dimensional gel electrophoresis.

    Merrick, B A; Patterson, R M; Witcher, L L; He, C; Selkirk, J K


    Strategies are needed for rapid protein isolation in order to identify disease-related proteins and facilitate the design of oligonucleotides for further molecular inquiry. In our laboratory, C3H10T1/2 murine fibroblasts have been found to express a variety of proteins in various subcellular fractions which are relevant to experimental transformation and carcinogenesis. Preparative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) procedures were developed to identify major cytoplasmic proteins by electroblotting and microsequencing. Isoelectric focusing tube gels were enlarged to 6 mm ID to accommodate larger protein loads at 0.5 to 2 mg protein. Separated proteins were electrotransferred from 6 mm thick slab gels onto 0.22 mu polyvinylidene difluoride membranes. Nearly 100 prominent blotted proteins were stained with Coomassie Brilliant Blue between pI 4.5-7.0 and 18-106 kDa and, of these, 27 prominent and well-resolved proteins were selected for sequencing. Sequences of 14 to 24 amino acid residues in length were obtained from 11 proteins which were identified from computerized databases. Some of these identified proteins had structural or enzymatic functions while others had only recently been discovered, including a newly reported Hsp 70 class member and a novel calcium-binding protein, reticulocalbin. The new heat shock protein has a molecular mass of 75 kDa and has been designated as Grp75, PBP74, CSA or p66mot-1 in mice and humans with purported roles in transformation and antigen processing. Reticulocalbin is an endoplasmic reticular protein which contains six domains of the EF-hand motif associated with high-affinity calcium-binding proteins. It may be involved in protein transport and luminal protein processing. In addition, sequences of 5 to 11 residues in length were also obtained from six other unidentified proteins. Thus, we have found that preparative 2-D PAGE serves as a powerful one-step purification method for protein isolation and

  20. Gene expression profile of androgen modulated genes in the murine fetal developing lung

    Côté Mélissa


    Full Text Available Abstract Background Accumulating evidences suggest that sex affects lung development. Indeed, a higher incidence of respiratory distress syndrome is observed in male compared to female preterm neonates at comparable developmental stage and experimental studies demonstrated an androgen-related delay in male lung maturation. However, the precise mechanisms underlying these deleterious effects of androgens in lung maturation are only partially understood. Methods To build up a better understanding of the effect of androgens on lung development, we analyzed by microarrays the expression of genes showing a sexual difference and those modulated by androgens. Lungs of murine fetuses resulting from a timely mating window of 1 hour were studied at gestational day 17 (GD17 and GD18, corresponding to the period of surge of surfactant production. Using injections of the antiandrogen flutamide to pregnant mice, we hunted for genes in fetal lungs which are transcriptionally modulated by androgens. Results Results revealed that 1844 genes were expressed with a sexual difference at GD17 and 833 at GD18. Many genes were significantly modulated by flutamide: 1597 at GD17 and 1775 at GD18. Datasets were analyzed by using in silico tools for reconstruction of cellular pathways. Between GD17 and GD18, male lungs showed an intensive transcriptional activity of proliferative pathways along with the onset of lung differentiation. Among the genes showing a sex difference or an antiandrogen modulation of their expression, we specifically identified androgen receptor interacting genes, surfactant related genes in particularly those involved in the pathway leading to phospholipid synthesis, and several genes of lung development regulator pathways. Among these latter, some genes related to Shh, FGF, TGF-beta, BMP, and Wnt signaling are modulated by sex and/or antiandrogen treatment. Conclusion Our results show clearly that there is a real delay in lung maturation between

  1. Sequential univariate gating approach to study the effects of erythropoietin in murine bone marrow.

    Achuthanandam, Ram; Quinn, John; Capocasale, Renold J; Bugelski, Peter J; Hrebien, Leonid; Kam, Moshe


    using SUG in less subjective, time efficient, biologically interpretable manner that corroborates with the expert analysis. The results suggest that basophilic erythroblasts cells or their immediate precursors are an important target for the effects of rHuEPO in murine bone marrow. The MATLAB implementation of the method described in the article, both experimental data and other supplemental materials are freely available at

  2. Deep sequencing of the murine olfactory receptor neuron transcriptome.

    Ninthujah Kanageswaran

    Full Text Available The ability of animals to sense and differentiate among thousands of odorants relies on a large set of olfactory receptors (OR and a multitude of accessory proteins within the olfactory epithelium (OE. ORs and related signaling mechanisms have been the subject of intensive studies over the past years, but our knowledge regarding olfactory processing remains limited. The recent development of next generation sequencing (NGS techniques encouraged us to assess the transcriptome of the murine OE. We analyzed RNA from OEs of female and male adult mice and from fluorescence-activated cell sorting (FACS-sorted olfactory receptor neurons (ORNs obtained from transgenic OMP-GFP mice. The Illumina RNA-Seq protocol was utilized to generate up to 86 million reads per transcriptome. In OE samples, nearly all OR and trace amine-associated receptor (TAAR genes involved in the perception of volatile amines were detectably expressed. Other genes known to participate in olfactory signaling pathways were among the 200 genes with the highest expression levels in the OE. To identify OE-specific genes, we compared olfactory neuron expression profiles with RNA-Seq transcriptome data from different murine tissues. By analyzing different transcript classes, we detected the expression of non-olfactory GPCRs in ORNs and established an expression ranking for GPCRs detected in the OE. We also identified other previously undescribed membrane proteins as potential new players in olfaction. The quantitative and comprehensive transcriptome data provide a virtually complete catalogue of genes expressed in the OE and present a useful tool to uncover candidate genes involved in, for example, olfactory signaling, OR trafficking and recycling, and proliferation.

  3. Temporal effect of local hyperthermia on murine contact hypersensitivity

    ZHANG Lan; WANG Yi-ru; HONG Yu-xiao; XU Ya-qin; ZHANG Li; LI Xiao-dong; XIAO Ting


    Background The sensitization and elicitation phases are involved in the immunopathogenesis of contact hypersensitivity (CHS).Langerhans cells (LCs) are believed to play pivotal roles in the sensitization stage of CHS.Local hyperthermia on skin induces the migration as well as maturation of epidermal LCs.Although fever-range whole body hyperthermia and local hyperthermia at 43℃ prior to sensitization were reported to suppress CHS,the effects of different temperatures and the timing sequence of local hyperthermia on CHS have not been tackled.Methods Local hyperthermia was applied to murine dorsal skin 3 days prior to,concurrent with,or 2 days post sensitization with fluorescein isothiocyanate (FITC) in BALB/c mice.Local hyperthermia temperatures at 37℃,39℃,41℃ and 43℃ were applied to mouse dorsal skin and the severity of CHS was calculated by measuring the swelling response of the challenged ears.Results Local hyperthermia at 39℃,41℃ and 43℃ prior to sensitization reduced the severity of CHS,as compared with that at 37℃.The suppression of CHS was temperature dependant in that higher temperature had a stronger effect.On the contrary,the hyperthermia treatments,either concurrent with or post-sensitization,resulted in an enhanced temperature-dependant ear swelling response.Conclusions The severity of murine CHS could be influenced by local hyperthermia at the sensitization stage in a temperature dependant manner.The temporal effect of local hyperthermia suggested a novel factor in interpreting the severity of allergic contact dermatitis.

  4. Gene expression in IFN-g-activated murine macrophages

    Pereira C.A.


    Full Text Available Macrophages are critical for natural immunity and play a central role in specific acquired immunity. The IFN-gamma activation of macrophages derived from A/J or BALB/c mice yielded two different patterns of antiviral state in murine hepatitis virus 3 infection, which were related to a down-regulation of the main virus receptor. Using cDNA hybridization to evaluate mRNA accumulation in the cells, we were able to identify several genes that are differently up- or down-regulated by IFN-gamma in A/J (267 and 266 genes, respectively, up- and down-regulated or BALB/c (297 and 58 genes, respectively, up- and down-regulated mouse macrophages. Macrophages from mice with different genetic backgrounds behave differently at the molecular level and comparison of the patterns of non-activated and IFN-gamma-activated A/J or BALB/c mouse macrophages revealed, for instance, an up-regulation and a down-regulation of genes coding for biological functions such as enzymatic reactions, nucleic acid synthesis and transport, protein synthesis, transport and metabolism, cytoskeleton arrangement and extracellular matrix, phagocytosis, resistance and susceptibility to infection and tumors, inflammation, and cell differentiation or activation. The present data are reported in order to facilitate future correlation of proteomic/transcriptomic findings as well as of results obtained from a classical approach for the understanding of biological phenomena. The possible implication of the role of some of the gene products relevant to macrophage biology can now be further scrutinized. In this respect, a down-regulation of the main murine hepatitis virus 3 receptor gene was detected only in IFN-gamma-activated macrophages of resistant mice.

  5. Animal experimentation.

    Kolar, Roman


    Millions of animals are used every year in often times extremely painful and distressing scientific procedures. Legislation of animal experimentation in modern societies is based on the supposition that this is ethically acceptable when certain more or less defined formal (e.g. logistical, technical) demands and ethical principles are met. The main parameters in this context correspond to the "3Rs" concept as defined by Russel and Burch in 1959, i.e. that all efforts to replace, reduce and refine experiments must be undertaken. The licensing of animal experiments normally requires an ethical evaluation process, often times undertaken by ethics committees. The serious problems in putting this idea into practice include inter alia unclear conditions and standards for ethical decisions, insufficient management of experiments undertaken for specific (e.g. regulatory) purposes, and conflicts of interest of ethics committees' members. There is an ongoing societal debate about ethical issues of animal use in science. Existing EU legislation on animal experimentation for cosmetics testing is an example of both the public will for setting clear limits to animal experiments and the need to further critically examine other fields and aspects of animal experimentation.

  6. Protein-RNA linkage and posttranslational modifications of feline calicivirus and murine norovirus VPg proteins

    Allan Olspert


    Full Text Available Members of the Caliciviridae family of positive sense RNA viruses cause a wide range of diseases in both humans and animals. The detailed characterization of the calicivirus life cycle had been hampered due to the lack of robust cell culture systems and experimental tools for many of the members of the family. However, a number of caliciviruses replicate efficiently in cell culture and have robust reverse genetics systems available, most notably feline calicivirus (FCV and murine norovirus (MNV. These are therefore widely used as representative members with which to examine the mechanistic details of calicivirus genome translation and replication. The replication of the calicivirus RNA genome occurs via a double-stranded RNA intermediate that is then used as a template for the production of new positive sense viral RNA, which is covalently linked to the virus-encoded protein VPg. The covalent linkage to VPg occurs during genome replication via the nucleotidylylation activity of the viral RNA-dependent RNA polymerase. Using FCV and MNV, we used mass spectrometry-based approach to identify the specific amino acid linked to the 5′ end of the viral nucleic acid. We observed that both VPg proteins are covalently linked to guanosine diphosphate (GDP moieties via tyrosine positions 24 and 26 for FCV and MNV respectively. These data fit with previous observations indicating that mutations introduced into these specific amino acids are deleterious for viral replication and fail to produce infectious virus. In addition, we also detected serine phosphorylation sites within the FCV VPg protein with positions 80 and 107 found consistently phosphorylated on VPg-linked viral RNA isolated from infected cells. This work provides the first direct experimental characterization of the linkage of infectious calicivirus viral RNA to the VPg protein and highlights that post-translational modifications of VPg may also occur during the viral life cycle.

  7. Co-culture: A quick approach for isolation of street rabies virus in murine neuroblastoma cells

    A. Sasikalaveni


    Full Text Available Background: Laboratory detection of rabies in most cases is based on detection of the antigen by fluorescent antibody test, however, in weak positive cases confirmative laboratory diagnosis depends on widely accepted mouse inoculation test. Cell lines like neuroblastoma have been used to isolate the virus with greater success not only to target for diagnosis, but also for molecular studies that determine the epidemiology of the circulating street rabies strains and in studies that look at the efficiency of the developed monoclonal antibodies to neutralize the different rabies strains. Due to the recent issues in obtaining ethical permission for mouse experimentation, and also the passages required in the cell lines to isolate the virus, we report herewith a co-culture protocol using the murine neuroblastoma (MNA cells, which enable quicker isolation of street rabies virus with minimum passages. Objective: This study is not to have an alternative diagnostic assay, but an approach to produce sufficient amount of rabies virus in minimum passages by a co-culture approach in MNA cells. Materials and Methods: The MNA cells are co-cultured by topping the normal cells with infected cells every 48 h and the infectivity was followed up by performing direct fluorescent-antibody test. Results: The co-culture approach results in 100% infectivity and hence the use of live mouse for experimentation could be avoided. Conclusion: Co-culture method provides an alternative for the situations with limited sample volume and for the quicker isolation of virus which warrants the wild type strains without much modification.

  8. Enteric salmonellosis disrupts the microbial ecology of the murine gastrointestinal tract

    Barman, Melissa; Unold, David; Shifley, Kathleen; Amir, Elad; Hung, Kueichun; Bos, Nicolaas; Salzman, Nita


    The commensal microbiota protects the murine host from enteric pathogens. Nevertheless, specific pathogens are able to colonize the intestinal tract and invade, despite the presence of an intact biota. Possibly, effective pathogens disrupt the indigenous microbiota, either directly through pathogen-

  9. Modeling Synergistic Drug Inhibition of Mycobacterium tuberculosis Growth in Murine Macrophages


    synergistic drug inhibition of Mycobacterium tuberculosis growth in murine macrophagesw Xin Fang, Anders Wallqvist and Jaques Reifman* Received 15th...inhibition of Mycobacterium tuberculosis in murine macrophage cells. We used it to simulate ex vivo bacterial growth inhibition due to 3-nitropropionate ( felt worldwide, with 9.4 million new cases and 1.8 million deaths in 2008.1,2 The causative agent of the disease, Mycobacterium tuberculosis

  10. Immunoenhancing activity of protopanaxatriol-type ginsenoside-F3 in murine spleen cells

    Jun-li YU; De-qiang DOU; Xiao-hong CHEN; Hong-zhen YANG; Na GUO; Gui-fang CHENG


    AIM: To investigate the immunoenhancing activity of ginsenoside-F3 in murine spleen cells and explore its mechanism.METHODS: The enhancing effect of ginsenoside-F3 on murine spleen cell proliferation was studied using [3H]thymidine incorporation assay. Effects of ginsenoside-F3 on the production of type 1 cytokines IL-2, IFN-γ, and type 2 cytokines IL-4 and IL-10 from murine spleen cells were detected by ELISA method. Effects of ginsenosideF3 on mRNA level of cytokines IL-4, IFN-γ, and transcription factors T-bet and GATA-3 were evaluated by RTPCR analysis. Effect of ginsenoside-F3 on NF-κB DNA binding activity in murine spleen cells was investigated by electrophoretic mobility shift assays (EMSA). RESULTS: Ginsenoside-F3 at 0.1-100μmol/L not only promoted the murine spleen cell proliferation, but also increased the production of IL-2 and IFN-γ, while decreased the production of IL-4 and IL-10 from murine spleen cells with the maximal effect at 10μmol/L. RT-PCR analysis displayed that ginsenoside-F3 enhanced the IFN-γ and T-bet gene expression and decreased IL-4 and GATA-3 gene expression. EMSA experiment showed that ginsenoside-F3 10μmol/L enhanced the NF-κB DNA binding activity induced by ConA in murine spleen cells. CONCLUSION: Ginsenoside-F3 has immunoenhancing activity by regulating production and gene expression of type 1 cytokines and type 2 cytokines in murine spleen cells.

  11. Sequencing and bacterial expression of a novel murine alpha interferon gene

    王焱; 王征宇; 周鸣南; 蔡菊娥; 孙兰英; 刘新垣; B.L.Daugherty; S.Pestka


    A murine new alpha interferon gene (mIFN-αB) was found by primer-based sequencing method in a murine genomic DNA library. The gene was cloned and its sequence was determined. It was expressed in Escherichia coli under the control of the PL promoter which resulted in antiviral activity on mouse L-cells. The sequence of mlFN-αB has been accepted by GENEBANK.

  12. 4T1 Murine Mammary Carcinoma Cells Enhance Macrophage-Mediated Innate Inflammatory Responses

    Laurence Madera; Anna Greenshields; Power Coombs, Melanie R.; Hoskin, David W.


    Tumor progression and the immune response are intricately linked. While it is known that cancers alter macrophage inflammatory responses to promote tumor progression, little is known regarding how cancers affect macrophage-dependent innate host defense. In this study, murine bone-marrow-derived macrophages (BMDM) were exposed to murine carcinoma-conditioned media prior to assessment of the macrophage inflammatory response. BMDMs exposed to 4T1 mammary carcinoma-conditioned medium demonstrated...

  13. Fundación del primer bioterio MPF funcional de Colombia Foundation of a functional murine pathogen free animal facility in Colombia

    Ómar Vesga


    Full Text Available Cualquier investigación donde se experimente con modelos animales exige que el estado microbiológico y genético de los mismos sea definido desde el principio y verificado periódicamente para garantizar la fiabilidad y certeza de los datos. Buscando producir ratones libres de patógenos murinos en Colombia, se importaron 173 reproductores no emparentados suizos albinos de la cepa ICR que se hospedaron bajo condiciones protocolizadas de aislamiento microbiológico en el Bioterio MPF de la Facultad de Medicina de la Universidad de Antioquia. Tras dos años de funcionamiento ininterrumpido, el control microbiológico demostró que los progenitores mantuvieron inalterada la flora con la cual fueron exportados a Colombia, que sus crías portan flora murina idéntica, y que la misma corresponde a la exigida internacionalmente. Diversas pruebas a la integridad de la barrera microbiológica condujeron a la detección y erradicación de un brote infeccioso por Parvovirus murino. El control reproductivo permitió mantener intacta la heterocigocidad de la cepa, denominada Udea:ICR(CD-1. In order to guarantee the accuracy of the data, experimentation with animal models requires well defined microbiological and genetic statuses with periodical verification. To produce murine pathogen free mice, an outbreeding program was set up by importing 173 non-related Swiss albino ICR breeders that were hosted under strict conditions of microbiological isolation in the animal facilities of the University of Antioquia Medical Faculty. After two years of uninterrupted reproduction, microbiological control demonstrated unaltered murine flora for both parents and offspring, the same with which original breeders were dispatched to Colombia, complying with current international standards. An outbreak of Murine Parvovirus (MPV was timely detected and erradicated thanks to strict testing of microbiological barriers. The breeding program allowed the production of animals free

  14. Murine AIDS: a model for the human disease or a distinct entity?

    Cunnigham, R K; Thacore, H R; Zhou, P; Terzian, R; Nakeeb, S; Zaleski, M B


    The LP-BM5 mixture of murine retroviruses elicits a disease in mice referred to as murine immunodeficiency syndrome (MAIDS) that is considered by some to be an animal homologue of human AIDS. In this article, we present and discuss some recent findings on the pathogenesis of the murine disease and their implications for the proposed homology between murine and human syndromes. The murine disease seems to display as many similarities to as it does differences from human AIDS. Among the latter are: definitive and exclusive viral etiology, a strong genetic effect on susceptibility to infection, expansion of the CD4+ cell population in spleen and peripheral blood, consistent transmissibility by a single transfusion of the minute amounts of blood or plasma from infected donors, and striking similarity between virus-induced alteration of the in vitro spleen cell proliferation and those caused by treatment with a protein kinase inhibitor K252a. With this in mind, the use of the noncommittal term retrovirus-induced murine lymphoproliferative disease instead of MAIDS appears to be more appropriate at this time.

  15. A role for smoothened during murine lens and cornea development.

    Janet J Y Choi

    Full Text Available Various studies suggest that Hedgehog (Hh signalling plays roles in human and zebrafish ocular development. Recent studies (Kerr et al., Invest Ophthalmol Vis Sci. 2012; 53, 3316-30 showed that conditionally activating Hh signals promotes murine lens epithelial cell proliferation and disrupts fibre differentiation. In this study we examined the expression of the Hh pathway and the requirement for the Smoothened gene in murine lens development. Expression of Hh pathway components in developing lens was examined by RT-PCR, immunofluorescence and in situ hybridisation. The requirement of Smo in lens development was determined by conditional loss-of-function mutations, using LeCre and MLR10 Cre transgenic mice. The phenotype of mutant mice was examined by immunofluorescence for various markers of cell cycle, lens and cornea differentiation. Hh pathway components (Ptch1, Smo, Gli2, Gli3 were detected in lens epithelium from E12.5. Gli2 was particularly localised to mitotic nuclei and, at E13.5, Gli3 exhibited a shift from cytosol to nucleus, suggesting distinct roles for these transcription factors. Conditional deletion of Smo, from ∼E12.5 (MLR10 Cre did not affect ocular development, whereas deletion from ∼E9.5 (LeCre resulted in lens and corneal defects from E14.5. Mutant lenses were smaller and showed normal expression of p57Kip2, c-Maf, E-cadherin and Pax6, reduced expression of FoxE3 and Ptch1 and decreased nuclear Hes1. There was normal G1-S phase but decreased G2-M phase transition at E16.5 and epithelial cell death from E14.5-E16.5. Mutant corneas were thicker due to aberrant migration of Nrp2+ cells from the extraocular mesenchyme, resulting in delayed corneal endothelial but normal epithelial differentiation. These results indicate the Hh pathway is required during a discrete period (E9.5-E12.5 in lens development to regulate lens epithelial cell proliferation, survival and FoxE3 expression. Defective corneal development occurs

  16. Bone marrow mononuclears from murine tibia after spaceflight on biosatellite

    Andreeva, Elena; Roe, Maria; Buravkova, Ludmila; Andrianova, Irina; Goncharova, Elena; Gornostaeva, Alexandra

    Elucidation of the space flight effects on the adult stem and progenitor cells is an important goal in space biology and medicine. A unique opportunity for this is provided by project "BION -M1". The purpose of this study was to evaluate the effects of a 30-day flight on biosatellite "BION - M1" and the subsequent 7-day recovery on the quantity, viability, immunophenotype of mononuclears from murine tibia bone marrow. Also the in vitro characterization of functional capacity of multipotent mesenchymal stromal cells (MSCs) was scheduled. Under the project, the S57black/6 mice were divided into groups: spaceflight/vivarium control, recovery after spaceflight/ vivarium control to recovery. Bone marrow mononuclears were isolated from the tibia and immunophenotyped using antibodies against CD45, CD34, CD90 on a flow cytometer Epics XL (Beckman Coulter). A part of the each pool was frozen for subsequent estimation of hematopoietic colony-forming units (CFU), the rest was used for the evaluation of fibroblast CFU (CFUf) number, MSC proliferative activity and osteogenic potency. The cell number in the flight group was significantly lower than in the vivarium control group. There were no differences in this parameter between flight and control groups after 7 days of recovery. The mononuclears viability was more than 95 percent in all examined groups. Flow cytometric analysis showed no differences in the bone marrow cell immunophenotype (CD45, CD34, CD90.1 (Thy1)), but the flight animals had more large-sized CD45+mononuclears, than the control groups of mice. There was no difference in the CFUf number between groups. After 7 days in vitro the MSC number in flight group was twice higher than in vivarium group, after 10 days - 4 times higher. These data may indicate a higher proliferative activity of MSCs after spaceflight. MSCs showed the same and high alkaline phosphatase activity, both in flight and in the control groups, suggesting no effect of spaceflight factors on early

  17. Assessment of carbon nanoparticle exposure on murine macrophage function

    Suro-Maldonado, Raquel M.

    There is growing concern about the potential cytotoxicity of nanoparticles. Exposure to respirable ultrafine particles (2.5uM) can adversely affect human health and have been implicated with episodes of increased respiratory diseases such as asthma and allergies. Nanoparticles are of particular interest because of their ability to penetrate into the lung and potentially elicit health effects triggering immune responses. Nanoparticles are structures and devises with length scales in the 1 to 100-nanometer range. Black carbon (BC) nanoparticles have been observed to be products of combustion, especially flame combustion and multi-walled carbon nanotubes (MWCNT) have been shown to be found in both indoor and outdoor air. Furthermore, asbestos, which have been known to cause mesothelioma as well as lung cancer, have been shown to be structurally identical to MWCNTs. The aims of these studies were to examine the effects of carbon nanoparticles on murine macrophage function and clearance mechanisms. Macrophages are immune cells that function as the first line of defense against invading pathogens and are likely to be amongst the first cells affected by nanoparticles. Our research focused on two manufactured nanoparticles, MWCNT and BC. The two were tested against murine-derived macrophages in a chronic contact model. We hypothesized that long-term chronic exposure to carbon nanoparticles would decrease macrophages ability to effectively respond to immunological challenge. Production of nitric oxide (NO), tumor necrosis factor alpha (TNF-alpha), cell surface macrophage; activation markers, reactive oxygen species formation (ROS), and antigen processing and presentation were examined in response to lipopolysaccharide (LPS) following a 144hr exposure to the particulates. Data demonstrated an increase in TNF-alpha, and NO production; a decrease in phagocytosis and antigen processing and presentation; and a decrease in the expression levels of cell surface macrophage

  18. Reverse genetics mediated recovery of infectious murine norovirus.

    Arias, Armando; Ureña, Luis; Thorne, Lucy; Yunus, Muhammad A; Goodfellow, Ian


    Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close person-to-person contact cannot be avoided (1, 2). During the last few years an increase in the incidence of outbreaks in hospitals has been reported, causing significant disruptions to their operational capacity as well as large economic losses. The identification of new antiviral approaches has been limited due to the inability of human noroviruses to complete a productive infection in cell culture (3). The recent isolation of a murine norovirus (MNV), closely related to human norovirus (4) but which can be propagated in cells (5) has opened new avenues for the investigation of these pathogens (6, 7). MNV replication results in the synthesis of new positive sense genomic and subgenomic RNA molecules, the latter of which corresponds to the last third of the viral genome (Figure 1). MNV contains four different open reading frames (ORFs), of which ORF1 occupies most of the genome and encodes seven non-structural proteins (NS1-7) released from a polyprotein precursor. ORF2 and ORF3 are contained within the subgenomic RNA region and encode the capsid proteins (VP1 and VP2, respectively) (Figure 1). Recently, we have identified that additional ORF4 overlapping ORF2 but in a different reading frame is functional and encodes for a mitochondrial localised virulence factor (VF1) (8). Replication for positive sense RNA viruses, including noroviruses, takes place in the cytoplasm resulting in the synthesis of new uncapped RNA genomes. To promote viral translation, viruses exploit different strategies aimed at recruiting the cellular protein synthesis machinery (9-11). Interestingly, norovirus translation is driven by the multifunctional viral protein-primer VPg covalently linked to the 5' end of both genomic and subgenomic RNAs (12-14). This sophisticated mechanism of translation is likely to be a major factor in the limited

  19. Novel Approach for Evaluation of Bacteroides fragilis Protective Role against Bartonella henselae Liver Damage in Immunocompromised Murine Model

    Pagliuca, Chiara; Cicatiello, Annunziata G.; Colicchio, Roberta; Greco, Adelaide; Cerciello, Raimondo; Auletta, Luigi; Albanese, Sandra; Scaglione, Elena; Pagliarulo, Caterina; Pastore, Gabiria; Mansueto, Gelsomina; Brunetti, Arturo; Avallone, Bice; Salvatore, Paola


    Bartonella henselae is a gram-negative facultative intracellular bacterium and is the causative agent of cat-scratch disease. Our previous data have established that Bacteroides fragilis colonization is able to prevent B. henselae damages through the polysaccharide A (PSA) in an experimental murine model. In order to determine whether the PSA is essential for the protection against pathogenic effects of B. henselae in immunocompromised hosts, SCID mice were co-infected with B. fragilis wild type or its mutant B. fragilis ΔPSA and the effects of infection on murine tissues have been observed by High-Frequency Ultrasound (HFUS), histopathological examination, and Transmission Electron Microscopy (TEM). For the first time, echostructure, hepatic lobes length, vascular alterations, and indirect signs of hepatic dysfunctions, routinely used as signs of disease in humans, have been analyzed in an immunocompromised murine model. Our findings showed echostructural alterations in all infected mice compared with the Phosphate Buffer Solution (PBS) control group; further, those infected with B. henselae and co-infected with B. henselae/B. fragilis ΔPSA presented the major echostructural alterations. Half of the mice infected with B. henselae and all those co-infected with B. henselae/B. fragilis ΔPSA have showed an altered hepatic echogenicity compared with the renal cortex. The echogenicity score of co-infected mice with B. henselae/B. fragilis ΔPSA differed significantly compared with the PBS control group (p < 0.05). Moreover the inflammation score of the histopathological evaluation was fairly concordant with ultrasound findings. Ultrastructural analysis performed by TEM revealed no significant alterations in liver samples of SCID mice infected with B. fragilis wild type while those infected with B. fragilis ΔPSA showed the presence of collagen around the main vessels compared with the PBS control group. The liver samples of mice infected with B. henselae showed

  20. Cloning and sequencing of murine T3 gamma cDNA from a subtractive cDNA library.

    Haser, W G; Saito, H; Koyama, T; Tonegawa, S


    The coding sequences of the murine and human T3 gamma chains are of identical length (182 amino acids) and contain a remarkable conservation of residues. The most striking observation is the high degree of homology between the murine and human cytosolic domains (89%), suggesting that the effector function of the T3 complex may be extremely similar or identical within human and murine lymphocytes. Both murine and human T lymphocytes can express two T3 gamma mRNA transcripts, suggesting that a second polyadenylation signal is present downstream. A poly(A) tail is not found in the 3' untranslated region of the murine gamma presented here, indicating that the murine clones analyzed represent mRNA generated by reading through the overlapping poly(A) signals at position 850-860 and possibly terminating at a position that would produce the 1.0 kb transcript.

  1. Experimental music for experimental physics

    Rosaria Marraffino


    Using the sonification technique, physicist and composer Domenico Vicinanza paid homage to CERN at its 60th anniversary ceremony. After months of hard work, he turned the CERN Convention and LHC data into music.   Click here to download the full score of the "LHChamber music". Every birthday deserves gifts and CERN’s 60th anniversary was no exception. Two gifts were very special, thanks to the hard work of Domenico Vicinanza, a physicist and composer. He created two experimental pieces by applying the sonification technique to the CERN Convention and to data recorded by the four LHC detectors during Run 1. “This technique allows us to ‘hear’ data using an algorithm that translates numbers or letters into notes. It keeps the same information enclosed in a graph or a document, but has a more aesthetic exposition,” explains Domenico Vicinanza. “The result is meant to be a metaphor for scientific cooperation, in which d...

  2. Immunotherapeutic effects on murine pancreatic carcinoma by β-elemene combined with dendritic cells modified with genes encoding interleukin-23

    TAN Guang; WANG Zhongyu; CHE Luanqing; YIN Shuo


    The dendritic cell vaccine is a treatment vaccine with potent clinical applications.Functional cytokines can enhance dendritic cell anti-tumor immune responses.This experiment was conducted to study the effects of bone marrow-derived dendritic cells (BM-DCs) modified with genes encoding murine interleukin-23 (IL-23) on murine pancreatic carcinoma,and effects of the treatment of pancreatic carcinoma with β-elemene combined with IL-23-modified den dritic cell vaccine.The mttrine IL-23 cDNA was sub-cloned into a dual-expression vector.DCs were pulsed with tumor cell lysate after being modified wth IL-23.Mice were divided into groups which were injected with IL-23-transduced DC vaccine,non-transduced DC vaccine and sodium respectively.The preventive immune and immunotherapeutic effects of DC vaccines on mice and cytokine release in vivo were then assessed.Results showed inhibitory effects on tumor cells and increased survival time in the experimental group treated with the vaccine combined with β-elemene.The IL-23 protein apparently increases the antigen presenting ability of DCs.After injection with DC vaccines,IFN-γ production in the treatment group was significantly increased as compared with that in the control group (P < 0.01),and IL-4 production was decreased as compared with that in the control group (P<0.05).Tumor size was obviously reduced,and survival time clearly prolonged in the group with β-elemene combined with DC vaccine,in comparison to the other treatment groups and the control (P<0.01).IL-23-modified dendritic cell vaccines enhance specific Th1-type and cytotoxic T lymphocyte (CTL) responses against pancreatic carcinoma cells,and induce not only auto-immune ability but also preventive immunity against pancreatic carcinoma implanted in mice.β-elemene has great anti-tumor collaborative functions.

  3. Bmp4 from the optic vesicle specifies murine retina formation.

    Huang, Jie; Liu, Ying; Oltean, Alina; Beebe, David C


    Previous studies of mouse embryos concluded that after the optic vesicle evaginates from the ventral forebrain and contacts the surface ectoderm, signals from the ectoderm specify the distal region of the optic vesicle to become retina and signals from the optic vesicle induce the lens. Germline deletion of Bmp4 resulted in failure of lens formation. We performed conditional deletion of Bmp4 from the optic vesicle to test the function of Bmp4 in murine eye development. The optic vesicle evaginated normally and contacted the surface ectoderm. Lens induction did not occur. The optic cup failed to form and the expression of retina-specific genes decreased markedly in the distal optic vesicle. Instead, cells in the prospective retina expressed genes characteristic of the retinal pigmented epithelium. We conclude that Bmp4 is required for retina specification in mice. In the absence of Bmp4, formation of the retinal pigmented epithelium is the default differentiation pathway of the optic vesicle. Differences in the signaling pathways required for specification of the retina and retinal pigmented epithelium in chicken and mouse embryos suggest major changes in signaling during the evolution of the vertebrate eye.

  4. Murine leukemia virus (MLV replication monitored with fluorescent proteins

    Bittner Alexandra


    Full Text Available Abstract Background Cancer gene therapy will benefit from vectors that are able to replicate in tumor tissue and cause a bystander effect. Replication-competent murine leukemia virus (MLV has been described to have potential as cancer therapeutics, however, MLV infection does not cause a cytopathic effect in the infected cell and viral replication can only be studied by immunostaining or measurement of reverse transcriptase activity. Results We inserted the coding sequences for green fluorescent protein (GFP into the proline-rich region (PRR of the ecotropic envelope protein (Env and were able to fluorescently label MLV. This allowed us to directly monitor viral replication and attachment to target cells by flow cytometry. We used this method to study viral replication of recombinant MLVs and split viral genomes, which were generated by replacement of the MLV env gene with the red fluorescent protein (RFP and separately cloning GFP-Env into a retroviral vector. Co-transfection of both plasmids into target cells resulted in the generation of semi-replicative vectors, and the two color labeling allowed to determine the distribution of the individual genomes in the target cells and was indicative for the occurrence of recombination events. Conclusions Fluorescently labeled MLVs are excellent tools for the study of factors that influence viral replication and can be used to optimize MLV-based replication-competent viruses or vectors for gene therapy.

  5. An in vitro model of murine middle ear epithelium

    Apoorva Mulay


    Full Text Available Otitis media (OM, or middle ear inflammation, is the most common paediatric disease and leads to significant morbidity. Although understanding of underlying disease mechanisms is hampered by complex pathophysiology it is clear that epithelial abnormalities underpin the disease. There is currently a lack of a well-characterised in vitro model of the middle ear (ME epithelium that replicates the complex cellular composition of the middle ear. Here, we report the development of a novel in vitro model of mouse middle ear epithelial cells (mMECs at an air–liquid interface (ALI that recapitulates the characteristics of the native murine ME epithelium. We demonstrate that mMECs undergo differentiation into the varied cell populations seen within the native middle ear. Proteomic analysis confirmed that the cultures secrete a multitude of innate defence proteins from their apical surface. We showed that the mMECs supported the growth of the otopathogen, nontypeable Haemophilus influenzae (NTHi, suggesting that the model can be successfully utilised to study host–pathogen interactions in the middle ear. Overall, our mMEC culture system can help to better understand the cell biology of the middle ear and improve our understanding of the pathophysiology of OM. The model also has the potential to serve as a platform for validation of treatments designed to reverse aspects of epithelial remodelling that underpin OM development.

  6. SERCA overexpression reduces hydroxyl radical injury in murine myocardium.

    Hiranandani, Nitisha; Bupha-Intr, Tepmanas; Janssen, Paul M L


    Hydroxyl radicals (*OH) are involved in the pathogenesis of ischemia-reperfusion injury and are observed in clinical situations, including acute heart failure, stroke, and myocardial infarction. Acute transient exposure to *OH causes an intracellular Ca(2+) overload and leads to impaired contractility. We investigated whether upregulation of sarcoplasmic reticulum Ca(2+)-ATPase function (SERCA) can attenuate *OH-induced dysfunction. Small, contracting right ventricular papillary muscles from wild-type (WT) SERCA1a-overexpressing (transgenic, TG) and SERCA2a heterogeneous knockout (HET) mice were directly exposed to *OH. This brief 2-min exposure led to a transient elevation of diastolic force (F(dia)) and depression of developed force (F(dev)). In WT mice, F(dia) increased to 485 +/- 49% and F(dev) decreased to 11 +/- 3%. In sharp contrast, in TG mice F(dia) increased only to 241 +/- 17%, whereas F(dev) decreased only to 51 +/- 5% (P group. The results indicate that SERCA overexpression can reduce the *OH-induced contractile dysfunction in murine myocardium, whereas a reduced SR Ca(2+)-ATPase activity aggravates this injury. Loss of pPLB levels at Ser16 likely amplifies the differences observed in injury response.

  7. Evaluation of murine models of permanent focal cerebral ischemia

    席刚明; 汪华侨; 何国厚; 黄朝芬; 魏国耀


    Background To date murine models of permanent focal cerebral ischemia have not been well characterized. The purposes of this paper were to compare three different permanent middle cerebral artery occlusion (MCAo) models with or without craniectomy, and to identify an ideal mouse model of permanent focal cerebral ischemia.Methods Experiments were performed on 45 healthy adult male Kunming mice, weighing 28 to 42 g. The animals were randomly assigned to three groups (n=15 in every group) based on surgical procedure: MCAo via the external carotid artery (ECA), MCAo via the common carotid artery (CCA), and direct ligation of the middle cerebral artery (MCA). Each day post-ischemia, the animals were scored using an eight-grade neurological function scale, and mortality was also recorded. Seven days post-ischemia, the brains were removed for lesion size determination using triphenyltetrazolium chloride staining. Correlation analysis of lesion volume and neurological score was carried out. Results Mortality in the group receiving direct MCA ligation was lowest among the three groups, and there was a significant difference between the direct MCA ligation group and the two intraluminal occlusion groups (P0.7, P<0.05), suggesting good reproducibility of lesion volume in the three groups, but the infarct volume was more constant in the direct MCA ligation group. Conclusion The direct ligation model of MCAo provides an optimal means of studying permanent focal cerebral ischemia, and is preferable to the models using intraluminal sutures.

  8. Gene expression of lactobacilli in murine forestomach biofilms.

    Schwab, Clarissa; Tveit, Alexander Tøsdal; Schleper, Christa; Urich, Tim


    Lactobacilli populate the gastro-intestinal tract of vertebrates, and are used in food fermentations and as probiotics. Lactobacilli are also major constituents of stable biofilms in the forestomach of rodents. In order to investigate the lifestyle of these biofilm lactobacilli in C57BL/6 mice, we applied metatranscriptomics to analyse gene expression (assessed by mRNA) and community composition (assessed by rRNA). Lactobacillales were the major biofilm inhabitants (62-82% of rRNA reads), followed by Clostridiales (8-31% of rRNA reads). To identify mRNA transcripts specific for the forestomach, we compared forestomach and hindgut metatranscriptomes. Gene expression of the biofilm microbiota was characterized by high abundance of transcripts related to glucose and maltose utilization, peptide degradation, and amino acid transport, indicating their major catabolic and anabolic pathways. The microbiota transcribed genes encoding pathways enhancing oxidative stress (glutathione synthesis) and acid tolerance. Various pathways, including metabolite formation (urea degradation, arginine pathway, γ-aminobutyrate) and cell wall modification (DltA, cyclopropane-fatty-acyl-phospholipid synthase), contributed to acid tolerance, as judged from the transcript profile. In addition, the biofilm microbiota expressed numerous genes encoding extracellular proteins involved in adhesion and/or biofilm formation (e.g. MucBP, glycosyl hydrolase families 68 and 70). This study shed light on the lifestyle and specific adaptations of lactobacilli in the murine forestomach that might also be relevant for lactobacilli biofilms in other vertebrates, including humans.

  9. Nardilysin regulates inflammation, metaplasia, and tumors in murine stomach

    Kimura, Yuto; Ikuta, Kozo; Kimura, Takeshi; Chiba, Tsutomu; Oshima, Hiroko; Oshima, Masanobu; Nishi, Eiichiro; Seno, Hiroshi


    Chronic inflammation contributes to a wide variety of human disorders. In the stomach, longstanding gastritis often results in structural alterations in the gastric mucosa, including metaplastic changes and gastric cancers. Therefore, it is important to elucidate factors that are involved in gastric inflammation. Nardilysin (N-arginine dibasic convertase; Nrdc) is a metalloendopeptidase of the M16 family that promotes ectodomain shedding of the precursor forms of various growth factors and cytokines by enhancing the protease activities of a disintegrin and metalloproteinase (ADAM) proteins. Here, we have demonstrated that Nrdc crucially regulates gastric inflammation caused by Helicobacter felis infection or forced expression of prostaglandin E2 in K19-C2mE mice. Metaplastic changes following gastric inflammation were suppressed by the deletion of Nrdc. Furthremore, the deletion of Nrdc significantly suppressed N-methyl-N-nitrosourea (MNU)-induced gastric tumorigenesis in the murine stomach. These data may lead to a global therapeutic approach against various gastric disorders by targeting Nrdc. PMID:28230087

  10. Dynamics of notch expression during murine prostate development and tumorigenesis.

    Shou, J; Ross, S; Koeppen, H; de Sauvage, F J; Gao, W Q


    Notch signaling has been widely demonstrated to be responsible for cell fate determination during normal development and implicated in human T-cell leukemia and mouse mammary carcinomas. Here we show that Notch signaling may be involved in prostatic development and cancer cell growth. In situ hybridization and reverse transcription-PCR analyses revealed that Notch1 was expressed in prostate epithelial cells during normal development and in prostate cancer cells. Characterization of Notch1-green fluorescent protein transgenic mice, in which the expression of reporter green fluorescent protein is under the control of the Notch1 promoter, indicated that Notch1-expressing cells were associated with the basal epithelial cell population in the prostate. Examination of the transgenic adenocarcinoma of the mouse prostate showed that expression of Notch1 was elevated in malignant prostatic epithelial cells of primary and metastatic tumors. Expression of Notch ligands, however, was low or undetectable in cultured prostate cancer cells or in malignant prostatic epithelial cells in transgenic adenocarcinoma of the mouse prostate. Furthermore, overexpression of a constitutively active form of Notch1 inhibited the proliferation of various prostate cancer cells, including DU145, LNCaP, and PC3 cells. Taken together, our data indicate for the first time that Notch signaling may play a role in murine prostatic development and tumorigenesis.

  11. Dystrophic spinal deformities in a neurofibromatosis type 1 murine model.

    Steven D Rhodes

    Full Text Available Despite the high prevalence and significant morbidity of spinal anomalies in neurofibromatosis type 1 (NF1, the pathogenesis of these defects remains largely unknown. Here, we present two murine models: Nf1flox/-;PeriCre and Nf1flox/-;Col.2.3Cre mice, which recapitulate spinal deformities seen in the human disease. Dynamic histomorphometry and microtomographic studies show recalcitrant bone remodeling and distorted bone microarchitecture within the vertebral spine of Nf1flox/-;PeriCre and Nf1flox/-;Col2.3Cre mice, with analogous histological features present in a human patient with dystrophic scoliosis. Intriguingly, 36-60% of Nf1flox/-;PeriCre and Nf1flox/-;Col2.3Cre mice exhibit segmental vertebral fusion anomalies with boney obliteration of the intervertebral disc (IVD. While analogous findings have not yet been reported in the NF1 patient population, we herein present two case reports of IVD defects and interarticular vertebral fusion in patients with NF1. Collectively, these data provide novel insights regarding the pathophysiology of dystrophic spinal anomalies in NF1, and provide impetus for future radiographic analyses of larger patient cohorts to determine whether IVD and vertebral fusion defects may have been previously overlooked or underreported in the NF1 patient population.

  12. Cysteine protease activation and apoptosis in Murine norovirus infection

    Ettayebi Khalil


    Full Text Available Abstract Background Noroviruses are the leading cause of viral gastroenteritis. Because a suitable in vitro culture system for the human virus has yet to be developed, many basic details of the infection process are unknown. Murine norovirus (MNV serves as a model system for the study of norovirus infection. Recently it was shown that infection of RAW 264.7 cells involved a novel apoptotic pathway involving survivin. Results Using a different set of approaches, the up-regulation of caspases, DNA condensation/fragmentation, and membrane blebbing, all of which are markers of apoptosis, were confirmed. Live cell imaging and activity-based protein profiling showed that activation of caspase-like proteases occurred within two hours of infection, followed by morphological changes to the cells. MNV infection in the presence of caspase inhibitors proceeded via a distinct pathway of rapid cellular necrosis and reduced viral production. Affinity purification of activity-based protein profiling targets and identification by peptide mass fingerprinting showed that the cysteine protease cathepsin B was activated early in infection, establishing this protein as an upstream activator of the intrinsic apoptotic pathway. Conclusion This work adds cathepsin B to the noncanonical programmed cell death induced by MNV, and provides data suggesting that the virus may induce apoptosis to expand the window of time for viral replication. This work also highlights the significant power of activity-based protein profiling in the study of viral pathogenesis.

  13. In silico whole genome association scan for murine prepulse inhibition.

    Bradley Todd Webb

    Full Text Available BACKGROUND: The complex trait of prepulse inhibition (PPI is a sensory gating measure related to schizophrenia and can be measured in mice. Large-scale public repositories of inbred mouse strain genotypes and phenotypes such as PPI can be used to detect Quantitative Trait Loci (QTLs in silico. However, the method has been criticized for issues including insufficient number of strains, not controlling for false discoveries, the complex haplotype structure of inbred mice, and failing to account for genotypic and phenotypic subgroups. METHODOLOGY/PRINCIPAL FINDINGS: We have implemented a method that addresses these issues by incorporating phylogenetic analyses, multilevel regression with mixed effects, and false discovery rate (FDR control. A genome-wide scan for PPI was conducted using over 17,000 single nucleotide polymorphisms (SNPs in 37 strains phenotyped. Eighty-nine SNPs were significant at a false discovery rate (FDR of 5%. After accounting for long-range linkage disequilibrium, we found 3 independent QTLs located on murine chromosomes 1 and 13. One of the PPI positives corresponds to a region of human chromosome 6p which includes DTNBP1, a gene implicated in schizophrenia. Another region includes the gene Tsn which alters PPI when knocked out. These genes also appear to have correlated expression with PPI. CONCLUSIONS/SIGNIFICANCE: These results support the usefulness of using an improved in silico mapping method to identify QTLs for complex traits such as PPI which can be then be used for to help identify loci influencing schizophrenia in humans.

  14. Klebsiella pneumoniae FimK Promotes Virulence in Murine Pneumonia.

    Rosen, David A; Hilliard, Julia K; Tiemann, Kristin M; Todd, Elizabeth M; Morley, S Celeste; Hunstad, David A


    Klebsiella pneumoniae, a chief cause of nosocomial pneumonia, is a versatile and commonly multidrug-resistant human pathogen for which further insight into pathogenesis is needed. We show that the pilus regulatory gene fimK promotes the virulence of K. pneumoniae strain TOP52 in murine pneumonia. This contrasts with the attenuating effect of fimK on urinary tract virulence, illustrating that a single factor may exert opposing effects on pathogenesis in distinct host niches. Loss of fimK in TOP52 pneumonia was associated with diminished lung bacterial burden, limited innate responses within the lung, and improved host survival. FimK expression was shown to promote serum resistance, capsule production, and protection from phagocytosis by host immune cells. Finally, while the widely used K. pneumoniae model strain 43816 produces rapid dissemination and death in mice, TOP52 caused largely localized pneumonia with limited lethality, thereby providing an alternative tool for studying K. pneumoniae pathogenesis and control within the lung.

  15. Immunological impact of magnetic nanoparticles (Ferucarbotran) on murine peritoneal macrophages

    Yeh, Chen-Hao; Hsiao, Jong-Kai; Wang, Jaw-Lin; Sheu, Fuu


    Ferucarbotran, a clinically used superparamagnetic iron oxide, is widely developed as a magnetic resonance imaging (MRI) contrast agent and has the potential to improve the monitoring of macrophage recirculation in vivo. However, the biological effect of Ferucarbotran or magnetic nanoparticles (MNPs) on macrophage is not clearly understood yet. This study is aimed to examine the immunological impact of Ferucarbotran toward murine peritoneal macrophages. Cells treated with Ferucarbotran demonstrated a dose-responsive increase of granularity in the cytoplasm. After 24 h of incubation, viability and cytotoxicity in macrophages treated with 200 μg Fe/mL of Ferucarbotran were not affected. Macrophages loaded with Ferucarbotran above 100 μg Fe/mL showed a significant ( p TNF-α, IL-1β, IL-6) secretion and mRNA expression, followed by nitric oxide (NO) secretion and iNOS mRNA expression. Chemotactic responses of Ferucarbotran-preloaded macrophages toward CX3CL1 were significantly ( p inflammatory cytokine secretion and NO production.

  16. Inactivation of murine norovirus and feline calicivirus during oyster fermentation.

    Seo, Dong Joo; Lee, Min Hwa; Seo, Jina; Ha, Sang-Do; Choi, Changsun


    Fermented seafood is popular in Asian countries. This study examined the survival of feline calicivirus (FCV) and murine norovirus (MNV) during oyster fermentation. Oysters spiked with FCV and MNV were fermented with 5% or 10% salt at 18 °C for 15 days, and MNV and FCV titers, lactic acid bacteria (LAB) populations, pH, and enzymatic activity were measured at 0, 1, 3, 5, 7, 10, and 15 days post-fermentation (DPF). Reductions in MNV and FCV were greater in 5% NaCl-supplemented oysters than in 10% NaCl-supplemented oysters. In 5% NaCl oysters, MNV and FCV titers significantly decreased by 1.60 log and 3.01 log, respectively, at 15 DPF. Populations of LAB increased from 3.62 log10 colony-forming units/g at 0 DPF to 8.77 log10 colony-forming units/g at 15 DPF during oyster fermentation supplemented with 5% NaCl supplementation, and the pH decreased gradually from 5.38 at 0 DPF to 4.17 at 15 DPF. During oyster fermentation, α-amylase, proteinase, and lipase were produced at higher levels in 5% salted oysters than in 10% salted oysters (P oysters could contribute to a reduction in foodborne viruses.

  17. Non-apoptotic toxicity of Pseudomonas aeruginosa toward murine cells.

    Sanhita Roy

    Full Text Available Although P. aeruginosa is especially dangerous in cystic fibrosis (CF, there is no consensus as to how it kills representative cell types that are of key importance in the lung. This study concerns the acute toxicity of the sequenced strain, PAO1, toward a murine macrophage cell line (RAW 264.7. Toxicity requires brief contact with the target cell, but is then delayed for more than 12 h. None of the classical toxic effectors of this organism is required and cell death occurs without phagocytosis or acute perturbation of the actin cytoskeleton. Apoptosis is not required for toxicity toward either RAW 264.7 cells or for alveolar macrophages. Transcriptional profiling shows that encounter between PAO1 and RAW 264.7 cells elicits an early inflammatory response, followed by growth arrest. As an independent strategy to understand the mechanism of toxicity, we selected variant RAW 264.7 cells that resist PAO1. Upon exposure to P. aeruginosa, they are hyper-responsive with regard to classical inflammatory cytokine production and show transient downregulation of transcripts that are required for cell growth. They do not show obvious morphologic changes. Although they do not increase interferon transcripts, when exposed to PAO1 they dramatically upregulate a subset of the responses that are characteristic of exposure to g-interferon, including several guanylate-binding proteins. The present observations provide a novel foundation for learning how to equip cells with resistance to a complex challenge.

  18. BCMA deficiency exacerbates lymphoproliferation and autoimmunity in murine lupus1

    Jiang, Chao; Loo, William M.; Greenley, Erin J.; Tung, Kenneth S.; Erickson, Loren D.


    Systemic lupus erythematosus (SLE) and its preclinical lupus-prone mouse models are autoimmune disorders involving the production of pathogenic autoantibodies. Genetic predisposition to SLE results in B cell hyperactivity, survival of self-reactive B cells, and differentiation to autoantibody-secreting plasma cells (PC). These corrupt B cell responses are, in part, controlled by excess levels of the cytokine B cell activation factor from the TNF family (BAFF) that normally maintains B cell homeostasis and self-tolerance through limited production. B cell maturation antigen (BCMA) is a receptor for BAFF that, under nonautoimmune conditions, is important for sustaining enduring antibody protection by mediating survival of long-lived PCs, but is not required for B cell maturation and homeostasis. Through analysis of two different lupus-prone mouse models deficient in BCMA, we identify BCMA as an important factor in regulating peripheral B cell expansion, differentiation, and survival. We demonstrate that a BCMA deficiency combined with the lpr mutation or the murine lupus susceptibility locus Nba2 cause dramatic B cell and PC lymphoproliferation, accelerated autoantibody production, and early lethality. This study unexpectedly reveals that BCMA works to control B cell homeostasis and self-tolerance in systemic autoimmunity. PMID:21536804

  19. Scrapie strain-specific interactions with endogenous murine leukaemia virus.

    Carp, R I; Meeker, H C; Caruso, V; Sersen, E


    The finding that a senescence-accelerated mouse (SAMP8) shows early brain ageing, with histopathological changes resembling those seen in scrapie, combined with the discovery of high levels of endogenous murine leukaemia virus (MuLV) in brains of SAMP8 mice prompted us to examine the effect of scrapie infection on MuLV titres in this strain and in one of its progenitors, the AKR strain. Three scrapie strains (ME7, 22L and 139A) that had a comparatively short incubation period in SAMP8 and AKR mice caused an increase in brain MuLV titres that was scrapie strain-specific: in each mouse strain, the greatest effect was with 1 39A, and the least with ME7. The 22A scrapie strain, which has a long incubation period in SAMP8 mice, did not affect MuLV titres in brains of this mouse strain. Previous analyses of scrapie incubation periods in AKR, SAMP8 and another strain derived from an AKR cross (SAMR1) showed an inverse relationship between brain MuLV titres and scrapie incubation periods. This finding, combined with the effect of scrapie on MuLV titres, suggests an interaction between the scrapie infectious process and MuLV replication.

  20. Hamster and Murine Models of Severe Destructive Lyme Arthritis

    Erik Munson


    Full Text Available Arthritis is a frequent complication of infection in humans with Borrelia burgdorferi. Weeks to months following the onset of Lyme borreliosis, a histopathological reaction characteristic of synovitis including bone, joint, muscle, or tendon pain may occur. A subpopulation of patients may progress to a chronic, debilitating arthritis months to years after infection which has been classified as severe destructive Lyme arthritis. This arthritis involves focal bone erosion and destruction of articular cartilage. Hamsters and mice are animal models that have been utilized to study articular manifestations of Lyme borreliosis. Infection of immunocompetent LSH hamsters or C3H mice results in a transient synovitis. However, severe destructive Lyme arthritis can be induced by infecting irradiated hamsters or mice and immunocompetent Borrelia-vaccinated hamsters, mice, and interferon-gamma- (IFN-γ- deficient mice with viable B. burgdorferi. The hamster model of severe destructive Lyme arthritis facilitates easy assessment of Lyme borreliosis vaccine preparations for deleterious effects while murine models of severe destructive Lyme arthritis allow for investigation of mechanisms of immunopathology.

  1. Transplantable NK cell progenitors in murine bone marrow.

    Moore, T; Bennett, M; Kumar, V


    Differentiation of NK cells from pluripotent hematopoietic stem cells is a poorly understood process. Although it is known that NK cells are bone marrow derived and dependent upon an intact bone marrow microenvironment for complete maturation, it is not known if they arise from an intermediate lymphoid stem cell or from progenitors exclusively committed to the NK lineage. To determine whether phenotypically distinct committed NK progenitor cells exist in murine bone marrow, we sorted cells capable of repopulating recipient mice with mature NK cells upon i.v. transfer. We identified a rare population of bone marrow cells with the phenotype Ly6+ Lin- c-kit+ CD43high Fall-3high TSA-1- AA4.1low Rh123high that is highly enriched for the ability to generate NK cells after transplantation. Although these cells are relatively depleted of Rh123low pluripotent stem cells, they are highly enriched for both lymphoid and myeloid repopulating ability. Thus, we have found no evidence to support the existence of a phenotypically distinct transplantable progenitor population in mouse bone marrow that is either exclusively committed to the NK cell lineage or exhibits the functional characteristics of a common lymphoid stem cell.

  2. TALEN-mediated somatic mutagenesis in murine models of cancer.

    Zhang, Shuyuan; Li, Lin; Kendrick, Sara L; Gerard, Robert D; Zhu, Hao


    Cancer genome sequencing has identified numerous somatic mutations whose biologic relevance is uncertain. In this study, we used genome-editing tools to create and analyze targeted somatic mutations in murine models of liver cancer. Transcription activator-like effector nucleases (TALEN) were designed against β-catenin (Ctnnb1) and adenomatous polyposis coli (Apc), two commonly mutated genes in hepatocellular carcinoma (HCC), to generate isogenic HCC cell lines. Both mutant cell lines exhibited evidence of Wnt pathway dysregulation. We asked whether these TALENs could create targeted somatic mutations after hydrodynamic transfection into mouse liver. TALENs targeting β-catenin promoted endogenous HCC carrying the intended gain-of-function mutations. However, TALENs targeting Apc were not as efficient in inducing in vivo homozygous loss-of-function mutations. We hypothesized that hepatocyte polyploidy might be protective against TALEN-induced loss of heterozygosity, and indeed Apc gene editing was less efficient in tetraploid than in diploid hepatocytes. To increase efficiency, we administered adenoviral Apc TALENs and found that we could achieve a higher mutagenesis rate in vivo. Our results demonstrate that genome-editing tools can enable the in vivo study of cancer genes and faithfully recapitulate the mosaic nature of mutagenesis in mouse cancer models. Cancer Res; 74(18); 5311-21. ©2014 AACR.

  3. Saliva suppresses osteoclastogenesis in murine bone marrow cultures.

    Caballé-Serrano, J; Cvikl, B; Bosshardt, D D; Buser, D; Lussi, A; Gruber, R


    Saliva can reach mineralized surfaces in the oral cavity; however, the relationship between saliva and bone resorption is unclear. Herein, we examined whether saliva affects the process of osteoclastogenesis in vitro. We used murine bone marrow cultures to study osteoclast formation. The addition of fresh sterile saliva eliminated the formation of multinucleated cells that stained positive for tartrate-resistant acid phosphatase (TRAP). In line with the histochemical staining, saliva substantially reduced gene expression of cathepsin K, calcitonin receptor, and TRAP. Addition of saliva led to considerably decreased gene expression of receptor activator of nuclear factor kappa-B (RANK) and, to a lesser extent, that of c-fms. The respective master regulators of osteoclastogenesis (c-fos and NFATc1) and the downstream cell fusion genes (DC-STAMP and Atp6v0d2) showed decreased expression after the addition of saliva. Among the costimulatory molecules for osteoclastogenesis, only OSCAR showed decreased expression. In contrast, CD40, CD80, and CD86-all costimulatory molecules of phagocytic cells-were increasingly expressed with saliva. The phagocytic capacity of the cells was confirmed by latex bead ingestion. Based on these in vitro results, it can be concluded that saliva suppresses osteoclastogenesis and leads to the development of a phagocytic cell phenotype.

  4. Amphotropic murine leukemia viruses induce spongiform encephalomyelopathy

    Münk, Carsten; Löhler, Jürgen; Prassolov, Vladimir; Just, Ursula; Stockschläder, Marcus; Stocking, Carol


    Recombinants of amphotropic murine leukemia virus (A-MuLV) have found widespread use in retroviral vector systems due to their ability to efficiently and stably infect cells of several different species, including human. Previous work has shown that replication-competent recombinants containing the amphotropic env gene, encoding the major SU envelope glycoprotein that determines host tropism, induce lymphomas in vivo. We show here that these viruses also induce a spongiform encephalomyelopathy in mice inoculated perinatally. This fatal central nervous system disease is characterized by noninflammatory spongiform lesions of nerve and glial cells and their processes, and is associated with moderate astro- and microgliosis. The first clinical symptoms are ataxia, tremor, and spasticity, progressing to complete tetraparesis and incontinence, and finally death of the animal. Sequences within the amphotropic env gene are necessary for disease induction. Coinfection of A-MuLV recombinants with nonneuropathogenic ecotropic or polytropic MuLV drastically increases the incidence, degree, and distribution of the neurodegenerative disorder. The consequence of these results in view of the use of A-MuLV recombinants in the clinic is discussed. PMID:9159161

  5. A comparative parasitologic study on Biomphalaria glabrata snail and C3H/He mice infected with human and murine isolates of Schistosoma mansoni derived from Sumidouro, Rio de Janeiro, Brazil

    Nilcéa Freire


    Full Text Available Experiments were carried out to analyze the biological characteristics of two sympatric isolates of Schistosoma mansoni derived from humans and murines in a low endemic transmission area (Sumidouro county, state of Rio de Janeiro, Brazil. Sympatric reared-laboratory Biomphalaria glabrata and C3H/He mice were used as experimental hosts. Parameters assessed comprised: precercarial period, infectivity and mortality (snails, prepatent period, infectivity (percentage of cercariae maturation into adult worm and intestinal egg count (mice. The murine isolate showed a shorter precercarial period and higher infectivity than human isolate (p 0.05. These data suggest that both isolates are local sub-populations, providing support for the hypotheses that in a same biotope mixed populations or sub-populations circulate among their main host (human beings and/or rodent as an anfixenous infection.

  6. Abnormal morphology of myelin and axon pathology in murine models of multiple sclerosis.

    Bando, Yoshio; Nomura, Taichi; Bochimoto, Hiroki; Murakami, Koichi; Tanaka, Tatsuhide; Watanabe, Tsuyoshi; Yoshida, Shigetaka


    Demyelination and axonal damage are responsible for neurological deficits in multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system. However, the pathology of axonal damage in MS is not fully understood. In this study, histological analysis of morphological changes of axonal organelles during demyelination in murine models was investigated by scanning electron microscopy (SEM) using an osmium-maceration method. In cuprizone-induced demyelination, SEM showed typical morphology of demyelination in the corpus callosum of mouse brain. In contrast, SEM displayed variations in ultrastructural abnormalities of myelin structures and axonal organelles in spinal cord white matter of experimental autoimmune encephalomyelitis (EAE) mice, an animal model of MS. Myelin detachment and excessive myelin formation were observed as typical morphological myelin abnormalities in EAE. In addition, well-developed axoplasmic reticulum-like structures and accumulated mitochondria were observed in tortuous degenerating/degenerated axons and the length of mitochondria in axons of EAE spinal cord was shorter compared with naïve spinal cord. Immunohistochemistry also revealed dysfunction of mitochondrial fusion/fission machinery in EAE spinal cord axons. Moreover, the number of Y-shaped mitochondria was significantly increased in axons of the EAE spinal cord. Axonal morphologies in myelin basic protein-deficient shiverer mice were similar to those in EAE. However, shiverer mice had "tortuous" (S-curve shaped mitochondria) and larger mitochondria compared with wild-type and EAE mice. Lastly, analysis of human MS patient autopsied brains also demonstrated abnormal myelin structures in demyelinating lesions. These results indicate that morphological abnormalities of myelin and axonal organelles play important role on the pathogenesis of axonal injury in demyelinating diseases.

  7. Treatment With Tetrahydrobiopterin Overcomes Brain Death–Associated Injury in a Murine Model of Pancreas Transplantation

    Oberhuber, R.; Ritschl, P.; Fabritius, C.; Nguyen, A.‐V.; Hermann, M.; Obrist, P.; Werner, E. R.; Maglione, M.; Flörchinger, B.; Ebner, S.; Resch, T.; Pratschke, J.


    Brain death (BD) has been associated with an immunological priming of donor organs and is thought to exacerbate ischemia reperfusion injury (IRI). Recently, we showed that the essential nitric oxide synthase co‐factor tetrahydrobiopterin (BH4) abrogates IRI following experimental pancreas transplantation. We therefore studied the effects of BD in a murine model of syngeneic pancreas transplantation and tested the therapeutic potential of BH4 treatment. Compared with sham‐operated controls, donor BD resulted in intragraft inflammation reflected by induced IL‐1ß, IL‐6, VCAM‐1, and P‐selectin mRNA expression levels and impaired microcirculation after reperfusion (p < 0.05), whereas pretreatment of the BD donor with BH4 significantly improved microcirculation after reperfusion (p < 0.05). Moreover, BD had a devastating impact on cell viability, whereas BH4‐treated grafts showed a significantly higher percentage of viable cells (p < 0.001). Early parenchymal damage in pancreatic grafts was significantly more pronounced in organs from BD donors than from sham or non‐BD donors (p < 0.05), but BH4 pretreatment significantly ameliorated necrotic lesions in BD organs (p < 0.05). Pretreatment of the BD donor with BH4 resulted in significant recipient survival (p < 0.05). Our data provide novel insights into the impact of BD on pancreatic isografts, further demonstrating the potential of donor pretreatment strategies including BH4 for preventing BD‐associated injury after transplantation. PMID:26104062

  8. Cannabidiol increases survival and promotes rescue of cognitive function in a murine model of cerebral malaria.

    Campos, A C; Brant, F; Miranda, A S; Machado, F S; Teixeira, A L


    Cerebral malaria (CM) is a severe complication resulting from Plasmodium falciparum infection that might cause permanent neurological deficits. Cannabidiol (CBD) is a nonpsychotomimetic compound of Cannabis sativa with neuroprotective properties. In the present work, we evaluated the effects of CBD in a murine model of CM. Female mice were infected with Plasmodium berghei ANKA (PbA) and treated with CBD (30mg/kg/day - 3 or 7days i.p.) or vehicle. On 5th day-post-infection (dpi), at the peak of the disease), animals were treated with single or repeated doses of Artesunate, an antimalarial drug. All groups were tested for memory impairment (Novel Object Recognition or Morris Water Maze) and anxiety-like behaviors (Open field or elevated plus maze test) in different stages of the disease (at the peak or after the complete clearance of the disease). Th1/Th2 cytokines and neurotrophins (brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF)) were measured in the prefrontal cortex and hippocampus of experimental groups. PbA-infected mice displayed memory deficits and exhibited increase in anxiety-like behaviors on the 5dpi or after the clearance of the parasitemia, effects prevented by CBD treatment. On 5dpi, TNF-α and IL-6 increased in the hippocampus, while only IL-6 increased in the prefrontal cortex. CBD treatment resulted in an increase in BDNF expression in the hippocampus and decreased levels of proinflammatory cytokines in the hippocampus (TNF-α) and prefrontal cortex (IL-6). Our results indicate that CBD exhibits neuroprotective effects in CM model and might be useful as an adjunctive therapy to prevent neurological symptoms following this disease.

  9. Pathogenicity of Trichosporon asahii in a murine model of disseminated trichosporonosis


    Background In recent years, superficial and deep mycoses caused by trichosporon were occasionally reported. In 2001, we reported the first case of disseminated trichosporonosis caused by Trichosporon asahii (T. asahii) in China. In this study, the pathogenicity of T. asahii was investigated in a murine model of disseminated trichosporonosis. Methods Seventy-five mice were randomly divided into 7 groups. Each group was inoculated with T. asahii, through intradermal, gastrointestinal tract or intravenous injection. The mice in the experimental groups were given an intraperitoneal injection of cyclophosphamide (CY) to induce granulocytopenia. Mice in the therapeutic group were given both liposomal amphotericin B and fluconazole. The main viscera of the mice were examined by means of tissue culture and pathologic sections. Results In the two intravenous inoculation groups, T. asahii was isolated from at least one organ in 10 of the 12 granulocytopenic mice and 2 of the 14 immunocompetent mice. Two of the 7 mice in the granulocytopenia group presented with lesions in the inoculation position, but none of the 30 mice in the granulocytopenia and the control group which were inoculated intradermally or through the gastrointestinal tract had viscera infection. In the therapeutic group, the ratio of consequently dead mice, the number of involved viscera, and the incidence of systemic infection were significantly less than the untreated group. Acute purulent inflammation and granulomatous inflammation were the main pathological changes in the course of the infection. Arthrospores and filaments were found in the focus. Conclusions T. asahii is an opportunistic pathogen that causes cutaneous and visceral infections in immunologically impaired hosts. An immunocompetent host was to be infected by the invading T. asahii. Several organs, namely the liver, lungs, kidneys, spleen and heart, were predisposed. The therapy of combining liposomal amphotericin B with fluconazole can

  10. Nanomechanical assessment of human and murine collagen fibrils via atomic force microscopy cantilever-based nanoindentation.

    Andriotis, Orestis G; Manuyakorn, Wiparat; Zekonyte, Jurgita; Katsamenis, Orestis L; Fabri, Sebastien; Howarth, Peter H; Davies, Donna E; Thurner, Philipp J


    The nanomechanical assessment of collagen fibrils via atomic force microscopy (AFM) is of increasing interest within the biomedical research community. In contrast to conventional nanoindentation there exists no common standard for conducting experiments and analysis of data. Currently used analysis approaches vary between studies and validation of quantitative results is usually not performed, which makes comparison of data from different studies difficult. Also there are no recommendations with regards to the maximum indentation depth that should not be exceeded to avoid substrate effects. Here we present a methodology and analysis approach for AFM cantilever-based nanoindentation experiments that allows efficient use of captured data and relying on a reference sample for determination of tip shape. Further we show experimental evidence that maximum indentation depth on collagen fibrils should be lower than 10-15% of the height of the fibril to avoid substrate effects and we show comparisons between our and other approaches used in previous works. While our analysis approach yields similar values for indentation modulus compared to the Oliver-Pharr method we found that Hertzian analysis yielded significantly lower values. Applying our approach we successfully and efficiently indented collagen fibrils from human bronchi, which were about 30 nm in size, considerably smaller compared to collagen fibrils obtained from murine tail-tendon. In addition, derived mechanical parameters of collagen fibrils are in agreement with data previously published. To establish a quantitative validation we compared indentation results from conventional and AFM cantilever-based nanoindentation on polymeric samples with known mechanical properties. Importantly we can show that our approach yields similar results when compared to conventional nanoindentation on polymer samples. Introducing an approach that is reliable, efficient and taking into account the AFM tip shape, we anticipate



    Objective: To investigate the therapeutic effects of the murine IL-12 (mIL-12) retrovirus packaging cell line on hepatoma injected locally. Methods: The retrovirus vector encoding mIL-12 gene was constructed and transfected into packaging cell line PA317. The cells were then used to treat the rats with experimental orthotopic hepatoma at different time. The therapeutic effects, immune functions of the hosts, pathological and toxicological responses were documented. Results: the results showed that the mIL-12 retrovirus packaging cell line could significantly inhibit the growth of the hepatoma cells injected locally to the hepatoma. The early treatment made the rats survive long, while the medium or late stage treatment could prolong the life time of the rats compared with the bland control group or bland vector control group, though the rats did not survive. The number of NK cells and T cells increased significantly in the treatment group. The effects of the early treatment were superior to those of the medium and late stage treatment. Moreover, the transfection of IL-12 gene locally in the hepatoma tissue could make the hepatoma disappear from other liver lobe. This phenomenon demonstrated that IL-12 could activate the immune cells of the host to kill the untransfected tumor cells. This is very important for IL-12 to be used in gene therapy clinically. Meanwhile, the hepatoma would not recur in the rats that had survived more than 2 months from the early treatment after being re-challenged with tumor cells. Conclusion: the results showed that IL-12 gene injected locally in the hepatoma tissue could enhance the anti-tumor immunity of the host.

  12. Detection of Neospora caninum in tissue sections using a murine monoclonal antibody.

    Cole, R A; Lindsay, D S; Dubey, J P; Blagburn, B L


    A murine monoclonal antibody (MAb 6G7), isotype IgG2a, produced against tachyzoites of Neospora caninum (isolate NC-1) reacted specifically with tachyzoites of N. caninum in an indirect immunofluorescent antibody test. MAb 6G7 did not react with tachyzoites of Toxoplasma gondii, sporozoites of Isospora suis, Eimeria bovis, or E. tenella, or merozoites of E. bovis in the indirect immunofluorescent antibody test. MAb 6G7 reacted positively with both tachyzoites and bradyzoites of N. caninum in an avidin-biotin peroxidase complex immunohistochemical test on formalin-fixed paraffin-embedded tissues. No reaction was observed with the following: tachyzoites and bradyzoites of T. gondii, T. gondii-like parasites, or Hammondia hammondi; bradyzoites of Frenkelia microti; schizonts and merozoites of Sarcocystis-like organisms; schizonts, sarcocysts, and oocysts/sporocysts of S. cruzi; schizonts and merozoites of S. canis; schizonts of S. hirsuta, S. tenella, and S. capracanis; merozoites of S. neurona and S. neurona-like organisms, E. bovis, or Haemoproteus sp.; bradyzoites and merozoites of S. montanaensis; bradyzoites of S. odocoileocanis, S. cruzi, and S. tenella; meronts, sexual stages, and caryocysts of Caryospora sp. and C. bigenetica; micromerozoites, macromerozoites, and schizonts of Hepatozoon canis; sporozoites, sexual stages, and oocysts of Cryptosporidium parvum and C. baileyi; trophozoites of Monocystis lumbrici, Tritrichomonas foetus, and Balantidium coli; tissue cysts and bradyzoites of Besnoitia sp. and B. jellisoni; amastigotes of Leishmania sp.; and trophic theronts of Ichthyopthirius multifilis. MAb 6G7 reacted with tachyzoites and bradyzoites of N. caninum in natural and experimental infections in dogs, cattle, mice, rats, sheep, and goats, indicating that host origin of the tissues did not affect the performance of the test.

  13. C/EBPβ is a transcriptional key regulator of IL-36α in murine macrophages.

    Nerlich, Andreas; Ruangkiattikul, Nanthapon; Laarmann, Kristin; Janze, Nina; Dittrich-Breiholz, Oliver; Kracht, Michael; Goethe, Ralph


    Interleukin (IL)-36α - one of the novel members of the IL-1 family of cytokines - is a potent regulator of dendritic and T cells and plays an important role in inflammatory processes like experimental skin inflammation in mice and in mouse models for human psoriasis. Here, we demonstrate that C/EBPβ, a transcription factor required for the selective expression of inflammatory genes, is a key activator of the Il36A gene in murine macrophages. RNAi-mediated suppression of C/EBPβ expression in macrophages (C/EBPβ(low) cells) significantly impaired Il36A gene induction following challenge with LPS. Despite the presence of five predicted C/EBP binding sites, luciferase reporter assays demonstrated that C/EBPβ confers responsiveness to LPS primarily through a half-CRE•C/EBP element in the proximal Il36A promoter. Electrophoretic mobility shift assays showed that C/EBPβ but not CREB proteins interact with this critical half-CRE•C/EBP element. In addition, overexpression of C/EBPβ in C/EBPβ(low) cells enhanced the expression of Il36A whereas CREB-1 had no effect. Finally, chromatin immunoprecipitation confirmed that C/EBPβ but neither CREB-1, ATF-2 nor ATF4 is directly recruited to the proximal promoter region of the Il36A gene. Together, these findings demonstrate an essential role of C/EBPβ in the regulation of the Il36A gene via the proximal half-CRE•C/EBP element in response to inflammatory stimuli.

  14. Membrane-dependent Activities of Human 15-LOX-2 and Its Murine Counterpart: IMPLICATIONS FOR MURINE MODELS OF ATHEROSCLEROSIS.

    Bender, Gunes; Schexnaydre, Erin E; Murphy, Robert C; Uhlson, Charis; Newcomer, Marcia E


    The enzyme encoded by the ALOX15B gene has been linked to the development of atherosclerotic plaques in humans and in a mouse model of hypercholesterolemia. In vitro, these enzymes, which share 78% sequence identity, generate distinct products from their substrate arachidonic acid: the human enzyme, a 15-S-hydroperoxy product; and the murine enzyme, an 8-S-product. We probed the activities of these enzymes with nanodiscs as membrane mimics to determine whether they can access substrate esterified in a bilayer and characterized their activities at the membrane interface. We observed that both enzymes transform phospholipid-esterified arachidonic acid to a 15-S-product. Moreover, when expressed in transfected HEK cells, both enzymes result in significant increases in the amounts of 15-hydroxyderivatives of eicosanoids detected. In addition, we show that 15-LOX-2 is distributed at the plasma membrane when the HEK293 cells are stimulated by the addition Ca(2+) ionophore and that cellular localization is dependent upon the presence of a putative membrane insertion loop. We also report that sequence differences between the human and mouse enzymes in this loop appear to confer distinct mechanisms of enzyme-membrane interaction for the homologues.

  15. Experimental evidence on the immunopathogenesis of primary biliary cirrhosis

    Selmi, Carlo; Meda, Francesca; Kasangian, Anaid; Invernizzi, Pietro; Tian, Zhigang; Lian, Zhexiong; Podda, Mauro; Gershwin, M Eric


    Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease for which an autoimmune pathogenesis is supported by clinical and experimental data, including the presence of autoantibodies and autoreactive T cells. The etiology remains to be determined, yet data suggest that both a susceptible genetic background and unknown environmental factors determine disease onset. Multiple infectious and chemical candidates have been proposed to trigger the disease in a genetically susceptible host, mostly by molecular mimicry. Most recently, several murine models have been reported, including genetically determined models as well as models induced by immunization with xenobiotics and bacteria. PMID:20029462

  16. Effect of resveratrol on cell cycle proteins in murine transplantable liver cancer

    Liang Yu; Zhong-Jie Sun; Sheng-Li Wu; Cheng-En Pan


    AIM: To study the antitumour activity of resveratrol and its effect on the expression of ceil cycle proteins including cyclin D1, cyclin B1 and p34cdc2 in transplanted liver cancer of murine.METHODS: Murine transplanted hepatoma H22 model was used to evaluate the in vivo antitumor activity of resveratrol.Following abdominal administration of resveratrol, the change in tumour size was recorded and the protein expression of cyclin D1, cyclin B1 and p34cdc2 in the tumor and adjacent noncancerous liver tissues were measured by immunohistochemistry.RESULTS: Following treatment of H22 tumour bearing mice with resveratrol at 10 or 15 mg/kg bodyweight for 10 days,the growth of murine transplantable liver cancer was inhibited by 36.3% or 49.3%, respectively. The inhibitory effect was significant compared to that in control group (P<0.05).The level of expression of cyclin B1 and p34cdc2 protein was decreased in the transplantable murine hepatoma 22treated with resveratrol whereas the expression of cyclin D1 protein did not change.CONCLUSION: Resveratrol exhibits anti-tumour activities on murine hepatoma H22. The underlying anti-tumour mechanism of resveratrol might involve the inhibition of the cell cycle progression by decreasing the expression of cyclinB1 and p34cdc2 protein.

  17. Enhanced detection and study of murine norovirus-1 using a more efficient microglial cell line

    Lu Yuanan


    Full Text Available Abstract Background Human Noroviruses are the predominant cause of non-bacterial gastroenteritis worldwide. To facilitate prevention and control, a norovirus isolated from mice can provide a model to understand human noroviruses. To establish optimal viral infectivity conditions for murine noroviruses, several cell lines of hematopoietic lineage, including murine BV-2, RAW 264.7, and TIB, as well as human CHME-5, were tested comparatively for their sensitivity to murine norovirus-1. Results Except for CHME-5, all three murine-derived cell lines were susceptible to MNV infection. Viral infection of these cells was confirmed by RT-PCR. Using both viral plaque and replication assays, BV-2 and RAW 264.7 cells were determined to have comparable sensitivities to MNV-1 infection. Comparisons of cell growth characteristics, general laboratory handling and potential in-field applications suggest the use of BV-2 to be more advantageous. Conclusion Results obtained from these studies demonstrate that an immortalized microglial cell line can support MNV-1 replication and provides a more efficient method to detect and study murine noroviruses, facilitating future investigations using MNV-1 as a model to study, detect, and control Human Norovirus.

  18. Identification and characterization of the inducible murine mast cell gene, imc-415.

    Cho, S H; Cho, J J; Kim, I S; Vliagoftis, H; Metcalfe, D D; Oh, C K


    Activation of mast cells results in the generation and release of bioactive mediators which in turn initiate allergic inflammation. Mast cell function is enhanced following stimulation in part because of the induction of specific genes and their products. To identify additional genes induced in mast cells that support this process, we thus constructed an activation-specific mast cell subtraction library. To date, we have isolated 26 novel inducible murine mast cell (imc) cDNA clones. Among them, a full-coding region of the murine gene imc-415 was found to have a greater than 90% nucleotide sequence homology and a 97.5% amino acid sequence homology to both a human beta4 integrin-binding protein (p27(BBP)) and a human translation initiation factor 6 (eIF6), which in turn are identical. In vitro translation of the imc-415 gene yielded a band of an approximately 26 kDa. This is the same as the calculated molecular weight of murine IMC-415 protein based on the predicted amino acid sequence and is the molecular weight of p27(BBP)/eIF6. Murine imc-415 message was also induced in inflamed lung tissues in a mouse model of asthma. These results suggest a role for murine imc-415 in allergic inflammation where it may enhance protein synthesis. Human eIF6/p27(BBP) may also play a role in allergic diseases based on the similarities in sequence and in gene expression patterns.

  19. Expression of heteromeric amino acid transporters along the murine intestine.

    Dave, Mital H; Schulz, Nicole; Zecevic, Marija; Wagner, Carsten A; Verrey, Francois


    Members of the new heterodimeric amino acid transporter family are composed of two subunits, a catalytic multitransmembrane spanning protein (light chain) and a type II glycoprotein (heavy chain). These transporters function as exchangers and thereby extend the transmembrane amino acid transport selectivity to specific amino acids. The heavy chain rBAT associates with the light chain b degrees (,+)AT to form a cystine and cationic amino acid transporter. The other heavy chain, 4F2hc, can interact with seven different light chains to form various transporters corresponding to systems L, y(+)L, asc or x(-)(c). The importance of some of these transporters in intestinal and renal (re)absorption of amino acids is highlighted by the fact that mutations in either the rBAT or b degrees (,+)AT subunit result in cystinuria whereas a defect in the y(+)-LAT1 light chain causes lysinuric protein intolerance. Here we investigated the localization of these transporters in intestine since both diseases are also characterized by altered intestinal amino acid absorption. Real time PCR showed organ-specific expression patterns for all transporter subunit mRNAs along the intestine and Western blotting confirmed these findings on the protein level. Immunohistochemistry demonstrated basolateral coexpression of 4F2hc, LAT2 and y(+)-LAT1 in stomach and small intestine, whereas rBAT and b degrees (,+)AT were found colocalizing on the apical side of small intestine epithelium. In stomach, 4F2hc and LAT2 were localized in H(+)/K(+)-ATPase-expressing parietal cells. The abundant expression of several members of the heterodimeric transporter family along the murine small intestine suggests their involvement in amino acids absorption. Furthermore, strong expression of rBAT, b degrees (,+)AT and y(+)-LAT1 in the small intestine explains the reduced intestinal absorption of some amino acid in patients with cystinuria or lysinuric protein intolerance.

  20. Murine fertilized ovum, blastomere and morula cells lacking SP phenotype


    In the field of stem cell research, SP (side population) phenotype is used to define the property that cells maintain a high efflux capability for some fluorescent dye, such as Hoechst 33342. Recently, many researches proposed that SP phenotype is a phenotype shared by some stem cells and some progenitor cells, and that SP phenotype is regarded as a candidate purification marker for stem cells. In this research, murine fertilized ova (including conjugate and single nucleus fertilized ova), 2-cell stage and 8-cell stage blastomeres, morulas and blastocysts were isolated and directly stained by Hoechst 33342 dye. The results show that fertilized ovum, blastomere and morula cells do not demonstrate any ability to efflux the dye. However, the inner cell mass (ICM) cells of blastocyst exhibit SP phenotype, which is consistent with the result of embryonic stem cells (ESCs) in vitro. These results indicate that the SP phenotype of ICM-derived ESCs is an intrinsic property and independent of the culture condition in vitro, and that SP phenotype is one of the characteristics of at least some pluripotent stem cells, but is not shared by totipotent stem cells. In addition, the result that the SP phenotype of ICM cells disappeared when the inhibitor verapamil was added into medium implies that the SP phenotype is directly associated with ABCG2. These results suggest that not all the stem cells demonstrate SP phenotype, and that SP phenotype might act as a purification marker for partial stem cells such as some pluripotent embryonic stem cells and multipotent adult stem cells, but not for all stem cells exampled by the totipotent stem cells in the very early stage of mouse embryos.

  1. A Murine Hypertrophic Cardiomyopathy Model: The DBA/2J Strain.

    Wenyuan Zhao

    Full Text Available Familial hypertrophic cardiomyopathy (HCM is attributed to mutations in genes that encode for the sarcomere proteins, especially Mybpc3 and Myh7. Genotype-phenotype correlation studies show significant variability in HCM phenotypes among affected individuals with identical causal mutations. Morphological changes and clinical expression of HCM are the result of interactions with modifier genes. With the exceptions of angiotensin converting enzyme, these modifiers have not been identified. Although mouse models have been used to investigate the genetics of many complex diseases, natural murine models for HCM are still lacking. In this study we show that the DBA/2J (D2 strain of mouse has sequence variants in Mybpc3 and Myh7, relative to widely used C57BL/6J (B6 reference strain and the key features of human HCM. Four-month-old of male D2 mice exhibit hallmarks of HCM including increased heart weight and cardiomyocyte size relative to B6 mice, as well as elevated markers for cardiac hypertrophy including β-myosin heavy chain (MHC, atrial natriuretic peptide (ANP, brain natriuretic peptide (BNP, and skeletal muscle alpha actin (α1-actin. Furthermore, cardiac interstitial fibrosis, another feature of HCM, is also evident in the D2 strain, and is accompanied by up-regulation of type I collagen and α-smooth muscle actin (SMA-markers of fibrosis. Of great interest, blood pressure and cardiac function are within the normal range in the D2 strain, demonstrating that cardiac hypertrophy and fibrosis are not secondary to hypertension, myocardial infarction, or heart failure. Because D2 and B6 strains have been used to generate a large family of recombinant inbred strains, the BXD cohort, the D2 model can be effectively exploited for in-depth genetic analysis of HCM susceptibility and modifier screens.

  2. Critical transition in tissue homeostasis accompanies murine lung senescence.

    Carla L Calvi

    Full Text Available BACKGROUND: Respiratory dysfunction is a major contributor to morbidity and mortality in aged populations. The susceptibility to pulmonary insults is attributed to "low pulmonary reserve", ostensibly reflecting a combination of age-related musculoskeletal, immunologic and intrinsic pulmonary dysfunction. METHODS/PRINCIPAL FINDINGS: Using a murine model of the aging lung, senescent DBA/2 mice, we correlated a longitudinal survey of airspace size and injury measures with a transcriptome from the aging lung at 2, 4, 8, 12, 16 and 20 months of age. Morphometric analysis demonstrated a nonlinear pattern of airspace caliber enlargement with a critical transition occurring between 8 and 12 months of age marked by an initial increase in oxidative stress, cell death and elastase activation which is soon followed by inflammatory cell infiltration, immune complex deposition and the onset of airspace enlargement. The temporally correlative transcriptome showed exuberant induction of immunoglobulin genes coincident with airspace enlargement. Immunohistochemistry, ELISA analysis and flow cytometry demonstrated increased immunoglobulin deposition in the lung associated with a contemporaneous increase in activated B-cells expressing high levels of TLR4 (toll receptor 4 and CD86 and macrophages during midlife. These midlife changes culminate in progressive airspace enlargement during late life stages. CONCLUSION/SIGNIFICANCE: Our findings establish that a tissue-specific aging program is evident during a presenescent interval which involves early oxidative stress, cell death and elastase activation, followed by B lymphocyte and macrophage expansion/activation. This sequence heralds the progression to overt airspace enlargement in the aged lung. These signature events, during middle age, indicate that early stages of the aging immune system may have important correlates in the maintenance of tissue morphology. We further show that time-course analyses of aging

  3. Skeletal muscle mitochondrial uncoupling in a murine cancer cachexia model.

    Tzika, A Aria; Fontes-Oliveira, Cibely Cristine; Shestov, Alexander A; Constantinou, Caterina; Psychogios, Nikolaos; Righi, Valeria; Mintzopoulos, Dionyssios; Busquets, Silvia; Lopez-Soriano, Francisco J; Milot, Sylvain; Lepine, Francois; Mindrinos, Michael N; Rahme, Laurence G; Argiles, Josep M


    Approximately half of all cancer patients present with cachexia, a condition in which disease-associated metabolic changes lead to a severe loss of skeletal muscle mass. Working toward an integrated and mechanistic view of cancer cachexia, we investigated the hypothesis that cancer promotes mitochondrial uncoupling in skeletal muscle. We subjected mice to in vivo phosphorous-31 nuclear magnetic resonance (31P NMR) spectroscopy and subjected murine skeletal muscle samples to gas chromatography/mass spectrometry (GC/MS). The mice used in both experiments were Lewis lung carcinoma models of cancer cachexia. A novel 'fragmented mass isotopomer' approach was used in our dynamic analysis of 13C mass isotopomer data. Our 31P NMR and GC/MS results indicated that the adenosine triphosphate (ATP) synthesis rate and tricarboxylic acid (TCA) cycle flux were reduced by 49% and 22%, respectively, in the cancer-bearing mice (p<0.008; t-test vs. controls). The ratio of ATP synthesis rate to the TCA cycle flux (an index of mitochondrial coupling) was reduced by 32% in the cancer-bearing mice (p=0.036; t-test vs. controls). Genomic analysis revealed aberrant expression levels for key regulatory genes and transmission electron microscopy (TEM) revealed ultrastructural abnormalities in the muscle fiber, consistent with the presence of abnormal, giant mitochondria. Taken together, these data suggest that mitochondrial uncoupling occurs in cancer cachexia and thus point to the mitochondria as a potential pharmaceutical target for the treatment of cachexia. These findings may prove relevant to elucidating the mechanisms underlying skeletal muscle wasting observed in other chronic diseases, as well as in aging.

  4. Functional dissection of the murine Ick distal promoter

    Wildin, R.S.; Wang, H.U.; Forbush, K.A. [Univ. of Washington, Seattle, WA (United States)] [and others


    The lymphocyte-specific proto-oncogene Ick is transcribed from two developmentally regulated, independently functioning promoters. The proximal promoter is used in thymocytes, but not in peripheral T lymphocytes. The distal promoter operates in all stages of T cell development, but predominates in more mature cells. Both promoters lack a TATAA element and they share little sequence similarity with each other. Using transgenic mice to locate in vivo functional cis-acting regions of the murine distal promoter, we defined a region from -1786 to -2913 that is essential for consistent insertion site-independent expression of a heterologous cDNA reporter. The transgene is lymphoid specific and expressed predominantly in T cells. One of four transgenic mice bearing a shortened distal promoter (-886 to +41) expressed the reporter in the expected developmental pattern, suggesting that important regulatory elements that require favorable flanking sequences for expression are present nearer the transcription start site. The DNA sequence from -4032 to +623 contains few consensus binding sites for previously described T lymphocyte-specific trans-acting factors, and their locations do not correlate well with the functional data. However, the locations of tissue-specific modifications of chromatin structure in the promoter region, manifest as sites of DNase hypersensitivity, correlated with these two functional regions in normal mice. The identification of Ick distal promoter regulatory regions provides a useful control element for deliberate expression of transgenes in mature T lymphocytes. In addition, these regulatory regions should assist in defining T cell-specific trans-acting factors.

  5. Endothelial pentraxin 3 contributes to murine ischemic acute kidney injury

    Chen, Jianlin; Matzuk, Martin M.; Zhou, Xin J.; Lu, Christopher Y.


    Toll-like receptor 4 (TLR4), a receptor forDamage Associated Molecular Pattern Molecules and also the lipopolysaccharide receptor, is required for early endothelial activation leading to maximal inflammation and injury during murine ischemic acute kidney injury. DNA microarray analysis of ischemic kidneys from TLR4-sufficient and deficient mice showed that pentraxin 3 (PTX3) was upregulated only on the former while transgenic knockout of PTX3 ameliorated acute kidney injury. PTX3 was expressed predominantly on peritubular endothelia of the outer medulla of the kidney in control mice. Acute kidney injury increased PTX3 protein in the kidney and the plasma where it may be a biomarker of the injury. Stimulation by hydrogen peroxide, or the TLR4 ligands recombinant human High-Mobility Group protein B1 or lipopolysaccharide, induced PTX3 expression in the Mile Sven 1 endothelial cell line and in primary renal endothelial cells suggesting that endothelial PTX3 was induced by pathways involving TLR4 and reactive oxygen species. This increase was inhibited by conditional endothelial knockout of Myeloid differentiation primary response gene 88, a mediator of a TLR4 intracellular signaling pathway. Compared to wild type mice, PTX3 knockout mice had decreased endothelial expression of cell adhesion molecules at 4 hours of reperfusion possibly contributing to a decreased early maladaptive inflammation in the kidneys of knockout mice. At 24 hours of reperfusion, PTX3 knockout increased expression of endothelial adhesion molecules when regulatory and reparative leukocytes enter the kidney. Thus, endothelial PTX3 plays a pivotal role in the pathogenesis of ischemic acute kidney injury. PMID:22895517

  6. PCR master mixes harbour murine DNA sequences. Caveat emptor!

    Philip W Tuke

    Full Text Available BACKGROUND: XMRV is the most recently described retrovirus to be found in Man, firstly in patients with prostate cancer (PC and secondly in 67% of patients with chronic fatigue syndrome (CFS and 3.7% of controls. Both disease associations remain contentious. Indeed, a recent publication has concluded that "XMRV is unlikely to be a human pathogen". Subsequently related but different polytropic MLV (pMLV sequences were also reported from the blood of 86.5% of patients with CFS. and 6.8% of controls. Consequently we decided to investigate blood donors for evidence of XMRV/pMLV. METHODOLOGY/PRINCIPAL FINDINGS: Testing of cDNA prepared from the whole blood of 80 random blood donors, generated gag PCR signals from two samples (7C and 9C. These had previously tested negative for XMRV by two other PCR based techniques. To test whether the PCR mix was the source of these sequences 88 replicates of water were amplified using Invitrogen Platinum Taq (IPT and Applied Biosystems Taq Gold LD (ABTG. Four gag sequences (2D, 3F, 7H, 12C were generated with the IPT, a further sequence (12D by ABTG re-amplification of an IPT first round product. Sequence comparisons revealed remarkable similarities between these sequences, endogeous MLVs and the pMLV sequences reported in patients with CFS. CONCLUSIONS/SIGNIFICANCE: Methodologies for the detection of viruses highly homologous to endogenous murine viruses require special caution as the very reagents used in the detection process can be a source of contamination and at a level where it is not immediately apparent. It is suggested that such contamination is likely to explain the apparent presence of pMLV in CFS.

  7. Helicobacter pylori impairs murine dendritic cell responses to infection.

    Ya-Hui Wang

    Full Text Available BACKGROUND: Helicobacter pylori, a human pathogen associated with chronic gastritis, peptic ulcer and gastric malignancies, is generally viewed as an extracellular microorganism. Here, we show that H. pylori replicates in murine bone marrow derived-dendritic cells (BMDCs within autophagosomes. METHODOLOGY/PRINCIPAL FINDINGS: A 10-fold increase of CFU is found between 2 h and 6 h p.i. in H. pylori-infected BMDCs. Autophagy is induced around the bacterium and participates at late time points of infection for the clearance of intracellular H. pylori. As a consequence of infection, LC3, LAMP1 and MHC class II molecules are retained within the H. pylori-containing vacuoles and export of MHC class II molecules to cell surface is blocked. However, formalin-fixed H. pylori still maintain this inhibitory activity in BMDC derived from wild type mice, but not in from either TLR4 or TLR2-deficient mice, suggesting the involvement of H. pylori-LPS in this process. TNF-alpha, IL-6 and IL-10 expression was also modulated upon infection showing a TLR2-specific dependent IL-10 secretion. No IL-12 was detected favoring the hypothesis of a down modulation of DC functions during H. pylori infection. Furthermore, antigen-specific T cells proliferation was also impaired upon infection. CONCLUSIONS/SIGNIFICANCE: H. pylori can infect and replicate in BMDCs and thereby affects DC-mediated immune responses. The implication of this new finding is discussed for the biological life cycle of H. pylori in the host.

  8. Expansion of intestinal epithelial stem cells during murine development.

    Jeffrey J Dehmer

    Full Text Available Murine small intestinal crypt development is initiated during the first postnatal week. Soon after formation, overall increases in the number of crypts occurs through a bifurcating process called crypt fission, which is believed to be driven by developmental increases in the number of intestinal stem cells (ISCs. Recent evidence suggests that a heterogeneous population of ISCs exists within the adult intestine. Actively cycling ISCs are labeled by Lgr5, Ascl2 and Olfm4; whereas slowly cycling or quiescent ISC are marked by Bmi1 and mTert. The goal of this study was to correlate the expression of these markers with indirect measures of ISC expansion during development, including quantification of crypt fission and side population (SP sorting. Significant changes were observed in the percent of crypt fission and SP cells consistent with ISC expansion between postnatal day 14 and 21. Quantitative real-time polymerase chain reaction (RT-PCR for the various ISC marker mRNAs demonstrated divergent patterns of expression. mTert surged earliest, during the first week of life as crypts are initially being formed, whereas Lgr5 and Bmi1 peaked on day 14. Olfm4 and Ascl2 had variable expression patterns. To assess the number and location of Lgr5-expressing cells during this period, histologic sections from intestines of Lgr5-EGFP mice were subjected to quantitative analysis. There was attenuated Lgr5-EGFP expression at birth and through the first week of life. Once crypts were formed, the overall number and percent of Lgr5-EGFP positive cells per crypt remain stable throughout development and into adulthood. These data were supported by Lgr5 in situ hybridization in wild-type mice. We conclude that heterogeneous populations of ISCs are expanding as measured by SP sorting and mRNA expression at distinct developmental time points.

  9. Chloroquine Inhibits Ca2+ Signaling in Murine CD4+ Thymocytes

    Jin-Chao Xu


    Full Text Available Background/Aims: Bitter-tasting chloroquine can suppress T cell activation by inhibiting Ca2+ signaling. However, the mechanism of inhibition remains largely unclear. Methods: In this study, CD4+ T cells were isolated from the thymus, and the calcium content of CD4+ thymocytes was measured using fura-2 AM and a TILL imaging system. Pyrazole-3 (Pyr3, thapsigargin (TG, and caffeine were used to assess the effects of chloroquine on the intracellular Ca2+ content of CD4+ T cells. Results: In murine CD4+ thymocytes, chloroquine decreased the TG-triggered intracellular Ca2+ increase in a dose-dependent manner. In the absence of chloroquine under Ca2+-free conditions (0 mM Ca2+ and 0.5 mM EGTA, TG induced a transient Ca2+ increase. After restoration of the extracellular Ca2+ concentration to 2 mM, a dramatic Ca2+ increase occurred. This elevation was completely blocked by chloroquine and was markedly inhibited by Pyr3, a selective antagonist of transient receptor potential C3 (TRPC3 channel and stromal interaction molecule (STIM/Orai channel. Furthermore, the TG-induced transient Ca2+ increase under Ca2+-free conditions was eliminated in the presence of chloroquine. Chloroquine also blocked the dialyzed inositol-1,4,5-trisphosphate (IP3-induced intracellular Ca2+ increase. However, chloroquine was not able to decrease the caffeine-induced Ca2+ increase. Conclusion: These data indicate that chloroquine inhibits the elevation of intracellular Ca2+ in thymic CD4+ T cells by inhibiting IP3 receptor-mediated Ca2+ release from intracellular stores and TRPC3 channel-mediated and/or STIM/Orai channel-mediated Ca2+ influx.

  10. Inactivation of murine norovirus by chemical biocides on stainless steel

    Steinmann Jörg


    Full Text Available Abstract Background Human norovirus (NoV causes more than 80% of nonbacterial gastroenteritis in Europe and the United States. NoV transmission via contaminated surfaces may be significant for the spread of viruses. Therefore, measures for prevention and control, such as surface disinfection, are necessary to interrupt the dissemination of human NoV. Murine norovirus (MNV as a surrogate for human NoV was used to study the efficacy of active ingredients of chemical disinfectants for virus inactivation on inanimate surfaces. Methods The inactivating properties of different chemical biocides were tested in a quantitative carrier test with stainless steel discs without mechanical action. Vacuum-dried MNV was exposed to different concentrations of alcohols, peracetic acid (PAA or glutaraldehyde (GDA for 5 minutes exposure time. Detection of residual virus was determined by endpoint-titration on RAW 264.7 cells. Results PAA [1000 ppm], GDA [2500 ppm], ethanol [50% (v/v] and 1-propanol [30% (v/v] were able to inactivate MNV under clean conditions (0.03% BSA on the carriers by ≥ 4 log10 within 5 minutes exposure time, whereas 2-propanol showed a reduced effectiveness even at 60% (v/v. Furthermore, there were no significant differences in virus reduction whatever interfering substances were used. When testing with ethanol, 1- and 2-propanol, results under clean conditions were nearly the same as in the presence of dirty conditions (0.3% BSA plus 0.3% erythrocytes. Conclusion Products based upon PAA, GDA, ethanol and 1-propanol should be used for NoV inactivation on inanimate surfaces. Our data provide valuable information for the development of strategies to control NoV transmission via surfaces.

  11. Induction and treatment of anergy in murine leprosy

    Juarez-Ortega, Mario; Hernandez, Víctor G; Arce-Paredes, Patricia; Villanueva, Enrique B; Aguilar-Santelises, Miguel; Rojas-Espinosa, Oscar


    Leprosy is a disease consisting of a spectrum of clinical, bacteriological, histopathological and immunological manifestations. Tuberculoid leprosy is frequently recognized as the benign polar form of the disease, while lepromatous leprosy is regarded as the malignant form. The different forms of leprosy depend on the genetic and immunological characteristics of the patient and on the characteristics of the leprosy bacillus. The malignant manifestations of lepromatous leprosy result from the mycobacterial-specific anergy that develops in this form of the disease. Using murine leprosy as a model of anergy in this study, we first induced the development of anergy to Mycobacterium lepraemurium (MLM) in mice and then attempted to reverse it by the administration of dialysable leucocyte extracts (DLE) prepared from healthy (HLT), BCG-inoculated and MLM-inoculated mice. Mice inoculated with either MLM or BCG developed a robust cell-mediated immune response (CMI) that was temporary in the MLM-inoculated group and long-lasting in the BCG-inoculated group. DLE were prepared from the spleens of MLM- and BCG-inoculated mice at the peak of CMI. Independent MLM intradermally-inoculated groups were treated every other day with HLT-DLE, BCG-DLE or MLM-DLE, and the effect was documented for 98 days. DLE administered at a dose of 1.0 U (1 × 106 splenocytes) did not affect the evolution of leprosy, while DLE given at a dose of 0.1 U showed beneficial effects regardless of the DLE source. The dose but not the specificity of DLE was the determining factor for reversing anergy. PMID:25529580

  12. Nuclear localization of Annexin A7 during murine brain development

    Noegel Angelika A


    Full Text Available Abstract Background Annexin A7 is a member of the annexin protein family, which is characterized by its ability to interact with phospholipids in the presence of Ca2+-ions and which is thought to function in Ca2+-homeostasis. Results from mutant mice showed altered Ca2+-wave propagation in astrocytes. As the appearance and distribution of Annexin A7 during brain development has not been investigated so far, we focused on the distribution of Annexin A7 protein during mouse embryogenesis in the developing central nervous system and in the adult mouse brain. Results Annexin A7 is expressed in cells of the developing brain where a change in its subcellular localization from cytoplasm to nucleus was observed. In the adult CNS, the subcellular distribution of Annexin A7 depends on the cell type. By immunohistochemistry analysis Annexin A7 was detected in the cytosol of undifferentiated cells at embryonic days E5–E8. At E11–E15 the protein is still present in the cytosol of cells predominantly located in the ventricular germinative zone surrounding the lateral ventricle. Later on, at embryonic day E16, Annexin A7 in cells of the intermediate and marginal zone of the neopallium translocates to the nucleus. Neuronal cells of all areas in the adult brain present Annexin A7 in the nucleus, whereas glial fibrillary acidic protein (GFAP-positive astrocytes exhibit both, a cytoplasmic and nuclear staining. The presence of nuclear Annexin A7 was confirmed by extraction of the nucleoplasm from isolated nuclei obtained from neuronal and astroglial cell lines. Conclusion We have demonstrated a translocation of Annexin A7 to nuclei of cells in early murine brain development and the presence of Annexin A7 in nuclei of neuronal cells in the adult animal. The role of Annexin A7 in nuclei of differentiating and mature neuronal cells remains elusive.

  13. Human Leucocyte Antigen-G (HLA-G) and Its Murine Functional Homolog Qa2 in the Trypanosoma cruzi Infection

    Dias, Fabrício C.; Mendes-Junior, Celso T.; Silva, Maria C.; Tristão, Fabrine S. M.; Dellalibera-Joviliano, Renata; Soares, Edson G.; Menezes, Jean G.; Schmidt, André; Dantas, Roberto O.; Marin-Neto, José A.; Silva, João S.; Donadi, Eduardo A.


    Genetic susceptibility factors, parasite strain, and an adequate modulation of the immune system seem to be crucial for disease progression after Trypanosoma cruzi infection. HLA-G and its murine functional homolog Qa2 have well-recognized immunomodulatory properties. We evaluated the HLA-G 3′ untranslated region (3′UTR) polymorphic sites (associated with mRNA stability and target for microRNA binding) and HLA-G tissue expression (heart, colon, and esophagus) in patients presenting Chagas disease, stratified according to the major clinical variants. Further, we investigated the transcriptional levels of Qa2 and other pro- and anti-inflammatory genes in affected mouse tissues during T. cruzi experimental acute and early chronic infection induced by the CL strain. Chagas disease patients exhibited differential HLA-G 3′UTR susceptibility allele/genotype/haplotype patterns, according to the major clinical variant (digestive/cardiac/mixed/indeterminate). HLA-G constitutive expression on cardiac muscle and colonic cells was decreased in Chagasic tissues; however, no difference was observed for Chagasic and non-Chagasic esophagus tissues. The transcriptional levels of Qa2 and other anti and proinflammatory (CTLA-4, PDCD1, IL-10, INF-γ, and NOS-2) genes were induced only during the acute T. cruzi infection in BALB/c and C57BL/6 mice. We present several lines of evidence indicating the role of immunomodulatory genes and molecules in human and experimental T. cruzi infection. PMID:25688175

  14. Metabolism under hypoxia in Tm1 murine melanoma cells is affected by the presence of galectin-3, a metabolomics approach.

    Bacchi, Pedro Starzynski; Bloise, Antonio Carlos; Bustos, Silvina Odete; Zimmermann, Lara; Chammas, Roger; Rabbani, Said Rahnamaye


    Metabolomics has proven an useful tool for systems biology. Here we have used a metabolomics approach to identify conditions in which de novo expression of an established tumor marker, galectin-3, would confer a potential selective advantage for melanoma growth and survival. A murine melanoma cell line (Tm1) that lacks galectin-3 was modified to express it or not (Tm1.G2 and Tm1.N3, respectively). These variant cell line were then exposed to conditions of controlled oxygen tensions and glucose levels. Metabolic profiling of intracellular metabolites of cells exposed to these conditions was obtained in steady state using high resolution (1)H Magnetic Resonance Spectroscopy ((1)H-MRS) and multivariate statistical analysis. The Nuclear Magnetic Resonance (NMR) spectra contained a large number of absorption lines from which we were able to distinguish 20 metabolites, 3 fatty acids and some absorption lines and clusters were not identified. Principal Components Analysis (PCA) allowed for the discrimination of 2 experimental conditions in which expression of the tumor marker galectin-3 may play a significant role, namely exposure of cells to hypoxia under high glucose. Interestingly, under all other experimental conditions tested, the cellular system was quite robust. Our results suggest that the Metabolomics approach can be used to access information about changes in many metabolic pathways induced in tumorigenic cells and to allow the evaluation of their behavior in controlled environmental conditions or selective pressures.

  15. Human Leucocyte Antigen-G (HLA-G and Its Murine Functional Homolog Qa2 in the Trypanosoma cruzi Infection

    Fabrício C. Dias


    Full Text Available Genetic susceptibility factors, parasite strain, and an adequate modulation of the immune system seem to be crucial for disease progression after Trypanosoma cruzi infection. HLA-G and its murine functional homolog Qa2 have well-recognized immunomodulatory properties. We evaluated the HLA-G 3′ untranslated region (3′UTR polymorphic sites (associated with mRNA stability and target for microRNA binding and HLA-G tissue expression (heart, colon, and esophagus in patients presenting Chagas disease, stratified according to the major clinical variants. Further, we investigated the transcriptional levels of Qa2 and other pro- and anti-inflammatory genes in affected mouse tissues during T. cruzi experimental acute and early chronic infection induced by the CL strain. Chagas disease patients exhibited differential HLA-G 3′UTR susceptibility allele/genotype/haplotype patterns, according to the major clinical variant (digestive/cardiac/mixed/indeterminate. HLA-G constitutive expression on cardiac muscle and colonic cells was decreased in Chagasic tissues; however, no difference was observed for Chagasic and non-Chagasic esophagus tissues. The transcriptional levels of Qa2 and other anti and proinflammatory (CTLA-4, PDCD1, IL-10, INF-γ, and NOS-2 genes were induced only during the acute T. cruzi infection in BALB/c and C57BL/6 mice. We present several lines of evidence indicating the role of immunomodulatory genes and molecules in human and experimental T. cruzi infection.

  16. A novel laser-Doppler flowmetry assisted murine model of acute hindlimb ischemia-reperfusion for free flap research.

    Tolga Taha Sönmez

    Full Text Available Suitable and reproducible experimental models of translational research in reconstructive surgery that allow in-vivo investigation of diverse molecular and cellular mechanisms are still limited. To this end we created a novel murine model of acute hindlimb ischemia-reperfusion to mimic a microsurgical free flap procedure. Thirty-six C57BL6 mice (n = 6/group were assigned to one control and five experimental groups (subject to 6, 12, 96, 120 hours and 14 days of reperfusion, respectively following 4 hours of complete hindlimb ischemia. Ischemia and reperfusion were monitored using Laser-Doppler Flowmetry. Hindlimb tissue components (skin and muscle were investigated using histopathology, quantitative immunohistochemistry and immunofluorescence. Despite massive initial tissue damage induced by ischemia-reperfusion injury, the structure of the skin component was restored after 96 hours. During the same time, muscle cells were replaced by young myotubes. In addition, initial neuromuscular dysfunction, edema and swelling resolved by day 4. After two weeks, no functional or neuromuscular deficits were detectable. Furthermore, upregulation of VEGF and tissue infiltration with CD34-positive stem cells led to new capillary formation, which peaked with significantly higher values after two weeks. These data indicate that our model is suitable to investigate cellular and molecular tissue alterations from ischemia-reperfusion such as occur during free flap procedures.

  17. Blood clot formation does not affect metastasis formation or tumor growth in a murine model of breast cancer.

    Stephanie Rossnagl

    Full Text Available Cancer is associated with increased fracture risk, due either to metastasis or associated osteoporosis. After a fracture, blood clots form. Because proteins of the coagulation cascade and activated platelets promote cancer development, a fracture in patients with cancer often raises the question whether it is a pathologic fracture or whether the fracture itself might promote the formation of metastatic lesions. We therefore examined whether blood clot formation results in increased metastasis in a murine model of experimental breast cancer metastasis. For this purpose, a clot was surgically induced in the bone marrow of the left tibia of immundeficient mice. Either one minute prior to or five minutes after clot induction, human cancer cells were introduced in the circulation by intracardiac injection. The number of cancer cells that homed to the intervention site was determined by quantitative real-time PCR and flow cytometry. Metastasis formation and longitudinal growth were evaluated by bioluminescence imaging. The number of cancer cells that homed to the intervention site after 24 hours was similar to the number of cells in the opposite tibia that did not undergo clot induction. This effect was confirmed using two more cancer cell lines. Furthermore, no difference in the number of macroscopic lesions or their growth could be detected. In the control group 72% developed a lesion in the left tibia. In the experimental groups with clot formation 79% and 65% developed lesions in the left tibia (p = ns when comparing each experimental group with the controls. Survival was similar too. In summary, the growth factors accumulating in a clot/hematoma are neither enough to promote cancer cell homing nor support growth in an experimental model of breast cancer bone metastasis. This suggests that blood clot formation, as occurs in traumatic fractures, surgical interventions, and bruises, does not increase the risk of metastasis formation.

  18. Identification and Characterisation of the Murine Homologue of the Gene Responsible for Cystinosis, Ctns

    Poras Isabelle


    Full Text Available Abstract Background Cystinosis is an autosomal recessive disorder characterised by an intralysosomal accumulation of cystine, and affected individuals progress to end-stage renal failure before the age of ten. The causative gene, CTNS, was cloned in 1998 and the encoded protein, cystinosin, was predicted to be a lysosomal membrane protein. Results We have cloned the murine homologue of CTNS, Ctns, and the encoded amino acid sequence is 92.6% similar to cystinosin. We localised Ctns to mouse chromosome 11 in a region syntenic to human chromosome 17 containing CTNS. Ctns is widely expressed in all tissues tested with the exception of skeletal muscle, in contrast to CTNS. Conclusions We have isolated, characterised and localised Ctns, the murine homologue of CTNS underlying cystinosis. Furthermore, our work has brought to light the existence of a differential pattern of expression between the human and murine homologues, providing critical information for the generation of a mouse model for cystinosis.

  19. Cloning of murine BRI3 gene and study on its function for inducing cell death


    To understand the molecular mechanism of TNFα effects, the cDNA of murine BRI3 gene was cloned from the total RNA of murine brain endothelial cells (bEnd.3)treated with hTNFα by using the suppression subtractive hybridization (SSH) and the RT-PCR method. The fusion expression vector harbouring BRI3 gene and enhanced green fluorescence protein (EGFP) thus obtained were designated as pEGFP/I3. Then pEGFP/I3 was transiently transfected into L929 cells and the fusion protein EGFP/I3 was localized in cytoplasm. It is found that the expression of EGFP/I3 could induce cell death in L929 cells detected by TUNEL method and flow cytometry. And the overexpression of Bci-2 in L929 cells can block cell death induced by EGFP/I3, indicating that murine BRI3 gene might related to the TNFα mediated cytotoxicity.

  20. ABC- and SLC-Transporters in Murine and Bovine Mammary Epithelium

    Yagdiran, Yagmur; Oskarsson, Agneta; Knight, Christopher H.


    Some chemicals are ligands to efflux transporters which may result in high concentrations in milk. Limited knowledge is available on the influence of maternal exposure to chemicals on the expression and function of transporters in the lactating mammary gland. We determined gene expression of ABC...... and SLC transporters in murine mammary tissue of different gestation and lactation stages, in murine mammary cells (HC11) featuring resting and secreting phenotypes and in bovine mammary tissue and cells (BME-UV). Effects on transporter expression and function of the imidazole fungicide prochloraz......, previously reported toinfluence BCRP in mammary cells, was investigated on transporter expression and functionin the two cell lines. Transporters studied were BCRP, MDR1, MRP1, OATP1A5/OATP1A2,OCTN1 and OCT1. Gene expressions of BCRP and OCT1 in murine mammary glandswere increased during gestation...

  1. Characterization of an immunodominant cancer-specific O-glycopeptide epitope in murine podoplanin (OTS8)

    Steentoft, Catharina; Schjoldager, Katrine T; Cló, Emiliano


    of the nature and timing of induction of auto-antibodies to distinct O-glycopeptide epitopes induced by cancer. The results demonstrate that truncated O-glycopeptides constitute highly distinct antibody epitopes with great potential as targets for biomarkers and immunotherapeutics.......Auto-antibodies induced by cancer represent promising sensitive biomarkers and probes to identify immunotherapeutic targets without immunological tolerance. Surprisingly few epitopes for such auto-antibodies have been identified to date. Recently, a cancer-specific syngeneic murine monoclonal...... antibody 237, developed to a spontaneous murine fibrosarcoma, was shown to be directed to murine podoplanin (OTS8) with truncated Tn O-glycans. Our understanding of such cancer-specific auto-antibodies to truncated glycoforms of glycoproteins is limited. Here we have investigated immunogenicity...

  2. Protective effects of astaxanthin from Paracoccus carotinifaciens on murine gastric ulcer models.

    Murata, Kenta; Oyagi, Atsushi; Takahira, Dai; Tsuruma, Kazuhiro; Shimazawa, Masamitsu; Ishibashi, Takashi; Hara, Hideaki


    The purpose of this study was to investigate the effect of astaxanthin extracted from Paracoccus carotinifaciens on gastric mucosal damage in murine gastric ulcer models. Mice were pretreated with astaxanthin for 1 h before ulcer induction. Gastric ulcers were induced in mice by oral administration of hydrochloride (HCl)/ethanol or acidified aspirin. The effect of astaxanthin on lipid peroxidation in murine stomach homogenates was also evaluated by measuring the level of thiobarbituric acid reactive substance (TBARS). The free radical scavenging activities of astaxanthin were also measured by electron spin resonance (ESR) measurements. Astaxanthin significantly decreased the extent of HCl/ethanol- and acidified aspirin-induced gastric ulcers. Astaxanthin also decreased the level of TBARS. The ESR measurement showed that astaxanthin had radical scavenging activities against the 1,1-diphenyl-2-picrylhydrazyl radical and the superoxide anion radical. These results suggest that astaxanthin has antioxidant properties and exerts a protective effect against ulcer formation in murine models.

  3. Antibody SPC-54 provides acute in vivo blockage of the murine protein C system.

    Burnier, Laurent; Fernández, José A; Griffin, John H


    Multiple protective effects of pharmacological activated protein C (APC) are reported in several organ pathologies. To help evaluate the endogenous murine PC system, we characterized a rat monoclonal anti-mouse PC antibody, SPC-54, which inhibited the amidolytic and anticoagulant activities of murine APC by>95%. SPC-54 blocked active site titration of purified APC using the active site titrant, biotinylated FPR-chloromethylketone, showing that SPC-54 blocks access to APC's active site to inhibit all enzymatic activity. A single injection of SPC-54 (10mg/kg) neutralized circulating PC in mice for at least 7days, and immunoblotting and immuno-precipitation with protein G-agarose confirmed that SPC-54 in vivo was bound to PC in plasma. Pre-infusion of SPC-54 in tissue factor-induced murine acute thromboembolism experiments caused a major decrease in mean survival time compared to controls (7min vs. 42.5min, P=0.0016). SPC-54 decreased lung perfusion in this model by 54% when monitored by vascular perfusion methodologies using infrared fluorescence of Evans blue dye. In LD50 endotoxemia murine models, SPC-54 infused at 7hr after endotoxin administration increased mortality from 42% to 100% (PSPC-54 ablates in vitro and in vivo APC protective functions and enzymatic activity. The ability of SPC-54 to block the endogenous PC/APC system provides a powerful tool to understand better the role of the endogenous PC system in murine injury models and in cell bioassays and also to neutralize the enzymatic activities of murine APC in any assay system.

  4. Identification of putative PPAR response elements in and around the murine UCP3 gene

    Siersbæk, Majken

    in thermogenesis in unerring, the physiological functions of UCP2 and UCP3 are at present not fully understood. Synthetic agonists for the peroxisome proliferator-activated receptors (PPARs) as well as fatty acids have been shown to increase murine UCP2 and UCP3 mRNA expression but response elements and mechanisms...... differentiation. Results from sequencing of chromatin immunoprecipitated (ChIP) material from 3T3-L1 adipocytes revealed three PPAR and retinoid X receptor (RXR) binding sites in and around the murine UCP3 gene. The recruitment of PPAR and RXR to the three potential PPREs was determined by ChIP combined...

  5. In vitro investigation of the roles of the proinflammatory cytokines tumor necrosis factor-α and interleukin-1 in murine osteoclastogenesis.

    Jules, Joel; Feng, Xu


    Whereas the monocyte/macrophage-colony stimulating factor (M-CSF) and the receptor activator of NF-кB ligand (RANKL) are essential and sufficient for osteoclastogenesis, a number of other cytokines including two proinflammatory cytokines, tumor necrosis factor-α (TNF-α), and interleukin-1 (IL-1), can exert profound effects on the osteoclastogenic process. However, the precise mode of action of TNF-α and IL-1 in osteoclastogenesis remains controversial. While some groups demonstrated that these two cytokines can promote murine osteoclastogenesis in vitro in the presence of M-CSF only, we and others showed that TNF-α-/IL-1-mediated osteoclastogenesis requires permissive levels of RANKL. This chapter describes the method that we have used to investigate the effects of TNF-α and IL-1 on osteoclast formation in in vitro osteoclastogenesis assays using primary murine bone marrow macrophages (BMMs). Detailed experimental conditions are provided and critical points are discussed to help the reader use the method to independently evaluate the roles of TNF-α and IL-1 in osteoclastogenesis in vitro. Moreover, this method can be used to further elucidate the signaling mechanisms by which these two cytokines act in concert with RANKL or with each other to modulate osteoclastogenesis.

  6. Transcriptome analysis of the ependymal barrier during murine neurocysticercosis

    Mishra Pramod


    to be upregulated at the protein level using immunofluorescence microcopy. This is important, because these molecules are members of the most significant pathways by IPA analyses. Conclusion Thus, our study indicates that ependymal cells actively express immune mediators and likely contribute to the observed immunopathogenesis during infection. Of particular interest is the major upregulation of antigen presentation pathway-related genes and chemokines/cytokines. This could explain how the ependyma is a prominent source of leukocyte infiltration into ventricles through the disrupted ependymal lining by way of pial vessels present in the internal leptomeninges in murine NCC.

  7. Evaluation of Antibody Responses Elicited by Immunization of Mice with a Pneumococcal Antigen Genetically Fused to Murine HSP70 and Murine Interleukin-4

    Dennis O. GOR; Salamatu S. MAMBULA


    The heat shock (stress) protein HSP70 has been shown to be a potent stimulator of cellular immune responses. In order to determine whether HSP70 has the ability to stimulate antibody responses, we constructed and expressed fusion proteins consisting of murine HSP70 or murine interleukin (IL)-4 covalently linked to a pneumococcal cell wall-associated protein antigen designated PpmA. Immunization of mice with the PpmA-HSP70 fusion protein (PpmA-70) failed to elicit an increased PpmA-specific serum antibody response. In contrast, mice immunized with PpmA fused to IL-4 (PpmA-IL4), or PpmA fused to both IL-4 and HSP70 (PpmA-IL4-70) fusion proteins elicited high levels of PpmA-specific antibody responses.These data suggest that HSP70 has a limited capacity to stimulate immune responses to heterologous antigens in vivo.


    朱康儿; KerryAtkinson


    We investigated the effects of mouse recombinant IL-4 on hematopoiesis in vitro and in vivo.IL-4 alone was found to be incapable of stimulating colony formation,but it inhibited both IL-3-and GM-CSF-induced colony for-mation by murine hematopoietic progenitor cells.In contrast,colony formation induced by G-CSF was enhanced in the presence of IL-4.We also studied the influence of IL-4 on hematopoietie reconstiution after allogeneic bone marrow transplantation in a murine model,and found that IL-4 and G-CSF was significantly suppressed by IL-4.The combination of IL-4 and GM-CSF caused a significant decrease in the absolute mumber of meutrophils.

  9. Murine Typhus and Leptospirosis as Causes of Acute Undifferentiated Fever, Indonesia

    Gasem, M.H.; Wagenaar, J.F.P.; Goris, M.G.A.; Adi, M.S.; Isbandrio, B.B.; Hartskeerl, R.A.; Rolain, J.M.; Raoult, D.; van Gorp, E.C.M.


    To investigate rickettsioses and leptospirosis among urban residents of Semarang, Indonesia, we tested the blood of 137 patients with fever. Evidence of Rickettsia typhi, the agent of murine typhus, was found in 9 patients. Another 9 patients showed inconclusive serologic results. Thirteen patients




    The therapeutic effect of a combination of liposomal muramyl dipeptide (MDP) and 5-fluorouracil (5FU) was studied in a murine tumor model of hepatic metastases of the tumor cell line C26, a colon adenocarcinoma. Liposomal MDP (250 mug/kg body wt) and a low, nontoxic, dose of 5FU (10 mg/kg body wt) w

  11. GPBAR1/TGR5 mediates bile acid-induced cytokine expression in murine Kupffer cells.

    Guiyu Lou

    Full Text Available GPBAR1/TGR5 is a novel plasma membrane-bound G protein-coupled bile acid (BA receptor. BAs are known to induce the expression of inflammatory cytokines in the liver with unknown mechanism. Here we show that without other external stimuli, TGR5 activation alone induced the expression of interleukin 1β (IL-1β and tumor necrosis factor-α (TNF-α in murine macrophage cell line RAW264.7 or murine Kupffer cells. The TGR5-mediated increase of pro-inflammatory cytokine expression was suppressed by JNK inhibition. Moreover, the induced pro-inflammatory cytokine expression in mouse liver by 1% cholic acid (CA diet was blunted in JNK-/- mice. TGR5 activation by its ligands enhanced the phosphorylation levels, DNA-binding and trans-activities of c-Jun and ATF2 transcription factors. Finally, the induced pro-inflammatory cytokine expression in Kupffer cells by TGR5 activation correlated with the suppression of Cholesterol 7α-hydroxylase (Cyp7a1 expression in murine hepatocytes. These results suggest that TGR5 mediates the BA-induced pro-inflammatory cytokine production in murine Kupffer cells through JNK-dependent pathway. This novel role of TGR5 may correlate to the suppression of Cyp7a1 expression in hepatocytes and contribute to the delicate BA feedback regulation.

  12. Successful implantation of intravenously administered stem cells correlates with severity of inflammation in murine myocarditis.

    Malek, S.; Kaplan, E.; Wang, J.F.; Ke, Q.; Rana, J.S.; Chen, Y.; Rahim, B.G.; Li, M.; Huang, Q.; Xiao, Y.F.; Verheugt, F.W.A.; Morgan, J.P.; Min, J.Y.


    The present study was designed to determine whether cardiac inflammation is important for the successful homing of stem cells to the heart after intravenous injection in a murine myocarditis model. Male Bagg albino/c mice were infected with encephalomyocarditis virus (EMCV) to produce myocarditis. S

  13. Phage-display libraries of murine and human antibody Fab fragments

    Engberg, J; Andersen, P S; Nielsen, L K


    We provide efficient and detailed procedures for construction, expression, and screening of comprehensive libraries of murine or human antibody Fab fragments displayed on the surface of filamentous phage. In addition, protocols for producing and using ultra-electrocompetent cells, for producing Fab...

  14. Murine gammaherpesvirus-68 (MHV-68) is not horizontally transmitted amongst laboratory mice by cage contact.

    Aligo, Jason; Brosnan, Kerry; Walker, Mindi; Emmell, Eva; Mikkelsen, S Rochelle; Burleson, Gary R; Burleson, Florence G; Volk, Amy; Weinstock, Daniel


    Murine gammaherpesvirus-68 (MHV-68), a natural pathogen of mice, is being evaluated as a model of Epstein Barr Virus (EBV) infection for use in investigation of the effects of immunomodulatory therapy on herpesvirus pathogenesis in humans. Immunosuppressive agents are used for treatment of a variety of autoimmune diseases as well as for prevention of tissue rejection after organ transplantation and can result in recrudescence of latent herpesvirus infections. Prior to examination of MHV-68 as a suitable model for EBV, better characterization of the MHV-68 model was desirable. Characterization of the MHV-68 model involved development of assays for detecting virus and for demonstration of safety when present in murine colonies. Limited information is available in the literature regarding MHV-68 transmission, although recent reports indicate the virus is not horizontally spread in research facilities. To further determine transmission potential, immunocompetent and immunodeficient mice were infected with MHV-68 and co-habitated with naïve animals. Molecular pathology assays were developed to characterize the MHV-68 model and to determine viral transmission. Horizontal transmission of virus was not observed from infected animals to naïve cagemates after fluorescence microscopy assays and quantitative PCR (qPCR). Serologic analysis complemented these studies and was used as a method of monitoring infection amongst murine colonies. Overall, these findings demonstrate that MHV-68 infection can be controlled and monitored in murine research facilities, and the potential for unintentional infection is low.

  15. Murine muscular dystrophy caused by a mutation in the laminin alpha 2 (Lama2) gene

    Xu, H; Wu, X R; Wewer, U M;


    The classic murine muscular dystrophy strain, dy, was first described almost 40 years ago. We have identified the molecular basis of an allele of dy, called dy2J, by detecting a mutation in the laminin alpha 2 chain gene--the first identified mutation in laminin-2. The G to A mutation in a splice...

  16. Effects of hypoxia on pluripotency in murine iPS cells.

    Sugimoto, Kouji; Yoshizawa, Yuu; Yamada, Shizuka; Igawa, Kazunari; Hayashi, Yoshihiko; Ishizaki, Hidetaka


    Retroviral transduction of four transcription factors (Oct4, Sox2, Klf4 and c-Myc) or three factors, excluding c-Myc, has been shown to initiate a reprogramming process that results in the transformation of murine fibroblasts to induced pluripotent stem (iPS) cells, and there has been a rapid increase in the number of iPS cell-based preclinical trials. In this study, the effects of these transcription factors were evaluated regarding the growth and differentiation of murine iPS cells under hypoxia. Based on the results of RT-PCR and alizarin red S staining, there were no statistical differences in the growth and differentiation of iPS cells or the induction of iPS cells to osteoblasts under hypoxia between the transcription factor groups. Furthermore, the function of hypoxia inducible factors (HIFs) in murine iPS cells under hypoxia was investigated in relation to the morphology and expression of transcription factors using RT-PCR and Western blotting. The HIF-2α knockdown group exhibited a decrease in the colony size of the iPS cells. The HIF-2α or -3α knockdown group demonstrated a statistically significant decrease in the transcription factor expression compared to that observed in the control group. These results demonstrate that HIF-2α among HIFs is the most influential candidate for the maintenance of the pluripotency of murine iPS cells.

  17. The interaction pattern of murine serum ficolin-A with microorganisms

    Hummelshøj, Tina; Ma, Ying Jie; Munthe-Fog, Lea;


    The ficolins are soluble pattern recognition molecules in the lectin pathway of complement, but the spectrum and mode of interaction with pathogens are largely unknown. In this study, we investigated the binding properties of the murine serum ficolin-A towards a panel of different clinical relevant...

  18. Characterization of murine SIRT3 transcript variants and corresponding protein products

    SIRT3 is one of the seven mammalian sirtuin homologs of the yeast SIR2 gene. SIRT3 possesses NAD(+)-dependent protein deacetylase activity. Recent studies indicate that the murine SIRT3 gene expresses different transcript variants, resulting in three possible SIRT3 protein isoforms with various leng...

  19. Fetal wound healing using a genetically modified murine model: the contribution of P-selectin

    During early gestation, fetal wounds heal with paucity of inflammation and absent scar formation. P-selectin is an adhesion molecule that is important for leukocyte recruitment to injury sites. We used a murine fetal wound healing model to study the specific contribution of P-selectin to scarless wo...

  20. A murine ESC-like state facilitates transgenesis and homologous recombination in human pluripotent stem cells

    C. Buecker (Christa); H.H. Chen; J.M. Polo (Jose); L. Daheron (Laurence); L. Bu (Lei); T.S. Barakat (Tahsin Stefan); P. Okwieka (Patricia); A. Porter (Andrew); J.H. Gribnau (Joost); K. Hochedlinger (Konrad); N. Geijsen (Niels)


    textabstractMurine pluripotent stem cells can exist in two functionally distinct states, LIF-dependent embryonic stem cells (ESCs) and bFGF-dependent epiblast stem cells (EpiSCs). However, human pluripotent cells so far seemed to assume only an epiblast-like state. Here we demonstrate that human iPS

  1. A Murine ESC-like State Facilitates Transgenesis and Homologous Recombination in Human Pluripotent Stem Cells

    Buecker, Christa; Chen, Hsu-Hsin; Polo, Jose Maria; Daheron, Laurence; Bu, Lei; Barakat, Tahsin Stefan; Okwieka, Patricia; Porter, Andrew; Gribnau, Joost; Hochedlinger, Konrad; Geijsen, Niels


    Murine pluripotent stem cells can exist in two functionally distinct states, LIF-dependent embryonic stem cells (ESCs) and bFGF-dependent epiblast stem cells (EpiSCs). However, human pluripotent cells so far seemed to assume only an epiblast-like state. Here we demonstrate that human iPSC reprogramm

  2. Midline 1 controls polarization and migration of murine cytotoxic T cells

    Boding, Lasse; Hansen, Ann K; Nielsen, Morten M;


    is strongly up-regulated in murine cytotoxic T lymphocytes (CTLs), and that it has a significant impact on exocytosis of lytic granules and the killing capacity of CTLs. The aims of the present study were to determine the localization of MID1 in migrating CTLs, and to investigate whether MID1 affects CTL...

  3. Bifidobacterium bifidum Actively Changes the Gene Expression Profile Induced by Lactobacillus acidophilus in Murine Dendritic Cells

    Weiss, Gudrun Margarethe; Rasmussen, Simon; Fink, Lisbeth Nielsen


    cytokine IL-12 in DC, whereas bifidobacteria do not induce IL-12 but inhibit the IL-12 production induced by lactobacilli. In the present study, genome-wide microarrays were used to investigate the gene expression pattern of murine DC stimulated with Lactobacillus acidophilus NCFM and Bifidobacterium...

  4. Transgene stability for three replication-competent murine leukemia virus vectors

    Duch, Mogens R.; Carrasco, Maria L; Hansen, Bettina Dencker


    cassette consisting of an internal ribosome entry site followed by the enhanced green fluorescent protein coding sequence inserted in different configurations into murine leukemia virus genomes. In two of the constructs, the insert was located in the upstream part of the U3 region while in the third...

  5. Evaluation of hypothalamic murine and human melanocortin 3 receptor transcript structure.

    Taylor-Douglas, Dezmond C; Basu, Arunabha; Gardner, Ryan M; Aspelund, Sender; Wen, Xin; Yanovski, Jack A


    The melanocortin 3 receptor (MC3R) is involved in regulation of energy homeostasis. However, its transcript structure is not well understood. We therefore studied initiation and termination sites for hypothalamic murine Mc3r and human MC3R transcripts. Rapid Amplification of cDNA Ends (RACE) was performed for the 5' and 3' ends of murine and human hypothalamic RNA. 5' RACE experiments using hypothalamic murine RNA indicated mouse hypothalamus expresses two major Mc3r transcription start sites: one with a 5' UTR approximately 368 bases in length and another previously unknown transcript with a 5' UTR approximately 440 bases in length. 5' RACE experiments using human hypothalamic RNA identified a 5' UTR beginning 533 bases upstream of the start codon with a 248 base splice. 3' RACE experiments using hypothalamic murine RNA indicated the 3' UTR terminates approximately 1286 bases after the translational stop codon, with a previously unknown 787 base splice between consensus splice donor and acceptor sites. 3' RACE experiments using human MC3R transcript indicated the 3' UTR terminates approximately 115-160 bases after the translational stop codon. These data provide insight into melanocortin 3 receptor transcript structure.

  6. In vitro activation of murine DRG neurons by CGRP-mediated mucosal mast cell degranulation

    De Jonge, F; De Laet, A; Van Nassauw, L; Miller, HRP; van Bogaert, PP; Timmermans, JP; Kroese, ABA


    Upregulation of CGRP-immunoreactive (IR) primary afferent nerve fibers accompanied by mastocytosis is characteristic for the Schistosoma mansoni-infected murine ileum. These mucosal mast cells (MMC) and CGRP-IR fibers, which originate from dorsal root (DRG) and nodose ganglia, are found in close app

  7. Rotenone inhibits primary murine myotube formation via Raf-1 and ROCK2

    Grefte, S.; Wagenaars, J.A.L.; Jansen, R.; Willems, P.H.G.M.; Koopman, W.J.H.


    Rotenone (ROT) is a widely used inhibitor of complex I (CI), the first complex of the mitochondrial oxidative phosphorylation (OXPHOS) system. However, particularly at high concentrations ROT was also described to display off-target effects. Here we studied how ROT affected in vitro primary murine m

  8. Fine mapping and functional activity of the adenosine deaminase origin in murine embryonic fibroblasts.

    Sibani, Sahar; Rampakakis, Emmanouil; Di Paola, Domenic; Zannis-Hadjopoulos, Maria


    DNA replication initiates at origins within the genome. The late-firing murine adenosine deaminase (mAdA) origin is located within a 2 kb fragment of DNA, making it difficult to examine by realtime technology. In this study, fine mapping of the mAdA region by measuring the abundance of nascent strand DNA identified two origins, mAdA-1 and mAdA-C, located 397 bp apart from each other. Both origins conferred autonomous replication to plasmids transfected in murine embryonic fibroblasts (MEFs), and exhibited similar activities in vivo and in vitro. Furthermore, both were able to recruit the DNA replication initiator proteins Cdc6 and Ku in vitro, similar to other bona fide replication origins. When tested in a murine Ku80(-/-) cell line, both origins exhibited replication activities comparable to those observed in wildtype cells, as did the hypoxanthine-guanine phosphoribosyltransferase (HPRT) and c-myc origins. This contrasts with previously published studies using Ku80-deficient human cells lines and suggests differences in the mechanism of initiation of DNA replication between the murine and human systems.

  9. Best practice for passaging murine embryonic enteric neuronal cell line before differentiation

    Rietdijk, Carmen D.; de Haan, Lydia; van Wezel, Richard J. A.; Garssen, Johan; Kraneveld, Aletta D.


    The enteric nervous system (ENS) is a complex network of neurons in the gut, regulating many local, vital functions of the gastro-intestinal tract. The ENS is also part of the bidirectional gut-brain axis. The murine immorto fetal enteric neuronal (IM-FEN) cell line was chosen as a model to study en

  10. An ES-Like pluripotent state in FGF-dependent murine iPS cells

    B. di Stefano (Bruno); C. Buecker (Christa); F. Ungaro (Federica); A. Prigione (Alessandro); H.H. Chen; M. Welling (Maaike); M. Eijpe (Maureen); G. Mostoslavsky (Gustavo); P. Tesar (Paul); J. Adjaye (James); N. Geijsen (Niels); V. Broccoli (Vania)


    textabstractRecent data demonstrates that stem cells can exist in two morphologically, molecularly and functionally distinct pluripotent states; a naïve LIF-dependent pluripotent state which is represented by murine embryonic stem cells (mESCs) and an FGFdependent primed pluripotent state represente

  11. Porcine humoral immune responses to multiple injections of murine monoclonal antibodies

    Lohse, Louise; Nielsen, Jens; Kamstrup, Søren;


    In humans and cattle, multiple injections of murine monoclonal antibodies (m-mAbs) induce anti-mouse antibody responses. The objectives of the present. study were to investigate whether a similar response could be seen when pigs were subjected to m-mAb therapy, and to study the kinetics of such a...

  12. Structure of the gene encoding the murine protein kinase CK2 beta subunit

    Boldyreff, B; Issinger, O G


    The mouse protein kinase CK2 beta subunit gene (Csnk2b) is composed of seven exons contained within 7874 bp. The exon and intron lengths extend from 76 to 321 and 111 to 1272 bp, respectively. The lengths of the murine coding exons correspond exactly to the lengths of the exons in the human CK2...


    The solely known function of Cu,Zn-superoxide dismutase (SOD1) is to catalyze the dismutation of superoxide anion into hydrogen peroxide. Our objective was to determine if SOD1 catalyzed murine liver protein nitration induced by acetaminophen (APAP) and lipopolysaccharide (LPS). Liver and plasma ...

  14. Addition of Caspofungin to Fluconazole Does Not Improve Outcome in Murine Candidiasis

    Graybill, John R.; Bocanegra, Rosie; Najvar, Laura K.; Hernandez, Steve; Larsen, Robert A.


    Caspofungin is a potent antifungal inhibiting glucan synthesis in Candida species. However, caspofungin is not 100% curative in candidiasis. Therefore, we evaluated combinations of fluconazole with caspofungin for murine candidemia. We could not show any benefit of combined therapy over individual antifungal drugs.

  15. Mechanisms of developmental control of transcription in the murine alpha- and beta-globin loci

    T. Trimborn (Tolleiv); J.H. Gribnau (Joost); P.J. Fraser (Peter); F.G. Grosveld (Frank)


    textabstractWe have characterized mRNA expression and transcription of the mouse alpha- and beta-globin loci during development. S1 nuclease and primary transcript in situ hybridization analyses demonstrate that all seven murine globin genes (zeta, alpha1, alpha2, epsil


    MALan; CAOXiao-Mei; BENKun-Long; CHENYun-Liang


    Clinical and experimental investigations have shown that secretory IgA antibodies to human sperm may bc one of the most important factors in immunological infertility. Studies on the effects of these antibodies on sperm function arc helpful to understand the role of

  17. Human pontine glioma cells can induce murine tumors

    Caretti, V.; Sewing, A.C.; Lagerweij, T.; Schellen, P.; Bugiani, M.; Jansen, M.H.; Vuurden, D.G. van; Navis, A.C.; Horsman, I.; Vandertop, W.P.; Noske, D.P.; Wesseling, P.; Kaspers, G.J.L.; Nazarian, J.; Vogel, H.; Hulleman, E.; Monje, M.; Wurdinger, T.


    Diffuse intrinsic pontine glioma (DIPG), with a median survival of only 9 months, is the leading cause of pediatric brain cancer mortality. Dearth of tumor tissue for research has limited progress in this disease until recently. New experimental models for DIPG research are now emerging. To develop


    张俊武; 张雪青; 陈平


    Objective. To examine the effect of isobutyramide synthesized in our laboratory on human and murine globingene expression and to test cell toxicity of the drug.Methods. ME L cells were transfected with the recombinant construct μLCRAγψβδβ3 and the stable transfor-mants were cultured in the medium with different concentrations of isobutyramide. The experimental mice and rab-bit were injected with different doses of isobutyramide. The globin mRNAs were analyzed by RNase protection as-say. The hematologdcal toxicity and electrolyte toxicity of the drug were tested.Results. An inducible and dose-dependent expression of the human γγ-, ββ- and mouse Aa-globin gene was ob-served in the transfected MEL cells. The induction of the human γ-globin gene is significant stronger than that ofthe ββ-globin gene. With 2.5 ~ 5 mmol/L isobutyramide, the induction of the human γ-globin gene is even moreeffective than that of mouse aa-globin gene. After a 15-day injection under the doses of 500 ~ 900mg * kg- 1 * d-1,the level of the mouse embryonic εy-globin mRNA could be significantly induced up to 3 ~ 4 fold of that of uninject-ed controls. The changes of hemoglobin(Hb), RBC, hematocrit(HCT), WBC, derived from mice injected withdifferent doses of isobutyramide at the interval of 24 hours for 2 ~4 weeks, were generally within the normalrange. In rabbits injected with isobutyramide in the same regiment for 2 weeks, the concentration of blood K+,Na +, C1- and CO.2 were all within normal range and serum ionic osmotic pressure remained stable as well.Conclusion. Our results suggested that isobutyramide is a weak inducer of cell differentiation, but it canselectively activate transcription of human γy-globin gene at a certain degree, and it can act on early stages of ery-throid progenitor differentiation in adult mice and activate transcription of embryonic εy-globin gene and have nohematological toxicity. Our results have further proved the potential value of

  19. Involvement of Nitric Oxide on Bothropoides insularis Venom Biological Effects on Murine Macrophages In Vitro.

    Ramon R P P B de Menezes

    Full Text Available Viperidae venom has several local and systemic effects, such as pain, edema, inflammation, kidney failure and coagulopathy. Additionally, bothropic venom and its isolated components directly interfere on cellular metabolism, causing alterations such as cell death and proliferation. Inflammatory cells are particularly involved in pathological envenomation mechanisms due to their capacity of releasing many mediators, such as nitric oxide (NO. NO has many effects on cell viability and it is associated to the development of inflammation and tissue damage caused by Bothrops and Bothropoides venom. Bothropoides insularis is a snake found only in Queimada Grande Island, which has markedly toxic venom. Thus, the aim of this work was to evaluate the biological effects of Bothropoides insularis venom (BiV on RAW 264.7 cells and assess NO involvement. The venom was submitted to colorimetric assays to identify the presence of some enzymatic components. We observed that BiV induced H2O2 production and showed proteolytic and phospholipasic activities. RAW 264.7 murine macrophages were incubated with different concentrations of BiV and then cell viability was assessed by MTT reduction assay after 2, 6, 12 and 24 hours of incubation. A time- and concentration-dependent effect was observed, with a tendency to cell proliferation at lower BiV concentrations and cell death at higher concentrations. The cytotoxic effect was confirmed after lactate dehydrogenase (LDH measurement in the supernatant from the experimental groups. Flow cytometry analyses revealed that necrosis is the main cell death pathway caused by BiV. Also, BiV induced NO release. The inhibition of both proliferative and cytotoxic effects with L-NAME were demonstrated, indicating that NO is important for these effects. Finally, BiV induced an increase in iNOS expression. Altogether, these results demonstrate that B. insularis venom have proliferative and cytotoxic effects on macrophages, with

  20. Static and dynamic mechanics of the murine lung after intratracheal bleomycin

    Papiris Spyridon


    Full Text Available Abstract Background Despite its widespread use in pulmonary fibrosis research, the bleomycin mouse model has not been thoroughly validated from a pulmonary functional standpoint using new technologies. Purpose of this study was to systematically assess the functional alterations induced in murine lungs by fibrogenic agent bleomycin and to compare the forced oscillation technique with quasi-static pressure-volume curves in mice following bleomycin exposure. Methods Single intratracheal injections of saline (50 μL or bleomycin (2 mg/Kg in 50 μL saline were administered to C57BL/6 (n = 40 and Balb/c (n = 32 mice. Injury/fibrosis score, tissue volume density (TVD, collagen content, airway resistance (RN, tissue damping (G and elastance coefficient (H, hysteresivity (η, and area of pressure-volume curve (PV-A were determined after 7 and 21 days (inflammation and fibrosis stage, respectively. Statistical hypothesis testing was performed using one-way ANOVA with LSD post hoc tests. Results Both C57BL/6 and Balb/c mice developed weight loss and lung inflammation after bleomycin. However, only C57BL/6 mice displayed cachexia and fibrosis, evidenced by increased fibrosis score, TVD, and collagen. At day 7, PV-A increased significantly and G and H non-significantly in bleomycin-exposed C57BL/6 mice compared to saline controls and further increase in all parameters was documented at day 21. G and H, but not PV-A, correlated well with the presence of fibrosis based on histology, TVD and collagen. In Balb/c mice, no change in collagen content, histology score, TVD, H and G was noted following bleomycin exposure, yet PV-A increased significantly compared to saline controls. Conclusions Lung dysfunction in the bleomycin model is more pronounced during the fibrosis stage rather than the inflammation stage. Forced oscillation mechanics are accurate indicators of experimental bleomycin-induced lung fibrosis. Quasi-static PV-curves may be more sensitive than

  1. Curcumin Ingestion Inhibits Mastocytosis and Suppresses Intestinal Anaphylaxis in a Murine Model of Food Allergy.

    Shannon R M Kinney

    Full Text Available IgE antibodies and mast cells play critical roles in the establishment of allergic responses to food antigens. Curcumin, the active ingredient of the curry spice turmeric, has anti-inflammatory properties, and thus may have the capacity to regulate Th2 cells and mucosal mast cell function during allergic responses. We assessed whether curcumin ingestion during oral allergen exposure can modulate the development of food allergy using a murine model of ovalbumin (OVA-induced intestinal anaphylaxis. Herein, we demonstrate that frequent ingestion of curcumin during oral OVA exposure inhibits the development of mastocytosis and intestinal anaphylaxis in OVA-challenged allergic mice. Intragastric (i.g. exposure to OVA in sensitized BALB/c mice induced a robust IgE-mediated response accompanied by enhanced OVA-IgE levels, intestinal mastocytosis, elevated serum mMCP-1, and acute diarrhea. In contrast, mice exposed to oral curcumin throughout the experimental regimen appeared to be normal and did not exhibit intense allergic diarrhea or a significant enhancement of OVA-IgE and intestinal mast cell expansion and activation. Furthermore, allergic diarrhea, mast cell activation and expansion, and Th2 responses were also suppressed in mice exposed to curcumin during the OVA-challenge phase alone, despite the presence of elevated levels of OVA-IgE, suggesting that curcumin may have a direct suppressive effect on intestinal mast cell activation and reverse food allergy symptoms in allergen-sensitized individuals. This was confirmed by observations that curcumin attenuated the expansion of both adoptively transferred bone marrow-derived mast cells (BMMCs, and inhibited their survival and activation during cell culture. Finally, the suppression of intestinal anaphylaxis by curcumin was directly linked with the inhibition of NF-κB activation in curcumin-treated allergic mice, and curcumin inhibited the phosphorylation of the p65 subunit of NF-κB in BMMCs. In

  2. Respiratory syncytial virus infection in immunocompetent and immunocompromised murine



    The purpose of this study is to distinguish respiratory syncytial virus (RSV) infection and immunology between immunocompetent and immunocompromised murine and to explore immune mechanism of RSV infection. At various time points after RSV infection of BALB/c mice and nude mice, pulmonary viral titers were assayed, RSV antigen was tested by direct immunofluorescent assay and immunohistochemistry. Pulmonary mRNA expressions of Toll like receptor (TLR)2 and TLR4 were assayed by RT-PCR. CD4+ cells and CD8+ cells in peripheral blood were examined by flow cytometry and plasma total IgE was assayed by ELISA. Leukocytes in bronchoalveolar lavage fluid (BALF) and pulmonary histology were identified to reflect airway inflammation. It was found that RSV titers of both mice peaked on the 3rd day post infection with a much higher level of viral titer in nude mice than in BALB/c mice and a longer viral duration in nude mice (over 9 days post infection) than in BALB/c mice (6 days post infection). RSV infection induced higher viral antigen expression in nude mice (0.267 ±0.045) than in BALB/c mice (0. 168 ± 0.031). RSV infection enhanced pulmonary TLR4 expression of BALB/c mice (51.96% ± 11.34%) and nude mice (48.96% ± 12.35%) compared with each control (34.04% ± 10.06% and 32.37% ± 9.87% respectively). CD4+ peripheral blood cells increased in RSV infected BALB/c mice (66.51% ± 2.09% ) compared with the control BALB/c mice (51.63% ± 5.90% ), and CD4+ cells and CD8+ cells were deficient in nude mice. RSV infection increased plasma total IgE in both mice, and BALB/c mice had a larger amount of IgE on the 7th day post infection (9.02 ng/ml ± 2.90 ng/ml) and on the 14th day post infection (12.76 ng/ml ± 4.15 ng/ml) than corresponding nude mice (3.72 ng/ml ± 1.06 ng/ml and 7.62 ng/ml ± 3.08 ng/ml respectively on the 7th and 14th day post infection). RSV infected nude mice had more severe airway inflammation than infected BALB/c mice. It is concluded that BALB/c mice and

  3. Inflammatory manifestations of experimental lymphatic insufficiency.

    Raymond Tabibiazar


    Full Text Available BACKGROUND: Sustained lymph stagnation engenders a pathological response that is complex and not well characterized. Tissue inflammation in lymphedema may reflect either an active or passive consequence of impaired immune traffic. METHODS AND FINDINGS: We studied an experimental model of acute post-surgical lymphedema in the tails of female hairless, immunocompetent SKH-1 mice. We performed in vivo imaging of impaired immune traffic in experimental, murine acquired lymphatic insufficiency. We demonstrated impaired mobilization of immunocompetent cells from the lymphedematous region. These findings correlated with histopathological alterations and large-scale transcriptional profiling results. We found intense inflammatory changes in the dermis and the subdermis. The molecular pattern in the RNA extracted from the whole tissue was dominated by the upregulation of genes related to acute inflammation, immune response, complement activation, wound healing, fibrosis, and oxidative stress response. CONCLUSIONS: We have characterized a mouse model of acute, acquired lymphedema using in vivo functional imaging and histopathological correlation. The model closely simulates the volume response, histopathology, and lymphoscintigraphic characteristics of human acquired lymphedema, and the response is accompanied by an increase in the number and size of microlymphatic structures in the lymphedematous cutaneous tissues. Molecular characterization through clustering of genes with known functions provides insights into processes and signaling pathways that compose the acute tissue response to lymph stagnation. Further study of genes identified through this effort will continue to elucidate the molecular mechanisms and lead to potential therapeutic strategies for lymphatic vascular insufficiency.

  4. Study on Association between Single Nucleotide Polymorphisms in Murine Double Minute 2 and Susceptibility of Hepatocellular Carcinoma

    Xia Wang


    Full Text Available Objective: To investigate the relationship between single nucleotide polymorphisms (SNP in murine double minute 2 (MDM2 and susceptibility and biological behavior of hepatocellularcarcinoma (HCC. Methods: MDM2 (rs2279744 site polymorphism in peripheral blood from 166 patients with HCC and 157 healthy controls were detected by SYBR GREEN PCR method and the relationship between MDM2 polymorphism and susceptibility and biological behavior of HCC was analyzed by comparing the differences of genotypes in two populations. Results: There was no statistical significance between two groups in terms of MDM2 allele distribution in research population (P = 0.753. The risk of HCC onset in individuals with GG+ TG genotype was 1.698 times of those with TT genotype in case group (95%CI = 1.027 -2.808. MDM2 SNP was associated with HBV infection and the degree of tumor differentiation (P< 0.05. The incidence of alleles in experimental group (T, 0.49; G, 0.51 was very different from that in control group (T, 0.59; G, 0.41 (P = 0.015. The incidence of GG genotype in patients with HCC (22.29% was significantly higher than those without HCC (13.38%. Compared with TT genotype, G allele or GG genotype had more correlation with HCC onset. Conclusion: Compared with TT genotype, MDM2 promoter SNP309 G allele or GG genotype is more associated with HCC onset in Chinese population.

  5. Activation of calpains, calpastatin and spectrin cleavage in the brain during the pathology of fatal murine cerebral malaria.

    Shukla, Meena; Rajgopal, Yadavalli; Babu, Phanithi Prakash


    Neuronal calpains appear to be activated uncontrollably by sustained elevation of cytosolic calcium levels under pathological conditions as well as neurodegenerative diseases. In the present study, we have characterized calpain activation in cytosolic extract of mice cerebral cortex and cerebellum using an experimental model of fatal murine cerebral malaria (FMCM). Pathology of FMCM resulted in the increase in activity of calpains in both cerebral cortex and cerebellum. Western blot analysis revealed an increase in the levels of mu-calpain (calpain-1) in the cytosolic fraction of infected cerebral cortex and cerebellum although a decrease in the level of m-calpain was observed in the cytosolic fraction of infected cerebellum and cerebral cortex. Calpain activation was further confirmed by monitoring the formation of calpain-specific spectrin breakdown products (SBDP). Protease-specific SBDP revealed the formation of calpain-generated 150kDa product in the infected cerebral cortex and cerebellum. The specific signature fragment of calpain activation and spectrin breakdown after Plasmodium berghei ANKA infection provide a strong evidence of the role of calpains during the cell death in cerebral cortex and cerebellum. Given the role of calpains in neurodegeneration and cell death, our results strongly suggest that calpains are important mediators of cell injury and neurological sequelae associated with FMCM.

  6. Effect of pecan phenolics on the release of nitric oxide from murine RAW 264.7 macrophage cells.

    Robbins, Katherine S; Greenspan, Phillip; Pegg, Ronald B


    Inflammation is linked to numerous chronic disease states. Phenolic compounds have attracted attention because a number of these compounds possess anti-inflammatory properties. A phenolic crude extract was prepared from pecans and separated by Sephadex LH-20 column chromatography into low- and high-molecular-weight (LMW/HMW) fractions. Anti-inflammatory properties of these fractions were assessed in LPS-stimulated RAW 264.7 murine macrophage cells. NO and reactive oxygen species (ROS) production was monitored after 3 different experimental protocols: (1) pre-treatment with Escherichia coli O111:B4 lipopolysaccharide (LPS); (2) pre-treatment with a pecan crude extract and its fractions; and (3) co-incubation of LPS with a pecan crude extract and its fractions. The LMW fraction displayed a dose-dependent decrease in NO production and a significant decrease from the LPS control in ROS production when cells were either co-incubated with or pre-treated with LPS. The phenolics were characterized by HPLC to help identify those responsible for the observed effect.

  7. Recombinant murine IL-12 promotes a protective Th1/cellular response in Mongolian gerbils infected with Sporothrix schenckii.

    Flores-García, Aurelio; Velarde-Félix, Jesús Salvador; Garibaldi-Becerra, Vicente; Rangel-Villalobos, Héctor; Torres-Bugarín, Olivia; Zepeda-Carrillo, Eloy Alfonso; Ruíz-Bernés, Salvador; Ochoa-Ramírez, Luis Antonio


    Sporotrichosis is a cutaneous fungal infection caused by Sporothrix schenckii. It is known to be mainly contained by Th1 responses. As IL-12 is crucial for Th1 response, we investigated if treatment with recombinant murine IL-12 (rmIL-12) promoted Th1 immunity and/or clinical improvement in an experimental sporotrichosis gerbil model. Gerbils were inoculated with S. schenckii in the footpad and treated with rmIL-12. Seven days post infection there was a significant increase in macrophage phagocytosis and oxidative burst, and in delayed-type hypersensitivity (DTH) reaction in rmIL-12 treated gerbils, as well as a ∼10-fold increase of serum IFN-gamma and a decrease of IL-4 and IL-10. Moreover, rmIL-12 substantially decreased (∼70%) S. schenckii burden in liver and spleen and improved the clinical outcome preventing footpad ulcer and tail nodules observed in untreated gerbils. Our study demonstrates that rmIL-12 promotes Th1 immune response against S. schenckii favouring its clearance and preventing clinical symptoms.

  8. The AOM/DSS murine model for the study of colon carcinogenesis: From pathways to diagnosis and therapy studies

    Robertis, Mariangela De; Massi, Emanuela; Poeta, Maria Luana; Carotti, Simone; Morini, Sergio; Cecchetelli, Loredana; Signori, Emanuela; Fazio, Vito Michele


    Colorectal cancer (CRC) is a major health problem in industrialized countries. Although inflammation-linked carcinogenesis is a well accepted concept and is often observed within the gastrointestinal tract, the underlying mechanisms remain to be elucidated. Inflammation can indeed provide initiating and promoting stimuli and mediators, generating a tumour-prone microenvironment. Many murine models of sporadic and inflammation-related colon carcinogenesis have been developed in the last decade, including chemically induced CRC models, genetically engineered mouse models, and xenoplants. Among the chemically induced CRC models, the combination of a single hit of azoxymethane (AOM) with 1 week exposure to the inflammatory agent dextran sodium sulphate (DSS) in rodents has proven to dramatically shorten the latency time for induction of CRC and to rapidly recapitulate the aberrant crypt foci–adenoma–carcinoma sequence that occurs in human CRC. Because of its high reproducibility and potency, as well as the simple and affordable mode of application, the AOM/DSS has become an outstanding model for studying colon carcinogenesis and a powerful platform for chemopreventive intervention studies. In this article we highlight the histopathological and molecular features and describe the principal genetic and epigenetic alterations and inflammatory pathways involved in carcinogenesis in AOM/DSS–treated mice; we also present a general overview of recent experimental applications and preclinical testing of novel therapeutics in the AOM/DSS model. PMID:21483655

  9. Chemokine binding protein M3 of murine gammaherpesvirus 68 modulates the host response to infection in a natural host.

    David J Hughes


    Full Text Available Murine γ-herpesvirus 68 (MHV-68 infection of Mus musculus-derived strains of mice is an attractive model of γ-herpesvirus infection. Surprisingly, however, ablation of expression of MHV-68 M3, a secreted protein with broad chemokine-binding properties in vitro, has no discernable effect during experimental infection via the respiratory tract. Here we demonstrate that M3 indeed contributes significantly to MHV-68 infection, but only in the context of a natural host, the wood mouse (Apodemus sylvaticus. Specifically, M3 was essential for two features unique to the wood mouse: virus-dependent inducible bronchus-associated lymphoid tissue (iBALT in the lung and highly organized secondary follicles in the spleen, both predominant sites of latency in these organs. Consequently, lack of M3 resulted in substantially reduced latency in the spleen and lung. In the absence of M3, splenic germinal centers appeared as previously described for MHV-68-infected laboratory strains of mice, further evidence that M3 is not fully functional in the established model host. Finally, analyses of M3's influence on chemokine and cytokine levels within the lungs of infected wood mice were consistent with the known chemokine-binding profile of M3, and revealed additional influences that provide further insight into its role in MHV-68 biology.

  10. Chemokine Binding Protein M3 of Murine Gammaherpesvirus 68 Modulates the Host Response to Infection in a Natural Host

    Hughes, David J.; Kipar, Anja; Leeming, Gail H.; Bennett, Elaine; Howarth, Deborah; Cummerson, Joanne A.; Papoula-Pereira, Rita; Flanagan, Brian F.; Sample, Jeffery T.; Stewart, James P.


    Murine γ-herpesvirus 68 (MHV-68) infection of Mus musculus-derived strains of mice is an attractive model of γ-herpesvirus infection. Surprisingly, however, ablation of expression of MHV-68 M3, a secreted protein with broad chemokine-binding properties in vitro, has no discernable effect during experimental infection via the respiratory tract. Here we demonstrate that M3 indeed contributes significantly to MHV-68 infection, but only in the context of a natural host, the wood mouse (Apodemus sylvaticus). Specifically, M3 was essential for two features unique to the wood mouse: virus-dependent inducible bronchus-associated lymphoid tissue (iBALT) in the lung and highly organized secondary follicles in the spleen, both predominant sites of latency in these organs. Consequently, lack of M3 resulted in substantially reduced latency in the spleen and lung. In the absence of M3, splenic germinal centers appeared as previously described for MHV-68-infected laboratory strains of mice, further evidence that M3 is not fully functional in the established model host. Finally, analyses of M3's influence on chemokine and cytokine levels within the lungs of infected wood mice were consistent with the known chemokine-binding profile of M3, and revealed additional influences that provide further insight into its role in MHV-68 biology. PMID:21445235

  11. Systems genetics of liver fibrosis: identification of fibrogenic and expression quantitative trait loci in the BXD murine reference population.

    Rabea A Hall

    Full Text Available The progression of liver fibrosis in response to chronic injury varies considerably among individual patients. The underlying genetics is highly complex due to large numbers of potential genes, environmental factors and cell types involved. Here, we provide the first toxicogenomic analysis of liver fibrosis induced by carbon tetrachloride in the murine 'genetic reference panel' of recombinant inbred BXD lines. Our aim was to define the core of risk genes and gene interaction networks that control fibrosis progression. Liver fibrosis phenotypes and gene expression profiles were determined in 35 BXD lines. Quantitative trait locus (QTL analysis identified seven genomic loci influencing fibrosis phenotypes (pQTLs with genome-wide significance on chromosomes 4, 5, 7, 12, and 17. Stepwise refinement was based on expression QTL mapping with stringent selection criteria, reducing the number of 1,351 candidate genes located in the pQTLs to a final list of 11 cis-regulated genes. Our findings demonstrate that the BXD reference population represents a powerful experimental resource for shortlisting the genes within a regulatory network that determine the liver's vulnerability to chronic injury.

  12. Inflammation-mediated memory dysfunction and effects of a ketogenic diet in a murine model of multiple sclerosis.

    Do Young Kim

    Full Text Available A prominent clinical symptom in multiple sclerosis (MS, a progressive disorder of the central nervous system (CNS due to heightened neuro-inflammation, is learning and memory dysfunction. Here, we investigated the effects of a ketogenic diet (KD on memory impairment and CNS-inflammation in a murine model of experimental autoimmune encephalomyelitis (EAE, using electrophysiological, behavioral, biochemical and in vivo imaging approaches. Behavioral spatial learning deficits were associated with motor disability in EAE mice, and were observed concurrently with brain inflammation. The KD improved motor disability in the EAE model, as well as CA1 hippocampal synaptic plasticity (long-term potentiation and spatial learning and memory (assessed with the Morris Water Maze. Moreover, hippocampal atrophy and periventricular lesions in EAE mice were reversed in KD-treated EAE mice. Finally, we found that the increased expression of inflammatory cytokines and chemokines, as well as the production of reactive oxygen species (ROS, in our EAE model were both suppressed by the KD. Collectively, our findings indicate that brain inflammation in EAE mice is associated with impaired spatial learning and memory function, and that KD treatment can exert protective effects, likely via attenuation of the robust immune response and increased oxidative stress seen in these animals.

  13. Chemokine receptor CCR7 regulates the intestinal TH1/TH17/Treg balance during Crohn's-like murine ileitis.

    McNamee, Eóin N; Masterson, Joanne C; Veny, Marisol; Collins, Colm B; Jedlicka, Paul; Byrne, Fergus R; Ng, Gordon Y; Rivera-Nieves, Jesús


    The regulation of T cell and DC retention and lymphatic egress within and from the intestine is critical for intestinal immunosurveillance; however, the cellular processes that orchestrate this balance during IBD remain poorly defined. With the use of a mouse model of TNF-driven Crohn's-like ileitis (TNF(Δ) (ARE)), we examined the role of CCR7 in the control of intestinal T cell and DC retention/egress during experimental CD. We observed that the frequency of CCR7-expressing TH1/TH17 effector lymphocytes increased during active disease in TNF(Δ) (ARE) mice and that ΔARE/CCR7(-/-) mice developed exacerbated ileitis and multiorgan inflammation, with a marked polarization and ileal retention of TH1 effector CD4(+) T cells. Furthermore, adoptive transfer of ΔARE/CCR7(-/-) effector CD4(+) into lymphopenic hosts resulted in ileo-colitis, whereas those transferred with ΔARE/CCR7(+/+) CD4(+) T cells developed ileitis. ΔARE/CCR7(-/-) mice had an acellular draining MLN, decreased CD103(+) DC, and decreased expression of RALDH enzymes and of CD4(+)CD25(+)FoxP3(+) Tregs. Lastly, a mAb against CCR7 exacerbated ileitis in TNF(Δ) (ARE) mice, phenocopying the effects of congenital CCR7 deficiency. Our data underscore a critical role for the lymphoid chemokine receptor CCR7 in orchestrating immune cell traffic and TH1 versus TH17 bias during chronic murine ileitis.

  14. Tendon mineralization is accelerated bilaterally and creep of contralateral tendons is increased after unilateral needle injury of murine achilles tendons.

    O'Brien, Etienne John Ogilvy; Shrive, Nigel G; Rosvold, Joshua M; Thornton, Gail M; Frank, Cyril B; Hart, David A


    Heterotopic mineralization may result in tendon weakness, but effects on other biomechanical responses have not been reported. We used a needle injury, which accelerates spontaneous mineralization of murine Achilles tendons, to test two hypotheses: that injured tendons would demonstrate altered biomechanical responses; and that unilateral injury would accelerate mineralization bilaterally. Mice underwent left hind (LH) injury (I; n = 11) and were euthanized after 20 weeks along with non-injured controls (C; n = 9). All hind limbs were examined by micro computed tomography followed by biomechanical testing (I = 7 and C = 6). No differences were found in the biomechanical responses of injured tendons compared with controls. However, the right hind (RH) tendons contralateral to the LH injury exhibited greater static creep strain and total creep strain compared with those LH tendons (p ≤ 0.045) and RH tendons from controls (p ≤ 0.043). RH limb lesions of injured mice were three times larger compared with controls (p = 0.030). Therefore, despite extensive mineralization, changes to the responses we measured were limited or absent 20 weeks postinjury. These results also suggest that bilateral occurrence should be considered where tendon mineralization is identified clinically. This experimental system may be useful to study the mechanisms of bilateral new bone formation in tendinopathy and other conditions.

  15. Murine dendritic cells generated under serum-free conditions have a mature phenotype and efficiently induce primary immune responses.

    Warncke, Max; Dodero, Anna; Dierbach, Heide; Follo, Marie; Veelken, Hendrik


    Vaccination with in vitro-generated dendritic cells (DC) that present tumor-associated antigens is a promising approach for immunotherapy of malignant tumors. For optimization of DC-based vaccination protocols, preclinical tumor models that mimic the clinical situation closely are highly desirable. Strong non-specific T cell activation was observed in experimental immunization of mice with syngeneic DC generated in standard FCS-supplemented culture medium. To avoid deviation of the immune response to FCS-derived antigens, a serum-free culture protocol for in vitro generation of murine DC from bone marrow progenitor cells was developed. In comparison to DC differentiated with FCS supplementation, DC generated under serum-free conditions (sfDC) have a more homogeneous phenotype with higher expression of IL-12 and the differentiation and activation markers CD11c, CD40, CD80, CD83, CD86, DEC-205, and MHC class II. Demonstration of strong uptake of protein and carbohydrate antigens and analysis of the in vivo migration behaviour of sfDC also indicated excellent APC function. Vaccination of mice with peptide-pulsed sfDC efficiently induced an antigen-specific T cell response as assessed by MHC tetramer staining, IFN-gamma ELISPOT and in vivo cytotoxicity assay. sfDC may therefore represent a valuable tool to improve active tumor immunotherapy in animal models.

  16. Functional and Structural Characterization of Novel Type of Linker Connecting Capsid and Nucleocapsid Protein Domains in Murine Leukemia Virus.

    Doležal, Michal; Hadravová, Romana; Kožíšek, Milan; Bednárová, Lucie; Langerová, Hana; Ruml, Tomáš; Rumlová, Michaela


    The assembly of immature retroviral particles is initiated in the cytoplasm by the binding of the structural polyprotein precursor Gag with viral genomic RNA. The protein interactions necessary for assembly are mediated predominantly by the capsid (CA) and nucleocapsid (NC) domains, which have conserved structures. In contrast, the structural arrangement of the CA-NC connecting region differs between retroviral species. In HIV-1 and Rous sarcoma virus, this region forms a rod-like structure that separates the CA and NC domains, whereas in Mason-Pfizer monkey virus, this region is densely packed, thus holding the CA and NC domains in close proximity. Interestingly, the sequence connecting the CA and NC domains in gammaretroviruses, such as murine leukemia virus (MLV), is unique. The sequence is called a charged assembly helix (CAH) due to a high number of positively and negatively charged residues. Although both computational and deletion analyses suggested that the MLV CAH forms a helical conformation, no structural or biochemical data supporting this hypothesis have been published. Using an in vitro assembly assay, alanine scanning mutagenesis, and biophysical techniques (circular dichroism, NMR, microcalorimetry, and electrophoretic mobility shift assay), we have characterized the structure and function of the MLV CAH. We provide experimental evidence that the MLV CAH belongs to a group of charged, E(R/K)-rich, single α-helices. This is the first single α-helix motif identified in viral proteins.

  17. Host responses to sepsis vary in different low-lethality murine models.

    Lori F Gentile

    Full Text Available Animal models for the study of sepsis are being increasingly scrutinized, despite their essential role for early translational research. In particular, recent studies have suggested that at the level of the leukocyte transcriptome, murine models of burns, trauma and endotoxemia markedly differ from their human equivalents, and are only weakly similar amongst themselves. We compared the plasma cytokine and leukocyte transcriptome responses between two different low-lethality murine models of polymicrobial intra-abdominal sepsis.Six to ten week male C57BL/6j mice underwent either the 'gold standard' cecal ligation and puncture (CLP model of intra-abdominal sepsis or administration of a cecal slurry (CS, where cecal contents are injected intraperitoneally. Surviving mice were euthanized at two hours, one or three days after sepsis.The murine leukocyte transcriptomic response to the CLP and CS models of sepsis was surprisingly dissimilar at two hours, one, and three days after sepsis. The Pearson correlation coefficient for the maximum change in expression for the entire leukocyte transcriptome that changed significantly over time (n = 19,071 was R = 0.54 (R2 = 0.297. The CS model resulted in greater magnitude of early inflammatory gene expression changes in response to sepsis with associated increased production of inflammatory chemokines and cytokines. Two hours after sepsis, CLP had more significant expression of genes associated with IL-10 signaling pathways, whereas CS had greater expression of genes related to CD28, apoptosis, IL-1 and T-cell receptor signaling. By three days, the changes in gene expression in both sepsis models were returning to baseline in surviving animals.These analyses reveal that the murine blood leukocyte response to sepsis is highly dependent on which model of intra-abdominal sepsis is employed, despite their similar lethality. It may be difficult to extrapolate findings from one murine model to another

  18. MarA, SoxS and Rob function as virulence factors in an Escherichia coli murine model of ascending pyelonephritis.

    Casaz, Paul; Garrity-Ryan, Lynne K; McKenney, David; Jackson, Caroline; Levy, Stuart B; Tanaka, S Ken; Alekshun, Michael N


    MarA, SoxS and Rob are transcription factors belonging to the AraC family. While these proteins have been associated historically with control of multiple antibiotic resistance, and tolerance to oxidative stress agents and organic solvents, only a paucity of experimental data support a role in regulating virulence. Clinical Escherichia coli isolates, and isogenic strains lacking marA, soxS and rob, were studied in a murine model of ascending pyelonephritis, which is a clinically relevant model of urinary tract infection. Organisms lacking all three transcription factors (triple knockouts) were significantly less virulent than parental strains, and complementation studies demonstrated that the addition of marA, soxS and rob individually restored wild-type virulence in the triple-knockout strain. Deletion of soxS or rob alone was more detrimental than the removal of marA. Thus, all three proteins contribute to virulence in vivo.

  19. Establishment of Experimental Murine Peritonitis Model with Hog Gastric Mucin for Carbapenem-Resistant Gram-Negative Bacteria

    Park, Chulmin; Chun, Hye-Sun; Byun, Ji-Hyun; Cho, Sung-Yeon


    Animal models are essential to studies of infectious diseases. The use of mice to test bacterial infection has been extensively reported. However, methods applied to clinical isolates, particularly for carbapenem-resistant bacteria, must be tailored according to the infection models and bacteria used. In this study, we infected 6-week-old female BALB/c mice intraperitoneally with different strains of resistant bacteria plus 3% hog gastric mucin. This method was found to be efficient and readily applicable for investigation of carbapenem-resisant Gram-negative pathogens (e.g., Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii) detected in Korea.

  20. 3-(3-Pyridylmethylidene-2-indolinone Reduces the Severity of Colonic Injury in a Murine Model of Experimental Colitis

    Kun-Ping Wang


    Full Text Available Nrf2 is the key transcription factor regulating the antioxidant response which is crucial for cytoprotection against extracellular stresses. Numerous in vivo studies indicate that Nrf2 plays a protective role in anti-inflammatory response. 3-(3-Pyridylmethylidene-2-indolinone (PMID is a synthesized derivative of 2-indolinone compounds. Our previous study suggested that PMID induces the activation of Nrf2/ARE pathway, then protecting against oxidative stress-mediated cell death. However, little is known regarding the anti-inflammatory properties of PMID in severe inflammatory phenotypes. In the present study we determined if PMID treatment protects mice from dextran sodium sulphate- (DSS- induced colitis. The result suggests that treatment with PMID prior to colitis induction significantly reduced body weight loss, shortened colon length, and decreased disease activity index compared to control mice. Histopathological analysis of the colon revealed attenuated inflammation in PMID pretreated animals. The levels of inflammatory markers in colon tissue and serum were reduced associated with inhibition of NF-κB activation. The expression levels of Nrf2-dependent genes such as HO-1, NQO1, and Nrf2 were increased in PMID pretreated mice. However, PMID pretreatment did not prevent DSS-induced colitis in Nrf2 knockout mice. These data indicate that PMID pretreatment in mice confers protection against DSS-induced colitis in Nrf2-dependent manner, suggesting a potential role of PMID in anti-inflammatory response.

  1. Forkhead Box O1 Regulates Macrophage Polarization Following Staphylococcus aureus Infection: Experimental Murine Data and Review of the Literature.

    Wang, Yu-Chen; Ma, Hong-Di; Yin, Xue-Ying; Wang, Yin-Hu; Liu, Qing-Zhi; Yang, Jing-Bo; Shi, Qing-Hua; Sun, Baolin; Gershwin, M Eric; Lian, Zhe-Xiong


    The functions of macrophages that lead to effective host responses are critical for protection against Staphylococcus aureus. Deep tissue-invading S. aureus initially countered by macrophages trigger macrophage accumulation and induce inflammatory responses through surface receptors, especially toll-like receptor 2 (TLR2). Here, we found that macrophages formed sporadic aggregates in the liver during infection. Within those aggregates, macrophages co-localized with T cells and were indispensable for their infiltration. In addition, we have focused on the mechanisms underlying the polarization of macrophages in Forkhead box transcription factor O1 (FoxO1) conditional knockout Lys (Cre/+) FoxO1 (fl/fl) mice following S. aureus infection and report herein that macrophage M1-M2 polarization via TLR2 is intrinsically regulated by FoxO1. Indeed, for effective FoxO1 activity, stimulation of TLR2 is essential. However, following S. aureus challenge, there was a decrease in macrophage FoxO1, with increased phosphorylation of FoxO1 because of TLR2-mediated activation of PI3K/Akt and c-Raf/MEK/ERK pathway. Following infection in Lys (Cre/+) FoxO1 (fl/fl) mice, mice became more susceptible to S. aureus with reduced macrophage aggregation in the liver and attenuated Th1 and Th17 responses. FoxO1 abrogation reduced M1 pro-inflammatory responses triggered by S. aureus and enhanced M2 polarization in macrophages. In contrast, overexpression of FoxO1 in macrophages increased pro-inflammatory mediators and functional surface molecule expression. In conclusion, macrophage FoxO1 is critical to promote M1 polarization and maintain a competent T cell immune response against S. aureus infection in the liver. FoxO1 regulates macrophage M1-M2 polarization downstream of TLR2 dynamically through phosphorylation.

  2. Ocular Hypotensive Effects of the ATP-Sensitive Potassium Channel Opener Cromakalim in Human and Murine Experimental Model Systems.

    Uttio Roy Chowdhury

    Full Text Available Elevated intraocular pressure (IOP is the most prevalent and only treatable risk factor for glaucoma, a leading cause of irreversible blindness worldwide. Unfortunately, all current therapeutics used to treat elevated IOP and glaucoma have significant and sometimes irreversible side effects necessitating the development of novel compounds. We evaluated the IOP lowering ability of the broad spectrum KATP channel opener cromakalim. Cultured human anterior segments when treated with 2 μM cromakalim showed a decrease in pressure (19.33 ± 2.78 mmHg at 0 hours to 13.22 ± 2.64 mmHg at 24 hours; p<0.001 when compared to vehicle treated controls (15.89 ± 5.33 mmHg at 0 h to 15.56 ± 4.88 mmHg at 24 hours; p = 0.89. In wild-type C57BL/6 mice, cromakalim reduced IOP by 18.75 ± 2.22% compared to vehicle treated contralateral eyes (17.01 ± 0.32 mmHg at 0 hours to 13.82 ± 0.37 mmHg at 24 hours; n = 10, p = 0.002. Cromakalim demonstrated an additive effect when used in conjunction with latanoprost free acid, a common ocular hypotensive drug prescribed to patients with elevated IOP. To examine KATP channel subunit specificity, Kir6.2(-/- mice were treated with cromakalim, but unlike wild-type animals, no change in IOP was noted. Histologic analysis of treated and control eyes in cultured human anterior segments and in mice showed similar cell numbers and extracellular matrix integrity within the trabecular meshwork, with no disruptions in the inner and outer walls of Schlemm's canal. Together, these studies suggest that cromakalim is a potent ocular hypotensive agent that lowers IOP via activation of Kir6.2 containing KATP channels, its effect is additive when used in combination with the commonly used glaucoma drug latanoprost, and is not toxic to cells and tissues of the aqueous humor outflow pathway, making it a candidate for future therapeutic development.

  3. Pomegranate (Punica granatum) peel is effective in a murine model of experimental Cryptosporidium parvum ultrastructural studies of the ileum.

    Al-Mathal, Ebtisam M; Alsalem, Afaf A


    The current treatments for cryptosporidiosis are ineffective, and there is an urgent need to search for more effective and safer alternatives. One such alternative may be treatments derived from natural resources. The pomegranate peel has been used effectively in traditional medicine to cure diarrhea and dysentery. The purpose of this study was to examine the effectiveness of a Punica granatum (pomegranate) peel suspension as a treatment for Cryptosporidium parvum infection. In this study, the effects of this treatment on the ultrastructure of both the intestinal epithelial layer of infected nursling mice and the parasite were observed with a transmission electron microscope. The histological study focused on the examination of the microvilli, columnar epithelium, goblet cells, lamina propria, and crypts of Lieberkuhn. Examination of the ileums of infected mice that received the pomegranate peel suspension demonstrated that the general structure of the ileal tissue of these mice was similar to that of the control group. In the infected mice treated with the suspension, but not the infected/untreated mice, there was an improvement in all ultrastructure aspects at 28days post-inoculation. The study of the ultrastructure of the parasite (C. parvum) in mice treated with the suspension showed that there was decomposition in the parasite to the extent that in some cases we were unable to identify the stage of the parasite due to the severe degeneration. Significant decomposition of the nutrition organ was also observed. Additionally, microgamonte and macrogamonte were not observed in the suspension-treated group, explaining the disappearance of the sexual phases of the parasite in the lumens of this group. In all, this examination demonstrated the restoration of the normal structures of villi and the disappearance of acute symptoms in the suspension-treated mice and showed that the suspension directly affected the parasite at various stages of its development and led to its decomposition and death.

  4. The effect of vitamin A supplementation and diphtheria-tetanus-pertussis vaccination on parasitaemia in an experimental murine malaria model

    Jørgensen, Mathias Jul; Hein-Kristensen, Line; Hempel, Casper;


    Abstract Background: Vitamin A supplementation (VAS) decreases overall child mortality in low-income countries. For logistical reasons, VAS has been linked to routine childhood immunizations. However, several recent studies have indicated that VAS may increase mortality and morbidity from infecti...... influence the outcome of malaria infection in mice, adding to the concerns about simultaneous VAS and DTP administration to children in low-income, malaria endemic countries.......Abstract Background: Vitamin A supplementation (VAS) decreases overall child mortality in low-income countries. For logistical reasons, VAS has been linked to routine childhood immunizations. However, several recent studies have indicated that VAS may increase mortality and morbidity from...... infectious diseases when given with the diphtheria-tetanus-pertussis (DTP) vaccine. The immunological effects of combining the 2 treatments are unknown. Methods: We studied the effect of treating C57BL/6 mice with VAS and DTP, 1 week prior to infection with Plasmodium berghei ANKA. The progression of disease...

  5. Combined anti-angiogenic therapy targeting PDGF and VEGF receptors lowers the interstitial fluid pressure in a murine experimental carcinoma.

    Agnieszka Kłosowska-Wardega

    Full Text Available Elevation of the interstitial fluid pressure (IFP of carcinoma is an obstacle in treatment of tumors by chemotherapy and correlates with poor drug uptake. Previous studies have shown that treatment with inhibitors of platelet-derived growth factor (PDGF or vascular endothelial growth factor (VEGF signaling lowers the IFP of tumors and improve chemotherapy. In this study, we investigated whether the combination of PDGFR and VEGFR inhibitors could further reduce the IFP of KAT-4 human carcinoma tumors. The tumor IFP was measured using the wick-in-needle technique. The combination of STI571 and PTK/ZK gave an additive effect on the lowering of the IFP of KAT-4 tumors, but the timing of the treatment was crucial. The lowering of IFP following combination therapy was accompanied by vascular remodeling and decreased vascular leakiness. The effects of the inhibitors on the therapeutic efficiency of Taxol were investigated. Whereas the anti-PDGF and anti-VEGF treatment did not significantly inhibit tumor growth, the inhibitors enhanced the effect of chemotherapy. Despite having an additive effect in decreasing tumor IFP, the combination therapy did not further enhance the effect of chemotherapy. Simultaneous targeting of VEGFR and PDGFR kinase activity may be a useful strategy to decrease tumor IFP, but the timing of the inhibitors should be carefully determined.

  6. [Capacity of triterpene glycosides from holothurians to stimulate antibacterial resistance in a model of experimental murine salmonellosis].

    Sedov, A M; Elkina, S I; Sergeev, V V; Kalina, N G; Sakandelidze, O G


    When injected intraperitoneally into mice in doses of 40.0-0.4 microgram, Cucumarioside, the preparation of triterpene glycosides obtained from sea cucumbers (Cusumaria japonica), enhanced the resistance of the animals to the subsequent challenge with Salmonella typhimurium. The study of the duration of the persistence of salmonellae in mice receiving the preparation in a dose of 0.001 microgram revealed a decrease in the contamination of their organs. The same dose of the preparation stimulated the phagocytic activity of peritoneal exudate cells with salmonellae showing decreased cytopathogenic action. This suggests that Cucumarioside enhances nonspecific protective factors, activates the macrophagal system and facilitates the development of complete phagocytosis.

  7. Effects of NSAIDs on Differentiation and Function of Human and Murine Osteoclasts – Crucial ‘Human Osteoclastology’

    Shigeru Kotake


    Full Text Available Osteoclasts play a critical role in both normal bone metabolism and bone resorption in the joints of patients with rheumatoid arthritis. It has been reported that non-steroidal anti-inflammatory drugs (NSAIDs inhibit murine osteoclastogenesis in vitro and murine arthritis models in vivo, but not the destruction of joints of patients with rheumatoid arthritis. In the current review article, we review the recent findings in the effect of NSAIDs on the formation and function of human and murine osteoclasts both in vitro and in vivo, underlining the importance of studies using human osteoclasts. Since 2009, we have suggested a novel term ‘human osteoclastology’.

  8. A GM-CSF and CD40L bystander vaccine is effective in a murine breast cancer model

    Soliman H


    Full Text Available Hatem Soliman,1 Melanie Mediavilla-Varela,2 Scott J Antonia,3 1Department of Women's Oncology and Experimental Therapeutics, 2Department of Immunology, 3Department of Thoracic Oncology, Moffitt Cancer Center, Tampa, FL, USA Background: There is increasing interest in using cancer vaccines to treat breast cancer patients in the adjuvant setting to prevent recurrence in high risk situations or in combination with other immunomodulators in the advanced setting. Current peptide vaccines are limited by immunologic compatibility issues, and engineered autologous cellular vaccines are difficult to produce on a large scale. Using standardized bystander cell lines modified to secrete immune stimulating adjuvant substances can greatly enhance the ability to produce immunogenic cellular vaccines using unmodified autologous cells or allogeneic medical grade tumor cell lines as targets. We investigated the efficacy of a cellular vaccine using B78H1 bystander cell lines engineered to secrete granulocyte macrophage-colony stimulating factor and CD40 ligand (BCG in a murine model of breast cancer. Methods: Five-week-old female BALB/c mice were injected orthotopically in the mammary fat pad with 4T1 tumor cells. Treatment consisted of irradiated 4T1 ± BCG cells given subcutaneously every 4 days and was repeated three times per mouse when tumors became palpable. Tumors were measured two to three times per week for 25 days. The vaccine's activity was confirmed in a second experiment using Lewis lung carcinoma (LLC cells in C57BL/6 mice to exclude a model specific effect. Interferon-γ (IFN-γ and interleukin-2 (IL-2 enzyme-linked immunospots (ELISPOTS were performed on splenic lymphocytes incubated with 4T1 lysates along with immunohistochemistry for CD3 on tumor sections. Results: Tumor growth was significantly inhibited in the 4T1-BCG and LLC-BCG treatment groups when compared to 4T1 and LLC treatment groups. There were higher levels of IL-2 and IFN

  9. Oral Administration of Polymyxin B Modulates the Activity of Lipooligosaccharide E. coli B against Lung Metastases in Murine Tumor Models.

    Jagoda Kicielińska

    Full Text Available Polymyxin B (PmB belongs to the group of cyclic peptide antibiotics, which neutralize the activity of LPS by binding to lipid A. The aim of this study was to analyze the effect of PmB on the biological activity of lipooligosaccharide (LOS E. coli B,rough form of LPS in vitro and in experimental metastasis models.Cultures of murine macrophage J774A.1 cells and murine bone marrow-derived dendritic cells (BM-DC stimulated in vitro with LOS and supplemented with PmB demonstrated a decrease in inflammatory cytokine production (IL-6, IL-10, TNF-α and down-regulation of CD40, CD80, CD86 and MHC class II molecule expression. Additionally, PmB suspended in drinking water was given to the C57BL/6 mice seven or five days prior to the intravenous injection of B16 or LLC cells and intraperitoneal application of LOS. This strategy of PmB administration was continued throughout the duration of the experiments (29 or 21 days. In B16 model, statistically significant decrease in the number of metastases in mice treated with PmB and LOS (p<0.01 was found on the 14th day of the experiments, whereas the most intensive changes in surface-antigen expression and ex vivo production of IL-6, IL-1β and TNF-α by peritoneal cells were observed 7 days earlier. By contrast, antigen expression and ex vivo production of IL-6, IL-10, IFN-γ by splenocytes remained relatively high and stable. Statistically significant decrease in LLC metastases number was observed after the application of LOS (p<0.01 and in the group of mice preconditioned by PmB and subsequently treated with LOS (LOS + PmB, p<0.01.In conclusion, prolonged in vivo application of PmB was not able to neutralize the LOS-induced immune cell activity but its presence in the organism of treated mice was important in modulation of the LOS-mediated response against the development of metastases.

  10. Anti-bacterial effects of enzymatically-isolated sialic acid from glycomacropeptide in a Helicobacter pylori-infected murine model

    Noh, Hye-Ji; Koh, Hong Bum; Kim, Hee-Kyoung; Cho, Hyang Hyun


    BACKGROUND/OBJECTIVES Helicobacter pylori (H. pylori) colonization of the stomach mucosa and duodenum is the major cause of acute and chronic gastroduodenal pathology in humans. Efforts to find effective anti-bacterial strategies against H. pylori for the non-antibiotic control of H. pylori infection are urgently required. In this study, we used whey to prepare glycomacropeptide (GMP), from which sialic acid (G-SA) was enzymatically isolated. We investigated the anti-bacterial effects of G-SA against H. pylori in vitro and in an H. pylori-infected murine model. MATERIALS/METHODS The anti-bacterial activity of G-SA was measured in vitro using the macrodilution method, and interleukin-8 (IL-8) production was measured in H. pylori and AGS cell co-cultures by ELISA. For in vivo study, G-SA 5 g/kg body weight (bw)/day and H. pylori were administered to mice three times over one week. After one week, G-SA 5 g/kg bw/day alone was administered every day for one week. Tumor necrosis factor-α (TNF-α), IL-1β, IL-6, and IL-10 levels were measured by ELISA to determine the anti-inflammatory effects of G-SA. In addition, real-time PCR was performed to measure the genetic expression of cytotoxin-associated gene A (cagA). RESULTS G-SA inhibited the growth of H. pylori and suppressed IL-8 production in H. pylori and in AGS cell co-cultures in vitro. In the in vivo assay, administration of G-SA reduced levels of IL-1β and IL-6 pro-inflammatory cytokines whereas IL-10 level increased. Also, G-SA suppressed the expression of cagA in the stomach of H. pylori-infected mice. CONCLUSION G-SA possesses anti-H. pylori activity as well as an anti-H. pylori-induced gastric inflammatory effect in an experimental H. pylori-infected murine model. G-SA has potential as an alternative to antibiotics for the prevention of H. pylori infection and H. pylori-induced gastric disease prevention. PMID:28194260

  11. Murine Models for Trypanosoma brucei gambiense disease progression--from silent to chronic infections and early brain tropism.

    Christiane Giroud

    Full Text Available Human African trypanosomiasis (HAT caused by Trypanosoma brucei gambiense remains highly prevalent in west and central Africa and is lethal if left untreated. The major problem is that the disease often evolves toward chronic or asymptomatic forms with low and fluctuating parasitaemia producing apparently aparasitaemic serological suspects who remain untreated because of the toxicity of the chemotherapy. Whether the different types of infections are due to host or parasite factors has been difficult to address, since T. b. gambiense isolated from patients is often not infectious in rodents thus limiting the variety of isolates.T. b. gambiense parasites were outgrown directly from the cerebrospinal fluid of infected patients by in vitro culture and analyzed for their molecular polymorphisms. Experimental murine infections showed that these isolates could be clustered into three groups with different characteristics regarding their in vivo infection properties, immune response and capacity for brain invasion. The first isolate induced a classical chronic infection with a fluctuating blood parasitaemia, an invasion of the central nervous system (CNS, a trypanosome specific-antibody response and death of the animals within 6-8 months. The second group induced a sub-chronic infection resulting in a single wave of parasitaemia after infection, followed by a low parasitaemia with no parasites detected by microscope observations of blood but detected by PCR, and the presence of a specific antibody response. The third isolate induced a silent infection characterised by the absence of microscopically detectable parasites throughout, but infection was detectable by PCR during the whole course of infection. Additionally, specific antibodies were barely detectable when mice were infected with a low number of this group of parasites. In both sub-chronic and chronic infections, most of the mice survived more than one year without major clinical symptoms

  12. The Relationship between the Antitumor Effect of the IL-12 Gene Therapy and the Expression of Th1 Cytokines in an HPV16-Positive Murine Tumor Model

    Flor García Paz


    Full Text Available Objective. The goal of the present study was to investigate the effect of IL-12 expressed in plasmid on the Th1 cytokine profile in an experimental HPV16-positive murine tumor model and the association with the IL-12’s antitumor effect. Methods. Mice were injected with BMK-16/myc cells to establish HPV16-positive tumor and then pNGVL3-mIL-12 plasmid; pcDNA3 plasmid or PBS was injected directly into tumor site. The antitumor effect of the treatment was evaluated and the cytokines expression profile in each tumor tissue was analyzed. Results. Treatment with pNGVL3-mIL-12 plasmid had a significant antitumor effect, and a Th2-Th3-type cytokines prolife was detected in the murine tumor model with expression of the cytokines IL-10, IL-4, and TGF-β1. However, after the tumor was treated with three intratumoral injections of plasmid containing IL-12 cDNA, it showed a cytokine profile associated with Th1 with expression of IL-2, IL-12, and IFN-γ cytokines and reduced expression of IL-10, IL-4, and TGF-β1. Conclusions. The treatment with the IL-12 gene in the experimental HPV16-positive tumor model promoted the activation of the cellular immune response via expression of a Th1-type cytokine profile and was associated with the inhibition of tumor growth. Thus, IL-12 treatment represents a novel approach for gene therapy against cervical cancer.

  13. Structural and functional characterization of human and murine C5a anaphylatoxins

    Schatz-Jakobsen, Janus Asbjørn; Yatime, Laure, E-mail:; Larsen, Casper [Aarhus University, Gustav Wieds Vej 10C, DK-8000 Aarhus (Denmark); Petersen, Steen Vang [Aarhus University, Bartholin Building, Wilhelm Meyers Allé 4, DK-8000 Aarhus (Denmark); Klos, Andreas [Medical School Hannover, Hannover (Germany); Andersen, Gregers Rom, E-mail: [Aarhus University, Gustav Wieds Vej 10C, DK-8000 Aarhus (Denmark)


    The structure of the human C5aR antagonist, C5a-A8, reveals a three-helix bundle conformation similar to that observed for human C5a-desArg, whereas murine C5a and C5a-desArg both form the canonical four-helix bundle. These conformational differences are discussed in light of the differential C5aR activation properties observed for the human and murine complement anaphylatoxins across species. Complement is an ancient part of the innate immune system that plays a pivotal role in protection against invading pathogens and helps to clear apoptotic and necrotic cells. Upon complement activation, a cascade of proteolytic events generates the complement effectors, including the anaphylatoxins C3a and C5a. Signalling through their cognate G-protein coupled receptors, C3aR and C5aR, leads to a wide range of biological events promoting inflammation at the site of complement activation. The function of anaphylatoxins is regulated by circulating carboxypeptidases that remove their C-terminal arginine residue, yielding C3a-desArg and C5a-desArg. Whereas human C3a and C3a-desArg adopt a canonical four-helix bundle fold, the conformation of human C5a-desArg has recently been described as a three-helix bundle. Here, the crystal structures of an antagonist version of human C5a, A8{sup Δ71–73}, and of murine C5a and C5a-desArg are reported. Whereas A8{sup Δ71–73} adopts a three-helix bundle conformation similar to human C5a-desArg, the two murine proteins form a four-helix bundle. A cell-based functional assay reveals that murine C5a-desArg, in contrast to its human counterpart, exerts the same level of activition as murine C5a on its cognate receptor. The role of the different C5a conformations is discussed in relation to the differential activation of C5a receptors across species.

  14. Efficacy of paracetamol on patent ductus arteriosus closure may be dose dependent: evidence from human and murine studies.

    El-Khuffash, Afif


    We evaluated the clinical effectiveness of variable courses of paracetamol on patent ductus arteriosus (PDA) closure and examined its effect on the in vitro term and preterm murine ductus arteriosus (DA).

  15. Multi-frequency, multi-technique pulsed EPR investigation of the copper binding site of murine amyloid β peptide.

    Kim, Donghun; Bang, Jeong Kyu; Kim, Sun Hee


    Copper-amyloid peptides are proposed to be the cause of Alzheimer's disease, presumably by oxidative stress. However, mice do not produce amyloid plaques and thus do not suffer from Alzheimer's disease. Although much effort has been focused on the structural characterization of the copper- human amyloid peptides, little is known regarding the copper-binding mode in murine amyloid peptides. Thus, we investigated the structure of copper-murine amyloid peptides through multi-frequency, multi-technique pulsed EPR spectroscopy in conjunction with specific isotope labeling. Based on our pulsed EPR results, we found that Ala2, Glu3, His6, and His14 are directly coordinated with the copper ion in murine amyloid β peptides at pH 8.5. This is the first detailed structural characterization of the copper-binding mode in murine amyloid β peptides. This work may advance the knowledge required for developing inhibitors of Alzheimer's disease.

  16. Expression of Prothrombinase/fibroleukin Gene fg12 in Lung Impairment in a Murine Severe Acute Respiratory Syndrome Model

    Wei-ming YAN; Jia-quan HUANG; Xiao-ping LUO; Qin NING


    To evaluate the role of murine fibrinogen like protein 2 (mfgl2) /fibroleukin in lung impairment in Severe acute respiratory syndrome (SARS), a murine SARS model induced by Murine hepatitis virus strain 3 (MHV-3) through trachea was established. Impressively, all the animals developed interstitial pneumonia with extensive hyaline membranes formation within alveoli, and presence of micro-vascular thrombosis in the pulmonary vessels. MHV-3 nucleocapsid gene transcripts were identified in multiple organs including lungs, spleen etc. As a representative proinflammatory gene, mfgl2 prothrombinase expression was evident in terminal and respiratory bronchioles, alveolar epithelia and infiltrated cells in the lungs associated with fibrin deposition and micro-vascular thrombosis. In summary, the established murine SARS model could mimic the pathologic characteristics of lungs in patients with SARS. Besides the physical damages due to virus replication in organs, the up-regulation of novel gene mfgl2 in lungs may play a vital role in the development of SARS associated lung damage.

  17. In vitro Staphylococcus aureus–induced oxidative stress in mice murine peritoneal macrophages: a duration–dependent approach

    Subhankari Prasad Chakraborty


    Conclusions: From this study, it may be summarized that in vitro VSSA infection not only generates excess free radical but also affects the antioxidant status and glutathione cycle in murine peritoneal macrophages.

  18. Intrapulmonary posaconazole penetration at the infection site in an immunosuppressed murine model of invasive pulmonary aspergillosis receiving oral prophylactic regimens

    Seyedmousavi Tasieh, S.; Bruggemann, R.J.M.; Melchers, W.J.G.; Verweij, P.E.; Mouton, J.W.


    Adequate penetration to the infection/colonization site is crucial to attain optimal efficacy of posaconazole against Aspergillus fumigatus diseases. We evaluated posaconazole exposure in pulmonary epithelial lining fluid (ELF) in a murine model of invasive pulmonary aspergillosis. The posaconazole

  19. Towards a Taenia solium Cysticercosis Vaccine: an Epitope Shared by Taenia crassiceps and Taenia solium Protects Mice against Experimental Cysticercosis

    Toledo, Andrea; Larralde, Carlos; Fragoso, Gladis; Gevorkian, Goar; Manoutcharian, Karen; Hernández, Marisela; Acero, Gonzalo; Rosas, Gabriela; López-Casillas, Fernando; Garfias, Carlos Kubli; Vázquez, Ricardo; Terrazas, Ignacio; Sciutto, Edda


    The Taenia crassiceps recombinant antigen KETc7 has been shown to be effective as a vaccine against experimental murine cysticercosis, a laboratory model used to test potentially promising molecules against porcine Taenia solium cysticercosis. Based on the deduced amino acid sequence of this proline-rich polypeptide, three fragments, GK-1, GK-2, and GK-3, were chemically synthesized in linear form. Of the three peptides, only GK-1 induced sterile protection against T. crassiceps cysticercosis...

  20. Probiotic yeasts: Anti-inflammatory potential of various non-pathogenic strains in experimental colitis in mice

    Benot; Foligné; Jo■lle; Dewulf; Pascal; Vandekerckove; Georges; Pignède; Bruno; Pot


    AIM: To evaluate the in vitro immunomodulation capacity of various non-pathogenic yeast strains and to investigate the ability of some of these food grade yeasts to prevent experimental colitis in mice.METHODS: In vitro immunomodulation was assessed by measuring cytokines [interleukin (IL)-12p70,IL-10,tumor necrosis factor and interferon γ] released by human peripheral blood mononuclear cells after 24 h stimulation with 6 live yeast strains (Saccharomyces ssp.) and with bacterial reference strains.A murine ...

  1. The nonpsychoactive cannabis constituent cannabidiol is an oral anti-arthritic therapeutic in murine collagen-induced arthritis

    Malfait, A. M.; Gallily, R; Sumariwalla, P. F.; Malik, A. S.; Andreakos, E; Mechoulam, R.; Feldmann, M


    The therapeutic potential of cannabidiol (CBD), the major nonpsychoactive component of cannabis, was explored in murine collagen-induced arthritis (CIA). CIA was elicited by immunizing DBA/1 mice with type II collagen (CII) in complete Freund's adjuvant. The CII used was either bovine or murine, resulting in classical acute CIA or in chronic relapsing CIA, respectively. CBD was administered after onset of clinical symptoms, and in both models of arthritis the treatment effectively blocked pro...

  2. Effects of silk fibroin in murine dry eye

    Kim, Chae Eun; Lee, Ji Hyun; Yeon, Yeung Kyu; Park, Chan Hum; Yang, Jaewook


    The study aimed to investigate the effects of silk fibroin in a mouse model of dry eye. The experimental dry eye mouse model was developed using more than twelve-weeks-old NOD.B10.H2b mice exposing them to 30–40% ambient humidity and injecting them with scopolamine hydrobromide for 10 days. Tear production and corneal irregularity score were measured by the instillation of phosphate buffered saline or silk fibroin. Corneal detachment and conjunctival goblet cell density were observed by hematoxylin and eosin or periodic acid Schiff staining in the cornea or conjunctiva. The expression of inflammatory markers was detected by immunohistochemistry in the lacrimal gland. The silk group tear production was increased, and corneal smoothness was improved. The corneal epithelial cells and conjunctival goblet cells were recovered in the silk groups. The expression of inflammatory factors was inhibited in the lacrimal gland of the silk group. These results show that silk fibroin improved the cornea, conjunctiva, and lacrimal gland in the mouse model of dry eye. These findings suggest that silk fibroin has anti-inflammatory effects in the experimental models of dry eye.

  3. The Effects of Targeted Deletion of Cannabinoid Receptors CB1 and CB2 on Intranasal Sensitization and Challenge with Adjuvant-Free Ovalbumin

    Kaplan, Barbara L. F.; Oberdick, Jody E.; Karmaus, Peer W. F.; Ngaotepprutaram, Thitirat; Birmingham, Neil P.; Harkema, Jack R.; Kaminski, Norbert E.


    The mechanisms by which cannabinoid receptors CB1 and CB2 modulate immune function are not fully elucidated. Critical tools for the determination of the role of both receptors in the immune system are CB1/CB2 double null mice (CB1/CB2 null), and previous studies have shown that CB1/CB2 null mice exhibit exaggerated responses to various immunological stimuli. The objective of these studies was to determine the magnitude to which CB1/CB2 null mice responded to the respiratory allergen ovalbumin...

  4. The effects of targeted deletion of cannabinoid receptors CB1 and CB2 on intranasal sensitization and challenge with adjuvant-free ovalbumin.

    Kaplan, Barbara L F; Lawver, Jody E; Karmaus, Peer W F; Ngaotepprutaram, Thitirat; Birmingham, Neil P; Harkema, Jack R; Kaminski, Norbert E


    The mechanisms by which cannabinoid receptors CB(1) and CB(2) modulate immune function are not fully elucidated. Critical tools for the determination of the role of both receptors in the immune system are CB(1)/CB(2) double null mice (CB(1)/CB(2) null), and previous studies have shown that CB(1)/CB(2) null mice exhibit exaggerated responses to various immunological stimuli. The objective of these studies was to determine the magnitude to which CB(1)/CB(2) null mice responded to the respiratory allergen ovalbumin (OVA) as compared with wild-type C57BL/6 mice. The authors determined that in the absence of adjuvant, both wild-type and CB(1)/CB(2) null mice mounted a marked response to intranasally instilled OVA as assessed by inflammatory cell infiltrate in the bronchoalveolar lavage fluid (BALF), eosinophilia, induction of mucous cell metaplasia, and IgE production. Many of the endpoints measured in response to OVA were similar in wild-type versus CB(1)/CB(2) null mice, with exceptions being modest reductions in OVA-induced IgE and attenuation of BALF neutrophilia in CB(1)/CB(2) null mice as compared with wild-type mice. These results suggest that T-cell responses are not universally exaggerated in CB(1)/CB(2) null mice.

  5. 1.8 Astroms Structure of Murine GITR Ligand Dimer Expressed in Drosophila Melanogaster S2 Cells

    Chattopadhyay, K.; Ramagopal, U; Nathenson, S; Almo, S


    Glucocorticoid-induced TNF receptor ligand (GITRL), a prominent member of the TNF superfamily, activates its receptor on both effector and regulatory T cells to generate critical costimulatory signals that have been implicated in a wide range of T-cell immune functions. The crystal structures of murine and human orthologs of GITRL recombinantly expressed in Escherichia coli have previously been determined. In contrast to all classical TNF structures, including the human GITRL structure, murine GITRL demonstrated a unique 'strand-exchanged' dimeric organization. Such a novel assembly behavior indicated a dramatic impact on receptor activation as well as on the signaling mechanism associated with the murine GITRL costimulatory system. In this present work, the 1.8 {angstrom} resolution crystal structure of murine GITRL expressed in Drosophila melanogaster S2 cells is reported. The eukaryotic protein-expression system allows transport of the recombinant protein into the extracellular culture medium, thus maximizing the possibility of obtaining correctly folded material devoid of any folding/assembly artifacts that are often suspected with E. coli-expressed proteins. The S2 cell-expressed murine GITRL adopts an identical 'strand-exchanged' dimeric structure to that observed for the E. coli-expressed protein, thus conclusively demonstrating the novel quaternary structure assembly behavior of murine GITRL.

  6. Mutated primer binding sites interacting with different tRNAs allow efficient murine leukemia virus replication

    Lund, Anders Henrik; Duch, M; Lovmand, J;


    can replicate by using various tRNA molecules as primers and propose primer binding site-tRNA primer interactions to be of major importance for tRNA primer selection. However, efficient primer selection does not require perfect Watson-Crick base pairing at all 18 positions of the primer binding site.......(Pro). Polymerase chain reaction amplification and sequence analysis of transduced proviruses confirmed the transfer of vectors with mutated primer binding sites and further showed that tRNA(Gln-2) may act efficiently in conjunction with the tRNA(Gln-1) primer binding site. We conclude that murine leukemia virus......Two Akv murine leukemia virus-based retroviral vectors with primer binding sites matching tRNA(Gln-1) and tRNA(Lys-3) were constructed. The transduction efficiency of these mutated vectors was found to be comparable to that of a vector carrying the wild-type primer binding site matching tRNA...

  7. Sensitivity to. gamma. rays of avian sarcoma and murine leukemia viruses. [/sup 60/Co, uv

    Toyoshima, K. (Osaka Univ., Japan); Niwa, O.; Yutsudo, M.; Sugiyama, H.; Tahara, S.; Sugahara, T.


    The direct inactivation of avian and murine oncoviruses by ..gamma.. rays was examined using /sup 60/Co as a ..gamma..-ray source. The inactivation of murine leukemia virus (M-MuLV) followed single-hit kinetics while the subgroup D Schmidt-Ruppin strain of avian sarcoma virus (SR-RSV D) showed multihit inactivation kinetics with an extrapolation number of 5. The two viruses showed similar uv-inactivation kinetics. The genomic RNA of the SR-RSV D strain was degraded by ..gamma.. irradiation faster than its infectivity, but viral clones isolated from the foci formed after ..gamma.. irradiation had a complete genome. These results suggest that SR-RSV D has a strong repair function, possibly connected with reverse transcriptase activity.

  8. Size-Dependent Accumulation of PEGylated Silane-Coated Magnetic Iron Oxide Nanoparticles in Murine Tumors

    Larsen, Esben Kjær Unmack; Nielsen, T.; Wittenborn, T.


    Magnetic nanoparticles (MNP) can be used as contrast-enhancing agents to visualize tumors by magnetic resonance imaging (MRI). Here we describe an easy synthesis method of magnetic nanoparticles coated with polyethylene glycol (PEG) and demonstrate size-dependent accumulation in murine tumors...... following intravenous injection. Biocompatible iron oxide MNPs coated with PEG were prepared by replacing oleic acid with a biocompatible and commercially available silane-PEG to provide an easy and effective method for chemical coating. The colloidal stable PEGylated MNPs were magnetically separated...... into two distinct size subpopulations of 20 and 40 nm mean diameters with increased phagocytic uptake observed for the 40 nm size range in vitro. MRI detection revealed greater iron accumulation in murine tumors for 40 nm nanoparticles after intravenous injection. The enhanced MRI contrast of the larger...

  9. Evidence for a precursor of the high-affinity metastasis-associated murine laminin receptor

    Rao, C N; Castronovo, V; Schmitt, M C;


    The high-affinity cellular receptor for the basement membrane component laminin is differentially expressed during tumor invasion and metastasis. A cDNA clone encoding the murine laminin receptor was isolated and identified on the basis of sequence homology to the human laminin receptor [Wewer et...... al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 7137-7141]. Primer extension experiments demonstrated that the clone contained the complete 5' sequence of the murine laminin receptor mRNA. RNA blot data demonstrated a single-sized laminin receptor mRNA, approximately 1400 bases long, in human, mouse......, and rat. The nascent laminin receptor predicted from the cDNA sequence is 295 amino acids long, with a molecular weight of 33,000, and contains one intradisulfide bridge, a short putative transmembrane domain, and an extracellular carboxy-terminal region which has abundant glutamic acid residues...

  10. Hepatic Differentiation from Murine and Human iPS Cells Using Nanofiber Scaffolds.

    Yamazoe, Taiji; Shiraki, Nobuaki; Kume, Shoen


    The induced pluripotent stem (iPS) cells of murine and human are capable to differentiate into any cell type of the body through recapitulating normal development, similarly as the embryonic stem (ES) cells. Lines of evidence support that both ES cells and iPS cells are induced to differentiate in vitro by sequential treatment of humoral cues such as growth factors and chemicals, combined with the use of certain microenvironments including extracellular matrices and scaffolds.Here, we describe the procedure to potentiate hepatic lineage cells differentiation from murine and human iPS cells, using growth factor cocktails and nanofiber scaffolds. Nanofiber scaffolds have a three-dimensional surface mimicking the fine structures of the basement membrane in vivo, allow the iPS cells to differentiate into the definitive endoderm and mature hepatocyte-like cells more efficiently than the two-dimensional conventional culture plates.

  11. Murine inner cell mass-derived lineages depend on Sall4 function

    Elling, Ulrich; Klasen, Christian; Eisenberger, Tobias; Anlag, Katrin; Treier, Mathias


    Sall4 is a mammalian Spalt transcription factor expressed by cells of the early embryo and germ cells, an expression pattern similar to that of both Oct4 and Sox2, which play essential roles during early murine development. We show that the activity of Sall4 is cell-autonomously required for the development of the epiblast and primitive endoderm from the inner cell mass. Furthermore, no embryonic or extraembryonic endoderm stem cell lines could be established from Sall4-deficient blastocysts. In contrast, neither the development of the trophoblast lineage nor the ability to generate trophoblast cell lines from murine blastocysts was impaired in the absence of Sall4. These data establish Sall4 as an essential transcription factor required for the early development of inner cell mass-derived cell lineages. PMID:17060609

  12. Hepatic differentiation of embryonic stem cells by murine fetal liver mesenchymal cells.

    Ishii, Takamichi; Yasuchika, Kentaro; Ikai, Iwao


    Hepatocytes derived from embryonic stem cells (ESCs) are a potential cell source for regenerative medicine. However, it has been technically difficult to differentiate ESCs into mature hepatocytes because the definitive growth factors and molecular mechanisms governing hepatocyte differentiation have not yet been well defined. The CD45(-)CD49f(+/-)Thy1(+)gp38(+) mesenchymal cells that reside in murine fetal livers induce hepatic progenitor cells to differentiate into mature hepatocytes by direct cell-cell contact. Utilizing these cells, we employ a two-step procedure for hepatic maturation of ESCs: first, ESCs are differentiated into endodermal cells or hepatic progenitor cells, and second, ESC-derived endodermal cells are matured into functional hepatocytes by coculture with murine fetal liver mesenchymal cells. The ESC-derived hepatocyte-like cells possess hepatic functions, including ammonia removal activity, albumin secretion ability, glycogen synthesis and storage, and cytochrome P450 enzymatic activity.

  13. Diet and specific microbial exposure trigger features of environmental enteropathy in a novel murine model.

    Brown, Eric M; Wlodarska, Marta; Willing, Benjamin P; Vonaesch, Pascale; Han, Jun; Reynolds, Lisa A; Arrieta, Marie-Claire; Uhrig, Marco; Scholz, Roland; Partida, Oswaldo; Borchers, Christoph H; Sansonetti, Philippe J; Finlay, B Brett


    Environmental enteropathy (EE) is a subclinical chronic inflammatory disease of the small intestine and has a profound impact on the persistence of childhood malnutrition worldwide. However, the aetiology of the disease remains unknown and no animal model exists to date, the creation of which would aid in understanding this complex disease. Here we demonstrate that early-life consumption of a moderately malnourished diet, in combination with iterative oral exposure to commensal Bacteroidales species and Escherichia coli, remodels the murine small intestine to resemble features of EE observed in humans. We further report the profound changes that malnutrition imparts on the small intestinal microbiota, metabolite and intraepithelial lymphocyte composition, along with the susceptibility to enteric infection. Our findings provide evidence indicating that both diet and microbes combine to contribute to the aetiology of EE, and describe a novel murine model that can be used to elucidate the mechanisms behind this understudied disease.

  14. Differential regulation of follicle stimulating hormone by activin A and TGFB1 in murine gonadotropes

    Miller William L


    Full Text Available Abstract Background Activins stimulate the synthesis of follicle stimulating hormone (FSH in pituitary gonadotropes, at least in part, by inducing transcription of its beta subunit (Fshb. Evidence from several laboratories studying transformed murine LbetaT2 gonadotropes indicates that activins signal through Smad-dependent and/or Smad-independent pathways, similar to those used by transforming growth factor beta-1 (TGFB1 in other cell types. Therefore, given common intracellular signaling mechanisms of these two ligands, we examined whether TGFBs can also induce transcription of Fshb in LbetaT2 cells as well as in purified primary murine gonadotropes. Methods Murine Fshb promoter-reporter (-1990/+1 mFshb-luc activity was measured in LbetaT2 cells treated with activin A or TGFB1, and in cells transfected with either activin or TGFB receptors. The ability of the ligands to stimulate phosphorylation of Smads 2 and 3 in LbetaT2 cells was measured by western blot analysis, and expression of TGFB type I and II receptors was assessed by reverse transcriptase polymerase chain reaction in both LbetaT2 cells and primary gonadotropes purified from male mice of different ages. Finally, regulation of endogenous murine Fshb mRNA levels by activin A and TGFB1 in purified gonadotropes and whole pituitary cultures was measured using quantitative RT-PCR. Results Activin A dose-dependently stimulated -1990/+1 mFshb-luc activity in LbetaT2 cells, but TGFB1 had no effect at doses up to 5 nM. Similarly, activin A, but not TGFB1, stimulated Smad 2 and 3 phosphorylation in these cells. Constitutively active forms of the activin (Acvr1b-T206D and TGFB (TGFBR1-T204D type I receptors strongly stimulated -1990/+1 mFshb-luc activity, showing that mechanisms down stream of Tgfbr1 seem to be intact in LbetaT2 cells. RT-PCR analysis of LbetaT2 cells and whole adult murine pituitaries indicated that both expressed Tgfbr1 mRNA, but that Tgfbr2 was not detected in LbetaT2 cells

  15. Host Characteristics and Bacterial Traits Predict Experimental Virulence for Escherichia coli Bloodstream Isolates From Patients With Urosepsis.

    Johnson, James R; Porter, Stephen; Johnston, Brian; Kuskowski, Michael A; Spurbeck, Rachel R; Mobley, Harry L T; Williamson, Deborah A


    Background.  Extraintestinal Escherichia coli infections are common, costly, and potentially serious. A better understanding of their pathogenesis is needed. Methods.  Sixty-seven E coli bloodstream isolates from adults with urosepsis (Seattle, WA; 1980s) underwent extensive molecular characterization and virulence assessment in 2 infection models (murine subcutaneous sepsis and moth larval lethality). Statistical comparisons were made among host characteristics, bacterial traits, and experimental virulence. Results.  The 67 source patients were diverse for age, sex, and underlying medical and urological conditions. The corresponding E coli isolates exhibited diverse phylogenetic backgrounds and virulence profiles. Despite the E coli isolates' common bloodstream origin, they exhibited a broad range of experimental virulence in mice and moth larvae, in patterns that (for the murine model only) corresponded significantly with host characteristics and bacterial traits. The most highly mouse-lethal strains were enriched with classic "urovirulence" traits and typically were from younger women with anatomically and functionally normal urinary tracts. The 2 animal models corresponded poorly with one another. Conclusions.  Host compromise, including older age and urinary tract abnormalities, allows comparatively low-virulence E coli strains to cause urosepsis. Multiple E coli traits predict both experimental and epidemiological virulence. The larval lethality model cannot be a substitute for the murine sepsis model.

  16. Metallothionein-1 and nitric oxide expression are inversely correlated in a murine model of Chagas disease


    Chagas disease, caused by Trypanosoma cruzi, represents an endemic among Latin America countries. The participation of free radicals, especially nitric oxide (NO), has been demonstrated in the pathophysiology of seropositive individuals with T. cruzi. In Chagas disease, increased NO contributes to the development of cardiomyopathy and megacolon. Metallothioneins (MTs) are efficient free radicals scavengers of NO in vitro and in vivo. Here, we developed a murine model of the chronic phase of C...

  17. Efficacy of Orally Delivered Cochleates Containing Amphotericin B in a Murine Model of Aspergillosis

    Delmas, G.; Park, S.; Chen, Z W; Tan, F.; Kashiwazaki, R.; Zarif, L.; Perlin, D. S.


    Cochleates containing amphotericin B (CAMB) were administered orally at doses ranging from 0 to 40 mg/kg of body weight/day for 14 days in a murine model of systemic aspergillosis. The administration of oral doses of CAMB (20 and 40 mg/kg/day) resulted in a survival rate of 70% and a reduction in colony counts of more than 2 logs in lungs, livers, and kidneys. Orally administered CAMB shows promise for the treatment of aspergillosis.

  18. Efficacy of orally delivered cochleates containing amphotericin B in a murine model of aspergillosis.

    Delmas, G; Park, S; Chen, Z W; Tan, F; Kashiwazaki, R; Zarif, L; Perlin, D S


    Cochleates containing amphotericin B (CAMB) were administered orally at doses ranging from 0 to 40 mg/kg of body weight/day for 14 days in a murine model of systemic aspergillosis. The administration of oral doses of CAMB (20 and 40 mg/kg/day) resulted in a survival rate of 70% and a reduction in colony counts of more than 2 logs in lungs, livers, and kidneys. Orally administered CAMB shows promise for the treatment of aspergillosis.

  19. Does murine spermatogenesis require WNT signalling? A lesson from Gpr177 conditional knockout mouse models

    Chen, Su-Ren; Tang, J-X; Cheng, J-M; Hao, X-X; Wang, Y-Q; Wang, X-X; Liu, Y-X


    Wingless-related MMTV integration site (WNT) proteins and several other components of the WNT signalling pathway are expressed in the murine testes. However, mice mutant for WNT signalling effector β-catenin using different Cre drivers have phenotypes that are inconsistent with each other. The complexity and overlapping expression of WNT signalling cascades have prevented researchers from dissecting their function in spermatogenesis. Depletion of the Gpr177 gene (the mouse orthologue of Droso...

  20. Simvastatin Treatment Improves Survival in a Murine Model of Burn Sepsis


    Infection is the most common and most serious complication of a major burn injury related to burn size. Despite improvements in antimicrobial therapies sepsis still accounts for 50–60% of deaths in burn patients. Given the acute onset and unpredictable nature of sepsis, primary prevention was rarely attempted in its management. However, recent studies have demonstrated that statin treatment can decrease mortality is a murine model of sepsis by preservation of cardiac function and reversal of ...

  1. Alternans in genetically modified Langendorff-perfused murine hearts modeling catecholaminergic polymorphic ventricular tachycardia

    Ian N Sabir


    Full Text Available The relationship between alternans and arrhythmogenicity was studied in genetically modified murine hearts modeling catecholaminergic polymorphic ventricular tachycardia (CPVT during Langendorff perfusion, before and after treatment with catecholamines and a β-adrenergic antagonist. Heterozygous (RyR2p/s and homozygous (RyR2s/s RyR2-P2328S hearts, and wild-type (WT controls, were studied before and after treatment with epinephrine (100 nM and 1 µM and propranolol (100 nM. Monophasic action potential recordings demonstrated significantly greater incidences of arrhythmia in RyR2s/p and RyR2s/s hearts as compared to WTs. Arrhythmogenicity in RyR2s/s hearts was associated with alternans, particularly at short baseline cycle lengths. Both phenomena were significantly accentuated by treatment with epinephrine and significantly diminished by treatment with propranolol, in full agreement with clinical expectations. These changes took place, however, despite an absence of changes in action potential durations, ventricular effective refractory periods or restitution curve characteristics. Furthermore pooled data from all hearts in which arrhythmia occurred demonstrated significantly greater alternans magnitudes, but similar restitution curve slopes, to hearts that did not demonstrate arrhythmia. These findings thus further validate the RyR2-P2328S murine heart as a model for human CPVT, confirming an alternans phenotype in common with murine genetic models of the Brugada syndrome and the congenital long-QT syndrome type 3. In contrast to these latter similarities, however, this report demonstrates the dissociation of alternans from changes in the properties of restitution curves for the first time in a murine model of a human arrhythmic syndrome.

  2. Antitumour Effects of Isocurcumenol Isolated from Curcuma zedoaria Rhizomes on Human and Murine Cancer Cells

    S. Lakshmi; G. Padmaja; Remani, P.


    Curcuma zedoaria belonging to the family Zingiberaceae has been used in the traditional system of medicine in India and Southwest Asia in treating many human ailments and is found to possess many biological activities. The rationale of the present study was to isolate, identify, and characterize antitumour principles from the rhizomes of Curcuma zedoaria, to assess its cytotoxic effects on human and murine cancer cells, to determine its apoptosis inducing capacity in cancer cells, and to eval...

  3. Interstitial Fibroblast-Like Cells Express Renin-Angiotensin System Components in a Fibrosing Murine Kidney

    Okada, Hirokazu; Inoue, Tsutomu; Kanno, Yoshihiko; Kobayashi, Tatsuya; Watanabe, Yusuke; Kopp, Jeffrey B; Carey, Robert M.; SUZUKI, HIROMICHI


    Recently, the renin-angiotensin system (RAS) was implicated in organ fibrosis. However, few studies have examined the localization of RAS components, such as angiotensin II receptors, renin (REN), angiotensinogen (AGTN), and angiotensin-converting enzyme (ACE), in the fibrosing kidney. To localize these components in the fibrosing kidney, we used a murine model of renal fibrosis that shows an enhanced expression of angiotensin II type 1A receptor (AT1AR) and AGTN. Our results indicate that th...

  4. Murine adenovirus infection of SCID mice induces hepatic lesions that resemble human Reye syndrome.

    Pirofski, L.; Horwitz, M S; Scharff, M. D.; Factor, S. M.


    Murine adenovirus type 1 (MAV-1) infection of CB-17 SCID mice (which are homozygous for the severe combined immunodeficiency mutation) induces hepatic histopathologic and ultrastructural features that are strikingly similar to human Reye syndrome. Gross pathologic examination of MAV-1-infected mice revealed only pale yellow liver tissue. Histopathologic studies of tissue from MAV-1-infected mice revealed diffuse hepatic injury manifested by microvesicular fatty degenerative changes of hepatoc...

  5. Homocysteine enhances MMP-9 production in murine macrophages via ERK and Akt signaling pathways

    Lee, Seung Jin; Lee, Yi Sle; Seo, Kyo Won; Bae, Jin Ung; Kim, Gyu Hee; Park, So Youn; Kim, Chi Dae, E-mail:


    Homocysteine (Hcy) at elevated levels is an independent risk factor of cardiovascular diseases, including atherosclerosis. In the present study, we investigated the effect of Hcy on the production of matrix metalloproteinases (MMP) in murine macrophages. Among the MMP known to regulate the activities of collagenase and gelatinase, Hcy exclusively increased the gelatinolytic activity of MMP-9 in J774A.1 cells as well as in mouse peritoneal macrophages. Furthermore, this activity was found to be correlated with Western blot findings in J774A.1 cells, which showed that MMP-9 expression was concentration- and time-dependently increased by Hcy. Inhibition of the ERK and Akt pathways led to a significant decrease in Hcy-induced MMP-9 expression, and combined treatment with inhibitors of the ERK and Akt pathways showed an additive effects. Activity assays for ERK and Akt showed that Hcy increased the phosphorylation of both, but these phosphorylation were not affected by inhibitors of the Akt and ERK pathways. In line with these findings, the molecular inhibition of ERK and Akt using siRNA did not affect the Hcy-induced phosphorylation of Akt and ERK, respectively. Taken together, these findings suggest that Hcy enhances MMP-9 production in murine macrophages by separately activating the ERK and Akt signaling pathways. -- Highlights: ► Homocysteine (Hcy) induced MMP-9 production in murine macrophages. ► Hcy induced MMP-9 production through ERK and Akt signaling pathways. ► ERK and Akt signaling pathways were activated by Hcy in murine macrophages. ► ERK and Akt pathways were additively act on Hcy-induced MMP-9 production. ► Hcy enhances MMP-9 production in macrophages via activation of ERK and Akt signaling pathways in an independent manner.

  6. Combination therapy of murine mucormycosis or aspergillosis with iron chelation, polyenes, and echinocandins.

    Ibrahim, Ashraf S; Gebremariam, Teclegiorgis; Luo, Guanpingsheng; Fu, Yue; French, Samuel W; Edwards, John E; Spellberg, Brad


    Liposomal amphotericin B (LAmB) combined wither either micafungin or deferasirox was synergistic in previous murine studies with mucormycosis or aspergillosis. We hypothesized that triple therapy using LAmB, micafungin, and deferasirox could further improve outcomes of mucormycosis or aspergillosis. Triple therapy improved survival and reduced tissue fungal burden of mice with mucormycosis and to a lesser extent with aspergillosis. Continued investigation into the use of triple therapy against mucormycosis and aspergillosis is warranted.

  7. Prefoldin 5 Is Required for Normal Sensory and Neuronal Development in a Murine Model*

    Lee, YongSuk; Smith, Richard S.; Jordan, Wanda; King, Benjamin L.; Won, Jungyeon; Valpuesta, Jose M.; Naggert, Jurgen K; Nishina, Patsy M.


    Molecular chaperones and co-chaperones are crucial for cellular development and maintenance as they assist in protein folding and stabilization of unfolded or misfolded proteins. Prefoldin (PFDN), a ubiquitously expressed heterohexameric co-chaperone, is necessary for proper folding of nascent proteins, in particular, tubulin and actin. Here we show that a genetic disruption in the murine Pfdn5 gene, a subunit of prefoldin, causes a syndrome characterized by photoreceptor degeneration, centra...

  8. High Throughput Quantitative Glycomics and Glycoform-focused Proteomics of Murine Dermis and Epidermis


    Despite recent advances in our understanding of the significance of the protein glycosylation, the throughput of protein glycosylation analysis is still too low to be applied to the exhaustive glycoproteomic analysis. Aiming to elucidate the N-glycosylation of murine epidermis and dermis glycoproteins, here we used a novel approach for focused proteomics. A gross N-glycan profiling (glycomics) of epidermis and dermis was first elucidated both qualitatively and quantitatively upon N-glycan der...

  9. Efficacy of Posaconazole in a Murine Model of Central Nervous System Aspergillosis

    Imai, Jackie K.; Singh, Gaurav; Clemons, Karl V.; Stevens, David A.


    Human central nervous system (CNS) aspergillosis has >90% mortality. We compared posaconazole with other antifungals for efficacy against murine CNS aspergillosis. All tested regimens of posaconazole were equivalent to those of amphotericin B and superior in prolonging survival and reducing CFU to those of itraconazole and caspofungin and to vehicle controls. No antifungal regimen effected cure. No toxicity was noted. Overall, posaconazole shows potential for treating CNS aspergillosis.

  10. Isolation and characterisation of Ebolavirus-specific recombinant antibody fragments from murine and shark immune libraries.

    Goodchild, Sarah A; Dooley, Helen; Schoepp, Randal J; Flajnik, Martin; Lonsdale, Stephen G


    Members of the genus Ebolavirus cause fulminating outbreaks of disease in human and non-human primate populations with a mortality rate up to 90%. To facilitate rapid detection of these pathogens in clinical and environmental samples, robust reagents capable of providing sensitive and specific detection are required. In this work recombinant antibody libraries were generated from murine (single chain variable domain fragment; scFv) and nurse shark, Ginglymostoma cirratum (IgNAR V) hosts immunised with Zaire ebolavirus. This provides the first recorded IgNAR V response against a particulate antigen in the nurse shark. Both murine scFv and shark IgNAR V libraries were panned by phage display technology to identify useful antibodies for the generation of immunological detection reagents. Two murine scFv were shown to have specificity to the Zaire ebolavirus viral matrix protein VP40. Two isolated IgNAR V were shown to bind to the viral nucleoprotein (NP) and to capture viable Zaire ebolavirus with a high degree of sensitivity. Assays developed with IgNAR V cross-reacted to Reston ebolavirus, Sudan ebolavirus and Bundibugyo ebolavirus. Despite this broad reactivity, neither of IgNAR V showed reactivity to Côte d'Ivoire ebolavirus. IgNAR V was substantially more resistant to irreversible thermal denaturation than murine scFv and monoclonal IgG in a comparative test. The demonstrable robustness of the IgNAR V domains may offer enhanced utility as immunological detection reagents in fieldable biosensor applications for use in tropical or subtropical countries where outbreaks of Ebolavirus haemorrhagic fever occur.

  11. Isolation of dual-tropic murine leukemia virus from radiation-induced thymoma in RF mouse

    Okumoto, M.; Nishikawa, R.; Takamori, Y.; Iwai, Y.; Iwai, M. (Radiation Center of Osaka Prefecture, Sakai (Japan))


    Two different murine leukemia viruses (MuLV) were isolated from radiation-induced thymoma in RF mouse by co-cultivation method with mink lung cells. One is ecotropic MuLV, which replicates efficiently in NIH Swiss mouse embryo (NIH ME) and SC-1 cells and was able to form XC plaques. The other was dualtropic MuLV, which replicates in both mink lung cells and NIH ME cells but was unable to form XC plaques.

  12. CD44 antibodies and immune thrombocytopenia in the amelioration of murine inflammatory arthritis.

    Patrick J Mott

    Full Text Available Antibodies to CD44 have been used to successfully ameliorate murine models of autoimmune disease. The most often studied disease model has been murine inflammatory arthritis, where a clear mechanism for the efficacy of CD44 antibodies has not been established. We have recently shown in a murine passive-model of the autoimmune disease immune thrombocytopenia (ITP that some CD44 antibodies themselves can induce thrombocytopenia in mice, and the CD44 antibody causing the most severe thrombocytopenia (IM7, also is known to be highly effective in ameliorating murine models of arthritis. Recent work in the K/BxN serum-induced model of arthritis demonstrated that antibody-induced thrombocytopenia reduced arthritis, causing us to question whether CD44 antibodies might primarily ameliorate arthritis through their thrombocytopenic effect. We evaluated IM7, IRAWB14.4, 5035-41.1D, KM201, KM114, and KM81, and found that while all could induce thrombocytopenia, the degree of protection against serum-induced arthritis was not closely related to the length or severity of the thrombocytopenia. CD44 antibody treatment was also able to reverse established inflammation, while thrombocytopenia induced by an anti-platelet antibody targeting the GPIIbIIIa platelet antigen, could not mediate this effect. While CD44 antibody-induced thrombocytopenia may contribute to some of its therapeutic effect against the initiation of arthritis, for established disease there are likely other mechanisms contributing to its efficacy. Humans are not known to express CD44 on platelets, and are therefore unlikely to develop thrombocytopenia after CD44 antibody treatment. An understanding of the relationship between arthritis, thrombocytopenia, and CD44 antibody treatment remains critical for continued development of CD44 antibody therapeutics.

  13. Inducible expression of endomorphins in murine dendritic cells.

    Yang, Xiaohuai; Xia, Hui; Chen, Yong; Liu, Xiaofen; Zhou, Cheng; Gao, Qin; Li, Zhenghong


    Bone marrow precursor cells were extracted from C57BL/6J mice aged 7-8 weeks, and dendritic cells were purified using anti-CD11c (a specific marker for dendritic cells) antibody-coated magnetic beads. Immunofluorescence staining revealed that the expression levels of endomorphin-1 and endomorphin-2 were upregulated in dendritic cells activated by lipopolysaccharide. An enzyme immunoassay showed that lipopolysaccharide and other Toll-like receptor ligands promoted the secretion of endomorphin-1 and endomorphin-2 from activated dendritic cells. [(3)H]-thymidine incorporation demonstrated that endomorphin-1 and endomorphin-2 both inhibited the proliferation of T lymphocyte induced by activated dendritic cells. Furthermore, this immunosuppressive effect was blocked by CTOP, a specific antagonist of µ-opioid receptors. Our experimental findings indicate that activated dendritic cells can induce the expression and secretion of endomorphins, and that endomorphins suppress T lymphocyte proliferation through activation of µ-opioid receptors.

  14. Inducible expression of endomorphins in murine dendritic cells

    Xiaohuai Yang; Hui Xia; Yong Chen; Xiaofen Liu; Cheng Zhou; Qin Gao; Zhenghong Li


    Bone marrow precursor cells were extracted from C57BL/6J mice aged 7–8 weeks, and dendritic cells were purified using anti-CD11c (a specific marker for dendritic cells) antibody-coated magnetic beads. Immunofluorescence staining revealed that the expression levels of endomorphin-1 and endomorphin-2 were upregulated in dendritic cells activated by lipopolysaccharide. An enzyme immunoassay showed that lipopolysaccharide and other Toll-like receptor ligands promoted the secretion of endomorphin-1 and endomorphin-2 from activated dendritic cells. [3H]-thymidine incorporation demonstrated that endomorphin-1 and endomorphin-2 both inhibited the proliferation of T lymphocyte induced by activated dendritic cells. Furthermore, this immunosuppressive effect was blocked by CTOP, a specific antagonist of μ-opioid receptors. Our experimental findings indicate that activated dendritic cells can induce the expression and secretion of endomorphins, and that endomorphins suppress T lymphocyte proliferation through activation of μ-opioid receptors.

  15. Effects of fenbendazole on the murine humoral immune system.

    Landin, Ana Marie; Frasca, Daniela; Zaias, Julia; Van der Put, Elaine; Riley, Richard L; Altman, Norman H; Blomberg, Bonnie B


    Pinworms are highly contagious parasites that have been effectively treated in laboratory rodents with fenbendazole (FBZ). Whether FBZ has any detrimental side effects that may compromise experimental results is unknown. Here we asked whether the immune systems from young and aged mice are altered under FBZ treatment. We compared control and FBZ-treated groups of young (age, 2 to 4 mo) and old (age, 22 to 24 mo) BALB/cN mice. The treated mice received a total of 4 wk (alternating-week treatment regimen) of FBZ-medicated feed. Spleen and bone marrow were collected for immunologic assays, and heart, stomach, intestines, kidneys, and liver were evaluated by histopathology. Our results indicate that FBZ treatment has significant effects on the immune systems of mice; these effects are greater in aged mice. FBZ treatment adversely affected mRNA and protein expression of E2A (a transcription factor crucial for B lymphocytes) in activated precursor B lymphocytes obtained from the bone marrow of young and old mice. These effects were reversed by 6 wk on regular feed after the end of treatment. Activated B lymphocytes from the spleens of young and old mice showed decreased function (cell proliferation, E2A mRNA and protein expression) through the last time point of FBZ treatment but recovered by 2 to 4 wk after treatment. Our findings suggest that FBZ treatment may alter sensitive immune and molecular measures as presented here, and postponing the experimental use of mice until at least 6 wk after treatment should be considered.

  16. Therapeutic effect of the YH6 phage in a murine hemorrhagic pneumonia model.

    Yang, Mei; Du, Chongtao; Gong, Pengjuan; Xia, Feifei; Sun, Changjiang; Feng, Xin; Lei, Liancheng; Song, Jun; Zhang, Lei; Wang, Bin; Xiao, Feng; Yan, Xinwu; Cui, Ziyin; Li, Xinwei; Gu, Jingmin; Han, Wenyu


    The treatment, in farmed mink, of hemorrhagic pneumonia caused by multidrug-resistant Pseudomonas aeruginosa strains has become increasingly difficult. This study investigated the potential use of phages as a therapy against hemorrhagic pneumonia caused by P. aeruginosa in a murine hemorrhagic pneumonia model. An N4-like phage designated YH6 was isolated using P. aeruginosa strain D9. YH6 is a virulent phage with efficient and broad host lytic activity against P. aeruginosa. No bacterial virulence- or lysogenesis-related ORF is present in the YH6 genome, making it eligible for use in phage therapy. In our murine experiments, a single intranasal administration of YH6 (2 × 10(7) PFU) 2 h after D9 intranasal injections at double minimum lethal dose was sufficient to protect mice against hemorrhagic pneumonia. The bacterial load in the lungs of YH6-protected mice was less than 10(3) CFU/g within 24 h after challenge and ultimately became undetectable, whereas the amount of bacteria in the lung tissue derived from unprotected mice was more than 10(8) CFU/g within 24 h after challenge. In view of its protective efficacy in this murine hemorrhagic pneumonia model, YH6 may serve as an alternative treatment strategy for infections caused by multidrug-resistant P. aeruginosa.

  17. Potent inhibition of Junín virus infection by interferon in murine cells.

    Huang, Cheng; Walker, Aida G; Grant, Ashley M; Kolokoltsova, Olga A; Yun, Nadezhda E; Seregin, Alexey V; Paessler, Slobodan


    The new world arenavirus Junín virus (JUNV) is the causative agent of Argentine hemorrhagic fever, a lethal human infectious disease. Adult laboratory mice are generally resistant to peripheral infection by JUNV. The mechanism underlying the mouse resistance to JUNV infection is largely unknown. We have reported that interferon receptor knockout mice succumb to JUNV infection, indicating the critical role of interferon in restricting JUNV infection in mice. Here we report that the pathogenic and vaccine strains of JUNV were highly sensitive to interferon in murine primary cells. Treatment with low concentrations of interferon abrogated viral NP protein expression in murine cells. The replication of both JUNVs was enhanced in IRF3/IRF7 deficient cells. In addition, the vaccine strain of JUNV displayed impaired growth in primary murine cells. Our data suggested a direct and potent role of host interferon response in restricting JUNV replication in mice. The defect in viral growth for vaccine JUNV might also partially explain its attenuation in mice.

  18. Humanized versus murine anti-human epidermal growth factor receptor monoclonal antibodies for immunoscintigraphic studies

    Morales, Alejo A. Morales; Duconge, Jorge; Alvarez-Ruiz, Daniel; Becquer-Viart, Maria de Los Angeles; Nunez-Gandolff, Gilda; Fernandez, Eduardo; Caballero-Torres, Idania; Iznaga-Escobar, Normando


    The anti-human epidermal growth factor receptor (EGF-R) humanized antibody h-R3 (IgG{sub 1}), which binds to an extracellular domain of EGF-R, was used to evaluate the biodistribution on nude mice xenografted with A431 epidermoid carcinoma cell line. Results are compared with its murine version ior egf/r3 monoclonal antibody (mAb). Twenty-one athymic female 4NMRI nu/nu mice were injected intravenously with 10 {mu}g/100 {mu}Ci of {sup 99m}Tc-labeled mAbs. The mAb ior C5 that recognizes an antigen expressed preferentially on the surface of malignant and cytoplasm of normal colorectal cells was used as negative control. Immunoreactivity of {sup 99m}Tc-labeled mAbs was measured by enzyme linked immunosorbent assay on A431 cell line and the immunoreactive fractions determined by Lindmo method. Among all organs significant accumulation was found in tumor (6.14{+-}2.50 %ID/g, 5.06{+-}2.61 %ID/g for murine and humanized mAbs, respectively) 4 h after injection. The immunoreactive fractions were found to be 0.88 and 0.81 for murine and humanized mAb, respectively. Thus, we expect better results using the humanized mAb h-R3 for diagnostic immunoscintigraphy.

  19. Murine metapodophalangeal sesamoid bones: morphology and potential means of mineralization underlying function.

    Doherty, Alison H; Lowder, Elizabeth M; Jacquet, Robin D; Landis, William J


    Normal murine metapodophalangeal sesamoid bones, closely associated with tendons, were examined in terms of their structure and mineralization with reference to their potential function following crystal deposition. This study utilized radiography, whole mount staining, histology, and conventional electron microscopy to establish a maturation timeline of mineral formation in 1- to 6-week-old metapodophalangeal sesamoids from CD-1 mice. An intimate cellular and structural relationship was documented in more detail than previously described between the sesamoid bone, tendon, and fibrocartilage enthesis at the metapodophalangeal joint. Sesamoid calcification began in 1-week lateral sesamoids of the murine metacarpophalangeal joint of the second digit. All sesamoids were completely calcified by 4 weeks. Transmission electron microscopy of 2-week metacarpophalangeal sesamoids revealed extensive Type I collagen in the associated tendon and fibrocartilage insertion sites and Type II collagen and proteoglycan networks in the interior of the sesamoid. No extracellular matrix vesicles were documented. The results demonstrate that murine sesamoid bones consist of cartilage elaborated by chondrocytes that predominantly synthesize and secrete Type II collagen and proteoglycan. Type II collagen and proteoglycans appear responsible for the onset and progression of mineral formation in this tissue. These data contribute to new understanding of the biochemistry, ultrastructure, and mineralization of sesamoids in relation to other bones and calcifying cartilage and tendon of vertebrates. They also reflect on the potentially important but currently uncertain function of sesamoids as serving as a fulcrum point along a tendon, foreshortening its length and altering advantageously its biomechanical properties with respect to tendon-muscle interaction.

  20. Murine protein H is comprised of 20 repeating units, 61 amino acids in length

    Kristensen, Torsten; Tack, B F


    A cDNA library constructed from size-selected (greater than 28 S) poly(A)+ RNA isolated from the livers of C57B10. WR mice was screened by using a 249-base-pair (bp) cDNA fragment encoding 83 amino acid residues of human protein H as a probe. Of 120,000 transformants screened, 30 hybridized...... with this cDNA probe. Ten positives were colony-purified, and the largest plasmid cDNA insert, MH8 (4.4 kb), was sequenced by the dideoxy chain termination method. MH8 contained the complete coding sequence for the precursor of murine complement protein factor H (3702 bp), 100 bp of 5'-untranslated sequence......, 448 bp of 3'-untranslated sequence, and a polyadenylylated tail of undetermined length. Murine pre-protein H was deduced to consist of an 18-amino acid signal peptide and 1216 residues of H-protein sequence. Murine H was composed of 20 repetitive units, each about 61 amino acid residues in length...

  1. Antileukemic Efficacy of Continuous vs Discontinuous Dexamethasone in Murine Models of Acute Lymphoblastic Leukemia.

    Laura B Ramsey

    Full Text Available Osteonecrosis is one of the most common, serious, toxicities resulting from the treatment of acute lymphoblastic leukemia. In recent years, pediatric acute lymphoblastic leukemia clinical trials have used discontinuous rather than continuous dosing of dexamethasone in an effort to reduce the incidence of osteonecrosis. However, it is not known whether discontinuous dosing would compromise antileukemic efficacy of glucocorticoids. Therefore, we tested the efficacy of discontinuous dexamethasone against continuous dexamethasone in murine models bearing human acute lymphoblastic leukemia xenografts (n = 8 patient samples or murine BCR-ABL+ acute lymphoblastic leukemia. Plasma dexamethasone concentrations (7.9 to 212 nM were similar to those achieved in children with acute lymphoblastic leukemia using conventional dosages. The median leukemia-free survival ranged from 16 to 59 days; dexamethasone prolonged survival from a median of 4 to 129 days in all seven dexamethasone-sensitive acute lymphoblastic leukemias. In the majority of cases (7 of 8 xenografts and the murine BCR-ABL model we demonstrated equal efficacy of the two dexamethasone dosing regimens; whereas for one acute lymphoblastic leukemia sample, the discontinuous regimen yielded inferior antileukemic efficacy (log-rank p = 0.002. Our results support the clinical practice of using discontinuous rather than continuous dexamethasone dosing in patients with acute lymphoblastic leukemia.

  2. Isolation of Murine Bone Marrow Derived Mesenchymal Stem Cells using Twist2 Cre Transgenic Mice

    Liu, Yaling; Wang, Liping; Fatahi, Reza; Kronenberg, Mark; Kalajzic, Ivo; Rowe, David; Li, Yingcui; Maye, Peter


    While human bone marrow derived mesenchymal stem cells (BMSCs) are of great interest for their potential therapeutic value, its murine equivalent remains an important basic research model that can provide critical insights into the biology of this progenitor cell population. Here we present a novel transgenic strategy that allowed for the selective identification and isolation of murine BMSCs at the early stages of stromal cell culture. This strategy involved crossing Twist2 –Cre mice with Cre reporter mice such as Z/EG or Ai9, which express EGFP or Tomato fluorescent protein, respectively, upon Cre mediated excision of a stop sequence. Using this approach, we identified an adherent fluorescent protein+ cell population (T2C+) that is present during the earliest stages of colony formation and by day 5 of culture represents ~20% of the total cell population. Cell surface profiling by flow cytometry showed that T2C+ cells are highly positive for SCA1 and CD29 and negative for CD45, CD117, TIE2, and TER119. Isolation of T2C+ cells by FACS selected for a cell population with skeletal potential that can be directed to differentiate into osteoblasts, adipocytes, or chondrocytes. We also demonstrated in a calvarial bone defect model that T2C+ cells retain a strong efficacy for osteogenic repair and can support a hematopoietic environment. Collectively, these studies provide evidence that the Twist2-Cre x Cre reporter breeding strategy can be used to positively identify and isolate multipotent murine BMSCs. PMID:20673822

  3. In vitro proteolytic cleavage of Gazdar murine sarcoma virus p65gag.

    Maxwell, S; Arlinghaus, R B


    Moloney murine leukemia virus, disrupted in concentrations of 0.1 to 0.5% Nonidet P-40, catalyzed the cleavage of p65, the gag gene polyprotein of the Gazdar strain of murine sarcoma virus, into polypeptides with sizes and antigenic determinants of murine leukemia virus-specified p30, p15, pp12, and p10. Cleavage performed in the presence of 0.15% Nonidet P-40 in water yielded polypeptides of approximately 40,000 (P40) and 25,000 (P25) Mr. In vitro cleavage performed in a buffered solution containing dithiothreitol in addition to 0.1% Nonidet P-40 allowed the efficient processing of P40 to p30 and a band migrating with p10. Immunoprecipitation with monospecific sera indicated that P40 contained p30 and p10, whereas P25 contained p15 and pp12 determinants. P40 and P25 are similar in size and antigenic properties to Pr40gag and Pr25gag observed in infected cells (Naso et al, J. Virol. 32:187-198, 1979).

  4. Characterization of the molecularly cloned murine alpha-globin transcription factor CP2.

    Lim, L C; Fang, L; Swendeman, S L; Sheffery, M


    We recently cloned human and murine cDNAs that encode CP2, a transcription factor that interacts with the murine alpha-globin promoter. In this report, we exploited our ability to express CP2 in bacteria and eukaryotic cells to further investigate factor activities in vitro and in vivo. CP2 expressed in bacteria was significantly enriched and used in a series of DNase I footprinting and electrophoretic gel shift assays. The results suggest that CP2 binds a hyphenated recognition sequence motif that spans one DNA helix turn. In addition, the enriched bacterial protein activated transcription of alpha-globin promoter templates approximately 3- to 4-fold in vitro. We then tested the effect of elevating CP2 levels 2.5- to 5.5-fold in vivo using both transient and stable transformation assays. When a reporter construct comprised of the intact murine alpha-globin promoter driving the bacterial chloramphenicol acetyltransferase (CAT) gene was introduced into these overexpressing cells, we observed a 3- to 6-fold increase in CAT activity when compared to cells expressing normal levels of CP2. These results define the CP2 factor binding site in more detail and help characterize the activities of the factor in vivo.

  5. Generation of a conditional knockout of murine glucocerebrosidase: utility for the study of Gaucher disease.

    Sinclair, Graham B; Jevon, Gareth; Colobong, Karen E; Randall, Derrick R; Choy, Francis Y M; Clarke, Lorne A


    Gaucher disease is a disorder of sphingolipid metabolism resulting from an inherited deficiency of the lysosomal hydrolase glucocerebrosidase. Affected individuals present with a spectrum of clinical symptoms ranging from hepatosplenomegaly, haematological abnormalities, and bone pain in type 1 disease, to severe neurodegeneration and premature death in types 2 and 3 disease. Although the basic biochemical defect is well characterized, there remains a poor understanding of the underlying pathophysiology of disease. In vitro studies suggest that macrophage glucocerebroside storage leads to tissue dysfunction through complex mechanisms involving altered intracellular calcium homeostasis and apoptosis. In order to study the pathogenic roles of these complex interactions, a viable animal model for Gaucher disease is needed. The complexity of this single gene disorder has been emphasized by the varied results of previous murine Gaucher models, ranging from perinatal lethality to phenotypically and biochemically asymptomatic animals. Recognizing the need to modulate the biochemical phenotype in mice to produce a relevant model, we have created a murine strain with key exons of the glucocerebrosidase gene flanked by loxP sites. We show that expression of Cre-recombinase in cells of hematopoietic and endothelial origin results in deficiency of glucocerebrosidase in the liver, spleen, bone marrow, and peripheral white cells. Glucocerebroside storage in this model leads to progressive splenomegaly with Gaucher cell infiltration and modest storage in the liver by 26 weeks of age. These results indicate the utility of this loxP GBA targeted murine strain for understanding the complex pathophysiology of Gaucher disease.

  6. Isolation of a mesenchymal cell population from murine dermis that contains progenitors of multiple cell lineages.

    Crigler, Lauren; Kazhanie, Amita; Yoon, Tae-Jin; Zakhari, Julia; Anders, Joanna; Taylor, Barbara; Virador, Victoria M


    The skin contains two known subpopulations of stem cells/epidermal progenitors: a basal keratinocyte population found in the interfollicular epithelium and cells residing in the bulge region of the hair follicle. The major role of the interfollicular basal keratinocyte population may be epidermal renewal, whereas the bulge population may only be activated and recruited to form a cutaneous epithelium in case of trauma. Using 3-dimensional cultures of murine skin under stress conditions in which only reserve epithelial cells would be expected to survive and expand, we demonstrate that a mesenchymal population resident in neonatal murine dermis has the unique potential to develop an epidermis in vitro. In monolayer culture, this dermal subpopulation has long-term survival capabilities in restricted serum and an inducible capacity to evolve into multiple cell lineages, both epithelial and mesenchymal, depending on culture conditions. When grafted subcutaneously, this dermal subpopulation gave rise to fusiform structures, reminiscent of disorganized muscle, that stained positive for smooth muscle actin and desmin; on typical epidermal grafts, abundant melanocytes appeared throughout the dermis that were not associated with hair follicles. The multipotential cells can be repeatedly isolated from neonatal murine dermis by a sequence of differential centrifugation and selective culture conditions. These results suggest that progenitors capable of epidermal differentiation exist in the mesenchymal compartment of an abundant tissue source and may have a function in mesenchymal-epithelial transition upon insult. Moreover, these cells could be available in sufficient quantities for lineage determination or tissue engineering applications.

  7. [Seromonitoring of laboratory mouse and rat colonies for common murine pathogens (author's transl)].

    Fujiwara, K; Tanishima, Y; Tanaka, M


    During a period from 1973 to 1978, 392 and 225 lots including 12,232 mouse and 8,044 rat individual sera, respectively, were examined for antibodies to murine hepatitis virus, Sendai virus, Bordetella bronchiseptica, Mycoplasma pulmonis, Tyzzer agents, Salmonella typhimurium and Corynebacterium kutscheri. Of mouse lots 94.5% and 39.3% from breeder and user colonies, respectively, were negative for all antibodies examined as well as 31.6% and 17.2% of rat breeder and user colonies, respectively. Among positive lots from mouse users, high positivity rates were seen with Senai virus (47.6%), M. pulmonis (19.0%), and murine hepatitis virus (JHM : 18.2%, MHV : 31.0%), while the rates were high in rat user lots with Sendai virus (24.4%), B. bronchiseptica (39.3%) M. pulmonis (12.5%), murine coronaviruses (JHM : 19.0%, MHV-2 : 28.0%) and tyzzer agents (MSK : 19.6%, RT : 17.9%). These pathogenes with high positivities should be monitored indispensably as a quality control of laboratory mice and rats.

  8. Adiponectin and leptin induce VCAM-1 expression in human and murine chondrocytes.

    Javier Conde

    Full Text Available BACKGROUND: Osteoarthritis (OA and rheumatoid arthritis (RA, the most common rheumatic diseases, are characterized by irreversible degeneration of the joint tissues. There are several factors involved in the pathogenesis of these diseases including pro-inflammatory cytokines, adipokines and adhesion molecules. OBJECTIVE: Up to now, the relationship between adipokines and adhesion molecules at cartilage level was not explored. Thus, the aim of this article was to study the effect of leptin and adiponectin on the expression of VCAM-1 in human and murine chondrocytes. For completeness, intracellular signal transduction pathway was also explored. METHODS: VCAM-1 expression was assessed by quantitative RT-PCR and western blot analysis upon treatment with leptin, adiponectin and other pertinent reagents in cultured human primary chondrocytes. Signal transduction pathways have been explored by using specific pharmacological inhibitors in the adipokine-stimulated human primary chondrocytes and ATDC5 murine chondrocyte cell line. RESULTS: Herein, we demonstrate, for the first time, that leptin and adiponectin increase VCAM-1 expression in human and murine chondrocytes. In addition, both adipokines have additive effect with IL-1β. Finally, we demonstrate that several kinases, including JAK2, PI3K and AMPK are at a play in the intracellular signalling of VCAM-1 induction. CONCLUSIONS: Taken together, our results suggest that leptin and adiponectin could perpetuate cartilage-degrading processes by inducing also factors responsible of leukocyte and monocyte infiltration at inflamed joints.

  9. The Immunomodulatory Activity of Jacaric Acid, a Conjugated Linolenic Acid Isomer, on Murine Peritoneal Macrophages.

    Wai Nam Liu

    Full Text Available This study aims at demonstrating the immunomodulatory property of jacaric acid, a conjugated linolenic acid (CLNA isomer that is present in jacaranda seed oil, on murine peritoneal macrophages. Our results showed that jacaric acid exhibited no significant cytotoxicity on the thioglycollate-elicited murine peritoneal macrophages as revealed by the neutral red uptake assay, but markedly increased their cytostatic activity on the T-cell lymphoma MBL-2 cells as measured by the fluorometric CyQuant® NF Cell Proliferation Assay Kit. Flow cytometric analysis indicated that jacaric acid could enhance the endocytic activity of macrophages and elevated their intracellular production of superoxide anion. Moreover, jacaric acid-treated macrophages showed an increase in the production of nitric oxide which was accompanied by an increase in the expression level of inducible nitric oxide synthase protein. In addition, the secretion of several pro-inflammatory cytokines, including interferon-γ, interleukin-1β and tumor necrosis factor-α, was up-regulated. Collectively, our results indicated that the naturally-occurring CLNA isomer, jacaric acid, could exhibit immunomodulating activity on the murine peritoneal macrophages in vitro, suggesting that this CLNA isomer may act as an immunopotentiator which can be exploited for the treatment of some immunological disorders with minimal toxicity and fewer side effects.

  10. Molecular phylogenetic characterization of common murine rodents from Manipur, Northeast India.

    Chingangbam, Dhananjoy S; Laishram, Joykumar M; Suzuki, Hitoshi


    The Indian subcontinent and Southeast Asia are hotspots of murine biodiversity, but no species from the Arakan Mountain system that demarcates the border between the two areas has been subjected to molecular phylogenetic analyses. We examined the mitochondrial cytochrome b gene sequences in six murine species (the Rattus rattus species complex, R. norvegicus, R. nitidus, Berylmys manipulus, Niviventer sp. and Mus musculus) from Manipur, which is located at the western foot of the mountain range. The sequences of B. manipulus and Niviventer sp. examined here were distinct from available congeneric sequences in the databases, with sequence divergences of 10-15%. Substantial degrees of intrapopulation divergence were detected in R. nitidus and the R. rattus species complex from Manipur, implying ancient habitation of the species in this region, while the recent introduction by modern and prehistoric human activities was suggested for R. norvegicus and M. musculus, respectively. In the nuclear gene Mc1r, also analyzed here, the R. rattus species complex from Manipur was shown to possess allelic sequences related to those from the Indian subcontinent in addition to those from East Asia. These results not only fill gaps in the phylogenetic knowledge of each taxon examined but also provide valuable insight to better understand the biogeographic importance of the Arakan Mountain system in generating the species and genetic diversity of murine rodents.

  11. Bicarbonate Secretion in the Murine Gallbladder - Lessons for the Treatment of Cystic Fibrosis

    Cuthbert AW


    Full Text Available The epithelium lining the gallbladder of mammalian species has absorptive and secretory functions. An important function is the secretion of a bicarbonate rich fluid that helps neutralise stomach acid and provides an appropriate environment for intestinal enzymes. In cystic fibrosis (CF this secretory function is lost. This study concerns the bicarbonate secreting activity of murine gallbladders in vitro using wild type and CF mice and four main questions are considered as follows: a Does the murine gallbladder secrete bicarbonate electrogenically and is this prevented in CF? b Can the secretory activity in CF gallbladders be restored by gene therapy or pharmacologically? c How is the cystic fibrosis transmembrane conductance regulator (CFTR involved in bicarbonate secretion? d Does the data offer prospects for the treatment of CF?. Work from both the author's laboratory and the literature will be reviewed. Consideration of the currently available data indicates that the wild type murine gallbladder does secrete bicarbonate electrogenically and that this is absent in CF mice. Further it has been demonstrated that bicarbonate secretory activity can be restored by both gene therapy and by the use of drugs. The role of CFTR in bicarbonate secretion remains equivocal. Much evidence suggests that CFTR can act as a channel for HCO(3(- ions as well as Cl(- ions, while others propose a parallel arrangement of CFTR with a Cl(-/HCO(3(- exchanger is necessary. The matter is further complicated by the regulatory role of CFTR on other transporting activities. Opportunities for possible application to man are discussed.

  12. Activation of P2X7 receptors decreases the proliferation of murine luteal cells.

    Wang, Jing; Liu, Shuangmei; Nie, Yijun; Wu, Bing; Wu, Qin; Song, Miaomiao; Tang, Min; Xiao, Li; Xu, Ping; Tan, Ximin; Zhang, Luyin; Li, Gang; Liang, Shangdong; Zhang, Chunping


    Extracellular ATP regulates cellular function in an autocrine or paracrine manner through activating purinergic signalling. Studies have shown that purinergic receptors were expressed in mammalian ovaries and they have been proposed as an intra-ovarian regulatory mechanism. P2X7 was expressed in porcine ovarian theca cells and murine and human ovarian surface epithelium and is involved in ATP-induced apoptotic cell death. However, the role of P2X7 in corpus luteum is still unclear. The aim of this study was to investigate the role of ATP signalling in murine luteal cells and the possible mechanism(s) involved. We found that P2X7 was highly expressed in murine small luteal cells. The agonists of P2X7, ATP and BzATP, inhibited the proliferation of luteal cells. P2X7 antagonist BBG reversed the inhibition induced by ATP and BzATP. Further studies showed that ATP and BzATP inhibited the expression of cell cycle regulators cyclinD2 and cyclinE2. ATP and BzATP also inhibited the p38-mitogen-activated protein kinase (MAPK) signalling pathway. These results reveal that P2X7 receptor activation is involved in corpus luteum formation and function.

  13. Lycium barbarum polysaccharides regulate phenotypic and functional maturation of murine dendritic cells.

    Zhu, Jie; Zhao, Lu-Hang; Zhao, Xiao-Ping; Chen, Zhi


    Lycium barbarum polysaccharides (LBPs) have been known to have a variety of immunomodulatory functions including activation of T cells, B cells and NK cells. Dendritic cells (DC) are potent antigen-presenting cells that play pivotal roles in the initiation of the primary immune response. However, little is known about the immunomodulatory effects of LBPs on murine bone marrow derived dendritic cells (BMDC). In the present study, the effects of LBPs on the phenotypic and functional maturation of murine BMDC were investigated in vitro. Compared to the BMDC that were only subjected to treatment with RPMI1640, the co-expression of I-A/I-E, CD11c and secretion of IL-12 p40 by BMDC stimulated with LBPs (100 microg/ml) were increased. In addition, the endocytosis of FITC-dextran by LBPs-treated BMDC (100 microg/ml) was impaired, whereas the activation of proliferation of allogenic lymphocytes by BMDC was enhanced. Our results strongly suggest that LBPs are capable of promoting both the phenotypic and functional maturation of murine BMDC in vitro.

  14. Role of Notch expression in premature senescence of murine bone marrow stromal cells

    Kejie Zhang; Lifang Huang; Hanying Sun; Yan Zhu; Yi Xiao; Mei Huang; Wenli Liu


    The aim of the present study was to investigate the role of the Notch signaling pathway in premature senescence of murine bone marrow stromal cells in vitro.The intracellular domain of Notch 1 (ICN) was transfected into cultured murine bone marrow stromal cells by lipofectamine transfection.After three days,the proliferation of transfected cells was measured by MTT assay.Cell cycle distribution was analyzed by flow cytometry.Senescence-associated beta-galactosidase (SA-beta-gal) was measured,and the percentage of positive cells was evaluated by assessing 1000 cells in random fields of view.The expressions of p53 and p21cip1/waf1 were analyzed by both RT-PCR and Western blot analysis.The results showed that activation of Notch signaling inhibited proliferation of murine bone marrow stromal cells with induction of G1 arrest,increased the percentage of SA-beta-gal positive cells,and upregulated p53 and p21Cip1/Waf1 mRNA and protein expression levels.Thus,the activated Notch signaling could induce premature senescence of bone marrow stromal cells through the p53-p21Cip1/waf1 pathway.(C) 2009 National Natural Science Foundation of China and Chinese Academy of Sciences.Published by Elsevier Limited and Science in China Press.All fights reserved.

  15. Antioxidative effects in vivo and colonization of Lactobacillus plantarum MA2 in the murine intestinal tract.

    Tang, Wei; Xing, Zhuqing; Hu, Wei; Li, Chao; Wang, Jinju; Wang, Yanping


    Lactobacillus plantarum MA2 was isolated from traditional Chinese Tibet kefir grains, which possess several excellent properties and functions. We previously demonstrated the antioxidant activities of this bacterium in vitro. However, the maintenance and survival of L. plantarum MA2 inside the murine intestinal tract, where it exerts its probiotic properties, and whether its effects are elicited directly on the host remain unknown. Therefore, this study investigated the mechanisms of L. plantarum MA2 in aging mice following D-galactose administration. The levels of malondialdehyde decreased significantly in the L. plantarum MA2 groups after oral ingestion compared to the D-galactose model group, and total antioxidant capacity and glutathione peroxidase and superoxide dismutase activities increased significantly in the serum and liver. We combined fluorescein isothiocyanate labeling and green fluorescent protein expression to dynamically monitor the colonization and distribution of L. plantarum MA2 in the murine intestinal tract. The results indicated that L. plantarum MA2 was detected in the ileum, colon, and feces after single and continuous oral administration at day 21 and was maintained at 10(4)-10(5) CFU/g. These results suggest that L. plantarum MA2 colonizes and survives in the murine intestinal tract to exert its antioxidative effects.

  16. Porphyromonas gingivalis peptidylarginine deiminase, a key contributor in the pathogenesis of experimental periodontal disease and experimental arthritis.

    Neville Gully

    Full Text Available To investigate the suggested role of Porphyromonas gingivalis peptidylarginine deiminase (PAD in the relationship between the aetiology of periodontal disease and experimentally induced arthritis and the possible association between these two conditions.A genetically modified PAD-deficient strain of P. gingivalis W50 was produced. The effect of this strain, compared to the wild type, in an established murine model for experimental periodontitis and experimental arthritis was assessed. Experimental periodontitis was induced following oral inoculation with the PAD-deficient and wild type strains of P. gingivalis. Experimental arthritis was induced via the collagen antibody induction process and was monitored by assessment of paw swelling and micro-CT analysis of the radio-carpal joints. Experimental periodontitis was monitored by micro CT scans of the mandible and histological assessment of the periodontal tissues around the mandibular molars. Serum levels of anti-citrullinated protein antibodies (ACPA and P. gingivalis were assessed by ELISA.The development of experimental periodontitis was significantly reduced in the presence of the PAD-deficient P. gingivalis strain. When experimental arthritis was induced in the presence of the PAD-deficient strain there was less paw swelling, less erosive bone damage to the joints and reduced serum ACPA levels when compared to the wild type P. gingivalis inoculated group.This study has demonstrated that a PAD-deficient strain of P. gingivalis was associated with significantly reduced periodontal inflammation. In addition the extent of experimental arthritis was significantly reduced in animals exposed to prior induction of periodontal disease through oral inoculation of the PAD-deficient strain versus the wild type. This adds further evidence to the potential role for P. gingivalis and its PAD in the pathogenesis of periodontitis and exacerbation of arthritis. Further studies are now needed to elucidate the

  17. Testing the Role of p21-Activated Kinases in Schwannoma Formation Using a Novel Genetically Engineered Murine Model that Closely Phenocopies Human NF2 Disease


    Kinases in Schwannoma Formation Using a Novel Genetically Engineered Murine Model that Closely Phenocopies Human NF2 Disease The views, opinions and...TITLE AND SUBTITLE Testing the Role of p21-Activated Kinases in Schwannoma Formation Using a Novel Genetically Engineered Murine Model that Closely...1.20 calendar Testing the Role of p21 Activated Kinases in Schwannoma Formation Using a Novel Genetically Engineered Murine Model that Closely

  18. Serological Surveillance of Scrub Typhus, Murine Typhus, and Leptospirosis in Small Mammals Captured at Firing Points 10 and 60, Gyeonggi Province, Republic of Korea, 2001-2005


    Scrub Typhus, Murine Typhus, and Leptospirosis in Small Mammals Captured at Firing Points 10 and 60, Gyeonggi Province, Republic of Korea, 2001-2005 t...These diseases include scrub typhus, murine typhus, and leptospirosis . Many of the training sites are rural or semi-rural, surrounded or co...murine typhus, and leptospirosis antibodies. Of the nine species of small mammals collected, Apodemus agrarius, the common striped field mouse and known

  19. Investigation of a possible interaction between the heme oxygenase/biliverdin reductase and nitric oxide synthase pathway in murine gastric fundus and jejunum.

    De Backer, Ole; Lefebvre, Romain A


    This study investigated the possible interaction between the heme oxygenase (HO)/biliverdin reductase (BVR) and nitric oxide synthase (NOS) pathway in murine gastric fundus and jejunum, since previous studies have shown that both HO-2 and BVR are expressed in interstitial cells of Cajal (ICCs) and co-localized with neuronal NOS in a large proportion of myenteric neurons along the gastrointestinal tract. Neither HO inhibition by chromium mesoporphyrin (CrMP) nor co-incubation with CO or biliverdin/bilirubin affected nitrergic neurotransmission - i.e. relaxations induced by non-adrenergic non-cholinergic (NANC) nerve stimulation or exogenous NO - under normal physiological conditions. However, biliverdin/bilirubin reversed the inhibitory effect of the superoxide generator LY83583 on exogenous NO-induced relaxations in both tissues. When gastric fundus muscle strips were depleted of the endogenous antioxidant Cu/Zn superoxide dismutase (SOD) by the Cu-chelator DETCA, electrically induced NANC relaxations were also affected by LY82583; however, biliverdin/bilirubin could not substitute for the loss of Cu/Zn SOD when this specific antioxidant enzyme was depleted. In jejunal muscle strips, the combination DETCA plus LY83583 nearly abolished contractile phasic activity and, hence, did not allow studying nitrergic relaxation in these experimental conditions. In conclusion, this study does not establish a role for HO/CO in inhibitory NANC neurotransmission in murine gastric fundus and jejunum under normal physiological conditions. However, the antioxidants biliverdin/bilirubin might play an important role in the protection of the nitrergic neurotransmitter against oxidative stress.

  20. HIRENASD Experimental Data

    National Aeronautics and Space Administration — Experimental Data for HIRENASD configuration Zip files below contain the experimental data for the pressure coefficients, both the static and the forced oscillation...

  1. The Involvement of Heat Shock Proteins in Murine Liver Regeneration

    Qing Shi; Zhongjun Dong; Haiming Wei


    Partial hepatectomy (PHx) in mammals is a very common experimental model to investigate the process of liver regeneration. The surgery itself could give birth to a series of stresses, such as the temporary raise of body temperature and the ischaemia-reperfusion injury. Heat shock proteins (HSPs) were a family of stress-inducible proteins involved in maintaining cell homeostasis and regulating the immune system. In our study, we intended to investigate the expression and role of HSPs in liver regeneration. Using RT-PCR and Western blotting, we determined the expression in regenerating liver of HSP27, HSP60, HSP70 and HSP90 in mRNA level and protein level, respectively, with mice treated with sham operation as controls. We also used quercertin as an inhibitior of HSPs to explore their effects on liver regeneration. We found that hepatic expression of HSPs increased at the early phase of liver regeneration and declined to the constitutively low level later. Moreover, quercetin pretreatment delayed the progress of liver regeneration in mice via inhibition of HSPs. The results indicated that HSPs played an important role in liver regeneration.

  2. The Proteome of the Murine Presynaptic Active Zone

    Melanie Laßek


    Full Text Available The proteome of the presynaptic active zone controls neurotransmitter release and the short- and long-term structural and functional dynamics of the nerve terminal. The proteinaceous inventory of the presynaptic active zone has recently been reported. This review will evaluate the subcellular fractionation protocols and the proteomic approaches employed. A breakthrough for the identification of the proteome of the presynaptic active zone was the successful employment of antibodies directed against a cytosolic epitope of membrane integral synaptic vesicle proteins for the immunopurification of synaptic vesicles docked to the presynaptic plasma membrane. Combining immunopurification and subsequent analytical mass spectrometry, hundreds of proteins, including synaptic vesicle proteins, components of the presynaptic fusion and retrieval machinery, proteins involved in intracellular and extracellular signaling and a large variety of adhesion molecules, were identified. Numerous proteins regulating the rearrangement of the cytoskeleton are indicative of the functional and structural dynamics of the presynapse. This review will critically discuss both the experimental approaches and prominent protein candidates identified. Many proteins have not previously been assigned to the presynaptic release sites and may be directly involved in the short- and long-term structural modulation of the presynaptic compartment. The identification of proteinaceous constituents of the presynaptic active zone provides the basis for further analyzing the interaction of presynaptic proteins with their targets and opens novel insights into the functional role of these proteins in neuronal communication.

  3. Ethics in Animal Experimentation

    Yusuf Ergun


    Full Text Available Experimental animals are frequently used to obtain information for primarily scientific reasons. In the present review, ethics in animal experimentation is examined. At first, the history of animal experimentation and animal rights is outlined. Thereafter, the terms in relation with the topic are defined. Finally, prominent aspects of 3Rs constituting scientific and ethical basis in animal experimentation are underlined. [Archives Medical Review Journal 2010; 19(4.000: 220-235

  4. Ethics in Animal Experimentation

    Yusuf Ergun


    Experimental animals are frequently used to obtain information for primarily scientific reasons. In the present review, ethics in animal experimentation is examined. At first, the history of animal experimentation and animal rights is outlined. Thereafter, the terms in relation with the topic are defined. Finally, prominent aspects of 3Rs constituting scientific and ethical basis in animal experimentation are underlined. [Archives Medical Review Journal 2010; 19(4.000): 220-235

  5. Microexperiencia Educativa (Microeducational Experimentation).

    Burton Meis, Roberto


    Experimentation for educational reform in Argentina is limited to specifically designated schools which are to be in a permanent state of experimentation. This article presents the official statements designating the experimental schools and includes remarks covering administration, evaluation, and supervision. (VM)

  6. A Proteomic Investigation of Hepatic Resistance to Ascaris in a Murine Model

    Deslyper, Gwendoline; Colgan, Thomas J.; Cooper, Andrew J. R.; Holland, Celia V.; Carolan, James C.


    The helminth Ascaris causes ascariasis in both humans and pigs. Humans, especially children, experience significant morbidity including respiratory complications, growth deficits and intestinal obstruction. Given that 800 million people worldwide are infected by Ascaris, this represents a significant global public health concern. The severity of the symptoms and associated morbidity are related to the parasite burden and not all hosts are infected equally. While the pathology of the disease has been extensively examined, our understanding of the molecular mechanisms underlying resistance and susceptibility to this nematode infection is poor. In order to investigate host differences associated with heavy and light parasite burden, an experimental murine model was developed utilising Ascaris-susceptible and -resistant mice strains, C57BL/6J and CBA/Ca, respectively, which experience differential burdens of migratory Ascaris larvae in the host lungs. Previous studies identified the liver as the site where this difference in susceptibility occurs. Using a label free quantitative proteomic approach, we analysed the hepatic proteomes of day four post infection C57BL/6J and CBA/Ca mice with and without Ascaris infection to identify proteins changes potentially linked to both resistance and susceptibility amongst the two strains, respectively. Over 3000 proteins were identified in total and clear intrinsic differences were elucidated between the two strains. These included a higher abundance of mitochondrial proteins, particularly those associated with the oxidative phosphorylation pathway and reactive oxygen species (ROS) production in the relatively resistant CBA/Ca mice. We hypothesise that the increased ROS levels associated with higher levels of mitochondrial activity results in a highly oxidative cellular environment that has a dramatic effect on the nematode’s ability to successfully sustain a parasitic association with its resistant host. Under infection, both

  7. Molecular dynamics study of lipid bilayers modeling the plasma membranes of normal murine thymocytes and leukemic GRSL cells.

    Andoh, Yoshimichi; Okazaki, Susumu; Ueoka, Ryuichi


    Molecular dynamics (MD) calculations for the plasma membranes of normal murine thymocytes and thymus-derived leukemic GRSL cells in water have been performed under physiological isothermal-isobaric conditions (310.15K and 1 atm) to investigate changes in membrane properties induced by canceration. The model membranes used in our calculations for normal and leukemic thymocytes comprised 23 and 25 kinds of lipids, respectively, including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophospholipids, and cholesterol. The mole fractions of the lipids adopted here were based on previously published experimental values. Our calculations clearly showed that the membrane area was increased in leukemic cells, and that the isothermal area compressibility of the leukemic plasma membranes was double that of normal cells. The calculated membranes of leukemic cells were thus considerably bulkier and softer in the lateral direction compared with those of normal cells. The tilt angle of the cholesterol and the conformation of the phospholipid fatty acid tails both showed a lower level of order in leukemic cell membranes compared with normal cell membranes. The lateral radial distribution function of the lipids also showed a more disordered structure in leukemic cell membranes than in normal cell membranes. These observations all show that, for the present thymocytes, the lateral structure of the membrane is considerably disordered by canceration. Furthermore, the calculated lateral self-diffusion coefficient of the lipid molecules in leukemic cell membranes was almost double that in normal cell membranes. The calculated rotational and wobbling autocorrelation functions also indicated that the molecular motion of the lipids was enhanced in leukemic cell membranes. Thus, here we have demonstrated that the membranes of thymocyte leukemic cells are more disordered and more fluid than normal cell membranes.

  8. Characterization of murine pituitary-derived cell lines Tpit/F1, Tpit/E and TtT/GF.

    Yoshida, Saishu; Higuchi, Masashi; Ueharu, Hiroki; Nishimura, Naoto; Tsuda, Mitsuyoshi; Yako, Hideji; Chen, Mo; Mitsuishi, Hideo; Sano, Yoshiya; Kato, Takako; Kato, Yukio


    The pituitary is an important endocrine tissue of the vertebrate that produces and secretes many hormones. Accumulating data suggest that several types of cells compose the pituitary, and there is growing interest in elucidating the origin of these cell types and their roles in pituitary organogenesis. Therein, the histogenous cell line is an extremely valuable experimental tool for investigating the function of derived tissue. In this study, we compared gene expression profiles by microarray analysis and real-time PCR for murine pituitary tumor-derived non-hormone-producing cell lines TtT/GF, Tpit/F1 and Tpit/E. Several genes are characteristically expressed in each cell line: Abcg2, Nestin, Prrx1, Prrx2, CD34, Eng, Cspg4 (Ng2), S100β and nNos in TtT/GF; Cxcl12, Raldh1, Msx1 and Twist1 in Tpit/F1; and Cxadr, Sox9, Cdh1, EpCAM and Krt8 in Tpit/E. Ultimately, we came to the following conclusions: TtT/GF cells show the most differentiated state, and may have some properties of the pituitary vascular endothelial cell and/or pericyte. Tpit/F1 cells show the epithelial and mesenchymal phenotypes with stemness still in a transiting state. Tpit/E cells have a phenotype of epithelial cells and are the most immature cells in the progression of differentiation or in the initial endothelial-mesenchymal transition (EMT). Thus, these three cell lines must be useful model cell lines for investigating pituitary stem/progenitor cells as well as organogenesis.

  9. Illumination of murine gammaherpesvirus-68 cycle reveals a sexual transmission route from females to males in laboratory mice.

    Sylvie François

    Full Text Available Transmission is a matter of life or death for pathogen lineages and can therefore be considered as the main motor of their evolution. Gammaherpesviruses are archetypal pathogenic persistent viruses which have evolved to be transmitted in presence of specific immune response. Identifying their mode of transmission and their mechanisms of immune evasion is therefore essential to develop prophylactic and therapeutic strategies against these infections. As the known human gammaherpesviruses, Epstein-Barr virus and Kaposi's Sarcoma-associated Herpesvirus are host-specific and lack a convenient in vivo infection model; related animal gammaherpesviruses, such as murine gammaherpesvirus-68 (MHV-68, are commonly used as general models of gammaherpesvirus infections in vivo. To date, it has however never been possible to monitor viral excretion or virus transmission of MHV-68 in laboratory mice population. In this study, we have used MHV-68 associated with global luciferase imaging to investigate potential excretion sites of this virus in laboratory mice. This allowed us to identify a genital excretion site of MHV-68 following intranasal infection and latency establishment in female mice. This excretion occurred at the external border of the vagina and was dependent on the presence of estrogens. However, MHV-68 vaginal excretion was not associated with vertical transmission to the litter or with horizontal transmission to female mice. In contrast, we observed efficient virus transmission to naïve males after sexual contact. In vivo imaging allowed us to show that MHV-68 firstly replicated in penis epithelium and corpus cavernosum before spreading to draining lymph nodes and spleen. All together, those results revealed the first experimental transmission model for MHV-68 in laboratory mice. In the future, this model could help us to better understand the biology of gammaherpesviruses and could also allow the development of strategies that could prevent

  10. Effect of doxorubicin/pluronic SP1049C on tumorigenicity, aggressiveness, DNA methylation and stem cell markers in murine leukemia.

    Daria Y Alakhova

    Full Text Available PURPOSE: Pluronic block copolymers are potent sensitizers of multidrug resistant cancers. SP1049C, a Pluronic-based micellar formulation of doxorubicin (Dox has completed Phase II clinical trial and demonstrated safety and efficacy in patients with advanced adenocarcinoma of the esophagus and gastroesophageal junction. This study elucidates the ability of SP1049C to deplete cancer stem cells (CSC and decrease tumorigenicity of cancer cells in vivo. EXPERIMENTAL DESIGN: P388 murine leukemia ascitic tumor was grown in BDF1 mice. The animals were treated with: (a saline, (b Pluronics alone, (c Dox or (d SP1049C. The ascitic cancer cells were isolated at different passages and examined for 1 in vitro colony formation potential, 2 in vivo tumorigenicity and aggressiveness, 3 development of drug resistance and Wnt signaling activation 4 global DNA methylation profiles, and 5 expression of CSC markers. RESULTS: SP1049C treatment reduced tumor aggressiveness, in vivo tumor formation frequency and in vitro clonogenic potential of the ascitic cells compared to drug, saline and polymer controls. SP1049C also prevented overexpression of BCRP and activation of Wnt-β-catenin signaling observed with Dox alone. Moreover, SP1049C significantly altered the DNA methylation profiles of the cells. Finally, SP1049C decreased CD133(+ P388 cells populations, which displayed CSC-like properties and were more tumorigenic compared to CD133(- cells. CONCLUSIONS: SP1049C therapy effectively suppresses the tumorigenicity and aggressiveness of P388 cells in a mouse model. This may be due to enhanced activity of SP1049C against CSC and/or altered epigenetic regulation restricting appearance of malignant cancer cell phenotype.

  11. The Effect of Noscapine on Oxygen-Glucose Deprivation on Primary Murine Cortical Neurons in High Glucose Condition.

    Vahabzadeh, Gelareh; Ebrahimi, Soltan-Ahmed; Rahbar-Roshandel, Nahid; Mahmoudian, Massoud


    In the present work we set out to investigate the neuroprotective effects of noscapine (0.5-2 µM) in presence of D-glucose on primary murine foetal cortical neurons after oxygen-glucose deprivation/24 h. recovery. Cell viability, nitric oxide production and intracellular calcium ((ca(2+))i) levels were evaluated by MTT assay, the modified Griess method and Fura-2 respectively. 25 and 100 mM D-glucose could, in a concentration dependent manner, improve cell viability and decrease NO production and (ca(2+))i level in neuronal cells after ischemic insult. Moreover, pre-incubation of cells with noscapine, noticeably enhanced protective effects of 25 and 100 mM D-glucose compared to similar conditions without noscapine pre-treatment. In fact, noscapine attenuated NO production in a dose-dependent fashion, after 30 minutes (min) OGD, during high-glucose (HG) condition in cortical neurons. Pretreatment with 2 μM noscapine and 25 or 100 mM D-glucose, was shown to decrease the rise in (ca(2+))i induced by Sodium azide/glucose deprivation (chemical OGD) model. These effects were more pronounced than that of 25 or 100 mM D-glucose alone. The present study demonstrated that the neuroprotective effects of HG before an ischemic insult were augmented by pre-treatment with noscapine. Our results also suggested that the neuroprotection offered by both HG and noscapine involve attenuation of NO production and (ca(2+))i levels stimulated by the experimental ischemia in cortical neurons.

  12. Cordyceps Militaris Alleviates Severity of Murine Acute Lung Injury Through miRNAs-Mediated CXCR2 Inhibition

    Sheng Liu


    Full Text Available Background/Aims: Acute lung injury (ALI and acute respiratory distress syndrome (ARDS are lethal diseases in humans, and the current treatments have limited therapeutic effects. Cordyceps militaris (CM is a caterpillar-grown traditional medicinal mushroom, and has been used as a natural invigorant for longevity, endurance, and vitality in China. Recently, purified extracts from CM have been shown to have beneficial effects on various diseases including cancer. Nevertheless, a role of CM in ALI has not been examined previously. Methods: Here, we used a bleomycin-induced ALI model to study the effects of CM on the severity of ALI in mice. The levels of CXCR2, a receptor for Interleukin 8 (IL-8 in pulmonary microvascular endothelial cells, were examined in different experimental groups. The levels of microRNA (miR-1321 and miR-3188 were also examined in lung samples and in CM. Adeno-associated viruses carrying miR-1321 and miR-3188 were injected into bleomycin-treated mice for evaluation their effects on the severity of ALI. Results: CM treatment significantly alleviated the severity of bleomycin-induced ALI in mice. The increases in lung CXCR2 by bleomycin were significantly reduced by CM at protein level, but not at mRNA level. CM contained high levels of 2 miRNAs (miR-1321 and miR-3188 that target 3'-UTR of CXCR2 mRNA to inhibit its expression. Overexpression of miR-1321 and miR-3188 in mouse lung through AAV-mediated gene therapy mimicked the effects of CM. Conclusion: CM may alleviate severity of murine ALI through miRNAs-mediated CXCR2 inhibition.

  13. Blockade of CCR2 reduces macrophage influx and development of chronic renal damage in murine renovascular hypertension.

    Kashyap, Sonu; Warner, Gina M; Hartono, Stella P; Boyilla, Rajendra; Knudsen, Bruce E; Zubair, Adeel S; Lien, Karen; Nath, Karl A; Textor, Stephen C; Lerman, Lilach O; Grande, Joseph P


    Renovascular hypertension (RVH) is a common cause of both cardiovascular and renal morbidity and mortality. In renal artery stenosis (RAS), atrophy in the stenotic kidney is associated with an influx of macrophages and other mononuclear cells. We tested the hypothesis that chemokine receptor 2 (CCR2) inhibition would reduce chronic renal injury by reducing macrophage influx in the stenotic kidney of mice with RAS. We employed a well-established murine model of RVH to define the relationship between macrophage infiltration and development of renal atrophy in the stenotic kidney. To determine the role of chemokine ligand 2 (CCL2)/CCR2 signaling in the development of renal atrophy, mice were treated with the CCR2 inhibitor RS-102895 at the time of RAS surgery and followed for 4 wk. Renal tubular epithelial cells expressed CCL2 by 3 days following surgery, a time at which no significant light microscopic alterations, including interstitial inflammation, were identified. Macrophage influx increased with time following surgery. At 4 wk, the development of severe renal atrophy was accompanied by an influx of inducible nitric oxide synthase (iNOS)+ and CD206+ macrophages that coexpressed F4/80, with a modest increase in macrophages coexpressing arginase 1 and F4/80. The CCR2 inhibitor RS-102895 attenuated renal atrophy and significantly reduced the number of dual-stained F4/80+ iNOS+ and F4/80+ CD206+ but not F4/80+ arginase 1+ macrophages. CCR2 inhibition reduces iNOS+ and CD206+ macrophage accumulation that coexpress F4/80 and renal atrophy in experimental renal artery stenosis. CCR2 blockade may provide a novel therapeutic approach to humans with RVH.

  14. Influence of the antifolate drug Methotrexate on the development of murine neural tube defects and genomic instability.

    Zhao, Jie; Guan, Tao; Wang, Jianhua; Xiang, Qian; Wang, Mingsheng; Wang, Xiuwei; Guan, Zhen; Xie, Qiu; Niu, Bo; Zhang, Ting


    Impaired folate metabolism is considered a risk factor for neural tube defects (NTDs). However, the relationship between folate deficiency and the risk of NTDs remains unclear, because experimentally induced dietary folate deficiency is insufficient to cause NTDs in non-mutant mice. Methotrexate (MTX) is a specific folate antagonist that competitively inhibits dihydrofolate reductase (DHFR) activity. The objective of this study was to develop a folate dysmetabolism murine model, and study the development of NTDs and its mechanism. Pregnant mice were injected with different doses of MTX [0, 0.5, 1.0, 3.0, 4.5 and 6.0 mg kg(-1) body weight (b/w) intraperitoneally (i.p.)] on gestational day 7.5 and sacrificed on gestational day 11.5. DHFR activity in embryonic tissues was detected, and folate concentrations were analyzed using LC/MS/MS. Copy number variations (CNVs) in neural tube tissues were detected using array comparative genomic hybridization (aCGH). A dose of MTX 4.5 mg kg(-1) b/w, resulted in the highest incidence of NTDs (31.4%) compared with the other groups, and DHFR activities, 5-MeTHF and 5-FoTHF concentrations in embryonic tissues decreased significantly after MTX injection. Furthermore, we found three high-confidence CNVs on chromosome X using aCGH, which was confirmed by RT-PCR and MassARRAY. These results indicate that MTX could cause a folate-associated dysmetabolism, which is similar to that of dietary folate deficiency in mice. The presence of CNVs in neural tube tissues was associated with the development of NTDs.

  15. Extracellular administration of BCL2 protein reduces apoptosis and improves survival in a murine model of sepsis.

    Akiko Iwata

    Full Text Available BACKGROUND: Severe sepsis and septic shock are major causes of morbidity and mortality worldwide. In experimental sepsis there is prominent apoptosis of various cell types, and genetic manipulation of death and survival pathways has been shown to modulate organ injury and survival. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the effect of extracellular administration of two anti-apoptotic members of the BCL2 (B-cell lymphoma 2 family of intracellular regulators of cell death in a murine model of sepsis induced by cecal ligation and puncture (CLP. We show that intraperitoneal injection of picomole range doses of recombinant human (rh BCL2 or rhBCL2A1 protein markedly improved survival as assessed by surrogate markers of death. Treatment with rhBCL2 or rhBCL2A1 protein significantly reduced the number of apoptotic cells in the intestine and heart following CLP, and this was accompanied by increased expression of endogenous mouse BCL2 protein. Further, mice treated with rhBCL2A1 protein showed an increase in the total number of neutrophils in the peritoneum following CLP with reduced neutrophil apoptosis. Finally, although neither BCL2 nor BCL2A1 are a direct TLR2 ligand, TLR2-null mice were not protected by rhBCL2A1 protein, indicating that TLR2 signaling was required for the protective activity of extracellularly adminsitered BCL2A1 protein in vivo. CONCLUSIONS/SIGNIFICANCE: Treatment with rhBCL2A1 or rhBCL2 protein protects mice from sepsis by reducing apoptosis in multiple target tissues, demonstrating an unexpected, potent activity of extracellularly administered BCL2 BH4-domain proteins.

  16. Acetamiprid Accumulates in Different Amounts in Murine Brain Regions

    Hayato Terayama


    Full Text Available Neonicotinoids such as acetamiprid (ACE belong to a new and widely used single class of pesticides. Neonicotinoids mimic the chemical structure of nicotine and share agonist activity with the nicotine acetylcholine receptor (nAchR. Neonicotinoids are widely considered to be safe in humans; however, they have recently been implicated in a number of human health disorders. A wide range of musculoskeletal and neuromuscular disorders associated with high doses of neonicotinoids administered to animals have also been reported. Consequently, we used a mouse model to investigate the response of the central nervous system to ACE treatment. Our results show that exposure to ACE-containing water for three or seven days (decuple and centuple of no observable adverse effect level (NOAEL/day caused a decrease in body weight in 10-week old A/JJmsSlc (A/J mice. However, the treatments did not affect brain histology or expression of CD34. ACE concentrations were significantly higher in the midbrain of ACE-treated mice than that of the normal and vehicle groups. Expression levels of α7, α4, and β2 nAChRs were found to be low in the olfactory bulb and midbrain of normal mice. Furthermore, in the experimental group (centuple ACE-containing water for seven days, β2 nAChR expression decreased in many brain regions. Information regarding the amount of accumulated ACE and expression levels of the acetylcholine receptor in each region of the brain is important for understanding any clinical symptoms that may be associated with ACE exposure.

  17. Typhonium flagelliforme decreases protein expression in murine breast cancer

    Chodidjah Chodidjah


    Full Text Available BACKGROUND Breast cancer treatment is still ineffective, having also various side effects. Breast cancer growth is affected by human epidermal growth factor receptor 2 (HER2/neu and B cell lymphoma 2 (BCL2 expression. In vitro studies on continuous culture of continuous culture of human lymphoblasts (CEMs showed that Typhonium flagelliforme (TF increases apoptosis. The objective of this study was to evaluate whether TF syrup (TFS could decrease HER2/ neu and BCL2 expression as well as breast cancer volume (BCV in mice. METHODS An experimental post-test only control group design was conducted on 18 C3H mice with breast cancers, randomly allocated to 3 groups of 6. Group 1 received 0.2 ml of distilled water. Group 2 and 3 animals were each given 0.2 ml of 40 mg/ml and 80 mg/ml TFS, respectively. The treatment was given orally once daily for 25 days. Assessment of HER2/neu and BCL2 expression was by immunohistochemistry, whereas BCV was measured by caliper. Anova and LSD were used for data analysis. RESULTS There was a significant difference in HER2/neu and BCL2 expression as well as in BCV among the treatment groups. LSD analysis showed that HER2/neu and BCL2 expression in group 3 (51.60%; 24.60% was significantly lower than in group 1 (245.40%; 114.40% as well as group 2 (235.50%; 54.20% (p=0.000. BCV in group 3 (4392.33 mm3 was significantly greater than BCV in group 1 (253.87 mm3 (p=0.002, but was not significantly different from BCV in group 2 (3667.16 mm3 (p=0.306. CONCLUSION Suplementation with TFS decreases HER2/neu and BCL2 expression. TF appears to be a promising plant demonstarting anti cancer activity.

  18. Acetamiprid Accumulates in Different Amounts in Murine Brain Regions

    Terayama, Hayato; Endo, Hitoshi; Tsukamoto, Hideo; Matsumoto, Koichi; Umezu, Mai; Kanazawa, Teruhisa; Ito, Masatoshi; Sato, Tadayuki; Naito, Munekazu; Kawakami, Satoshi; Fujino, Yasuhiro; Tatemichi, Masayuki; Sakabe, Kou


    Neonicotinoids such as acetamiprid (ACE) belong to a new and widely used single class of pesticides. Neonicotinoids mimic the chemical structure of nicotine and share agonist activity with the nicotine acetylcholine receptor (nAchR). Neonicotinoids are widely considered to be safe in humans; however, they have recently been implicated in a number of human health disorders. A wide range of musculoskeletal and neuromuscular disorders associated with high doses of neonicotinoids administered to animals have also been reported. Consequently, we used a mouse model to investigate the response of the central nervous system to ACE treatment. Our results show that exposure to ACE-containing water for three or seven days (decuple and centuple of no observable adverse effect level (NOAEL)/day) caused a decrease in body weight in 10-week old A/JJmsSlc (A/J) mice. However, the treatments did not affect brain histology or expression of CD34. ACE concentrations were significantly higher in the midbrain of ACE-treated mice than that of the normal and vehicle groups. Expression levels of α7, α4, and β2 nAChRs were found to be low in the olfactory bulb and midbrain of normal mice. Furthermore, in the experimental group (centuple ACE-containing water for seven days), β2 nAChR expression decreased in many brain regions. Information regarding the amount of accumulated ACE and expression levels of the acetylcholine receptor in each region of the brain is important for understanding any clinical symptoms that may be associated with ACE exposure. PMID:27669271




    An overview is given of the potential for neutrino physics studies through parasitic use of the intense high energy neutrino beams that would be produced at future many-TeV muon colliders. Neutrino experiments clearly cannot compete with the collider physics. Except at the very highest energy muon colliders, the main thrust of the neutrino physics program would be to improve on the measurements from preceding neutrino experiments at lower energy muon colliders, particularly in the fields of B physics, quark mixing and CP violation. Muon colliders at the 10 TeV energy scale might already produce of order 10{sup 8} B hadrons per year in a favorable and unique enough experimental environment to have some analytical capabilities beyond any of the currently operating or proposed B factories. The most important of the quark mixing measurements at these energies might well be the improved measurements of the important CKM matrix elements {vert_bar}V{sub ub}{vert_bar} and {vert_bar}V{sub cb}{vert_bar} and, possibly, the first measurements of {vert_bar}V{sub td}{vert_bar} in the process of flavor changing neutral current interactions involving a top quark loop. Muon colliders at the highest center-of-mass energies that have been conjectured, 100--1,000 TeV, would produce neutrino beams for neutrino-nucleon interaction experiments with maximum center-of-mass energies from 300--1,000 GeV. Such energies are close to, or beyond, the discovery reach of all colliders before the turn-on of the LHC. In particular, they are comparable to the 314 GeV center-of-mass energy for electron-proton scattering at the currently operating HERA collider and so HERA provides a convenient benchmark for the physics potential. It is shown that these ultimate terrestrial neutrino experiments, should they eventually come to pass, would have several orders of magnitude more luminosity than HERA. This would potentially open up the possibility for high statistics studies of any exotic particles, such as

  20. Murine cysticercosis model: influence of the infection time and the time of treatment on the cysticidal efficacy of albendazole and praziquantel.

    Palomares-Alonso, Francisca; Palencia Hernández, Guadalupe; Rojas-Tomé, Irma Susana; Jung-Cook, Helgi; Pinzón-Estrada, Enrique


    In the search of new alternatives for neurocysticercosis treatment, Taenia crassiceps ORF strain cysticerci have been used instead of T. solium for in vitro studies. Up to date, the main criteria for the use of the murine cysticercosis model for drug efficacy evaluation have not been assessed. The aim of the present study was to evaluate the influence of two of the main variables related to the in vivo efficacy: the length of drug treatment and the starting time of treatment after experimental infection, using albendazole (ABZ) and praziquantel (PZQ) as test drugs. Additionally, the relationship between the number of cysts and the parasite weight was assessed. For the study, female BALB/c mice were experimentally infected with T. crassiceps cysts. Three different post-infection periods (10, 20 and 30 days) and three different lengths of treatment with ABZ or PZQ (10, 20 and 30 days) were selected. The efficacy of each treatment was evaluated by comparison with a control group. Our results show that for in vivo efficacy studies, the best time to start the drug treatment is 10 days post-infection and that a minimum of 20 days of treatment is required when ABZ or PZQ are used as positive control. Moreover, in this model the parasite weight can be used as a rapid tool to measure the in vivo drug activity.

  1. CCR5 knockout suppresses experimental autoimmune encephalomyelitis in C57BL/6 mice


    Multiple sclerosis (MS) is an inflammatory disease in which myelin in the spinal cord is damaged. C-C chemokine receptor type 5 (CCR5) is implicated in immune cell migration and cytokine release in central nervous system (CNS). We investigated whether CCR5 plays a role in MS progression using a murine model, experimental autoimmune encephalomyelitis (EAE), in CCR5 deficient (CCR5−/−) mice. CCR5−/− and CCR5+/+ (wild-type) mice were immunized with myelin oligodendrocyte glycoprotein 35-55 (MOG3...

  2. An elegant technique for ex vivo imaging in experimental research—Optical coherence tomography (OCT)

    Tschernig, T.; Thrane, Lars; Jørgensen, Thomas Martini


    Optical coherence tomography (OCT) is an elegant technology for imaging of tissues and organs and has been established for clinical use for around a decade. Thus, it is used in vivo but can also serve as a valuable ex vivo imaging tool in experimental research. Here, a brief overview is given...... with a focus on an ex vivo application of OCT. Image and video examples of freshly obtained murine lungs are included. The main advantage of OCT for ex vivo analysis is the non-contact, non-invasive, and non-destructive fast acquisition of a three-dimensional data set with micrometer-resolution....

  3. Immunogenicity of the P-8 amastigote antigen in the experimental model of canine visceral leishmaniasis

    Carrillo, E.; S. Ahmed; Goldsmith-Pestana, K.; Nieto, J; Y Osorio; Travi,B; Moreno, J; McMahon-Pratt, D


    The P-8 proteoglycolipid complex (P-8 PGLC), an amastigote antigen of L. pifanoi, has been demonstrated to induce protection in mouse models, as well as to induce Tc1/Th1-like cellular responses in American cutaneous leishmaniasis patients. Because the immunization with P-8 PGLC in the murine model does not appear to be genetically restricted, we have studied the reactivity of the P-8 PGLC in L. infantum infected dogs. In this study, it is shown that PBMC from experimentally infected dogs (as...

  4. Is animal experimentation fundamental?

    d'Acampora, Armando José; Rossi, Lucas Félix; Ely, Jorge Bins; de Vasconcellos, Zulmar Acciolli


    The understanding about the utilization of experimental animals in scientific research and in teaching is many times a complex issue. Special attention needs to be paid to attain the understanding by the general public of the importance of animal experimentation in experimental research and in undergraduate medical teaching. Experimental teaching and research based on the availability of animals for experimentation is important and necessary for the personal and scientific development of the physician-to-be. The technological arsenal which intends to mimic experimentation animals and thus fully replace their use many times does not prove to be compatible with the reality of the living animal. The purpose of this paper is to discuss aspects concerning this topic, bringing up an issue which is complex and likely to arouse in-depth reflections.

  5. Mapping the global mRNA transcriptome during development of the murine first molar

    Maria A. eLandin


    Full Text Available The main objective of this study was to map global gene expression in order to provide information about the populations of mRNA species participating in murine tooth development at 24 h intervals, starting at the eleventh embryonic day (E11.5 up to the seventh post-natal day (P7. The levels of RNA species expressed during murine tooth development were mesured using a total of 58 deoxyoligonucleotide microarrays. Microarray data was validated using real-time RT-PCR. Differentially expressed genes (p<0.05 were subjected to bioinformatic analysis to identify cellular activities significantly associated with these genes. Using ANOVA the microarray data yielded 4362 genes as being differentially expressed from the elleventh embryonic day (E11.5 up to seven days post-natal (P7, 1921 of these being genes without known functions. The remaining 2441 genes were subjected to further statistical analysis using a supervised procedure.Bioinformatic analysis results for each time-point studied suggests that the main molecular functions associated with genes expressed at the early pre-natal stages (E12.5-E18.5 studied were cell cycle progression, cell morphology, lipid metabolism, cellular growth, proliferation, senescence and apoptosis, whereas most genes expressed at post-natal and secretory stages (P0- P7 were significantly associated with regulation of cell migration, biosynthesis, differentiation, oxidative stress, polarization and cell death. Differentially expressed genes (DE not described earlier during murine tooth development; Inositol 1, 4, 5-triphosphate receptor 3 (Itpr3, metallothionein 1(Mt1, cyclin-dependent kinase 4 (Cdk4, cathepsin D (Ctsd, keratin complex 2, basic, gene 6a (Krt2-6a, cofilin 1, non-muscle (Cfl1, cyclin 2 (Ccnd2, were verified by real-time RT-PCR.

  6. Oatp-associated uptake and toxicity of microcystins in primary murine whole brain cells.

    Feurstein, D; Holst, K; Fischer, A; Dietrich, D R


    Microcystins (MCs) are naturally occurring cyclic heptapeptides that exhibit hepato-, nephro- and possibly neurotoxic effects in mammals. Organic anion transporting polypeptides (rodent Oatp/human OATP) appear to be specifically required for active uptake of MCs into hepatocytes and kidney epithelial cells. Based on symptoms of neurotoxicity in MC-intoxicated patients and the presence of Oatp/OATP at the blood-brain-barrier (BBB) and blood-cerebrospinal-fluid-barrier (BCFB) it is hypothesized that MCs can be transported across the BBB/BCFB in an Oatp/OATP-dependent manner and can induce toxicity in brain cells via inhibition of protein phosphatase (PP). To test these hypotheses, the presence of murine Oatp (mOatp) in primary murine whole brain cells (mWBC) was investigated at the mRNA and protein level. MC transport was tested by exposing mWBCs to three different MC-congeners (MC-LR, -LW, -LF) with/without co-incubation with the OATP/Oatp-substrates taurocholate (TC) and bromosulfophthalein (BSP). Uptake of MCs and cytotoxicity was demonstrated via MC-Western blot analysis, immunocytochemistry, cell viability and PP inhibition assays. All MC congeners bound covalently and inhibited mWBC PP. MC-LF was the most cytotoxic congener followed by -LW and -LR. The lowest toxin concentration significantly reducing mWBC viability after 48 h exposure was 400 nM (MC-LF). Uptake of MCs into mWBCs was inhibited via co-incubation with excess TC (50 and 500 microM) and BSP (50 microM). MC-Western blot analysis demonstrated a concentration-dependent accumulation of MCs. In conclusion, the in vitro data support the assumed MC-congener-dependent uptake in a mOatp-associated manner and cytotoxicity of MCs in primary murine whole brain cells.

  7. Novel murine dendritic cell lines: a powerful auxiliary tool for dendritic cell research

    Silvia A Fuertes Marraco


    Full Text Available Research in vitro facilitates discovery, screening and pilot experiments, often preceding research in vivo. Several technical difficulties render Dendritic Cell (DC research particularly challenging, including the low frequency of DC in vivo, thorough isolation requirements, and the vulnerability of DC ex vivo. Critically, there is not as yet a widely accepted human or murine DC line and in vitro systems of DC research are limited. In this study, we report the generation of new murine DC lines, named MutuDC, originating from cultures of splenic CD8α conventional DC (cDC tumors. By direct comparison to normal WT splenic cDC subsets, we describe the phenotypic and functional features of the MutuDC lines and show that they have retained all the major features of their natural counterpart in vivo, the splenic CD8α cDC. These features include expression of surface markers Clec9A, DEC205, and CD24, positive response to TLR3 and TLR9 but not TLR7 stimuli, secretion of cytokines and chemokines upon activation, as well as cross-presentation capacity. In addition to the close resemblance to normal splenic CD8α cDC, a major advantage is the ease of derivation and maintenance of the MutuDC lines, using standard culture medium and conditions, importantly without adding supplementary growth factors or maturation-inducing stimuli to the medium. Furthermore, genetically modified MutuDC lines have been successfully obtained either by lentiviral transduction or by culture of DC tumors originating from genetically modified mice.In view of the current lack of stable and functional DC lines, these novel murine DC lines have the potential to serve as an important auxiliary tool for DC research.

  8. Modulatory Effects and Action Mechanisms of Tryptanthrin on Murine Myeloid Leukemia Cells

    Hoi-Ling Chan; Hon-Yan Yip; Nai-Ki Mak; Kwok-Nam Leung


    Leukemia is the disorder of hematopoietic cell development and is characterized by an uncoupling of cell proliferation and differentiation. There is a pressing need for the development of novel tactics for leukemia therapy as conventional treatments often have severe adverse side effects. Tryptanthrin (6,12-dihydro-6,12-dioxoindolo-(2,1-b)-quinazoline) is a naturally-occurring, weakly basic alkaloid isolated from the dried roots of medicinal indigo plants (Ban-Lan-Gen). It has been reported to have various biological and pharmacological activities, including anti-microbial, anti-inflammatory, immunomodulatory and anti-tumor effects. However, its modulatory effects and action mechanisms on myeloid cells remain poorly understood. In this study, tryptanthrin was shown to suppress the proliferation of the murine myeloid leukemia WEHI-3B JCS cells in a dose- and time-dependent manner. It also significantly reduced the growth of WEHI-3B JCS cells in vivo in syngeneic BALB/c mice. However, it exhibited no significant direct cytotoxicity on normal murine peritoneal macrophages. Flow cytometric analysis showed an obvious cell cycle arrest of the tryptanthrin-treated WEHI-3B JCS cells at the G0/G1 phase. The expression of cyclin D2,D3, Cdk 2, 4 and 6 genes in WEHI-3B JCS cells was found to be down-regulated at 24 h as measured by RT-PCR. Morphological and functional studies revealed that tryptanthrin could induce differentiation in WEHI-3B JCS cells, as shown by the increases in vacuolation, cellular granularity and NBT-reducing activity in tryptanthrin-treated cells. Collectively, our findings suggest that tryptanthrin might exert its anti-tumor effect on the murine myelomonocytic leukemia WEHI-3B JCS cells by causing cell cycle arrest and by triggering cell differentiation. Cellular & Molecular Immunology. 2009;6(5):335-342.

  9. Interferon-Gamma-Induced Nitric Oxide Inhibits the Proliferation of Murine Renal Cell Carcinoma Cells

    David J. Tate Jr., John R. Patterson, Cruz Velasco-Gonzalez, Emily N. Carroll, Janie Trinh, Daniel Edwards, Ashok Aiyar, Beatriz Finkel-Jimenez, Arnold H. Zea


    Full Text Available Renal cell carcinoma (RCC remains one of the most resistant tumors to systemic chemotherapy, radiotherapy, and immunotherapy. Despite great progress in understanding the basic biology of RCC, the rate of responses in animal models and clinical trials using interferons (IFNs has not improved significantly. It is likely that the lack of responses can be due to the tumor's ability to develop tumor escape strategies. Currently, the use of targeted therapies has improved the clinical outcomes of patients with RCC and is associated with an increase of Th1-cytokine responses (IFNγ, indicating the importance of IFNγ in inhibiting tumor proliferation. Thus, the present study was designed to investigate a new mechanism by which IFNγ mediates direct anti-proliferative effects against murine renal cell carcinoma cell lines. When cultured RCC cell lines were exposed to murine recombinant IFNγ, a dose dependent growth inhibition in CL-2 and CL-19 cells was observed; this effect was not observed in Renca cells. Growth inhibition in CL-2 and CL-19 cell lines was associated with the intracellular induction of nitric oxide synthase (iNOS protein, resulting in a sustained elevation of nitric oxide (NO and citrulline, and a decrease in arginase activity. The inhibition of cell proliferation appears to be due to an arrest in the cell cycle. The results indicate that in certain RCC cell lines, IFNγ modulates L-arginine metabolism by shifting from arginase to iNOS activity, thereby developing a potent inhibitory mechanism to encumber tumor cell proliferation and survival. Elucidating the cellular events triggered by IFNγ in murine RCC cell lines will permit anti-tumor effects to be exploited in the development of new combination therapies that interfere with L-arginine metabolism to effectively combat RCC in patients.

  10. Immune tolerance induction using fetal directed placental injection in rodent models: a murine model.

    Kei Takahashi

    Full Text Available Induction of the immune response is a major problem in replacement therapies for inherited protein deficiencies. Tolerance created in utero can facilitate postnatal treatment. In this study, we aimed to induce immune tolerance towards a foreign protein with early gestational cell transplantation into the chorionic villi under ultrasound guidance in the murine model.Pregnant C57BL/6 (B6 mice on day 10 of gestation were anesthetized and imaged by high resolution ultrasound. Murine embryos and their placenta were positioned to get a clear view in B-mode with power mode of the labyrinth, which is the equivalent of chorionic villi in the human. Bone marrow cells (BMCs from B6-Green Fluorescence Protein (B6GFP transgenic mice were injected into the fetal side of the placenta which includes the labyrinth with glass microcapillary pipettes. Each fetal mouse received 2 x 105 viable GFP-BMCs. After birth, we evaluated the humoral and cell-mediated immune response against GFP.Bone marrow transfer into fetal side of placenta efficiently distributed donor cells to the fetal mice. The survival rate of this procedure was 13.5%(5 out of 37. Successful engraftment of the B6-GFP donor skin grafts was observed in all recipient (5 out of 5 mice 6 weeks after birth. Induction of anti-GFP antibodies was completely inhibited. Cytotoxic immune reactivity of thymic cells against cells harboring GFP was suppressed by ELISPOT assay.In this study, we utilized early gestational placental injection targeting the murine fetus, to transfer donor cells carrying a foreign protein into the fetal circulation. This approach is sufficient to induce both humoral and cell-mediated immune tolerance against the foreign protein.

  11. Development of renal atrophy in murine 2 kidney 1 clip hypertension is strain independent.

    Kashyap, Sonu; Boyilla, Rajendra; Zaia, Paula J; Ghossan, Roba; Nath, Karl A; Textor, Stephen C; Lerman, Lilach O; Grande, Joseph P


    The murine 2-kidney 1-clip (2K1C) model has been used to identify mechanisms underlying chronic renal disease in human renovascular hypertension. Although this model recapitulates many of the features of human renovascular disease, strain specific variability in renal outcomes and animal-to-animal variation in the degree of arterial stenosis are well recognized limitations. In particular, the C57BL/6J strain is considered to be resistant to chronic renal damage in other models. Our objectives were to determine strain dependent variations in renal disease progression and to identify parameters that predict renal atrophy in murine 2K1C hypertension. We used a 0.20mm polytetrafluoroethylene cuff to establish RAS in 3 strains of mice C57BL/6J (N=321), C57BLKS/J (N=177) and129Sv (N=156). The kidneys and hearts were harvested for histopathologic analysis after 3days or after 1, 2, 4, 6, 7, 11 or 17weeks. We performed multivariate analysis to define associations between blood pressure, heart and kidney weights, ratio of stenotic kidney/contralateral kidney (STK/CLK) weight, percent atrophy (% atrophy) and plasma renin content. The STK of all 3 strains showed minimal histopathologic alterations after 3days, but later developed progressive interstitial fibrosis, tubular atrophy, and inflammation. The STK weight negatively correlated with maximum blood pressure and % atrophy, and positively correlated with STK/CLK ratio. RAS produces severe chronic renal injury in the STK of all murine strains studied, including C57BL/6J. Systolic blood pressure is negatively associated with STK weight, STK/CLK ratio and positively with atrophy and may be used to assess adequacy of vascular stenosis in this model.

  12. Cytokine combinations on the potential for ex vivo expansion of murine hematopoietic stem cells.

    Lui, Wing Chi; Chan, Yuen Fan; Chan, Li Chong; Ng, Ray Kit


    Hematopoietic stem cell (HSC) is a rare cell population, which is capable of self-renewal and differentiation to all blood lineages. The clinical potential of HSCs for treating hematological disorders has led to the use of cytokine stimulation for ex vivo expansion. However, little is known about the molecular features of the HSC populations expanded under different cytokine combinations. We studied the expansion of murine HSCs cultured with six different cytokine combinations under serum-containing or serum-free conditions for 14days. We found that all the cytokine combinations promoted expansion of murine HSCs. Although SCF/IL-3/IL-6 induced the highest expansion of the immunophenotypic Lineage(-)Sca-1(+)c-Kit(+) (LSK) cells at day 14, over 90% of them were FcεRIα(+) mast cells. In contrast, the serum-free medium with SCF/Flt3-L/IL-11 effectively promoted the expansion of LSK/FcεRIα(-) HSCs by over 50-fold. HSCs expanded by SCF/Flt3-L/IL-11 combination formed compact hematopoietic colonies and demonstrated a higher degree of multipotency compared to the HSCs cultured with other cytokine combinations. Surprisingly, despite the same LSK/FcεRIα(-) immunophenotype, HSCs cultured with different cytokine combinations demonstrated differential patterns of hematopoietic gene expression. HSCs cultured with SCF/Flt3-L/IL-11 maintained a transcription profile resembling that of freshly isolated HSCs. We propose that serum-free medium supplemented with SCF/Flt3-L/IL-11 is the optimal culture condition to maintain the stemness of ex vivo expanded HSCs. This study used molecular characterization of cytokine-expanded murine HSCs to facilitate the selection of cytokine combinations that could induce fully competent HSC for clinical applications.

  13. Correction of murine hemoglobinopathies by prenatal tolerance induction and postnatal nonmyeloablative allogeneic BM transplants.

    Peranteau, William H; Hayashi, Satoshi; Abdulmalik, Osheiza; Chen, Qiukan; Merchant, Aziz; Asakura, Toshio; Flake, Alan W


    Sickle cell disease (SCD) and thalassemias (Thal) are common congenital disorders, which can be diagnosed early in gestation and result in significant morbidity and mortality. Hematopoietic stem cell transplantation, the only curative therapy for SCD and Thal, is limited by the absence of matched donors and treatment-related toxicities. In utero hematopoietic stem cell transplantation (IUHCT) is a novel nonmyeloablative transplant approach that takes advantage of the immunologic immaturity and normal developmental properties of the fetus to achieve mixed allogeneic chimerism and donor-specific tolerance (DST). We hypothesized that a combined strategy of IUHCT to induce DST, followed by postnatal nonmyeloablative same donor "booster" bone marrow (BM) transplants in murine models of SCD and Thal would result in high levels of allogeneic engraftment and donor hemoglobin (Hb) expression with subsequent phenotypic correction of SCD and Thal. Our results show that: (1) IUHCT is associated with DST and low levels of allogeneic engraftment in the murine SCD and Thal models; (2) low-level chimerism following IUHCT can be enhanced to high-level chimerism and near complete Hb replacement with normal donor Hb with this postnatal "boosting" strategy; and (3) high-level chimerism following IUHCT and postnatal "boosting" results in phenotypic correction in the murine Thal and SCD models. This study supports the potential of IUHCT, combined with a postnatal nonmyelablative "boosting" strategy, to cure Thal and SCD without the toxic conditioning currently required for postnatal transplant regimens while expanding the eligible transplant patient population due to the lack of a restricted donor pool.

  14. Distinct regulation of murine lupus susceptibility genes by the IRF5/Blimp-1 axis.

    Panchanathan, Ravichandran; Liu, Hongzhu; Liu, Hongqi; Fang, Chee-Mun; Erickson, Loren D; Pitha, Paula M; Choubey, Divaker


    Genome-wide association studies have identified lupus susceptibility genes such as IRF5 and PRDM1 (encoding for IFN regulatory factor 5 [IRF]5 and Blimp-1) in the human genome. Accordingly, the murine Irf5 and Prdm1 genes have been shown to play a role in lupus susceptibility. However, it remains unclear how IRF5 and Blimp-1 (a transcriptional target of IRF5) contribute to lupus susceptibility. Given that the murine lupus susceptibility locus Nba2 includes the IFN-regulated genes Ifi202 (encoding for the p202 protein), Aim2 (encoding for the Aim2 protein), and Fcgr2b (encoding for the FcγRIIB receptor), we investigated whether the IRF5/Blimp-1 axis could regulate the expression of these genes. We found that an Irf5 deficiency in mice decreased the expression of Blimp-1 and reduced the expression of the Ifi202. However, the deficiency increased the expression of Aim2 and Fcgr2b. Correspondingly, increased expression of IRF5 in cells increased levels of Blimp-1 and p202 protein. Moreover, Blimp-1 expression increased the expression of Ifi202, whereas it reduced the expression of Aim2. Interestingly, an Aim2 deficiency in female mice increased the expression of IRF5. Similarly, the Fcgr2b-deficient mice expressed increased levels of IRF5. Moreover, increased expression of IRF5 and Blimp-1 in lupus-prone C57BL/6.Nba2, New Zealand Black, and C57BL/6.Sle123 female mice (as compared with age-matched C57BL/6 female mice) was associated with increased levels of the p202 protein. Taken together, our observations demonstrate that the IRF5/Blimp-1 axis differentially regulates the expression of Nba2 lupus susceptibility genes, and they suggest an important role for the IRF5/Blimp-1/p202 axis in murine lupus susceptibility.

  15. Distinct regulation of murine lupus susceptibility genes by the IRF5-Blimp-1 axis

    Panchanathan, Ravichandran; Liu, Hongzhu; Liu, Hongqi; Fang, Chee-Mun; Erickson, Loren D.; Pitha, Paula M.; Choubey, Divaker


    Genome-wide association studies have identified lupus susceptibility genes such as IRF5 and PRDM1 (encoding for the IRF5 and Blimp-1) in the human genome. Accordingly, the murine Irf5 and Prdm1 genes have been shown to play a role in lupus susceptibility. However, it remains unclear how IRF5 and Blimp-1 (a transcriptional target of IRF5) contribute to lupus susceptibility. Given that the murine lupus susceptibility locus Nba2 includes the interferon-regulated genes Ifi202 (encoding for the p202 protein), Aim2 (encoding for the Aim2 protein), and Fcgr2b (encoding for the FcγRIIB receptor), we investigated whether the IRF5-Blimp-1 axis could regulate the expression of these genes. We found that an Irf5-deficiency in mice decreased the expression of Blimp-1 and reduced the expression of the Ifi202. However, the deficiency increased the expression of Aim2 and Fcgr2b. Correspondingly, increased expression of IRF5 in cells increased levels of Blimp-1 and p202 protein. Moreover, Blimp-1 expression increased the expression of Ifi202, whereas it reduced the expression of Aim2. Interestingly, an Aim2-deficiency in female mice increased the expression of IRF5. Similarly, the Fcgr2b-deficient mice expressed increased levels of IRF5. Moreover, increased expression of IRF5 and Blimp-1 in lupus-prone B6.Nba2, NZB, and B6.Sle123 female mice (as compared to age-matched C57BL/6 female mice) was associated with increased levels of the p202 protein. Together, our observations demonstrate that the IRF5-Blimp-1 axis differentially regulates the expression of Nba2 lupus susceptibility genes, and suggest an important role for the IRF5-Blimp-1-p202 axis in murine lupus susceptibility. PMID:22116829

  16. Multivariate modelling with 1H NMR of pleural effusion in murine cerebral malaria

    Ghosh Soumita


    Full Text Available Abstract Background Cerebral malaria is a clinical manifestation of Plasmodium falciparum infection. Although brain damage is the predominant pathophysiological complication of cerebral malaria (CM, respiratory distress, acute lung injury, hydrothorax/pleural effusion are also observed in several cases. Immunological parameters have been assessed in pleural fluid in murine models; however there are no reports of characterization of metabolites present in pleural effusion. Methods 1H NMR of the sera and the pleural effusion of cerebral malaria infected mice were analyzed using principal component analysis, orthogonal partial least square analysis, multiway principal component analysis, and multivariate curve resolution. Results It has been observed that there was 100% occurrence of pleural effusion (PE in the mice affected with CM, as opposed to those are non-cerebral and succumbing to hyperparasitaemia (NCM/HP. An analysis of 1H NMR and SDS-PAGE profile of PE and serum samples of each of the CM mice exhibited a similar profile in terms of constituents. Multivariate analysis on these two classes of biofluids was performed and significant differences were detected in concentrations of metabolites. Glucose, creatine and glutamine contents were high in the PE and lipids being high in the sera. Multivariate curve resolution between sera and pleural effusion showed that changes in PE co-varied with that of serum in CM mice. The increase of glucose in PE is negatively correlated to the glucose in serum in CM as obtained from the result of multiway principal component analysis. Conclusions This study reports for the first time, the characterization of metabolites in pleural effusion formed during murine cerebral malaria. The study indicates that the origin of PE metabolites in murine CM may be the serum. The loss of the components like glucose, glutamine and creatine into the PE may worsen the situation of patients, in conjunction with the enhanced

  17. Adiponectin and plant-derived mammalian adiponectin homolog exert a protective effect in murine colitis

    Arsenescu, Violeta


    Background: Hypoadiponectinemia has been associated with states of chronic inflammation in humans. Mesenteric fat hypertrophy and low adiponectin have been described in patients with Crohn\\'s disease. We investigated whether adiponectin and the plant-derived homolog, osmotin, are beneficial in a murine model of colitis. Methods: C57BL/6 mice were injected (i.v.) with an adenoviral construct encoding the full-length murine adiponectin gene (AN+DSS) or a reporter-LacZ (Ctr and V+DSS groups) prior to DSS colitis protocol. In another experiment, mice with DSS colitis received either osmotin (Osm+DSS) or saline (DSS) via osmotic pumps. Disease progression and severity were evaluated using body weight, stool consistency, rectal bleeding, colon lengths, and histology. In vitro experiments were carried out in bone marrow-derived dendritic cells. Results: Mice overexpressing adiponectin had lower expression of proinflammatory cytokines (TNF, IL-1β), adipokines (angiotensin, osteopontin), and cellular stress and apoptosis markers. These mice had higher levels of IL-10, alternative macrophage marker, arginase 1, and leukoprotease inhibitor. The plant adiponectin homolog osmotin similarly improved colitis outcome and induced robust IL-10 secretion. LPS induced a state of adiponectin resistance in dendritic cells that was reversed by treatment with PPARγ agonist and retinoic acid. Conclusion: Adiponectin exerted protective effects during murine DSS colitis. It had a broad activity that encompassed cytokines, chemotactic factors as well as processes that assure cell viability during stressful conditions. Reducing adiponectin resistance or using plant-derived adiponectin homologs may become therapeutic options in inflammatory bowel disease. © 2011 Springer Science+Business Media, LLC.

  18. Preliminary characterization of a murine model for 1-bromopropane neurotoxicity: Role of cytochrome P450.

    Zong, Cai; Garner, C Edwin; Huang, Chinyen; Zhang, Xiao; Zhang, Lingyi; Chang, Jie; Toyokuni, Shinya; Ito, Hidenori; Kato, Masashi; Sakurai, Toshihiro; Ichihara, Sahoko; Ichihara, Gaku


    Neurotoxicity of 1-bromopropane (1-BP) has been reported in both human cases and animal studies. To date, neurotoxicity of 1-BP has been induced in rats but not in mice due to the lethal hepatotoxicity of 1-BP. Oxidization by cytochromes P450 and conjugation with glutathione (GSH) are two critical metabolism pathways of 1-BP and play important roles in toxicity of 1-BP. The aim of the present study was to establish a murine model of 1-BP neurotoxicity, by reducing the hepatotoxicity of 1-BP with 1-aminobenzotriazole (1-ABT); a commonly used nonspecific P450s inhibitor. The results showed that subcutaneous or intraperitoneal injection of 1-ABT at 50mg/kg body weight BID (100mg/kg BW/day) for 3days, inhibited about 92-96% of hepatic microsomal CYP2E1 activity, but only inhibited about 62-64% of CYP2E1 activity in brain microsomes. Mice treated with 1-ABT survived even after exposure to 1200ppm 1-BP for 4 weeks and histopathological studies showed that treatment with 1-ABT protected mice from 1-BP-induced hepatic necrosis, hepatocyte degeneration, and hemorrhage. After 4-week exposure to 1-BP, the brain weight of 1-ABT(+)/1200ppm 1-BP group was decreased significantly. In 1-ABT-treated groups, expression of hippocampal Ran protein and cerebral cortical GRP78 was dose-dependently increased by exposure to 1-BP. We conclude that the control of hepatic P450 activity allows the observation of effects of 1-BP on the murine brain at a higher concentration by reduction of hepatotoxicity. The study suggests that further experiments with liver-specific control of P450 activity using gene technology might provide better murine models for 1-bromopropane-induced neurotoxicity.

  19. Immunotherapeutic targeting of shared melanoma-associated antigens in a murine glioma model.

    Prins, Robert M; Odesa, Sylvia K; Liau, Linda M


    Immune-based treatments for central nervous system gliomas have traditionally lagged behind those of more immunogenic tumors such as melanoma. The relative paucity of defined glioma-associated antigens that can be targeted by the immune system may partially account for this situation. Antigens present on melanomas have been extensively characterized, both in humans and in murine preclinical models. Melanocytes and astrocytes are both derived embryologically from the neural ectoderm. Their neoplastic counterparts, malignant melanomas and gliomas, have been shown in humans to share common antigens at the RNA level. However, little is known concerning whether gliomas can be targeted by immune-based strategies that prime T cells to epitopes from melanoma-associated antigens (MAAs). In this study, we provide evidence that two common murine glioma cell lines (GL26 and GL261) express the melanoma antigens gp100 and tyrosinase-related protein 2 (TRP-2). To understand the immunogenicity of murine gliomas to CD8(+) T cells, we examined the ability of a MAA-specific CTL cell line to lyse the glioma cells, as well as the in vivo expansion of MAA-specific CD8(+) T cells in animals harboring gliomas. Both glioma cell lines were lysed by a human gp100-specific CTL cell line in vitro. Mice harboring s.c. GL26 gliomas possessed TRP-2-specific CD8(+) T cells, providing further evidence that these gliomas express the protein products in the context of MHC class I. Furthermore, MAA peptide-pulsed dendritic cells could prime T cells that specifically recognize GL26 glioma cells in vitro. Lastly, mice that were prevaccinated with human gp100 and TRP-2 peptide-pulsed dendritic cells had significantly extended survival when challenged with tumor cells in the brain, resulting in >50% long-term survival. These results suggest that shared MAAs on gliomas can be targeted immunotherapeutically, pointing the way to a new potential treatment option for patients with malignant gliomas.

  20. SuperSILAC Quantitative Proteome Profiling of Murine Middle Ear Epithelial Cell Remodeling with NTHi.

    Stéphanie Val

    Full Text Available Chronic Otitis Media with effusion (COME develops after sustained inflammation and is characterized by secretory middle ear epithelial metaplasia and effusion, most frequently mucoid. Non-typeable Haemophilus influenzae (NTHi, the most common acute Otitis Media (OM pathogen, is postulated to promote middle ear epithelial remodeling in the progression of OM from acute to chronic. The goals of this study were to examine histopathological and quantitative proteomic epithelial effects of NTHi challenge in a murine middle ear epithelial cell line.NTHi lysates were generated and used to stimulate murine epithelial cells (mMEEC cultured at air-liquid interface over 48 hours- 1 week. Conditional quantitative Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC of cell lysates was performed to interrogate the global protein production in the cells, using the SuperSILAC technique. Histology of the epithelium over time was done to measure bacterial dependent remodeling.Mass spectrometry analysis identified 2,565 proteins across samples, of which 74 exhibited differential enrichment or depletion in cell lysates (+/-2.0 fold-change; p value<0.05. The key molecular functions regulated by NTHi lysates exposure were related to cell proliferation, death, migration, adhesion and inflammation. Finally, chronic exposure induced significant epithelial thickening of cells grown at air liquid interface.NTHi lysates drive pathways responsible of cell remodeling in murine middle ear epithelium which likely contributes to observed epithelial hyperplasia in vitro. Further elucidation of these mediators will be critical in understanding the progression of OM from acute to chronic at the molecular level.