Anatomy and Histology of the Human and Murine Prostate.

PubMed

Ittmann, Michael

2018-05-01

The human and murine prostate glands have similar functional roles in the generation of seminal fluid to assist in reproduction. There are significant differences in the anatomy and histology of murine and human prostate and knowledge of the normal anatomy and histology of the murine prostate is essential to interpreting changes in genetically engineered mouse models. In this review, the normal anatomy and histology of both human and mouse prostate will be described. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

Role of the POZ Zinc Finger Transcription Factor FBI-1 in Human and Murine Adipogenesis

PubMed Central

Laudes, Matthias; Christodoulides, Constantinos; Sewter, Ciaran; Rochford, Justin J.; Considine, Robert V.; Sethi, Jaswinder K.; Vidal-Puig, Antonio; O’Rahilly, Stephen

2015-01-01

Poxvirus zinc finger (POZ) zinc finger domain transcription factors have been shown to play a role in the control of growth arrest and differentiation in several types of mesenchymal cells but not, as yet, adipocytes. We found that a POZ domain protein, factor that binds to inducer of short transcripts-1 (FBI-1), was induced during both murine and human preadipocyte differentiation with maximal expression levels seen at days 2–4. FBI-1 mRNA was expressed in human adipose tissue with the highest levels found in samples from morbidly obese subjects. Murine cell lines constitutively expressing FBI-1 showed evidence for accelerated adipogenesis with earlier induction of markers of differentiation and enhanced lipid accumulation, suggesting that FBI-1 may be an active participant in the differentiation process. Consistent with the properties of this family of proteins in other cell systems, 3T3L1 cells stably overexpressing FBI-1 showed reduced DNA synthesis and reduced expression of cyclin A, cyclin-dependent kinase 2, and p107, proteins known to be involved in the regulation of mitotic clonal expansion. In addition, FBI-1 reduced the transcriptional activity of the cyclin A promoter. Thus, FBI-1, a POZ zinc finger transcription factor, is induced during the early phases of human and murine preadipocyte differentiation where it may contribute to adipogenesis through influencing the switch from cellular proliferation to terminal differentiation. PMID:14701838

Antibody blockade of IL-17 family cytokines in immunity to acute murine oral mucosal candidiasis.

PubMed

Whibley, Natasha; Tritto, Elaine; Traggiai, Elisabetta; Kolbinger, Frank; Moulin, Pierre; Brees, Dominique; Coleman, Bianca M; Mamo, Anna J; Garg, Abhishek V; Jaycox, Jillian R; Siebenlist, Ulrich; Kammüller, Michael; Gaffen, Sarah L

2016-06-01

Antibodies targeting IL-17A or its receptor, IL-17RA, are approved to treat psoriasis and are being evaluated for other autoimmune conditions. Conversely, IL-17 signaling is critical for immunity to opportunistic mucosal infections caused by the commensal fungus Candida albicans, as mice and humans lacking the IL-17R experience chronic mucosal candidiasis. IL-17A, IL-17F, and IL-17AF bind the IL-17RA-IL-17RC heterodimeric complex and deliver qualitatively similar signals through the adaptor Act1. Here, we used a mouse model of acute oropharyngeal candidiasis to assess the impact of blocking IL-17 family cytokines compared with specific IL-17 cytokine gene knockout mice. Anti-IL-17A antibodies, which neutralize IL-17A and IL-17AF, caused elevated oral fungal loads, whereas anti-IL-17AF and anti-IL-17F antibodies did not. Notably, there was a cooperative effect of blocking IL-17A, IL-17AF, and IL-17F together. Termination of anti-IL-17A treatment was associated with rapid C. albicans clearance. IL-17F-deficient mice were fully resistant to oropharyngeal candidiasis, consistent with antibody blockade. However, IL-17A-deficient mice had lower fungal burdens than anti-IL-17A-treated mice. Act1-deficient mice were much more susceptible to oropharyngeal candidiasis than anti-IL-17A antibody-treated mice, yet anti-IL-17A and anti-IL-17RA treatment caused equivalent susceptibilities. Based on microarray analyses of the oral mucosa during infection, only a limited number of genes were associated with oropharyngeal candidiasis susceptibility. In sum, we conclude that IL-17A is the main cytokine mediator of immunity in murine oropharyngeal candidiasis, but a cooperative relationship among IL-17A, IL-17AF, and IL-17F exists in vivo. Susceptibility displays the following hierarchy: IL-17RA- or Act1-deficiency > anti-IL-17A + anti-IL-17F antibodies > anti-IL-17A or anti-IL-17RA antibodies > IL-17A deficiency. © Society for Leukocyte Biology.

Atomic structure of the murine norovirus protruding domain and sCD300lf receptor complex.

PubMed

Kilic, Turgay; Koromyslova, Anna; Malak, Virginie; Hansman, Grant S

2018-03-21

Human noroviruses are the leading cause of acute gastroenteritis in human. Noroviruses also infect animals such as cow, mice, cat, and dog. How noroviruses bind and enter host cells is still incompletely understood. Recently, the type I transmembrane protein CD300lf was recently identified as the murine norovirus receptor, yet it is unclear how the virus capsid and receptor interact at the molecular level. In this study, we determined the X-ray crystal structure of the soluble CD300lf (sCD300lf) and murine norovirus capsid-protruding domain complex at 2.05 Å resolution. We found that the sCD300lf binding site is located on the topside of the protruding domain and involves a network of hydrophilic and hydrophobic interactions. The sCD300lf locked nicely into a complementary cavity on the protruding domain that is additionally coordinated with a positive surface charge on the sCD300lf and a negative surface charge on the protruding domain. Five of six protruding domain residues interacting with sCD300lf were maintained between different murine norovirus strains, suggesting that the sCD300lf was capable of binding to a highly conserved pocket. Moreover, a sequence alignment with other CD300 paralogs showed that the sCD300lf interacting residues were partially conserved in CD300ld, but variable in other CD300 family members, consistent with previously reported infection selectivity. Overall, these data provide insights into how a norovirus engages a protein receptor and will be important for a better understanding of selective recognition and norovirus attachment and entry mechanisms. IMPORTANCE Noroviruses exhibit exquisite host-range specificity due to species-specific interactions between the norovirus capsid protein and host molecules. Given this strict host-range restriction it has been unclear how the viruses are maintained within a species between relatively sporadic epidemics. While much data demonstrates that noroviruses can interact with carbohydrates

Murine norovirus infection in Brazilian animal facilities

PubMed Central

Rodrigues, Daniele Masselli; Moreira, Josélia Cristina de Oliveira; Lancellotti, Marcelo; Gilioli, Rovilson; Corat, Marcus Alexandre Finzi

2016-01-01

Murine norovirus (MNV) is a single-stranded positive-sense RNA virus of the Caliciviridae family. MNV has been reported to infect laboratory mice with the ability to cause lethal infections in strains lacking components of the innate immune response. Currently, MNV is considered the most prevalent infectious agent detected in laboratory mouse facilities. In this study, mice in 22 laboratory animal facilities within Brazil were analyzed for MNV infection. Using primers targeting a conserved region of the viral capsid, MNV was detected by RT-PCR in 137 of 359 mice from all 22 facilities. Nucleotide sequencing and phylogenetic analysis of the capsid region from the viral genome showed identity ranging from 87% to 99% when compared to reported MNV sequences. In addition, RAW264.7 cells inoculated with a mouse fecal suspension displayed cytopathic effect after the fifth passage. This study represents the first report of MNV in mouse colonies in Brazilian laboratory animal facilities, emphasizing the relevance of a health surveillance program in such environments. PMID:28049885

Three-dimensional crystal structure of recombinant murine interferon-beta.

PubMed Central

Senda, T; Shimazu, T; Matsuda, S; Kawano, G; Shimizu, H; Nakamura, K T; Mitsui, Y

1992-01-01

The crystal structure of recombinant murine interferon-beta (IFN-beta) has been solved by the multiple isomorphous replacement method and refined to an R-factor of 20.5% against 2.6 A X-ray diffraction data. The structure shows a variant of the alpha-helix bundle with a new chain-folding topology, which seems to represent a basic structural framework of all the IFN-alpha and IFN-beta molecules belonging to the type I family. Functionally important segments of the polypeptide chain, as implied through numerous gene manipulation studies carried out so far, are spatially clustered indicating the binding site(s) to the receptor(s). Comparison of the present structure with those of other alpha-helical cytokine proteins, including porcine growth hormone, interleukin 2 and interferon gamma, indicated either a topological similarity in chain folding or a similar spatial arrangement of the alpha-helices. Images PMID:1505514

Analysis of cardiomyocyte movement in the developing murine heart

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hashimoto, Hisayuki; Yuasa, Shinsuke, E-mail: yuasa@a8.keio.jp; Tabata, Hidenori

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 cellmore » 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.« less

Diversity in genomic organisation, developmental regulation and distribution of the murine PR72/B" subunits of protein phosphatase 2A

PubMed Central

Zwaenepoel, Karen; Louis, Justin V; Goris, Jozef; Janssens, Veerle

2008-01-01

Background Protein phosphatase 2A (PP2A) is a serine/threonine-specific phosphatase displaying vital functions in growth and development through its role in various signalling pathways. PP2A holoenzymes comprise a core dimer composed of a catalytic C and a structural A subunit, which can associate with a variable B-type subunit. The importance of the B-type subunits for PP2A regulation cannot be overestimated as they determine holoenzyme localisation, activity and substrate specificity. Three B-type subunit families have been identified: PR55/B, PR61/B' and PR72/B", of which the latter is currently the least characterised. Results We deduced the sequences and genomic organisation of the different murine PR72/B" isoforms: three genes encode nine isoforms, five of which are abundantly expressed and give rise to genuine PP2A subunits. Thereby, one novel subunit was identified. Using Northern blotting, we examined the tissue-specific and developmental expression of these subunits. All subunits are highly expressed in heart, suggesting an important cardiac function. Immunohistochemical analysis revealed a striated expression pattern of PR72 and PR130 in heart and skeletal muscle, but not in bladder smooth muscle. The subcellular localisation and cell cycle regulatory ability of several PR72/B" isoforms were determined, demonstrating differences as well as similarities. Conclusion In contrast to PR55/B and PR61/B', the PR72/B" family seems evolutionary more divergent, as only two of the murine genes have a human orthologue. We have integrated these results in a more consistent nomenclature of both human and murine PR72/B" genes and their transcripts/proteins. Our results provide a platform for the future generation of PR72/B" knockout mice. PMID:18715506

Purification and Crystallization of Murine Myostatin: A Negative Regulator of Muscle Mass

NASA Technical Reports Server (NTRS)

Hong, Young S.; Adamek, Daniel; Bridge, Kristi; Malone, Christine C.; Young, Ronald B.; Miller, Teresa; Karr, Laurel

2004-01-01

Myostatin (MSTN) has been crystallized and its preliminary X-ray diffraction data were collected. MSTN is a negative regulator of muscle growt/differentiation and suppressor of fat accumulation. It is a member of TGF-b family of proteins. Like other members of this family, the regulation of MSTN is critically tied to its process of maturation. This process involves the formation of a homodimer followed by two proteolytic steps. The first proteolytic cleavage produces a species where the n-terminal portion of the dimer is covalently separated from, but remains non-covalently bound to, the c-terminal, functional, portion of the protein. The protein is activated upon removal of the n-terminal "pro-segment" by a second n-terminal proteolytic cut by BMP-1 in vivo, or by acid treatment in vitro. Understanding the structural nature and physical interactions involved in these regulatory processes is the objective of our studies. Murine MSTN was purified from culture media of genetically engineered Chinese Hamster Ovary cells by multicolumn purification process and crystallized using the vapor diffusion method.

Murine models of osteosarcoma: A piece of the translational puzzle.

PubMed

Walia, Mannu K; Castillo-Tandazo, Wilson; Mutsaers, Anthony J; Martin, Thomas John; Walkley, Carl R

2018-06-01

Osteosarcoma (OS) is the most common cancer of bone in children and young adults. Despite extensive research efforts, there has been no significant improvement in patient outcome for many years. An improved understanding of the biology of this cancer and how genes frequently mutated contribute to OS may help improve outcomes for patients. While our knowledge of the mutational burden of OS is approaching saturation, our understanding of how these mutations contribute to OS initiation and maintenance is less clear. Murine models of OS have now been demonstrated to be highly valid recapitulations of human OS. These models were originally based on the frequent disruption of p53 and Rb in familial OS syndromes, which are also common mutations in sporadic OS. They have been applied to significantly improve our understanding about the functions of recurrently mutated genes in disease. The murine models can be used as a platform for preclinical testing and identifying new therapeutic targets, in addition to testing the role of additional mutations in vivo. Most recently these models have begun to be used for discovery based approaches and screens, which hold significant promise in furthering our understanding of the genetic and therapeutic sensitivities of OS. In this review, we discuss the mouse models of OS that have been reported in the last 3-5 years and newly identified pathways from these studies. Finally, we discuss the preclinical utilization of the mouse models of OS for identifying and validating actionable targets to improve patient outcome. © 2017 Wiley Periodicals, Inc.

In vitro and in vivo effects of CpG-Oligodeoxynucleotides (CpG-ODN) on murine transitional cell carcinoma and on the native murine urinary bladder wall.

PubMed

Olbert, Peter Jochen; Schrader, Andres Jan; Simon, Corinna; Dalpke, Alexander; Barth, Peter; Hofmann, Rainer; Hegele, Axel

2009-06-01

Intravesical BCG instillation is established and efficient in the prophylaxis of recurrent transitional cell carcinoma. A Th-1 biased immune response is postulated. Recent work has proven the efficacy of synthetic CpG-Oligodeoxynucleotides (ODN) as inducers and adjuvants for a strong Th1-response and there is evidence for a direct and/or adjuvant anti-neoplastic effect. The purpose of this study was to examine the local effects of CpG-ODN on the murine bladder wall after intravesical instillation and the effects on cytokine expression in an orthotopic murine bladder cancer model. Histopathology, immunohistochemistry and fluorescence microscopy were performed after different instillation schedules of stimulatory, non-stimulatory biotinylized and FITC-labelled CpG-ODN into the murine bladder. MB-49 murine bladder cancer cells were tested for TLR-9 expression to exclude a potential direct responsiveness to CpG-ODN. Furthermore induction of apoptosis was tested by annexin V staining and FACS analysis of CpG-ODN stimulated tumor cells. In an orthotopic C57/Bl6 murine bladder cancer model, the expressions of IL-12, IFNgamma, IL-10 and TGF-beta were evaluated after repeated CpG-ODN treatment. Single and repeated instillation of CpG-ODN induced subepithelial and urothelial lymphocytic infiltrations with consecutive apoptoses. PBS and non-stimulative ODN induced no visible reaction. Bladder submucosa stained positive for biotin. Controls showed no endogenic biotin staining. FITC-labelled ODN adhered to the bladder mucosa and penetration of the mucosal barrier was not detected. MB-49 TCC cells did not express TLR-9 and CpG-ODN did not induce apoptosis in these cells. Repeated intravesical instillations of CpG-ODN in orthotopic murine tumor bearing urinary bladders resulted in significant up-regulation of both Th-1 and Th-2 cytokines. CpG-ODNs have promising anti-neoplastic potential. They exert a pronounced immunological response both in the native murine urinary bladder and

Decrease in level of APG-2, a member of the heat shock protein 110 family, in murine brain following systemic administration of kainic acid.

PubMed

Ogita, K; Takagi, R; Oyama, N; Okuda, H; Ito, F; Okui, M; Shimizu, N; Yoneda, Y

2001-09-01

APG-2 belongs to the heat shock protein 110 family. Although kainic acid (KA)-induced seizures are known to elicit expression of inducible heat shock protein 70 (HSP70) in the brain, no investigation has been carried out on the APG-2 level after excitatory amino acid-induced seizures. By means of an immunoblot assay, we determined the levels of HSP70 and APG-2 in discrete brain structures of mice after a single intraperitoneal injection of KA or N-methyl-D-aspartic acid (NMDA). APG-2 level was significantly decreased in frontal cortex, hippocampus, and striatum three days after the administration of KA, while HSP70 level was increased in these regions following the administration. In any of these regions, APG-2 levels were returned to the control levels 10 days after the administration. However, no significant changes were observed in levels of both HSP70 and APG-2 in hypothalamus, midbrain, medulla-pons, and cerebellum of the mice. By contrast, NMDA administration did not significantly affect both levels in any of the regions examined. These findings indicate that the transient decrease in APG-2 expression is one of the intracellular events elicited by signals peculiar to KA, but not by those peculiar to NMDA, in telencephalon of murine brain.

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

PubMed

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

2017-02-23

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 E 2 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.

Distinct transcripts are recognized by sense and antisense riboprobes for a member of the murine HSP70 gene family, HSP70.2, in various reproductive tissues

NASA Technical Reports Server (NTRS)

Murashov, A. K.; Wolgemuth, D. J.

1996-01-01

The expression of hsp70.2, an hsp70 gene family member, originally characterized by its high levels of expression in germ cells in the adult mouse testis, was detected in several other reproductive tissues, including epididymis, prostate, and seminal vesicles, as well as in extraembryonic tissues of mid-gestation fetuses. In addition, hybridization with RNA probes transcribed in the sense orientation surprisingly indicated the presence of slightly larger "antisense" transcripts in several tissues. The levels of antisense transcripts varied among the tissues, with the highest signal detected in the prostate and no signal being detectable in the testis. Consistent with these results, in situ hybridization analysis clearly localized the sense-orientation transcripts to pachytene spermatocytes, while no antisense-orientation transcripts were observed in adjacent sections of the same tubules. Our findings have thus shown that although hsp70.2 was expressed abundantly and in a highly stage-specific manner in the male germ line, it was also expressed in other murine tissues. Furthermore, we have made the surprising observation of antisense transcription of the hsp70.2 gene in several mouse tissues, revealing another level of complexity in the regulation and function of heat shock proteins.

Antibody ligation of murine Ly-6G induces neutropenia, blood flow cessation, and death via complement-dependent and independent mechanisms.

PubMed

Abbitt, Katherine B; Cotter, Matthew J; Ridger, Victoria C; Crossman, David C; Hellewell, Paul G; Norman, Keith E

2009-01-01

Ly-6G is a member of the Ly-6 family of GPI-linked proteins, which is expressed on murine neutrophils. Antibodies against Ly-6G cause neutropenia, and fatal reactions also develop if mice are primed with TNF-alpha prior to antibody treatment. We have investigated the mechanisms behind these responses to Ly-6G ligation in the belief that similar mechanisms may be involved in neutropenia and respiratory disorders associated with alloantibody ligation of the related Ly-6 family member, NB1, in humans. Neutrophil adhesion, microvascular obstruction, breathing difficulties, and death initiated by anti-Ly-6G antibodies in TNF-alpha-primed mice were shown to be highly complement-dependent, partly mediated by CD11b, CD18, and FcgammaR and associated with clustering of Ly-6G. Neutrophil depletion, on the other hand, was only partly complement-dependent and was not altered by blockade of CD11b, CD18, or FcgammaR. Unlike other neutrophil-activating agents, Ly-6G ligation did not induce neutropenia via sequestration in the lungs. Cross-linking Ly-6G mimicked the responses seen with whole antibody in vivo and also activated murine neutrophils in vitro. Although this suggests that the responses are, in part, mediated by nonspecific properties of antibody ligation, neutrophil depletion requires an additional mechanism possibly specific to the natural function of Ly-6G.

A murine model of autosomal dominant neurohypophyseal diabetes insipidus reveals progressive loss of vasopressin-producing neurons

PubMed Central

Russell, Theron A.; Ito, Masafumi; Ito, Mika; Yu, Richard N.; Martinson, Fred A.; Weiss, Jeffrey; Jameson, J. Larry

2003-01-01

Familial neurohypophyseal diabetes insipidus (FNDI) is an autosomal dominant disorder caused by mutations in the arginine vasopressin (AVP) precursor. The pathogenesis of FNDI is proposed to involve mutant protein–induced loss of AVP-producing neurons. We established murine knock-in models of two different naturally occurring human mutations that cause FNDI. A mutation in the AVP signal sequence [A(–1)T] is associated with a relatively mild phenotype or delayed presentation in humans. This mutation caused no apparent phenotype in mice. In contrast, heterozygous mice expressing a mutation that truncates the AVP precursor (C67X) exhibited polyuria and polydipsia by 2 months of age and these features of DI progressively worsened with age. Studies of the paraventricular and supraoptic nuclei revealed induction of the chaperone protein BiP and progressive loss of AVP-producing neurons relative to oxytocin-producing neurons. In addition, Avp gene products were not detected in the neuronal projections, suggesting retention of WT and mutant AVP precursors within the cell bodies. In summary, this murine model of FNDI recapitulates many features of the human disorder and demonstrates that expression of the mutant AVP precursor leads to progressive neuronal cell loss. PMID:14660745

Telomere sister chromatid exchange in telomerase deficient murine cells.

PubMed

Wang, Yisong; Giannone, Richard J; Liu, Yie

2005-10-01

We have recently demonstrated that several types of genomic rearrangements (i.e., telomere sister chromatid exchange (T-SCE), genomic-SCE, or end-to-end fusions) were more often detected in long-term cultured murine telomerase deficient embryonic stem (ES) cells than in freshly prepared murine splenocytes, even through they possessed similar frequencies of critically short telomeres. The high rate of genomic rearrangements in telomerase deficient ES cells, when compared to murine splenocytes, may reflect the cultured cells' gained ability to protect chromosome ends with eroded telomeres allowing them to escape "end crisis". However, the possibility that ES cells were more permissive to genomic rearrangements than other cell types or that differences in the microenvironment or genetic background of the animals might consequentially determine the rate of T-SCEs or other genomic rearrangements at critically short telomeres could not be ruled out.

Telomere sister chromatid exchange in telomerase deficient murine cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Yisong; Giannone, Richard J; Liu, Yie

2005-01-01

We have recently demonstrated that several types of genomic rearrangements (i.e., telomere sister chromatid exchange (T-SCE), genomic-SCE, or end-to-end fusions) were more often detected in long-term cultured murine telomerase deficient embryonic stem (ES) cells than in freshly prepared murine splenocytes, even through they possessed similar frequencies of critically short telomeres. The high rate of genomic rearrangements in telomerase deficient ES cells, when compared to murine splenocytes, may reflect the cultured cells' gained ability to protect chromosome ends with eroded telomeres allowing them to escape 'end crisis'. However, the possibility that ES cells were more permissive to genomic rearrangements than othermore » cell types or that differences in the microenvironment or genetic background of the animals might consequentially determine the rate of T-SCEs or other genomic rearrangements at critically short telomeres could not be ruled out.« less

Current Translational Research and Murine Models For Duchenne Muscular Dystrophy

PubMed Central

Rodrigues, Merryl; Echigoya, Yusuke; Fukada, So-ichiro; Yokota, Toshifumi

2016-01-01

Duchenne muscular dystrophy (DMD) is an X-linked genetic disorder characterized by progressive muscle degeneration. Mutations in the DMD gene result in the absence of dystrophin, a protein required for muscle strength and stability. Currently, there is no cure for DMD. Since murine models are relatively easy to genetically manipulate, cost effective, and easily reproducible due to their short generation time, they have helped to elucidate the pathobiology of dystrophin deficiency and to assess therapies for treating DMD. Recently, several murine models have been developed by our group and others to be more representative of the human DMD mutation types and phenotypes. For instance, mdx mice on a DBA/2 genetic background, developed by Fukada et al., have lower regenerative capacity and exhibit very severe phenotype. Cmah-deficient mdx mice display an accelerated disease onset and severe cardiac phenotype due to differences in glycosylation between humans and mice. Other novel murine models include mdx52, which harbors a deletion mutation in exon 52, a hot spot region in humans, and dystrophin/utrophin double-deficient (dko), which displays a severe dystrophic phenotype due the absence of utrophin, a dystrophin homolog. This paper reviews the pathological manifestations and recent therapeutic developments in murine models of DMD such as standard mdx (C57BL/10), mdx on C57BL/6 background (C57BL/6-mdx), mdx52, dystrophin/utrophin double-deficient (dko), mdxβgeo, Dmd-null, humanized DMD (hDMD), mdx on DBA/2 background (DBA/2-mdx), Cmah-mdx, and mdx/mTRKO murine models. PMID:27854202

Convection enhanced delivery of carmustine to the murine brainstem: a feasibility study.

PubMed

Sewing, A Charlotte P; Caretti, Viola; Lagerweij, Tonny; Schellen, Pepijn; Jansen, Marc H A; van Vuurden, Dannis G; Idema, Sander; Molthoff, Carla F M; Vandertop, W Peter; Kaspers, Gertjan J L; Noske, David P; Hulleman, Esther

2014-12-30

Systemic delivery of therapeutic agents remains ineffective against diffuse intrinsic pontine glioma (DIPG), possibly due to an intact blood-brain-barrier (BBB) and to dose-limiting toxicity of systemic chemotherapeutic agents. Convection-enhanced delivery (CED) into the brainstem may provide an effective local delivery alternative for DIPG patients. The aim of this study is to develop a method to perform CED into the murine brainstem and to test this method using the chemotherapeutic agent carmustine (BiCNU). To this end, a newly designed murine CED catheter was tested in vitro and in vivo. After determination of safety and distribution, mice bearing VUMC-DIPG-3 and E98FM-DIPG brainstem tumors were treated with carmustine dissolved in DW 5% or carmustine dissolved in 10% ethanol. Our results show that CED into the murine brainstem is feasible and well tolerated by mice with and without brainstem tumors. CED of carmustine dissolved in 5% DW increased median survival of mice with VUMC-DIPG-3 and E98FM-DIPG tumors with 35% and 25% respectively. Dissolving carmustine in 10% ethanol further improved survival to 45% in mice with E98FM-DIPG tumors. Since genetically engineered and primary DIPG models are currently only available in mice, murine CED studies have clear advantages over CED studies in other animals. CED in the murine brainstem can be performed safely, is well tolerated and can be used to study efficacy of chemotherapeutic agents orthotopically. These results set the foundation for more CED studies in murine DIPG models. Copyright © 2014 Elsevier B.V. All rights reserved.

Isolation and characterization of recombinant murine Wnt3a.

PubMed

Witkowski, Andrzej; Krishnamoorthy, Aparna; Su, Betty; Beckstead, Jennifer A; Ryan, Robert O

2015-02-01

Wnt proteins are a family of morphogens that possess potent biological activity. Structure-function studies have been impeded by poor yield of biologically active recombinant Wnt as well as a propensity of isolated Wnt to self-associate in the absence of detergent. Using stably transfected Drosophila S2 cells, studies have been conducted to improve recovery of recombinant murine Wnt3a, establish conditions for a detergent-free Wnt preparation and examine the effects of limited proteolysis. S2 cell culture conditioned media was subjected to a 3-step protocol including dye-ligand chromatography, immobilized metal affinity chromatography and gel filtration chromatography. Through selective pooling of column fractions, homogeneous and purified Wnt3a preparations were obtained. Limited proteolysis of Wnt3a with thrombin resulted in site-specific cleavage within the N-terminal saposin-like motif. To generate detergent-free protein, Wnt3a was immobilized on Cu(2+)-charged, iminodiacetic acid-derivatized Sepharose beads, detergent-free buffer was applied and Wnt3a eluted from the beads with buffer containing imidazole plus 30mM methyl-ß-cyclodextrin (MßCD). Wnt3a recovered in MßCD-containing buffer was soluble and biologically active. Insofar as MßCD is a member of a family of non-toxic, low molecular weight compounds capable of binding and solubilizing small hydrophobic ligands, Wnt-cyclodextrin complexes may facilitate structure-activity studies in the absence of adverse detergent effects. Copyright © 2014 Elsevier Inc. All rights reserved.

Isolation and characterization of recombinant murine Wnt3a

PubMed Central

Witkowski, Andrzej; Krishnamoorthy, Aparna; Su, Betty; Beckstead, Jennifer A.; Ryan, Robert O.

2014-01-01

Wnt proteins are a family of morphogens that possess potent biological activity. Structure – function studies have been impeded by poor yield of biologically active recombinant Wnt as well as a propensity of isolated Wnt to self-associate in the absence of detergent. Using stably transfected Drosophila S2 cells, studies have been conducted to improve recovery of recombinant murine Wnt3a, establish conditions for a detergent-free Wnt preparation and examine the effects of limited proteolysis. S2 cell culture conditioned media was subjected to a 3-step protocol including dye-ligand chromatography, immobilized metal affinity chromatography and gel filtration chromatography. Through selective pooling of column fractions, homogeneous and purified Wnt3a preparations were obtained. Limited proteolysis of Wnt3a with thrombin resulted in site-specific cleavage within the N-terminal saposin-like motif. To generate detergent-free protein, Wnt3a was immobilized on Cu2+-charged, iminodiacetic acid-derivatized Sepharose beads, detergent-free buffer was applied and Wnt3a eluted from the beads with buffer containing imidazole plus 30 mM methyl-β-cyclodextrin (MβCD). Wnt3a recovered in MβCD-containing buffer was soluble and biologically active. Insofar as MβCD is a member of a family of non-toxic, low molecular weight compounds capable of binding and solubilizing small hydrophobic ligands, Wnt-cyclodextrin complexes may facilitate structure-activity studies in the absence of adverse detergent effects. PMID:25448592

Behavior of a cloned murine interferon alpha/beta receptor expressed in homospecific or heterospecific background.

PubMed

Uzé, G; Lutfalla, G; Bandu, M T; Proudhon, D; Mogensen, K E

1992-05-15

A murine interferon (IFN) alpha/beta receptor was cloned from the IFN-sensitive L1210 cell line on the basis of its homology with the human receptor. A combination of methods that includes the screening of random-primed and oligo(dT)-primed cDNA libraries and polymerase chain reactions with a single-side specificity was used. At the amino acid level, the murine IFN-alpha/beta shows 46% identity with its human counterpart. Both human WISH cells presenting a low sensitivity to mouse IFN and a murine L1210 mutant subline that does not express the receptor have been stably transfected with the murine IFN-alpha/beta receptor. Whereas transfected human cells became sensitive to a limited number of mouse IFN-alpha/beta subtypes, the transfected murine L1210 mutant was found to be fully complemented and became sensitive to all mouse IFN-alpha/beta subtypes tested, including those that were not active on transfected human cells. These results strongly suggest that the receptor described here is implicated in the mediation of the activities of all murine IFN-alpha/beta subtypes.

Vitamin E attenuates myocardial ischemia-reperfusion injury in murine AIDS.

PubMed

Chen, Yinhong; Davis-Gorman, Grace; Watson, Ronald Ross; McDonagh, Paul F

2002-01-01

The incidence of myocardial infarction in patients who have the aquired immunodeficiency syndrome (AIDS) is increasing. However, no effective therapeutic agents have been discovered to reduce myocardial ischemia-reperfusion (I/R) injury in pathologies associated with AIDS. The aim of this study was to determine if infarct size is increased in murine AIDS after I/R injury and if I/R injury could be attenuated with vitamin E supplementation. Three groups of mice were studied: control, murine AIDS, and murine AIDS with vitamin E supplementation. Anesthetized mice were subjected to 30 min of left anterior descending coronary artery occlusion and 120 min of reperfusion. The hearts in mice that had murine AIDS had a larger infarct size compared to controls after I/R injury. Vitamin E supplementation significantly reduced infarct size and inhibited polymorphonuclear neutrophil (PMN) CD11b expression (p < 0.05). However, vitamin E supplementation did not affect PMN reactive oxygen species (ROS) production and platelet CD62p expression. These results suggest that the reduction of myocardial I/R injury with vitamin E supplementation may be the result of the inhibition of PMN CD11b expression. Vitamin E may be a promising prophylactic agent for the reduction of the severity of myocardial I/R injury in patients who have AIDS.

Miniature Microwave Applicator for Murine Bladder Hyperthermia Studies

PubMed Central

Salahi, Sara; Maccarini, Paolo F.; Rodrigues, Dario B.; Etienne, Wiguins; Landon, Chelsea D.; Inman, Brant A.; Dewhirst, Mark W.; Stauffer, Paul R.

2012-01-01

Purpose Novel combinations of heat with chemotherapeutic agents are often studied in murine tumor models. Currently, no device exists to selectively heat small tumors at depth in mice. In this project, we modelled, built and tested a miniature microwave heat applicator, the physical dimensions of which can be scaled to adjust the volume and depth of heating to focus on the tumor volume. Of particular interest is a device that can selectively heat murine bladder. Materials and Methods Using Avizo® segmentation software, we created a numerical mouse model based on micro-MRI scan data. The model was imported into HFSS™ simulation software and parametric studies were performed to optimize the dimensions of a water-loaded circular waveguide for selective power deposition inside a 0.15ml bladder. A working prototype was constructed operating at 2.45GHz. Heating performance was characterized by mapping fiber-optic temperature sensors along catheters inserted at depths of 0-1mm (subcutaneous), 2-3mm (vaginal), and 4-5mm (rectal) below the abdominal wall, with the mid-depth catheter adjacent to the bladder. Core temperature was monitored orally. Results Thermal measurements confirm the simulations which demonstrate that this applicator can provide local heating at depth in small animals. Measured temperatures in murine pelvis show well-localized bladder heating to 42-43°C while maintaining normothermic skin and core temperatures. Conclusions Simulation techniques facilitate the design optimization of microwave antennas for use in pre-clinical applications such as localized tumor heating in small animals. Laboratory measurements demonstrate the effectiveness of a new miniature water-coupled microwave applicator for localized heating of murine bladder. PMID:22690856

Miniature microwave applicator for murine bladder hyperthermia studies.

PubMed

Salahi, Sara; Maccarini, Paolo F; Rodrigues, Dario B; Etienne, Wiguins; Landon, Chelsea D; Inman, Brant A; Dewhirst, Mark W; Stauffer, Paul R

2012-01-01

Novel combinations of heat with chemotherapeutic agents are often studied in murine tumour models. Currently, no device exists to selectively heat small tumours at depth in mice. In this project we modelled, built and tested a miniature microwave heat applicator, the physical dimensions of which can be scaled to adjust the volume and depth of heating to focus on the tumour volume. Of particular interest is a device that can selectively heat murine bladder. Using Avizo(®) segmentation software, we created a numerical mouse model based on micro-MRI scan data. The model was imported into HFSS™ (Ansys) simulation software and parametric studies were performed to optimise the dimensions of a water-loaded circular waveguide for selective power deposition inside a 0.15 mL bladder. A working prototype was constructed operating at 2.45 GHz. Heating performance was characterised by mapping fibre-optic temperature sensors along catheters inserted at depths of 0-1 mm (subcutaneous), 2-3 mm (vaginal), and 4-5 mm (rectal) below the abdominal wall, with the mid depth catheter adjacent to the bladder. Core temperature was monitored orally. Thermal measurements confirm the simulations which demonstrate that this applicator can provide local heating at depth in small animals. Measured temperatures in murine pelvis show well-localised bladder heating to 42-43°C while maintaining normothermic skin and core temperatures. Simulation techniques facilitate the design optimisation of microwave antennas for use in pre-clinical applications such as localised tumour heating in small animals. Laboratory measurements demonstrate the effectiveness of a new miniature water-coupled microwave applicator for localised heating of murine bladder.

Essential roles for Cdx in murine primitive hematopoiesis.

PubMed

Brooke-Bisschop, Travis; Savory, Joanne G A; Foley, Tanya; Ringuette, Randy; Lohnes, David

2017-02-15

The Cdx transcription factors play essential roles in primitive hematopoiesis in the zebrafish where they exert their effects, in part, through regulation of hox genes. Defects in hematopoiesis have also been reported in Cdx mutant murine embryonic stem cell models, however, to date no mouse model reflecting the zebrafish Cdx mutant hematopoietic phenotype has been described. This is likely due, in part, to functional redundancy among Cdx members and the early lethality of Cdx2 null mutants. To circumvent these limitations, we used Cre-mediated conditional deletion to assess the impact of concomitant loss of Cdx1 and Cdx2 on murine primitive hematopoiesis. We found that Cdx1/Cdx2 double mutants exhibited defects in primitive hematopoiesis and yolk sac vasculature concomitant with reduced expression of several genes encoding hematopoietic transcription factors including Scl/Tal1. Chromatin immunoprecipitation analysis revealed that Scl was occupied by Cdx2 in vivo, and Cdx mutant hematopoietic yolk sac differentiation defects could be rescued by expression of exogenous Scl. These findings demonstrate critical roles for Cdx members in murine primitive hematopoiesis upstream of Scl. Copyright © 2017 Elsevier Inc. All rights reserved.

The murine Cd48 gene: allelic polymorphism in the IgV-like region.

PubMed

Cabrero, J G; Freeman, G J; Reiser, H

1998-12-01

The murine CD48 molecule is a member of the immunoglobulin superfamily which regulates the activation of T lymphocytes. prior cloning experiments using mRNA from two different mouse strains had yielded discrepant sequences within the IgV-like domain of murine CD48. To resolve this issue, we have directly sequenced genomic DNA of 10 laboratory strains and two inbred strains of wild origin. The results of our analysis reveal an allelic polymorphism within the IgV-like domain of murine CD48.

An evolutionarily conserved gene family encodes proton-selective ion channels.

PubMed

Tu, Yu-Hsiang; Cooper, Alexander J; Teng, Bochuan; Chang, Rui B; Artiga, Daniel J; Turner, Heather N; Mulhall, Eric M; Ye, Wenlei; Smith, Andrew D; Liman, Emily R

2018-03-02

Ion channels form the basis for cellular electrical signaling. Despite the scores of genetically identified ion channels selective for other monatomic ions, only one type of proton-selective ion channel has been found in eukaryotic cells. By comparative transcriptome analysis of mouse taste receptor cells, we identified Otopetrin1 (OTOP1), a protein required for development of gravity-sensing otoconia in the vestibular system, as forming a proton-selective ion channel. We found that murine OTOP1 is enriched in acid-detecting taste receptor cells and is required for their zinc-sensitive proton conductance. Two related murine genes, Otop2 and Otop3 , and a Drosophila ortholog also encode proton channels. Evolutionary conservation of the gene family and its widespread tissue distribution suggest a broad role for proton channels in physiology and pathophysiology. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Murine models of H. pylori-induced gastritis and gastric adenocarcinoma.

PubMed

Krueger, Sabine; Roessner, Albert; Kuester, Doerthe

2011-10-15

Laboratory mice have become one of the best animal species for mechanistic studies in gastrointestinal research. Their abundant genetic information, the way of causing carcinogenesis easily by transgenic and gene knockout techniques, limited effort in time and costs, and their practicability provide advantages over other animal models. Meanwhile, several murine practical models have been established for the investigation of the initiation, expansion, and progression of gastritis and gastric carcinoma, for assessing the effects of bacterial, genetic and environmental factors, and for evaluating therapeutic and preventive strategies in gastric diseases. This article gives a review of murine models of gastritis and gastric cancer, placing emphasis on the models associated with Helicobacter pylori infection and techniques used in our laboratory. We discuss matters of murine gastric anatomy, as well as techniques of infection, tissue preparation, and histology. Copyright © 2011 Elsevier GmbH. All rights reserved.

Behavior of a cloned murine interferon alpha/beta receptor expressed in homospecific or heterospecific background.

PubMed Central

Uzé, G; Lutfalla, G; Bandu, M T; Proudhon, D; Mogensen, K E

1992-01-01

A murine interferon (IFN) alpha/beta receptor was cloned from the IFN-sensitive L1210 cell line on the basis of its homology with the human receptor. A combination of methods that includes the screening of random-primed and oligo(dT)-primed cDNA libraries and polymerase chain reactions with a single-side specificity was used. At the amino acid level, the murine IFN-alpha/beta shows 46% identity with its human counterpart. Both human WISH cells presenting a low sensitivity to mouse IFN and a murine L1210 mutant subline that does not express the receptor have been stably transfected with the murine IFN-alpha/beta receptor. Whereas transfected human cells became sensitive to a limited number of mouse IFN-alpha/beta subtypes, the transfected murine L1210 mutant was found to be fully complemented and became sensitive to all mouse IFN-alpha/beta subtypes tested, including those that were not active on transfected human cells. These results strongly suggest that the receptor described here is implicated in the mediation of the activities of all murine IFN-alpha/beta subtypes. Images PMID:1533935

The CpG Island in the Murine Foxl2 Proximal Promoter Is Differentially Methylated in Primary and Immortalized Cells

PubMed Central

Tran, Stella; Wang, Ying; Lamba, Pankaj; Zhou, Xiang; Boehm, Ulrich; Bernard, Daniel J.

2013-01-01

Forkhead box L2 (Foxl2), a member of the forkhead transcription factor family, plays important roles in pituitary follicle-stimulating hormone synthesis and in ovarian maintenance and function. Mutations in the human FOXL2 gene cause eyelid malformations and premature ovarian failure. FOXL2/Foxl2 is expressed in pituitary gonadotrope and thyrotrope cells, the perioptic mesenchyme of the developing eyelid, and ovarian granulosa cells. The mechanisms governing this cell-restricted expression have not been described. We mapped the Foxl2 transcriptional start site in immortalized murine gonadotrope-like cells, LβT2, by 5’ rapid amplification of cDNA ends and then PCR amplified approximately 1 kb of 5’ flanking sequence from murine genomic DNA. When ligated into a reporter plasmid, the proximal promoter conferred luciferase activity in both homologous (LβT2) and, unexpectedly, heterologous (NIH3T3) cells. In silico analyses identified a CpG island in the proximal promoter and 5’ untranslated region, suggesting that Foxl2 transcription might be regulated epigenetically. Indeed, pyrosequencing and quantitative analysis of DNA methylation using real-time PCR revealed Foxl2 proximal promoter hypomethylation in homologous compared to some, though not all, heterologous cell lines. The promoter was also hypomethylated in purified murine gonadotropes. In vitro promoter methylation completely silenced reporter activity in heterologous and homologous cells. Collectively, the data suggest that differential proximal promoter DNA methylation may contribute to cell-specific Foxl2 expression in some cellular contexts. However, gonadotrope-specific expression of the gene cannot be explained by promoter hypomethylation alone. PMID:24098544

Growth and metabolism of murine and bovine embryos in bovine uterine flushing-supplemented culture media.

PubMed Central

Rondeau, M; Guay, P; Goff, A K; Cooke, G M

1996-01-01

The aim of this study was to compare the development and metabolic activity of cultured murine and bovine embryos in 2 standard media (HAM F-10 and RPMI) in the presence or absence of bovine uterine flushings. Murine morulae (n = 653) and day 7 bovine embryos (n = 273) were cultured for 18 h or 36 h in either HAM F-10 or RPMI in the presence or absence of bovine uterine flushings. After culture, the development, quality, and metabolic activity (glucose utilization or methionine uptake and incorporation) of embryos was assessed. It was found that HAM F-10 (without uterine flushings) was a more suitable medium than RPMI for optimal development and metabolism of murine and bovine embryos. Poor quality and development, as well as decreased metabolism, were evident after culture of murine embryos in RPMI; in contrast, this medium had no adverse effects on bovine embryos in culture. Supplementation of HAM F-10 with bovine uterine flushings improved the growth of murine embryos and the protein synthesis (as measured by an increased methionine incorporation) for both murine and bovine embryos. However, supplementation with bovine uterine flushings could not overcome deficiencies of an inappropriate medium (RPMI) for murine embryos. Supplementation of a well-defined culture medium with uterine flushings increased metabolism of embryos in culture, and thus might help to increase pregnancy rates after transfer of such embryos to recipient cows. PMID:8825988

Structural Heterogeneity and Functional Domains of Murine Immunoglobulin G Fc Receptors

NASA Astrophysics Data System (ADS)

Ravetch, Jeffrey V.; Luster, Andrew D.; Weinshank, Richard; Kochan, Jarema; Pavlovec, Amalia; Portnoy, Daniel A.; Hulmes, Jeffrey; Pan, Yu-Ching E.; Unkeless, Jay C.

1986-11-01

Binding of antibodies to effector cells by way of receptors to their constant regions (Fc receptors) is central to the pathway that leads to clearance of antigens by the immune system. The structure and function of this important class of receptors on immune cells is addressed through the molecular characterization of Fc receptors (FcR) specific for the murine immunoglobulin G isotype. Structural diversity is encoded by two genes that by alternative splicing result in expression of molecules with highly conserved extracellular domains and different transmembrane and intracytoplasmic domains. The proteins encoded by these genes are members of the immunoglobulin supergene family, most homologous to the major histocompatibility complex molecule Eβ. Functional reconstitution of ligand binding by transfection of individual FcR genes demonstrates that the requirements for ligand binding are encoded in a single gene. These studies demonstrate the molecular basis for the functional heterogeneity of FcR's, accounting for the possible transduction of different signals in response to a single ligand.

Diagnostic imaging advances in murine models of colitis.

PubMed

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

2016-01-21

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.

Regulation of murine cystic fibrosis transmembrane conductance regulator Cl− channels expressed in Chinese hamster ovary cells

PubMed Central

Lansdell, K A; Kidd, J F; Delaney, S J; Wainwright, B J; Sheppard, D N

1998-01-01

We investigated the effect of protein kinases and phosphatases on murine cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channels, expressed in Chinese hamster ovary (CHO) cells, using iodide efflux and the excised inside-out configuration of the patch-clamp technique.The protein kinase C (PKC) activator, phorbol dibutyrate, enhanced cAMP-stimulated iodide efflux. However, PKC did not augment the single-channel activity of either human or murine CFTR Cl− channels that had previously been activated by protein kinase A.Fluoride, a non-specific inhibitor of protein phosphatases, stimulated both human and murine CFTR Cl− channels. However, calyculin A, a potent inhibitor of protein phosphatases 1 and 2A, did not enhance cAMP-stimulated iodide efflux.The alkaline phosphatase inhibitor, (−)-bromotetramisole augmented cAMP-stimulated iodide efflux and, by itself, stimulated a larger efflux than that evoked by cAMP agonists. However, (+)-bromotetramisole, the inactive enantiomer, had the same effect. For murine CFTR, neither enantiomer enhanced single-channel activity. In contrast, both enantiomers increased the open probability (Po) of human CFTR, suggesting that bromotetramisole may promote the opening of human CFTR.As murine CFTR had a low Po and was refractory to stimulation by activators of human CFTR, we investigated whether murine CFTR may open to a subconductance state. When single-channel records were filtered at 50 Hz, a very small subconductance state of murine CFTR was observed that had a Po greater than that of human CFTR. The occupancy of this subconductance state may explain the differences in channel regulation observed between human and murine CFTR. PMID:9769419

Significance of major international seaports in the distribution of murine typhus in Taiwan.

PubMed

Kuo, Chi-Chien; Wardrop, Nicola; Chang, Chung-Te; Wang, Hsi-Chieh; Atkinson, Peter M

2017-03-01

International seaports are hotspots for disease invasion and pathogens can persist in seaports even after ports are abandoned. Transmitted by fleas infected by Rickettsia typhi, murine typhus, a largely neglected and easily misdiagnosed disease, is known to occur primarily in large seaports. However, the significance of seaports in the occurrence of murine typhus has never been validated quantitatively. We studied the spatial distribution of murine typhus, a notifiable disease, in Taiwan. We investigated whether risk of infection was correlated with distance to international seaports and a collection of environmental and socioeconomic factors, using a Bayesian negative binomial conditionally autoregressive model, followed with geographically weighted regression. Seaports that are currently in use and those that operated in the 19th century for trade with China, but were later abandoned due to siltation were analyzed. A total of 476 human cases of murine typhus were reported during 2000-2014 in the main island of Taiwan, with spatial clustering in districts in southwest and central-west Taiwan. A higher incidence rate (case/population) was associated with a smaller distance to currently in-use international seaports and lower rainfall and temperature, but was uncorrelated with distance to abandoned ports. Geographically weighted regression revealed a geographic heterogeneity in the importance of distance to in-use seaports near the four international seaports of Taiwan. Our study suggests that murine typhus is associated with international seaports, especially for those with large trading volume. Thus, one of the costs of global trade in Taiwan might be elevated risks of murine typhus. Globalization has accelerated the spread of infectious diseases, but the burden of disease varies geographically, with regions surrounding major international seaports warranting particular surveillance.

Murine typhus in two travelers returning from Bali, Indonesia: an underdiagnosed disease.

PubMed

Takeshita, Nozomi; Imoto, Kazuya; Ando, Shuji; Yanagisawa, Kunio; Ohji, Goh; Kato, Yasuyuki; Sakata, Akiko; Hosokawa, Naoto; Kishimoto, Toshio

2010-01-01

Two Japanese travelers from Bali were diagnosed with murine typhus in Japan during the same period. Although one had only mild illness, the other experienced liver and kidney dysfunction. Murine typhus may be missed not only in endemic areas around the world, but also in travelers, especially those returning from marine resorts in these areas. © 2010 International Society of Travel Medicine.

Increased Cerebral Tff1 Expression in Two Murine Models of Neuroinflammation.

PubMed

Znalesniak, Eva B; Fu, Ting; Guttek, Karina; Händel, Ulrike; Reinhold, Dirk; Hoffmann, Werner

2016-01-01

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. 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. 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. 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. © 2016 The Author(s) Published by S. Karger AG, Basel.

Iron regulatory protein 1 is not required for the modulation of ferritin and transferrin receptor expression by iron in a murine pro-B lymphocyte cell line

PubMed Central

Schalinske, Kevin L.; Blemings, Kenneth P.; Steffen, Daniel W.; Chen, Opal S.; Eisenstein, Richard S.

1997-01-01

Iron regulatory proteins (IRPs) are cytoplasmic RNA binding proteins that are central components of a sensory and regulatory network that modulates vertebrate iron homeostasis. IRPs regulate iron metabolism by binding to iron responsive element(s) (IREs) in the 5′ or 3′ untranslated region of ferritin or transferrin receptor (TfR) mRNAs. Two IRPs, IRP1 and IRP2, have been identified previously. IRP1 exhibits two mutually exclusive functions as an RNA binding protein or as the cytosolic isoform of aconitase. We demonstrate that the Ba/F3 family of murine pro-B lymphocytes represents the first example of a mammalian cell line that fails to express IRP1 protein or mRNA. First, all of the IRE binding activity in Ba/F3-gp55 cells is attributable to IRP2. Second, synthesis of IRP2, but not of IRP1, is detectable in Ba/F3-gp55 cells. Third, the Ba/F3 family of cells express IRP2 mRNA at a level similar to other murine cell lines, but IRP1 mRNA is not detectable. In the Ba/F3 family of cells, alterations in iron status modulated ferritin biosynthesis and TfR mRNA level over as much as a 20- and 14-fold range, respectively. We conclude that IRP1 is not essential for regulation of ferritin or TfR expression by iron and that IRP2 can act as the sole IRE-dependent mediator of cellular iron homeostasis. PMID:9380695

An anisotropic, hyperelastic model for skin: experimental measurements, finite element modelling and identification of parameters for human and murine skin.

PubMed

Groves, Rachel B; Coulman, Sion A; Birchall, James C; Evans, Sam L

2013-02-01

The mechanical characteristics of skin are extremely complex and have not been satisfactorily simulated by conventional engineering models. The ability to predict human skin behaviour and to evaluate changes in the mechanical properties of the tissue would inform engineering design and would prove valuable in a diversity of disciplines, for example the pharmaceutical and cosmetic industries, which currently rely upon experiments performed in animal models. The aim of this study was to develop a predictive anisotropic, hyperelastic constitutive model of human skin and to validate this model using laboratory data. As a corollary, the mechanical characteristics of human and murine skin have been compared. A novel experimental design, using tensile tests on circular skin specimens, and an optimisation procedure were adopted for laboratory experiments to identify the material parameters of the tissue. Uniaxial tensile tests were performed along three load axes on excised murine and human skin samples, using a single set of material parameters for each skin sample. A finite element model was developed using the transversely isotropic, hyperelastic constitutive model of Weiss et al. (1996) and was embedded within a Veronda-Westmann isotropic material matrix, using three fibre families to create anisotropic behaviour. The model was able to represent the nonlinear, anisotropic behaviour of the skin well. Additionally, examination of the optimal material coefficients and the experimental data permitted quantification of the mechanical differences between human and murine skin. Differences between the skin types, most notably the extension of the skin at low load, have highlighted some of the limitations of murine skin as a biomechanical model of the human tissue. The development of accurate, predictive computational models of human tissue, such as skin, to reduce, refine or replace animal models and to inform developments in the medical, engineering and cosmetic fields, is a

Clinical and laboratory features of murine typhus in central Tunisia: a report of seven cases.

PubMed

Letaïef, A Omezzine; Kaabia, N; Chakroun, M; Khalifa, M; Bouzouaia, N; Jemni, L

2005-11-01

Murine or endemic typhus, caused by Rickettsia typhi, has been reported in all continents. In the 1970s, no cases of murine typhus were diagnosed in Tunisia. The clinico-epidemiological characteristics of seven cases of murine typhus diagnosed at our hospitals since 1993 are reported. Diagnosis was confirmed by indirect fluorescence assay detecting specific R. typhi antibodies. Murine typhus occurred in all ages from 18-80 years during the hot season in rural areas. Clinical features were: sudden onset of fever and absence of eschar in all cases, with maculo-papular rash (five cases), prostration (four cases), meningism (three cases) and pneumonia (four cases). Frequent laboratory findings were moderate thrombopenia (four cases) and elevated transaminases (four cases). Before the results of serology, clinical diagnoses were Mediterranean Spotted Fever (four cases), Q fever (one case), pneumonia (one case), and lymphocytic meningitis (one case). Serology confirmed all diagnoses with cross-reactivity with Rickettsia conorii. Murine typhus exists in Tunisia and its prevalence is underestimated. Further, more specific studies are needed to evaluate the true prevalence.

The Murine Factor H-Related Protein FHR-B Promotes Complement Activation.

PubMed

Cserhalmi, Marcell; Csincsi, Ádám I; Mezei, Zoltán; Kopp, Anne; Hebecker, Mario; Uzonyi, Barbara; Józsi, Mihály

2017-01-01

Factor H-related (FHR) proteins consist of varying number of complement control protein domains that display various degrees of sequence identity to respective domains of the alternative pathway complement inhibitor factor H (FH). While such FHR proteins are described in several species, only human FHRs were functionally investigated. Their biological role is still poorly understood and in part controversial. Recent studies on some of the human FHRs strongly suggest a role for FHRs in enhancing complement activation via competing with FH for binding to certain ligands and surfaces. The aim of the current study was the functional characterization of a murine FHR, FHR-B. To this end, FHR-B was expressed in recombinant form. Recombinant FHR-B bound to human C3b and was able to compete with human FH for C3b binding. FHR-B supported the assembly of functionally active C3bBb alternative pathway C3 convertase via its interaction with C3b. This activity was confirmed by demonstrating C3 activation in murine serum. In addition, FHR-B bound to murine pentraxin 3 (PTX3), and this interaction resulted in murine C3 fragment deposition due to enhanced complement activation in mouse serum. FHR-B also induced C3 deposition on C-reactive protein, the extracellular matrix (ECM) extract Matrigel, and endothelial cell-derived ECM when exposed to mouse serum. Moreover, mouse C3 deposition was strongly enhanced on necrotic Jurkat T cells and the mouse B cell line A20 by FHR-B. FHR-B also induced lysis of sheep erythrocytes when incubated in mouse serum with FHR-B added in excess. Altogether, these data demonstrate that, similar to human FHR-1 and FHR-5, mouse FHR-B modulates complement activity by promoting complement activation via interaction with C3b and via competition with murine FH.

Cryoprotective effects of low-density lipoproteins, trehalose and soybean lecithin on murine spermatogonial stem cells.

PubMed

Wang, Peng; Li, Ying; Hu, Xiao-Chen; Cai, Xiao-Li; Hou, Li-Peng; Wang, Yan-Feng; Hu, Jian-Hong; Li, Qing-Wang; Suo, Li-Juan; Fan, Zhi-Guo; Zhang, Bo

2014-05-01

Spermatogonial stem cells (SSCs) have the ability to self-renew and offer a pathway for genetic engineering of the male germ line. Cryopreservation of SSCs has potential value for the treatment of male infertility, spermatogonial transplantation, and so on. In order to investigate the cryopreservation effects of different cryoprotectants on murine SSCs, 0.2 M of low-density lipoproteins (LDL), trehalose and soybean lecithin were added to the cryoprotective medium, respectively, and the murine SSCs were frozen at -80°C or -196°C. The results indicated that the optimal recovery rates of murine SSCs in the cryoprotective medium supplemented with LDL, trehalose and soybean lecithin were 92.53, 76.35 and 75.48% at -80°C, respectively. Compared with freezing at -196°C, the optimum temperature for improvement of recovery rates of frozen murine SSCs, cryopreservation in three different cryoprotectants at -80°C, were 17.11, 6.68 and 10.44% respectively. The recovery rates of murine SSCs in the cryoprotective medium supplemented with 0.2 M LDL were significantly higher than that of other cryoprotectants (P < 0.05). Moreover, the recovery rates were demonstrated to be greater at -80°C compared with at -196°C (P < 0.05). In conclusion, 0.2 M of LDL could significantly protect murine SSCs at -80°C. In the freezing-thawing process, LDL is responsible for the cryopreservation of murine SSCs because it can form a protective film at the surface of membranes. However, more research is needed to evaluate and understand the precise role of LDL during the freezing-thawing of SSCs.

Characterization of Ribosomal Frameshifting in Theiler's Murine Encephalomyelitis Virus.

PubMed

Finch, Leanne K; Ling, Roger; Napthine, Sawsan; Olspert, Allan; Michiels, Thomas; Lardinois, Cécile; Bell, Susanne; Loughran, Gary; Brierley, Ian; Firth, Andew E

2015-08-01

Theiler's murine encephalomyelitis virus (TMEV) is a member of the genus Cardiovirus in the Picornaviridae, a family of positive-sense single-stranded RNA viruses. Previously, we demonstrated that in the related cardiovirus, Encephalomyocarditis virus, a programmed-1 ribosomal frameshift (1 PRF) occurs at a conserved G_GUU_UUU sequence within the 2B-encoding region of the polyprotein open reading frame (ORF). Here we show that-1 PRF occurs at a similar site during translation of the TMEV genome. In addition, we demonstrate that a predicted 3= RNA stem-loop structure at a noncanonical spacing downstream of the shift site is required for efficient frameshifting in TMEV and that frameshifting also requires virus infection. Mutating the G_GUU_UUU shift site to inhibit frameshifting results in an attenuated virus with reduced growth kinetics and a small-plaque phenotype. Frameshifting in the virus context was found to be extremely efficient at 74 to 82%, which, to our knowledge, is the highest frameshifting efficiency recorded to date for any virus. We propose that highly efficient-1 PRF in TMEV provides a mechanism to escape the confines of equimolar expression normally inherent in the single-polyprotein expression strategy of picornaviruses.

Significance of major international seaports in the distribution of murine typhus in Taiwan

PubMed Central

Wardrop, Nicola; Chang, Chung-Te; Wang, Hsi-Chieh; Atkinson, Peter M.

2017-01-01

Background International seaports are hotspots for disease invasion and pathogens can persist in seaports even after ports are abandoned. Transmitted by fleas infected by Rickettsia typhi, murine typhus, a largely neglected and easily misdiagnosed disease, is known to occur primarily in large seaports. However, the significance of seaports in the occurrence of murine typhus has never been validated quantitatively. Methodology/Principal findings We studied the spatial distribution of murine typhus, a notifiable disease, in Taiwan. We investigated whether risk of infection was correlated with distance to international seaports and a collection of environmental and socioeconomic factors, using a Bayesian negative binomial conditionally autoregressive model, followed with geographically weighted regression. Seaports that are currently in use and those that operated in the 19th century for trade with China, but were later abandoned due to siltation were analyzed. A total of 476 human cases of murine typhus were reported during 2000–2014 in the main island of Taiwan, with spatial clustering in districts in southwest and central-west Taiwan. A higher incidence rate (case/population) was associated with a smaller distance to currently in-use international seaports and lower rainfall and temperature, but was uncorrelated with distance to abandoned ports. Geographically weighted regression revealed a geographic heterogeneity in the importance of distance to in-use seaports near the four international seaports of Taiwan. Conclusions/Significance Our study suggests that murine typhus is associated with international seaports, especially for those with large trading volume. Thus, one of the costs of global trade in Taiwan might be elevated risks of murine typhus. Globalization has accelerated the spread of infectious diseases, but the burden of disease varies geographically, with regions surrounding major international seaports warranting particular surveillance. PMID

Genomic alterations in spontaneous and carcinogen-induced murine melanoma cell lines.

PubMed

Melnikova, Vladislava O; Bolshakov, Svetlana V; Walker, Christopher; Ananthaswamy, Honnavara N

2004-03-25

We have conducted an analysis of genetic alterations in spontaneous murine melanoma cell line B16F0 and its two metastatic clones, B16F1 and B16F10 and the carcinogen-induced murine melanoma cell lines CM519, CM3205, and K1735. We found that unlike human melanomas, the murine melanoma cell lines did not have activating mutations in the Braf oncogene at exon 11 or 15. However, there were distinct patterns of alterations in the ras, Ink4a/Arf, and p53 genes in the two melanoma groups. In the spontaneous B16 melanoma cell lines, expression of p16Ink4a and p19Arf tumor suppressor proteins was lost as a consequence of a large deletion spanning Ink4a/Arf exons 1alpha, 1beta, and 2. In contrast, the carcinogen-induced melanoma cell lines expressed p16Ink4a but had inactivating mutations in either p19Arf (K1735) or p53 (CM519 and CM3205). Inactivation of p19Arf or p53 in carcinogen-induced melanomas was accompanied by constitutive activation of mitogen-activated protein kinases (MAPKs) and/or mutation-associated activation of N-ras. These results indicate that genetic alterations in p16Ink4a/p19Arf, p53 and ras-MAPK pathways can cooperate in the development of murine melanoma.

DEPDC5 takes a second hit in familial focal epilepsy.

PubMed

Anderson, Matthew P

2018-04-30

Loss-of-function mutations in a single allele of the gene encoding DEP domain-containing 5 protein (DEPDC5) are commonly linked to familial focal epilepsy with variable foci; however, a subset of patients presents with focal cortical dysplasia that is proposed to result from a second-hit somatic mutation. In this issue of the JCI, Ribierre and colleagues provide several lines of evidence to support second-hit DEPDC5 mutations in this disorder. Moreover, the authors use in vivo, in utero electroporation combined with CRISPR-Cas9 technology to generate a murine model of the disease that recapitulates human manifestations, including cortical dysplasia-like changes, focal seizures, and sudden unexpected death. This study provides important insights into familial focal epilepsy and provides a preclinical model for evaluating potential therapies.

1.8 Astroms Structure of Murine GITR Ligand Dimer Expressed in Drosophila Melanogaster S2 Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chattopadhyay, K.; Ramagopal, U; Nathenson, S

2009-01-01

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 themore » 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.« less

Immunosuppressive Effect of B7-H4 Pathway in a Murine Systemic Lupus Erythematosus Model.

PubMed

Xiao, Ze Xiu; Zheng, Xu; Hu, Li; Wang, Julie; Olsen, Nancy; Zheng, Song Guo

2017-01-01

B7-H4, one of the co-stimulatory molecules of the B7 family, has been shown to play an important role in negatively regulating the adaptive immune response by inhibiting the proliferation, activation, and cytokine production of T cells. In this study, we investigate the role of B7-H4 in development of systemic lupus erythematosus (SLE). We investigated a murine model of SLE using transfer of bone marrow-derived dendritic cells (BMDCs) that were incubated with activated syngeneic lymphocyte-derived DNA. The recipient mouse produced anti-ds-DNA antibodies as well as displayed splenomegaly and lymphadenopathy as shown by significantly increased weights, and the kidneys showed lupus-like pathological changes include urine protein and glomerulonephritis with hyperplasia in glomeruli and increased mesangial cells and vasculitis with perivascular cell infiltration, glomerular deposition of IgG and complement C3. We showed that B7-H4 deficiency in BMDCs could cause greater production of anti-ds-DNA antibodies in transferred mice, and the lymph tissue swelling and the kidney lesions were also exacerbated with B7-H4 deficiency. Treatment with a B7-H4 antagonist antibody also aggravated the lupus model. Conversely, B7-H4 Ig alleviated the lupus manifestations. Therefore, we conclude that B7-H4 is a negative check point for the development of SLE in this murine model. These results suggest that this approach may have a clinical potential in treating human SLE.

Immunosuppressive Effect of B7-H4 Pathway in a Murine Systemic Lupus Erythematosus Model

PubMed Central

Xiao, Ze Xiu; Zheng, Xu; Hu, Li; Wang, Julie; Olsen, Nancy; Zheng, Song Guo

2017-01-01

B7-H4, one of the co-stimulatory molecules of the B7 family, has been shown to play an important role in negatively regulating the adaptive immune response by inhibiting the proliferation, activation, and cytokine production of T cells. In this study, we investigate the role of B7-H4 in development of systemic lupus erythematosus (SLE). We investigated a murine model of SLE using transfer of bone marrow-derived dendritic cells (BMDCs) that were incubated with activated syngeneic lymphocyte-derived DNA. The recipient mouse produced anti-ds-DNA antibodies as well as displayed splenomegaly and lymphadenopathy as shown by significantly increased weights, and the kidneys showed lupus-like pathological changes include urine protein and glomerulonephritis with hyperplasia in glomeruli and increased mesangial cells and vasculitis with perivascular cell infiltration, glomerular deposition of IgG and complement C3. We showed that B7-H4 deficiency in BMDCs could cause greater production of anti-ds-DNA antibodies in transferred mice, and the lymph tissue swelling and the kidney lesions were also exacerbated with B7-H4 deficiency. Treatment with a B7-H4 antagonist antibody also aggravated the lupus model. Conversely, B7-H4 Ig alleviated the lupus manifestations. Therefore, we conclude that B7-H4 is a negative check point for the development of SLE in this murine model. These results suggest that this approach may have a clinical potential in treating human SLE. PMID:29321778

Biomechanical Properties of Murine Meniscus Surface via AFM-based Nanoindentation

PubMed Central

Li, Qing; Doyran, Basak; Gamer, Laura W.; Lu, X. Lucas; Qin, Ling; Ortiz, Christine; Grodzinsky, Alan J.; Rosen, Vicki; Han, Lin

2015-01-01

This study aimed to quantify the biomechanical properties of murine meniscus surface. Atomic force microscopy (AFM)-based nanoindentation was performed on the central region, proximal side of menisci from 6- to 24-week old male C57BL/6 mice using microspherical tips (Rtip ≈ 5 μm) in PBS. A unique, linear correlation between indentation depth, D, and response force, F, was found on menisci from all age groups. This non-Hertzian behavior is likely due to the dominance of tensile resistance by the collagen fibril bundles on meniscus surface that are mostly aligned along the circumferential direction observed on 12-week old menisci. The indentation resistance was calculated as both the effective stiffness, Sind = dF/dD, and the effective modulus, Eind, via the isotropic Hertz model. Values of Sind and Eind were found to depend on indentation rate, suggesting the existence of poro-viscoelasticity. These values do not significantly vary with anatomical sites, lateral versus medial compartments, or mouse age. In addition, Eind of meniscus surface (e.g., 6.1 ± 0.8 MPa for 12 weeks of age, mean ± SEM, n = 13) was found to be significantly higher than those of meniscus surfaces in other species, and of murine articular cartilage surface (1.4 ± 0.1 MPa, n = 6). In summary, these results provided the first direct mechanical knowledge of murine knee meniscus tissues. We expect this understanding to serve as a mechanics-based benchmark for further probing the developmental biology and osteoarthritis symptoms of meniscus in various murine models. PMID:25817332

Severe interstitial pneumonia due to murine typhus in a patient returning from Bali.

PubMed

Malheiro, Luís; Ceia, Filipa; Alves, João; Carvalho, Ana Cláudia; Sobrinho-Simões, Joana; Sousa, Rita; Sarmento, António; Santos, Lurdes

2017-01-01

Murine typhus has been increasingly reported as a cause of fever in returning travelers from Southeast Asia. We report a case of a previously healthy traveler returning from Bali with an non-specific febrile illness which quickly progressed to a severe form of interstitial pneumonia. After a careful epidemiological evaluation and laboratory analysis, murine typhus was diagnosed.

[Virulence of Sporothrix globosa in murine models].

PubMed

Cruz Choappa, Rodrigo; Pérez Gaete, Salomón; Rodríguez Badilla, Valentina; Vieille Oyarzo, Peggy; Opazo Sanchez, Héctor

The sporothricosis disease is an infection caused by species included in Sporothrix schenkii complex. Verify the virulence of a strain of S. globosa using two different concentrations of inoculum by intraperitoneally and subcutaneously, into a mouse model. Nonrandomized pilot study, in murine inoculated with a strain of S. globosa (CBS 14.076M) by intraperitoneally and subcutaneously with inoculum concentrations of 0.5 and 4 McFarland. For this purpose 18 rodents CF-1 (ISP, Santiago, Chile) were used. The studied strain did not induce illness or injury on animals, they all survived and neither the tissue culture nor the histopathological analysis showed fungal growth or suggestive infection by organ abnormalities. The S. globosa strain did not present any virulence enough to cause disease at 0.5 and 4.0 McFarland concentration inoculum when inoculated in both intraperitoneally and subcutaneously, in murine models. Copyright © 2016 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

Three-dimensional alginate spheroid culture system of murine osteosarcoma.

PubMed

Akeda, Koji; Nishimura, Akinobu; Satonaka, Haruhiko; Shintani, Ken; Kusuzaki, Katsuyuki; Matsumine, Akihiko; Kasai, Yuichi; Masuda, Koichi; Uchida, Atsumasa

2009-11-01

Osteosarcoma (OS) is the most common primary malignant tumor of the bone and often forms pulmonary metastases, which are the most important prognostic factor. For further elucidation of the mechanism underlying the progression and metastasis of human OS, a culture system mimicking the microenvironment of the tumor in vivo is needed. We report a novel three-dimensional (3D) alginate spheroid culture system of murine osteosarcoma. Two different metastatic clones, the parental Dunn and its derivative line LM8, which has a higher metastatic potential to the lungs, were encapsulated in alginate beads to develop the 3D culture system. The beads containing murine OS cells were also transplanted into mice to determine their metastatic potential in vivo. In this culture system, murine OS cells encapsulated in alginate beads were able to grow in a 3D structure with cells detaching from the alginate environment. The number of detaching cells was higher in the LM8 cell line than the Dunn cell line. In the in vivo alginate bead transplantation model, the rate of pulmonary metastasis was higher with LM8 cells compared with that of Dunn cells. The cell characteristics and kinetics in this culture system closely reflect the original malignant potential of the cells in vivo.

Evaluation of a Murine Single-Blood-Injection SAH Model

PubMed Central

Sommer, Clemens; Steiger, Hans-Jakob; Schneider, Toni; Hänggi, Daniel

2014-01-01

The molecular pathways underlying the pathogenesis after subarachnoid haemorrhage (SAH) are poorly understood and continue to be a matter of debate. A valid murine SAH injection model is not yet available but would be the prerequisite for further transgenic studies assessing the mechanisms following SAH. Using the murine single injection model, we examined the effects of SAH on regional cerebral blood flow (rCBF) in the somatosensory (S1) and cerebellar cortex, neuro-behavioural and morphological integrity and changes in quantitative electrocorticographic and electrocardiographic parameters. Micro CT imaging verified successful blood delivery into the cisterna magna. An acute impairment of rCBF was observed immediately after injection in the SAH and after 6, 12 and 24 hours in the S1 and 6 and 12 hours after SAH in the cerebellum. Injection of blood into the foramen magnum reduced telemetric recorded total ECoG power by an average of 65%. Spectral analysis of ECoGs revealed significantly increased absolute delta power, i.e., slowing, cortical depolarisations and changes in ripples and fast ripple oscillations 12 hours and 24 hours after SAH. Therefore, murine single-blood-injection SAH model is suitable for pathophysiological and further molecular analysis following SAH. PMID:25545775

Systematic Characterization of the Murine Mitochondrial Proteome Using Functionally Validated Cardiac Mitochondria

PubMed Central

Zhang, Jun; Li, Xiaohai; Mueller, Michael; Wang, Yueju; Zong, Chenggong; Deng, Ning; Vondriska, Thomas M.; Liem, David A.; Yang, Jeong-In; Korge, Paavo; Honda, Henry; Weiss, James N.; Apweiler, Rolf; Ping, Peipei

2009-01-01

Mitochondria play essential roles in cardiac pathophysiology and the murine model has been extensively used to investigate cardiovascular diseases. In the present study, we characterized murine cardiac mitochondria using an LC/MS/MS approach. We extracted and purified cardiac mitochondria; validated their functionality to ensure the final preparation contains necessary components to sustain their normal function; and subjected these validated organelles to LC/MS/MS-based protein identification. A total of 940 distinct proteins were identified from murine cardiac mitochondria, among which, 480 proteins were not previously identified by major proteomic profiling studies. The 940 proteins consist of functional clusters known to support oxidative phosphorylation, metabolism and biogenesis. In addition, there are several other clusters--including proteolysis, protein folding, and reduction/oxidation signaling-which ostensibly represent previously under-appreciated tasks of cardiac mitochondria. Moreover, many identified proteins were found to occupy other subcellular locations, including cytoplasm, ER, and golgi, in addition to their presence in the mitochondria. These results provide a comprehensive picture of the murine cardiac mitochondrial proteome and underscore tissue- and species-specification. Moreover, the use of functionally intact mitochondria insures that the proteomic observations in this organelle are relevant to its normal biology and facilitates decoding the interplay between mitochondria and other organelles. PMID:18348319

Disruption of Canonical TGFβ-signaling in Murine Coronary Progenitor Cells by Low Level Arsenic

PubMed Central

Allison, Patrick; Huang, Tianfang; Broka, Derrick; Parker, Patti; Barnett, Joey V.; Camenisch, Todd D.

2013-01-01

Exposure to arsenic results in several types of cancers as well as heart disease. A major contributor to ischemic heart pathologies is coronary artery disease, however the influences by environmental arsenic in this disease process are not known. Similarly, the impact of toxicants on blood vessel formation and function during development has not been studied. During embryogenesis, the epicardium undergoes proliferation, migration, and differentiation into several cardiac cell types including smooth muscle cells which contribute to the coronary vessels. The TGFβ family of ligands and receptors are essential for developmental cardiac epithelial to mesenchymal transition (EMT) and differentiation into coronary smooth muscle cells. In this in vitrostudy, 18 hour exposure to 1.34 μMarsenite disrupted developmental EMT programming in murine epicardial cells causing a deficit in cardiac mesenchyme. The expression of EMT genes including TGFβ2, TGFβ receptor-3, Snail, and Has-2 are decreased in a dose-dependent manner following exposure to arsenite. TGFβ2 cell signaling is abrogated as detected by decreases in phosphorylated Smad2/3 when cells are exposed to 1.34 μMarsenite. There is also loss of nuclear accumulation pSmad due to arsenite exposure. These observations coincide with a decrease invimentinpositive mesenchymal cells invading three-dimensional collagen gels. However, arsenite does not block TGFβ2 mediated smooth muscle cell differentiation by epicardial cells. Overall these results show that arsenic exposure blocks developmental EMT gene programming in murine coronary progenitor cells by disrupting TGFβ2 signals and Smad activation, and that smooth muscle cell differentiation is refractory to this arsenic toxicity. PMID:23732083

Mesenchymal stromal cell derived extracellular vesicles rescue radiation damage to murine marrow hematopoietic cells

PubMed Central

Wen, Sicheng; Dooner, Mark; Cheng, Yan; Papa, Elaine; Del Tatto, Michael; Pereira, Mandy; Deng, Yanhui; Goldberg, Laura; Aliotta, Jason; Chatterjee, Devasis; Stewart, Connor; Carpanetto, Andrea; Collino, Federica; Bruno, Stefania; Camussi, Giovanni; Quesenberry, Peter

2016-01-01

Mesenchymal stromal cells (MSC) have been shown to reverse radiation damage to marrow stem cells. We have evaluated the capacity of MSC-derived extracellular vesicles (MSC-EVs) to mitigate radiation injury to marrow stem cells at 4 hours to 7 days after irradiation. Significant restoration of marrow stem cell engraftment at 4, 24 and 168 hours post-irradiation by exposure to MSC-EVs was observed at 3 weeks to 9 months after transplant and further confirmed by secondary engraftment. Intravenous injection of MSC-EVs to 500cGy exposed mice led to partial recovery of peripheral blood counts and restoration of the engraftment of marrow. The murine hematopoietic cell line, FDC-P1 exposed to 500 cGy, showed reversal of growth inhibition, DNA damage and apoptosis on exposure to murine or human MSC-EVs. Both murine and human MSC-EVs reverse radiation damage to murine marrow cells and stimulate normal murine marrow stem cell/progenitors to proliferate. A preparation with both exosomes and microvesicles was found to be superior to either microvesicles or exosomes alone. Biologic activity was seen in freshly isolated vesicles and in vesicles stored for up to 6 months in 10% DMSO at −80°C. These studies indicate that MSC-EVs can reverse radiation damage to bone marrow stem cells. PMID:27150009

Integrin Based Isolation Enables Purification of Murine Lineage Committed Cardiomyocytes

PubMed Central

Tarnawski, Laura; Xian, Xiaojie; Monnerat, Gustavo; Macaulay, Iain C.; Malan, Daniela; Borgman, Andrew; Wu, Sean M.; Fleischmann, Bernd K.; Jovinge, Stefan

2015-01-01

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. PMID:26323090

[Effects of traditional tibetan medicine, Fructus Lonicerae microphyllae on phagecytosis and cytokines production of murine macrophages].

PubMed

Wang, Ju-Le; Sun, Yang; Zhou, Hui-Ying; Xu, Qiang; Dun, Zhu

2006-01-01

To explore the effects of traditional Tibetan medicine, Fructus Lonicerae microphyllae (FLM) on phagecytosis and cytokines production of murine macrophages. The phagecytosis of murine macrophages was analyzed by neutral red phagecytosis assay. The activities of IL-1 and TNF-alpha were measured by biological methods. The mRNA of TNF-alpha and INF-gamma expressed by macrophages was detected by RT-PCR. The phagecytosis of murine macrophages was significantly enhanced by FLM at a concentration from 1 microg x mL(-1) to 100 microg x mL(-1) and the secretions of IL-1, and TNF-alpha from macrophages were markedly induced by FLM. Meanwhile, FLM also increased the expression of TNF-alpha mRNA and INF-gamma mRHA from macrophages in vitro. FLM could promote phagecytosis and cytokines production of murine macrophages.

Magnetic resonance imaging and spectroscopy of the murine cardiovascular system.

PubMed

Akki, Ashwin; Gupta, Ashish; Weiss, Robert G

2013-03-01

Magnetic resonance imaging (MRI) has emerged as a powerful and reliable tool to noninvasively study the cardiovascular system in clinical practice. Because transgenic mouse models have assumed a critical role in cardiovascular research, technological advances in MRI have been extended to mice over the last decade. These have provided critical insights into cardiac and vascular morphology, function, and physiology/pathophysiology in many murine models of heart disease. Furthermore, magnetic resonance spectroscopy (MRS) has allowed the nondestructive study of myocardial metabolism in both isolated hearts and in intact mice. This article reviews the current techniques and important pathophysiological insights from the application of MRI/MRS technology to murine models of cardiovascular disease.

Novel Application of Micro-Computerized Tomography for Morphologic Characterization of the Murine Penis.

PubMed

O'Neill, Marisol; Huang, Gene O; Lamb, Dolores J

2017-12-01

The murine penis model has enriched our understanding of anomalous penile development. The morphologic characterization of the murine penis using conventional serial sectioning methods is labor intensive and prone to errors. To develop a novel application of micro-computerized tomography (micro-CT) with iodine staining for rapid, non-destructive morphologic study of murine penis structure. Penises were dissected from 10 adult wild-type mice and imaged using micro-CT with iodine staining. Images were acquired at 5-μm spatial resolution on a Bruker SkyScan 1272 micro-CT system. After images were acquired, the specimens were washed of any remaining iodine and embedded in paraffin for conventional histologic examination. Histologic and micro-CT measurements for all specimens were made by 2 independent observers. Measurements of penile structures were made on virtual micro-CT sections and histologic slides. The Lin concordance correlation coefficient demonstrated almost perfect strength of agreement for interobserver variability for histologic section (0.9995, 95% CI = 0.9990-0.9997) and micro-CT section (0.9982, 95% CI = 0.9963-0.9991) measurements. Bland-Altman analysis for agreement between the 2 modalities of measurement demonstrated mean differences of -0.029, 0.022, and -0.068 mm for male urogenital mating protuberance, baculum, and penile glans length, respectively. There did not appear to be a bias for overestimation or underestimation of measured lengths and limits of agreement were narrow. The enhanced ability offered by micro-CT to phenotype the murine penis has the potential to improve translational studies examining the molecular pathways contributing to anomalous penile development. The present study describes the first reported use of micro-CT with iodine staining for imaging the murine penis. Producing repeated histologic sections of identical orientation was limited by inherent imperfections in mounting and tissue sectioning, but this was

A novel murine model for evaluating bovine papillomavirus prophylactics/therapeutics for equine sarcoid-like tumours

PubMed Central

Bogaert, Lies; Woodham, Andrew W.; Da Silva, Diane M.; Martens, Ann; Meyer, Evelyne

2015-01-01

Equine sarcoids are highly recurrent bovine papillomavirus (BPV)-induced fibroblastic neoplasms that are the most common skin tumours in horses. In order to facilitate the study of potential equine sarcoid prophylactics or therapeutics, which can be a slow and costly process in equines, a murine model for BPV-1 protein-expressing equine sarcoid-like tumours was developed in mice through stable transfection of BPV-1 E5 and E6 in a murine fibroblast tumour cell line (K-BALB). Like equine sarcoids, these murine tumour cells (BPV-KB) were of fibroblast origin, were tumorigenic and expressed BPV-1 proteins. As an initial investigation of the preclinical potential of this tumour model for equine sarcoids prophylactics, mice were immunized with BPV-1 E5E6 Venezuelan equine encephalitis virus replicon particles, prior to BPV-KB challenge, which resulted in an increased tumour-free period compared with controls, indicating that the BPV-KB murine model may be a valuable preclinical alternative to equine clinical trials. PMID:26044793

A novel murine model for evaluating bovine papillomavirus prophylactics/therapeutics for equine sarcoid-like tumours.

PubMed

Bogaert, Lies; Woodham, Andrew W; Da Silva, Diane M; Martens, Ann; Meyer, Evelyne; Kast, W Martin

2015-09-01

Equine sarcoids are highly recurrent bovine papillomavirus (BPV)-induced fibroblastic neoplasms that are the most common skin tumours in horses. In order to facilitate the study of potential equine sarcoid prophylactics or therapeutics, which can be a slow and costly process in equines, a murine model for BPV-1 protein-expressing equine sarcoid-like tumours was developed in mice through stable transfection of BPV-1 E5 and E6 in a murine fibroblast tumour cell line (K-BALB). Like equine sarcoids, these murine tumour cells (BPV-KB) were of fibroblast origin, were tumorigenic and expressed BPV-1 proteins. As an initial investigation of the preclinical potential of this tumour model for equine sarcoids prophylactics, mice were immunized with BPV-1 E5E6 Venezuelan equine encephalitis virus replicon particles, prior to BPV-KB challenge, which resulted in an increased tumour-free period compared with controls, indicating that the BPV-KB murine model may be a valuable preclinical alternative to equine clinical trials.

Detection of adulterated murine components in meat products by TaqMan© real-time PCR.

PubMed

Fang, Xin; Zhang, Chi

2016-02-01

Using murine meat to substitute mutton has been identified as a new type of meat fraud in China, yet no detection method for murine species has been reported. Here, three kinds of rodent were used as target species to establish a murine-specific real-time PCR method of detection. The mitochondrial cytochrome b gene (cytb) of each target was sequenced and a TaqMan probe was designed based on the cytb. Simultaneously, an internal positive control (IPC) plasmid along with its respective probe were designed to monitor the PCR reaction. As a result, the duplex real-time PCR system was verified to be specific. The limit of detection (LOD) was lower than 1 pg of DNA per reaction and 0.1% murine contamination in meat mixtures. Standard curves were generated for a quantitative analysis. Thus, this study provided a new tool to control the quality of meat products for official and third-party laboratories. Copyright © 2015. Published by Elsevier Ltd.

Characterization of Ribosomal Frameshifting in Theiler's Murine Encephalomyelitis Virus

PubMed Central

Finch, Leanne K.; Ling, Roger; Napthine, Sawsan; Olspert, Allan; Michiels, Thomas; Lardinois, Cécile; Bell, Susanne; Loughran, Gary; Brierley, Ian

2015-01-01

ABSTRACT Theiler's murine encephalomyelitis virus (TMEV) is a member of the genus Cardiovirus in the Picornaviridae, a family of positive-sense single-stranded RNA viruses. Previously, we demonstrated that in the related cardiovirus, Encephalomyocarditis virus, a programmed −1 ribosomal frameshift (−1 PRF) occurs at a conserved G_GUU_UUU sequence within the 2B-encoding region of the polyprotein open reading frame (ORF). Here we show that −1 PRF occurs at a similar site during translation of the TMEV genome. In addition, we demonstrate that a predicted 3′ RNA stem-loop structure at a noncanonical spacing downstream of the shift site is required for efficient frameshifting in TMEV and that frameshifting also requires virus infection. Mutating the G_GUU_UUU shift site to inhibit frameshifting results in an attenuated virus with reduced growth kinetics and a small-plaque phenotype. Frameshifting in the virus context was found to be extremely efficient at 74 to 82%, which, to our knowledge, is the highest frameshifting efficiency recorded to date for any virus. We propose that highly efficient −1 PRF in TMEV provides a mechanism to escape the confines of equimolar expression normally inherent in the single-polyprotein expression strategy of picornaviruses. IMPORTANCE Many viruses utilize programmed −1 ribosomal frameshifting (−1 PRF) to produce different protein products at a defined ratio, or to translate overlapping ORFs to increase coding capacity. With few exceptions, −1 PRF occurs on specific “slippery” heptanucleotide sequences and is stimulated by RNA structure beginning 5 to 9 nucleotides (nt) downstream of the slippery site. Here we describe an unusual case of −1 PRF in Theiler's murine encephalomyelitis virus (TMEV) that is extraordinarily efficient (74 to 82% of ribosomes shift into the alternative reading frame) and, in stark contrast to other examples of −1 PRF, is dependent upon a stem-loop structure beginning 14 nt downstream of

Osmotic characteristics and fertility of murine spermatozoa collected in different solutions.

PubMed

Si, Wei; Men, Hongsheng; Benson, James D; Critser, John K

2009-02-01

Osmotic stress is an important factor that can result in cell damage during cryopreservation. Before ejaculation or collection for cryopreservation, murine spermatozoa are stored in epididymal fluid, a physiologically hyperosmotic environment (approximately 415 mmol/kg). The objectives of this study were to determine the osmotic tolerance limits of sperm motion parameters of ICR and C57BL/6 mouse spermatozoa collected in isosmotic (290 mmol/kg) and hyperosmotic (415 mmol/kg) media, and the effect of the osmolality of sperm collection media on sperm fertility after cryopreservation. Our results indicate that murine spermatozoa collected in media with different osmolalities (290 and 415 mmol/kg Dulbecco's phosphate buffered saline (DPBS)) appeared to have different osmotic tolerances for the maintenance of sperm motility and other motion parameters in both mouse strains. The hypo- and hyperosmotic treatments decreased motility and affected other motion parameters of spermatozoa collected in 290 mmol/kg DPBS. The extent of the change of motion parameters after treatments corresponded with the levels of osmotic stress. However, for spermatozoa collected in 415 mmol/kg DPBS, exposure to 290 mmol/kg DPBS tended to increase sperm motility and the quality of their motion parameters. The osmolality of sperm collection medium can affect murine sperm fertility. Spermatozoa collected in 415 mmol/kg medium showed higher fertility compared with spermatozoa collected in 290 mmol/kg as assessed by IVF. Results characterizing murine sperm osmotic tolerance collected in media with different osmolalities from different strains and the effect of collection media osmolality on sperm fertility after cryopreservation will be useful in designing cryopreservation protocols.

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

PubMed

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

2012-08-01

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. Copyright © 2011 John Wiley & Sons, Ltd.

Human CD22 Inhibits Murine B Cell Receptor Activation in a Human CD22 Transgenic Mouse Model.

PubMed

Bednar, Kyle J; Shanina, Elena; Ballet, Romain; Connors, Edward P; Duan, Shiteng; Juan, Joana; Arlian, Britni M; Kulis, Michael D; Butcher, Eugene C; Fung-Leung, Wai-Ping; Rao, Tadimeti S; Paulson, James C; Macauley, Matthew S

2017-11-01

CD22, a sialic acid-binding Ig-type lectin (Siglec) family member, is an inhibitory coreceptor of the BCR with established roles in health and disease. The restricted expression pattern of CD22 on B cells and most B cell lymphomas has made CD22 a therapeutic target for B cell-mediated diseases. Models to better understand how in vivo targeting of CD22 translates to human disease are needed. In this article, we report the development of a transgenic mouse expressing human CD22 (hCD22) in B cells and assess its ability to functionally substitute for murine CD22 (mCD22) for regulation of BCR signaling, Ab responses, homing, and tolerance. Expression of hCD22 on transgenic murine B cells is comparable to expression on human primary B cells, and it colocalizes with mCD22 on the cell surface. Murine B cells expressing only hCD22 have identical calcium (Ca 2+ ) flux responses to anti-IgM as mCD22-expressing wild-type B cells. Furthermore, hCD22 transgenic mice on an mCD22 -/- background have restored levels of marginal zone B cells and Ab responses compared with deficiencies observed in CD22 -/- mice. Consistent with these observations, hCD22 transgenic mice develop normal humoral responses in a peanut allergy oral sensitization model. Homing of B cells to Peyer's patches was partially rescued by expression of hCD22 compared with CD22 -/- B cells, although not to wild-type levels. Notably, Siglec-engaging antigenic liposomes formulated with an hCD22 ligand were shown to prevent B cell activation, increase cell death, and induce tolerance in vivo. This hCD22 transgenic mouse will be a valuable model for investigating the function of hCD22 and preclinical studies targeting hCD22. Copyright © 2017 by The American Association of Immunologists, Inc.

Magnetic resonance imaging and spectroscopy of the murine cardiovascular system

PubMed Central

Akki, Ashwin; Gupta, Ashish

2013-01-01

Magnetic resonance imaging (MRI) has emerged as a powerful and reliable tool to noninvasively study the cardiovascular system in clinical practice. Because transgenic mouse models have assumed a critical role in cardiovascular research, technological advances in MRI have been extended to mice over the last decade. These have provided critical insights into cardiac and vascular morphology, function, and physiology/pathophysiology in many murine models of heart disease. Furthermore, magnetic resonance spectroscopy (MRS) has allowed the nondestructive study of myocardial metabolism in both isolated hearts and in intact mice. This article reviews the current techniques and important pathophysiological insights from the application of MRI/MRS technology to murine models of cardiovascular disease. PMID:23292717

First steps to define murine amniotic fluid stem cell microenvironment.

PubMed

Bertin, E; Piccoli, M; Franzin, C; Spiro, G; Donà, S; Dedja, A; Schiavi, F; Taschin, E; Bonaldo, P; Braghetta, P; De Coppi, P; Pozzobon, M

2016-11-15

Stem cell niche refers to the microenvironment where stem cells reside in living organisms. Several elements define the niche and regulate stem cell characteristics, such as stromal support cells, gap junctions, soluble factors, extracellular matrix proteins, blood vessels and neural inputs. In the last years, different studies demonstrated the presence of cKit + cells in human and murine amniotic fluid, which have been defined as amniotic fluid stem (AFS) cells. Firstly, we characterized the murine cKit + cells present both in the amniotic fluid and in the amnion. Secondly, to analyze the AFS cell microenvironment, we injected murine YFP + embryonic stem cells (ESC) into the amniotic fluid of E13.5 wild type embryos. Four days after transplantation we found that YFP + sorted cells maintained the expression of pluripotency markers and that ESC adherent to the amnion were more similar to original ESC in respect to those isolated from the amniotic fluid. Moreover, cytokines evaluation and oxygen concentration analysis revealed in this microenvironment the presence of factors that are considered key regulators in stem cell niches. This is the first indication that AFS cells reside in a microenvironment that possess specific characteristics able to maintain stemness of resident and exogenous stem cells.

Getting away with murder: how do the BCL-2 family of proteins kill with immunity?

PubMed Central

Renault, Thibaud T.; Chipuk, Jerry E.

2013-01-01

About 1 million per second is the number of white blood cells the adult human body produces. However, only a small fraction of them will survive as the majority is eliminated through a genetically controlled form of cell death referred to as apoptosis. This review places into perspective recent studies pertaining to the BCL-2 family of proteins as critical regulators of the development and function of the immune system, with particular attention on B cell and T cell biology. Here we discuss how elegant murine model systems have revealed the major contributions of the BCL-2 family in establishing an effective immune system. Moreover, we highlight some key regulatory pathways that influence the expression, function, and stability of individual BCL-2 family members, and discuss their role in immunity. From deadly methods to more gentle manners, the final portion of the review discusses the non-apoptotic functions of the BCL-2 family and how they pertain to the control of immunity. PMID:23527542

1.8 Å structure of murine GITR ligand dimer expressed in Drosophila melanogaster S2 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chattopadhyay, Kausik; Ramagopal, Udupi A.; Nathenson, Stanley G., E-mail: nathenso@aecom.yu.edu

2009-05-01

1.8 Å X-ray crystal structure of mouse GITRL expressed in D. melanogaster S2 cells shows an identical ‘strand-exchanged’ dimeric assembly similar to that observed previously for the E. coli-expressed protein. 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,more » 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 Å 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.« less

IL-33 signaling protects from murine oxazolone colitis by supporting intestinal epithelial function

PubMed Central

Waddell, Amanda; Vallance, Jefferson E; Moore, Preston D; Hummel, Amy T; Wu, David; Shanmukhappa, Shiva K; Fei, Lin; Washington, M Kay; Minar, Phillip; Coburn, Lori A; Nakae, Susumu; Wilson, Keith T; Denson, Lee A; Hogan, Simon P; Rosen, Michael J

2015-01-01

Background IL-33, a member of the IL-1 cytokine family that signals through ST2, is upregulated in ulcerative colitis (UC); however, the role of IL-33 in colitis remains unclear. IL-33 augments type 2 immune responses, which have been implicated in UC pathogenesis. We sought to determine the role of IL-33 signaling in oxazolone (OXA) colitis, a type 2 cytokine-mediated murine model of UC. Methods Colon mucosal IL-33 expression was compared between pediatric and adult UC and non-IBD patients using immunohistochemistry and real-time PCR. OXA colitis was induced in WT, IL-33−/− and ST2−/− mice, and histopathology, cytokine levels, and goblet cells were assessed. Transepithelial resistance (TER) was measured across IL-33-treated T84 cell monolayers. Results Colon mucosal IL-33 was increased in pediatric patients with active UC and in OXA colitis. IL-33−/− and ST2−/− OXA mice exhibited increased disease severity compared to WT OXA mice. OXA induced a mixed mucosal cytokine response, but few differences were observed between OXA WT and IL-33−/− or ST2−/− mice. Goblet cells were significantly decreased in IL-33−/− and ST2−/− OXA compared to WT OXA mice. IL-33 augmented TER in T84 cells, and this effect was blocked by the ERK1/2 inhibitor PD98,059. Conclusions OXA colitis is exacerbated in IL-33−/− and ST2−/− mice. Increased mucosal IL-33 in human UC and murine colitis may be a homeostatic response to limit inflammation, potentially through effects on epithelial barrier function. Further investigation of IL-33 protective mechanisms would inform the development of novel therapeutic approaches. PMID:26313694

Characterization of Human and Murine T-Cell Immunoglobulin Mucin Domain 4 (TIM-4) IgV Domain Residues Critical for Ebola Virus Entry.

PubMed

Rhein, Bethany A; Brouillette, Rachel B; Schaack, Grace A; Chiorini, John A; Maury, Wendy

2016-07-01

Phosphatidylserine (PtdSer) receptors that are responsible for the clearance of dying cells have recently been found to mediate enveloped virus entry. Ebola virus (EBOV), a member of the Filoviridae family of viruses, utilizes PtdSer receptors for entry into target cells. The PtdSer receptors human and murine T-cell immunoglobulin mucin (TIM) domain proteins TIM-1 and TIM-4 mediate filovirus entry by binding to PtdSer on the virion surface via a conserved PtdSer binding pocket within the amino-terminal IgV domain. While the residues within the TIM-1 IgV domain that are important for EBOV entry are characterized, the molecular details of virion-TIM-4 interactions have yet to be investigated. As sequences and structural alignments of the TIM proteins suggest distinct differences in the TIM-1 and TIM-4 IgV domain structures, we sought to characterize TIM-4 IgV domain residues required for EBOV entry. Using vesicular stomatitis virus pseudovirions bearing EBOV glycoprotein (EBOV GP/VSVΔG), we evaluated virus binding and entry into cells expressing TIM-4 molecules mutated within the IgV domain, allowing us to identify residues important for entry. Similar to TIM-1, residues in the PtdSer binding pocket of murine and human TIM-4 (mTIM-4 and hTIM-4) were found to be important for EBOV entry. However, additional TIM-4-specific residues were also found to impact EBOV entry, with a total of 8 mTIM-4 and 14 hTIM-4 IgV domain residues being critical for virion binding and internalization. Together, these findings provide a greater understanding of the interaction of TIM-4 with EBOV virions. With more than 28,000 cases and over 11,000 deaths during the largest and most recent Ebola virus (EBOV) outbreak, there has been increased emphasis on the development of therapeutics against filoviruses. Many therapies under investigation target EBOV cell entry. T-cell immunoglobulin mucin (TIM) domain proteins are cell surface factors important for the entry of many enveloped viruses

Characterization of Human and Murine T-Cell Immunoglobulin Mucin Domain 4 (TIM-4) IgV Domain Residues Critical for Ebola Virus Entry

PubMed Central

Rhein, Bethany A.; Brouillette, Rachel B.; Schaack, Grace A.; Chiorini, John A.

2016-01-01

ABSTRACT Phosphatidylserine (PtdSer) receptors that are responsible for the clearance of dying cells have recently been found to mediate enveloped virus entry. Ebola virus (EBOV), a member of the Filoviridae family of viruses, utilizes PtdSer receptors for entry into target cells. The PtdSer receptors human and murine T-cell immunoglobulin mucin (TIM) domain proteins TIM-1 and TIM-4 mediate filovirus entry by binding to PtdSer on the virion surface via a conserved PtdSer binding pocket within the amino-terminal IgV domain. While the residues within the TIM-1 IgV domain that are important for EBOV entry are characterized, the molecular details of virion–TIM-4 interactions have yet to be investigated. As sequences and structural alignments of the TIM proteins suggest distinct differences in the TIM-1 and TIM-4 IgV domain structures, we sought to characterize TIM-4 IgV domain residues required for EBOV entry. Using vesicular stomatitis virus pseudovirions bearing EBOV glycoprotein (EBOV GP/VSVΔG), we evaluated virus binding and entry into cells expressing TIM-4 molecules mutated within the IgV domain, allowing us to identify residues important for entry. Similar to TIM-1, residues in the PtdSer binding pocket of murine and human TIM-4 (mTIM-4 and hTIM-4) were found to be important for EBOV entry. However, additional TIM-4-specific residues were also found to impact EBOV entry, with a total of 8 mTIM-4 and 14 hTIM-4 IgV domain residues being critical for virion binding and internalization. Together, these findings provide a greater understanding of the interaction of TIM-4 with EBOV virions. IMPORTANCE With more than 28,000 cases and over 11,000 deaths during the largest and most recent Ebola virus (EBOV) outbreak, there has been increased emphasis on the development of therapeutics against filoviruses. Many therapies under investigation target EBOV cell entry. T-cell immunoglobulin mucin (TIM) domain proteins are cell surface factors important for the entry of many

Murine recombinant angiotensin-converting enzyme 2 attenuates kidney injury in experimental Alport syndrome.

PubMed

Bae, Eun Hui; Fang, Fei; Williams, Vanessa R; Konvalinka, Ana; Zhou, Xiaohua; Patel, Vaibhav B; Song, Xuewen; John, Rohan; Oudit, Gavin Y; Pei, York; Scholey, James W

2017-06-01

Angiotensin-converting enzyme 2 (ACE2) is a monocarboxypeptidase in the renin-angiotensin system that catalyzes the breakdown of angiotensin II to angiotensin 1-7. We have reported that ACE2 expression in the kidney is reduced in experimental Alport syndrome but the impact of this finding on disease progression has not been studied. Accordingly, we evaluated effects of murine recombinant ACE2 treatment in Col4a3 knockout mice, a model of Alport syndrome characterized by proteinuria and progressive renal injury. Murine recombinant ACE2 (0.5 mg/kg/day) was administered from four to seven weeks of age via osmotic mini-pump. Pathological changes were attenuated by murine recombinant ACE2 treatment which ameliorated kidney fibrosis as shown by decreased expression of COL1α1 mRNA, less accumulation of extracellular matrix proteins, and inhibition of transforming growth factor-β signaling. Further, increases in proinflammatory cytokine expression, macrophage infiltration, inflammatory signaling pathway activation, and heme oxygenase-1 levels in Col4a3 knockout mice were also reduced by murine recombinant ACE2 treatment. Lastly, murine recombinant ACE2 influenced the turnover of renal ACE2, as it suppressed the expression of tumor necrosis factor-α converting enzyme, a negative regulator of ACE2. Thus, treatment with exogenous ACE2 alters angiotensin peptide metabolism in the kidneys of Col4a3 knockout mice and attenuates the progression of Alport syndrome nephropathy. Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Glutathione S-transferase pi isoform (GSTP1) expression in murine retina increases with developmental maturity.

PubMed

Lee, Wen-Hsiang; Joshi, Pratibha; Wen, Rong

2014-01-01

Glutathione S-transferase pi isoform (GSTP1) is an intracellular detoxification enzyme that catalyzes reduction of chemically reactive electrophiles and is a zeaxanthin-binding protein in the human macula. We have previously demonstrated that GSTP1 levels are decreased in human age-related macular degeneration (AMD) retina compared to normal controls (Joshi et al., Invest Ophthalmol Vis Sci, e-abstract, 2009). We also showed that GSTP1 levels parallel survival of human retinal pigment epithelial (RPE) cells exposed to ultraviolet (UV) light, and GSTP1 over-expression protects them against UV light damage (Joshi et al., Invest Ophthalmol Vis Sci, e-abstract, 2010). In the present work, we determined the developmental time course of GSTP1 expression in murine retina and in response to light challenge. Eyes from BALB/c mice at postnatal day 20, 1 month, and 2 months of age were prepared for retinal protein extraction and cryo sectioning, and GSTP1 levels in the retina were analyzed by Western blot and immunohistochemistry (IHC). Another group of BALB/c mice with the same age ranges was exposed to 1000 lx of white fluorescent light for 24 h, and their retinas were analyzed for GSTP1 expression by Western blot and IHC in a similar manner. GSTP1 levels in the murine retina increased in ascending order from postnatal day 20, 1 month, and 2 months of age. Moreover, GSTP1 expression in murine retina at postnatal day 20, 1 month, and 2 months of age increased in response to brief light exposure compared to age-matched controls under normal condition. GSTP1 expression in retina increases with developmental age in mice and accompanies murine retinal maturation. Brief exposure to light induces GSTP1 expression in the murine retina across various developmental ages. GSTP1 induction may be a protective response to light-induced oxidative damage in the murine retina.

Glutathione S-Transferase Pi Isoform (GSTP1) Expression in Murine Retina Increases with Developmental Maturity

PubMed Central

Lee, Wen-Hsiang; Joshi, Pratibha; Wen, Rong

2014-01-01

Background and Aims Glutathione S-transferase pi isoform (GSTP1) is an intracellular detoxification enzyme that catalyzes reduction of chemically reactive electrophiles and is a zeaxanthin-binding protein in the human macula. We have previously demonstrated that GSTP1 levels are decreased in human age-related macular degeneration (AMD) retina compared to normal controls [1]. We also showed that GSTP1 levels parallel survival of human retinal pigment epithelial (RPE) cells exposed to UV light, and GSTP1 over-expression protects them against UV light damage [2]. In the present work, we determined the developmental time course of GSTP1 expression in murine retina and in response to light challenge. Methods Eyes from BALB/c mice at post-natal day 20, 1 month, and 2 months of age were prepared for retinal protein extraction and cryo sectioning, and GSTP1 levels in the retina were analyzed by Western blot and immunohistochemistry (IHC). Another group of BALB/c mice with the same age ranges was exposed to 1000 lux of white fluorescent light for 24 hours, and their retinas were analyzed for GSTP1 expression by Western blot and IHC in a similar manner. Results GSTP1 levels in the murine retina increased in ascending order from post-natal day 20, 1 month, and 2 months of age. Moreover, GSTP1 expression in murine retina at post-natal day 20, 1 month, and 2 months of age increased in response to brief light exposure compared to age-matched controls under normal condition. Conclusions GSTP1 expression in retina increases with developmental age in mice and accompanies murine retinal maturation. Brief exposure to light induces GSTP1 expression in the murine retina across various developmental ages. GSTP1 induction may be a protective response to light-induced oxidative damage in the murine retina. PMID:24664677

Correction of murine mucopolysaccharidosis VII by a human. beta. -glucuronidase transgene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kyle, J.W.; Vogler, C.; Hoffmann, J.W.

1990-05-01

The authors recently described a murine model for mucopolysaccharidosis VII in mice that have an inherited deficiency of {beta}-glucuronidase. Affected mice, of genotype gus{sup mps}/gus{sup mps}, present clinical manifestations similar to those of humans with mucopolysaccharidosis VII (Sly syndrome) and are shown here to have secondary elevations of other lysosomal enzymes. The mucopolysaccharidosis VII phenotype in both species includes dwarfism, skeletal deformities, and premature death. Lysosome storage is visualized within enlarged vesicles and correlates biochemically with accumulation of undegraded and partially degraded glycosaminoglycans. In this report they describe the consequences of introducing the human {beta}-glucuronidase gene, GUSB, into gus{sup mps}/gus{supmore » mps} mice that produce virtually no murine {beta}-glucuronidase. Transgenic mice homozygous for the mucopolysaccharidosis VII mutation expressed high levels of human {beta}-glucuronidase activity in all tissues examined and were phenotypically normal. Biochemically, both the intralysosomal storage of glycosaminoglycans and the secondary elevation of other acid hydrolases were corrected. These findings demonstrate that the GUSB transgene is expressed in gus{sup mps}/gus{sup mps} mice and that human {beta}-glucuronidase corrects the murine mucopolysaccharidosis storage disease.« less

Murine aggregation chimeras and wholemount imaging in airway stem cell biology.

PubMed

Rosewell, Ian R; Giangreco, Adam

2012-01-01

Local tissue stem cells are known to exist in mammalian lungs but their role in epithelial maintenance remains unclear. We therefore developed murine aggregation chimera and wholemount imaging techniques to assess the contribution of these cells to lung homeostasis and repair. In this chapter we provide further details regarding the generation of murine aggregation chimera mice and their subsequent use in wholemount lung imaging. We also describe methods related to the interpretation of this data that allows for quantitative assessment of airway stem cell activation versus quiescence. Using these techniques, it is possible to compare the growth and differentiation capacity of various lung epithelial cells in normal, repairing, and diseased states.

Restriction by APOBEC3 proteins of endogenous retroviruses with an extracellular life cycle: ex vivo effects and in vivo "traces" on the murine IAPE and human HERV-K elements

PubMed Central

Esnault, Cécile; Priet, Stéphane; Ribet, David; Heidmann, Odile; Heidmann, Thierry

2008-01-01

Background APOBEC3 cytosine deaminases have been demonstrated to restrict infectivity of a series of retroviruses, with different efficiencies depending on the retrovirus. In addition, APOBEC3 proteins can severely restrict the intracellular transposition of a series of retroelements with a strictly intracellular life cycle, including the murine IAP and MusD LTR-retrotransposons. Results Here we show that the IAPE element, which is the infectious progenitor of the strictly intracellular IAP elements, and the infectious human endogenous retrovirus HERV-K are restricted by both murine and human APOBEC3 proteins in an ex vivo assay for infectivity, with evidence in most cases of strand-specific G-to-A editing of the proviruses, with the expected signatures. In silico analysis of the naturally occurring genomic copies of the corresponding endogenous elements performed on the mouse and human genomes discloses "traces" of APOBEC3-editing, with the specific signature of the murine APOBEC3 and human APOBEC3G enzymes, respectively, and to a variable extent depending on the family member. Conclusion These results indicate that the IAPE and HERV-K elements, which can only replicate via an extracellular infection cycle, have been restricted at the time of their entry, amplification and integration into their target host genomes by definite APOBEC3 proteins, most probably acting in evolution to limit the mutagenic effect of these endogenized extracellular parasites. PMID:18702815

MicroRNA-125 family members exert a similar role in the regulation of murine hematopoiesis.

PubMed

Wojtowicz, Edyta E; Walasek, Marta A; Broekhuis, Mathilde J C; Weersing, Ellen; Ritsema, Martha; Ausema, Albertina; Bystrykh, Leonid V; de Haan, Gerald

2014-10-01

MicroRNAs (miRNAs) are crucial for proper functioning of hematopoietic stem and progenitor cells (HSPCs). Members of the miRNA-125 family (consisting of miR-125a, miR-125b1, and miR-125b2) are known to confer a proliferative advantage on cells upon overexpression, to decrease the rate of apoptosis by targeting proapoptotic genes, and to promote differentiation toward the myeloid lineage in mice. However, many distinct biological effects of the three miR-125 species have been reported as well. In the current study, we set out to assess whether the three miRNA-125s that carry identical seed sequences could be functionally different. Our data show that overexpression of each of the three miR-125 family members preserves HSPCs in a primitive state in vitro, results in a competitive advantage upon serial transplantation, and promotes skewing toward the myeloid lineage. All miR-125 family members decreased the pool of phenotypically defined Lin(-)Sca(+)Kit(+)CD48(-)CD150(+) long-term hematopoietic stem cells, simultaneously increasing the self-renewal activity upon secondary transplantation. The downregulation of miR-125s in hematopoietic stem cells abolishes these effects and impairs long-term contribution to blood cell production. The introduction of a point mutation within the miRNA-125 seed sequence abolishes all abovementioned effects and leads to the restoration of normal hematopoiesis. Our results show that all miR-125 family members are similar in function, they likely operate in a seed-sequence-dependent manner, and they induce a highly comparable hematopoietic phenotype. Copyright © 2014 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Murine models of breast cancer bone metastasis

PubMed Central

Wright, Laura E; Ottewell, Penelope D; Rucci, Nadia; Peyruchaud, Olivier; Pagnotti, Gabriel M; Chiechi, Antonella; Buijs, Jeroen T; Sterling, Julie A

2016-01-01

Bone metastases cause significant morbidity and mortality in late-stage breast cancer patients and are currently considered incurable. Investigators rely on translational models to better understand the pathogenesis of skeletal complications of malignancy in order to identify therapeutic targets that may ultimately prevent and treat solid tumor metastasis to bone. Many experimental models of breast cancer bone metastases are in use today, each with its own caveats. In this methods review, we characterize the bone phenotype of commonly utilized human- and murine-derived breast cell lines that elicit osteoblastic and/or osteolytic destruction of bone in mice and report methods for optimizing tumor-take in murine models of bone metastasis. We then provide protocols for four of the most common xenograft and syngeneic inoculation routes for modeling breast cancer metastasis to the skeleton in mice, including the intra-cardiac, intra-arterial, orthotopic and intra-tibial methods of tumor cell injection. Recommendations for in vivo and ex vivo assessment of tumor progression and bone destruction are provided, followed by discussion of the strengths and limitations of the available tools and translational models that aid investigators in the study of breast cancer metastasis to bone. PMID:27867497

A flavone derivative from Sesbania sesban leaves and its cytotoxicity against murine leukemia P-388 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dianhar, Hanhan, E-mail: liadewi@chem.itb.ac.id; Syah, Yana Maolana, E-mail: liadewi@chem.itb.ac.id; Mujahidin, Didin, E-mail: liadewi@chem.itb.ac.id

2014-03-24

Sesbania sesban, locally named as Jayanti, is one of Indonesia plants belonging to Fabaceae family. This species is traditionally used by Indonesian people to cure digestive disorders, fever, or headache. Jayanti can grow well in tropical to subtropical region, such as in Asia and Africa. Based on literature, qualitative analysis of the methanol extract of leaves of S. sesban showed that it contained flavonoids, alkaloids, saponins and glycosides. In addition, the activity assay of extracts of different tissues of this species showed antitumor, antimalarial, and antidiabetic activityies (leaves and seed extracts), antioxidants (flower extract), and analgesic (wood extract). Though themore » extracts of S. sesban parts showed interesting activities, chemical study of those extracts have not been widely reported. Therefore, the objective of this research was to isolate the secondary metabolites from methanol extract of leaves of S. sesban and to determine their cytotoxicity against murine leukemia P-388 cells. One compound has been obtained and identified as 3-hydroxy-4',7-dimethoxyflavone (1), a new isolated compound from nature. This compound was obtained through separation of methanol extract using various chromatographic techniques, such as vacuum liquid chromatography and radial chromatography. The structure elucidation of isolated compound was based on 1D NMR ({sup 1}H-NMR and {sup 13}C-NMR) and 2D NMR (HMBC). The cytotoxicity of methanol extract and compound 1 against murine leukemia P-388 cells examined through MTT assay showed IC{sub 50} value of 60.04 μg/mL and 5.40 μg/mL, respectively.« less

A flavone derivative from Sesbania sesban leaves and its cytotoxicity against murine leukemia P-388 cells

NASA Astrophysics Data System (ADS)

Dianhar, Hanhan; Syah, Yana Maolana; Mujahidin, Didin; Hakim, Euis Holisotan; Juliawaty, Lia Dewi

2014-03-01

Sesbania sesban, locally named as Jayanti, is one of Indonesia plants belonging to Fabaceae family. This species is traditionally used by Indonesian people to cure digestive disorders, fever, or headache. Jayanti can grow well in tropical to subtropical region, such as in Asia and Africa. Based on literature, qualitative analysis of the methanol extract of leaves of S. sesban showed that it contained flavonoids, alkaloids, saponins and glycosides. In addition, the activity assay of extracts of different tissues of this species showed antitumor, antimalarial, and antidiabetic activityies (leaves and seed extracts), antioxidants (flower extract), and analgesic (wood extract). Though the extracts of S. sesban parts showed interesting activities, chemical study of those extracts have not been widely reported. Therefore, the objective of this research was to isolate the secondary metabolites from methanol extract of leaves of S. sesban and to determine their cytotoxicity against murine leukemia P-388 cells. One compound has been obtained and identified as 3-hydroxy-4',7-dimethoxyflavone (1), a new isolated compound from nature. This compound was obtained through separation of methanol extract using various chromatographic techniques, such as vacuum liquid chromatography and radial chromatography. The structure elucidation of isolated compound was based on 1D NMR (1H-NMR and 13C-NMR) and 2D NMR (HMBC). The cytotoxicity of methanol extract and compound 1 against murine leukemia P-388 cells examined through MTT assay showed IC50 value of 60.04 μg/mL and 5.40 μg/mL, respectively.

Effect of cooling rate on human and murine hemopoietic precursor cell recovery

DOE Office of Scientific and Technical Information (OSTI.GOV)

Niskanen, E.; Pirsch, G.

1983-08-01

The effect of cooling rate on recovery of human and murine hemopoietic precursor cells was studied. In the presence of 10% Me2SO, a cooling rate of 7 degrees C/min from -4 to -30 degrees C was optimal for recovery of both human and murine precursor cells which give rise to colonies in diffusion chambers implanted in mice (CFU-DG). Cooling of human marrow at a rate between 3 and 7 degrees C/min resulted in the best CFU-C recovery, although no good correlation between the cooling rate and murine CFU-C recovery was demonstrated. These data suggest that recovery of the primitive hemopoieticmore » precursor cells can be improved by changing the standard cryopreservation programs used presently. However, improved recovery of CFU-DG does not necessarily translate into faster reconstitution of hemopoiesis. No significant difference was observed in overall recovery of bone marrow cellularity in lethally irradiated mice following injection of untreated marrow and marrow cooled at a rate of 1 and 7 degrees C/min.« less

In vivo laser confocal microscopic analysis of murine cornea and lens microstructures.

PubMed

Yuasa, Masashi; Kobayashi, Akira; Yokogawa, Hideaki; Sugiyama, Kazuhisa

2008-01-01

The purpose of the current study is to investigate in vivo microstructures of anterior segments of normal murine eyes by new-generation in vivo laser confocal microscopy. Twenty-six corneas and lenses from 13 mice were analyzed by in vivo laser confocal microscopy. Murine corneal superficial cells formed a polygonal cell pattern, with a mean cell density of 577 +/- 115 cells/mm2 (mean +/- standard deviation). Corneal basal epithelial cells had dark cytoplasm and were closely organized (9,312 +/- 1,777 cells/mm2). Sub-basal nerve fiber bundles were arranged in a whorl pattern, with both clockwise and counter-clockwise patterns. In the stroma, keratocytes were observed as numerous reflective stellate structures. The endothelial cells were organized in a honeycomb pattern (2,463 +/- 292 cells/mm2). Deeper inside the eye, murine lens epithelial cells were organized in a regular pattern (4,168 +/- 636 cells/mm2) and numerous lens fibers were observed. In vivo laser confocal microscopy can provide high-resolution images of all corneal layers and lens structures of mice without sacrificing animals or tissue preparation.

Trichothecenes NIV and DON modulate the maturation of murine dendritic cells.

PubMed

Luongo, D; Severino, L; Bergamo, P; D'Arienzo, R; Rossi, M

2010-01-01

Nivalenol (NIV) and Deoxynivalenol (DON), mycotoxins of the trichothecene family are considered very common food contaminants. In this work, we investigated whether the immunotoxic effects ascribed to these trichothecenes may be mediated by perturbations in the activity of dendritic cells (DCs). Murine bone marrow-derived DCs were used to evaluate the effects of NIV and DON on the LPS-induced maturation process. We found that the expression of the class II MHC and of the accessory CD11c molecules, but not of the costimulatory CD86 marker, was down-regulated by NIV and DON exposure in LPS-treated DCs, as well as nitric oxide (NO) production. Interestingly, NIV, but not DON, induced DC necrosis. Moreover, the analysis of the cytokine pattern showed that IL-12 and IL-10 expressions induced by LPS exposure were suppressed by both trichothecenes in a dose-dependent fashion. On the other hand, the secretion of the proinflammatory cytokine TNF-alpha was increased as a direct consequence of DON and NIV exposure. Taken together, our data indicated that the immunotoxicity of NIV and DON was related to the capacity of both trichothecenes to interfere with phenotypic and functional features of maturing DCs.

Use of the KlADH3 promoter for the quantitative production of the murine PDE5A isoforms in the yeast Kluyveromyces lactis.

PubMed

Cardarelli, Silvia; Giorgi, Mauro; Naro, Fabio; Malatesta, Francesco; Biagioni, Stefano; Saliola, Michele

2017-09-22

Phosphodiesterases (PDE) are a superfamily of enzymes that hydrolyse cyclic nucleotides (cAMP/cGMP), signal molecules in transduction pathways regulating crucial aspects of cell life. PDEs regulate the intensity and duration of the cyclic nucleotides signal modulating the downstream biological effect. Due to this critical role associated with the extensive distribution and multiplicity of isozymes, the 11 mammalian families (PDE1 to PDE11) constitute key therapeutic targets. PDE5, one of these cGMP-specific hydrolysing families, is the molecular target of several well known drugs used to treat erectile dysfunction and pulmonary hypertension. Kluyveromyces lactis, one of the few yeasts capable of utilizing lactose, is an attractive host alternative to Saccharomyces cerevisiae for heterologous protein production. Here we established K. lactis as a powerful host for the quantitative production of the murine PDE5 isoforms. Using the promoter of the highly expressed KlADH3 gene, multicopy plasmids were engineered to produce the native and recombinant Mus musculus PDE5 in K. lactis. Yeast cells produced large amounts of the purified A1, A2 and A3 isoforms displaying K m , V max and Sildenafil inhibition values similar to those of the native murine enzymes. PDE5 whose yield was nearly 1 mg/g wet weight biomass for all three isozymes (30 mg/L culture), is well tolerated by K. lactis cells without major growth deficiencies and interferences with the endogenous cAMP/cGMP signal transduction pathways. To our knowledge, this is the first time that the entire PDE5 isozymes family containing both regulatory and catalytic domains has been produced at high levels in a heterologous eukaryotic organism. K. lactis has been shown to be a very promising host platform for large scale production of mammalian PDEs for biochemical and structural studies and for the development of new specific PDE inhibitors for therapeutic applications in many pathologies.

Characterization of airway and vascular responses in murine lungs

PubMed Central

Held, Heinz-Dieter; Martin, Christian; Uhlig, Stefan

1999-01-01

We characterized the responses of murine airways and pulmonary vessels to a variety of endogenous mediators in the isolated perfused and ventilated mouse lung (IPL) and compared them with those in precision-cut lung slices. Airways: The EC50 (μM) for contractions of airways in IPL/slices was methacholine (Mch), 6.1/1.5>serotonin, 0.7/2.0>U46619 (TP-receptor agonist), 0.1/0.06>endothelin-1, 0.1/0.05. In the IPL, maximum increase in airway resistance (RL) was 0.6, 0.4, 0.8 and 11 cmH2O s ml−1, respectively. Adenosine (⩽1 mM), bombesin (⩽100 μM), histamine (⩽10 mM), LTC4 (⩽1 μM), PAF (0.25 μM) and substance P (⩽100 μM) had only weak effects (<5% of Mch) on RL. Vessels: The EC50 (μM) for vasoconstriction in the IPL was LTC4, 0.06>U46619, 0.05Murine precision-cut lung slices maintain important characteristics of the whole organ. The maximum reagibility of murine airways to endogenous mediators is serotoninmurine pulmonary vasculature is serotonin

Properties of murine embryonic stem cells maintained on human foreskin fibroblasts without LIF.

PubMed

Meng, G L; Zur Nieden, N I; Liu, S Y; Cormier, J T; Kallos, M S; Rancourt, D E

2008-04-01

In embryonic stem (ES) cells, leukemia inhibitory factor (LIF)/STAT3, wnt and nodal/activin signaling are mainly active to control pluripotency during expansion. To maintain pluripotency, ES cells are typically cultured on feeder cells of varying origins. Murine ES cells are commonly cultured on murine embryonic fibroblasts (MEFs), which senesce early and must be frequently prepared. This process is laborious and leads to batch variation presenting a challenge for high-throughput ES cell expansion. Although some cell lines can be sustained by exogenous LIF, this method is costly. We present here a novel and inexpensive culture method for expanding murine ES cells on human foreskin fibroblast (HFF) feeders. After 20 passages on HFFs without LIF, ES cell lines showed normal expression levels of pluripotency markers, maintained a normal karyotype and retained the ability to contribute to the germline. As HFFs do not senesce for at least 62 passages, they present a vast supply of feeders. Copyright 2007 Wiley-Liss, Inc.

Murine Intracisternal A Type Particles Fail to Separate from the Membrane of the Endoplasmic Reticulum

PubMed Central

Perk, Kalman; Dahlberg, John E.

1974-01-01

Analysis of serial sections of murine cells containing intracisternal A particles revealed that over 99% of all A particles remain in a budding configuration. This indicates that these particles fail to detach from the membrane of the endoplasmic reticulum. This observation explains how, despite their intracellular abundance in certain murine tumors, no extracellular A-type particles can be found. Images PMID:4431082

Isometachromin, a new cytotoxic sesquiterpenoid from a deep water sponge of the family Spongiidae.

PubMed

McConnell, O J; Longley, R; Gunasekera, M

1992-09-15

Isometachromin (1), a new sesquiterpene-quinone that is related structurally to metachromin C (2), and the known compounds ilimaquinone (3) and 5-epi-ilimaquinone (4), were isolated from a deep water sponge in the family Spongiidae; the structure of isometachromin was elucidated by spectral methods. Isometachromin exhibits in vitro cytotoxicity against the human lung cancer cell line A549 (IC50 = 2.6 micrograms/ml), but not against P388 murine leukemia (IC 50 > or equal to 10 micrograms/ml) and also exhibits antimicrobial activity.

A murine monoclonal antibody directed against the carboxyl-terminal domain of GRP78 suppresses melanoma growth in mice.

PubMed

de Ridder, Gustaaf G; Ray, Rupa; Pizzo, Salvatore V

2012-06-01

The HSP70 family member GRP78 is a selective tumor marker upregulated on the surface of many tumor cell types, including melanoma, where it acts as a growth factor receptor-like protein. Receptor-recognized forms of the proteinase inhibitor α2-macroglobulin (α2M*) are the best-characterized ligands for GRP78, but in melanoma and other cancer patients, autoantibodies arise against the NH2-terminal domain of GRP78 that react with tumor cell-surface GRP78. This causes the activation of signaling cascades that are proproliferative and antiapoptotic. Antibodies directed against the COOH-terminal domain of GRP78, however, upregulate p53-mediated proapoptotic signaling, leading to cell death. Here, we describe the binding characteristics, cell signaling properties, and downstream cellular effects of three novel murine monoclonal antibodies. The NH2-terminal domain-reactive antibody, N88, mimics α2M* as a ligand and drives PI 3-kinase-dependent activation of Akt and the subsequent stimulation of cellular proliferation in vitro. The COOH-terminal domain-reactive antibody, C38, acts as an antagonist of both α2M* and N88, whereas another, C107, directly induces apoptosis in vitro. In a murine B16F1 melanoma flank tumor model, we demonstrate the acceleration of tumor growth by treatment with N88, whereas C107 significantly slowed tumor growth whether administered before (P<0.005) or after (P<0.05) tumor implantation.

An automated system using spatial oversampling for optical mapping in murine atria. Development and validation with monophasic and transmembrane action potentials.

PubMed

Yu, Ting Yue; Syeda, Fahima; Holmes, Andrew P; Osborne, Benjamin; Dehghani, Hamid; Brain, Keith L; Kirchhof, Paulus; Fabritz, Larissa

2014-08-01

We developed and validated a new optical mapping system for quantification of electrical activation and repolarisation in murine atria. The system makes use of a novel 2nd generation complementary metal-oxide-semiconductor (CMOS) camera with deliberate oversampling to allow both assessment of electrical activation with high spatial and temporal resolution (128 × 2048 pixels) and reliable assessment of atrial murine repolarisation using post-processing of signals. Optical recordings were taken from isolated, superfused and electrically stimulated murine left atria. The system reliably describes activation sequences, identifies areas of functional block, and allows quantification of conduction velocities and vectors. Furthermore, the system records murine atrial action potentials with comparable duration to both monophasic and transmembrane action potentials in murine atria. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Lactoferrin Expression in Human and Murine Ocular Tissue.

PubMed

Rageh, Abrar A; Ferrington, Deborah A; Roehrich, Heidi; Yuan, Ching; Terluk, Marcia R; Nelson, Elizabeth F; Montezuma, Sandra R

2016-07-01

Lactoferrin (LF) is a multifunctional protein known to provide innate defense due to its antimicrobial and anti-inflammatory properties. In the eye, LF has been identified in the tears and vitreous humor. Its presence in other ocular tissues has not been determined. Our aim is to assess the presence of LF in the cornea, iris, retina and retinal pigment epithelium (RPE) of humans and mice. To test for the endogenous production of LF, reverse transcription polymerase chain reaction was performed in cultured human cells from the cornea and RPE and in murine tissues. To confirm LF localization in specific ocular tissue, immunohistochemistry was performed on flat mounts of cornea, retina and RPE in human donor eyes. The presence of LF was assessed by western blotting in human and mouse ocular tissue and human culture cells (cornea and RPE). To verify antibody specificity, purified human LF and transferrin (TF) were used on 1D and 2D western blots. LF gene expression was confirmed in the cornea and RPE cell cultures from humans, suggesting that LF is an endogenously produced protein. PCR results from mouse ocular tissue showed LF expression in cornea, iris, RPE, but not in retina. These results were also consistent with immunohistochemical localization of LF in human donor tissue. Antibody reaction for human LF was specific and western blotting showed its presence in the cornea, iris and RPE tissues. A faint reaction for the retina was observed but was likely due to contamination from other ocular tissues. Multiple commercially available antibodies for murine LF cross-reacted with TF, so no reliable results were obtained for murine western blot. LF is expressed in multiple eye tissues of humans and mice. This widespread expression and multifunctional activity of LF suggests that it may play an important role in protecting eye tissues from inflammation-associated diseases.

Practical Murine Hematopathology: A Comparative Review and Implications for Research

PubMed Central

O'Connell, Karyn E; Mikkola, Amy M; Stepanek, Aaron M; Vernet, Andyna; Hall, Christopher D; Sun, Chia C; Yildirim, Eda; Staropoli, John F; Lee, Jeannie T; Brown, Diane E

2015-01-01

Hematologic parameters are important markers of disease in human and veterinary medicine. Biomedical research has benefited from mouse models that recapitulate such disease, thus expanding knowledge of pathogenetic mechanisms and investigative therapies that translate across species. Mice in health have many notable hematologic differences from humans and other veterinary species, including smaller erythrocytes, higher percentage of circulating reticulocytes or polychromasia, lower peripheral blood neutrophil and higher peripheral blood and bone marrow lymphocyte percentages, variable leukocyte morphologies, physiologic splenic hematopoiesis and iron storage, and more numerous and shorter-lived erythrocytes and platelets. For accurate and complete hematologic analyses of disease and response to investigative therapeutic interventions, these differences and the unique features of murine hematopathology must be understood. Here we review murine hematology and hematopathology for practical application to translational investigation. PMID:25926395

Viwithan, a Standardized Withania somnifera Root Extract Induces Apoptosis in Murine Melanoma Cells.

PubMed

Sudeep, H V; Gouthamchandra, K; Venkatesh, B J; Prasad, K Shyam

2018-01-01

cytotoxicity against B16F1 murine melanoma cellsWe have investigated the in vitro cytotoxicity of Viwithan in different human and murine cancer cells. Interestingly, we found that Viwithan was particularly very effective against B16F1 melanoma cells with a half maximal inhibitory concentration value of 220 μg/mlThe microscopic observations following acridine orange/ethidium bromide staining and DNA fragmentation assays clearly indicated that Viwithan might initiate late apoptosis in B16F1 cellsThe binding affinity of withanolides in Viwithan with antiapoptotic proteins of B-cell lymphoma 2 family was predicted using AutoDock tool. The results from in silico studies indicated a plausible synergistic effect of withanolides attributing to the Viwithan-induced apoptosis through suppression of intrinsic pathway for carcinogenesis. Abbreviations used: MTT: Thiazolyl blue tetrazolium blue; DMSO: Dimethyl sulfoxide; BSA: Bovine serum albumin; DMEM: Dulbecco's minimum essential medium; NCCS: National Centre for Cell Science; PBS: Phosphate-Buffered Saline; HepG2: Liver hepatocellular carcinoma; HeLa: Henrietta Lacks cervical carcinoma cells; HCT-116: Human colorectal carcinoma cell line; EAC: Ehrlich ascites carcinoma cells; IC50: Half maximal inhibitory concentration; AO/EB: Acridine orange/Ethidium bromide; BCL-2: B-cell lymphoma 2; BCL-XL: B-cell lymphoma-extra large; MCL-1: Myeloid cell leukemia 1; PDB: Protein Data Bank; ANOVA: Analysis of variance.

Three-dimensional optical coherence tomography of the embryonic murine cardiovascular system

NASA Astrophysics Data System (ADS)

Luo, Wei; Marks, Daniel L.; Ralston, Tyler S.; Boppart, Stephen A.

2006-03-01

Optical coherence tomography (OCT) is an emerging high-resolution real-time biomedical imaging technology that has potential as a novel investigational tool in developmental biology and functional genomics. In this study, murine embryos and embryonic hearts are visualized with an OCT system capable of 2-µm axial and 15-µm lateral resolution and with real-time acquisition rates. We present, to our knowledge, the first sets of high-resolution 2- and 3-D OCT images that reveal the internal structures of the mammalian (murine) embryo (E10.5) and embryonic (E14.5 and E17.5) cardiovascular system. Strong correlations are observed between OCT images and corresponding hematoxylin- and eosin-stained histological sections. Real-time in vivo embryonic (E10.5) heart activity is captured by spectral-domain optical coherence tomography, processed, and displayed at a continuous rate of five frames per second. With the ability to obtain not only high-resolution anatomical data but also functional information during cardiovascular development, the OCT technology has the potential to visualize and quantify changes in murine development and in congenital and induced heart disease, as well as enable a wide range of basic in vitro and in vivo research studies in functional genomics.

Taci Is a Traf-Interacting Receptor for Tall-1, a Tumor Necrosis Factor Family Member Involved in B Cell Regulation

PubMed Central

Xia, Xing-Zhong; Treanor, James; Senaldi, Giorgio; Khare, Sanjay D.; Boone, Tom; Kelley, Michael; Theill, Lars E.; Colombero, Anne; Solovyev, Irina; Lee, Frances; McCabe, Susan; Elliott, Robin; Miner, Kent; Hawkins, Nessa; Guo, Jane; Stolina, Marina; Yu, Gang; Wang, Judy; Delaney, John; Meng, Shi-Yuan; Boyle, William J.; Hsu, Hailing

2000-01-01

We and others recently reported tumor necrosis factor (TNF) and apoptosis ligand–related leukocyte-expressed ligand 1 (TALL-1) as a novel member of the TNF ligand family that is functionally involved in B cell proliferation. Transgenic mice overexpressing TALL-1 have severe B cell hyperplasia and lupus-like autoimmune disease. Here, we describe expression cloning of a cell surface receptor for TALL-1 from a human Burkitt's lymphoma RAJI cell library. The cloned receptor is identical to the previously reported TNF receptor (TNFR) homologue transmembrane activator and calcium modulator and cyclophilin ligand (CAML) interactor (TACI). Murine TACI was subsequently isolated from the mouse B lymphoma A20 cells. Human and murine TACI share 54% identity overall. Human TACI exhibits high binding affinities to both human and murine TALL-1. Soluble TACI extracellular domain protein specifically blocks TALL-1–mediated B cell proliferation without affecting CD40- or lipopolysaccharide-mediated B cell proliferation in vitro. In addition, when injected into mice, soluble TACI inhibits antibody production to both T cell–dependent and –independent antigens. By yeast two-hybrid screening of a B cell library with TACI intracellular domain, we identified that, like many other TNFR family members, TACI intracellular domain interacts with TNFR-associated factor (TRAF)2, 5, and 6. Correspondingly, TACI activation in a B cell line results in nuclear factor κB and c-Jun NH2-terminal kinase activation. The identification and characterization of the receptor for TALL-1 provides useful information for the development of a treatment for B cell–mediated autoimmune diseases such as systemic lupus erythematosus. PMID:10880535

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

PubMed

Zhao, Wenyuan; Zhao, Tieqiang; Chen, Yuanjian; Zhao, Fengbo; Gu, Qingqing; Williams, Robert W; Bhattacharya, Syamal K; Lu, Lu; Sun, Yao

2015-01-01

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.

Enhanced Cultivation Of Stimulated Murine B Cells

NASA Technical Reports Server (NTRS)

Sammons, David W.

1994-01-01

Method of in vitro cultivation of large numbers of stimulated murine B lymphocytes. Cells electrofused with other cells to produce hybridomas and monoclonal antibodies. Offers several advantages: polyclonally stimulated B-cell blasts cultivated for as long as 14 days, hybridomas created throughout culture period, yield of hybridomas increases during cultivation, and possible to expand polyclonally in vitro number of B cells specific for antigenic determinants first recognized in vivo.

Selective uptake of multi-walled carbon nanotubes by tumor macrophages in a murine glioma model.

PubMed

VanHandel, Michelle; Alizadeh, Darya; Zhang, Leying; Kateb, Babak; Bronikowski, Michael; Manohara, Harish; Badie, Behnam

2009-03-31

Carbon nantotubes (CNTs) are emerging as a new family of nanovectors for drug and gene delivery into biological systems. To evaluate potential application of this technology for brain tumor therapy, we studied uptake and toxicity of multi-walled CNTs (MWCNTs) in the GL261 murine intracranial glioma model. Within 24 h of a single intratumoral injection of labeled MWCNTs (5 microg), nearly 10-20% of total cells demonstrated CNT internalization. Most CNT uptake, however, occurred by tumor-associated macrophages (MP), which accounted for most (75%) MWCNT-positive cells. Within 24 h of injection, nearly 30% of tumor MP became MWCNT-positive. Despite a transient increase in inflammatory cell infiltration into both normal and tumor-bearing brains following MWCNT injection, no significant toxicity was noted in mice, and minor changes in tumor cytokine expression were observed. This study suggests that MWCNTs could potentially be used as a novel and non-toxic vehicle for targeting MP in brain tumors.

Antitumour Effects of Isocurcumenol Isolated from Curcuma zedoaria Rhizomes on Human and Murine Cancer Cells

PubMed Central

Lakshmi, S.; Padmaja, G.; Remani, P.

2011-01-01

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 evaluate its tumour reducing properties in in vivo mice models. Isocurcumenol was characterized as the active compound by spectroscopy and was found to inhibit the proliferation of cancer cells without inducing significant toxicity to the normal cells. Fluorescent staining exhibited the morphological features of apoptosis in the compound-treated cancer cells. In vivo tumour reduction studies revealed that a dose of 35.7 mg/kg body weight significantly reduced the ascitic tumour in DLA-challenged mice and increased the lifespan with respect to untreated control mice. PMID:27429805

Effects of the murine skull in optoacoustic brain microscopy.

PubMed

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

2016-01-01

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. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Prostaglandin E/sub 2/ localization and receptor identification within the developing murine secondary palate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jones, J.

1986-01-01

Transient elevations in murine secondary palatal adenosine 3',5'-monophosphate (cAMP) levels occur during palate ontogeny. Since palatal processes exposed to dibutyryl cAMP differentiate precociously, increases in palatal cAMP levels are of interest. Prostaglandin E/sub 2/ (PGE/sub 2/), which is synthesized by murine embryonic palate mesenchyme cells (MEPM), regulates cAMP levels in adult tissues via specific membrane bound receptors coupled to adenylate cyclase. Therefore, a PGE/sub 2/ receptor-adenylate cyclase systems was proposed in the developing murine secondary palate. Utilizing a radioligand binding assay, it was determined that murine palatal tissue on day 13 of gestation contained PGE/sub 2/ receptors that were saturable,more » of high affinity and low capacity. Specific (/sup 3/H)-PGE/sub 2/ binding was reversible by 30 min. The order of prostanoid binding affinity at specific PGE/sub 2/ binding sites was E/sub 2/ > F/sub 2//sub ..cap alpha../ > A/sub 2/ > E/sub 1/ = D/sub 2/ indicating specificity of the receptor for PGE/sub 2/. The ability of MEPM cells to respond to PGE/sub 2/ with dose-dependent accumulations of intracellular cAMP demonstrated the functional nature of these binding sites. Analysis of palatal PGE/sub 2/ receptor characteristics on days 12 and 14 of palate development indicated temporal alterations in receptor affinity and density during palate ontogeny.« less

Invasion of the Placenta during Murine Listeriosis

PubMed Central

Le Monnier, Alban; Join-Lambert, Olivier F.; Jaubert, Francis; Berche, Patrick; Kayal, Samer

2006-01-01

Feto-placental infections due to Listeria monocytogenes represent a major threat during pregnancy, and the underlying mechanisms of placental invasion remain poorly understood. Here we used a murine model of listeriosis (pregnant mice, infected at day 14 of gestation) to investigate how this pathogen invades and grows within the placenta to ultimately infect the fetus. When L. monocytogenes is injected intravenously, the invasion of the placenta occurs early after the initial bacteremia, allowing the placental growth of the bacteria, which is an absolute requirement for vertical transmission to the fetus. Kinetically, bacteria first target the cells lining the central arterial canal of the placenta, which stain positively with cytokeratin, demonstrating their fetal trophoblast origin. Bacteria then disseminate rapidly to the other trophoblastic structures, like syncytiotrophoblast cells lining the villous core in the labyrinthine zone of placenta. Additionally, we found that an inflammatory reaction predominantly constituted of polymorphonuclear cells occurs in the villous placenta and participates in the control of infection. Altogether, our results suggest that the infection of murine placenta is dependent, at the early phase, on circulating bacteria and their interaction with endovascular trophoblastic cells. Subsequently, the bacteria spread to the other trophoblastic cells before crossing the placental barrier. PMID:16369023

Survival and characteristics of murine leukaemic and normal stem cells after hyperthermia: a murine model for human bone marrow purging.

PubMed

Gidáli, J; Szamosvölgyi, S; Fehér, I; Kovács, P

1990-01-01

The effect of hyperthermia in vitro on the survival and leukaemogenic effectiveness of WEHI 3-B cells and on the survival and transplantation efficiency of bone marrow cells was compared in a murine model system. Normal murine clonogenic haemopoietic cells (day 9 CFU-S and CFU-GM) proved to be significantly less sensitive to 42.5 degrees C hyperthermia (Do values: 54.3 and 41.1 min, respectively) than leukaemic clonogenic cells (CFU-L) derived from suspension culture or from bone marrow of leukaemic mice (Do: 17.8 min). Exposure for 120 min to 42.5 degrees C reduced the surviving fraction of CFU-L to 0.002 and that of CFU-S to 0.2. If comparable graft sizes were transplanted from normal or heat exposed bone marrow, 60-day survival of supralethally irradiated mice was similar. Surviving WEHI 3-B cells were capable of inducing leukaemia in vivo. The two log difference in the surviving fraction of CFU-L and CFU-S after 120 min exposure to 42.5 degrees C suggests that hyperthermia ex vivo may be a suitable purging method for autologous bone marrow transplantation.

STRAIN-DEPENDENT SUSCEPTIBILITY TO TRANSPLACENTALLY-INDUCED MURINE LUNG TUMORS

EPA Science Inventory

STRAIN-DEPENDENT SUSCEPTIBILITY TO TRANSPLACENTALLY-INDUCED MURINE LUNG TUMORS
M S Miller, J E Moore, M Xu, G B Nelson, S T Dance, N D Kock, J A Ross Wake Forest University, Winston-Salem, NC and USEPA, Research Triangle Park, NC

Previously, our laboratory demonstrated...

Structural studies on Rauscher murine leukemia virus: isolation and characterization of viral envelopes.

PubMed Central

van de Ven, W J; Vermorken, A J; Onnekink, C; Bloemers, H P; Bloemendal, H

1978-01-01

A preparative method for isolating pure viral envelopes from a type-C RNA tumor virus, Rauscher murine leukemia virus, is described. Fractionation of virions of Rauscher murine leukemia virus was studied after disruption of the virions with the detergents sodium dodecyl sulfate of Nonidet P-40 in combination with ether. Fractionation was performed through flotation in a discontinuous sucrose gradient and, as appeared from electron microscopic examination, a pure viral envelope fraction was obtained in this way. By use of sensitive competition radioimmunoassays or sodium dodecyl sulfate-polyacrylamide gel electrophoresis after immunoprecipitation with polyvalent and monospecific antisera directed against Rauscher murine leukemia virus proteins, the amount of the gag and env gene-encoded structural polypeptides in the virions and the isolated envelope fraction was compared. The predominant viral structural polypeptides in the purified envelope fraction were the env gene-encoded polypeptides gp70, p15(E), and p12(E), whereas, except for p15, there was only a relatively small amount of the gag gene-encoded structural polypeptides in this fraction. Images PMID:702639

Aortic iron overload with oxidative stress and inflammation in human and murine abdominal aortic aneurysm.

PubMed

Sawada, Hisashi; Hao, Hiroyuki; Naito, Yoshiro; Oboshi, Makiko; Hirotani, Shinichi; Mitsuno, Masataka; Miyamoto, Yuji; Hirota, Seiichi; Masuyama, Tohru

2015-06-01

Although iron is an essential element for maintaining physiological function, excess iron leads to tissue damage caused by oxidative stress and inflammation. Oxidative stress and inflammation play critical roles for the development of abdominal aortic aneurysm (AAA). However, it has not been investigated whether iron plays a role in AAA formation through oxidative stress and inflammation. We, therefore, examined whether iron is involved in the pathophysiology of AAA formation using human AAA walls and murine AAA models. Human aortic walls were collected from 53 patients who underwent cardiovascular surgery (non-AAA=34; AAA=19). Murine AAA was induced by infusion of angiotensin II to apolipoprotein E knockout mice. Iron was accumulated in human and murine AAA walls compared with non-AAA walls. Immunohistochemistry showed that both 8-hydroxy-2'-deoxyguanosine and CD68-positive areas were increased in AAA walls compared with non-AAA walls. The extent of iron accumulated area positively correlated with that of 8-hydroxy-2'-deoxyguanosine expression area and macrophage infiltration area in human and murine AAA walls. We next investigated the effects of dietary iron restriction on AAA formation in mice. Iron restriction reduced the incidence of AAA formation with attenuation of oxidative stress and inflammation. Aortic expression of transferrin receptor 1, intracellular iron transport protein, was increased in human and murine AAA walls, and transferrin receptor 1-positive area was similar to areas where iron accumulated and F4/80 were positive. Iron is involved in the pathophysiology of AAA formation with oxidative stress and inflammation. Dietary iron restriction could be a new therapeutic strategy for AAA progression. © 2015 American Heart Association, Inc.

Comparison of vectorial ion transport in primary murine airway and human sinonasal air-liquid interface cultures, models for studies of cystic fibrosis, and other airway diseases.

PubMed

Zhang, Shaoyan; Fortenberry, James A; Cohen, Noam A; Sorscher, Eric J; Woodworth, Bradford A

2009-01-01

The purpose of this study was to compare vectorial ion transport within murine trachea, murine nasal septa, and human sinonasal cultured epithelium. Our hypothesis is that murine septal epithelium, rather than trachea, will more closely mimic the electrophysiology properties of human sinonasal epithelium. Epithelium from murine trachea, murine septa, and human sinonasal tissue were cultured at an air-liquid interface to confluence and full differentiation. A limited number of homozygous dF508 epithelia were also cultured. Monolayers were mounted in modified Ussing chambers to investigate pharmacologic manipulation of ion transport. The change in forskolin-stimulated current (delta-I(SC), expressed as micro-A/cm(2)) in murine septal (n = 19; 16.84 +/- 2.09) and human sinonasal (n = 18; 12.15 +/- 1.93) cultures was significantly increased over murine tracheal cultures (n = 15; 6.75 +/- 1.35; p = 0.035 and 0.0005, respectively). Forskolin-stimulated I(SC) was inhibited by the specific cystic fibrosis transmembrane regulator (CFTR) inhibitor INH-172 (5 microM). No forskolin-stimulated I(SC) was shown in cultures of dF508 homozygous murine septal epithelium (n = 3). Murine septal I(SC) was largely inhibited by amiloride (12.03 +/- 0.66), whereas human sinonasal cultures had a very limited response (0.70 +/- 0.47; p < 0.0001). The contribution of CFTR to stimulated chloride current as measured by INH-172 was highly significantly different between all groups (murine septa, 19.51 +/- 1.28; human sinonasal, 11.12 +/- 1.58; murine trachea, 4.85 +/- 0.49; p < 0.0001). Human sinonasal and murine septal epithelial cultures represent a useful model for studying CFTR activity and may provide significant advantages over lower airway tissues for investigating upper and lower respiratory pathophysiology.

Ascofuranone stimulates expression of adiponectin and peroxisome proliferator activated receptor through the modulation of mitogen activated protein kinase family members in 3T3-L1, murine pre-adipocyte cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, Young-Chae, E-mail: ycchang@cu.ac.kr; Cho, Hyun-Ji, E-mail: hjcho.dr@gmail.com

2012-06-08

Highlights: Black-Right-Pointing-Pointer Ascofuranone increases expression of adiponectin and PPAR{gamma}. Black-Right-Pointing-Pointer Inhibitors for MEK and JNK increased the expression of adiponectin and PPAR{gamma}. Black-Right-Pointing-Pointer Ascofuranone significantly suppressed phosho-ERK, while increasing phospho-p38. -- Abstract: Ascofuranone, an isoprenoid antibiotic, was originally isolated as a hypolipidemic substance from a culture broth of the phytopathogenic fungus, Ascochyta visiae. Adiponectin is mainly synthesized by adipocytes. It relieves insulin resistance by decreasing the plasma triglycerides and improving glucose uptake, and has anti-atherogenic properties. Here, we found that ascofuranone increases expression of adiponectin and PPAR{gamma}, a major transcription factor for adiponectin, in 3T3-L1, murine pre-adipocytes cell line, withoutmore » promoting accumulation of lipid droplets. Ascofuranone induced expression of adiponectin, and increases the promoter activity of adiponectin and PPRE, PPAR response element, as comparably as a PPAR{gamma} agonist, rosiglitazone, that stimulates lipid accumulation in the preadipocyte cell line. Moreover, inhibitors for MEK and JNK, like ascofuranone, considerably increased the expression of adiponectin and PPAR{gamma}, while a p38 inhibitor significantly suppressed. Ascofuranone significantly suppressed ERK phosphorylation, while increasing p38 phosphorylation, during adipocyte differentiation program. These results suggest that ascofuranone regulates the expression of adiponectin and PPAR{gamma} through the modulation of MAP kinase family members.« less

[Establishment of the retrovirus-mediated murine model with MLL-AF9 leukemia].

PubMed

Xu, Si-Miao; Yang, Yang; Zhou, Mi; Zhao, Xue-Jiao; Qin, Yu; Zhang, Pei-Ling; Yuan, Rui-Feng; Zhou, Jian-Feng; Fang, Yong

2013-10-01

This study was purposed to establish a retrovirus-mediated murine model with MLL-AF9 leukemia, so as to provide a basis for further investigation of the pathogenesis and therapeutic strategy of MLL associated leukemia. Murine (CD45.2) primary hematopoietic precursor positively selected for expression of the progenitor marker c-Kit by means of MACS were transduced with a retrovirus carrying MLL-AF9 fusion gene. After cultured in vitro, the transduced cells were injected intravenously through the tail vein into the lethally irradiated mice (CD45.1). PCR, flow cytometry and morphological observation were employed to evaluate the murine leukemia model system. The results showed that MLL-AF9 fusion gene was expressed in the infected cells, and the cells had a dramatically enhanced potential to generate myeloid colonies with primitive and immature morphology. Flow cytometric analysis revealed that the immortalized cells highly expressed myeloid lineage surface markers Gr-1 and Mac-1. Moreover, the expression levels of Hoxa9 and Meis1 mRNA were significantly higher in the MLL-AF9 cells than that in control. The mice transplanted with MLL-AF9 cells displayed typical signs of leukemia within 6-12 weeks. Extensive infiltration leukemic cells was observed in the Wright-Giemsa stained peripheral blood smear and bone marrow, and also in the histology of liver and spleen. Flow cytometric analysis of the bone marrow and spleen cells demonstrated that the CD45.2 populations expressed highly myeloid markers Gr-1 and Mac-1. The leukemic mice died within 12 weeks. It is concluded that the retrovirus-mediated murine model with MLL-AF9 leukemia is successfully established, which can be applied in the subsequent researches.

Tissue distribution, regulation and intracellular localization of murine CD1 molecules.

PubMed

Mandal, M; Chen, X R; Alegre, M L; Chiu, N M; Chen, Y H; Castaño, A R; Wang, C R

1998-06-01

CD1 molecules are MHC-unlinked class Ib molecules consisting of classical (human CD 1a-c) and non-classical subsets (human CD1d and murine CD1). The characterization of non-classical subsets of CD1 is limited due to the lack of reagents. In this study, we have generated two new anti-mouse CD1 monoclonal antibodies, 3H3 and 5C6, by immunization of hamsters with purified CD1 protein. These antibodies recognize CD1-transfected cells and have no reactivity to cells isolated from CD1-/- mice. Both antibodies precipitate the 52 kDa heavy chain and 12 kDa beta2m from thymocytes and splenocytes by radio-immunoprecipitation. Deglycosylation of CD1 reduces molecular mass of the heavy chain by 7.5 kDa, which can be detected by 3H3 but not 5C6. 3H3 and 5C6 detect surface CD1 expression on cells from the thymus, spleen, lymph node and bone marrow, but not on intestinal epithelial cells. Developmentally, CD1 is expressed on thymocytes prior to TCR rearrangement and remains constant throughout thymic development. CD1 is expressed early in the fetal liver (day 14) and remains expressed in hepatocytes postnatally. These data support evidence of a role for CD1 in the selection and/or expansion of NK1- T cells of both thymic origin and extrathymic origin. Unlike classical class I molecules, murine CD1 levels are not affected by IFN-gamma, but like human CD1b can be up-regulated by IL-4 and GM-CSF although only moderately. Similar to human CD1b, murine CD1 is found by immunofluorescence microscopy on the cell surface, and in various intracellular vesicles, including early and late endosomes. Localization in endocytic compartments indicates that murine CD1 may be capable of binding endocytosed antigens.

Endothelial SRF/MRTF ablation causes vascular disease phenotypes in murine retinae

PubMed Central

Weinl, Christine; Riehle, Heidemarie; Park, Dongjeong; Stritt, Christine; Beck, Susanne; Huber, Gesine; Wolburg, Hartwig; Olson, Eric N.; Seeliger, Mathias W.; Adams, Ralf H.; Nordheim, Alfred

2013-01-01

Retinal vessel homeostasis ensures normal ocular functions. Consequently, retinal hypovascularization and neovascularization, causing a lack and an excess of vessels, respectively, are hallmarks of human retinal pathology. We provide evidence that EC-specific genetic ablation of either the transcription factor SRF or its cofactors MRTF-A and MRTF-B, but not the SRF cofactors ELK1 or ELK4, cause retinal hypovascularization in the postnatal mouse eye. Inducible, EC-specific deficiency of SRF or MRTF-A/MRTF-B during postnatal angiogenesis impaired endothelial tip cell filopodia protrusion, resulting in incomplete formation of the retinal primary vascular plexus, absence of the deep plexi, and persistence of hyaloid vessels. All of these features are typical of human hypovascularization-related vitreoretinopathies, such as familial exudative vitreoretinopathies including Norrie disease. In contrast, conditional EC deletion of Srf in adult murine vessels elicited intraretinal neovascularization that was reminiscent of the age-related human pathologies retinal angiomatous proliferation and macular telangiectasia. These results indicate that angiogenic homeostasis is ensured by differential stage-specific functions of SRF target gene products in the developing versus the mature retinal vasculature and suggest that the actin-directed MRTF-SRF signaling axis could serve as a therapeutic target in the treatment of human vascular retinal diseases. PMID:23563308

Endothelial SRF/MRTF ablation causes vascular disease phenotypes in murine retinae.

PubMed

Weinl, Christine; Riehle, Heidemarie; Park, Dongjeong; Stritt, Christine; Beck, Susanne; Huber, Gesine; Wolburg, Hartwig; Olson, Eric N; Seeliger, Mathias W; Adams, Ralf H; Nordheim, Alfred

2013-05-01

Retinal vessel homeostasis ensures normal ocular functions. Consequently, retinal hypovascularization and neovascularization, causing a lack and an excess of vessels, respectively, are hallmarks of human retinal pathology. We provide evidence that EC-specific genetic ablation of either the transcription factor SRF or its cofactors MRTF-A and MRTF-B, but not the SRF cofactors ELK1 or ELK4, cause retinal hypovascularization in the postnatal mouse eye. Inducible, EC-specific deficiency of SRF or MRTF-A/MRTF-B during postnatal angiogenesis impaired endothelial tip cell filopodia protrusion, resulting in incomplete formation of the retinal primary vascular plexus, absence of the deep plexi, and persistence of hyaloid vessels. All of these features are typical of human hypovascularization-related vitreoretinopathies, such as familial exudative vitreoretinopathies including Norrie disease. In contrast, conditional EC deletion of Srf in adult murine vessels elicited intraretinal neovascularization that was reminiscent of the age-related human pathologies retinal angiomatous proliferation and macular telangiectasia. These results indicate that angiogenic homeostasis is ensured by differential stage-specific functions of SRF target gene products in the developing versus the mature retinal vasculature and suggest that the actin-directed MRTF-SRF signaling axis could serve as a therapeutic target in the treatment of human vascular retinal diseases.

Human-murine transthyretin heterotetramers are kinetically stable and non-amyloidogenic. A lesson in the generation of transgenic models of diseases involving oligomeric proteins.

PubMed

Reixach, Natàlia; Foss, Ted R; Santelli, Eugenio; Pascual, Jaime; Kelly, Jeffery W; Buxbaum, Joel N

2008-01-25

The transthyretin amyloidoses appear to be caused by rate-limiting tetramer dissociation and partial monomer unfolding of the human serum protein transthyretin, resulting in aggregation and extracellular deposition of amorphous aggregates and amyloid fibrils. Mice transgenic for few copies of amyloid-prone human transthyretin variants, including the aggressive L55P mutant, failed to develop deposits. Silencing the murine transthyretin gene in the presence of the L55P human gene resulted in enhanced tissue deposition. To test the hypothesis that the murine protein interacted with human transthyretin, preventing the dissociation and partial unfolding required for amyloidogenesis, we produced recombinant murine transthyretin and human/murine transthyretin heterotetramers and compared their structures and biophysical properties to recombinant human transthyretin. We found no significant differences between the crystal structures of murine and human homotetramers. Murine transthyretin is not amyloidogenic because the native homotetramer is kinetically stable under physiologic conditions and cannot dissociate into partially unfolded monomers, the misfolding and aggregation precursor. Heterotetramers composed of murine and human subunits are also kinetically stable. These observations explain the lack of transthyretin deposition in transgenics carrying a low copy number of human transthyretin genes. The incorporation of mouse subunits into tetramers otherwise composed of human amyloid-prone transthyretin subunits imposes kinetic stability, preventing dissociation and subsequent amyloidogenesis.

Human and Murine Innate Immune Cell Populations Display Common and Distinct Response Patterns during Their In Vitro Interaction with the Pathogenic Mold Aspergillus fumigatus.

PubMed

Hellmann, Anna-Maria; Lother, Jasmin; Wurster, Sebastian; Lutz, Manfred B; Schmitt, Anna Lena; Morton, Charles Oliver; Eyrich, Matthias; Czakai, Kristin; Einsele, Hermann; Loeffler, Juergen

2017-01-01

Aspergillus fumigatus is the main cause of invasive fungal infections occurring almost exclusively in immunocompromised patients. An improved understanding of the initial innate immune response is key to the development of better diagnostic tools and new treatment options. Mice are commonly used to study immune defense mechanisms during the infection of the mammalian host with A. fumigatus . However, little is known about functional differences between the human and murine immune response against this fungal pathogen. Thus, we performed a comparative functional analysis of human and murine dendritic cells (DCs), macrophages, and polymorphonuclear cells (PMNs) using standardized and reproducible working conditions, laboratory protocols, and readout assays. A. fumigatus did not provoke identical responses in murine and human immune cells but rather initiated relatively specific responses. While human DCs showed a significantly stronger upregulation of their maturation markers and major histocompatibility complex molecules and phagocytosed A. fumigatus more efficiently compared to their murine counterparts, murine PMNs and macrophages exhibited a significantly stronger release of reactive oxygen species after exposure to A. fumigatus . For all studied cell types, human and murine samples differed in their cytokine response to conidia or germ tubes of A. fumigatus . Furthermore, Dectin-1 showed inverse expression patterns on human and murine DCs after fungal stimulation. These specific differences should be carefully considered and highlight potential limitations in the transferability of murine host-pathogen interaction studies.

Human and Murine Innate Immune Cell Populations Display Common and Distinct Response Patterns during Their In Vitro Interaction with the Pathogenic Mold Aspergillus fumigatus

PubMed Central

Hellmann, Anna-Maria; Lother, Jasmin; Wurster, Sebastian; Lutz, Manfred B.; Schmitt, Anna Lena; Morton, Charles Oliver; Eyrich, Matthias; Czakai, Kristin; Einsele, Hermann; Loeffler, Juergen

2017-01-01

Aspergillus fumigatus is the main cause of invasive fungal infections occurring almost exclusively in immunocompromised patients. An improved understanding of the initial innate immune response is key to the development of better diagnostic tools and new treatment options. Mice are commonly used to study immune defense mechanisms during the infection of the mammalian host with A. fumigatus. However, little is known about functional differences between the human and murine immune response against this fungal pathogen. Thus, we performed a comparative functional analysis of human and murine dendritic cells (DCs), macrophages, and polymorphonuclear cells (PMNs) using standardized and reproducible working conditions, laboratory protocols, and readout assays. A. fumigatus did not provoke identical responses in murine and human immune cells but rather initiated relatively specific responses. While human DCs showed a significantly stronger upregulation of their maturation markers and major histocompatibility complex molecules and phagocytosed A. fumigatus more efficiently compared to their murine counterparts, murine PMNs and macrophages exhibited a significantly stronger release of reactive oxygen species after exposure to A. fumigatus. For all studied cell types, human and murine samples differed in their cytokine response to conidia or germ tubes of A. fumigatus. Furthermore, Dectin-1 showed inverse expression patterns on human and murine DCs after fungal stimulation. These specific differences should be carefully considered and highlight potential limitations in the transferability of murine host–pathogen interaction studies. PMID:29270175

[Evaluation of Fusarium spp. pathogenicity in plant and murine models].

PubMed

Forero-Reyes, Consuelo M; Alvarado-Fernández, Angela M; Ceballos-Rojas, Ana M; González-Carmona, Lady C; Linares-Linares, Melva Y; Castañeda-Salazar, Rubiela; Pulido-Villamarín, Adriana; Góngora-Medina, Manuel E; Cortés-Vecino, Jesús A; Rodríguez-Bocanegra, María X

The genus Fusarium is widely recognized for its phytopathogenic capacity. However, it has been reported as an opportunistic pathogen in immunocompetent and immunocompromised patients. Thus, it can be considered a microorganism of interest in pathogenicity studies on different hosts. Therefore, this work evaluated the pathogenicity of Fusarium spp. isolates from different origins in plants and animals (murine hosts). Twelve isolates of Fusarium spp. from plants, animal superficial mycoses, and human superficial and systemic mycoses were inoculated in tomato, passion fruit and carnation plants, and in immunocompetent and immunosuppressed BALB/c mice. Pathogenicity tests in plants did not show all the symptoms associated with vascular wilt in the three plant models; however, colonization and necrosis of the vascular bundles, regardless of the species and origin of the isolates, showed the infective potential of Fusarium spp. in different plant species. Moreover, the pathogenicity tests in the murine model revealed behavioral changes. It was noteworthy that only five isolates (different origin and species) caused mortality. Additionally, it was observed that all isolates infected and colonized different organs, regardless of the species and origin of the isolates or host immune status. In contrast, the superficial inoculation test showed no evidence of epidermal injury or colonization. The observed results in plant and murine models suggest the pathogenic potential of Fusarium spp. isolates in different types of hosts. However, further studies on pathogenicity are needed to confirm the multihost capacity of this genus. Copyright © 2017 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

Transcriptional profiling of murine osteoblast differentiation based on RNA-seq expression analyses.

PubMed

Khayal, Layal Abo; Grünhagen, Johannes; Provazník, Ivo; Mundlos, Stefan; Kornak, Uwe; Robinson, Peter N; Ott, Claus-Eric

2018-04-11

Osteoblastic differentiation is a multistep process characterized by osteogenic induction of mesenchymal stem cells, which then differentiate into proliferative pre-osteoblasts that produce copious amounts of extracellular matrix, followed by stiffening of the extracellular matrix, and matrix mineralization by hydroxylapatite deposition. Although these processes have been well characterized biologically, a detailed transcriptional analysis of murine primary calvaria osteoblast differentiation based on RNA sequencing (RNA-seq) analyses has not previously been reported. Here, we used RNA-seq to obtain expression values of 29,148 genes at four time points as murine primary calvaria osteoblasts differentiate in vitro until onset of mineralization was clearly detectable by microscopic inspection. Expression of marker genes confirmed osteogenic differentiation. We explored differential expression of 1386 protein-coding genes using unsupervised clustering and GO analyses. 100 differentially expressed lncRNAs were investigated by co-expression with protein-coding genes that are localized within the same topologically associated domain. Additionally, we monitored expression of 237 genes that are silent or active at distinct time points and compared differential exon usage. Our data represent an in-depth profiling of murine primary calvaria osteoblast differentiation by RNA-seq and contribute to our understanding of genetic regulation of this key process in osteoblast biology. Copyright © 2018 Elsevier Inc. All rights reserved.

Sex differences in the MB49 syngeneic, murine model of bladder cancer.

PubMed

White-Gilbertson, Shai; Davis, Megan; Voelkel-Johnson, Christina; Kasman, Laura M

The MB49 syngeneic, murine model of bladder cancer has been widely used for more than 35 years. In humans, bladder cancer is one third as prevalent in women as in men, with a trend toward lower prevalence in parous compared to nulliparous women. Our objective was to determine if the MB49 bladder cancer model reproduces the sex differences observed in humans, and to determine its sensitivity to testosterone and the pregnancy hormone, human chorionic gonadotropin (hCG). Male and female C57BL/6 mice were implanted with MB49 murine bladder cancer cells, and observed for tumor growth. MB49 dose responses to hCG and dihydrotestosterone were determined in vitro . MB49 tumor growth was significantly greater in male mice than female mice. Pregnancy did not affect MB49 tumor growth in female mice. MB49 cells did not proliferate in response to hCG in vitro and the functional receptor for gonadotropins was absent. Dihydrotestosterone strongly stimulated growth of MB49 cells in vitro . The MB49 murine model of bladder cancer reproduced some aspects of the sex differences observed in humans. Our results suggest that testosterone may stimulate MB49 cell proliferation, which may explain the more rapid MB49 tumor growth observed in male mice.

Viwithan, a Standardized Withania somnifera Root Extract Induces Apoptosis in Murine Melanoma Cells

PubMed Central

Sudeep, H.V.; Gouthamchandra, K.; Venkatesh, B. J.; Prasad, K. Shyam

2017-01-01

Viwithan, a standardized 5% Withania somnifera root extract, has potent cytotoxicity against B16F1 murine melanoma cellsWe have investigated the in vitro cytotoxicity of Viwithan in different human and murine cancer cells. Interestingly, we found that Viwithan was particularly very effective against B16F1 melanoma cells with a half maximal inhibitory concentration value of 220 μg/mlThe microscopic observations following acridine orange/ethidium bromide staining and DNA fragmentation assays clearly indicated that Viwithan might initiate late apoptosis in B16F1 cellsThe binding affinity of withanolides in Viwithan with antiapoptotic proteins of B-cell lymphoma 2 family was predicted using AutoDock tool. The results from in silico studies indicated a plausible synergistic effect of withanolides attributing to the Viwithan-induced apoptosis through suppression of intrinsic pathway for carcinogenesis. Abbreviations used: MTT: Thiazolyl blue tetrazolium blue; DMSO: Dimethyl sulfoxide; BSA: Bovine serum albumin; DMEM: Dulbecco's minimum essential medium; NCCS: National Centre for Cell Science; PBS: Phosphate-Buffered Saline; HepG2: Liver hepatocellular carcinoma; HeLa: Henrietta Lacks cervical carcinoma cells; HCT-116: Human colorectal carcinoma cell line; EAC: Ehrlich ascites carcinoma cells; IC50: Half maximal inhibitory concentration; AO/EB: Acridine orange/Ethidium bromide; BCL-2: B-cell lymphoma 2; BCL-XL: B-cell lymphoma-extra large; MCL-1: Myeloid cell leukemia 1; PDB: Protein Data Bank; ANOVA: Analysis of variance. PMID:29491636

Spatial and temporal expression of the Grainyhead-like transcription factor family during murine development.

PubMed

Auden, Alana; Caddy, Jacinta; Wilanowski, Tomasz; Ting, Stephen B; Cunningham, John M; Jane, Stephen M

2006-10-01

The Drosophila transcription factor Grainyhead (grh) is expressed in ectoderm-derived tissues where it regulates several key developmental events including cuticle formation, tracheal elongation and dorsal closure. Our laboratory has recently identified three novel mammalian homologues of the grh gene, Grainyhead-like 1, -2 and -3 (Grhl1-3) that rewrite the phylogeny of this family. Using gene targeting in mice, we have shown that Grhl3 is essential for neural tube closure, skin barrier formation and wound healing. Despite their extensive sequence homology, Grhl1 and Grhl2 are unable to compensate for loss of Grhl3 in these developmental processes. To explore this lack of redundancy, and to gain further insights into the functions of this gene family in mammalian development we have performed an extensive in situ hybridisation analysis. We demonstrate that, although all three Grhl genes are highly expressed in the developing epidermis, they display subtle differences in the timing and level of expression. Surprisingly, we also demonstrate differential expression patterns in non-ectoderm-derived tissues, including the heart, the lung, and the metanephric kidney. These findings expand our understanding of the unique role of Grhl3 in neurulation and epidermal morphogenesis, and provide a focus for further functional analysis of the Grhl genes during mouse embryogenesis.

Giardia Alters Commensal Microbial Diversity throughout the Murine Gut

PubMed Central

Barash, N. R.; Maloney, J. G.

2017-01-01

ABSTRACT Giardia lamblia is the most frequently identified protozoan cause of intestinal infection. Over 200 million people are estimated to have acute or chronic giardiasis, with infection rates approaching 90% in areas where Giardia is endemic. Despite its significance in global health, the mechanisms of pathogenesis associated with giardiasis remain unclear, as the parasite neither produces a known toxin nor induces a robust inflammatory response. Giardia colonization and proliferation in the small intestine of the host may, however, disrupt the ecological homeostasis of gastrointestinal commensal microbes and contribute to diarrheal disease associated with giardiasis. To evaluate the impact of Giardia infection on the host microbiota, we used culture-independent methods to quantify shifts in the diversity of commensal microbes throughout the gastrointestinal tract in mice infected with Giardia. We discovered that Giardia's colonization of the small intestine causes a systemic dysbiosis of aerobic and anaerobic commensal bacteria. Specifically, Giardia colonization is typified by both expansions in aerobic Proteobacteria and decreases in anaerobic Firmicutes and Melainabacteria in the murine foregut and hindgut. Based on these shifts, we created a quantitative index of murine Giardia-induced microbial dysbiosis. This index increased at all gut regions during the duration of infection, including both the proximal small intestine and the colon. Giardiasis could be an ecological disease, and the observed dysbiosis may be mediated directly via the parasite's unique anaerobic fermentative metabolism or indirectly via parasite induction of gut inflammation. This systemic alteration of murine gut commensal diversity may be the cause or the consequence of inflammatory and metabolic changes throughout the gut. Shifts in the commensal microbiota may explain observed variations in giardiasis between hosts with respect to host pathology, degree of parasite colonization

TIM-family proteins inhibit HIV-1 release

PubMed Central

Li, Minghua; Ablan, Sherimay D.; Miao, Chunhui; Zheng, Yi-Min; Fuller, Matthew S.; Rennert, Paul D.; Maury, Wendy; Johnson, Marc C.; Freed, Eric O.; Liu, Shan-Lu

2014-01-01

Accumulating evidence indicates that T-cell immunoglobulin (Ig) and mucin domain (TIM) proteins play critical roles in viral infections. Herein, we report that the TIM-family proteins strongly inhibit HIV-1 release, resulting in diminished viral production and replication. Expression of TIM-1 causes HIV-1 Gag and mature viral particles to accumulate on the plasma membrane. Mutation of the phosphatidylserine (PS) binding sites of TIM-1 abolishes its ability to block HIV-1 release. TIM-1, but to a much lesser extent PS-binding deficient mutants, induces PS flipping onto the cell surface; TIM-1 is also found to be incorporated into HIV-1 virions. Importantly, TIM-1 inhibits HIV-1 replication in CD4-positive Jurkat cells, despite its capability of up-regulating CD4 and promoting HIV-1 entry. In addition to TIM-1, TIM-3 and TIM-4 also block the release of HIV-1, as well as that of murine leukemia virus (MLV) and Ebola virus (EBOV); knockdown of TIM-3 in differentiated monocyte-derived macrophages (MDMs) enhances HIV-1 production. The inhibitory effects of TIM-family proteins on virus release are extended to other PS receptors, such as Axl and RAGE. Overall, our study uncovers a novel ability of TIM-family proteins to block the release of HIV-1 and other viruses by interaction with virion- and cell-associated PS. Our work provides new insights into a virus-cell interaction that is mediated by TIMs and PS receptors. PMID:25136083

TGF-β mimic proteins form an extended gene family in the murine parasite Heligmosomoides polygyrus.

PubMed

Smyth, Danielle J; Harcus, Yvonne; White, Madeleine P J; Gregory, William F; Nahler, Janina; Stephens, Ian; Toke-Bjolgerud, Edward; Hewitson, James P; Ivens, Alasdair; McSorley, Henry J; Maizels, Rick M

2018-04-01

We recently reported the discovery of a new parasite-derived protein that functionally mimics the immunosuppressive cytokine transforming growth factor (TGF)-β. The Heligmosomoides polygyrus TGF-β Mimic (Hp-TGM) shares no homology to any TGF-β family member, however it binds the mammalian TGF-β receptor and induces expression of Foxp3, the canonical transcription factor of both mouse and human regulatory T cells. Hp-TGM consists of five atypical Complement Control Protein (CCP, Pfam 00084) domains, each lacking certain conserved residues and 12-15 amino acids longer than the 60-70 amino acids consensus domain, but with a recognizable 3-cysteine, tryptophan, cysteine motif. We now report on the identification of a family of nine related Hp-TGM homologues represented in the secreted proteome and transcriptome of H. polygyrus. Recombinant proteins from five of the nine new TGM members were tested for TGF-β activity, but only two were functionally active in an MFB-F11 reporter assay, and by the induction of T cell Foxp3 expression. Sequence comparisons reveal that proteins with functional activity are similar or identical to Hp-TGM across the first three CCP domains, but more variable in domains 4 and 5. Inactive proteins diverged in all domains, or lacked some domains entirely. Testing truncated versions of Hp-TGM confirmed that domains 1-3 are essential for full activity in vitro, while domains 4 and 5 are not required. Further studies will elucidate whether these latter domains fulfill other functions in promoting host immune regulation during infection and if the more divergent family members play other roles in immunomodulation. Copyright © 2018. Published by Elsevier Ltd.

Murine Electrophysiological Models of Cardiac Arrhythmogenesis

PubMed Central

2016-01-01

Cardiac arrhythmias can follow disruption of the normal cellular electrophysiological processes underlying excitable activity and their tissue propagation as coherent wavefronts from the primary sinoatrial node pacemaker, through the atria, conducting structures and ventricular myocardium. These physiological events are driven by interacting, voltage-dependent, processes of activation, inactivation, and recovery in the ion channels present in cardiomyocyte membranes. Generation and conduction of these events are further modulated by intracellular Ca2+ homeostasis, and metabolic and structural change. This review describes experimental studies on murine models for known clinical arrhythmic conditions in which these mechanisms were modified by genetic, physiological, or pharmacological manipulation. These exemplars yielded molecular, physiological, and structural phenotypes often directly translatable to their corresponding clinical conditions, which could be investigated at the molecular, cellular, tissue, organ, and whole animal levels. Arrhythmogenesis could be explored during normal pacing activity, regular stimulation, following imposed extra-stimuli, or during progressively incremented steady pacing frequencies. Arrhythmic substrate was identified with temporal and spatial functional heterogeneities predisposing to reentrant excitation phenomena. These could arise from abnormalities in cardiac pacing function, tissue electrical connectivity, and cellular excitation and recovery. Triggering events during or following recovery from action potential excitation could thereby lead to sustained arrhythmia. These surface membrane processes were modified by alterations in cellular Ca2+ homeostasis and energetics, as well as cellular and tissue structural change. Study of murine systems thus offers major insights into both our understanding of normal cardiac activity and its propagation, and their relationship to mechanisms generating clinical arrhythmias. PMID:27974512

Amelioration of tissue fibrosis by toll-like receptor 4 knockout in murine models of systemic sclerosis.

PubMed

Takahashi, Takehiro; Asano, Yoshihide; Ichimura, Yohei; Toyama, Tetsuo; Taniguchi, Takashi; Noda, Shinji; Akamata, Kaname; Tada, Yayoi; Sugaya, Makoto; Kadono, Takafumi; Sato, Shinichi

2015-01-01

Bleomycin-induced fibrosis and the tight skin (TSK/+) mouse are well-established experimental murine models of human systemic sclerosis (SSc). Growing evidence has demonstrated the pivotal role of Toll-like receptors (TLRs) in several autoimmune inflammatory diseases, including SSc. This study was undertaken to determine the role of TLR-4 in the fibrotic processes in these murine models. We generated a murine model of bleomycin-induced SSc using TLR-4(-/-) mice and TLR-4(-/-) ;TSK/+ mice. The mechanisms by which TLR-4 contributes to pathologic tissue fibrosis were investigated in these 2 models by histologic examination, hydroxyproline assay, enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and flow cytometry. Dermal and lung fibrosis was attenuated in bleomycin-treated TLR-4(-/-) mice compared with their wild-type counterparts. Inflammatory cell infiltration, expression of various inflammatory cytokines, and pathologic angiogenesis induced by bleomycin treatment were suppressed with TLR-4 deletion. Furthermore, the increased expression of interleukin-6 (IL-6) in fibroblasts, endothelial cells, and immune cells in response to bleomycin in vivo and to lipopolysaccharide in vitro was notably abrogated in the absence of TLR-4. Moreover, TLR-4 deletion was associated with alleviated B cell activation and skew toward a Th2/Th17 response against bleomycin treatment. Importantly, in TSK/+ mice, another SSc murine model, TLR-4 abrogation attenuated hypodermal fibrosis. These results indicate the pivotal contribution of TLR-4 to the pathologic tissue fibrosis of SSc murine models. Our results indicate the critical role of TLR-4 signaling in the development of tissue fibrosis, suggesting that biomolecular TLR-4 targeting might be a potential therapeutic approach to SSc. Copyright © 2015 by the American College of Rheumatology.

I-309/T cell activation gene-3 chemokine protects murine T cell lymphomas against dexamethasone-induced apoptosis.

PubMed

Van Snick, J; Houssiau, F; Proost, P; Van Damme, J; Renauld, J C

1996-09-15

We have previously reported that cytokines such as IL-9, IL-4, and IL-6 protect murine thymic lymphoma cell lines against dexamethasone-induced apoptosis. A similar activity, which could not be ascribed to any of these factors, was found in a number of human T cell supernatants that enabled mouse BW5147 thymic lymphoma not only to escape apoptosis but also to maintain proliferation. The protein responsible for this activity was purified to homogeneity from the culture medium of activated leukemic T cells and was found to be identical with the I-309 chemokine. Half-maximal anti-apoptotic activity was obtained with approximately 1 ng/ml, a concentration considerably lower than that required for the monocyte chemotactic activity of this molecule, as measured on THP-1 cells. The purified I-309 also improved the survival of two other mouse thymic lymphoma cell lines. This activity was as potent as that of IL-9, which was the strongest anti-apoptotic factor found to date for these cells. Similar results were obtained for BW5147 cells with recombinant I-309 and with T cell activation gene-3, the murine homologue of I-309, but not with other members of the chemokine family, including IL-8, neutrophil-activating peptide-2, granulocyte chemotactic protein-2, macrophage inflammatory protein-1a, RANTES (regulated upon activation, normal T cell expressed and secreted), monocyte chemotactic protein-1 (MCP-1), and MCP-2. MCP-3, however, showed a minor, but significant effect in this model. Unlike that of IL-9, the activity of I-309 was completely inhibited in the presence of pertussis toxin, indicating the involvement of a G protein in this process.

Murine Toxicity of Cochliobolus carbonum1

PubMed Central

Hamilton, Pat B.; Nelson, R. R.; Harris, B. S. H.

1968-01-01

Seventeen wild-type strains of the phytopathogenic fungus Cochliobolus carbonum, tested by intraperitoneal injection into mice, were lethal within 48 hr. The lethal effect appeared to be a toxic rather than an infectious process, because death occurred within 3 hr after injection of two of the isolates and heat-killed cultures were lethal. Assays of ascospore progeny from two crosses involving three isolates indicated that the toxic metabolites were under genetic control and quantitative regulation. Studies of the toxicological, cultural, and chemical characteristics of these three strains indicated that more than one murine toxin was present. PMID:16349821

ERP, a new member of the ets transcription factor/oncoprotein family: cloning, characterization, and differential expression during B-lymphocyte development.

PubMed

Lopez, M; Oettgen, P; Akbarali, Y; Dendorfer, U; Libermann, T A

1994-05-01

The ets gene family encodes a group of proteins which function as transcription factors under physiological conditions and, if aberrantly expressed, can cause cellular transformation. We have recently identified two regulatory elements in the murine immunoglobulin heavy-chain (IgH) enhancer, pi and microB, which exhibit striking similarity to binding sites for ets-related proteins. To identify ets-related transcriptional regulators expressed in pre-B lymphocytes that may interact with either the pi or the microB site, we have used a PCR approach with degenerate oligonucleotides encoding conserved sequences in all members of the ets family. We have cloned the gene for a new ets-related transcription factor, ERP (ets-related protein), from the murine pre-B cell line BASC 6C2 and from mouse lung tissue. The ERP protein contains a region of high homology with the ETS DNA-binding domain common to all members of the ets transcription factor/oncoprotein family. Three additional smaller regions show homology to the ELK-1 and SAP-1 genes, a subgroup of the ets gene family that interacts with the serum response factor. Full-length ERP expresses only negligible DNA-binding activity by itself. Removal of the carboxy terminus enables ERP to interact with a variety of ets-binding sites including the E74 site, the IgH enhancer pi site, and the lck promoter ets site, suggesting a carboxy-terminal negative regulatory domain. At least three ERP-related transcripts are expressed in a variety of tissues. However, within the B-cell lineage, ERP is highly expressed primarily at early stages of B-lymphocyte development, and expression declines drastically upon B-cell maturation, correlating with the enhancer activity of the IgH pi site. These data suggest that ERP might play a role in B-cell development and in IgH gene regulation.

Dynamic Determination of Oxygenation and Lung Compliance in Murine Pneumonectomy

PubMed Central

Gibney, Barry; Lee, Grace S.; Houdek, Jan; Lin, Miao; Miele, Lino; Chamoto, Kenji; Konerding, Moritz A.; Tsuda, Akira; Mentzer, Steven J.

2012-01-01

Thoracic surgical procedures in mice have been applied to a wide range of investigations, but little is known about the murine physiologic response to pulmonary surgery. Using continuous arterial oximetry monitoring and the FlexiVent murine ventilator, we investigated the effect of anesthesia and pneumonectomy on mouse oxygen saturation and lung mechanics. Sedation resulted in a dose-dependent decline of oxygen saturation that ranged from 55–82%. Oxygen saturation was restored by mechanical ventilation with increased rate and tidal volumes. In the mouse strain studied, optimal ventilatory rates were a rate of 200/minute and a tidal volume of 10ml/kg. Sustained inflation pressures, referred to as a "recruitment maneuver," improved lung volumes, lung compliance and arterial oxygenation. In contrast, positive end expiratory pressure (PEEP) had a detrimental effect on oxygenation; an effect that was ameliorated after pneumonectomy. Our results confirm that lung volumes in the mouse are dynamically determined and suggest a threshold level of mechanical ventilation to maintain perioperative oxygen saturation. PMID:21574875

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

PubMed

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

2016-07-01

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. Copyright © 2016 Elsevier Ltd. All rights reserved.

Responses of the Murine Myeloid Colony-Forming Cell to Ansamycin Antibiotics

PubMed Central

Horoszewicz, Julius S.; Carter, William A.

1974-01-01

The in vitro susceptibility of murine myeloid colony-forming cells to the antiproliferative activities of three ansamycin antibiotics was determined. These cells were found to be 10- to 40-fold more susceptible than the corresponding human ones. PMID:4151701

Characterization of a Novel Murine Model to Study Zika Virus.

PubMed

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

2016-06-01

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 ∼10(7) 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. © The American Society of Tropical Medicine and Hygiene.

Sex differences in the MB49 syngeneic, murine model of bladder cancer

PubMed Central

White-Gilbertson, Shai; Davis, Megan; Voelkel-Johnson, Christina; Kasman, Laura M.

2016-01-01

OBJECTIVE The MB49 syngeneic, murine model of bladder cancer has been widely used for more than 35 years. In humans, bladder cancer is one third as prevalent in women as in men, with a trend toward lower prevalence in parous compared to nulliparous women. Our objective was to determine if the MB49 bladder cancer model reproduces the sex differences observed in humans, and to determine its sensitivity to testosterone and the pregnancy hormone, human chorionic gonadotropin (hCG). METHODS Male and female C57BL/6 mice were implanted with MB49 murine bladder cancer cells, and observed for tumor growth. MB49 dose responses to hCG and dihydrotestosterone were determined in vitro. RESULTS MB49 tumor growth was significantly greater in male mice than female mice. Pregnancy did not affect MB49 tumor growth in female mice. MB49 cells did not proliferate in response to hCG in vitro and the functional receptor for gonadotropins was absent. Dihydrotestosterone strongly stimulated growth of MB49 cells in vitro. CONCLUSIONS The MB49 murine model of bladder cancer reproduced some aspects of the sex differences observed in humans. Our results suggest that testosterone may stimulate MB49 cell proliferation, which may explain the more rapid MB49 tumor growth observed in male mice. PMID:26998503

Optical monitoring of glucose demand and vascular delivery in a preclinical murine model

NASA Astrophysics Data System (ADS)

Frees, Amy; Rajaram, Narasimhan; McCachren, Sam; Vaz, Alex; Dewhirst, Mark; Ramanujam, Nimmi

2014-03-01

Targeted therapies such as PI3K inhibition can affect tumor vasculature, and hence delivery of imaging agents like FDG, while independently modifying intrinsic glucose demand. Therefore, it is important to identify whether perceived changes in glucose uptake are caused by vascular or true metabolic changes. This study sought to develop an optical strategy for quantifying tissue glucose uptake free of cross-talk from tracer delivery effects. Glucose uptake kinetics were measured using a fluorescent D-glucose derivative 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-deoxy-Dglucose (2-NBDG), and 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-deoxy-L-glucose (2-NBDLG) was used as a control to report on non-specific uptake. Vascular oxygenation (SO2) was calculated from wavelength-dependent hemoglobin absorption. We have previously shown that the rate of 2-NBDG delivery in vivo profoundly affects perceived demand. In this study, we investigated the potential of the ratio of 2-NBDG uptake to the rate of delivery (2-NBDG60/RD) to report on 2-NBDG demand in vivo free from confounding delivery effects. In normal murine tissue, we show that 2-NBDG60/RD can distinguish specific uptake from non-specific cell membrane binding, whereas fluorescence intensity alone cannot. The ratio 2-NBDG60/RD also correlates with blood glucose more strongly than 2-NBDG60 does in normal murine tissue. Additionally, 2-NBDG60/RD can distinguish normal murine tissue from a murine metastatic tumor across a range of SO2 values. The results presented here indicate that the ratio of 2-NBDG uptake to the rate of 2-NBDG delivery (2- NBDG60/RD) is superior to 2-NBDG intensity alone for quantifying changes in glucose demand.

The pharmacokinetics and metabolism of lumiracoxib in chimeric humanized and murinized FRG mice.

PubMed

Dickie, A P; Wilson, C E; Schreiter, K; Wehr, R; Wilson, E M; Bial, J; Scheer, N; Wilson, I D; Riley, R J

2017-07-01

The pharmacokinetics and metabolism of lumiracoxib were studied, after administration of single 10mg/kg oral doses to chimeric liver-humanized and murinized FRG mice. In the chimeric humanized mice, lumiracoxib reached peak observed concentrations in the blood of 1.10±0.08μg/mL at 0.25-0.5h post-dose with an AUC inf of 1.74±0.52μgh/mL and an effective half-life for the drug of 1.42±0.72h (n=3). In the case of the murinized animals peak observed concentrations in the blood were determined as 1.15±0.08μg/mL at 0.25h post-dose with an AUC inf of 1.94±0.22μgh/mL and an effective half-life of 1.28±0.02h (n=3). Analysis of blood indicated only the presence of unchanged lumiracoxib. Metabolic profiling of urine, bile and faecal extracts revealed a complex pattern of metabolites for both humanized and murinized animals with, in addition to unchanged parent drug, a variety of hydroxylated and conjugated metabolites detected. The profiles obtained in humanized mice were different compared to murinized animals with e.g., a higher proportion of the dose detected in the form of acyl glucuronide metabolites and much reduced amounts of taurine conjugates. Comparison of the metabolic profiles obtained from the present study with previously published data from C57bl/6J mice and humans, revealed a greater though not complete match between chimeric humanized mice and humans, such that the liver-humanized FRG model may represent a useful approach to assessing the biotransformation of such compounds in humans. Copyright © 2017 Elsevier Inc. All rights reserved.

Improved Murine Blastocyst Quality and Development in a Single Culture Medium Compared to Sequential Culture Media

PubMed Central

Hennings, Justin M.; Zimmer, Randall L.; Nabli, Henda; Davis, J. Wade; Sutovsky, Peter; Sutovsky, Miriam; Sharpe-Timms, Kathy L.

2015-01-01

Objective: Validate single versus sequential culture media for murine embryo development. Design: Prospective laboratory experiment. Setting: Assisted Reproduction Laboratory. Animals: Murine embryos. Interventions: Thawed murine zygotes cultured for 3 or 5 days (d3 or d5) in single or sequential embryo culture media developed for human in vitro fertilization. Main Outcome Measures: On d3, zygotes developing to the 8 cell (8C) stage or greater were quantified using 4’,6-diamidino-2-phenylindole (DAPI), and quality was assessed by morphological analysis. On d5, the number of embryos reaching the blastocyst stage was counted. DAPI was used to quantify total nuclei and inner cell mass nuclei. Localization of ubiquitin C-terminal hydrolase L1 (UCHL1) and ubiquitin C-terminal hydrolase L3 (UCHL3) was reference points for evaluating cell quality. Results: Comparing outcomes in single versus to sequential media, the odds of embryos developing to the 8C stage on d3 were 2.34 time greater (P = .06). On d5, more embryos reached the blastocyst stage (P = <.0001), hatched, and had significantly more trophoblast cells (P = .005) contributing to the increased total cell number. Also at d5, localization of distinct cytoplasmic UCHL1 and nuclear UCHL3 was found in high-quality hatching blastocysts. Localization of UCHL1 and UCHL3 was diffuse and inappropriately dispersed throughout the cytoplasm in low-quality nonhatching blastocysts. Conclusions: Single medium yields greater cell numbers, an increased growth rate, and more hatching of murine embryos. Cytoplasmic UCHL1 and nuclear UHCL3 localization patterns were indicative of embryo quality. Our conclusions are limited to murine embryos but one might speculate that single medium may also be more beneficial for human embryo culture. Human embryo studies are needed. PMID:26668049

Improved Murine Blastocyst Quality and Development in a Single Culture Medium Compared to Sequential Culture Media.

PubMed

Hennings, Justin M; Zimmer, Randall L; Nabli, Henda; Davis, J Wade; Sutovsky, Peter; Sutovsky, Miriam; Sharpe-Timms, Kathy L

2016-03-01

Validate single versus sequential culture media for murine embryo development. Prospective laboratory experiment. Assisted Reproduction Laboratory. Murine embryos. Thawed murine zygotes cultured for 3 or 5 days (d3 or d5) in single or sequential embryo culture media developed for human in vitro fertilization. On d3, zygotes developing to the 8 cell (8C) stage or greater were quantified using 4',6-diamidino-2-phenylindole (DAPI), and quality was assessed by morphological analysis. On d5, the number of embryos reaching the blastocyst stage was counted. DAPI was used to quantify total nuclei and inner cell mass nuclei. Localization of ubiquitin C-terminal hydrolase L1 (UCHL1) and ubiquitin C-terminal hydrolase L3 (UCHL3) was reference points for evaluating cell quality. Comparing outcomes in single versus to sequential media, the odds of embryos developing to the 8C stage on d3 were 2.34 time greater (P = .06). On d5, more embryos reached the blastocyst stage (P = <.0001), hatched, and had significantly more trophoblast cells (P = .005) contributing to the increased total cell number. Also at d5, localization of distinct cytoplasmic UCHL1 and nuclear UCHL3 was found in high-quality hatching blastocysts. Localization of UCHL1 and UCHL3 was diffuse and inappropriately dispersed throughout the cytoplasm in low-quality nonhatching blastocysts. Single medium yields greater cell numbers, an increased growth rate, and more hatching of murine embryos. Cytoplasmic UCHL1 and nuclear UHCL3 localization patterns were indicative of embryo quality. Our conclusions are limited to murine embryos but one might speculate that single medium may also be more beneficial for human embryo culture. Human embryo studies are needed. © The Author(s) 2015.

Disruption of canonical TGFβ-signaling in murine coronary progenitor cells by low level arsenic

DOE Office of Scientific and Technical Information (OSTI.GOV)

Allison, Patrick; Huang, Tianfang; Broka, Derrick

2013-10-01

Exposure to arsenic results in several types of cancers as well as heart disease. A major contributor to ischemic heart pathologies is coronary artery disease, however the influences by environmental arsenic in this disease process are not known. Similarly, the impact of toxicants on blood vessel formation and function during development has not been studied. During embryogenesis, the epicardium undergoes proliferation, migration, and differentiation into several cardiac cell types including smooth muscle cells which contribute to the coronary vessels. The TGFβ family of ligands and receptors is essential for developmental cardiac epithelial to mesenchymal transition (EMT) and differentiation into coronarymore » smooth muscle cells. In this in vitro study, 18 hour exposure to 1.34 μM arsenite disrupted developmental EMT programming in murine epicardial cells causing a deficit in cardiac mesenchyme. The expression of EMT genes including TGFβ2, TGFβ receptor-3, Snail, and Has-2 are decreased in a dose-dependent manner following exposure to arsenite. TGFβ2 cell signaling is abrogated as detected by decreases in phosphorylated Smad2/3 when cells are exposed to 1.34 μM arsenite. There is also loss of nuclear accumulation pSmad due to arsenite exposure. These observations coincide with a decrease in vimentin positive mesenchymal cells invading three-dimensional collagen gels. However, arsenite does not block TGFβ2 mediated smooth muscle cell differentiation by epicardial cells. Overall these results show that arsenic exposure blocks developmental EMT gene programming in murine coronary progenitor cells by disrupting TGFβ2 signals and Smad activation, and that smooth muscle cell differentiation is refractory to this arsenic toxicity. - Highlights: • Arsenic blocks TGFβ2 induced expression of EMT genes. • Arsenic blocks TGFβ2 triggered Smad2/3 phosphorylation and nuclear translocation. • Arsenic blocks epicardial cell differentiation into cardiac

The development of a murine model for Forcipomyia taiwana (biting midge) allergy.

PubMed

Lee, Mey-Fann; Yang, Kai-Jei; Wang, Nancy M; Chiu, Yung-Tsung; Chen, Pei-Chih; Chen, Yi-Hsing

2014-01-01

Forcipomyia taiwana (biting midge) allergy is the most prevalent biting insect allergy in Taiwan. An animal model corresponding to the human immuno-pathologic features of midge allergy is needed for investigating the mechanisms and therapies. This study successfully developed a murine model of Forcipomyia taiwana allergy. BALB/c mice were sensitized intra-peritoneally with midge extract on days 0, 7, 14, 21 then intra-dermally on days 28, 31 and 35. Serum midge-specific IgE, IgG1, and IgG2a were measured every 14 days by indirect ELISA. The mice were challenged intradermally with midge extract at day 40 and then sacrificed. Proliferation and cytokine production of splenocytes after stimulation with midge extract were determined by MTT assay and ELISA, respectively. The cytokine mRNA expression in response to midge stimulation was analyzed by RT-PCR. Serum IgE, total IgG, and IgG1 antibody levels against midge extract were significantly higher in the midge-sensitized mice than in the control mice. After the two-step sensitization, all mice in the midge-sensitized group displayed immediate itch and plasma extravasation reactions in response to challenge with midge extract. Skin histology from midge-sensitized mice showed marked eosinophil and lymphocyte infiltrations similar to that observed in humans. Stimulation of murine splenocytes with midge extract elicited significant proliferation, IL-4, IL-10, IL-13 and IFN-γ protein production, and up-regulation of mRNA in a dose-dependent manner in the midge-sensitized group, but not in the control group. A murine model of midge bite allergy has been successfully developed using a two-step sensitization protocol. The sensitized mice have very similar clinical and immunologic reactions to challenge with midge proteins as the reactions of human to midge bites. This murine model may be a useful platform for future research and the development of treatment strategies for insect bite allergy.

mEBT: multiple-matching Evidence-based Translator of Murine Genomic Responses for Human Immunity Studies.

PubMed

Tae, Donghyun; Seok, Junhee

2018-05-29

In this paper, we introduce multiple-matching Evidence-based Translator (mEBT) to discover genomic responses from murine expression data for human immune studies, which are significant in the given condition of mice and likely have similar responses in the corresponding condition of human. mEBT is evaluated over multiple data sets and shows improved inter-species agreement. mEBT is expected to be useful for research groups who use murine models to study human immunity. http://cdal.korea.ac.kr/mebt/. jseok14@korea.ac.kr. Supplementary data are available at Bioinformatics online.

Local IL-23 expression in murine vaginal candidiasis and its relationship with infection and immune status.

PubMed

Wu, Yan; Tan, Zhijian; Liu, Zhixiang; Xia, Dechao; Li, Jiawen

2006-01-01

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 estrogen-treated 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 IL-23p19mRNA 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.

Stable long-term blood formation by stem cells in murine steady-state hematopoiesis.

PubMed

Zavidij, Oksana; Ball, Claudia R; Herbst, Friederike; Oppel, Felix; Fessler, Sylvia; Schmidt, Manfred; von Kalle, Christof; Glimm, Hanno

2012-09-01

Hematopoietic stem cells (HSCs) generate all mature blood cells during the whole lifespan of an individual. However, the clonal contribution of individual HSC and progenitor cells in steady-state hematopoiesis is poorly understood. To investigate the activity of HSCs under steady-state conditions, murine HSC and progenitor cells were genetically marked in vivo by integrating lentiviral vectors (LVs) encoding green fluorescent protein (GFP). Hematopoietic contribution of individual marked clones was monitored by determination of lentiviral integration sites using highly sensitive linear amplification-mediated-polymerase chain reaction. A remarkably stable small proportion of hematopoietic cells expressed GFP in LV-injected animals for up to 24 months, indicating stable marking of murine steady-state hematopoiesis. Analysis of the lentiviral integration sites revealed that multiple hematopoietic clones with both myeloid and lymphoid differentiation potential contributed to long-term hematopoiesis. In contrast to intrafemoral vector injection, intravenous administration of LV preferentially targeted short-lived progenitor cells. Myelosuppressive treatment of mice prior to LV-injection did not affect the marking efficiency. Our study represents the first continuous analysis of clonal behavior of genetically marked hematopoietic cells in an unmanipulated system, providing evidence that multiple clones are simultaneously active in murine steady-state hematopoiesis. Copyright © 2012 AlphaMed Press.

The effects of nandrolone, testosterone and their decanoate esters on murine lupus.

PubMed Central

Verheul, H A; Stimson, W H; den Hollander, F C; Schuurs, A H

1981-01-01

Treatment of NZB/NZW F1 (B/W) female and castrated male mice with testosterone or 19-nortestosterone (nandrolone), either by implantation in silastic tubing or by subcutaneous injections of their decanoate esters, reduced in a dose-dependent manner symptoms associated with murine lupus (proteinuria, IgG antibodies to DNA) and prolonged survival. These phenomena were observed under both prophylactic (start at 3-4 weeks) and therapeutic treatments (start 27-29 weeks). Nandrolone and its decanoate ester were at least as potent as testosterone and testosterone decanoate. As the unwanted androgenic properties of nandrolone and its ester are significantly less pronounced than those of testosterone and its ester, also in these NZB/NZW mice, the beneficial effect on murine lupus does not seem to be associated with these properties. PMID:6973425

Thrombopoietin inhibits murine mast cell differentiation

PubMed Central

Martelli, Fabrizio; Ghinassi, Barbara; Lorenzini, Rodolfo; Vannucchi, Alessandro M; Rana, Rosa Alba; Nishikawa, Mitsuo; Partamian, Sandra; Migliaccio, Giovanni; Migliaccio, Anna Rita

2009-01-01

We have recently shown that Mpl, the thrombopoietin receptor, is expressed on murine mast cells and on their precursors and that targeted deletion of the Mpl gene increases mast cell differentiation in mice. Here we report that treatment of mice with thrombopoietin, or addition of this growth factor to bone marrow-derived mast cell cultures, severely hampers the generation of mature cells from their precursors by inducing apoptosis. Analysis of the expression profiling of mast cells obtained in the presence of thrombopoietin suggests that thrombopoietin induces apoptosis of mast cells by reducing expression of the transcription factor Mitf and its target anti-apoptotic gene Bcl2. PMID:18276801

IL-25 promotes the function of CD4+CD25+ T regulatory cells and prolongs skin-graft survival in murine models.

PubMed

Tang, Jiayou; Zhou, Xiaohui; Liu, Jie; Meng, Qingshu; Han, Yang; Wang, Zhulin; Fan, Huimin; Liu, Zhongmin

2015-10-01

Interleukin (IL)-25, also known as IL-17E, belongs to the IL-17 family of cytokines. Unlike other IL-17 family members, IL-25 promotes Th2-type immune responses, stimulating IL-4, IL-5, and IL-13 production. Here, we employed murine models of skin graft to explore the role of IL-25 in suppression of graft rejection. We found that IL-25 expression is increased during allograft rejection, and allograft rejection was enhanced in IL-25 KO mice. IL-25 KO was associated with down-regulation of Foxp3 expression in CD4+ T cells. Further, while adoptive transfer of WT regulatory T cells (Tregs) protected against allograft rejection, adoptive transfer of IL-25 deficient Tregs failed to protect against allograft rejection. Exogenous IL-25 restored Foxp3 expression and Treg function in vitro. Moreover, IL-25 promoted phosphorylation of NFAT2. Thus, IL-25 may enhance Treg function by up-regulating NFAT2 phosphorylation. Our findings suggest that IL-25 can sustain Foxp3 expression, enhance the suppressive function of Tregs, and prolong skin-graft survival. Copyright © 2015 Elsevier B.V. All rights reserved.

Biological properties in vitro of a combination of recombinant murine interleukin-3 and granulocyte-macrophage colony-stimulating factor.

PubMed

Riklis, I; Kletter, Y; Bleiberg, I; Fabian, I

1989-04-01

The effect of recombinant murine interleukin-3 (rIL-3) and recombinant murine granulocyte-macrophage colony-stimulating factor (rGM-CSF) on in vitro murine myeloid progenitor cell (CFU-C) growth and on the function of murine resident peritoneal macrophages was investigated. Both rIL-3 and rGM-CSF are known to support the growth of CFU-C and, when combined, were found to act synergistically to induce the development of an increased number of CFU-C. The distribution pattern of myeloid colonies in the presence of these two growth factors was in general similar to that in the presence of rGM-CSF alone. Both rGM-CSF and rIL-3 enhanced the phagocytosis of Candida albicans (CA) by mature macrophages producing an increase in the percentage of phagocytosing cells as well as an increase in the number of yeast particles ingested per cell. No additive effect on the phagocytosis was observed when the two growth factors were added concurrently. rGM-CSF, but not rIL-3, enhanced the killing of CA by macrophages. This killing was inhibited by scavengers of oxygen radicals.

The mutational landscape of MYCN, Lin28b and ALK F1174L driven murine neuroblastoma mimics human disease.

PubMed

De Wilde, Bram; Beckers, Anneleen; Lindner, Sven; Kristina, Althoff; De Preter, Katleen; Depuydt, Pauline; Mestdagh, Pieter; Sante, Tom; Lefever, Steve; Hertwig, Falk; Peng, Zhiyu; Shi, Le-Ming; Lee, Sangkyun; Vandermarliere, Elien; Martens, Lennart; Menten, Björn; Schramm, Alexander; Fischer, Matthias; Schulte, Johannes; Vandesompele, Jo; Speleman, Frank

2018-02-02

Genetically engineered mouse models have proven to be essential tools for unraveling fundamental aspects of cancer biology and for testing novel therapeutic strategies. To optimally serve these goals, it is essential that the mouse model faithfully recapitulates the human disease. Recently, novel mouse models for neuroblastoma have been developed. Here, we report on the further genomic characterization through exome sequencing and DNA copy number analysis of four of the currently available murine neuroblastoma model systems ( ALK, Th- MYCN, Dbh- MYCN and Lin28b ). The murine tumors revealed a low number of genomic alterations - in keeping with human neuroblastoma - and a positive correlation of the number of genetic lesions with the time to onset of tumor formation was observed. Gene copy number alterations are the hallmark of both murine and human disease and frequently affect syntenic genomic regions. Despite low mutational load, the genes mutated in murine disease were found to be enriched for genes mutated in human disease. Taken together, our study further supports the validity of the tested mouse models for mechanistic and preclinical studies of human neuroblastoma.

Comparison of optical projection tomography and optical coherence tomography for assessment of murine embryonic development

NASA Astrophysics Data System (ADS)

Singh, Manmohan; Nair, Achuth; Vadakkan, Tegy; Piazza, Victor; Udan, Ryan; Frazier, Michael V.; Janecek, Trevor; Dickinson, Mary E.; Larin, Kirill V.

2015-03-01

The murine model is a common model for studying developmental diseases. In this study, we compare the performance of the relatively new method of Optical Projection Tomography (OPT) to the well-established technique of Optical Coherence Tomography (OCT) to assess murine embryonic development at three stages, 9.5, 11.5, and 13.5 days post conception. While both methods can provide spatial resolution at the micrometer scale, OPT can provide superior imaging depth compared to OCT. However, OPT requires samples to be fixed, placed in an immobilization media such as agar, and cleared before imaging. Because OCT does not require fixing, it can be used to image embryos in vivo and in utero. In this study, we compare the efficacy of OPT and OCT for imaging murine embryonic development. The data demonstrate the superior capability of OPT for imaging fine structures with high resolution in optically-cleared embryos while only OCT can provide structural and functional imaging of live embryos ex vivo and in utero with micrometer scale resolution.

Sulforaphane promotes murine hair growth by accelerating the degradation of dihydrotestosterone.

PubMed

Sasaki, Mari; Shinozaki, Shohei; Shimokado, Kentaro

2016-03-25

Dihydrotestosterone (DHT) causes the regression of human hair follicles in the parietal scalp, leading to androgenic alopecia (AGA). Sulforaphane (SFN) increases the expression of DHT degrading enzymes, such as 3α-hydroxysteroid dehydrogenases (3α-HSDs), and, therefore, SFN treatment may improve AGA. To determine the effects of SFN on hair growth, we administered SFN (10 mg/kg BW, IP) or vehicle (DMSO) to ob/ob mice for six weeks and examined hair regeneration and the plasma levels of testosterone and DHT. We also tested the effects of SFN on the expression of two forms of 3α-HSD, aldo-keto reductase 1c21 and dehydrogenase/reductase (SDR family) member 9, both in vitro and in vivo. SNF significantly enhanced hair regeneration in ob/ob mice. The mice treated with SFN showed lower plasma levels of testosterone and DHT than those treated with vehicle. SFN increased the mRNA and protein levels of the two forms of 3α-HSD in the liver of the mice and in cultured murine hepatocyte Hepa1c1c7 cells. These results suggest that SFN treatment increases the amount of 3α-HSDs in the liver, accelerates the degradation of blood DHT, and subsequently blocks the suppression of hair growth by DHT. Copyright © 2016 Elsevier Inc. All rights reserved.

Fv1-like restriction of N-tropic replication-competent murine leukaemia viruses in mCAT-1-expressing human cells.

PubMed

Aagaard, Lars; Mikkelsen, Jacob Giehm; Warming, Søren; Duch, Mogens; Pedersen, Finn Skou

2002-02-01

To study the replication of murine leukaemia viruses in human cells we have used full-length as well as EGFP-tagged ecotropic viruses in combination with mCAT-1-expressing human cells. We present results showing that N-tropic murine leukaemia viruses are restricted in both infection and replication in such cells while B-tropic viruses, modified at capsid position 110, escape restriction. These results support a recently reported Fv1-like restriction in mammalian cells. We extend the analysis of Fv1-like restriction by demonstrating that NB-tropic viruses also escape restriction and human mCAT-1-expressing cells are thus similar to murine Fv1(b) cells with respect to infection though the ecotropic receptor pathway.

Genotoxicity of nimesulide in murine bone marrow cells.

PubMed

Khan, P K; Amod, K; Haque, M; Nath, A

2003-01-01

The genotoxic potentiality of nimesulide was evaluated in vivo in murine bone marrow cells. The human equivalent prophylactic dose of nimesulide (5 mg/kg body wt/day) was given to animals orally, once daily for seven consecutive days. Metaphase chromosome analyses revealed the significant increase in the incidence of chromosomal aberrations with preference to structural over the numerical ones. It therefore suggested the clastogenic effect of the nimesulide. The molecular mechanism of mutagenesis is yet to be determined.

Nanoliposomal artemisinin for the treatment of murine visceral leishmaniasis.

PubMed

Want, Muzamil Y; Islammudin, Mohammad; Chouhan, Garima; Ozbak, Hani A; Hemeg, Hassan A; Chattopadhyay, Asoke P; Afrin, Farhat

2017-01-01

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 IC 50 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 percentage inhibition of 82.4%±3.8% in the liver and 77.6%±5.5% in spleen at the highest dose of 20 mg/kg body weight with modulation of cell-mediated immunity towards protective Th1 type. This study is the first report on the use of a liposomal drug delivery system for artemisinin as a promising alternative intervention against VL.

Nanoliposomal artemisinin for the treatment of murine visceral leishmaniasis

PubMed Central

Want, Muzamil Y; Islammudin, Mohammad; Chouhan, Garima; Ozbak, Hani A; Hemeg, Hassan A; Chattopadhyay, Asoke P; Afrin, Farhat

2017-01-01

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 percentage inhibition of 82.4%±3.8% in the liver and 77.6%±5.5% in spleen at the highest dose of 20 mg/kg body weight with modulation of cell-mediated immunity towards protective Th1 type. This study is the first report on the use of a liposomal drug delivery system for artemisinin as a promising alternative intervention against VL. PMID:28356736

Diagnosing hypoxia in murine models of rheumatoid arthritis from reflectance multispectral images

NASA Astrophysics Data System (ADS)

Glinton, Sophie; Naylor, Amy J.; Claridge, Ela

2017-07-01

Spectra computed from multispectral images of murine models of Rheumatoid Arthritis show a characteristic decrease in reflectance within the 600-800nm region which is indicative of the reduction in blood oxygenation and is consistent with hypoxia.

Targeted destruction of murine macrophage cells with bioconjugated gold nanorods

NASA Astrophysics Data System (ADS)

Pissuwan, Dakrong; Valenzuela, Stella M.; Killingsworth, Murray C.; Xu, Xiaoda; Cortie, Michael B.

2007-12-01

Gold nanorods manifest a readily tunable longitudinal plasmon resonance with light and consequently have potential for use in photothermal therapeutics. Recent work by others has shown how gold nanoshells and rods can be used to target cancer cells, which can then be destroyed using relatively high power laser radiation (˜1×105 to 1×1010 W/m2). Here we extend this concept to demonstrate how gold nanorods can be modified to bind to target macrophage cells, and show that high intensity laser radiation is not necessary, with even 5×102 W/m2 being sufficient, provided that a total fluence of ˜30 J/cm2 is delivered. We used the murine cell line RAW 264.7 and the monoclonal antibody CD11b, raised against murine macrophages, as our model system and a 5 mW solid state diode laser as our energy source. Exposure of the cells labeled with gold nanorods to a laser fluence of 30 J/cm2 resulted in 81% cell death compared to only 0.9% in the control, non-labeled cells.

Efficacy and immunological actions of FAHF-2 in a murine model of multiple food allergies.

PubMed

Srivastava, Kamal D; Bardina, Ludmilla; Sampson, Hugh A; Li, Xiu-Min

2012-05-01

Food Allergy Herbal Formula-2 (FAHF-2) prevents anaphylaxis in a murine model of peanut allergy. Multiple food allergies (MFA) are common and associated with a higher risk of anaphylaxis. No well-characterized murine model of sensitization to multiple food allergens exists, and no satisfactory therapy for MFA is currently available. To determine the effect of FAHF-2 in a murine model of MFA. C3H/HeJ mice were orally sensitized to peanut, codfish, and egg concurrently. Oral FAHF-2 treatment commenced 1 day after completing sensitization and continued daily for 7 weeks. Mice were subsequently orally challenged with each allergen. Antibodies in sera from mice simultaneously sensitized with peanut, codfish, and egg recognized major allergens of all 3 foods, demonstrating sensitization to multiple unrelated food allergens (MFA mice). Sham-treated MFA mice exhibited anaphylactic symptoms accompanied by elevation of plasma histamine and hypothermia. In contrast, FAHF-2-treated MFA mice showed no anaphylactic symptoms, normal body temperature, and histamine levels after challenge with each allergen. Protection was accompanied by reduction in allergen-specific immunoglobulin E levels. Allergen-stimulated Th2 cytokine interleukin-4 and interleukin-13 production levels decreased, whereas the Th1 cytokine interferon-γ levels were elevated in cultured splenocytes and mesenteric lymph node cells in FAHF-2-treated mice. We established the first murine model of MFA. FAHF-2 prevents peanut, egg, and fish-induced anaphylactic reactions in this model, suggesting that FAHF-2 may have potential for treating human MFA. Copyright © 2012 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

TRIMELLITIC ANHYDRIDE-INDUCED EOSINOPHILIA IN A MURINE MODEL OF OCCUPATIONAL ASTHMA

EPA Science Inventory

TRIMELLITIC ANHYDRIDE-INDUCED EOSINOPHILIA IN A MURINE MODEL OF OCCUPATIONAL ASTHMA. J F Regal, ME Mohrman, E Boykin and D Sailstad. Dept. of Pharmacology, University of Minnesota, Duluth, MN, USA and NHEERL, ORD, US EPA, RTP, NC, USA.
Trimellitic anhydride (TMA) is a small m...

[Establishment of systemic lupus erythematosus-like murine model with Sm mimotope].

PubMed

Xie, Hong-Fu; Feng, Hao; Zeng, Hai-Yan; Li, Ji; Shi, Wei; Yi, Mei; Wu, Bin

2007-04-01

To establish systemic lupus erythematosus (SLE) -like murine model by immunizing BALB/C mice with Sm mimotope. Sm mimotope was identified by screening a 12-mer random peptide library with monoclonal anti-Smith antibody. Sm mimotope was initially defined with sandwich ELISA, DNA sequencing, and deduced amino acid sequence; and BALB/C mice were subcutaneously injected with mixture phages clones. Sera Sm antibody, anti-double stranded DNA (dsDNA) antibody, and antinuclear antibody (ANA) of mice were detected using direct immunofluorescence; kidney histological changes were examined by HE staining. Five randomly selected peptides were sequenced and the amino acid sequences IR, SQ, and PP were detected in a higher frequency. High-titer IgG autoantibodies of dsDNA, Sm, and ANA in the sera of experiment group were detected by ELISA 28 days after having been immunized by Sm mimotope. Proteinuria was detected 33 days later; immune complex and nephritis were observed in kidney specimens. SLE-like murine model can be successfully induced by Sm phage mimotope.

Immunologic features of a carcinogen-induced murine bladder cancer: in vivo and in vitro studies.

PubMed

Javadpour, N; Hyatt, C L; Soares, T

1979-01-01

Certain in vivo and in vitro immunologic features of carcinogen-induced murine bladder cancer have been studied. The consistency of tumor induction, its natural history, and immunogenicity both in vivo and in vitro render this syngeneic murine bladder tumor a suitable model for immunologic studies. Pre-immunization of strain C3H/Hen mice with mid-gestational fetal cells did not protect the animals from tumor challenge. Sera of mice immunized with mid-gestational fetal cells were not cytotoxic to cultured tumor cells in a microcytotoxicity assay indicative of dissimilarity between the tumor associated antigen and the syngeneic mid-gestational fetal antigen.

Clinical and Epidemiological Characteristics of Scrub Typhus and Murine Typhus among Hospitalized Patients with Acute Undifferentiated Fever in Northern Vietnam

PubMed Central

Hamaguchi, Sugihiro; Cuong, Ngo Chi; Tra, Doan Thu; Doan, Yen Hai; Shimizu, Kenta; Tuan, Nguyen Quang; Yoshida, Lay-Myint; Mai, Le Quynh; Duc-Anh, Dang; Ando, Shuji; Arikawa, Jiro; Parry, Christopher M.; Ariyoshi, Koya; Thuy, Pham Thanh

2015-01-01

A descriptive study on rickettsiosis was conducted at the largest referral hospital in Hanoi, Vietnam, to identify epidemiological and clinical characteristics of specific rickettsiosis. Between March 2001 and February 2003, we enrolled 579 patients with acute undifferentiated fever (AUF), excluding patients with malaria, dengue fever, and typhoid fever, and serologically tested for Orientia tsutsugamushi and Rickettsia typhi. Of the patients, 237 (40.9%) and 193 (33.3%) had scrub and murine typhus, respectively, and 149 (25.7%) had neither of them (non–scrub and murine typhus [non-ST/MT]). The proportion of murine typhus was highest among patients living in Hanoi whereas that of scrub typhus was highest in national or regional border areas. The presence of an eschar, dyspnea, hypotension, and lymphadenopathy was significantly associated with a diagnosis of scrub typhus (OR = 46.56, 10.90, 9.01, and 7.92, respectively). Patients with murine typhus were less likely to have these findings but more likely to have myalgia, rash, and relative bradycardia (OR = 1.60, 1.56, and 1.45, respectively). Scrub typhus and murine typhus were shown to be common causes of AUF in northern Vietnam although the occurrence of spotted fever group rickettsiae was not determined. Clinical and epidemiological information may help local clinicians make clinical diagnosis of specific rickettsioses in a resource-limited setting. PMID:25778504

Intratumoral Infection with Murine Cytomegalovirus Synergizes with PD-L1 Blockade to Clear Melanoma Lesions and Induce Long-term Immunity

PubMed Central

Erkes, Dan A; Xu, Guangwu; Daskalakis, Constantine; Zurbach, Katherine A; Wilski, Nicole A; Moghbeli, Toktam; Hill, Ann B; Snyder, Christopher M

2016-01-01

Cytomegalovirus is an attractive cancer vaccine platform because it induces strong, functional CD8+ T-cell responses that accumulate over time and migrate into most tissues. To explore this, we used murine cytomegalovirus expressing a modified gp100 melanoma antigen. Therapeutic vaccination by the intraperitoneal and intradermal routes induced tumor infiltrating gp100-specific CD8+ T-cells, but provided minimal benefit for subcutaneous lesions. In contrast, intratumoral infection of established tumor nodules greatly inhibited tumor growth and improved overall survival in a CD8+ T-cell-dependent manner, even in mice previously infected with murine cytomegalovirus. Although murine cytomegalovirus could infect and kill B16F0s in vitro, infection was restricted to tumor-associated macrophages in vivo. Surprisingly, the presence of a tumor antigen in the virus only slightly increased the efficacy of intratumoral infection and tumor-specific CD8+ T-cells in the tumor remained dysfunctional. Importantly, combining intratumoral murine cytomegalovirus infection with anti-PD-L1 therapy was synergistic, resulting in tumor clearance from over half of the mice and subsequent protection against tumor challenge. Thus, while a murine cytomegalovirus-based vaccine was poorly effective against established subcutaneous tumors, direct infection of tumor nodules unexpectedly delayed tumor growth and synergized with immune checkpoint blockade to promote tumor clearance and long-term protection. PMID:27434584

Micro-computed tomography in murine models of cerebral cavernous malformations as a paradigm for brain disease.

PubMed

Girard, Romuald; Zeineddine, Hussein A; Orsbon, Courtney; Tan, Huan; Moore, Thomas; Hobson, Nick; Shenkar, Robert; Lightle, Rhonda; Shi, Changbin; Fam, Maged D; Cao, Ying; Shen, Le; Neander, April I; Rorrer, Autumn; Gallione, Carol; Tang, Alan T; Kahn, Mark L; Marchuk, Douglas A; Luo, Zhe-Xi; Awad, Issam A

2016-09-15

Cerebral cavernous malformations (CCMs) are hemorrhagic brain lesions, where murine models allow major mechanistic discoveries, ushering genetic manipulations and preclinical assessment of therapies. Histology for lesion counting and morphometry is essential yet tedious and time consuming. We herein describe the application and validations of X-ray micro-computed tomography (micro-CT), a non-destructive technique allowing three-dimensional CCM lesion count and volumetric measurements, in transgenic murine brains. We hereby describe a new contrast soaking technique not previously applied to murine models of CCM disease. Volumetric segmentation and image processing paradigm allowed for histologic correlations and quantitative validations not previously reported with the micro-CT technique in brain vascular disease. Twenty-two hyper-dense areas on micro-CT images, identified as CCM lesions, were matched by histology. The inter-rater reliability analysis showed strong consistency in the CCM lesion identification and staging (K=0.89, p<0.0001) between the two techniques. Micro-CT revealed a 29% greater CCM lesion detection efficiency, and 80% improved time efficiency. Serial integrated lesional area by histology showed a strong positive correlation with micro-CT estimated volume (r(2)=0.84, p<0.0001). Micro-CT allows high throughput assessment of lesion count and volume in pre-clinical murine models of CCM. This approach complements histology with improved accuracy and efficiency, and can be applied for lesion burden assessment in other brain diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

Murine model of long term obstructive jaundice

PubMed Central

Aoki, Hiroaki; Aoki, Masayo; Yang, Jing; Katsuta, Eriko; Mukhopadhyay, Partha; Ramanathan, Rajesh; Woelfel, Ingrid A.; Wang, Xuan; Spiegel, Sarah; Zhou, Huiping; Takabe, Kazuaki

2016-01-01

Background With the recent emergence of conjugated bile acids as signaling molecules in cancer, a murine model of obstructive jaundice by cholestasis with long-term survival is in need. Here, we investigated the characteristics of 3 murine models of obstructive jaundice. Methods C57BL/6J mice were used for total ligation of the common bile duct (tCL), partial common bile duct ligation (pCL), and ligation of left and median hepatic bile duct with gallbladder removal (LMHL) models. Survival was assessed by Kaplan-Meier method. Fibrotic change was determined by Masson-Trichrome staining and Collagen expression. Results 70% (7/10) of tCL mice died by Day 7, whereas majority 67% (10/15) of pCL mice survived with loss of jaundice. 19% (3/16) of LMHL mice died; however, jaundice continued beyond Day 14, with survival of more than a month. Compensatory enlargement of the right lobe was observed in both pCL and LMHL models. The pCL model demonstrated acute inflammation due to obstructive jaundice 3 days after ligation but jaundice rapidly decreased by Day 7. The LHML group developed portal hypertension as well as severe fibrosis by Day 14 in addition to prolonged jaundice. Conclusion The standard tCL model is too unstable with high mortality for long-term studies. pCL may be an appropriate model for acute inflammation with obstructive jaundice but long term survivors are no longer jaundiced. The LHML model was identified to be the most feasible model to study the effect of long-term obstructive jaundice. PMID:27916350

Serological differentiation of murine typhus and epidemic typhus using cross-adsorption and Western blotting.

PubMed

La Scola, B; Rydkina, L; Ndihokubwayo, J B; Vene, S; Raoult, D

2000-07-01

Differentiation of murine typhus due to Rickettsia typhi and epidemic typhus due to Rickettsia prowazekii is critical epidemiologically but difficult serologically. Using serological, epidemiological, and clinical criteria, we selected sera from 264 patients with epidemic typhus and from 44 patients with murine typhus among the 29,188 tested sera in our bank. These sera cross-reacted extensively in indirect fluorescent antibody assays (IFAs) against R. typhi and R. prowazekii, as 42% of the sera from patients with epidemic typhus and 34% of the sera from patients with murine typhus exhibited immunoglobulin M (IgM) and/or IgG titers against the homologous antigen (R. prowazekii and R. typhi, respectively) that were more than one dilution higher than those against the heterologous antigen. Serum cross-adsorption studies and Western blotting were performed on sera from 12 selected patients, 5 with murine typhus, 5 with epidemic typhus, and 2 suffering from typhus of undetermined etiology. Differences in IFA titers against R. typhi and R. prowazekii allowed the identification of the etiological agent in 8 of 12 patients. Western blot studies enabled the identification of the etiological agent in six patients. When the results of IFA and Western blot studies were considered in combination, identification of the etiological agent was possible for 10 of 12 patients. Serum cross-adsorption studies enabled the differentiation of the etiological agent in all patients. Our study indicates that when used together, Western blotting and IFA are useful serological tools to differentiate between R. prowazekii and R. typhi exposures. While a cross-adsorption study is the definitive technique to differentiate between infections with these agents, it was necessary in only 2 of 12 cases (16.7%), and the high costs of such a study limit its use.

Serological Differentiation of Murine Typhus and Epidemic Typhus Using Cross-Adsorption and Western Blotting

PubMed Central

La Scola, Bernard; Rydkina, Lena; Ndihokubwayo, Jean-Bosco; Vene, Sirkka; Raoult, Didier

2000-01-01

Differentiation of murine typhus due to Rickettsia typhi and epidemic typhus due to Rickettsia prowazekii is critical epidemiologically but difficult serologically. Using serological, epidemiological, and clinical criteria, we selected sera from 264 patients with epidemic typhus and from 44 patients with murine typhus among the 29,188 tested sera in our bank. These sera cross-reacted extensively in indirect fluorescent antibody assays (IFAs) against R. typhi and R. prowazekii, as 42% of the sera from patients with epidemic typhus and 34% of the sera from patients with murine typhus exhibited immunoglobulin M (IgM) and/or IgG titers against the homologous antigen (R. prowazekii and R. typhi, respectively) that were more than one dilution higher than those against the heterologous antigen. Serum cross-adsorption studies and Western blotting were performed on sera from 12 selected patients, 5 with murine typhus, 5 with epidemic typhus, and 2 suffering from typhus of undetermined etiology. Differences in IFA titers against R. typhi and R. prowazekii allowed the identification of the etiological agent in 8 of 12 patients. Western blot studies enabled the identification of the etiological agent in six patients. When the results of IFA and Western blot studies were considered in combination, identification of the etiological agent was possible for 10 of 12 patients. Serum cross-adsorption studies enabled the differentiation of the etiological agent in all patients. Our study indicates that when used together, Western blotting and IFA are useful serological tools to differentiate between R. prowazekii and R. typhi exposures. While a cross-adsorption study is the definitive technique to differentiate between infections with these agents, it was necessary in only 2 of 12 cases (16.7%), and the high costs of such a study limit its use. PMID:10882661

Homocysteine enhances MMP-9 production in murine macrophages via ERK and Akt signaling pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Seung Jin; Lee, Yi Sle; Seo, Kyo Won

2012-04-01

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 ledmore » 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.« less

Characterization of murine SIRT3 transcript variants and corresponding protein products

USDA-ARS?s Scientific Manuscript database

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...

Irradiation Design for an Experimental Murine Model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ballesteros-Zebadua, P.; Moreno-Jimenez, S.; Suarez-Campos, J. E.

2010-12-07

In radiotherapy and stereotactic radiosurgery, small animal experimental models are frequently used, since there are still a lot of unsolved questions about the biological and biochemical effects of ionizing radiation. This work presents a method for small-animal brain radiotherapy compatible with a dedicated 6MV Linac. This rodent model is focused on the research of the inflammatory effects produced by ionizing radiation in the brain. In this work comparisons between Pencil Beam and Monte Carlo techniques, were used in order to evaluate accuracy of the calculated dose using a commercial planning system. Challenges in this murine model are discussed.

Efficacy of posaconazole in murine experimental sporotrichosis.

PubMed

Fernández-Silva, Fabiola; Capilla, Javier; Mayayo, Emilio; Guarro, Josep

2012-05-01

We developed a murine model of systemic sporotrichosis by using three strains of each of the two commonest species causing sporotrichosis, i.e., Sporothrix schenckii sensu stricto and Sporothrix brasiliensis, in order to evaluate the efficacy of posaconazole (PSC). The drug was administered at a dose of 2.5 or 5 mg/kg of body weight twice a day by gavage, and one group was treated with amphotericin B (AMB) as a control treatment. Posaconazole, especially at 5 mg/kg, showed good efficacy against all the strains tested, regardless of their MICs, as measured by prolonged survival, tissue burden reduction, and histopathology.

A model system for testing gene vectors using murine tumor cells on the chorioallantoic membrane of the chick embryo.

PubMed

Dani, Sergio U; Espindola, Rachel

2002-06-30

We developed a model system for testing gene vectors, based on the growth of murine tumors on the chorioallantoic membrane (CAM) of embryonic chickens. The ability of selected murine cells to grow on the CAM was rated according to the following criteria: i) formation of tumor masses; ii) metastasis formation; iii) reproducibility; iv) yield, indicated as the number of embryos surviving to assessment time with visible tumors on the CAM; v) maintainability of the cell, both in the original host and the embryonic chick, or 'shuttle maintainability'; vi) detection by the naked eye, and vii) cost/benefit relation. The murine melanoma cell lineage, B16F10, which efficiently forms distinct, pigmented tumor masses and metastases on the CAM, performed better in this model than the murine B61 cell line. In vitro transduction of B16F10 cells with a recombinant adenovirus carrying a construct of the E. coli LacZ gene followed by inoculation onto the CAM resulted in beta-galactosidase expression in the tumor mass growing on the CAM. This model is potentially applicable to preclinical evaluation of gene vectors, especially for gene therapy of cancer.

Effect of Erythromycin on Chronic Respiratory Infection Caused by Pseudomonas aeruginosa with Biofilm Formation in an Experimental Murine Model

PubMed Central

Nagata, Towako; Mukae, Hiroshi; Kadota, Junichi; Hayashi, Tomayoshi; Fujii, Takeshi; Kuroki, Misuzu; Shirai, Ryo; Yanagihara, Katsunori; Tomono, Kazunori; Koji, Takehiko; Kohno, Shigeru

2004-01-01

Diffuse panbronchiolitis (DPB) is a chronic lower respiratory tract infection commonly associated with persistent late-stage Pseudomonas aeruginosa infection. However, low-dose long-term therapy with certain macrolides is effective in most patients with DPB. The present study was designed to examine the effects of long-term erythromycin (ERY) therapy by using our established murine model of chronic respiratory P. aeruginosa infection. ERY or saline was administered from day 80 after intubation with a P. aeruginosa-precoated tube for the subsequent 10, 20, 40, and 80 days. Bacteriologic and histologic analyses of the murine lungs and electron microscopy of the intubated tube were performed. In the murine model, treatment with ERY for 80 days significantly reduced the number of viable P. aeruginosa organisms in the lungs (P < 0.05). The biofilm formed in situ by P. aeruginosa on the inner wall of the inoculation tube placed into the murine bronchus became significantly thinner after 80 days of ERY treatment. We conclude that the clinical efficacy of macrolides in DPB may be due at least in part to the reduction in P. aeruginosa biofilm formation. PMID:15155229

Ribonucleases 6 and 7 have antimicrobial function in the human and murine urinary tract

PubMed Central

Becknell, Brian; Eichler, Tad; Beceiro, Susana; Li, Birong; Easterling, Robert; Carpenter, Ashley R.; James, Cindy; McHugh, Kirk M.; Hains, David S.; Partida-Sanchez, Santiago; Spencer, John David

2014-01-01

Recent evidence suggests antimicrobial peptides protect the urinary tract from infection. Ribonuclease 7 (RNase 7), a member of the RNase A superfamily, is a potent epithelial-derived protein that maintains human urinary tract sterility. RNase 7 expression is restricted to primates, limiting evaluation of its antimicrobial activity in vivo. Here we identified Ribonuclease 6 (RNase 6) as the RNase A Superfamily member present in humans and mice that is most conserved at the amino acid level relative to RNase 7. Like RNase 7, recombinant human and murine RNase 6 has potent antimicrobial activity against uropathogens. Quantitative real-time PCR and immunoblot analysis indicate that RNase 6 mRNA and protein are up-regulated in the human and murine urinary tract during infection. Immunostaining located RNase 6 to resident and infiltrating monocytes, macrophages, and neutrophils. Uropathogenic E. coli induces RNase 6 peptide expression in human CD14+ monocytes and murine bone marrow derived macrophages. Thus, RNase 6 is an inducible, myeloid-derived protein with markedly different expression from the epithelial-derived RNase 7 but with equally potent antimicrobial activity. Our studies suggest RNase 6 serves as an evolutionarily conserved antimicrobial peptide that participates in the maintenance of urinary tract sterility. PMID:25075772

Antileukemic Efficacy of Continuous vs Discontinuous Dexamethasone in Murine Models of Acute Lymphoblastic Leukemia

PubMed Central

Ramsey, Laura B.; Janke, Laura J.; Payton, Monique A.; Cai, Xiangjun; Paugh, Steven W.; Karol, Seth E.; Kamdem, Landry Kamdem; Cheng, Cheng; Williams, Richard T.; Jeha, Sima; Pui, Ching-Hon; Evans, William E.; Relling, Mary V.

2015-01-01

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. PMID:26252865

Porphyrin-substrate binding to murine ferrochelatase: effect on the thermal stability of the enzyme

PubMed Central

2004-01-01

Ferrochelatase (EC 4.99.1.1), the terminal enzyme of the haem biosynthetic pathway, catalyses the chelation of Fe(II) into the protoporphyrin IX ring. The energetics of the binding between murine ferrochelatase and mesoporphyrin were determined using isothermal titration calorimetry, which revealed a stoichiometry of one molecule of mesoporphyrin bound per protein monomer. The binding is strongly exothermic, with a large intrinsic enthalpy (ΔH=−97.1 kJ · mol−1), and is associated with the uptake of two protons from the buffer. This proton transfer suggests that hydrogen bonding between ferrochelatase and mesoporphyrin is a key factor in the thermodynamics of the binding reaction. Differential scanning calorimetry thermograms indicated a co-operative two-state denaturation process with a single transition temperature of 56 °C for wild-type murine ferrochelatase. An increase in the thermal stability of ferrochelatase is dependent upon mesoporphyrin binding. Similarly, murine ferrochelatase variants, in which the active site Glu-289 was replaced by either glutamine or alanine and, when purified, contained specifically-bound protoporphyrin, exhibited enhanced protein stability when compared with wild-type ferrochelatase. However, in contrast with the wild-type enzyme, the thermal denaturation of ferrochelatase variants was best described as a non-co-operative denaturation process. PMID:15496139

Baicalein induces cell death in murine T cell lymphoma via inhibition of thioredoxin system.

PubMed

Patwardhan, Raghavendra S; Pal, Debojyoti; Checker, Rahul; Sharma, Deepak; Sandur, Santosh K

2017-10-01

We have earlier demonstrated the radioprotective potential of baicalein using murine splenic lymphocytes. Here, we have studied the effect of baicalein on murine T cell lymphoma EL4 cells and investigated the underlying mechanism of action. We observed that baicalein induced a dose dependent cell death in EL4 cells in vitro and significantly reduced the frequency of cancer stem cells. Previously, we have reported that murine and human T cell lymphoma cells have increased oxidative stress tolerance capacity due to active thioredoxin system. Hence, we monitored the effect of baicalein on thioredoxin system in EL4 cells. Docking studies revealed that baicalein could bind to the active site of thioredoxin reductase. Baicalein treatment led to significant reduction in the activity of thioredoxin reductase and nuclear levels of thioredoxin-1 thereby increasing ASK1 levels and caspase-3 activity. Interestingly, CRISPR-Cas9 based knock-out of ASK1 or over-expression of thioredoxin-1 abolished anti-tumor effects of baicalein in EL4 cells. Further, baicalein administration significantly reduced intra-peritoneal tumor burden of EL4 cells in C57BL/6 mice. Thus, our study describes anti-tumor effects of baicalein in EL4 cells via inhibition of thioredoxin system. Copyright © 2017 Elsevier Ltd. All rights reserved.

Orthotopic lung cancer murine model by nonoperative transbronchial approach.

PubMed

Nakajima, Takahiro; Anayama, Takashi; Matsuda, Yasushi; Hwang, David M; McVeigh, Patrick Z; Wilson, Brian C; Zheng, Gang; Keshavjee, Shaf; Yasufuku, Kazuhiro

2014-05-01

The aim of this work was to establish a novel orthotopic human non-small cell lung cancer (NSCLC) murine xenograft model by a nonsurgical, transbronchial approach. Male athymic nude mice and human NSCLC cell lines, including A549, H460, and H520 were used. Under direct visualization of the vocal cords, a 23-gauge blunt-tip slightly curved metal catheter was introduced into the trachea to the bronchus, and 2.5×10(5) tumor cells mixed with Matrigel (BD Biosciences, Mississauga, Ontario, Canada) were administered into the lung. Mice were monitored using weekly microcomputed tomography scans for tumor formation. When the tumor size reached more than 4 mm in diameter, the animals were euthanized, and the tumor tissue was evaluated histopathologically. Of 37 mice studied, 34 were confirmed to have tumor formation: 29 developed solitary tumors and 5 had multifocal lesions. There was no evidence of extrapleural dissemination or effusion. Transbronchial delivery of tumor cells enabled the establishment of a novel orthotopic human NSCLC murine xenograft model. This clinically relevant preclinical model bearing a solitary nodule is of value for a variety of in vivo research studies. Copyright © 2014 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.

Isolation and characterisation of Ebolavirus-specific recombinant antibody fragments from murine and shark immune libraries.

PubMed

Goodchild, Sarah A; Dooley, Helen; Schoepp, Randal J; Flajnik, Martin; Lonsdale, Stephen G

2011-09-01

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. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

Targeting of a Nuclease to Murine Leukemia Virus Capsids Inhibits Viral Multiplication

NASA Astrophysics Data System (ADS)

Natsoulis, Georges; Seshaiah, Partha; Federspiel, Mark J.; Rein, Alan; Hughes, Stephen H.; Boeke, Jef D.

1995-01-01

Capsid-targeted viral inactivation is an antiviral strategy in which toxic fusion proteins are targeted to virions, where they inhibit viral multiplication by destroying viral components. These fusion proteins consist of a virion structural protein moiety and an enzymatic moiety such as a nuclease. Such fusion proteins can severely inhibit transposition of yeast retrotransposon Ty1, an element whose transposition mechanistically resembles retroviral multiplication. We demonstrate that expression of a murine retrovirus capsid-staphylococcal nuclease fusion protein inhibits multiplication of the corresponding murine leukemia virus by 30- to 100-fold. Staphylococcal nuclease is apparently inactive intracellularly and hence nontoxic to the host cell, but it is active extracellularly because of its requirement for high concentrations of Ca2+ ions. Virions assembled in and shed from cells expressing the fusion protein contain very small amounts of intact viral RNA, as would be predicted for nuclease-mediated inhibition of viral multiplication.

A fluorescence model of the murine lung for optical detection of pathogenic bacteria

NASA Astrophysics Data System (ADS)

Durkee, Madeleine S.; Cirillo, Jeffrey D.; Maitland, Kristen C.

2017-07-01

We present a computer model of intravital excitation and external fluorescence detection in the murine lungs validated with a three-dimensional lung tissue phantom. The model is applied to optical detection of pulmonary tuberculosis infection.

Isolation and (111)In-Oxine Labeling of Murine NK Cells for Assessment of Cell Trafficking in Orthotopic Lung Tumor Model.

PubMed

Malviya, Gaurav; Nayak, Tapan; Gerdes, Christian; Dierckx, Rudi A J O; Signore, Alberto; de Vries, Erik F J

2016-04-04

A noninvasive in vivo imaging method for NK cell trafficking is essential to gain further understanding of the pathogenesis of NK cell mediated immune response to the novel cancer treatment strategies, and to discover the homing sites and physiological distribution of NK cells. Although human NK cells can be labeled for in vivo imaging, little is known about the murine NK cell labeling and its application in animal models. This study describes the isolation and ex vivo radiolabeling of murine NK cells for the evaluation of cell trafficking in an orthotopic model of human lung cancer in mice. Scid-Tg(FCGR3A)Blt transgenic SCID mice were used to isolate NK cells from mouse splenocytes using the CD49b (DX5) MicroBeads positive selection method. The purity and viability of the isolated NK cells were confirmed by FACS analysis. Different labeling buffers and incubation times were evaluated to optimize (111)In-oxine labeling conditions. Functionality of the radiolabeled NK cell was assessed by (51)Cr-release assay. We evaluated physiological distribution of (111)In-oxine labeled murine NK cells in normal SCID mice and biodistribution in irradiated and nonirradiated SCID mice with orthotopic A549 human lung tumor lesions. Imaging findings were confirmed by histology. Results showed that incubation with 0.011 MBq of (111)In-oxine per million murine NK cells in PBS (pH 7.4) for 20 min is the best condition that provides optimum labeling efficiency without affecting cell viability and functionality. Physiological distribution in normal SCID mice demonstrated NK cells homing mainly in the spleen, while (111)In released from NK cells was excreted via kidneys into urine. Biodistribution studies demonstrated a higher lung uptake in orthotopic lung tumor-bearing mice than control mice. In irradiated mice, lung tumor uptake of radiolabeled murine NK cells decreased between 24 h and 72 h postinjection (p.i.), which was accompanied by tumor regression, while in nonirradiated mice

GLUT4, GLUT1, and GLUT8 are the dominant GLUT transcripts expressed in the murine left ventricle

PubMed Central

2012-01-01

Background The heart derives energy from a wide variety of substrates including fatty acids, carbohydrates, ketones, and amino acids. The healthy heart generates up to 30% of its ATP from glucose. Under conditions of cardiac injury or stress, the heart relies even more heavily on glucose as a source of fuel. Glucose is transported into the heart by members of the family of facilitative glucose transporters (GLUTs). While research examining the transport of glucose into the heart has primarily focused on the roles of the classical glucose transporters GLUT1 and GLUT4, little is known about the functions of more newly identified GLUT isoforms in the myocardium. Methods In this study the presence and relative RNA message abundance of each of the known GLUT isoforms was determined in left ventricular tissue from two commonly used inbred laboratory mouse strains (C57BL/6J and FVB/NJ) by quantitative real time PCR. Relative message abundance was also determined in GLUT4 null mice and in murine models of dilated and hypertrophic cardiomyopathy. Results GLUT4, GLUT1, and GLUT8 were found to be the most abundant GLUT transcripts in the normal heart, while GLUT3, GLUT10, and GLUT12 are present at relatively lower levels. Assessment of relative GLUT expression in left ventricular myocardium from mice with dilated cardiomyopathy revealed increased expression of GLUT1 with reduced levels of GLUT4, GLUT8, and GLUT12. Compensatory increase in the expression of GLUT12 was observed in genetically altered mice lacking GLUT4. Conclusions Glucose transporter expression varies significantly among murine models of cardiac dysfunction and involves several of the class III GLUT isoforms. Understanding how these more newly identified GLUT isoforms contribute to regulating myocardial glucose transport will enhance our comprehension of the normal physiology and pathophysiology of the heart. PMID:22681646

GLUT4, GLUT1, and GLUT8 are the dominant GLUT transcripts expressed in the murine left ventricle.

PubMed

Aerni-Flessner, Lauren; Abi-Jaoude, Melissa; Koenig, Amanda; Payne, Maria; Hruz, Paul W

2012-06-08

The heart derives energy from a wide variety of substrates including fatty acids, carbohydrates, ketones, and amino acids. The healthy heart generates up to 30% of its ATP from glucose. Under conditions of cardiac injury or stress, the heart relies even more heavily on glucose as a source of fuel. Glucose is transported into the heart by members of the family of facilitative glucose transporters (GLUTs). While research examining the transport of glucose into the heart has primarily focused on the roles of the classical glucose transporters GLUT1 and GLUT4, little is known about the functions of more newly identified GLUT isoforms in the myocardium. In this study the presence and relative RNA message abundance of each of the known GLUT isoforms was determined in left ventricular tissue from two commonly used inbred laboratory mouse strains (C57BL/6J and FVB/NJ) by quantitative real time PCR. Relative message abundance was also determined in GLUT4 null mice and in murine models of dilated and hypertrophic cardiomyopathy. GLUT4, GLUT1, and GLUT8 were found to be the most abundant GLUT transcripts in the normal heart, while GLUT3, GLUT10, and GLUT12 are present at relatively lower levels. Assessment of relative GLUT expression in left ventricular myocardium from mice with dilated cardiomyopathy revealed increased expression of GLUT1 with reduced levels of GLUT4, GLUT8, and GLUT12. Compensatory increase in the expression of GLUT12 was observed in genetically altered mice lacking GLUT4. Glucose transporter expression varies significantly among murine models of cardiac dysfunction and involves several of the class III GLUT isoforms. Understanding how these more newly identified GLUT isoforms contribute to regulating myocardial glucose transport will enhance our comprehension of the normal physiology and pathophysiology of the heart.

Sodium Thiosulfate Prevents Chondrocyte Mineralization and Reduces the Severity of Murine Osteoarthritis

PubMed Central

Nasi, Sonia; Ea, Hang-Korng; Lioté, Frédéric; So, Alexander; Busso, Nathalie

2016-01-01

Objectives Calcium-containing crystals participate in the pathogenesis of OA. Sodium thiosulfate (STS) has been shown to be an effective treatment in calcification disorders such as calciphylaxis and vascular calcification. This study investigated the effects and mechanisms of action of STS in a murine model of OA and in chondrocyte calcification. Methods Hydroxyapatite (HA) crystals-stimulated murine chondrocytes and macrophages were treated with STS. Mineralization and cellular production of IL-6, MCP-1 and reactive oxygen species (ROS) were assayed. STS's effects on genes involved in calcification, inflammation and cartilage matrix degradation were studied by RT-PCR. STS was administered in the menisectomy model of murine OA, and the effect on periarticular calcific deposits and cartilage degeneration was investigated by micro-CT-scan and histology. Results In vitro, STS prevented in a dose-dependent manner calcium crystal deposition in chondrocytes and inhibited Annexin V gene expression. In addition, there was a reduction in crystal-induced IL-6 and MCP-1 production. STS also had an antioxidant effect, diminished HA-induced ROS generation and abrogated HA-induced catabolic responses in chondrocytes. In vivo, administration of STS reduced the histological severity of OA, by limiting the size of new periarticular calcific deposits and reducing the severity of cartilage damage. Conclusions STS reduces the severity of periarticular calcification and cartilage damage in an animal model of OA via its effects on chondrocyte mineralization and its attenuation of crystal-induced inflammation as well as catabolic enzymes and ROS generation. Our study suggests that STS may be a disease-modifying drug in crystal-associated OA. PMID:27391970

Sodium Thiosulfate Prevents Chondrocyte Mineralization and Reduces the Severity of Murine Osteoarthritis.

PubMed

Nasi, Sonia; Ea, Hang-Korng; Lioté, Frédéric; So, Alexander; Busso, Nathalie

2016-01-01

Calcium-containing crystals participate in the pathogenesis of OA. Sodium thiosulfate (STS) has been shown to be an effective treatment in calcification disorders such as calciphylaxis and vascular calcification. This study investigated the effects and mechanisms of action of STS in a murine model of OA and in chondrocyte calcification. Hydroxyapatite (HA) crystals-stimulated murine chondrocytes and macrophages were treated with STS. Mineralization and cellular production of IL-6, MCP-1 and reactive oxygen species (ROS) were assayed. STS's effects on genes involved in calcification, inflammation and cartilage matrix degradation were studied by RT-PCR. STS was administered in the menisectomy model of murine OA, and the effect on periarticular calcific deposits and cartilage degeneration was investigated by micro-CT-scan and histology. In vitro, STS prevented in a dose-dependent manner calcium crystal deposition in chondrocytes and inhibited Annexin V gene expression. In addition, there was a reduction in crystal-induced IL-6 and MCP-1 production. STS also had an antioxidant effect, diminished HA-induced ROS generation and abrogated HA-induced catabolic responses in chondrocytes. In vivo, administration of STS reduced the histological severity of OA, by limiting the size of new periarticular calcific deposits and reducing the severity of cartilage damage. STS reduces the severity of periarticular calcification and cartilage damage in an animal model of OA via its effects on chondrocyte mineralization and its attenuation of crystal-induced inflammation as well as catabolic enzymes and ROS generation. Our study suggests that STS may be a disease-modifying drug in crystal-associated OA.

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

NASA Astrophysics Data System (ADS)

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

2010-01-01

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 < 0.01) increase in cytokine (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 < 0.05) lower than those of untreated macrophages. Taking together, Ferucarbotran at high dose (100 μg Fe/mL) could induce murine peritoneal macrophages activation in pro-inflammatory cytokine secretion and NO production.

Correlation between experimental human and murine skin sensitization induction thresholds.

PubMed

Api, Anne Marie; Basketter, David; Lalko, Jon

2015-01-01

Quantitative risk assessment for skin sensitization is directed towards the determination of levels of exposure to known sensitizing substances that will avoid the induction of contact allergy in humans. A key component of this work is the predictive identification of relative skin sensitizing potency, achieved normally by the measurement of the threshold (the "EC3" value) in the local lymph node assay (LLNA). In an extended series of studies, the accuracy of this murine induction threshold as the predictor of the absence of a sensitizing effect has been verified by conduct of a human repeated insult patch test (HRIPT). Murine and human thresholds for a diverse set of 57 fragrance chemicals spanning approximately four orders of magnitude variation in potency have been compared. The results confirm that there is a useful correlation, with the LLNA EC3 value helping particularly to identify stronger sensitizers. Good correlation (with half an order of magnitude) was seen with three-quarters of the dataset. The analysis also helps to identify potential outlier types of (fragrance) chemistry, exemplified by hexyl and benzyl salicylates (an over-prediction) and trans-2-hexenal (an under-prediction).

Adaptive Immunity Restricts Replication of Novel Murine Astroviruses

PubMed Central

Yokoyama, Christine C.; Loh, Joy; Zhao, Guoyan; Stappenbeck, Thaddeus S.; Wang, David; Huang, Henry V.

2012-01-01

The mechanisms of astrovirus pathogenesis are largely unknown, in part due to a lack of a small-animal model of disease. Using shotgun sequencing and a custom analysis pipeline, we identified two novel astroviruses capable of infecting research mice, murine astrovirus (MuAstV) STL1 and STL2. Subsequent analysis revealed the presence of at least two additional viruses (MuAstV STL3 and STL4), suggestive of a diverse population of murine astroviruses in research mice. Complete genomic characterization and subsequent phylogenetic analysis showed that MuAstV STL1 to STL4 are members of the mamastrovirus genus and are likely members of a new mamastrovirus genogroup. Using Rag1−/− mice deficient in B and T cells, we demonstrate that adaptive immunity is required to control MuAstV infection. Furthermore, using Stat1−/− mice deficient in innate signaling, we demonstrate a role for the innate immune response in the control of MuAstV replication. Our results demonstrate that MuAstV STL permits the study of the mechanisms of astrovirus infection and host-pathogen interactions in a genetically manipulable small-animal model. Finally, we detected MuAstV in commercially available mice, suggesting that these viruses may be present in academic and commercial research mouse facilities, with possible implications for interpretation of data generated in current mouse models of disease. PMID:22951832

Murine model of long-term obstructive jaundice.

PubMed

Aoki, Hiroaki; Aoki, Masayo; Yang, Jing; Katsuta, Eriko; Mukhopadhyay, Partha; Ramanathan, Rajesh; Woelfel, Ingrid A; Wang, Xuan; Spiegel, Sarah; Zhou, Huiping; Takabe, Kazuaki

2016-11-01

With the recent emergence of conjugated bile acids as signaling molecules in cancer, a murine model of obstructive jaundice by cholestasis with long-term survival is in need. Here, we investigated the characteristics of three murine models of obstructive jaundice. C57BL/6J mice were used for total ligation of the common bile duct (tCL), partial common bile duct ligation (pCL), and ligation of left and median hepatic bile duct with gallbladder removal (LMHL) models. Survival was assessed by Kaplan-Meier method. Fibrotic change was determined by Masson-Trichrome staining and Collagen expression. Overall, 70% (7 of 10) of tCL mice died by day 7, whereas majority 67% (10 of 15) of pCL mice survived with loss of jaundice. A total of 19% (3 of 16) of LMHL mice died; however, jaundice continued beyond day 14, with survival of more than a month. Compensatory enlargement of the right lobe was observed in both pCL and LMHL models. The pCL model demonstrated acute inflammation due to obstructive jaundice 3 d after ligation but jaundice rapidly decreased by day 7. The LHML group developed portal hypertension and severe fibrosis by day 14 in addition to prolonged jaundice. The standard tCL model is too unstable with high mortality for long-term studies. pCL may be an appropriate model for acute inflammation with obstructive jaundice, but long-term survivors are no longer jaundiced. The LHML model was identified to be the most feasible model to study the effect of long-term obstructive jaundice. Copyright © 2016 Elsevier Inc. All rights reserved.

Abnormal cardiac response to exercise in a murine model of familial hypertrophic cardiomyopathy.

PubMed

Nguyen, Lan; Chung, Jessica; Lam, Lien; Tsoutsman, Tatiana; Semsarian, Christopher

2007-07-10

Clinical outcome in familial hypertrophic cardiomyopathy (FHC) may be influenced by modifying factors such as exercise. Transgenic mice which overexpress the human disease-causing cTnI gene mutation, Gly203Ser (designated cTnI-G203S), develop all the characteristic phenotypic features of FHC. To study the modifying effect of exercise in early disease, mice underwent swimming exercise at an early age prior to the development of the FHC phenotype. In non-transgenic and cTnI-wt mice, swimming resulted in a significant increase in left ventricular wall thickness and contractility on echocardiography, consistent with a physiological hypertrophic response to exercise. In contrast, cTnI-G203S mice showed no increase in these parameters, indicating an abnormal response to exercise. The lack of a physiological response to exercise may indicate an important novel mechanistic insight into the role of exercise in triggering adverse events in FHC.

A novel method for murine intrahepatic islet transplantation via cecal vein.

PubMed

Byun, Nari; Kim, Hyun-Je; Min, Byoung-Hoon; Shin, Jun-Seop; Yoon, Il-Hee; Kim, Jong-Min; Kim, Yong-Hee; Park, Chung-Gyu

2015-12-01

Islet transplantation is one of the most beneficial treatment modality to treat type 1 diabetic patients with frequent hypoglycemic unawareness. In clinical setting, human islets are infused via portal vein and are settled in the end-portal venules in the liver. However, mouse islets are transplanted into kidney subcapsule or liver through direct portal vein. These conventional transplantation methods have several drawbacks such as different physiological environments around the transplanted islets in kidney subcapsule from the liver and high mortality rate in direct portal vein approach. In this study, we introduced murine intrahepatic islet transplantation method via cecal vein to have the same surgical operation route in humans as well as guaranteeing low mortality rate after islet transplantation. With this protocol, consistent normoglycemia can be obtained in diabetic mice, while keeping operation-related mortality extremely low. This approach with easier accessibility and low mortality will make murine intrahepatic islet transplantation a useful model for studying immunological mechanisms such as strong innate and adaptive immune responses that occur in human islet transplantation. Copyright © 2015 Elsevier B.V. All rights reserved.

Molecular cloning of a murine homologue of membrane cofactor protein (CD46): preferential expression in testicular germ cells.

PubMed Central

Tsujimura, A; Shida, K; Kitamura, M; Nomura, M; Takeda, J; Tanaka, H; Matsumoto, M; Matsumiya, K; Okuyama, A; Nishimune, Y; Okabe, M; Seya, T

1998-01-01

Human membrane cofactor protein (MCP, CD46) has been suggested, although no convincing evidence has been proposed, to be a fertilization-associated protein, in addition to its primary functions as a complement regulator and a measles virus receptor. We have cloned a cDNA encoding the murine homologue of MCP. This cDNA showed 45% identity in deduced protein sequence and 62% identity in nucleotide sequence with human MCP. Its ectodomains were four short consensus repeats and a serine/threonine-rich domain, and it appeared to be a type 1 membrane protein with a 23-amino acid transmembrane domain and a short cytoplasmic tail. The protein expressed on Chinese hamster ovary cell transfectants was 47 kDa on SDS/PAGE immunoblotting, approximately 6 kDa larger than the murine testis MCP. It served as a cofactor for factor I-mediated inactivation of the complement protein C3b in a homologous system and, to a lesser extent, in a human system. Strikingly, the major message of murine MCP was 1.5 kb and was expressed predominantly in the testis. It was not detected in mice defective in spermatogenesis or with immature germ cells (until 23 days old). Thus, murine MCP may be a sperm-dominant protein the message of which is expressed selectively in spermatids during germ-cell differentiation. PMID:9461505

Identification and characterization of a silencer regulatory element in the 3'-flanking region of the murine CD46 gene.

PubMed Central

Nomura, M; Tsujimura, A; Begum, N A; Matsumoto, M; Wabiko, H; Toyoshima, K; Seya, T

2000-01-01

The murine membrane cofactor protein (CD46) gene is expressed exclusively in testis, in contrast to human CD46, which is expressed ubiquitously. To elucidate the mechanism of differential CD46 gene expression among species, we cloned entire murine CD46 genomic DNA and possible regulatory regions were placed in the flanking region of the luciferase reporter gene. The reporter gene assay revealed a silencing activity not in the promoter, but in the 3'-flanking region of the gene and the silencer-like element was identified within a 0.2-kb region between 0.6 and 0.8 kb downstream of the stop codon. This silencer-like element was highly similar to that of the pig MHC class-I gene. The introduction of a mutation into this putative silencer element of murine CD46 resulted in an abrogation of the silencing effect. Electrophoretic mobility-shift assay indicated the presence of the binding molecule(s) for this silencer sequence in murine cell lines and tissues. A size difference of the protein-silencer-element complex was observed depending upon the solubilizers used for preparation of the nuclear extracts. A mutated silencer sequence failed to interact with the binding molecules. The level of the binding factor was lower in the testicular germ cells compared with other organs. Thus the silencer element and its binding factor may play a role in transcriptional regulation of murine CD46 gene expression. These results imply that the effects of the CD46 silencer element encompass the innate immune and reproductive systems, and in mice may determine the testicular germ-cell-dominant expression of CD46. PMID:11023821

Efficacy of Posaconazole in Murine Experimental Sporotrichosis

PubMed Central

Fernández-Silva, Fabiola; Capilla, Javier; Mayayo, Emilio

2012-01-01

We developed a murine model of systemic sporotrichosis by using three strains of each of the two commonest species causing sporotrichosis, i.e., Sporothrix schenckii sensu stricto and Sporothrix brasiliensis, in order to evaluate the efficacy of posaconazole (PSC). The drug was administered at a dose of 2.5 or 5 mg/kg of body weight twice a day by gavage, and one group was treated with amphotericin B (AMB) as a control treatment. Posaconazole, especially at 5 mg/kg, showed good efficacy against all the strains tested, regardless of their MICs, as measured by prolonged survival, tissue burden reduction, and histopathology. PMID:22330929

Murine mesenchymal and embryonic stem cells express a similar Hox gene profile.

PubMed

Phinney, Donald G; Gray, Andrew J; Hill, Katy; Pandey, Amitabh

2005-12-30

Using degenerate oligonucleotide primers targeting the homeobox domain, we amplified by PCR and sequenced 723 clones from five murine cell populations and lines derived from embryonic mesoderm and adult bone marrow. Transcripts from all four vertebrate Hox clusters were expressed by the different populations. Hierarchical clustering of the data revealed that mesenchymal stem cells (MSCs) and the embryonic stem (ES) cell line D3 shared a similar Hox expression profile. These populations exclusively expressed Hoxb2, Hoxb5, Hoxb7, and Hoxc4, transcripts regulating self-renewal and differentiation of other stem cells. Additionally, Hoxa7 transcript quantified by real-time PCR strongly correlated (r2=0.89) with the number of Hoxa7 clones identified by sequencing, validating that data from the PCR screen reflects differences in Hox mRNA abundance between populations. This is the first study to catalogue Hox transcripts in murine MSCs and by comparative analyses identify specific Hox genes that may contribute to their stem cell character.

Cancer immunotherapeutic effects of novel CpG ODN in murine tumor model.

PubMed

Cho, Hyeon Cheol; Kim, Bo Hwan; Kim, Kyunghoon; Park, Ju Youn; Chang, Jae-Ho; Kim, Soo-Ki

2008-10-01

While CpG oligodeoxynucleotides (ODN) are excellent candidates for cancer immunotherapeutics, the numbers of usable CpG ODNs are limited in current clinical settings. To resolve this, we investigated whether novel CpG ODN (KSK-CpG) would be an effective immunotherapeutic in a murine tumor model by affecting in vivo and in vitro parameters, such as survival span, the number of tumor nodules, natural killer (NK) cell and cytotoxic T lymphocyte (CTL) activity and interleukin (IL)-6 or IL-12 cytokine release in splenocytes. We found that KSK-CpG was effective in the murine cancer model by way of prolonging survival span, reducing the number of tumor nodules, augmenting NK cell and CTL cytotoxicity, as well as evoking IL-6 and IL-12 cytokine release in splenocytes. Collectively, these data demonstrate that KSK-CpG is active against the highly malignant B16BL6 and EL4 tumor mouse model via innate immune augmentation.

Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models

PubMed Central

Lever, Teresa E.; Braun, Sabrina M.; Brooks, Ryan T.; Harris, Rebecca A.; Littrell, Loren L.; Neff, Ryan M.; Hinkel, Cameron J.; Allen, Mitchell J.; Ulsas, Mollie A.

2015-01-01

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research. Successful outcomes are dependent upon three critical components: test chambers that permit self-feeding while standing unrestrained in a confined space, recipes that mask the aversive taste/odor of commercially-available oral contrast agents, and a step-by-step test protocol that permits quantification of swallow physiology. Elimination of one or more of these components will have a detrimental impact on the study results. Moreover, the energy level capability of the fluoroscopy system will determine which swallow parameters can be investigated. Most research centers have high energy fluoroscopes designed for use with people and larger animals, which results in exceptionally poor image quality when testing mice and other small rodents. Despite this limitation, we have identified seven VFSS parameters that are consistently quantifiable in mice when using a high energy fluoroscope in combination with the new murine VFSS protocol. We recently obtained a low energy fluoroscopy system with exceptionally high imaging resolution and magnification capabilities that was designed for use with mice and other small rodents. Preliminary work using this new system, in combination with the new murine VFSS protocol, has identified 13 swallow parameters that are consistently quantifiable in mice, which is nearly double the number obtained using conventional (i.e., high energy) fluoroscopes. Identification of additional swallow parameters is expected as we optimize the capabilities of this new system. Results thus far demonstrate the utility of using a low energy fluoroscopy system to detect and quantify subtle changes in swallow physiology that may otherwise be overlooked when using high energy fluoroscopes to investigate murine disease models. PMID:25866882

Dynamic changes in high and low mammographic density human breast tissues maintained in murine tissue engineering chambers during various murine peripartum states and over time.

PubMed

Chew, G L; Huang, D; Huo, C W; Blick, T; Hill, P; Cawson, J; Frazer, H; Southey, M D; Hopper, J L; Henderson, M A; Haviv, I; Thompson, E W

2013-07-01

Mammographic density (MD) is a strong heritable risk factor for breast cancer, and may decrease with increasing parity. However, the biomolecular basis for MD-associated breast cancer remains unclear, and systemic hormonal effects on MD-associated risk is poorly understood. This study assessed the effect of murine peripartum states on high and low MD tissue maintained in a xenograft model of human MD. Method High and low MD human breast tissues were precisely sampled under radiographic guidance from prophylactic mastectomy specimens of women. The high and low MD tissues were maintained in separate vascularised biochambers in nulliparous or pregnant SCID mice for 4 weeks, or mice undergoing postpartum involution or lactation for three additional weeks. High and low MD biochamber material was harvested for histologic and radiographic comparisons during various murine peripartum states. High and low MD biochamber tissues in nulliparous mice were harvested at different timepoints for histologic and radiographic comparisons. Results High MD biochamber tissues had decreased stromal (p = 0.0027), increased adipose (p = 0.0003) and a trend to increased glandular tissue areas (p = 0.076) after murine postpartum involution. Stromal areas decreased (p = 0.042), while glandular (p = 0.001) and adipose areas (p = 0.009) increased in high MD biochamber tissues during lactation. A difference in radiographic density was observed in high (p = 0.0021) or low MD biochamber tissues (p = 0.004) between nulliparous, pregnant and involution groups. No differences in tissue composition were observed in high or low MD biochamber tissues maintained for different durations, although radiographic density increased over time. Conclusion High MD biochamber tissues had measurable histologic changes after postpartum involution or lactation. Alterations in radiographic density occurred in biochamber tissues between different peripartum states and over time. These findings

Lactobacillus delbrueckii subsp. lactis (strain CIDCA 133) stimulates murine macrophages infected with Citrobacter rodentium.

PubMed

Hugo, Ayelén A; Rolny, Ivanna S; Romanin, David; Pérez, Pablo F

2017-03-01

Citrobacter rodentium is a specific murine enteropathogen which causes diarrheal disease characterized by colonic hyperplasia and intestinal inflammation. Recruitment of neutrophils and macrophages constitute a key step to control the infection. Since modulation of the activity of professional phagocytic cells could contribute to improve host´s defences against C. rodentium, we investigated the effect of Lactobacillus delbrueckii subsp. lactis (strain CIDCA 133) on the interaction between murine macrophages (RAW 264.7) and C. rodentium. Phagocytosis, surface molecules and inducible nitric oxide synthase (iNOs) expression were determined by flow cytometry. Reactive oxygen species (ROS) were assessed by fluorescence microscopy. The presence of lactobacilli increased phagocytosis of C. rodentium whereas C. rodentium had no effect on lactobacilli internalization. Survival of internalized C. rodentium diminished when strain CIDCA 133 was present. CD-86, MHCII, iNOs expression and nitrite production were increased when C. rodentium and lactobacilli were present even though strain CIDCA 133 alone had no effect. Strain CIDCA 133 led to a strong induction of ROS activity which was not modified by C. rodentium. Lactobacillus delbrueckii subsp. lactis (strain CIDCA 133) is able to increase the activation of murine macrophages infected with C. rodentium. The sole presence of lactobacilli is enough to modify some stimulation markers (e.g. ROS induction) whereas other markers require the presence of both bacteria; thus, indicating a synergistic effect.

A pancreas specificity results from the combination of polyomavirus and Moloney murine leukemia virus enhancer.

PubMed Central

Rochford, R; Campbell, B A; Villarreal, L P

1987-01-01

An infectious recombinant polyomavirus was constructed in which a regulatory region of its genome, the B enhancer region (nucleotides 5128-5265) has been replaced with the 72- or 73-base-pair repeat enhancer from the Moloney murine leukemia virus genome. We show that this recombinant polyomavirus displays a strong tissue specificity for the pancreas of mice. This organ was not permissive for either the parental polyomavirus, which is predominantly kidney and salivary gland specific, or the Moloney murine leukemia virus, which is lymphotropic. This result indicated that tissue specificity can be achieved by a combination of apparently modular elements. Some of the implications of a modular mechanism of tissue specificity are considered. Images PMID:3025873

Differential roles for pathogenicity islands SPI-13 and SPI-8 in the interaction of Salmonella Enteritidis and Salmonella Typhi with murine and human macrophages.

PubMed

Espinoza, Rodrigo A; Silva-Valenzuela, Cecilia A; Amaya, Fernando A; Urrutia, Ítalo M; Contreras, Inés; Santiviago, Carlos A

2017-02-15

Salmonella pathogenicity island (SPI)-13 is conserved in many serovars of S. enterica, including S. Enteritidis, S. Typhimurium and S. Gallinarum. However, it is absent in typhoid serovars such as S. Typhi and Paratyphi A, which carry SPI-8 at the same genomic location. Because the interaction with macrophages is a critical step in Salmonella pathogenicity, in this study we investigated the role played by SPI-13 and SPI-8 in the interaction of S. Enteritidis and S. Typhi with cultured murine (RAW264.7) and human (THP-1) macrophages. Our results showed that SPI-13 was required for internalization of S. Enteritidis in murine but not human macrophages. On the other hand, SPI-8 was not required for the interaction of S. Typhi with human or murine macrophages. Of note, the presence of an intact copy of SPI-13 in a S. Typhi mutant carrying a deletion of SPI-8 did not improve its ability to be internalized by, or survive in human or murine macrophages. Altogether, our results point out to different roles for SPI-13 and SPI-8 during Salmonella infection. While SPI-13 contributes to the interaction of S. Enteritidis with murine macrophages, SPI-8 is not required in the interaction of S. Typhi with murine or human macrophages. We hypothesized that typhoid serovars have lost SPI-13 and maintained SPI-8 to improve their fitness during another phase of human infection.

Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kusabuka, Hotaka; Fujiwara, Kento; Tokunaga, Yusuke

Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period ofmore » pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. - Highlights: • We established highly efficient gene transduction protocols for murine T cells. • CD8{sup +} CAR-T cells had antigen-specific cytotoxic activity. • CD4{sup +} CAR-T cells secreted multiple cytokines by antigen stimulation. • This finding can contribute to the development of T-cell biology and immunotherapy.« less

Activation of DNA damage repair pathways by murine polyomavirus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heiser, Katie; Nicholas, Catherine; Garcea, Robert

Nuclear replication of DNA viruses activates DNA damage repair (DDR) pathways, which are thought to detect and inhibit viral replication. However, many DNA viruses also depend on these pathways in order to optimally replicate their genomes. We investigated the relationship between murine polyomavirus (MuPyV) and components of DDR signaling pathways including CHK1, CHK2, H2AX, ATR, and DNAPK. We found that recruitment and retention of DDR proteins at viral replication centers was independent of H2AX, as well as the viral small and middle T-antigens. Additionally, infectious virus production required ATR kinase activity, but was independent of CHK1, CHK2, or DNAPK signaling.more » ATR inhibition did not reduce the total amount of viral DNA accumulated, but affected the amount of virus produced, indicating a defect in virus assembly. These results suggest that MuPyV may utilize a subset of DDR proteins or non-canonical DDR signaling pathways in order to efficiently replicate and assemble. -- Highlights: •Murine polyomavirus activates and recruits DNA damage repair (DDR) proteins to replication centers. •Large T-antigen mediates recruitment of DDR proteins to viral replication centers. •Inhibition or knockout of CHK1, CHK2, DNA-PK or H2AX do not affect viral titers. •Inhibition of ATR activity reduces viral titers, but not viral DNA accumulation.« less

The accessory gene regulator (agr) controls Staphylococcus aureus virulence in a murine intracranial abscesses model.

PubMed

Gong, Jian; Li, Dongzhi; Yan, Jun; Liu, Yu; Li, Di; Dong, Jie; Gao, Yaping; Sun, Tao; Yang, Guang

2014-01-01

Intracranial abscesses are associated with high mortality. Staphylococcus aureus is one of the main pathogens that cause intracranial infection. Until now, there is no report to identify the key effectors of S. aureus during the intracranial infection. The murine intracranial abscesses model induced by S. aureus was constructed. The vital sign and survival rate of mice were observed to evaluate the infection. Histological examination was used to diagnose the pathological alterations of mouse tissues. The sensitivity of S. aureus to whole blood was evaluated by whole-blood killing assay. In murine intracranial abscesses model, it was shown that the mortality caused by the accessory gene regulator (agr) locus deficient strain was significant decreased compared with its parent strain. Moreover, we found that RNAIII, the effector of agr system, was essential for the intracranial infection caused by S. aureus. In the further investigation, it was shown that restoration the expression of α-toxin in agr deficient strain could partially recover the mortality in the murine intracranial abscesses model. Our data suggested that the agr system of S. aureus is an important virulence determinant in the induction and mortality of intracranial abscesses in mice. Copyright © 2014 Elsevier Editora Ltda. All rights reserved.

5-HT4 receptors facilitate cholinergic neurotransmission throughout the murine gastrointestinal tract.

PubMed

Pauwelyn, V; Lefebvre, R A

2017-08-01

In the gastrointestinal tract of several species, facilitating 5-HT 4 receptors were proposed on myenteric cholinergic neurons innervating smooth muscle by in vitro study of the effect of the selective 5-HT 4 receptor agonist prucalopride on submaximal cholinergic contractions. This was not yet established in the murine gastrointestinal tract. In circular smooth muscle strips from murine fundus, jejunum and colon, contractions were induced by electrical field stimulation in the presence of guanethidine, L-NAME and for colon also MRS 2500. Submaximal contractions were induced to study the influence of prucalopride. Electrical field stimulation at reduced voltage induced reproducible submaximal neurogenic and cholinergic contractions as the contractions were abolished by tetrodotoxin and atropine. Hexamethonium had no systematic inhibitory effect but mecamylamine reduced the responses, suggesting that part of the cholinergic response is due to activation of preganglionic neurons. Prucalopride concentration-dependently increased the submaximal cholinergic contractions in the three tissue types, reaching maximum from 0.03 μmol/L onwards. The facilitation in the different series with 0.03 μmol/L prucalopride ranged from 41% to 104%, 30% to 76% and 24% to 74% in fundus, jejunum, and colon, respectively. The effect of 0.03 μmol/L prucalopride was concentration-dependently inhibited by GR 113808. In the murine gastrointestinal tract, activation of 5-HT 4 receptors with prucalopride enhances cholinergic contractions, illustrating facilitation of myenteric cholinergic neurotransmission. The degree of enhancement with prucalopride is of similar magnitude as previously reported in other species, but the effective concentrations are lower than those needed in the gastrointestinal tract of other species. © 2017 John Wiley & Sons Ltd.

Expression and purification of soluble murine CD40L monomers and polymers in yeast Pichia pastoris

PubMed Central

Hermanrud, Christina E.; Lucas, Carrie L.; Sykes, Megan; Huang, Christene A.; Wang, Zhirui

2010-01-01

The anti-murine CD40L monoclonal antibody MR1 has been widely used in immunology research to block the CD40-CD40L interaction for induction of transplantation tolerance and to abrogate autoimmune diseases. The availability of recombinant CD40L with high binding capacity for MR1 would provide a valuable immunological research tool. In this study, we constructed the single chain murine soluble CD40L monomer, dimer, trimer and successfully expressed them in yeast Pichia pastoris under the control of the alcohol oxidase promoter. The secreted single chain murine soluble CD40L monomers, dimers, and trimers were initially enriched through histidine tag capture by Ni-Sepharose 6 fast flow resin and further purified on a cation exchange resin. Purity reached more than 95% for the monomer and dimer forms and more than 90% for the trimer. Protein yield following purification was 16 mg/L for the monomer and dimer, and 8 mg/L for the trimer. ELISA analysis demonstrated that the CD40L dimers and trimers correctly folded in conformations exposing the MR1 antigenic determinant. PMID:21074618

Simultaneous Targeting of RGD-Integrins and Dual Murine Double Minute Proteins in Glioblastoma Multiforme.

PubMed

Merlino, Francesco; Daniele, Simona; La Pietra, Valeria; Di Maro, Salvatore; Di Leva, Francesco Saverio; Brancaccio, Diego; Tomassi, Stefano; Giuntini, Stefano; Cerofolini, Linda; Fragai, Marco; Luchinat, Claudio; Reichart, Florian; Cavallini, Chiara; Costa, Barbara; Piccarducci, Rebecca; Taliani, Sabrina; Da Settimo, Federico; Martini, Claudia; Kessler, Horst; Novellino, Ettore; Marinelli, Luciana

2018-05-18

In the fight against Glioblastoma Multiforme, recent literature data have highlighted that integrin α5β1 and p53 are part of convergent pathways in the control of glioma apoptosis. This observation prompted us to seek a molecule able to simultaneously modulate both target families. Analyzing the results of a previous virtual screening against murine double minute 2 protein (MDM2), we envisaged that Arg-Gly-Asp (RGD)-mimetic molecules could be inhibitors of MDM2/4. Herein we present the discovery of compound 7, which inhibits both MDM2/4 and α5β1/αvβ3 integrins. A lead optimization campaign was carried out on 7 with aim to preserve the activities on integrins while improving those on MDM proteins. Compound 9 turned out to be a potent MDM2/4, and α5β1/αvβ3 blocker. In p53-wild type glioma cells, 9 arrested cell cycle and proliferation and strongly reduced cell invasiveness, emerging as the first molecule of a novel class of integrin/MDM inhibitors, which might be especially useful in subpopulations of patients with glioblastoma expressing a functional p53 concomitantly with a high level of α5β1 integrin.

Expression and secretion of cathelicidin antimicrobial peptides in murine mammary glands and human milk.

PubMed

Murakami, Masamoto; Dorschner, Robert A; Stern, Lauren J; Lin, Kenneth H; Gallo, Richard L

2005-01-01

Mammalian milk possesses inherent antimicrobial properties that have been attributed to several diverse molecules. Recently, antimicrobial peptides that belong to the cathelicidin gene family have been found to be important to the mammalian immune response. This antimicrobial is expressed in several tissues and increased in neonatal skin, possibly to compensate for an immature adaptive immune response. We hypothesized that the mammary gland could produce and secrete cathelicidin onto the epithelial surface and into milk. Human cathelicidin hCAP18/LL-37 mRNA was detected in human milk cells by PCR. Quantitative real-time PCR demonstrated an increase in relative expression levels at 30 and 60 d after parturition. Immunohistochemistry of mouse breast tissue identified the murine cathelicidin-related antimicrobial peptide in lobuloacinar and ductules. Western blot analysis of human milk showed that LL-37 was secreted and present in the mature peptide form. The antimicrobial activity of LL-37 against Staphylococcus aureus, group A Streptococcus, and enteroinvasive Escherichia coli O29 in the human milk ionic environment was confirmed by solution colony-forming assay using synthetic peptide. These results indicate that cathelicidin is secreted in mammary gland and human milk, has antimicrobial activity against both Gram-positive and Gram-negative bacteria, and can contribute to the anti-infectious properties of milk.

A Murine Herpesvirus Closely Related to Ubiquitous Human Herpesviruses Causes T-Cell Depletion

PubMed Central

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

2017-01-01

ABSTRACT The human roseoloviruses human herpesvirus 6A (HHV-6A), HHV-6B, and HHV-7 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 174-kb genome sequence containing 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, HHV-6A, HHV-6B, and HHV-7, than to 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 HHV-6A, HHV-6B, and HHV-7. 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 herpesviruses 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

A Murine Herpesvirus Closely Related to Ubiquitous Human Herpesviruses Causes T-Cell Depletion.

PubMed

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

2017-05-01

The human roseoloviruses human herpesvirus 6A (HHV-6A), HHV-6B, and HHV-7 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 174-kb genome sequence containing 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, HHV-6A, HHV-6B, and HHV-7, than to 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 HHV-6A, HHV-6B, and HHV-7. 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 herpesviruses 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

Newly Identified TLR9 Stimulant, M6-395 Is a Potent Polyclonal Activator for Murine B Cells.

PubMed

Park, Mi-Hee; Jung, Yu-Jin; Kim, Pyeung-Hyeun

2012-02-01

Toll-like receptors (TLRs) have been extensively studied in recent years. However, functions of these molecules in murine B cell biology are largely unknown. A TLR4 stimulant, LPS is well known as a powerful polyclonal activator for murine B cells. In this study, we explored the effect of a murine TLR9 stimulant, M6-395 (a synthetic CpG ODNs) on B cell proliferation and Ig production. First, M6-395 was much more potent than LPS in augmenting B cell proliferation. As for Ig expression, M6-395 facilitated the expression of both TGF-β1-induced germ line transcript α (GLTα) and IL-4-induced GLTγ1 as levels as those by LPS and Pam3CSK4 (TLR1/2 agonist) : a certain Ig GLT expression is regarded as an indicative of the corresponding isotype switching recombination. However, IgA and IgG1 secretion patterns were quite different--these Ig isotype secretions by M6-395 were much less than those by LPS and Pam3CSK4. Moreover, the increase of IgA and IgG1 production by LPS and Pam3CSK4 was virtually abrogated by M6-395. The same was true for the secretion of IgG3. We found that this unexpected phenomena provoked by M6-395 is attributed, at least in part, to its excessive mitogenic nature. Taken together, these results suggest that M6-395 can act as a murine polyclonal activator but its strong mitogenic activity is unfavorable to Ig isotype switching.

The SRL peptide of Rhesus Rotavirus VP4 protein governs cholangiocyte infection and the murine model of biliary atresia

PubMed Central

Mohanty, Sujit K.; Donnelly, Bryan; Lobeck, Inna; Walther, Ashley; Dupree, Phylicia; Coots, Abigail; Meller, Jaroslaw; McNeal, Monica; Sestak, Karol; Tiao, Greg

2016-01-01

Biliary atresia (BA) is a neonatal obstructive cholangiopathy which progresses to end stage liver disease, often requiring transplantation. The murine model of BA, employing rhesus rotavirus (RRV), parallels human disease and has been used to elucidate mechanistic aspects of a virus induced biliary cholangiopathy. We previously reported that RRV VP4 gene plays an integral role in activating the immune system and induction of BA. Utilizing rotavirus binding and blocking assays, this study elucidated how RRV VP4 protein governs cholangiocyte susceptibility to infection both in vitro and in vivo in the murine model of BA. We identified the amino acid sequence on VP4 and its cholangiocyte binding protein, finding that the sequence is specific to those rotavirus strains which cause an obstructive cholangiopathy. Pretreatment of murine and human cholangiocytes with this VP4 derived peptide (TRTRVSRLY), significantly reduced RRV’s ability to bind and infect the cells. However, the peptide did not block cholangiocyte binding of TUCH and Ro1845, strains which do not induce murine BA. The SRL sequence within TRTRVSRLY is required for cholangiocyte binding and viral replication. The cholangiocyte membrane protein bound by SRL was found to be Hsc70. Inhibition of Hsc70 by siRNAs reduced RRV’s ability to infect cholangiocytes. This virus-cholangiocyte interaction is also seen in vivo in the murine model of BA, where inoculation of mice with TRTRVSRLY peptide significantly reduced symptoms and mortality in RRV-injected mice. Conclusion The tri-peptide SRL on RRV VP4 binds to the cholangiocyte membrane protein Hsc70 defining a novel binding site governing VP4 attachment. Investigations are underway to determine the cellular response following this interaction to understand how it contributes to the pathogenesis of BA. PMID:27859498

Mouse cysteine-rich secretory protein 4 (CRISP4): a member of the Crisp family exclusively expressed in the epididymis in an androgen-dependent manner.

PubMed

Jalkanen, Jenni; Huhtaniemi, Ilpo; Poutanen, Matti

2005-05-01

The final maturation of spermatozoa produced in the testis takes place during their passage through the epididymis. In this process, the proteins secreted into the epididymal lumen along with changes in the pH and salt composition of the epididymal fluid cause several biochemical changes and remodeling of the sperm plasma membrane. The Crisp family is a group of cysteine-rich secretory proteins that previously consisted of three members, one of which-CRISP1-is an epididymal protein shown to attach to the sperm surface in the epididymal lumen and to inhibit gamete membrane fusion. In the present paper, we introduce a new member of the Crisp protein family, CRISP4. The new gene was discovered through in silico analysis of the epididymal expressed sequence tag library deposited in the UniGene database. The peptide sequence of CRISP4 has a signal sequence suggesting that it is secreted into the epididymal lumen and might thus interact with sperm. Unlike the other members of the family, Crisp4 is located on chromosome 1 in a cluster of genes encoding for cysteine-rich proteins. Crisp4 is expressed in the mouse exclusively in epithelial cells of the epididymis in an androgen-dependent manner, and the expression of the gene starts at puberty along with the onset of sperm maturation. The identified murine CRISP4 peptide has high homology with human CRISP1, and the homology is higher than that between murine and human CRISP1, suggesting that CRISP4 represents the mouse counterpart of human CRISP1 and could have similar effects on sperm membrane as mouse and human CRISP1.

Flexibility damps macromolecular crowding effects on protein folding dynamics: Application to the murine prion protein (121-231)

NASA Astrophysics Data System (ADS)

Bergasa-Caceres, Fernando; Rabitz, Herschel A.

2014-01-01

A model of protein folding kinetics is applied to study the combined effects of protein flexibility and macromolecular crowding on protein folding rate and stability. It is found that the increase in stability and folding rate promoted by macromolecular crowding is damped for proteins with highly flexible native structures. The model is applied to the folding dynamics of the murine prion protein (121-231). It is found that the high flexibility of the native isoform of the murine prion protein (121-231) reduces the effects of macromolecular crowding on its folding dynamics. The relevance of these findings for the pathogenic mechanism are discussed.

VARIATION IN MORBIDITY AND MORTALITY OF MURINE TYPHUS INFECTION IN MICE WITH CHANGES IN THE ENVIRONMENTAL TEMPERATURE

PubMed Central

Moragues, Vicente; Pinkerton, Henry

1944-01-01

Murine typhus rickettsiae injected intraperitoneally in mice of the dba strain caused a uniformly fatal rickettsial peritonitis if the animals were kept at a room temperature ranging from 65–73°F. or from 70–80°F. With an environmental temperature range of 85–98°F., a mortality of less than 25 per cent was observed. By utilizing different strains of mice and controlling the environmental temperature, conditions may be created under which murine typhus will have any desired degree of mortality. Such conditions have obvious advantages for the evaluation of therapeutic measures in typhus infection. PMID:19871351

Examination of West Nile Virus Neuroinvasion and Neuropathogenesis in the Central Nervous System of a Murine Model.

PubMed

Sultana, Hameeda

2016-01-01

West Nile virus (WNV) is a neurotropic virus that causes inflammation and neuronal loss in the Central Nervous System leading to encephalitis and death. In this chapter, detailed methods to detect WNV in the murine brain tissue by quantitative real-time polymerase chain reaction and viral plaque assays are described. Determination of WNV neuropathogenesis by Hematoxylin and Eosin staining and immunohistochemical procedures are provided. In addition, TUNEL assays to determine neuronal loss during WNV neuropathogenesis are discussed in detail. Collectively, the methods mentioned in this chapter provide an overview to understand neuroinvasion and neuropathogenesis in a murine model of WNV infection.

2-O, 3-O desulfated heparin mitigates murine chemotherapy- and radiation-induced thrombocytopenia

PubMed Central

Tkaczynski, Elizabeth; Arulselvan, Abinaya; Tkaczynski, John; Avery, Stephen; Xiao, Liqing; Torok-Storb, Beverly; Abrams, Kraig; Rao, Narayanam V.; Johnson, Gregory; Poncz, Mortimer

2018-01-01

Thrombocytopenia is a significant complication of chemotherapy and radiation therapy. Platelet factor 4 (PF4; CXCL4) is a negative paracrine of megakaryopoiesis. We have shown that PF4 levels are inversely related to steady-state platelet counts, and to the duration and severity of chemotherapy- and radiation-induced thrombocytopenia (CIT and RIT, respectively). Murine studies suggest that blocking the effect of PF4 improves megakaryopoiesis, raising nadir platelet counts and shortening the time to platelet count recovery. We examined the ability of 2-O, 3-O desulfated heparin (ODSH), a heparin variant with little anticoagulant effects, to neutralize PF4’s effects on megakaryopoiesis. Using megakaryocyte colony assays and liquid cultures, we show that ODSH restored megakaryocyte proliferation in PF4-treated Cxcl4−/− murine and human CD34+-derived megakaryocyte cultures (17.4% megakaryocyte colonies, P < .01 compared with PF4). In murine CIT and RIT models, ODSH, started 24 hours after injury, was examined for the effect on hematopoietic recovery demonstrating higher platelet count nadirs (9% ± 5% treated vs 4% ± 4% control) and significantly improved survival in treated animals (73% treated vs 36% control survival). Treatment with ODSH was able to reduce intramedullary free PF4 concentrations by immunohistochemical analysis. In summary, ODSH mitigated CIT and RIT in mice by neutralizing the intramedullary negative paracrine PF4. ODSH, already in clinical trials in humans as an adjuvant to chemotherapy, may be an important, clinically relevant therapeutic for CIT and RIT. PMID:29599195

Murine homeobox-containing gene, Msx-1: analysis of genomic organization, promoter structure, and potential autoregulatory cis-acting elements.

PubMed

Kuzuoka, M; Takahashi, T; Guron, C; Raghow, R

1994-05-01

Detailed molecular organization of the coding and upstream regulatory regions of the murine homeodomain-containing gene, Msx-1, is reported. The protein-encoding portion of the gene is contained in two exons, 590 and 1214 bp in length, separated by a 2107-bp intron; the homeodomain is located in the second exon. The two-exon organization of the murine Msx-1 gene resembles a number of other homeodomain-containing genes. The 5'-(GTAAGT) and 3'-(CCCTAG) splicing junctions and the mRNA polyadenylation signal (UAUAA) of the murine Msx-1 gene are also characteristic of other vertebrate genes. By nuclease protection and primer extension assays, the start of transcription of the Msx-1 gene was located 256 bp upstream of the first AUG. Computer analysis of the promoter proximal 1280-bp sequence revealed a number of potentially important cis-regulatory sequences; these include the recognition elements for Ap-1, Ap-2, Ap-3, Sp-1, a possible binding site for RAR:RXR, and a number of TCF-1 consensus motifs. Importantly, a perfect reverse complement of (C/G)TTAATTG, which was recently shown to be an optimal binding sequence for the homeodomain of Msx-1 protein (K.M. Catron, N. Iler, and C. Abate (1993) Mol. Cell. Biol. 13:2354-2365), was also located in the murine Msx-1 promoter. Binding of bacterially expressed Msx-1 homeodomain polypeptide to Msx-1-specific oligonucleotide was experimentally demonstrated, raising a distinct possibility of autoregulation of this developmentally regulated gene.

Lessons learned: Optimization of a murine small bowel resection model

PubMed Central

Taylor, Janice A.; Martin, Colin A.; Nair, Rajalakshmi; Guo, Jun; Erwin, Christopher R.; Warner, Brad W.

2008-01-01

Background/Purpose Central to the use of murine models of disease is the ability to derive reproducible data. The purpose of this study was to determine factors contributing to variability in our murine model of small bowel resection (SBR). Methods Male C57Bl/6 mice were randomized to sham or 50% SBR. The effect of housing type (pathogen-free versus standard housing), nutrition (reconstituted powder versus tube feeding formulation), and correlates of intestinal morphology with gene expression changes were investigated Multiple linear regression modeling or one-way ANOVA was used for data analysis. Results Pathogen-free mice had significantly shorter ileal villi at baseline and demonstrated greater villus growth after SBR compared to mice housed in standard rooms. Food type did not affect adaptation. Gene expression changes were more consistent and significant in isolated crypt cells that demonstrated adaptive growth when compared with crypts that did not deepen after SBR. Conclusion Maintenance of mice in pathogen-free conditions and restricting gene expression analysis to individual animals exhibiting morphologic adaptation enhances sensitivity and specificity of data derived from this model. These refinements will minimize experimental variability and lead to improved understanding of the complex process of intestinal adaptation. PMID:18558176

Modulation of proinflammatory NF-κB signaling by ectromelia virus in RAW 264.7 murine macrophages.

PubMed

Struzik, Justyna; Szulc-Dąbrowska, Lidia; Papiernik, Diana; Winnicka, Anna; Niemiałtowski, Marek

2015-09-01

Macrophages are antigen-presenting cells (APCs) that play a crucial role in the innate immune response and may be involved in both clearance and spread of viruses. Stimulation of macrophages via Toll-like receptors (TLRs) results in activation of nuclear factor κB (NF-κB) and synthesis of proinflammatory cytokines. In this work, we show modulation of proinflammatory NF-κB signaling by a member of the family Poxviridae, genus Orthopoxvirus--ectromelia virus (ECTV)--in RAW 264.7 murine macrophages. ECTV interfered with p65 NF-κB nuclear translocation induced by TLR ligands such as lipopolysaccharide (LPS) (TLR4), polyinosinic-polycytidylic acid (poly(I:C)) (TLR3) and diacylated lipopeptide Pam2CSK4 (TLR2/6). We observed that ECTV modulates phosphorylation of Ser32 of inhibitor of κB (IκBα) and Ser536 of p65. Interference of ECTV with TLR signaling pathways implied that proinflammatory cytokine synthesis was inhibited. Our studies provide new insights into the strategies of proinflammatory signaling modulation by orthopoxviruses during their replication cycle in immune cells. Understanding important immune interactions between viral pathogens and APCs might contribute to the identification of drug targets and the development of vaccines.

Corn silk induced cyclooxygenase-2 in murine macrophages.

PubMed

Kim, Kyung A; Shin, Hyun-Hee; Choi, Sang Kyu; Choi, Hye-Seon

2005-10-01

Stimulation of murine macrophages with corn silk induced cyclooxygenase (COX)-2 with secretion of PGE2. Expression of COX-2 was inhibited by pyrolidine dithiocarbamate (PDTC), and increased DNA binding by nuclear factor kappa B (NF-kappaB), indicating that COX-2 induction proceeds also via the NF-kappaB signaling pathway. A specific inhibitor of COX-2 decreased the expression level of inducible nitric oxide synthase (iNOS) stimulated by corn silk. PGE2 elevated the expression level of iNOS, probably via EP2 and EP4 receptors on the surface of the macrophages.

Cloning and characterization of murine fanconi anemia group A gene: Fanca protein is expressed in lymphoid tissues, testis, and ovary.

PubMed

van de Vrugt, H J; Cheng, N C; de Vries, Y; Rooimans, M A; de Groot, J; Scheper, R J; Zhi, Y; Hoatlin, M E; Joenje, H; Arwert, F

2000-04-01

Fanconi anemia (FA) is an autosomal recessive disorder in humans characterized by bone marrow failure, cancer predisposition, and cellular hypersensitivity to cross-linking agents such as mitomycin C and diepoxybutane. FA genes display a caretaker function essential for maintenance of genomic integrity. We have cloned the murine homolog of FANCA, the gene mutated in the major FA complementation group (FA-A). The full-length mouse Fanca cDNA consists of 4503 bp and encodes a protein with a predicted molecular weight of 161 kDa. The deduced Fanca mouse protein shares 81% amino acid sequence similarity and 66% identity with the human protein. The nuclear localization signal and partial leucine zipper consensus motifs found in the human FANCA protein were also present in the murine homolog. In spite of the species difference, the murine Fanca cDNA was capable of correcting the cross-linker sensitive phenotype of human FA-A cells, suggesting functional conservation. Based on Northern as well as Western blots, Fanca was mainly expressed in lymphoid tissues, testis, and ovary. This expression pattern correlates with some of the clinical symptoms observed in FA patients. The availability of the murine Fanca cDNA now allows the gene to be studied in experimental mouse models.

ROLE OF COPPER,ZINC-SUPEROXIDE DISMUTASE IN CATALYZING NITROTYROSINE FORMATION IN MURINE LIVER

USDA-ARS?s Scientific Manuscript database

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 ...

COMPARATIVE TOXICITY OF DIFFERENT EMISSION PARTICLES IN MURINE PULMONARY EPITHELIAL CELLS AND MACROPHAGES

EPA Science Inventory

Comparative Toxicity of Different Emission Particles in Murine Pulmonary Epithelial Cells and Macrophages. T Stevens1, M Daniels2, P Singh2, M I Gilmour2. 1 UNC, Chapel Hill 27599 2Experimental Toxicology Division, NHEERL, RTP, NC 27711

Epidemiological studies have shown ...

Sensitivity to. gamma. rays of avian sarcoma and murine leukemia viruses. [/sup 60/Co, uv

DOE Office of Scientific and Technical Information (OSTI.GOV)

Toyoshima, K.; Niwa, O.; Yutsudo, M.

1980-09-01

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 strongmore » repair function, possibly connected with reverse transcriptase activity.« less

Murine AIDS Protects Mice Against Experimental Cerebral Malaria: Down-Regulation by Interleukin 10 a T-Helper Type 1 CD4^+ Cell-Mediated Pathology

NASA Astrophysics Data System (ADS)

Eckwalanga, Michel; Marussig, Myriam; Dias Tavares, Marisa; Bouanga, Jean Claude; Hulier, Elisabeth; Henriette Pavlovitch, Jana; Minoprio, Paola; Portnoi, Denis; Renia, Laurent; Mazier, Dominique

1994-08-01

The retrovirus LP-BM5 murine leukemia virus induces murine AIDS in C57BL/6 mice that has many similarities with human AIDS; Plasmodium berghei ANKA causes experimental cerebral malaria in the same strain of mice. The outcome of malaria infection was studied in mice concurrently infected with the two pathogens. The retrovirus significantly reduced the gravity of the neurological manifestations associated with Plasmodium berghei ANKA infection. The protection against experimental cerebral malaria induced by murine AIDS increased with duration of viral infection and, hence, with the severity of the immunodeficiency. Interleukin 10, principally from splenic T cells, was shown to play a crucial role in this protection.

Identification of the Muscarinic Acetylcholine Receptor Subtype Mediating Cholinergic Vasodilation in Murine Retinal Arterioles

PubMed Central

Sniatecki, Jan J.; Goloborodko, Evgeny; Steege, Andreas; Zavaritskaya, Olga; Vetter, Jan M.; Grus, Franz H.; Patzak, Andreas; Wess, Jürgen; Pfeiffer, Norbert

2011-01-01

Purpose. To identify the muscarinic acetylcholine receptor subtype that mediates cholinergic vasodilation in murine retinal arterioles. Methods. Muscarinic receptor gene expression was determined in murine retinal arterioles using real-time PCR. To assess the functional relevance of muscarinic receptors for mediating vascular responses, retinal vascular preparations from muscarinic receptor–deficient mice were studied in vitro. Changes in luminal arteriole diameter in response to muscarinic and nonmuscarinic vasoactive substances were measured by video microscopy. Results. Only mRNA for the M3 receptor was detected in retinal arterioles. Thus, M3 receptor–deficient mice (M3R−/−) and respective wild-type controls were used for functional studies. Acetylcholine concentration-dependently dilated retinal arterioles from wild-type mice. In contrast, vasodilation to acetylcholine was almost completely abolished in retinal arterioles from M3R−/− mice, whereas responses to the nitric oxide (NO) donor nitroprusside were retained. Carbachol, an acetylcholinesterase-resistant analog of acetylcholine, also evoked dilation in retinal arterioles from wild-type, but not from M3R−/−, mice. Vasodilation responses from wild-type mice to acetylcholine were negligible after incubation with the non–subtype-selective muscarinic receptor blocker atropine or the NO synthase inhibitor Nω-nitro-l-arginine methyl ester, and were even reversed to contraction after endothelial damage with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Conclusions. These findings provide evidence that endothelial M3 receptors mediate cholinergic vasodilation in murine retinal arterioles via activation of NO synthase. PMID:21873683

Accessing the genomic effects of naked nanoceria in murine neuronal cells.

PubMed

Lee, Tin-Lap; Raitano, Joan M; Rennert, Owen M; Chan, Siu-Wai; Chan, Wai-Yee

2012-07-01

Cerium oxide nanoparticles (nanoceria) are engineered nanoparticles whose versatility is due to their unique redox properties. We and others have demonstrated that naked nanoceria can act as antioxidants to protect cells against oxidative damage. Although the redox properties may be beneficial, the genome-wide effects of nanoceria on gene transcription and associated biological processes remain elusive. Here we applied a functional genomic approach to examine the genome-wide effects of nanoceria on global gene transcription and cellular functions in mouse neuronal cells. Importantly, we demonstrated that nanoceria induced chemical- and size-specific changes in the murine neuronal cell transcriptome. The nanoceria contributed more than 83% of the population of uniquely altered genes and were associated with a unique spectrum of genes related to neurological disease, cell cycle control, and growth. These observations suggest that an in-depth assessment of potential health effects of naked nanoceria and other naked nanoparticles is both necessary and imminent. Cerium oxide nanoparticles are important antioxidants, with potential applications in neurodegenerative conditions. This team of investigators demonstrated the genomic effects of nanoceria, showing that it induced chemical- and size-specific changes in the murine neuronal cell transcriptome. Published by Elsevier Inc.

Lactobacillus reuteri 100-23 Modulates Urea Hydrolysis in the Murine Stomach

PubMed Central

Wilson, Charlotte M.; Loach, Diane; Lawley, Blair; Bell, Tracey; Sims, Ian M.; O'Toole, Paul W.; Zomer, Aldert

2014-01-01

Comparisons of in vivo (mouse stomach) and in vitro (laboratory culture) transcriptomes of Lactobacillus reuteri strain 100-23 were made by microarray analysis. These comparisons revealed the upregulation of genes associated with acid tolerance, including urease production, in the mouse stomach. Inactivation of the ureC gene reduced the acid tolerance of strain 100-23 in vitro, and the mutant was outcompeted by the wild type in the gut of ex-Lactobacillus-free mice. Urine analysis showed that stable isotope-labeled urea, administered by gavage, was metabolized to a greater extent in Lactobacillus-free mice than animals colonized by strain 100-23. This surprising observation was associated with higher levels of urease activity and fecal-type bacteria in the stomach digesta of Lactobacillus-free mice. Despite the modulation of urea hydrolysis in the stomach, recycling of urea nitrogen in the murine host was not affected since the essential amino acid isoleucine, labeled with a stable isotope, was detected in the livers of both Lactobacillus-free and 100-23-colonized animals. Therefore, our experiments reveal a new and unexpected impact of Lactobacillus colonization on urea hydrolysis in the murine gut. PMID:25063664

Lactobacillus reuteri 100-23 modulates urea hydrolysis in the murine stomach.

PubMed

Wilson, Charlotte M; Loach, Diane; Lawley, Blair; Bell, Tracey; Sims, Ian M; O'Toole, Paul W; Zomer, Aldert; Tannock, Gerald W

2014-10-01

Comparisons of in vivo (mouse stomach) and in vitro (laboratory culture) transcriptomes of Lactobacillus reuteri strain 100-23 were made by microarray analysis. These comparisons revealed the upregulation of genes associated with acid tolerance, including urease production, in the mouse stomach. Inactivation of the ureC gene reduced the acid tolerance of strain 100-23 in vitro, and the mutant was outcompeted by the wild type in the gut of ex-Lactobacillus-free mice. Urine analysis showed that stable isotope-labeled urea, administered by gavage, was metabolized to a greater extent in Lactobacillus-free mice than animals colonized by strain 100-23. This surprising observation was associated with higher levels of urease activity and fecal-type bacteria in the stomach digesta of Lactobacillus-free mice. Despite the modulation of urea hydrolysis in the stomach, recycling of urea nitrogen in the murine host was not affected since the essential amino acid isoleucine, labeled with a stable isotope, was detected in the livers of both Lactobacillus-free and 100-23-colonized animals. Therefore, our experiments reveal a new and unexpected impact of Lactobacillus colonization on urea hydrolysis in the murine gut. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Potent and reversible lentiviral vector restriction in murine induced pluripotent stem cells.

PubMed

Geis, Franziska K; Galla, Melanie; Hoffmann, Dirk; Kuehle, Johannes; Zychlinski, Daniela; Maetzig, Tobias; Schott, Juliane W; Schwarzer, Adrian; Goffinet, Christine; Goff, Stephen P; Schambach, Axel

2017-05-31

Retroviral vectors are derived from wild-type retroviruses, can be used to study retrovirus-host interactions and are effective tools in gene and cell therapy. However, numerous cell types are resistant or less permissive to retrovirus infection due to the presence of active defense mechanisms, or the absence of important cellular host co-factors. In contrast to multipotent stem cells, pluripotent stem cells (PSC) have potential to differentiate into all three germ layers. Much remains to be elucidated in the field of anti-viral immunity in stem cells, especially in PSC. In this study, we report that transduction with HIV-1-based, lentiviral vectors (LV) is impaired in murine PSC. Analyses of early retroviral events in induced pluripotent stem cells (iPSC) revealed that the restriction is independent of envelope choice and does not affect reverse transcription, but perturbs nuclear entry and proviral integration. Proteasomal inhibition by MG132 could not circumvent the restriction. However, prevention of cyclophilin A (CypA) binding to the HIV-1 capsid via use of either a CypA inhibitor (cyclosporine A) or CypA-independent capsid mutants improved transduction. In addition, application of higher vector doses also increased transduction. Our data revealed a CypA mediated restriction in iPSC, which was acquired during reprogramming, associated with pluripotency and relieved upon subsequent differentiation. We showed that murine PSC and iPSC are less susceptible to LV. The block observed in iPSC was CypA-dependent and resulted in reduced nuclear entry of viral DNA and proviral integration. Our study helps to improve transduction of murine pluripotent cells with HIV-1-based vectors and contributes to our understanding of retrovirus-host interactions in PSC.

Development of an ex vivo BrdU labeling procedure for the murine LLNA

EPA Science Inventory

The murine local lymph node assay (LLNA) is widely used to identify chemicals that may cause allergic contact dermatitis. Exposure to a dermal sensitizer results in proliferation of local lymph node T cells, which has traditionally been measured by in vivo incorporation of [3H]m...

Decay-accelerating factor 1 deficiency exacerbates Trypanosoma cruzi-induced murine chronic myositis.

PubMed

Solana, María E; Ferrer, María F; Novoa, María Mercedes; Song, Wen-Chao; Gómez, Ricardo M

2012-10-01

Murine infection with Trypanosoma cruzi (Tc) has been used to study the role of T-cells in the pathogenesis of human inflammatory idiopathic myositis. Absence of decay-accelerating factor 1 (Daf1) has been shown to enhance murine T-cell responses and autoimmunity. To determine whether Daf1 deficiency can exacerbate Tc-induced myositis, C57BL/6 DAF(+/+) and DAF(-/-) mice were inoculated with 5 × 10(4) trypomastigotes, and their morbidity, parasitemia, parasite burden, histopathology, and T-cell expansion were studied in the acute and chronic stages. DAF(-/-) mice had lower parasitemia and parasite burden but higher morbidity, muscle histopathology, and increased number of CD44(+) (activated/memory phenotype) splenic CD4(+) and CD8(+) T-cells. An enhanced CD8(+) T-cell immune-specific response may explain the lower parasitemia and parasite burden levels and the increase in histopathological lesions. We propose that Tc-inoculated DAF(-/-) mice are a useful model to study T-cell mediated immunity in skeletal muscle tissues. Copyright © 2012 Wiley Periodicals, Inc.

Effect of MPS1 Inhibition on Genotoxic Stress Responses in Murine Tumour Cells.

PubMed

Suzuki, Motofumi; Yamamori, Tohru; Yasui, Hironobu; Inanami, Osamu

2016-06-01

The monopolar spindle 1 (MPS1) is a serine/threonine kinase that plays an important role in spindle assembly checkpoint signaling. To determine the possible relationship between MPS1 inhibition and genotoxic stress responses, herein we examined whether MPS1 inhibition influences cellular susceptibility towards two genotoxic treatments, etoposide and ionizing radiation (IR). Two murine tumour cell lines, SCCVII and EMT6, were used. The effect of genotoxic treatments with or without two novel MPS1 inhibitors, NMS-P715 and AZ3146, on cellular survival, cell-cycle distribution, centrosome status and mitotic catastrophe (MC) was evaluated. MPS1 inhibition sensitized murine tumour cells to etoposide but not to IR. In addition, MPS1 inhibition altered cell-cycle progression and exacerbated centrosome abnormalities, resulting in enhanced MC induced by etoposide but not by IR. MPS1 inhibition promotes the etoposide-induced aberrant mitosis and, consequently, the induction of tumour cell death. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Preventive effect of ultraviolet radiation on murine chronic sclerodermatous graft-versus-host disease.

PubMed

Mermet, Isabelle; Kleinclauss, François; Marandin, Aliette; Guérrini, Jean Sébastien; Angonin, Régis; Tiberghien, Pierre; Saas, Philippe; Aubin, François

2007-12-27

Although previous studies have demonstrated the efficient modulatory effects of ultraviolet radiation B (UVB) on cutaneous graft-versus-host disease (GVHD), most animal research on GVHD has been performed in murine models of acute GVHD. Here, we studied the preventive effect of UVB radiation on the occurrence of chronic sclerodermatous (Scl) GVHD in a murine model. Scl GVHD was induced by transplanting lethally irradiated BALB/c mice with B10.D2 bone marrow and spleen cells. Recipient mice were exposed to UVB before or after bone marrow and spleen cell infusion. Histological and clinical evaluation of GVHD was performed, in association with the characterization of epidermal Langerhans cells. UVB irradiation of recipients after, and more remarkably before, transplantation induced a decrease of Scl GVHD severity associated with epidermal Langerhans cells depletion. We conclude that UVB irradiation of recipient before or after transplantation has a preventive effect on cutaneous Scl GVHD and may represent an effective strategy for prevention of Scl GVHD.

Heat shock protein 90 functions to stabilize and activate the testis-specific serine/threonine kinases, a family of kinases essential for male fertility.

PubMed

Jha, Kula N; Coleman, Alyssa R; Wong, Lily; Salicioni, Ana M; Howcroft, Elizabeth; Johnson, Gibbes R

2013-06-07

Spermiogenesis is characterized by a profound morphological differentiation of the haploid spermatid into spermatozoa. The testis-specific serine/threonine kinases (TSSKs) comprise a family of post-meiotic kinases expressed in spermatids, are critical to spermiogenesis, and are required for male fertility in mammals. To explore the role of heat shock protein 90 (HSP90) in regulation of TSSKs, the stability and catalytic activity of epitope-tagged murine TSSKs were assessed in 293T and COS-7 cells. TSSK1, -2, -4, and -6 (small serine/threonine kinase) were all found to associate with HSP90, and pharmacological inhibition of HSP90 function using the highly specific drugs 17-AAG, SNX-5422, or NVP-AUY922 reduced TSSK protein levels in cells. The attenuation of HSP90 function abolished the catalytic activities of TSSK4 and -6 but did not significantly alter the specific activities of TSSK1 and -2. Inhibition of HSP90 resulted in increased TSSK ubiquitination and proteasomal degradation, indicating that HSP90 acts to control ubiquitin-mediated catabolism of the TSSKs. To study HSP90 and TSSKs in germ cells, a mouse primary spermatid culture model was developed and characterized. Using specific antibodies against murine TSSK2 and -6, it was demonstrated that HSP90 inhibition resulted in a marked decrease of the endogenous kinases in spermatids. Together, our findings demonstrate that HSP90 plays a broad and critical role in stabilization and activation of the TSSK family of protein kinases.

β-Arrestin2 mediates progression of murine primary myelofibrosis.

PubMed

Rein, Lindsay Am; Wisler, James W; Kim, Jihee; Theriot, Barbara; Huang, LiYin; Price, Trevor; Yang, Haeyoon; Chen, Minyong; Chen, Wei; Sipkins, Dorothy; Fedoriw, Yuri; Walker, Julia Kl; Premont, Richard T; Lefkowitz, Robert J

2017-12-21

Primary myelofibrosis is a myeloproliferative neoplasm associated with significant morbidity and mortality, for which effective therapies are lacking. β-Arrestins are multifunctional adaptor proteins involved in developmental signaling pathways. One isoform, β-arrestin2 (βarr2), has been implicated in initiation and progression of chronic myeloid leukemia, another myeloproliferative neoplasm closely related to primary myelofibrosis. Accordingly, we investigated the relationship between βarr2 and primary myelofibrosis. In a murine model of MPLW515L-mutant primary myelofibrosis, mice transplanted with donor βarr2-knockout (βarr2-/-) hematopoietic stem cells infected with MPL-mutant retrovirus did not develop myelofibrosis, whereas controls uniformly succumbed to disease. Although transplanted βarr2-/- cells homed properly to marrow, they did not repopulate long-term due to increased apoptosis and decreased self-renewal of βarr2-/- cells. In order to assess the effect of acute loss of βarr2 in established primary myelofibrosis in vivo, we utilized a tamoxifen-induced Cre-conditional βarr2-knockout mouse. Mice that received Cre (+) donor cells and developed myelofibrosis had significantly improved survival compared with controls. These data indicate that lack of antiapoptotic βarr2 mediates marrow failure of murine hematopoietic stem cells overexpressing MPLW515L. They also indicate that βarr2 is necessary for progression of primary myelofibrosis, suggesting that it may serve as a novel therapeutic target in this disease.

β-Arrestin2 mediates progression of murine primary myelofibrosis

PubMed Central

Rein, Lindsay A.M.; Wisler, James W.; Kim, Jihee; Theriot, Barbara; Huang, LiYin; Price, Trevor; Yang, Haeyoon; Chen, Wei; Sipkins, Dorothy; Fedoriw, Yuri; Walker, Julia K.L.; Premont, Richard T.; Lefkowitz, Robert J.

2017-01-01

Primary myelofibrosis is a myeloproliferative neoplasm associated with significant morbidity and mortality, for which effective therapies are lacking. β-Arrestins are multifunctional adaptor proteins involved in developmental signaling pathways. One isoform, β-arrestin2 (βarr2), has been implicated in initiation and progression of chronic myeloid leukemia, another myeloproliferative neoplasm closely related to primary myelofibrosis. Accordingly, we investigated the relationship between βarr2 and primary myelofibrosis. In a murine model of MPLW515L-mutant primary myelofibrosis, mice transplanted with donor βarr2-knockout (βarr2–/–) hematopoietic stem cells infected with MPL-mutant retrovirus did not develop myelofibrosis, whereas controls uniformly succumbed to disease. Although transplanted βarr2–/– cells homed properly to marrow, they did not repopulate long-term due to increased apoptosis and decreased self-renewal of βarr2–/– cells. In order to assess the effect of acute loss of βarr2 in established primary myelofibrosis in vivo, we utilized a tamoxifen-induced Cre-conditional βarr2-knockout mouse. Mice that received Cre (+) donor cells and developed myelofibrosis had significantly improved survival compared with controls. These data indicate that lack of antiapoptotic βarr2 mediates marrow failure of murine hematopoietic stem cells overexpressing MPLW515L. They also indicate that βarr2 is necessary for progression of primary myelofibrosis, suggesting that it may serve as a novel therapeutic target in this disease. PMID:29263312

Newly Identified TLR9 Stimulant, M6-395 Is a Potent Polyclonal Activator for Murine B Cells

PubMed Central

Park, Mi-Hee; Jung, Yu-Jin

2012-01-01

Background Toll-like receptors (TLRs) have been extensively studied in recent years. However, functions of these molecules in murine B cell biology are largely unknown. A TLR4 stimulant, LPS is well known as a powerful polyclonal activator for murine B cells. Methods In this study, we explored the effect of a murine TLR9 stimulant, M6-395 (a synthetic CpG ODNs) on B cell proliferation and Ig production. Results First, M6-395 was much more potent than LPS in augmenting B cell proliferation. As for Ig expression, M6-395 facilitated the expression of both TGF-β1-induced germ line transcript α (GLTα) and IL-4-induced GLTγ1 as levels as those by LPS and Pam3CSK4 (TLR1/2 agonist) : a certain Ig GLT expression is regarded as an indicative of the corresponding isotype switching recombination. However, IgA and IgG1 secretion patterns were quite different--these Ig isotype secretions by M6-395 were much less than those by LPS and Pam3CSK4. Moreover, the increase of IgA and IgG1 production by LPS and Pam3CSK4 was virtually abrogated by M6-395. The same was true for the secretion of IgG3. We found that this unexpected phenomena provoked by M6-395 is attributed, at least in part, to its excessive mitogenic nature. Conclusion Taken together, these results suggest that M6-395 can act as a murine polyclonal activator but its strong mitogenic activity is unfavorable to Ig isotype switching. PMID:22536167

A Common Ancestral Mutation in CRYBB3 Identified in Multiple Consanguineous Families with Congenital Cataracts

PubMed Central

Irum, Bushra; Khan, Arif O.; Wang, Qiwei; Li, David; Khan, Asma A.; Husnain, Tayyab; Akram, Javed; Riazuddin, Sheikh

2016-01-01

Purpose This study was performed to investigate the genetic determinants of autosomal recessive congenital cataracts in large consanguineous families. Methods Affected individuals underwent a detailed ophthalmological examination and slit-lamp photographs of the cataractous lenses were obtained. An aliquot of blood was collected from all participating family members and genomic DNA was extracted from white blood cells. Initially, a genome-wide scan was performed with genomic DNAs of family PKCC025 followed by exclusion analysis of our familial cohort of congenital cataracts. Protein-coding exons of CRYBB1, CRYBB2, CRYBB3, and CRYBA4 were sequenced bidirectionally. A haplotype was constructed with SNPs flanking the causal mutation for affected individuals in all four families, while the probability that the four familial cases have a common founder was estimated using EM and CHM-based algorithms. The expression of Crybb3 in the developing murine lens was investigated using TaqMan assays. Results The clinical and ophthalmological examinations suggested that all affected individuals had nuclear cataracts. Genome-wide linkage analysis localized the causal phenotype in family PKCC025 to chromosome 22q with statistically significant two-point logarithm of odds (LOD) scores. Subsequently, we localized three additional families, PKCC063, PKCC131, and PKCC168 to chromosome 22q. Bidirectional Sanger sequencing identified a missense variation: c.493G>C (p.Gly165Arg) in CRYBB3 that segregated with the disease phenotype in all four familial cases. This variation was not found in ethnically matched control chromosomes, the NHLBI exome variant server, or the 1000 Genomes or dbSNP databases. Interestingly, all four families harbor a unique disease haplotype that strongly suggests a common founder of the causal mutation (p<1.64E-10). We observed expression of Crybb3 in the mouse lens as early as embryonic day 15 (E15), and expression remained relatively steady throughout

Murine CD103+ dendritic cells protect against steatosis progression towards steatohepatitis.

PubMed

Heier, Eva-Carina; Meier, Anna; Julich-Haertel, Henrike; Djudjaj, Sonja; Rau, Monica; Tschernig, Thomas; Geier, Andreas; Boor, Peter; Lammert, Frank; Lukacs-Kornek, Veronika

2017-06-01

Non-alcoholic fatty liver (NAFL) is the hepatic consequence of metabolic syndrome and can progress to non-alcoholic steatohepatitis (NASH). The identification of molecular and cellular factors that determine the progression of NASH and lead to irreversible hepatocellular damage are crucial. Dendritic cells (DCs) represent a heterogeneous cell population among which CD103 + DCs play a significant role in immunity and tolerance. We aimed to clarify the role of this DC subset in the pathomechanism of NASH. Steatosis progression towards steatohepatitis was analysed using multicolor FACS analyses, cytokine and qPCR array in high sucrose diet (HSD) and methionine and choline deficient diet (MCD) fed wild-type and basic leucine zipper transcription factor, ATF-Like-3 (Batf3) deficient animals, which lack CD103 + DCs (classical type-1 DC, cDC1s). Metabolic challenge of Batf3 -/- animals resulted in the progression of steatosis towards steatohepatitis, manifesting by an increased influx of inflammatory cells into the liver and elevated inflammatory cytokine production of myeloid cells upon innate stimuli. However, the lack of cDC1s did not affect cellular apoptosis and fibrosis progression but altered genes involved in lipid metabolism. The adoptive transfer of CD103 + cDC1s to Batf3 deficient animals reversed these observed changes and more importantly could attenuate cellular damage and inflammation in established murine steatohepatitis. Here, we have identified the murine CD103 + cDC1s as a protective DC subtype that influences the pro-anti-inflammatory balance and protects the liver from metabolic damage. As guardians of liver integrity, they play a key role in the inflammatory process during the development of steatohepatitis in mice. Non-alcoholic fatty liver (NAFL) is the hepatic consequence of metabolic syndrome and can lead to non-alcoholic steatohepatitis (NASH). The current study demonstrated that a specific murine dendritic cell subtype possesses a potent

Murine Endogenous Retroviruses Are Detectable in Patient-Derived Xenografts but Not in Patient-Individual Cell Lines of Human Colorectal Cancer.

PubMed

Bock, Stephanie; Mullins, Christina S; Klar, Ernst; Pérot, Philippe; Maletzki, Claudia; Linnebacher, Michael

2018-01-01

Endogenous retroviruses are remnants of retroviral infections. In contrast to their human counterparts, murine endogenous retroviruses (mERV) still can synthesize infectious particles and retrotranspose. Xenotransplanted human cells have occasionally been described to be mERV infected. With genetic engineered mice and patient-derived xenografts (PDXs) on the rise as eminent research tools, we here systematically investigated, if different tumor models harbor mERV infections. Relevant mERV candidates were first preselected by next generation sequencing (NGS) analysis of spontaneous lymphomas triggered by colorectal cancer (CRC) PDX tissue. Two primer systems were designed for each of these candidates (AblMLV, EcoMLV, EndoPP, MLV, and preXMRV) and implemented in an quantitative real-time (RT-qPCR) screen using murine tissues ( n = 11), PDX-tissues ( n = 22), PDX-derived cell lines ( n = 13), and patient-derived tumor cell lines ( n = 14). The expression levels of mERV varied largely both in the PDX samples and in the mouse tissues. No mERV signal was, however, obtained from cDNA or genomic DNA of CRC cell lines. Expression of EcoMLV was higher in PDX than in murine tissues; for EndoPP it was the opposite. These two were thus further investigated in 40 additional PDX. In addition, four patient-derived cell lines free of any mERV expression were subcutaneously injected into immunodeficient mice. Outgrowing cell-derived xenografts barely expressed EndoPP. In contrast, the expression of EcoMLV was even higher than in surrounding mouse tissues. This expression gradually vanished within few passages of re-cultivated cells. In summary, these results strongly imply that: (i) PDX and murine tissues in general are likely to be contaminated by mERV, (ii) mERV are expressed transiently and at low level in fresh PDX-derived cell cultures, and (iii) mERV integration into the genome of human cells is unlikely or at least a very rare event. Thus, mERVs are stowaways present in

Antioxidative effects in vivo and colonization of Lactobacillus plantarum MA2 in the murine intestinal tract.

PubMed

Tang, Wei; Xing, Zhuqing; Hu, Wei; Li, Chao; Wang, Jinju; Wang, Yanping

2016-08-01

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.

The ZIP family zinc transporters support the virulence of Cryptococcus neoformans

PubMed Central

Do, Eunsoo; Hu, Guanggan; Caza, Mélissa; Kronstad, James W.; Jung, Won Hee

2016-01-01

Zinc is an essential element in living organisms and a cofactor for various metalloproteins. To disseminate and survive, a pathogenic microbe must obtain zinc from the host, which is an environment with extremely limited zinc availability. In this study, we investigated the roles of the ZIP family zinc transporters Zip1 and Zip2 in the human pathogenic fungus Cryptococcus neoformans. Zip1 and Zip2 are homologous to Zrt1 and Zrt2 of the model fungus, Saccharomyces cerevisiae, respectively. We found that the expression of ZIP1 was regulated by the zinc concentration in the environment. Furthermore, the mutant lacking ZIP1 displayed a severe growth defect under zinc-limited conditions, while the mutant lacking ZIP2 displayed normal growth. Inductively coupled plasma–atomic emission spectroscopy analysis showed that the absence of Zip1 expression significantly reduced total cellular zinc levels relative to that in the wild type, while overexpression of Zip1 was associated with increased cellular zinc levels. These findings suggested that Zip1 plays roles in zinc uptake in C. neoformans. We also constructed a Zip1-FLAG fusion protein and found, by immunofluorescence, not only that the protein was localized to the periphery implying it is a membrane transporter, but also that the protein was N-glycosylated. Furthermore, the mutant lacking ZIP1 showed attenuated virulence in a murine inhalation model of cryptococcosis and reduced survival within murine macrophages. Overall, our data suggest that Zip1 plays essential roles in zinc transport and the virulence of C. neoformans. PMID:27118799

Murine cell glycolipids customization by modular expression of glycosyltransferases.

PubMed

Cid, Emili; Yamamoto, Miyako; Buschbeck, Marcus; Yamamoto, Fumiichiro

2013-01-01

Functional analysis of glycolipids has been hampered by their complex nature and combinatorial expression in cells and tissues. We report an efficient and easy method to generate cells with specific glycolipids. In our proof of principle experiments we have demonstrated the customized expression of two relevant glycosphingolipids on murine fibroblasts, stage-specific embryonic antigen 3 (SSEA-3), a marker for stem cells, and Forssman glycolipid, a xenoantigen. Sets of genes encoding glycosyltansferases were transduced by viral infection followed by multi-color cell sorting based on coupled expression of fluorescent proteins.

Suppression of methylmercury-induced MIP-2 expression by N-acetyl-L-cysteine in murine RAW264.7 macrophage cell line.

PubMed

David, Juliet; Nandakumar, Athira; Muniroh, Muflihatul; Akiba, Suminori; Yamamoto, Megumi; Koriyama, Chihaya

2017-11-09

The aim of this study is to examine the inflammatory-cytokine expressions in the presence of non-cytotoxic dose of methylmercury (MeHg) in murine macrophages, which is suspected to play an important role in brain damage caused by MeHg exposure. We focused on murine macrophage inflammatory protein-2 (MIP-2), keratinocyte chemoattractant (KC), and monocyte chemoattractant protein-5 (MCP-5). MIP-2 and KC are murine functional homologues of human IL-8 and MCP-5 for human MCP-1. Furthermore, we examined the suppressive effect of N-acetyl-L-cysteine (NAC) on the MeHg-induced inflammatory cytokines. In a murine RAW264.7 macrophage cell line, MeHg-induced cytokine expressions were measured using real-time PCR. The suppressive effect of NAC was examined by putting it into the culture medium together with MeHg (co-treatment). In addition, pre- and post-treatment experiments were conducted, in which the cells were treated with NAC before and after MeHg exposure, respectively. Exposure to a non-cytotoxic dose of MeHg up-regulated the mRNA expression of MIP-2 and MCP-5. On the other hand, KC expression was not induced in the presence of MeHg. Effect of MeHg on MIP-2 expressions was suppressed by pre-, co-, and post-treatment with NAC. However, the suppressive effect of pre-treatment was less than the post-treatment, which was as effective as co-treatment. In functional homologues of human IL-8, only MIP-2 expression, not KC, was activated in the presence of non-cytotoxic dose of MeHg in murine RAW264.7 macrophage cell line. The more evident inhibitory effect of NAC observed in post-treatment experiments suggests a possible involvement of intracellular activities such as antioxidant effects.

Efficacy of Histatin5 in a murine model of vulvovaginal candidiasis caused by Candida albicans.

PubMed

Liao, Hong; Liu, Shanling; Wang, He; Su, Hang; Liu, Zhenjun

2017-08-31

Histatin5 (Hst-5) is a member of the histatin family of antimicrobial peptides secreted by human parotid and submandibular glands. With the natural antibacterial activity, it plays an important role in the first-line barrier of oral cave against pathogens, especially for the fungal intrusion. In this study, we explored the utility of Hst-5 in the treatment of vulvovaginal candidiasis, a common condition of women of the childbearing age. We used a synthesized Hst-5 over five consecutive days as the topical treatment in a murine model of vulvovaginal candidiasis. According to the fungal colony counts, fungal burden in the vagina lavage dropped remarkably after treatment with Hst-5. Furthermore, cytological analysis of the lavage fluid indicated that the number of cast-off cells including cornified epithelial cells and inflammatory cells also decreased; histological evaluation of the vagina tissue revealed less fungi adhering to the vaginal wall in treated animals than in controls. Combined, these results suggested for the first time the potential utility of Hst-5 as a topical treatment for vulvovaginal candidiasis, uncovering the possibility of exploiting the natural antibiotic peptides in other aspects. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Redundant roles of the phosphatidate phosphatase family in triacylglycerol synthesis in human adipocytes.

PubMed

Temprano, Ana; Sembongi, Hiroshi; Han, Gil-Soo; Sebastián, David; Capellades, Jordi; Moreno, Cristóbal; Guardiola, Juan; Wabitsch, Martin; Richart, Cristóbal; Yanes, Oscar; Zorzano, Antonio; Carman, George M; Siniossoglou, Symeon; Miranda, Merce

2016-09-01

In mammals, the evolutionary conserved family of Mg(2+)-dependent phosphatidate phosphatases (PAP1), involved in phospholipid and triacylglycerol synthesis, consists of lipin-1, lipin-2 and lipin-3. While mutations in the murine Lpin1 gene cause lipodystrophy and its knockdown in mouse 3T3-L1 cells impairs adipogenesis, deleterious mutations of human LPIN1 do not affect adipose tissue distribution. However, reduced LPIN1 and PAP1 activity has been described in participants with type 2 diabetes. We aimed to characterise the roles of all lipin family members in human adipose tissue and adipogenesis. The expression of the lipin family was analysed in adipose tissue in a cross-sectional study. Moreover, the effects of lipin small interfering RNA (siRNA)-mediated depletion on in vitro human adipogenesis were assessed. Adipose tissue gene expression of the lipin family is altered in type 2 diabetes. Depletion of every lipin family member in a human Simpson-Golabi-Behmel syndrome (SGBS) pre-adipocyte cell line, alters expression levels of adipogenic transcription factors and lipid biosynthesis genes in early stages of differentiation. Lipin-1 knockdown alone causes a 95% depletion of PAP1 activity. Despite the reduced PAP1 activity and alterations in early adipogenesis, lipin-silenced cells differentiate and accumulate neutral lipids. Even combinatorial knockdown of lipins shows mild effects on triacylglycerol accumulation in mature adipocytes. Overall, our data support the hypothesis of alternative pathways for triacylglycerol synthesis in human adipocytes under conditions of repressed lipin expression. We propose that induction of alternative lipid phosphate phosphatases, along with the inhibition of lipid hydrolysis, contributes to the maintenance of triacylglycerol content to near normal levels.

Whole exome sequencing of an asbestos-induced wild-type murine model of malignant mesothelioma.

PubMed

Sneddon, Sophie; Patch, Ann-Marie; Dick, Ian M; Kazakoff, Stephen; Pearson, John V; Waddell, Nicola; Allcock, Richard J N; Holt, Robert A; Robinson, Bruce W S; Creaney, Jenette

2017-06-02

Malignant mesothelioma (MM) is an aggressive cancer of the pleural and peritoneal cavities caused by exposure to asbestos. Asbestos-induced mesotheliomas in wild-type mice have been used extensively as a preclinical model because they are phenotypically identical to their human counterpart. However, it is not known if the genetic lesions in these mice tumours are similar to in the human disease, a prerequisite for any new preclinical studies that target genetic abnormalities. We performed whole exome sequencing of fifteen asbestos-induced murine MM tumour cell lines from BALB/c, CBA and C57BL/6 mouse strains and compared the somatic mutations and copy number variations with those recurrently reported in human MM. We then catalogued and characterised the mutational landscape of the wild-type murine MM tumours. Quantitative RT-PCR was used to interrogate the expression of key MM genes of interest in the mRNA. Consistent with human MM tumours, we identified homozygous loss of the tumour suppressor Cdkn2a in 14/15 tumours. One tumour retained the first exon of both of the p16INK4a and p19ARF isoforms though this tumour also contained genetic amplification of Myc resulting in increased expression of the c-Myc proto-oncogene in the mRNA. There were no chromosomal losses in either the Bap1 or Nf2 regions. One tumour harbored homozygous loss of Trp53 in the DNA. Mutation rates were similar in tumours generated in the CBA and C57BL/6 strains when compared to human MM. Interestingly, all BALB/c tumour lines displayed high mutational loads, consistent with the known mutator phenotype of the host strain. The Wnt, MAPK and Jak-STAT signaling pathways were found to be the most commonly affected biological pathways. Mutations and copy number deletions also occurred in the Hedgehog and Hippo pathways. These data suggest that in the wild-type murine model asbestos causes mesotheliomas in a similar way to in human MM. This further supports the notion that the murine model of MM

The virucidal effects against murine norovirus and feline calicivirus F4 as surrogates for human norovirus by the different additive concentrations of ethanol-based sanitizers.

PubMed

Akasaka, Tempei; Shimizu-Onda, Yuko; Hayakawa, Satoshi; Ushijima, Hiroshi

2016-03-01

Since human norovirus is non-cultivable, murine norovirus and feline calicivirus have been used as surrogates. In this study, the virucidal effects of ethanol-based sanitizers with different concentrations of additives (malic acid/sodium malate, glycerin-fatty acid ester) against murine norovirus and feline calicivirus F4 were examined. The ethanol-based sanitizers at pH 7 showed sufficient virucidal effects, but glycerin-fatty acid ester included in ethanol-based sanitizers at pH 4 or 6 reduced the virucidal effects against murine norovirus. The ethanol-based sanitizers containing malic acid/sodium malate inactivated feline calicivirus F4 in shorter time, but there is no difference between ethanol-based sanitizers with and without glycerin-fatty acid ester. Traditionally, feline calicivirus has been used for long time as a surrogate virus for human norovirus. However, this study suggested that murine norovirus and feline calicivirus F4 had different sensitivity with the additive components of ethanol-based sanitizers. Therefore, using feline calicivirus alone as a surrogate for human norovirus may not be sufficient to evaluate the virucidal effect of sanitizers on food-borne infections caused by human norovirus. Sanitizers having virucidal effects against at least both murine norovirus and feline calicivirus may be more suitable to inactivate human norovirus. Copyright © 2015. Published by Elsevier Ltd.

Usefulness of the murine model to study the immune response against Histoplasma capsulatum infection.

PubMed

Sahaza, Jorge H; Pérez-Torres, Armando; Zenteno, Edgar; Taylor, Maria Lucia

2014-05-01

The present paper is an overview of the primary events that are associated with the histoplasmosis immune response in the murine model. Valuable data that have been recorded in the scientific literature have contributed to an improved understanding of the clinical course of this systemic mycosis, which is caused by the dimorphic fungus Histoplasma capsulatum. Data must be analyzed carefully, given that misinterpretation could be generated because most of the available information is based on experimental host-parasite interactions that used inappropriate proceedings, i.e., the non-natural route of infection with the parasitic and virulent fungal yeast-phase, which is not the usual infective phase of the etiological agent of this mycosis. Thus, due to their versatility, complexity, and similarities with humans, several murine models have played a fundamental role in exploring the host-parasite interaction during H. capsulatum infection. Copyright © 2014 Elsevier Ltd. All rights reserved.

Retinal Ultrastructure of Murine Models of Dry Age-related Macular Degeneration (AMD)

PubMed Central

Ramkumar, Hema L.; Zhang, Jun; Chan, Chi-Chao

2010-01-01

Age-related macular degeneration (AMD) is the most prevalent form of irreversible blindness worldwide in the elderly population. The pathology of dry AMD consists of degeneration of photoreceptors and the RPE, lipofuscin (A2E) accumulation, and drusen formation. Mice have been widely used for generating models that simulate human AMD features for investigating the pathogenesis, treatment and prevention of the disease. Although the mouse has no macula, focal atrophy of photorecptors and RPE, lipofuscin accumulation, and increased A2E can develop in aged mouse eyes. However, drusen are rarely seen in mice because of their simpler Bruch’s membrane and different process of lipofuscin extrusion compared with humans. Thus, analyzing basal deposits at the ultrastructural level and understanding the ultrastructural pathologic differences between various mouse AMD models are critical to comprehending the significance of research findings and response to possible therapeutic options for dry AMD. Based on the multifactorial pathogenesis of AMD, murine dry AMD models can be classified into three groups. First, genetically engineered mice that target genes related to juvenile macular dystrophies are the most common models, and they include abcr−/− (Stargardt disease), transgenic ELOVL4 (Stargardt-3 dominant inheritary disease), Efemp1R345W/R345W (Doyne honeycomb retinal dystrophy), and Timp3S156C/S156C (Sorsby fundus dystrophy) mice. Other murine models target genes relevant to AMD, including inflammatory genes such as Cfh−/−, Ccl2−/−, Ccr2−/−, Cx3cr1−/−, and Ccl2−/−/cx3cr1−/−, oxidative stress associated genes such as Sod1−/− and Sod2 knockdown, metabolic pathway genes such as neprilysin −/− (amyloid β), transgenic mcd/mcd (cathepsin D), Cp−/−/Heph−/Y (ferroxidase ceruloplasmin/hepaestin, iron metabolism), and transgenic ApoE4 on high fat and high cholesterol diet (lipid metabolism). Second, mice have also been immunologically

Altered Innate and Lymphocytic Immunity in Murine Splenocytes Following Short-Duration Spaceflight

NASA Technical Reports Server (NTRS)

Crucian, Brian E.; Hwang, Shen-An; Actor, Jeffrey K.; Quiriarte, Heather; Sams, Clarence F.

2011-01-01

Immune dysregulation has been demonstrated following spaceflight of varying durations and limited in-flight studies indicate this phenomenon may persist during spaceflight. Causes may include microgravity, physiological stress, isolation, confinement and disrupted circadian rhythms. To further investigate the mechanisms associated with flight-associated immune changes, murine splenocytes immune parameters were assessed following 14 day space flight on Space Shuttle mission STS-135.

Gender and dose dependent ovalbumin induced hypersensitivity responses in murine model of food allergy

USDA-ARS?s Scientific Manuscript database

While federal regulations mandate the labeling of major food allergens, allowable food allergen thresholds have yet to be determined. Therefore the aim of this project was to identify the lowest egg allergen ovalbumin (OVA) dose causing hypersensitization using a validated murine model. Mice were or...

Gender and dose dependent ovalbumin induced hypersensitivity responses in murine model of food allergy

USDA-ARS?s Scientific Manuscript database

While federal regulations mandate the labeling of major food allergens, allowable food allergen thresholds have yet to be determined. Therefore the aim of this project was to identify the lowest egg allergen ovalbumin (OVA) dose causing hypersensitization using a validated murine model. Mice were o...

Effects of oxaliplatin and oleic acid Gc-protein-derived macrophage-activating factor on murine and human microglia.

PubMed

Branca, Jacopo J V; Morucci, Gabriele; Malentacchi, Francesca; Gelmini, Stefania; Ruggiero, Marco; Pacini, Stefania

2015-09-01

The biological properties and characteristics of microglia in rodents have been widely described, but little is known about these features in human microglia. Several murine microglial cell lines are used to investigate neurodegenerative and neuroinflammatory conditions; however, the extrapolation of the results to human conditions is frequently met with criticism because of the possibility of species-specific differences. This study compares the effects of oxaliplatin and of oleic acid Gc-protein-derived macrophage-activating factor (OA-GcMAF) on two microglial cell lines, murine BV-2 cells and human C13NJ cells. Cell viability, cAMP levels, microglial activation, and vascular endothelial growth factor (VEGF) expression were evaluated. Our data demonstrate that oxaliplatin induced a significant decrease in cell viability in BV-2 and in C13NJ cells and that this effect was not reversed with OA-GcMAF treatment. The signal transduction pathway involving cAMP/VEGF was activated after treatment with oxaliplatin and/or OA-GcMAF in both cell lines. OA-GcMAF induced a significant increase in microglia activation, as evidenced by the expression of the B7-2 protein, in BV-2 as well as in C13NJ cells that was not associated with a concomitant increase in cell number. Furthermore, the effects of oxaliplatin and OA-GcMAF on coculture morphology and apoptosis were evaluated. Oxaliplatin-induced cell damage and apoptosis were nearly completely reversed by OA-GcMAF treatment in both BV-2/SH-SY5Y and C13NJ/SH-SY5Y cocultures. Our data show that murine and human microglia share common signal transduction pathways and activation mechanisms, suggesting that the murine BV-2 cell line may represent an excellent model for studying human microglia. © 2015 Wiley Periodicals, Inc.

A functional murine model of hindlimb demand ischemia.

PubMed

Peck, Michael A; Crawford, Robert S; Abularrage, Christopher J; Patel, Virendra I; Conrad, Mark F; Yoo, Jin Hyung; Watkins, Michael T; Albadawi, Hassan

2010-05-01

To date, murine models of treadmill exercise have been used to study general exercise physiology and angiogenesis in ischemic hindlimbs. The purpose of these experiments was to develop a murine model of demand ischemia in an ischemic limb to mimic claudication in humans. The primary goal was to determine whether treadmill exercise reflected a hemodynamic picture which might be consistent with the hyperemic response observed in humans. Aged hypercholesterolemic ApoE null mice (ApoE(-/-), n = 13) were subjected to femoral artery ligation (FAL) and allowed to recover from the acute ischemic response. Peripheral perfusion of the hindlimbs at rest was determined by serial evaluation using laser Doppler imaging (LDI) on days 0, 7, and 14 following FAL. During the experiments, mice were also assessed on an established five-point clinical ischemic score, which assessed the degree of digital amputation, necrosis, and cyanosis compared to the nonischemic contralateral limb. After stabilization of the LDI ratio (ischemic limb flux/contralateral nonischemic limb flux) and clinical ischemic score, mice underwent 2 days of treadmill training (10 min at 10 m/min, incline of 10 degrees ) followed by 60 min of daily treadmill exercise (13 m/min, incline of 10 degrees ) through day 25. An evaluation of preexercise and postexercise perfusion using LDI was performed on two separate occasions following the onset of daily exercise. During the immediate 15 min postexercise evaluation, LDI scanning was obtained in quadruplicate, to allow identification of peak flux ratios. Statistical analysis included unpaired t-tests and analysis of variance. After FAL, the LDI flux ratio reached a nadir between days 1 and 2, then stabilized by day 14 and remained stable through day 25. The clinical ischemic score stabilized at day 7 and remained stable throughout the rest of the experiment. Based on stabilization of both the clinical ischemic score and LDI ratio, exercise training began on day 15. The

Synchronous down-modulation of miR-17 family members is an early causative event in the retinal angiogenic switch.

PubMed

Nunes, Diana N; Dias-Neto, Emmanuel; Cardó-Vila, Marina; Edwards, Julianna K; Dobroff, Andrey S; Giordano, Ricardo J; Mandelin, Jami; Brentani, Helena P; Hasselgren, Catrin; Yao, Virginia J; Marchiò, Serena; Pereira, Carlos A B; Passetti, Fabio; Calin, George A; Sidman, Richard L; Arap, Wadih; Pasqualini, Renata

2015-03-24

Six members of the microRNA-17 (miR-17) family were mapped to three different chromosomes, although they share the same seed sequence and are predicted to target common genes, among which are those encoding hypoxia-inducible factor-1α (HIF1A) and VEGFA. Here, we evaluated the in vivo expression profile of the miR-17 family in the murine retinopathy of prematurity (ROP) model, whereby Vegfa expression is highly enhanced at the early stage of retinal neovascularization, and we found simultaneous reduction of all miR-17 family members at this stage. Using gene reporter assays, we observed binding of these miRs to specific sites in the 3' UTRs of Hif1a and Vegfa. Furthermore, overexpression of these miRs decreased HIF1A and VEGFA expression in vitro. Our data indicate that this miR-17 family elicits a regulatory synergistic down-regulation of Hif1a and Vegfa expression in this biological model. We propose the existence of a coordinated regulatory network, in which diverse miRs are synchronously regulated to target the Hif1a transcription factor, which in turn, potentiates and reinforces the regulatory effects of the miRs on Vegfa to trigger and sustain a significant physiological response.

Murine Leukemia Viruses: Objects and Organisms

PubMed Central

Rein, Alan

2011-01-01

Murine leukemia viruses (MLVs) are among the simplest retroviruses. Prototypical gammaretroviruses encode only the three polyproteins that will be used in the assembly of progeny virus particles. These are the Gag polyprotein, which is the structural protein of a retrovirus particle, the Pol protein, comprising the three retroviral enzymes—protease, which catalyzes the maturation of the particle, reverse transcriptase, which copies the viral RNA into DNA upon infection of a new host cell, and integrase, which inserts the DNA into the chromosomal DNA of the host cell, and the Env polyprotein, which induces the fusion of the viral membrane with that of the new host cell, initiating infection. In general, a productive MLV infection has no obvious effect upon host cells. Although gammaretroviral structure and replication follow the same broad outlines as those of other retroviruses, we point out a number of significant differences between different retroviral genera. PMID:22312342

Murine leukemia viruses: objects and organisms.

PubMed

Rein, Alan

2011-01-01

Murine leukemia viruses (MLVs) are among the simplest retroviruses. Prototypical gammaretroviruses encode only the three polyproteins that will be used in the assembly of progeny virus particles. These are the Gag polyprotein, which is the structural protein of a retrovirus particle, the Pol protein, comprising the three retroviral enzymes-protease, which catalyzes the maturation of the particle, reverse transcriptase, which copies the viral RNA into DNA upon infection of a new host cell, and integrase, which inserts the DNA into the chromosomal DNA of the host cell, and the Env polyprotein, which induces the fusion of the viral membrane with that of the new host cell, initiating infection. In general, a productive MLV infection has no obvious effect upon host cells. Although gammaretroviral structure and replication follow the same broad outlines as those of other retroviruses, we point out a number of significant differences between different retroviral genera.

Murinization of internalin extends its receptor repertoire, altering Listeria monocytogenes cell tropism and host responses.

PubMed

Tsai, Yu-Huan; Disson, Olivier; Bierne, Hélène; Lecuit, Marc

2013-01-01

Listeria monocytogenes (Lm) is an invasive foodborne pathogen that leads to severe central nervous system and maternal-fetal infections. Lm ability to actively cross the intestinal barrier is one of its key pathogenic properties. Lm crosses the intestinal epithelium upon the interaction of its surface protein internalin (InlA) with its host receptor E-cadherin (Ecad). InlA-Ecad interaction is species-specific, does not occur in wild-type mice, but does in transgenic mice expressing human Ecad and knock-in mice expressing humanized mouse Ecad. To study listeriosis in wild-type mice, InlA has been "murinized" to interact with mouse Ecad. Here, we demonstrate that, unexpectedly, murinized InlA (InlA(m)) mediates not only Ecad-dependent internalization, but also N-cadherin-dependent internalization. Consequently, InlA(m)-expressing Lm targets not only goblet cells expressing luminally-accessible Ecad, as does Lm in humanized mice, but also targets villous M cells, which express luminally-accessible N-cadherin. This aberrant Lm portal of entry results in enhanced innate immune responses and intestinal barrier damage, both of which are not observed in wild-type Lm-infected humanized mice. Murinization of InlA therefore not only extends the host range of Lm, but also broadens its receptor repertoire, providing Lm with artifactual pathogenic properties. These results challenge the relevance of using InlA(m)-expressing Lm to study human listeriosis and in vivo host responses to this human pathogen.

MMTV insertional mutagenesis identifies genes, gene families and pathways involved in mammary cancer.

PubMed

Theodorou, Vassiliki; Kimm, Melanie A; Boer, Mandy; Wessels, Lodewyk; Theelen, Wendy; Jonkers, Jos; Hilkens, John

2007-06-01

We performed a high-throughput retroviral insertional mutagenesis screen in mouse mammary tumor virus (MMTV)-induced mammary tumors and identified 33 common insertion sites, of which 17 genes were previously not known to be associated with mammary cancer and 13 had not previously been linked to cancer in general. Although members of the Wnt and fibroblast growth factors (Fgf) families were frequently tagged, our exhaustive screening for MMTV insertion sites uncovered a new repertoire of candidate breast cancer oncogenes. We validated one of these genes, Rspo3, as an oncogene by overexpression in a p53-deficient mammary epithelial cell line. The human orthologs of the candidate oncogenes were frequently deregulated in human breast cancers and associated with several tumor parameters. Computational analysis of all MMTV-tagged genes uncovered specific gene families not previously associated with cancer and showed a significant overrepresentation of protein domains and signaling pathways mainly associated with development and growth factor signaling. Comparison of all tagged genes in MMTV and Moloney murine leukemia virus-induced malignancies showed that both viruses target mostly different genes that act predominantly in distinct pathways.

L1-mediated retrotransposition of murine B1 and B2 SINEs recapitulated in cultured cells.

PubMed

Dewannieux, Marie; Heidmann, Thierry

2005-06-03

SINEs are short interspersed nucleotide elements with transpositional activity, present at a high copy number (up to a million) in mammalian genomes. They are 80-400 bp long, non-coding sequences which derive either from the 7SL RNA (e.g. human Alus, murine B1s) or tRNA (e.g. murine B2s) polymerase III-driven genes. We have previously demonstrated that Alus very efficiently divert the enzymatic machinery of the autonomous L1 LINE (long interspersed nucleotide element) retrotransposons to transpose at a high rate. Here we show, using an ex vivo assay for transposition, that both B1 and B2 SINEs can be mobilized by murine LINEs, with the hallmarks of a bona fide retrotransposition process, including target site duplications of varying lengths and integrations into A-rich sequences. Despite different phylogenetic origins, transposition of the tRNA-derived B2 sequences is as efficient as that of the human Alus, whereas that of B1s is 20-100-fold lower despite a similar high copy number of these elements in the mouse genome. We provide evidence, via an appropriate nucleotide substitution within the B1 sequence in a domain essential for its intracellular targeting, that the current B1 SINEs are not optimal for transposition, a feature most probably selected for the host sake in the course of evolution.

Stable integration of recombinant adeno-associated virus vector genomes after transduction of murine hematopoietic stem cells.

PubMed

Han, Zongchao; Zhong, Li; Maina, Njeri; Hu, Zhongbo; Li, Xiaomiao; Chouthai, Nitin S; Bischof, Daniela; Weigel-Van Aken, Kirsten A; Slayton, William B; Yoder, Mervin C; Srivastava, Arun

2008-03-01

We previously reported that among single-stranded adeno-associated virus (ssAAV) vectors, serotypes 1 through 5, ssAAV1 is the most efficient in transducing murine hematopoietic stem cells (HSCs), but viral second-strand DNA synthesis remains a rate-limiting step. Subsequently, using double-stranded, self-complementary AAV (scAAV) vectors, serotypes 7 through 10, we observed that scAAV7 vectors also transduce murine HSCs efficiently. In the present study, we used scAAV1 and scAAV7 shuttle vectors to transduce HSCs in a murine bone marrow serial transplant model in vivo, which allowed examination of the AAV proviral integration pattern in the mouse genome, as well as recovery and nucleotide sequence analyses of AAV-HSC DNA junction fragments. The proviral genomes were stably integrated, and integration sites were localized to different mouse chromosomes. None of the integration sites was found to be in a transcribed gene, or near a cellular oncogene. None of the animals, monitored for up to 1 year, exhibited pathological abnormalities. Thus, AAV proviral integration-induced risk of oncogenesis was not found in our study, which provides functional confirmation of stable transduction of self-renewing multipotential HSCs by scAAV vectors as well as promise for the use of these vectors in the potential treatment of disorders of the hematopoietic system.

Nanobiotechnological Nanocapsules Containing Polyhemoglobin-Tyrosinase: Effects on Murine B16F10 Melanoma Cell Proliferation and Attachment

PubMed Central

Wang, Yun; Chang, Thomas M. S.

2012-01-01

We have reported previously that daily intravenous infusions of a soluble nanobiotechnological complex, polyhemoglobin-tyrosinase [polyHb-Tyr], can suppress the growth of murine B16F10 melanoma in a mouse model. In order to avoid the need for daily intravenous injections, we have now extended this further as follows. We have prepared two types of biodegradable nanocapsules containing [polyHb-Tyr]. One type is to increase the circulation time and decrease the frequency of injection and is based on polyethyleneglycol-polylactic acid (PEG-PLA) nanocapsules containing [polyHb-Tyr]. The other type is to allow for intratumoural or local injection and is based on polylactic acid (PLA) nanocapsules containing [polyHb-Tyr]. Cell culture studies show that it can inhibit the proliferation of murine B16F10 melanoma cells in the “proliferation model”. It can also inhibit the attachment of murine B16F10 melanoma cells in the “attachment model.” This could be due to the action of tyrosinase on the depletion of tyrosine or the toxic effect of tyrosine metabolites. The other component, polyhemoglobin (polyHb), plays a smaller role in nanocapsules containing [polyHb-Tyr], and this is most likely by its depletion of nitric oxide needed for melanoma cell growth. PMID:23209910

Hepatocyte Transplantation Improves Phenotype and Extends Survival in a Murine Model of Intermediate Maple Syrup Urine Disease

PubMed Central

Skvorak, Kristen J; Paul, Harbhajan S; Dorko, Kenneth; Marongiu, Fabio; Ellis, Ewa; Chace, Donald; Ferguson, Carolyn; Gibson, K Michael; Homanics, Gregg E; Strom, Stephen C

2009-01-01

Maple syrup urine disease (MSUD; OMIM 248600) is an inborn error of metabolism of the branched chain α-ketoacid dehydrogenase (BCKDH) complex that is treated primarily by dietary manipulation of branched-chain amino acids (BCAA). Dietary restriction is lifelong and compliance is difficult. Liver transplantation significantly improves outcomes; however, alternative therapies are needed. To test novel therapies such as hepatocyte transplantation (HTx), we previously created a murine model of intermediate MSUD (iMSUD), which closely mimics human iMSUD. LacZ-positive murine donor hepatocytes were harvested and directly injected (105 cells/50 µl) into liver of iMSUD mice (two injections at 1–10 days of age). Donor hepatocytes engrafted into iMSUD recipient liver, increased liver BCKDH activity, improved blood total BCAA/alanine ratio, increased body weight at weaning, and extended the lifespan of HTx-treated iMSUD mice compared to phosphate-buffered saline (PBS)–treated and untreated iMSUD mice. Based on these data demonstrating partial metabolic correction of iMSUD in a murine model, coupled to the fact that multiple transplants are possible to enhance these results, we suggest that HTx represents a promising therapeutic intervention for MSUD that warrants further investigation. PMID:19436271

Oxaliplatin but Not Irinotecan Impairs Posthepatectomy Liver Regeneration in a Murine Model

PubMed Central

Soriano, Perry A.; Liu, Nian; Castillo, Erick; Foster, Brock; Artinyan, Avo; Kim, Joseph; Huang, Wendong; Wagman, Lawrence D.

2011-01-01

Introduction. We examined the murine hepatectomy model of liver regeneration (LR) in the setting of neoadjuvant chemotherapy. Methods. C57BL/6 mice were randomized to receive neoadjuvant intraperitoneal (IP) injections of a control, oxaliplatin (15 mg/kg), or irinotecan (100 mg/Kg or 250 mg/Kg) solution. Hepatectomy (70%) was performed 14 days after the final IP treatment. Animals were sacrificed at postoperative day (D) 0, 1, 2, 3, and 7. Liver remnants and serum were collected for analysis. T-tests for independent samples were used for statistical comparisons. Results. For oxaliplatin, percent LR did not differ at D1 or D2 but was significantly less at D3 (89.0% versus 70.0%, P = 0.048) with no difference on D7 (P = 0.21). Irinotecan-treated mice at both dose levels (100 mg/Kg and 250 mg/Kg) showed no significant differences in LR. BrdU incorporation was significantly decreased in oxaliplatin-treated animals (D1,2,3). Conclusions. Neoadjuvant oxaliplatin but not irinotecan impairs early LR in a posthepatectomy murine model which correlates with decreased DNA synthesis. PMID:22164336

Contribution of murine innate serum inhibitors toward interference within influenza virus immune assays

PubMed Central

Cwach, Kevin T.; Sandbulte, Heather R.; Klonoski, Joshua M.; Huber, Victor C.

2011-01-01

Please cite this paper as: Cwach et al. (2011) Contribution of murine innate serum inhibitors toward interference within influenza virus immune assays. Influenza and Other Respiratory Viruses DOI: 10.1111/j.1750‐2659.2011.00283.x. Background  Prior to detection of an antibody response toward influenza viruses using the hemagglutination inhibition assay (HAI), sera are routinely treated to inactivate innate inhibitors using both heat inactivation (56°C) and recombinant neuraminidase [receptor‐destroying enzyme (RDE)]. Objectives  We revisited the contributions of innate serum inhibitors toward interference with influenza viruses in immune assays, using murine sera, with emphasis on the interactions with influenza A viruses of the H3N2 subtype. Methods  We used individual serum treatments: 56°C alone, RDE alone, or RDE + 56°C, to treat sera prior to evaluation within HAI, microneutralization, and macrophage uptake assays. Results  Our data demonstrate that inhibitors present within untreated murine sera interfere with the HAI assay in a manner that is different from that seen for the microneutralization assay. Specifically, the γ class inhibitor α2‐Macroglobulin (A2‐M) can inhibit H3N2 viruses within the HAI assay, but not in the microneutralization assay. Based on these findings, we used a macrophage uptake assay to demonstrate that these inhibitors can increase uptake by macrophages when the influenza viruses express an HA from a 1968 H3N2 virus isolate, but not a 1997 H3N2 isolate. Conclusions  The practice of treating sera to inactivate innate inhibitors of influenza viruses prior to evaluation within immune assays has allowed us to effectively detect influenza virus‐specific antibodies for decades. However, this practice has yielded an under‐appreciation for the contribution of innate serum inhibitors toward host immune responses against these viruses, including contributions toward neutralization and macrophage uptake. PMID

A mutual activation loop between breast cancer cells and myeloid-derived suppressor cells facilitates spontaneous metastasis through IL-6 trans-signaling in a murine model.

PubMed

Oh, Keunhee; Lee, Ok-Young; Shon, Suh Youn; Nam, Onyou; Ryu, Po Mee; Seo, Myung Won; Lee, Dong-Sup

2013-01-01

Tumor cell interactions with the microenvironment, especially those of bone-marrow-derived myeloid cells, are important in various aspects of tumor metastasis. Myeloid-derived suppressor cells (MDSCs) have been suggested to constitute tumor-favoring microenvironments. In this study, we elucidated a novel mechanism by which the MDSCs can mediate spontaneous distant metastasis of breast cancer cells. Murine breast cancer cells, 4T1 and EMT6, were orthotopically grafted into the mammary fat pads of syngeneic BALB/c mice. CD11b(+)Gr-1(+) MDSCs in the spleen, liver, lung and primary tumor mass were analyzed. To evaluate the role of MDSCs in the distant metastasis, MDSCs were depleted or reconstituted in tumor-bearing mice. To evaluate whether MDSCs in the metastasizing tumor microenvironment affect breast cancer cell behavior, MDSCs and cancer cells were co-cultivated. To investigate the role of MDSCs in in vivo metastasis, we blocked the interactions between MDSCs and cancer cells. Using a murine breast cancer cell model, we showed that murine breast cancer cells with high IL-6 expression recruited more MDSCs and that the metastasizing capacity of cancer cells paralleled MDSC recruitment in tumor-bearing mice. Metastasizing, but not non-metastasizing, tumor-derived factors induced MDSCs to increase IL-6 production and full activation of recruited MDSCs occurred in the primary tumor site and metastatic organ in the vicinity of metastasizing cancer cells, but not in lymphoid organs. In addition, tumor-expanded MDSCs expressed Adam-family proteases, which facilitated shedding of IL-6 receptor, thereby contributing to breast cancer cell invasiveness and distant metastasis through IL-6 trans-signaling. The critical role of IL-6 trans-signaling was confirmed in both the afferent and efferent pathways of metastasis. In this study, we showed that metastasizing cancer cells induced higher MDSCs infiltration and prompted them to secret exaggerated IL-6 as well as soluble IL-6Ra

Oroxylin A Inhibits Allergic Airway Inflammation in Ovalbumin (OVA)-Induced Asthma Murine Model.

PubMed

Zhou, De-Gang; Diao, Bao-Zhong; Zhou, Wen; Feng, Jia-Long

2016-04-01

Oroxylin A, a natural flavonoid isolated from the medicinal herb Scutellaria baicalensis Georgi, has been reported to have anti-inflammatory property. In this study, we aimed to investigate the protective effects and mechanism of oroxylin A on allergic inflammation in OVA-induced asthma murine model. BABL/c mice were sensitized and airway-challenged with OVA to induce asthma. Oroxylin A (15, 30, and 60 mg/kg) was administered by oral gavage 1 h before the OVA treatment on day 21 to 23. The results showed that oroxylin A attenuated OVA-induced lung histopathologic changes, airway hyperresponsiveness, and the number of inflammatory cells. Oroxylin A also inhibited the levels of IL-4, IL-5, IL-13, and OVA-specific IgE in BALF. Furthermore, oroxylin A significantly inhibited OVA-induced NF-κB activation. In conclusion, these results suggested that oroxylin A inhibited airway inflammation in OVA-induced asthma murine model by inhibiting NF-κB activation. These results suggested that oroxylin A was a potential therapeutic drug for treating allergic asthma.

Anti-Inflammatory Effects of Rebamipide Eyedrop Administration on Ocular Lesions in a Murine Model of Primary Sjögren's Syndrome

PubMed Central

Arakaki, Rieko; Eguchi, Hiroshi; Yamada, Akiko; Kudo, Yasusei; Iwasa, Akihiko; Enkhmaa, Tserennadmid; Hotta, Fumika; Mitamura-Aizawa, Sayaka; Mitamura, Yoshinori; Hayashi, Yoshio; Ishimaru, Naozumi

2014-01-01

Background Topical therapy is effective for dry eye, and its prolonged effects should help in maintaining the quality of life of patients with dry eye. We previously reported that the oral administration of rebamipide (Reb), a mucosal protective agent, had a potent therapeutic effect on autoimmune lesions in a murine model of Sjögren's syndrome (SS). However, the effects of topical treatment with Reb eyedrops on the ocular lesions in the murine model of SS are unknown. Methods and Finding Reb eyedrops were administered to the murine model of SS aged 4–8 weeks four times daily. Inflammatory lesions of the extraorbital and intraorbital lacrimal glands and Harderian gland tissues were histologically evaluated. The direct effects of Reb on the lacrimal glands were analyzed using cultured lacrimal gland cells. Tear secretions of Reb-treated mice were significantly increased compared with those of untreated mice. In addition to the therapeutic effect of Reb treatment on keratoconjunctivitis, severe inflammatory lesions of intraorbital lacrimal gland tissues in this model of SS were resolved. The mRNA expression levels of IL-10 and mucin 5Ac in conjunctival tissues from Reb-treated mice was significantly increased compared with those of control mice. Moreover, lactoferrin production from lacrimal gland cells was restored by Reb treatment. Conclusion Topical Reb administration had an anti-inflammatory effect on the ocular autoimmune lesions in the murine model of SS and a protective effect on the ocular surfaces. PMID:24866156

Biotin deficiency up-regulates TNF-alpha production in murine macrophages.

PubMed

Kuroishi, Toshinobu; Endo, Yasuo; Muramoto, Koji; Sugawara, Shunji

2008-04-01

Biotin, a water-soluble vitamin of the B complex, functions as a cofactor of carboxylases that catalyze an indispensable cellular metabolism. Although significant decreases in serum biotin levels have been reported in patients with chronic inflammatory diseases, the biological roles of biotin in inflammatory responses are unclear. In this study, we investigated the effects of biotin deficiency on TNF-alpha production. Mice were fed a basal diet or a biotin-deficient diet for 8 weeks. Serum biotin levels were significantly lower in biotin-deficient mice than biotin-sufficient mice. After i.v. administration of LPS, serum TNF-alpha levels were significantly higher in biotin-deficient mice than biotin-sufficient mice. A murine macrophage-like cell line, J774.1, was cultured in a biotin-sufficient or -deficient medium for 4 weeks. Cell proliferation and biotinylation of intracellular proteins were decreased significantly in biotin-deficient cells compared with biotin-sufficient cells. Significantly higher production and mRNA expression of TNF-alpha were detected in biotin-deficient J774.1 cells than biotin-sufficient cells in response to LPS and even without LPS stimulation. Intracellular TNF-alpha expression was inhibited by actinomycin D, indicating that biotin deficiency up-regulates TNF-alpha production at the transcriptional level. However, the expression levels of TNF receptors, CD14, and TLR4/myeloid differentiation protein 2 complex were similar between biotin-sufficient and -deficient cells. No differences were detected in the activities of the NF-kappaB family or AP-1. The TNF-alpha induction by biotin deficiency was down-regulated by biotin supplementation in vitro and in vivo. These results indicate that biotin deficiency may up-regulate TNF-alpha production or that biotin excess down-regulates TNF-alpha production, suggesting that biotin status may influence inflammatory diseases.

Enhancers Are Major Targets for Murine Leukemia Virus Vector Integration

PubMed Central

De Ravin, Suk See; Su, Ling; Theobald, Narda; Choi, Uimook; Macpherson, Janet L.; Poidinger, Michael; Symonds, Geoff; Pond, Susan M.; Ferris, Andrea L.; Hughes, Stephen H.

2014-01-01

ABSTRACT Retroviral vectors have been used in successful gene therapies. However, in some patients, insertional mutagenesis led to leukemia or myelodysplasia. Both the strong promoter/enhancer elements in the long terminal repeats (LTRs) of murine leukemia virus (MLV)-based vectors and the vector-specific integration site preferences played an important role in these adverse clinical events. MLV integration is known to prefer regions in or near transcription start sites (TSS). Recently, BET family proteins were shown to be the major cellular proteins responsible for targeting MLV integration. Although MLV integration sites are significantly enriched at TSS, only a small fraction of the MLV integration sites (<15%) occur in this region. To resolve this apparent discrepancy, we created a high-resolution genome-wide integration map of more than one million integration sites from CD34+ hematopoietic stem cells transduced with a clinically relevant MLV-based vector. The integration sites form ∼60,000 tight clusters. These clusters comprise ∼1.9% of the genome. The vast majority (87%) of the integration sites are located within histone H3K4me1 islands, a hallmark of enhancers. The majority of these clusters also have H3K27ac histone modifications, which mark active enhancers. The enhancers of some oncogenes, including LMO2, are highly preferred targets for integration without in vivo selection. IMPORTANCE We show that active enhancer regions are the major targets for MLV integration; this means that MLV preferentially integrates in regions that are favorable for viral gene expression in a variety of cell types. The results provide insights for MLV integration target site selection and also explain the high risk of insertional mutagenesis that is associated with gene therapy trials using MLV vectors. PMID:24501411

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

USDA-ARS?s Scientific Manuscript database

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...

Development of Murine Cyp3a Knockout Chimeric Mice with Humanized Liver.

PubMed

Kato, Kota; Ohbuchi, Masato; Hamamura, Satoko; Ohshita, Hiroki; Kazuki, Yasuhiro; Oshimura, Mitsuo; Sato, Koya; Nakada, Naoyuki; Kawamura, Akio; Usui, Takashi; Kamimura, Hidetaka; Tateno, Chise

2015-08-01

We developed murine CYP3A knockout ko chimeric mice with humanized liver expressing human P450S similar to those in humans and whose livers and small intestines do not express murine CYP3A this: approach may overcome effects of residual mouse metabolic enzymes like Cyp3a in conventional chimeric mice with humanized liver, such as PXB-mice [urokinase plasminogen activator/severe combined immunodeficiency (uPA/SCID) mice repopulated with over 70% human hepatocytes] to improve the prediction of drug metabolism and pharmacokinetics in humans. After human hepatocytes were transplanted into Cyp3a KO/uPA/SCID host mice, human albumin levels logarithmically increased until approximately 60 days after transplantation, findings similar to those in PXB-mice. Quantitative real-time-polymerase chain reaction analyses showed that hepatic human P450s, UGTs, SULTs, and transporters mRNA expression levels in Cyp3a KO chimeric mice were also similar to those in PXB-mice and confirmed the absence of Cyp3a11 mRNA expression in mouse liver and intestine. Findings for midazolam and triazolam metabolic activities in liver microsomes were comparable between Cyp3a KO chimeric mice and PXB-mice. In contrast, these activities in the intestine of Cyp3a KO chimeric mice were attenuated compared with PXB-mice. Owing to the knockout of murine Cyp3a, hepatic Cyp2b10 and 2c55 mRNA levels in Cyp3a KO/uPA/SCID mice (without hepatocyte transplants) were 8.4- and 61-fold upregulated compared with PXB-mice, respectively. However, human hepatocyte transplantation successfully restored Cyp2b10 level nearly fully and Cyp2c55 level partly (still 13-fold upregulated) compared with those in PXB-mice. Intestinal Cyp2b10 and 2c55 were also repressed by human hepatocyte transplantation in Cyp3a KO chimeric mice. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Clinical and molecular genetic characterisation of a family segregating autosomal dominant retinitis pigmentosa and sensorineural deafness.

PubMed

Kenna, P; Mansergh, F; Millington-Ward, S; Erven, A; Kumar-Singh, R; Brennan, R; Farrar, G J; Humphries, P

1997-03-01

To characterise clinically a large kindred segregating retinitis pigmentosa and sensorineural hearing impairment in an autosomal dominant pattern and perform genetic linkage studies in this family. Extensive linkage analysis in this family had previously excluded the majority of loci shown to be involved in the aetiologies of RP, some other forms of inherited retinal degeneration, and inherited deafness. Members of the family were subjected to detailed ophthalmic and audiological assessment. In addition, some family members underwent skeletal muscle biopsy, electromyography, and electrocardiography. Linkage analysis using anonymous microsatellite markers was performed on DNA samples from all living members of the pedigree. Patients in this kindred have a retinopathy typical of retinitis pigmentosa in addition to a hearing impairment. Those members of the pedigree examined demonstrated a subclinical myopathy, as evidence by abnormal skeletal muscle histology, electromyography, and electrocardiography. LOD scores of Zmax = 3.75 (theta = 0.10), Zmax = 3.41 (theta = 0.10), and Zmax = 3.25 (theta = 0.15) respectively were obtained with the markers D9S118, D9S121, and ASS, located on chromosome 9q34-qter, suggesting that the causative gene in this family may lie on the long arm (q) of chromosome 9. These data indicate that the gene responsible for the phenotype in this kindred is located on chromosome 9 q. These data, together with evidence that a murine deafness gene is located in a syntenic area of the mouse genome, should direct the research community to consider this area as a candidate region for retinopathy and/or deafness genes.

Maleimide conjugation markedly enhances the immunogenicity of both human and murine idiotype-KLH vaccines

PubMed Central

Kafi, Kamran; Betting, David J.; Yamada, Reiko E.; Bacica, Michael; Steward, Kristopher K.; Timmerman, John M.

2009-01-01

The collection of epitopes present within the variable regions of the tumor-specific clonal immunoglobulin expressed by B cell lymphomas (idiotype, Id) can serve as a target for active immunotherapy. Traditionally, tumor-derived Id protein is chemically-conjugated to the immunogenic foreign carrier protein keyhole limpet hemocyanin (KLH) using glutaraldehyde to serve as a therapeutic vaccine. While this approach offered promising results for some patients treated in early clinical trials, glutaraldehyde Id-KLH vaccines have failed to induce immune and clinical responses in many vaccinated subjects. We recently described an alternative conjugation method employing maleimide-sulfhydryl chemistry that significantly increased the therapeutic efficacy of Id-KLH vaccines in three different murine B cell lymphoma models, with protection mediated by either CD8+ T cells or antibodies. We now define in detail the methods and parameters critical for enhancing the in vivo immunogenicity of human as well as murine Id-KLH conjugate vaccines. Optimal conditions for Id sulfhydryl pre-reduction were determined, and maleimide Id-KLH conjugates maintained stability and potency even after prolonged storage. Field flow fractionation analysis of Id-KLH particle size revealed that maleimide conjugates were far more uniform in size than glutaraldehyde conjugates. Under increasingly stringent conditions, maleimide Id-KLH vaccines maintained superior efficacy over glutaraldehyde Id-KLH in treating established, disseminated murine lymphoma. More importantly, human maleimide Id-KLH conjugates were consistently superior to glutaraldehyde Id-KLH conjugates in inducing Id-specific antibody and T cell responses. The described methods should be easily adaptable to the production of clinical grade vaccines for human trials in B cell malignancies. PMID:19046770

Cloning and Characterization of the Genes Encoding the Murine Homologues of the Human Melanoma Antigens MART1 and gp100

PubMed Central

Zhai, Yifan; Yang, James C.; Spiess, Paul; Nishimura, Michael I.; Overwijk, Willem W.; Roberts, Bruce; Restifo, Nicholas P.; Rosenberg, Steven A.

2008-01-01

The recent identification of genes encoding melanoma-associated antigens has opened new possibilities for the development of cancer vaccines designed to cause the rejection of established tumors. To develop a syngeneic animal model for evaluating antigen-specific vaccines in cancer therapy, the murine homologues of the human melanoma antigens MART1 and gp 100, which were specifically recognized by tumor-infiltrating lymphocytes from patients with melanoma, were cloned and sequenced from a murine B16 melanoma cDNA library. The open reading frames of murine MART1 and gp 100 encode proteins of 113- and 626-amino acids with 68.8 and 77% identity to the respective human proteins. Comparison of the DNA sequences of the murine MART1 genes, derived from normal melanocytes, the immortalized nontumorgenic melanocyte line Melan-a and the B16 melanoma, showed all to be identical. Northern and Western blot analyses confirmed that both genes encoded products that were melanocyte lineage proteins. Mice immunized with murine MART1 or gp 100 using recombinant vaccinia virus failed to produce any detectable T-cell responses or protective immunity against B16 melanoma. In contrast, immunization of mice with human gp 100 using recombinant adenoviruses elicited T cells specific for hgp100, but these T cells also cross reacted with B16 tumor in vitro and induced significant but weak protection against B16 challenge. Immunization with human and mouse gp100 together [adenovirus type 2 (Ad2)-hep100 plus recombinant vaccinia virus (rVV)-mgp100], or immunization with human gp100 (Ad2-hgp100) and boosting with heterologous vector (rVV-hgp100 or rVV-mgp100) or homologous vector (Ad2-hgp100), did not significantly enhance the protective response against B16 melanoma. These results may suggest that immunization with heterologous tumor antigen, rather than self, may be more effective as an immunotherapeutic reagent in designing antigen-specific cancer vaccines. PMID:9101410

Differential chemokine responses in the murine brain following lyssavirus infection.

PubMed

Hicks, D J; Núñez, A; Banyard, A C; Williams, A; Ortiz-Pelaez, A; Fooks, A R; Johnson, N

2013-11-01

The hallmark of lyssavirus infection is lethal encephalomyelitis. Previous studies have reported distinct lyssavirus isolate-related differences in severity of cellular recruitment into the encephalon in a murine model of infection following peripheral inoculation with rabies virus (RABV) and European bat lyssavirus (EBLV)-1 and -2. In order to understand the role of chemokines in this process, comparative studies of the chemokine pattern, distribution and production in response to infection with these lyssaviruses were undertaken. Expression of CCL2, CCL5 and CXCL10 was observed throughout the murine brain with a distinct caudal bias in distribution, similar to both inflammatory changes and virus antigen distribution. CCL2 immunolabelling was localized to neuronal and astroglial populations. CCL5 immunolabelling was only detected in the astroglia, while CXCL10 labelling, although present in the astroglia, was more prominent in neurons. Isolate-dependent differences in the amount of chemokine immunolabelling in specific brain regions and chemokine production by neurons in vitro were observed, with a greater expression of CCL5 in vivo and CXCL10 production in vitro after EBLV infection. Additionally, strong positive associations between chemokine immunolabelling and perivascular cuffing and, to a lesser extent, virus antigen score were also observed. These differences in chemokine expression may explain the variation in severity of encephalitic changes observed in animals infected with different lyssavirus isolates. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

Inhibition of soluble epoxide hydrolase contributes to the anti-inflammatory effect of antimicrobial triclocarban in a murine model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu Junyan; Qiu Hong; Morisseau, Christophe

The increasing use of the antimicrobial triclocarban (TCC) in personal care products (PCPs) has resulted in concern regarding environmental pollution. TCC is a potent inhibitor of soluble epoxide hydrolase (sEH). Inhibitors of sEH (sEHIs) are anti-inflammatory, anti-hypertensive and cardio-protective in multiple animal models. However, the in vivo effects anticipated from a sEHI have not been reported for TCC. Here we demonstrated the anti-inflammatory effects in vivo of TCC in a murine model. TCC was employed in a lipopolysaccharide (LPS)-challenged murine model. Systolic blood pressure, plasma levels of several inflammatory cytokines and chemokine, and metabolomic profile of plasma oxylipins were determined.more » TCC significantly reversed LPS-induced morbid hypotension in a time-dependent manner. TCC significantly repressed the increased release of inflammatory cytokines and chemokine caused by LPS. Furthermore, TCC significantly shifted the oxylipin profile in vivo in a time-dependent manner towards resolution of inflammation as expected from a sEHI. These results demonstrated that at the doses used TCC is anti-inflammatory in the murine model. This study suggests that TCC may provide some benefits in humans in addition to its antimicrobial activities due to its potent inhibition of sEH. It may be a promising starting point for developing new low volume high value applications of TCC. However these biological effects also caution against the general over use of TCC in PCPs. - Graphical abstract: Display Omitted Research Highlights: > Anti-microbial triclocarban (TCC) is anti-inflammatory in a murine model. > TCC significantly shifted the oxylipin profile in vivo as expected from a sEHI. > TCC significantly reversed LPS-induced morbid hypotension in a time-dependent manner. > TCC significantly repressed LPS-induced increased release of inflammatory cytokines.« less

Angiogenic effect of platelet-rich plasma combined with gelatin hydrogel granules injected into murine subcutis.

PubMed

Kakudo, Natsuko; Morimoto, Naoki; Ogawa, Takeshi; Hihara, Masakatsu; Notodihardjo, Priscilla Valentin; Matsui, Makoto; Tabata, Yasuhiko; Kusumoto, Kenji

2017-07-01

Platelet-rich plasma (PRP), which contains highly concentrated platelets, is produced by centrifuging whole blood. It is a safe and readily available source of a wide range of growth factors necessary for angiogenesis. Gelatin hydrogel granules have been designed and prepared for the controlled release of many growth factors. The angiogenic effect of human PRP was examined in vitro, and the effect of its subcutaneous injection with gelatin hydrogel granules into murine subcutis was evaluated. Human PRP was prepared using a double-spin method. The concentration of growth factors and the platelet count were examined in PRP and in vitro, and the angiogenic activity of human umbilical vein endothelial cells (HUVECs) in co-culture with human dermal fibroblast cells (NHDFs) in the presence and absence of PRP was evaluated. Then, in vivo, PRP, either free or with gelatin hydrogel granules, was injected subcutaneously into tiebacks on mice. Using a microscope and Kurabo angiogenesis image analyser software, the area containing newly formed capillaries was evaluated histologically and the microvascular network score was calculated. PRP was shown to contain high concentrations of PDGF, VEGF and TGFβ and had an angiogenic effect on the co-culture system. PRP with gelatin hydrogel granules significantly enlarged the area containing newly formed capillaries and promoted the microvascular network in murine subcutaneous tissue. PRP encapsulated in gelatin hydrogel microspheres shows promise for enhancing angiogenic effects in murine subcutis and could represent a potential therapeutic combination for the treatment of ischaemic disorders. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

A mixture of amino acids and other small molecules present in the serum suppresses the growth of murine and human tumors in vivo

PubMed Central

Kulcsár, Gyula; Gaál, Dezső; Kulcsár, Péter I; Schulcz, Ákos; Czömpöly, Tamás

2013-01-01

Previously we have hypothesized that the small molecules which are selectively accumulated in cancer cells might participate in a non-immunological antitumor surveillance mechanism. We demonstrated earlier that a mixture of experimentally selected substances (“active mixture”, AM: l-arginine, l-histidine, l-methionine, l-phenylalanine, l-tyrosine, l-tryptophan, l-ascorbate, d-biotin, pyridoxine, riboflavin, adenine, l(-)malate) possesses a selective toxic effect in vitro on a variety of tumor cell lines, and we have shown that the AM selectively induces apoptosis of cancer cells in vitro. To explore the in vivo significance of our earlier findings we examined the antitumor effect of AM in Colon 26 murine colorectal adenocarcinoma, B16 murine melanoma, MXT murine mammary carcinoma, S180 murine sarcoma, P388 murine lymphoid leukemia, HL-60 human promyeloid leukemia, PC-3 human prostate carcinoma, and HT-29 human colon carcinoma tumor models. Treatment of tumor bearing mice with AM inhibited the growth of the tumors investigated, with an inhibitory effect ranging from 40 to 69%. The AM had a comparable antitumor effect with 5-fluorouracil and cisplatin in the Colon-26 tumor model, and combined treatment with AM and 5-fluorouracil or cisplatin resulted in an enhanced tumor growth inhibitory effect. The AM induced apoptosis through the mitochondrial pathway and induced G1 arrest in PC-3 cells and increased the number of apoptotic cells in PC-3 xenografts. These findings suggest that the AM might offer an interesting perspective in the treatment of cancer and in combination with other treatments may offer hope for a more effective cancer therapy. PMID:22858865

A mixture of amino acids and other small molecules present in the serum suppresses the growth of murine and human tumors in vivo.

PubMed

Kulcsár, Gyula; Gaál, Dezső; Kulcsár, Péter I; Schulcz, Ákos; Czömpöly, Tamás

2013-03-01

Previously we have hypothesized that the small molecules which are selectively accumulated in cancer cells might participate in a non-immunological antitumor surveillance mechanism. We demonstrated earlier that a mixture of experimentally selected substances ("active mixture", AM: L-arginine, L-histidine, L-methionine, L-phenylalanine, L-tyrosine, L-tryptophan, L-ascorbate, D-biotin, pyridoxine, riboflavin, adenine, L(-)malate) possesses a selective toxic effect in vitro on a variety of tumor cell lines, and we have shown that the AM selectively induces apoptosis of cancer cells in vitro. To explore the in vivo significance of our earlier findings we examined the antitumor effect of AM in Colon 26 murine colorectal adenocarcinoma, B16 murine melanoma, MXT murine mammary carcinoma, S180 murine sarcoma, P388 murine lymphoid leukemia, HL-60 human promyeloid leukemia, PC-3 human prostate carcinoma, and HT-29 human colon carcinoma tumor models. Treatment of tumor bearing mice with AM inhibited the growth of the tumors investigated, with an inhibitory effect ranging from 40 to 69%. The AM had a comparable antitumor effect with 5-fluorouracil and cisplatin in the Colon-26 tumor model, and combined treatment with AM and 5-fluorouracil or cisplatin resulted in an enhanced tumor growth inhibitory effect. The AM induced apoptosis through the mitochondrial pathway and induced G1 arrest in PC-3 cells and increased the number of apoptotic cells in PC-3 xenografts. These findings suggest that the AM might offer an interesting perspective in the treatment of cancer and in combination with other treatments may offer hope for a more effective cancer therapy. Copyright © 2012 UICC.

Electrocautery effect on intestinal vascularisation in a murine model.

PubMed

Tremblay, Jean-François; Sideris, Lucas; Leblond, François A; Trépanier, Jean-Sébastien; Badrudin, David; Drolet, Pierre; Mitchell, Andrew; Dubé, Pierre

2016-09-01

The use of electrocautery devices is associated with complications such as perforation or fistulisation when used near intestinal structures. This is likely due to its effect on vascularisation of the bowel wall. To test this hypothesis we established a murine model to quantify the effect of electrocautery injury on the intestinal microvascularisation. Sprague-Dawley rats were subjected to five electrocautery injuries on the small bowel in coagulation mode (30 W intensity) and in cut mode (40 W, 80 W and 200 W intensities) for durations of 1, 2 and 5 s. 5 mg/kg of fluorescein was injected intravenously, the injured bowel segments harvested and the rat sacrificed. The segments were analysed to measure the fluorescence of injured bowel compared to adjacent unharmed tissue. A significant decrease in bowel wall microvascularisation occurred with increasing intensity (coag 30 W/cut 40 W versus cut 200 W 1 s: p < 0.05) and duration of electrocautery injury (cut 40 W 1/2 s versus 5 s: p < 0.05). There was a 40% perforation rate when decreased bowel wall microvascularisation was 25% or more. Despite similar electrocautery injury, a significantly greater microvascularisation decrease was observed in jejunum compared to ileum (p < 0.05). We successfully established a murine model to quantify the decrease of bowel wall microvascularisation associated with electrocautery use. Unsurprisingly, the decrease in microvascularisation is greater with higher intensity and duration of electrocautery and is associated with more perforations in the experimental model. The jejunum seems more vulnerable to electrocautery injury than the ileum. These observations support caution when using electrocautery devices near intestinal structures.

Ginseng ameliorates chronic histopathologic changes in a murine model of asthma.

PubMed

Babayigit, Arzu; Olmez, Duygu; Karaman, Ozkan; Bagriyanik, H Alper; Yilmaz, Osman; Kivcak, Bijen; Erbil, Guven; Uzuner, Nevin

2008-01-01

Currently, asthma therapies are effective in reducing inflammation but airway remodeling is poorly responsive to these agents. New therapeutic options that have fewer side effects and reverse chronic changes in the lungs are essential. This study aimed to determine the efficacy of oral administration of ginseng on lung histopathology in a murine model of chronic asthma. BALB/c mice were divided into four groups: control, placebo, ginseng, and dexamethasone. All mice except those in the control group were sensitized and challenged with ovalbumin. Then, mice in the ginseng group were given 2 gr/kg per day of ginseng and mice in the dexamethasone group received 1 mg/kg per day of dexamethasone via orogastic gavage once daily for 1 week. Lung histopathology was evaluated by using light and electron microscopy in all groups. All of the chronic changes of airways in the ginseng group were significantly ameliorated when compared with the placebo group. When compared with the dexamethasone group, the ginseng group had significantly lower numbers of mast cell count. Thicknesses of basement membrane, epithelium, and subepithelial smooth muscle were not statistically different between the ginseng and dexamethasone groups. Goblet cell numbers were much more reduced in the dexamethasone group. Ginseng is effective in resolving the established chronic histopathological changes of the lungs in the murine model of asthma.

Modulation of fibronectin-mediated Bacillus Calmette-Guérin attachment to murine bladder mucosa by drugs influencing the coagulation pathways.

PubMed

Hudson, M A; Brown, E J; Ritchey, J K; Ratliff, T L

1991-07-15

Adjuvant intravesical Bacillus Calmette-Guérin (BCG) has proved to be an effective treatment for superficial bladder cancer. Intraluminal attachment of BCG organisms via binding to the extracellular matrix protein, fibronectin (FN), appears to be required for expression of the antitumor efficacy of BCG against a murine bladder tumor. Initial studies demonstrated that radiolabeled FN localized to the acutely injured urothelium but not to intact urothelium. These studies also demonstrated that exogenous administration of FN enhanced BCG attachment to the injured but not to the intact urothelium. Because FN has been shown to be an integral part of clot formation at sites of urothelial injury, drugs known to affect fibrin clot formation were tested for their effects on BCG attachment and antitumor efficacy in a murine bladder tumor model. A stabilizer of fibrin clot formation was shown to enhance both BCG attachment and antitumor efficacy in the same model. An increased number of BCG organisms were also retained in the lymph nodes and spleens of mice receiving fibrin clot stabilizers, suggesting indirectly that immunological mechanisms are involved in the antitumor efficacy of BCG. The data presented herein provide further support for the hypothesis that BCG attachment to the injured bladder is mediated by FN. Furthermore, modulation of BCG-FN attachment is demonstrated to be possible with drugs influencing the coagulation pathway. This attachment is shown to be required for the antitumor efficacy in a murine bladder tumor model, and thus modulation of BCG-FN attachment appears to have significant influence on the antitumor efficacy of BCG in the murine bladder tumor model.

Antileukemic effect of zerumbone-loaded nanostructured lipid carrier in WEHI-3B cell-induced murine leukemia model

PubMed Central

Rahman, Heshu Sulaiman; Rasedee, Abdullah; How, Chee Wun; Zeenathul, Nazariah Allaudin; Chartrand, Max Stanley; Yeap, Swee Keong; Abdul, Ahmad Bustamam; Tan, Sheau Wei; Othman, Hemn Hassan; Ajdari, Zahra; Namvar, Farideh; Arulselvan, Palanisamy; Fakurazi, Sharida; Mehrbod, Parvaneh; Daneshvar, Nasibeh; Begum, Hasina

2015-01-01

Cancer nanotherapy is progressing rapidly with the introduction of many innovative drug delivery systems to replace conventional therapy. Although the antitumor activity of zerumbone (ZER) has been reported, there has been no information available on the effect of ZER-loaded nanostructured lipid carrier (NLC) (ZER-NLC) on murine leukemia cells. In this study, the in vitro and in vivo effects of ZER-NLC on murine leukemia induced with WEHI-3B cells were investigated. The results from 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Hoechst 33342, Annexin V, cell cycle, and caspase activity assays showed that the growth of leukemia cells in vitro was inhibited by ZER-NLC. In addition, outcomes of histopathology, transmission electron microscopy, and Tdt-mediated dUTP nick-end labeling analyses revealed that the number of leukemia cells in the spleen of BALB/c leukemia mice significantly decreased after 4 weeks of oral treatment with various doses of ZER-NLC. Western blotting and reverse-transcription quantitative polymerase chain reaction assays confirmed the antileukemia effects of ZER-NLC. In conclusion, ZER-NLC was shown to induce a mitochondrial-dependent apoptotic pathway in murine leukemia. Loading of ZER in NLC did not compromise the anticancer effect of the compound, suggesting ZER-NLC as a promising and effective delivery system for treatment of cancers. PMID:25767386

Antileukemic effect of zerumbone-loaded nanostructured lipid carrier in WEHI-3B cell-induced murine leukemia model.

PubMed

Rahman, Heshu Sulaiman; Rasedee, Abdullah; How, Chee Wun; Zeenathul, Nazariah Allaudin; Chartrand, Max Stanley; Yeap, Swee Keong; Abdul, Ahmad Bustamam; Tan, Sheau Wei; Othman, Hemn Hassan; Ajdari, Zahra; Namvar, Farideh; Arulselvan, Palanisamy; Fakurazi, Sharida; Mehrbod, Parvaneh; Daneshvar, Nasibeh; Begum, Hasina

2015-01-01

Cancer nanotherapy is progressing rapidly with the introduction of many innovative drug delivery systems to replace conventional therapy. Although the antitumor activity of zerumbone (ZER) has been reported, there has been no information available on the effect of ZER-loaded nanostructured lipid carrier (NLC) (ZER-NLC) on murine leukemia cells. In this study, the in vitro and in vivo effects of ZER-NLC on murine leukemia induced with WEHI-3B cells were investigated. The results from 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Hoechst 33342, Annexin V, cell cycle, and caspase activity assays showed that the growth of leukemia cells in vitro was inhibited by ZER-NLC. In addition, outcomes of histopathology, transmission electron microscopy, and Tdt-mediated dUTP nick-end labeling analyses revealed that the number of leukemia cells in the spleen of BALB/c leukemia mice significantly decreased after 4 weeks of oral treatment with various doses of ZER-NLC. Western blotting and reverse-transcription quantitative polymerase chain reaction assays confirmed the antileukemia effects of ZER-NLC. In conclusion, ZER-NLC was shown to induce a mitochondrial-dependent apoptotic pathway in murine leukemia. Loading of ZER in NLC did not compromise the anticancer effect of the compound, suggesting ZER-NLC as a promising and effective delivery system for treatment of cancers.

A novel intermediate in processing of murine leukemia virus envelope glycoproteins. Proteolytic cleavage in the late Golgi region.

PubMed

Bedgood, R M; Stallcup, M R

1992-04-05

The intracellular processing of the murine leukemia virus envelope glycoprotein precursor Pr85 to the mature products gp70 and p15e was analyzed in the mouse T-lymphoma cell line W7MG1. Kinetic (pulse-chase) analysis of synthesis and processing, coupled with endoglycosidase (endo H) and neuraminidase digestions revealed the existence of a novel high molecular weight processing intermediate, gp95, containing endo H-resistant terminally glycosylated oligosaccharide chains. In contrast to previously published conclusions, our data indicate that proteolytic cleavage of the envelope precursor occurs after the acquisition of endo H-resistant chains and terminal glycosylation and thus after the mannosidase II step. In the same W7MG1 cell line, the type and order of murine leukemia virus envelope protein processing events was identical to that for the mouse mammary tumor virus envelope protein. Interestingly, complete mouse mammary tumor virus envelope protein processing requires the addition of glucocorticoid hormone, whereas murine leukemia virus envelope protein processing occurs constitutively in these W7MG1 cells. We propose that all retroviral envelope proteins share a common processing pathway in which proteolytic processing is a late event that follows acquisition of endo H resistance and terminal glycosylation.

Association of murine lupus and thymic full-length endogenous retroviral expression maps to a bone marrow stem cell

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krieg, A.M.; Gourley, M.F.; Steinberg, A.D.

1991-05-01

Recent studies of thymic gene expression in murine lupus have demonstrated 8.4-kb (full-length size) modified polytropic (Mpmv) endogenous retroviral RNA. In contrast, normal control mouse strains do not produce detectable amounts of such RNA in their thymuses. Prior studies have attributed a defect in experimental tolerance in murine lupus to a bone marrow stem cell rather than to the thymic epithelium; in contrast, infectious retroviral expression has been associated with the thymic epithelium, rather than with the bone marrow stem cell. The present study was designed to determine whether the abnormal Mpmv expression associated with murine lupus mapped to thymicmore » epithelium or to a marrow precursor. Lethally irradiated control and lupus-prone mice were reconstituted with T cell depleted bone marrow; one month later their thymuses were studied for endogenous retroviral RNA and protein expression. Recipients of bone marrow from nonautoimmune donors expressed neither 8.4-kb Mpmv RNA nor surface MCF gp70 in their thymuses. In contrast, recipients of bone marrow from autoimmune NZB or BXSB donors expressed thymic 8.4-kb Mpmv RNA and mink cell focus-forming gp70. These studies demonstrate that lupus-associated 8.4-kb Mpmv endogenous retroviral expression is determined by bone marrow stem cells.« less

Murine and Human Tissue-Engineered Esophagus Form from Sufficient Stem/Progenitor Cells and Do Not Require Microdesigned Biomaterials

PubMed Central

Spurrier, Ryan Gregory; Speer, Allison L.; Hou, Xiaogang; El-Nachef, Wael N.

2015-01-01

Purpose: Tissue-engineered esophagus (TEE) may serve as a therapeutic replacement for absent foregut. Most prior esophagus studies have favored microdesigned biomaterials and yielded epithelial growth alone. None have generated human TEE with mesenchymal components. We hypothesized that sufficient progenitor cells might only require basic support for successful generation of murine and human TEE. Materials and Methods: Esophageal organoid units (EOUs) were isolated from murine or human esophagi and implanted on a polyglycolic acid/poly-l-lactic acid collagen-coated scaffold in adult allogeneic or immune-deficient mice. Alternatively, EOU were cultured for 10 days in vitro prior to implantation. Results: TEE recapitulated all key components of native esophagus with an epithelium and subjacent muscularis. Differentiated suprabasal and proliferative basal layers of esophageal epithelium, muscle, and nerve were identified. Lineage tracing demonstrated that multiple EOU could contribute to the epithelium and mesenchyme of a single TEE. Cultured murine EOU grew as an expanding sphere of proliferative basal cells on a neuromuscular network that demonstrated spontaneous peristalsis in culture. Subsequently, cultured EOU generated TEE. Conclusions: TEE forms after transplantation of mouse and human organ-specific stem/progenitor cells in vivo on a relatively simple biodegradable scaffold. This is a first step toward future human therapies. PMID:25298083

Molecular characterisation of murine acute myeloid leukaemia induced by 56Fe ion and 137Cs gamma ray irradiation.

PubMed

Steffen, Leta S; Bacher, Jeffery W; Peng, Yuanlin; Le, Phuong N; Ding, Liang-Hao; Genik, Paula C; Ray, F Andrew; Bedford, Joel S; Fallgren, Christina M; Bailey, Susan M; Ullrich, Robert L; Weil, Michael M; Story, Michael D

2013-01-01

Exposure to sparsely ionising gamma- or X-ray irradiation is known to increase the risk of leukaemia in humans. However, heavy ion radiotherapy and extended space exploration will expose humans to densely ionising high linear energy transfer (LET) radiation for which there is currently no understanding of leukaemia risk. Murine models have implicated chromosomal deletion that includes the hematopoietic transcription factor gene, PU.1 (Sfpi1), and point mutation of the second PU.1 allele as the primary cause of low-LET radiation-induced murine acute myeloid leukaemia (rAML). Using array comparative genomic hybridisation, fluorescence in situ hybridisation and high resolution melt analysis, we have confirmed that biallelic PU.1 mutations are common in low-LET rAML, occurring in 88% of samples. Biallelic PU.1 mutations were also detected in the majority of high-LET rAML samples. Microsatellite instability was identified in 42% of all rAML samples, and 89% of samples carried increased microsatellite mutant frequencies at the single-cell level, indicative of ongoing instability. Instability was also observed cytogenetically as a 2-fold increase in chromatid-type aberrations. These data highlight the similarities in molecular characteristics of high-LET and low-LET rAML and confirm the presence of ongoing chromosomal and microsatellite instability in murine rAML.

Diminished potential for B-lymphoid differentiation after murine leukemia virus infection in vivo and in EML hematopoietic progenitor cells.

PubMed

Finstad, Samantha L; Rosenberg, Naomi; Levy, Laura S

2007-07-01

Infection with a recombinant murine-feline gammaretrovirus, MoFe2, or with the parent virus, Moloney murine leukemia virus, caused significant reduction in B-lymphoid differentiation of bone marrow at 2 to 8 weeks postinfection. The suppression was selective, in that myeloid potential was significantly increased by infection. Analysis of cell surface markers and immunoglobulin H gene rearrangements in an in vitro model demonstrated normal B-lymphoid differentiation after infection but significantly reduced viability of differentiating cells. This reduction in viability may confer a selective advantage on undifferentiated lymphoid progenitors in the bone marrow of gammaretrovirus-infected animals and thereby contribute to the establishment of a premalignant state.

First transplantation of isolated murine follicles in alginate.

PubMed

Vanacker, Julie; Dolmans, Marie-Madeleine; Luyckx, Valérie; Donnez, Jacques; Amorim, Christiani A

2014-01-01

Our aim is to develop an artificial ovary allowing survival and growth of isolated follicles and ovarian cells, to restore fertility in women diagnosed with pathologies at high risk of ovarian involvement. For this, alginate beads containing isolated preantral follicles and ovarian cells were autografted to immunocompetent mice. One week after grafting, the beads were invaded by proliferating murine cells (12.1%) and capillaries. The recovery rate of follicles per graft ranged from 0% to 35.5%. Of the analyzed follicles, 77% were Ki67-positive and 81%, TUNEL-negative. Three antral follicles were also identified, evidencing their ability to grow in the matrix. Our results suggest that an artificial ovary is now conceivable, opening new perspectives to restore fertility in women.

Melanocyte pigmentation stiffens murine cardiac tricuspid valve leaflet

PubMed Central

Balani, Kantesh; Brito, Flavia C.; Kos, Lidia; Agarwal, Arvind

2009-01-01

Pigmentation of murine cardiac tricuspid valve leaflet is associated with melanocyte concentration, which affects its stiffness. Owing to its biological and viscoelastic nature, estimation of the in situ stiffness measurement becomes a challenging task. Therefore, quasi-static and nanodynamic mechanical analysis of the leaflets of the mouse tricuspid valve is performed in the current work. The mechanical properties along the leaflet vary with the degree of pigmentation. Pigmented regions of the valve leaflet that contain melanocytes displayed higher storage modulus (7–10 GPa) than non-pigmented areas (2.5–4 GPa). These results suggest that the presence of melanocytes affects the viscoelastic properties of the mouse atrioventricular valves and are important for their proper functioning in the organism. PMID:19586956

The kin17 Protein in Murine Melanoma Cells

PubMed Central

Ramos, Anelise C.; Gaspar, Vanessa P.; Kelmer, Sabrina M. G.; Sellani, Tarciso A.; Batista, Ana G. U.; De Lima Neto, Quirino A.; Rodrigues, Elaine G.; Fernandez, Maria A.

2015-01-01

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. PMID:26610484

Family Structure and Family Processes in Mexican American Families

PubMed Central

Zeiders, Katharine H.; Roosa, Mark W.; Tein, Jenn-Yun

2010-01-01

Despite increases in single-parent families among Mexican Americans (MA), few studies have examined the association of family structure and family adjustment. Utilizing a diverse sample of 738 Mexican American families (21.7% single parent), the current study examined differences across family structure on early adolescent outcomes, family functioning, and parent-child relationship variables. Results revealed that early adolescents in single parent families reported greater school misconduct, CD/ODD and MDD symptoms, and greater parent-child conflict than their counterparts in two parent families. Single parent mothers reported greater economic hardship, depression and family stress. Family stress and parent-child conflict emerged as significant mediators of the association between family structure and early adolescent outcomes, suggesting important processes linking MA single parent families and adolescent adjustment. PMID:21361925

Murine Norovirus Increases Atherosclerotic Lesion Size and Macrophages in Ldlr−/− Mice

PubMed Central

Paik, Jisun; Fierce, Yvette; Mai, Phuong-Oanh; Phelps, Susan R; McDonald, Thomas; Treuting, Piper; Drivdahl, Rolf; Brabb, Thea; LeBoeuf, Renee; O'Brien, Kevin D

2011-01-01

Murine norovirus (MNV) is prevalent in rodent facilities in the United States. Because MNV has a tropism for macrophages and dendritic cells, we hypothesized that it may alter phenotypes of murine models of inflammatory diseases, such as obesity and atherosclerosis. We examined whether MNV infection influences phenotypes associated with diet-induced obesity and atherosclerosis by using Ldlr−/− mice. Male Ldlr−/− mice were maintained on either a diabetogenic or high-fat diet for 16 wk, inoculated with either MNV or vehicle, and monitored for changes in body weight, blood glucose, glucose tolerance, and insulin sensitivity. Influence of MNV on atherosclerosis was analyzed by determining aortic sinus lesion area. Under both dietary regimens, MNV-infected and control mice gained similar amounts of weight and developed similar degrees of insulin resistance. However, MNV infection was associated with significant increases in aortic sinus lesion area and macrophage content in Ldlr−/− mice fed a high-fat diet but not those fed a diabetogenic diet. In conclusion, MNV infection exacerbates atherosclerosis in Ldlr−/− mice fed a high-fat diet but does not influence obesity- and diabetes-related phenotypes. Increased lesion size was associated with increased macrophages, suggesting that MNV may influence macrophage activation or accumulation in the lesion area. PMID:22330248

Silencing of diphthamide synthesis 3 (Dph3) reduces metastasis of murine melanoma.

PubMed

Wang, Lei; Shi, Yu; Ju, Peijun; Liu, Rui; Yeo, Siok Ping; Xia, Yinyan; Owlanj, Hamed; Feng, Zhiwei

2012-01-01

Melanoma is the most dangerous skin cancer due to its highly metastatic potential and resistance to chemotherapy. Currently, there is no effective treatment for melanoma once it is progressed to metastatic stage. Therefore, further study to elucidate the molecular mechanism underlying the metastasis of melanoma cells is urgently required for the improvement of melanoma treatment. In the present study, we found that diphthamide synthesis 3 (Dph3) is involved in the metastasis of B16F10 murine melanoma cells by insertional mutagenesis. We demonstrated that Dph3 disruption impairs the migration of B16F10 murine melanoma cells. The requirement of Dph3 in the migration of melanoma cells was further confirmed by gene silencing with siRNA in vitro. In corresponding to this result, overexpression of Dph3 significantly promoted the migratory ability of B16F10 and B16F0 melanoma cells. Moreover, down regulation of Dph3 expression in B16F10 melanoma cells strikingly inhibits their cellular invasion and metastasis in vivo. Finally, we found that Dph3 promotes melanoma migration and invasion through the AKT signaling pathway. To conclude, our findings suggest a novel mechanism underlying the metastasis of melanoma cells which might serve as a new intervention target for the treatment of melanoma.

Establishment of immortalized murine mesothelial cells and a novel mesothelioma cell line.

PubMed

Blum, Walter; Pecze, László; Felley-Bosco, Emanuela; Worthmüller-Rodriguez, Janine; Wu, Licun; Vrugt, Bart; de Perrot, Marc; Schwaller, Beat

2015-08-01

Mesothelial cells are susceptible to asbestos fiber-induced cytotoxicity and on longer time scales to transformation; the resulting mesothelioma is a highly aggressive neoplasm that is considered as incurable at the present time Zucali et al. (Cancer Treatment Reviews 37:543-558, 2011). Only few murine cell culture models of immortalized mesothelial cells and mesothelioma cell lines exist to date. We generated SV40-immortalized cell lines derived from wild-type (WT) and neurofibromatosis 2 (merlin) heterozygote (Nf2+/-) mice, both on a commonly used genetic background, C57Bl/6J. All immortalized mesothelial clones consistently grow in DMEM supplemented with fetal bovine serum. Cells can be passaged for more than 40 times without any signs of morphological changes or a decrease in proliferation rate. The tumor suppressor gene NF2 is one of the most frequently mutated genes in human mesothelioma, but its detailed function is still unknown. Thus, these genotypically distinct cell lines likely relevant for malignant mesothelioma formation are expected to serve as useful in vitro models, in particular to compare with in vivo studies in mice of the same genotype. Furthermore, we generated a novel murine mesothelioma cell line RN5 originating from an Nf2+/- mouse subjected to repeated crocidolite exposure. RN5 cells are highly tumorigenic.

Accumulation of murine amyloid-β mimics early Alzheimer's disease.

PubMed

Krohn, Markus; Bracke, Alexander; Avchalumov, Yosef; Schumacher, Toni; Hofrichter, Jacqueline; Paarmann, Kristin; Fröhlich, Christina; Lange, Cathleen; Brüning, Thomas; von Bohlen Und Halbach, Oliver; Pahnke, Jens

2015-08-01

Amyloidosis mouse models of Alzheimer's disease are generally established by transgenic approaches leading to an overexpression of mutated human genes that are known to be involved in the generation of amyloid-β in Alzheimer's families. Although these models made substantial contributions to the current knowledge about the 'amyloid hypothesis' of Alzheimer's disease, the overproduction of amyloid-β peptides mimics only inherited (familiar) Alzheimer's disease, which accounts for <1% of all patients with Alzheimer's disease. The inherited form is even regarded a 'rare' disease according to the regulations for funding of the European Union (www.erare.eu). Here, we show that mice that are double-deficient for neprilysin (encoded by Mme), one major amyloid-β-degrading enzyme, and the ABC transporter ABCC1, a major contributor to amyloid-β clearance from the brain, develop various aspects of sporadic Alzheimer's disease mimicking the clinical stage of mild cognitive impairment. Using behavioural tests, electrophysiology and morphological analyses, we compared different ABC transporter-deficient animals and found that alterations are most prominent in neprilysin × ABCC1 double-deficient mice. We show that these mice have a reduced probability to survive, show increased anxiety in new environments, and have a reduced working memory performance. Furthermore, we detected morphological changes in the hippocampus and amygdala, e.g. astrogliosis and reduced numbers of synapses, leading to defective long-term potentiation in functional measurements. Compared to human, murine amyloid-β is poorly aggregating, due to changes in three amino acids at N-terminal positions 5, 10, and 13. Interestingly, our findings account for the action of early occurring amyloid-β species/aggregates, i.e. monomers and small amyloid-β oligomers. Thus, neprilysin × ABCC1 double-deficient mice present a new model for early effects of amyloid-β-related mild cognitive impairment that allows

Modeling of Chronic Myeloid Leukemia: An Overview of In Vivo Murine and Human Xenograft Models

PubMed Central

Vellenga, Edo

2016-01-01

Over the past years, a wide variety of in vivo mouse models have been generated in order to unravel the molecular pathology of Chronic Myeloid Leukemia (CML) and to develop and improve therapeutic approaches. These models range from (conditional) transgenic models, knock-in models, and murine bone marrow retroviral transduction models followed by transplantation. With the advancement of immunodeficient xenograft models, it has become possible to use human stem/progenitor cells for in vivo studies as well as cells directly derived from CML patients. These models not only mimic CML but also have been instrumental in uncovering various fundamental mechanisms of CML disease progression and tyrosine kinase inhibitor (TKI) resistance. With the availability of iPSC technology, it has become feasible to derive, maintain, and expand CML subclones that are at least genetically identical to those in patients. The following review provides an overview of all murine as well as human xenograft models for CML established till date. PMID:27642303

Key Role of MicroRNA in the Regulation of Granulocyte Macrophage Colony-stimulating Factor Expression in Murine Alveolar Epithelial Cells during Oxidative Stress*

PubMed Central

Sturrock, Anne; Mir-Kasimov, Mustafa; Baker, Jessica; Rowley, Jesse; Paine, Robert

2014-01-01

GM-CSF is an endogenous pulmonary cytokine produced by normal alveolar epithelial cells (AEC) that is a key defender of the alveolar space. AEC GM-CSF expression is suppressed by oxidative stress through alternations in mRNA turnover, an effect that is reversed by treatment with recombinant GM-CSF. We hypothesized that specific microRNA (miRNA) would play a key role in AEC GM-CSF regulation. A genome-wide miRNA microarray identified 19 candidate miRNA altered in primary AEC during oxidative stress with reversal by treatment with GM-CSF. Three of these miRNA (miR 133a, miR 133a*, and miR 133b) are also predicted to bind the GM-CSF 3′-untranslated region (UTR). PCR for the mature miRNA confirmed induction during oxidative stress that was reversed by treatment with GM-CSF. Experiments using a GM-CSF 3′-UTR reporter construct demonstrated that miR133a and miR133b effects on GM-CSF expression are through interactions with the GM-CSF 3′-UTR. Using lentiviral transduction of specific mimics and inhibitors in primary murine AEC, we determined that miR133a and miR133b suppress GM-CSF expression and that their inhibition both reverses oxidant-induced suppression of GM-CSF expression and increases basal expression of GM-CSF in cells in normoxia. In contrast, these miRNAs are not active in regulation of GM-CSF expression in murine EL4 T cells. Thus, members of the miR133 family play key roles in regulation of GM-CSF expression through effects on mRNA turnover in AEC during oxidative stress. Increased understanding of GM-CSF gene regulation may provide novel miRNA-based interventions to augment pulmonary innate immune defense in lung injury. PMID:24371146

Improved BM212 MmpL3 Inhibitor Analogue Shows Efficacy in Acute Murine Model of Tuberculosis Infection

PubMed Central

Alfonso, Salvatore; Cocozza, Martina; Porretta, Giulio Cesare; Ballell, Lluís; Rullas, Joaquin; Ortega, Fátima; De Logu, Alessandro; Agus, Emanuela; La Rosa, Valentina; Pasca, Maria Rosalia; De Rossi, Edda; Wae, Baojie; Franzblau, Scott G.; Manetti, Fabrizio; Botta, Maurizio; Biava, Mariangela

2013-01-01

1,5-Diphenyl pyrroles were previously identified as a class of compounds endowed with high in vitro efficacy against M. tuberculosis. To improve the physical chemical properties and drug-like parameters of this class of compounds, a medicinal chemistry effort was undertaken. By selecting the optimal substitution patterns for the phenyl rings at N1 and C5 and by replacing the thiomorpholine moiety with a morpholine one, a new series of compounds was produced. The replacement of the sulfur with oxygen gave compounds with lower lipophilicity and improved in vitro microsomal stability. Moreover, since the parent compound of this family has been shown to target MmpL3, mycobacterial mutants resistant to two compounds have been isolated and characterized by sequencing the mmpL3 gene; all the mutants showed point mutations in this gene. The best compound identified to date was progressed to dose-response studies in an acute murine TB infection model. The resulting ED99 of 49 mg/Kg is within the range of commonly employed tuberculosis drugs, demonstrating the potential of this chemical series. The in vitro and in vivo target validation evidence presented here adds further weight to MmpL3 as a druggable target of interest for anti-tubercular drug discovery. PMID:23437287

Kir 4.1 inward rectifier potassium channel is upregulated in astrocytes in a murine multiple sclerosis model.

PubMed

Mercado, Francisco; Almanza, Angélica; Rubio, Nazario; Soto, Enrique

2018-06-11

Multiple sclerosis (MS) is a high prevalence degenerative disease characterized at the cellular level by glial and neuronal cell death. The causes of cell death during the disease course are not fully understood. In this work we demonstrate that in a MS model induced by Theiler's murine encephalomyelitis virus (TMEV) infection, the inward rectifier (Kir) 4.1 potassium channel subunit is overexpressed in astrocytes. In voltage clamp experiments the inward current density from TMEV-infected astrocytes was significantly larger than in mock-infected ones. The cRNA hybridization analysis from mock- and TMEV-infected cells showed an upregulation of a potassium transport channel coding sequence. We validated this mRNA increase by RT-PCR and quantitative PCR using Kir 4.1 specific primers. Western blotting experiments confirmed the upregulation of Kir 4.1, and alignment between sequences provided the demonstration that the over-expressed gene encodes for a Kir family member. Flow cytometry showed that the Kir 4.1 protein is located mainly in the cell membrane in mock and TMEV-infected astrocytes. Our results demonstrate an increase in K + inward current in TMEV-infected glial cells, this increment may reduce the neuronal depolarization, contributing to cell resilience mechanisms. Copyright © 2018 Elsevier B.V. All rights reserved.

Ex vivo micro-CT imaging of murine brain models using non-ionic iodinated contrast

NASA Astrophysics Data System (ADS)

Salas Bautista, N.; Martínez-Dávalos, A.; Rodríguez-Villafuerte, M.; Murrieta-Rodríguez, T.; Manjarrez-Marmolejo, J.; Franco-Pérez, J.; Calvillo-Velasco, M. E.

2014-11-01

Preclinical investigation of brain tumors is frequently carried out by means of intracranial implantation of brain tumor xenografts or allografts, with subsequent analysis of tumor growth using conventional histopathology. However, very little has been reported on the use contrast-enhanced techniques in micro-CT imaging for the study of malignant brain tumors in small animal models. The aim of this study has been to test a protocol for ex vivo imaging of murine brain models of glioblastoma multiforme (GBM) after treatment with non-ionic iodinated solution, using an in-house developed laboratory micro-CT. We have found that the best compromise between acquisition time and image quality is obtained using a 50 kVp, 0.5 mAs, 1° angular step on a 360 degree orbit acquisition protocol, with 70 μm reconstructed voxel size using the Feldkamp algorithm. With this parameters up to 4 murine brains can be scanned in tandem in less than 15 minutes. Image segmentation and analysis of three sample brains allowed identifying tumor volumes as small as 0.4 mm3.

Dietary intake of plant sterols stably increases plant sterol levels in the murine brain.

PubMed

Vanmierlo, Tim; Weingärtner, Oliver; van der Pol, Susanne; Husche, Constanze; Kerksiek, Anja; Friedrichs, Silvia; Sijbrands, Eric; Steinbusch, Harry; Grimm, Marcus; Hartmann, Tobias; Laufs, Ulrich; Böhm, Michael; de Vries, Helga E; Mulder, Monique; Lütjohann, Dieter

2012-04-01

Plant sterols such as sitosterol and campesterol are frequently administered as cholesterol-lowering supplements in food. Recently, it has been shown in mice that, in contrast to the structurally related cholesterol, circulating plant sterols can enter the brain. We questioned whether the accumulation of plant sterols in murine brain is reversible. After being fed a plant sterol ester-enriched diet for 6 weeks, C57BL/6NCrl mice displayed significantly increased concentrations of plant sterols in serum, liver, and brain by 2- to 3-fold. Blocking intestinal sterol uptake for the next 6 months while feeding the mice with a plant stanol ester-enriched diet resulted in strongly decreased plant sterol levels in serum and liver, without affecting brain plant sterol levels. Relative to plasma concentrations, brain levels of campesterol were higher than sitosterol, suggesting that campesterol traverses the blood-brain barrier more efficiently. In vitro experiments with brain endothelial cell cultures showed that campesterol crossed the blood-brain barrier more efficiently than sitosterol. We conclude that, over a 6-month period, plant sterol accumulation in murine brain is virtually irreversible.

Sealing effect of a polysaccharide nanosheet for murine cecal puncture.

PubMed

Fujie, Toshinori; Kinoshita, Manabu; Shono, Satoshi; Saito, Akihiro; Okamura, Yosuke; Saitoh, Daizoh; Takeoka, Shinji

2010-07-01

Recent developments in nanobiotechnology have led us to develop a method of producing a free-standing polymer nanosheet composed of polysaccharides (ie, polysaccharide nanosheet) with a thickness of tens of nanometers. Owing to its enormous aspect ratio, the polysaccharide nanosheet is semi-absorbent and has a physical adhesive strength 7.5-fold greater than that of conventional films of >1 microm thickness. Herein, we have investigated the therapeutic sealing effect of this polysaccharide nanosheet on murine cecal puncture as a wound dressing material. Murine cecum was punctured and then overlapped with the polysaccharide nanosheet. Thereafter, we evaluated its sealing effect on bacterial peritonitis as well as the protection offered by the polysaccharide nanosheet against bacterial permeability using an in vitro transmembrane assay. The 39-nm-thick polysaccharide nanosheet overlapped tightly the perforated cecum. No adhering agents were required because of the ability of the polysaccharide nanosheet to adhere to the tissue surface by physical adsorption (eg, van der Waals interaction). Sealing the perforated cecum with the polysaccharide nanosheet increased survival rate without postoperative adhesion by comparison with untreated mice (90 vs 30%; P < .01). These data were supported by the improvement in peritonitis related to bacterial counts, white blood cell counts, and the serum tumor necrosis factor level. Moreover, using an in vitro transmembrane assay, we showed that the polysaccharide nanosheet inhibited effectively bacterial penetration. We have demonstrated the potential clinical benefits of the nanosheet-type biomaterial that can be used for repairing a cecal colotomy without chemical bonding agents. Copyright 2010 Mosby, Inc. All rights reserved.

Tenascin-C Prevents Articular Cartilage Degeneration in Murine Osteoarthritis Models.

PubMed

Matsui, Yuriyo; Hasegawa, Masahiro; Iino, Takahiro; Imanaka-Yoshida, Kyoko; Yoshida, Toshimichi; Sudo, Akihiro

2018-01-01

Objective The objective of this study was to determine whether intra-articular injections of tenascin-C (TNC) could prevent cartilage damage in murine models of osteoarthritis (OA). Design Fluorescently labeled TNC was injected into knee joints and its distribution was examined at 1 day, 4 days, 1 week, 2 weeks, and 4 weeks postinjection. To investigate the effects of TNC on cartilage degeneration after surgery to knee joints, articular spaces were filled with 100 μg/mL (group I), 10 μg/mL (group II) of TNC solution, or control (group III). TNC solution of 10 μg/mL was additionally injected twice after 3 weeks (group IV) or weekly after 1 week, 2 weeks, and 3 weeks (group V). Joint tissues were histologically assessed using the Mankin score and the modified Chambers system at 2 to 8 weeks after surgery. Results Exogenous TNC was maintained in the cartilage and synovium for 1 week after administration. Histological scores in groups I and II were better than scores in group III at 4 and 6 weeks, but progressive cartilage damage was seen in all groups 8 weeks postoperatively. Sequential TNC injections (groups IV and V) showed significantly better Mankin score than single injection (group II) at 8 weeks. Conclusion TNC administered exogenously remained in the cartilage of knee joints for 1 week, and could decelerate articular cartilage degeneration in murine models of OA. We also showed that sequential administration of TNC was more effective than a single injection. TNC could be an important molecule for prevention of articular cartilage damage.

Murine Sarcoma Virus Gene Expression: Transformants Which Express Viral Envelope Glycoprotein In The Absence Of The Major Internal Protein And Infectious Particles

PubMed Central

Bilello, John A.; Strand, Mette; August, J. T.

1974-01-01

Expression of the major internal protein and the envelope glycoprotein of murine C-type viruses in focus-derived lines of normal rat kidney cells infected with Kirsten murine sarcoma virus was measured by radioimmunoassay. Of the clones selected, which do not produce virus particles or the major viral structural protein, approximately half express the viral envelope glycoprotein at concentrations found in productively infected cells. Expression of the envelope glycoprotein did not appear to alter significantly the properties of the transformed cells in culture. PMID:4370209

Cryo-EM structure of the cytoplasmic domain of murine transient receptor potential cation channel subfamily C member 6 (TRPC6).

PubMed

Azumaya, Caleigh M; Sierra-Valdez, Francisco; Cordero-Morales, Julio F; Nakagawa, Terunaga

2018-05-11

The kidney maintains the internal milieu by regulating the retention and excretion of proteins, ions, and small molecules. The glomerular podocyte forms the slit diaphragm of the ultrafiltration filter, whose damage leads to progressive kidney failure and focal segmental glomerulosclerosis (FSGS). The canonical transient receptor potential 6 (TRPC6) ion channel is expressed in the podocyte and mutations in its cytoplasmic domain cause FSGS in humans. In vitro evaluation of disease-causing mutations in TRPC6 has revealed that these genetic alterations result in abnormal ion channel gating. However, the mechanism whereby the cytoplasmic domain modulates TRPC6 function is largely unknown. Here we report a cryoEM structure of the cytoplasmic domain of murine TRPC6 at 3.8Å resolution. The cytoplasmic fold of TRPC6 is characterized by an inverted dome-like chamber pierced by four radial horizontal helices that converge into a vertical coiled-coil at the central axis. Unlike in other TRP channels, TRPC6 displays a unique domain swap that occurs at the junction of the horizontal helices and coiled-coil. Multiple FSGS mutations converge at the buried interface between the vertical coiled-coil and the ankyrin repeats, which form the dome, suggesting these regions are critical for allosteric gating modulation. This functionally critical interface is a potential target for drug design. Importantly, dysfunction in other family members leads to learning deficits (TRPC1/4/5) and ataxia (TRPC3). Our data provide a structural framework for the mechanistic investigation of the TRPC family. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Epigallocatechin-3-gallate (EGCG) up-regulates miR-15b expression thus attenuating store operated calcium entry (SOCE) into murine CD4+ T cells and human leukaemic T cell lymphoblasts.

PubMed

Zhang, Shaqiu; Al-Maghout, Tamer; Bissinger, Rosi; Zeng, Ni; Pelzl, Lisann; Salker, Madhuri S; Cheng, Anchun; Singh, Yogesh; Lang, Florian

2017-10-27

CD4 + T cells are key elements in immune responses and inflammation. Activation of T cell receptors in CD4 + T cells triggers cytosolic Ca 2+ release with subsequent store operated Ca 2+ entry (SOCE), which is accomplished by the pore forming Ca 2+ release activated Ca 2+ (CRAC) channel Orai1 and its regulator stromal cell-interaction molecule 2 (STIM2). Green tea polyphenol epigallocatechin-3-gallate (EGCG) acts as a potent anti-inflammatory and anti-oxidant agent for various types of cells including immune cells. However, how post-transcriptional gene regulators such as miRNAs are involved in the regulation of Ca 2+ influx into murine CD4 + T cells and human Jurkat T cells through EGCG is not defined. EGCG treatment of murine CD4 + T cells significantly down-regulated the expression of STIM2 and Orai1 both at mRNA and protein levels. Furthermore, EGCG significantly decreased SOCE in both murine and human T cells. EGCG treatment increased miRNA-15b (miR-15b) abundance in both murine and human T cells. Bioinformatics analysis reveals that miR-15b, which has a STIM2 binding site, is involved in the down-regulation of SOCE. Overexpression of miR-15b significantly decreased the mRNA and protein expression of STIM2 and Orai1 in murine T cells. Treatment of Jurkat T cells with 10 μM EGCG further decreased mTOR and PTEN protein levels. EGCG decreased mitochondrial membrane potential (MMP) in both human and murine T cells. In conclusion, the observations suggest that EGCG inhibits the Ca 2+ entry into murine and human T cells, an effect accomplished at least in part by up-regulation of miR-15b.

Murine but not human basophil undergoes cell-specific proteolysis of a major endoplasmic reticulum chaperone.

PubMed

Liu, Bei; Staron, Matthew; Li, Zihai

2012-01-01

Basophil has been implicated in anti-parasite defense, allergy and in polarizing T(H)2 response. Mouse model has been commonly used to study basophil function although the difference between human and mouse basophils is underappreciated. As an essential chaperone for multiple Toll-like receptors and integrins in the endoplasmic reticulum, gp96 also participates in general protein homeostasis and in the ER unfolded protein response to ensure cell survival during stress. The roles of gp96 in basophil development are unknown. We genetically delete gp96 in mice and examined the expression of gp96 in basophils by Western blot and flow cytometry. We compared the expression pattern of gp96 between human and mouse basophils. We found that gp96 was dispensable for murine basophil development. Moreover, gp96 was cleaved by serine protease(s) in murine but not human basophils leading to accumulation of a nun-functional N-terminal ∼50 kDa fragment and striking induction of the unfolded protein response. The alteration of gp96 was unique to basophils and was not observed in any other cell types including mast cells. We also demonstrated that the ectopic expression of a mouse-specific tryptase mMCP11 does not lead to gp96 cleavage in human basophils. Our study revealed a remarkable biochemical event of gp96 silencing in murine but not human basophils, highlighting the need for caution in using mouse models to infer the function of basophils in human immune response. Our study also reveals a novel mechanism of shutting down gp96 post-translationally in regulating its function.

Ochronosis in a murine model of alkaptonuria is synonymous to that in the human condition

PubMed Central

Taylor, A.M.; Preston, A.J.; Paulk, N.K.; Sutherland, H.; Keenan, C.M.; Wilson, P.J.M.; Wlodarski, B.; Grompe, M.; Ranganath, L.R.; Gallagher, J.A.; Jarvis, J.C.

2012-01-01

Objective Alkaptonuria (AKU) is a rare genetic disease which results in severe early onset osteoarthropathy. It has recently been shown that the subchondral interface is of key significance in disease pathogenesis. Human surgical tissues are often beyond this initial stage and there is no published murine model of pathogenesis, to study the natural history of the disease. The murine genotype exists but it has been reported not to demonstrate ochronotic osteoarthropathy consistent with the human disease. Recent anecdotal evidence of macroscopic renal ochronosis in a mouse model of tyrosinaemia led us to perform histological analysis of tissues of these mice that are known to be affected in human AKU. Design The homogentisate 1,2-dioxygenase Hgd+/−Fah−/− mouse can model either hereditary tyrosinaemia type I (HT1) or AKU depending on selection conditions. Mice having undergone Hgd reversion were sacrificed at various time points, and their tissues taken for histological analysis. Sections were stained with haematoxylin eosin (H&E) and Schmorl’s reagent. Results Early time point observations at 8 months showed no sign of macroscopic ochronosis of tissues. Macroscopic examination at 13 months revealed ochronosis of the kidneys. Microscopic analysis of the kidneys revealed large pigmented nodules displaying distinct ochre colouration. Close microscopic examination of the distal femur and proximal fibula at the subchondral junctions revealed the presence of numerous pigmented chondrocytes. Conclusions Here we present the first data showing ochronosis of tissues in a murine model of AKU. These preliminary histological observations provide a stimulus for further studies into the natural history of the disease to provide a greater understanding of this class of arthropathy. PMID:22542924

Adoptive transfer of syngeneic T cells transduced with a chimeric antigen receptor that recognizes murine CD19 can eradicate lymphoma and normal B cells.

PubMed

Kochenderfer, James N; Yu, Zhiya; Frasheri, Dorina; Restifo, Nicholas P; Rosenberg, Steven A

2010-11-11

Adoptive T-cell therapy with anti-CD19 chimeric antigen receptor (CAR)-expressing T cells is a new approach for treating advanced B-cell malignancies. To evaluate anti-CD19-CAR-transduced T cells in a murine model of adoptive T-cell therapy, we developed a CAR that specifically recognized murine CD19. We used T cells that were retrovirally transduced with this CAR to treat mice bearing a syngeneic lymphoma that naturally expressed the self-antigen murine CD19. One infusion of anti-CD19-CAR-transduced T cells completely eliminated normal B cells from mice for at least 143 days. Anti-CD19-CAR-transduced T cells eradicated intraperitoneally injected lymphoma cells and large subcutaneous lymphoma masses. The antilymphoma efficacy of anti-CD19-CAR-transduced T cells was critically dependent on irradiation of mice before anti-CD19-CAR-transduced T-cell infusion. Anti-CD19-CAR-transduced T cells had superior antilymphoma efficacy compared with the anti-CD19 monoclonal antibody from which the anti-CD19 CAR was derived. Our results demonstrated impressive antilymphoma activity and profound destruction of normal B cells caused by anti-CD19-CAR-transduced T cells in a clinically relevant murine model.

Feasibility and scalability of spring parameters in distraction enterogenesis in a murine model.

PubMed

Huynh, Nhan; Dubrovsky, Genia; Rouch, Joshua D; Scott, Andrew; Stelzner, Matthias; Shekherdimian, Shant; Dunn, James C Y

2017-07-01

Distraction enterogenesis has been investigated as a novel treatment for short bowel syndrome (SBS). With variable intestinal sizes, it is critical to determine safe, translatable spring characteristics in differently sized animal models before clinical use. Nitinol springs have been shown to lengthen intestines in rats and pigs. Here, we show spring-mediated intestinal lengthening is scalable and feasible in a murine model. A 10-mm nitinol spring was compressed to 3 mm and placed in a 5-mm intestinal segment isolated from continuity in mice. A noncompressed spring placed in a similar fashion served as a control. Spring parameters were proportionally extrapolated from previous spring parameters to accommodate the smaller size of murine intestines. After 2-3 wk, the intestinal segments were examined for size and histology. Experimental group with spring constants, k = 0.2-1.4 N/m, showed intestinal lengthening from 5.0 ± 0.6 mm to 9.5 ± 0.8 mm (P < 0.0001), whereas control segments lengthened from 5.3 ± 0.5 mm to 6.4 ± 1.0 mm (P < 0.02). Diameter increased similarly in both groups. Isolated segment perforation was noted when k ≥ 0.8 N/m. Histologically, lengthened segments had increased muscularis thickness and crypt depth in comparison to normal intestine. Nitinol springs with k ≤ 0.4 N/m can safely yield nearly 2-fold distraction enterogenesis in length and diameter in a scalable mouse model. Not only does this study derive the safe ranges and translatable spring characteristics in a scalable murine model for patients with short bowel syndrome, it also demonstrates the feasibility of spring-mediated intestinal lengthening in a mouse, which can be used to study underlying mechanisms in the future. Copyright © 2017 Elsevier Inc. All rights reserved.

Shikonin inhibits maturation of bone marrow-derived dendritic cells and suppresses allergic airway inflammation in a murine model of asthma

PubMed Central

Lee, Chen-Chen; Wang, Chien-Neng; Lai, Yu-Ting; Kang, Jaw-Jou; Liao, Jiunn-Wang; Chiang, Bor-Luen; Chen, Hui-Chen; Cheng, Yu-Wen

2010-01-01

BACKGROUND AND PURPOSE Shikonin exhibits a wide range of anti-inflammatory actions. Here, we assessed its effects on maturation of murine bone marrow-derived dendritic cells (BM-DCs) and on allergic reactions in a murine model of asthma. EXPERIMENTAL APPROACH Cultured murine BM-DCs were used to investigate the effects of shikonin on expression of cell surface markers and their stimulation of T-cell proliferation and cytokine production. The therapeutic potential of shikonin was evaluated in a model of allergic airway disease. KEY RESULTS Shikonin dose-dependently inhibited expression of major histocompatibility complex class II, CD80, CD86, CCR7 and OX40L on BM-DCs, induced by a mixture of ovalbumin (OVA; 100 µg·mL−1) and thymic stromal lymphopoietin (TSLP; 20 ng·mL−1). Shikonin-treated BM-DCs were poor stimulators of CD4+ T lymphocyte and induced lower levels of interleukin (IL)-4, IL-5, IL-13 and tumour necrosis factor (TNF)-α release by responding T-cells. After intratracheal instillation of shikonin in OVA-immunized mice, OVA challenge induced lower IL-4, IL-5, IL-13, TNF-α and eotaxin release in bronchial alveolar lavage fluid, lower IL-4 and IL-5 production in lung cells and mediastinal lymph node cells and attenuated OVA-induced lung eosinophilia and airway hyperresponsiveness. CONCLUSION AND IMPLICATIONS Shikonin effectively suppressed OVA + TSLP-induced BM-DC maturation in vitro and inhibited allergic inflammation and airway hyperresponsiveness in a murine model of asthma, showing good potential as a treatment for allergic asthma. Also, our model provides a novel platform for screening drugs for allergic diseases. PMID:20735407

Hyperlipidemia affects multiscale structure and strength of murine femur.

PubMed

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

2014-07-18

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. Copyright © 2014 Elsevier Ltd. All rights reserved.

The Influence of Flightless I on Toll-Like-Receptor-Mediated Inflammation in a Murine Model of Diabetic Wound Healing

PubMed Central

Ruzehaji, Nadira; Mills, Stuart J.; Melville, Elizabeth; Arkell, Ruth; Fitridge, Robert; Cowin, Allison J.

2013-01-01

Impaired wound healing and ulceration represent a serious complication of both type 1 and type 2 diabetes. Cytoskeletal protein Flightless I (Flii) is an important inhibitor of wound repair, and reduced Flii gene expression in fibroblasts increased migration, proliferation, and adhesion. As such it has the ability to influence all phases of wound healing including inflammation, remodelling and angiogenesis. Flii has the potential to modulate inflammation through its interaction with MyD88 which it an adaptor protein for TLR4. To assess the effect of Flii on the inflammatory response of diabetic wounds, we used a murine model of streptozocin-induced diabetes and Flii genetic mice. Increased levels of Flii were detected in Flii transgenic murine wounds resulting in impaired healing which was exacerbated when diabetes was induced. When Flii levels were reduced in diabetic wounds of Flii-deficient mice, healing was improved and decreased levels of TLR4 were observed. In contrast, increasing the level of Flii in diabetic mouse wounds led to increased TLR4 and NF-κB production. Treatment of murine diabetic wounds with neutralising antibodies to Flii led to an improvement in healing with decreased expression of TLR4. Decreasing the level of Flii in diabetic wounds may therefore reduce the inflammatory response and improve healing. PMID:23555084

TGF-β induces surface LAP expression on murine CD4 T cells independent of Foxp3 induction.

PubMed

Oida, Takatoku; Weiner, Howard L

2010-11-24

It has been reported that human FOXP3(+) CD4 Tregs express GARP-anchored surface latency-associated peptide (LAP) after activation, based on the use of an anti-human LAP mAb. Murine CD4 Foxp3(+) Tregs have also been reported to express surface LAP, but these studies have been hampered by the lack of suitable anti-mouse LAP mAbs. We generated anti-mouse LAP mAbs by immunizing TGF-β(-/-) animals with a mouse Tgfb1-transduced P3U1 cell line. Using these antibodies, we demonstrated that murine Foxp3(+) CD4 Tregs express LAP on their surface. In addition, retroviral transduction of Foxp3 into mouse CD4(+)CD25(-) T cells induced surface LAP expression. We then examined surface LAP expression after treating CD4(+)CD25(-) T cells with TGF-β and found that TGF-β induced surface LAP not only on T cells that became Foxp3(+) but also on T cells that remained Foxp3(-) after TGF-β treatment. GARP expression correlated with the surface LAP expression, suggesting that surface LAP is GARP-anchored also in murine T cells. Unlike human CD4 T cells, surface LAP expression on mouse CD4 T cells is controlled by Foxp3 and TGF-β. Our newly described anti-mouse LAP mAbs will provide a useful tool for the investigation and functional analysis of T cells that express LAP on their surface.

Characterization of the gag/fusion protein encoded by the defective Duplan retrovirus inducing murine acquired immunodeficiency syndrome.

PubMed Central

Huang, M; Jolicoeur, P

1990-01-01

Murine acquired immunodeficiency syndrome is induced by a defective retrovirus. Sequencing of this defective viral genome revealed a long open reading frame which encodes a putative gag/fusion protein, N-MA-p12-CA-NC-COOH, (D. C. Aziz, Z. Hanna, and P. Jolicoeur, Nature (London) 338:505-508, 1989). We raised a specific antibody to the unique p12 domain of this gag fusion precursor, Pr60gag. We found that Pr60gag was indeed encoded by the defective viral genome both in cell-free translation reticulocyte extracts and in infected mouse fibroblasts. Pr60gag was found to be myristylated, phosphorylated, and attached to the cell membrane, like other helper murine leukemia virus (MuLV) gag precursors. Pr60gag was not substantially cleaved within the nonproducer cells and was not released from these cells. However, in the presence of helper MuLV proteins, it formed phenotypically mixed particles. In these particles, Pr60gag was only partially cleaved. In helper MuLV-producing cells harboring the defective virus, a gag-related p40 intermediate was generated both intracellularly and extracellularly. In these cells, Pr60gag appeared to behave as a dominant negative mutant, interfering with proper cleavage of helper Pr65gag. Our data indicate that Pr60gag is a major (and possibly the only) gene product of the defective murine acquired immunodeficiency syndrome virus and is likely to harbor some determinants of pathogenicity of this virus. Images PMID:2243376

Novel Markers to Delineate Murine M1 and M2 Macrophages

PubMed Central

Jablonski, Kyle A.; Amici, Stephanie A.; Webb, Lindsay M.; Ruiz-Rosado, Juan de Dios; Popovich, Phillip G.; Partida-Sanchez, Santiago; Guerau-de-Arellano, Mireia

2015-01-01

Classically (M1) and alternatively activated (M2) macrophages exhibit distinct phenotypes and functions. It has been difficult to dissect macrophage phenotypes in vivo, where a spectrum of macrophage phenotypes exists, and also in vitro, where low or non-selective M2 marker protein expression is observed. To provide a foundation for the complexity of in vivo macrophage phenotypes, we performed a comprehensive analysis of the transcriptional signature of murine M0, M1 and M2 macrophages and identified genes common or exclusive to either subset. We validated by real-time PCR an M1-exclusive pattern of expression for CD38, G-protein coupled receptor 18 (Gpr18) and Formyl peptide receptor 2 (Fpr2) whereas Early growth response protein 2 (Egr2) and c-Myc were M2-exclusive. We further confirmed these data by flow cytometry and show that M1 and M2 macrophages can be distinguished by their relative expression of CD38 and Egr2. Egr2 labeled more M2 macrophages (~70%) than the canonical M2 macrophage marker Arginase-1, which labels 24% of M2 macrophages. Conversely, CD38 labeled most (71%) in vitro M1 macrophages. In vivo, a similar CD38+ population greatly increased after LPS exposure. Overall, this work defines exclusive and common M1 and M2 signatures and provides novel and improved tools to distinguish M1 and M2 murine macrophages. PMID:26699615

Local expression of vaginal Th1 and Th2 cytokines in murine vaginal candidiasis under different immunity conditions.

PubMed

Chen, Shanjuan; Li, Shaohua; Wu, Yan; Liu, Zhixiang; Li, Jiawen

2008-08-01

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-beta1) 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-2 mRNA was increased during early stage of infection, while IL-10 was increased transiently during late stage of infection. TGF-beta1 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-beta1 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.

Testing the Role of p21 Activated Kinases in Schwannoma Formation Using a Novel Genetically Engineered Murine Model that Closely Phenocopies Human NF2 Disease

DTIC Science & Technology

2017-06-01

Kinases in Schwannoma Formation Using a Novel Genetically Engineered Murine Model that Closely Phenocopies Human NF2 Disease The views, opinions and...Role of p21 Activated Kinases in Schwannoma Formation Using a Novel Genetically Engineered Murine Model that Closely Phenocopies Human NF2 Disease Form...NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. The major goal of this research project was to genetically and pharmacologically test the requirement of PAK

A Chitin-Like Component on Sclerotic Cells of Fonsecaea pedrosoi Inhibits Dectin-1-Mediated Murine Th17 Development by Masking β-Glucans

PubMed Central

Li, Ruoyu; Chen, Sharon C.-A.; Liu, Weihuang; Liu, Wei; Chen, Liuqing; Chen, Yao; Zhang, Xu; Tong, Zhongsheng; Xia, Yun; Xia, Ping; Wang, Yan; Duan, Yiqun

2014-01-01

Fonsecaea pedrosoi (F. pedrosoi), a major agent of chromoblastomycosis, has been shown to be recognized primarily by C-type lectin receptors (CLRs) in a murine model of chromoblastomycosis. Specifically, the β-glucan receptor, Dectin-1, mediates Th17 development and consequent recruitment of neutrophils, and is evidenced to have the capacity to bind to saprophytic hyphae of F. pedrosoi in vitro. However, when embedded in tissue, most etiological agents of chromoblastomycosis including F. pedrosoi will transform into the sclerotic cells, which are linked to the greatest survival of melanized fungi in tissue. In this study, using immunocompetent and athymic (nu/nu) murine models infected subcutaneously or intraperitoneally with F. pedrosoi, we demonstrated that T lymphocytes play an active role in the resolution of localized footpad infection, and there existed a significantly decreased expression of Th17-defining transcription factor Rorγt and inefficient recruitment of neutrophils in chronically infected spleen where the inoculated mycelium of F. pedrosoi transformed into the sclerotic cells. We also found that Dectin-1-expressing histocytes and neutrophils participated in the enclosure of transformed sclerotic cells in the infectious foci. Furthermore, we induced the formation of sclerotic cells in vitro, and evidenced a significantly decreased binding capacity of human or murine-derived Dectin-1 to the induced sclerotic cells in comparison with the saprophytic mycelial forms. Our analysis of β-glucans-masking components revealed that it is a chitin-like component, but not the mannose moiety on the sclerotic cells, that interferes with the binding of β-glucans by human or murine Dectin-1. Notably, we demonstrated that although Dectin-1 contributed to the development of IL-17A-producing CD3+CD4+ murine splenocytes upon in vitro-stimulation by saprophytic F. pedrosoi, the masking effect of chitin components partly inhibited Dectin-1-mediated Th17 development

Activin A increases phagocytosis of Escherichia coli K1 by primary murine microglial cells activated by toll-like receptor agonists.

PubMed

Diesselberg, Catharina; Ribes, Sandra; Seele, Jana; Kaufmann, Annika; Redlich, Sandra; Bunkowski, Stephanie; Hanisch, Uwe-Karsten; Michel, Uwe; Nau, Roland; Schütze, Sandra

2018-06-07

Bacterial meningitis is associated with high mortality and long-term neurological sequelae. Increasing the phagocytic activity of microglia could improve the resistance of the CNS against infections. We studied the influence of activin A, a member of the TGF-β family with known immunoregulatory and neuroprotective effects, on the functions of microglial cells in vitro. Primary murine microglial cells were treated with activin A (0.13 ng/ml-13 μg/ml) alone or in combination with agonists of TLR2, 4, and 9. Phagocytosis of Escherichia coli K1 as well as release of TNF-α, IL-6, CXCL1, and NO was assessed. Activin A dose-dependently enhanced the phagocytosis of Escherichia coli K1 by microglial cells activated by agonists of TLR2, 4, and 9 without further increasing NO and proinflammatory cytokine release. Cell viability of microglial cells was not affected by activin A. Priming of microglial cells with activin A could increase the elimination of bacteria in bacterial CNS infections. This preventive strategy could improve the resistance of the brain to infections, particularly in elderly and immunocompromised patients.

Silencing of Diphthamide Synthesis 3 (Dph3) Reduces Metastasis of Murine Melanoma

PubMed Central

Wang, Lei; Shi, Yu; Ju, Peijun; Liu, Rui; Yeo, Siok Ping; Xia, Yinyan; Owlanj, Hamed; Feng, Zhiwei

2012-01-01

Melanoma is the most dangerous skin cancer due to its highly metastatic potential and resistance to chemotherapy. Currently, there is no effective treatment for melanoma once it is progressed to metastatic stage. Therefore, further study to elucidate the molecular mechanism underlying the metastasis of melanoma cells is urgently required for the improvement of melanoma treatment. In the present study, we found that diphthamide synthesis 3 (Dph3) is involved in the metastasis of B16F10 murine melanoma cells by insertional mutagenesis. We demonstrated that Dph3 disruption impairs the migration of B16F10 murine melanoma cells. The requirement of Dph3 in the migration of melanoma cells was further confirmed by gene silencing with siRNA in vitro. In corresponding to this result, overexpression of Dph3 significantly promoted the migratory ability of B16F10 and B16F0 melanoma cells. Moreover, down regulation of Dph3 expression in B16F10 melanoma cells strikingly inhibits their cellular invasion and metastasis in vivo. Finally, we found that Dph3 promotes melanoma migration and invasion through the AKT signaling pathway. To conclude, our findings suggest a novel mechanism underlying the metastasis of melanoma cells which might serve as a new intervention target for the treatment of melanoma. PMID:23185508

Modifying murine von Willebrand factor A1 domain for in vivo assessment of human platelet therapies.

PubMed

Chen, Jianchun; Tan, Kui; Zhou, Hairu; Lo, Hsuan-Fu; Tronik-Le Roux, Diana; Liddington, Robert C; Diacovo, Thomas G

2008-01-01

The A1 domain of von Willebrand factor (VWF-A1) plays a crucial role in hemostasis and thrombosis by initiating platelet adhesion at sites of arterial injury through interactions with the platelet receptor glycoprotein Ib alpha (GPIbalpha). Here we report that murine VWF-A1 supports limited binding of human platelets. However, atomic models of GPIbalpha-VWF-A1 complexes identified an electrostatic 'hot-spot' that, when mutated in murine VWF-A1, switches its binding specificity from mouse to human GPIbalpha. Furthermore, mice expressing this mutant VWF-A1 display a bleeding phenotype that can be corrected by infusion of human platelets. Mechanistically, human platelets correct the phenotype by forming occlusive thrombi, an event that can be abrogated by blockade of GPIbalpha or by the preadministration of inhibitors of platelet activation or adhesion (clopidogrel (Plavix) and abciximab (ReoPro), respectively). Thus, by modifying a protein interface, we have generated a potential biological platform for preclinical screening of antithrombotics that specifically target human platelets.

Clonogenic colony-forming ability of flow cytometrically isolated hepatic progenitor cells in the murine fetal liver.

PubMed

Taniguchi, H; Kondo, R; Suzuki, A; Zheng, Y W; Takada, Y; Fukunaga, K; Seino, K; Yuzawa, K; Otsuka, M; Fukao, K; Nakauchi, H

2000-01-01

Stem cells are defined as cells having multilineage differentiation potential and self-renewal capability. Hepatic stem cells have aroused considerable interest not only because of their developmental importance but also for their therapeutic potential. However, their presence in the liver has not yet been demonstrated. With the use of a fluorescence-activated cell sorter (FACS) and monoclonal antibodies, we attempted to ascertain whether hepatic stem cells are present in the murine fetal liver. For this purpose, we optimized a cell isolation technique for FACS sorting of fetal liver cells. When isolated CD45 TER119 cells (the non-blood cell fraction in the fetal liver) were tested for their clonogenic colony-forming ability, mechanical dissociation (pipetting) was the most suitable cell isolation technique for FACS sorting. We confirmed that these colonies contained not only cells expressing hepatocyte markers but also cells expressing cholangiocyte markers. To identify hepatic stem cells, studies must focus on CD45TER119- cells in the murine fetal liver.

Cytokine-like factor-1, a novel soluble protein, shares homology with members of the cytokine type I receptor family.

PubMed

Elson, G C; Graber, P; Losberger, C; Herren, S; Gretener, D; Menoud, L N; Wells, T N; Kosco-Vilbois, M H; Gauchat, J F

1998-08-01

In this report we describe the identification, cloning, and expression pattern of human cytokine-like factor 1 (hCLF-1) and the identification and cloning of its murine homologue. They were identified from expressed sequence tags using amino acid sequences from conserved regions of the cytokine type I receptor family. Human CLF-1 and murine CLF-1 shared 96% amino acid identity and significant homology with many cytokine type I receptors. CLF-1 is a secreted protein, suggesting that it is either a soluble subunit within a cytokine receptor complex, like the soluble form of the IL-6R alpha-chain, or a subunit of a multimeric cytokine, e.g., IL-12 p40. The highest levels of hCLF-1 mRNA were observed in lymph node, spleen, thymus, appendix, placenta, stomach, bone marrow, and fetal lung, with constitutive expression of CLF-1 mRNA detected in a human kidney fibroblastic cell line. In fibroblast primary cell cultures, CLF-1 mRNA was up-regulated by TNF-alpha, IL-6, and IFN-gamma. Western blot analysis of recombinant forms of hCLF-1 showed that the protein has the tendency to form covalently linked di- and tetramers. These results suggest that CLF-1 is a novel soluble cytokine receptor subunit or part of a novel cytokine complex, possibly playing a regulatory role in the immune system and during fetal development.

A homozygous CARD9 mutation in a family with susceptibility to fungal infections.

PubMed

Glocker, Erik-Oliver; Hennigs, Andre; Nabavi, Mohammad; Schäffer, Alejandro A; Woellner, Cristina; Salzer, Ulrich; Pfeifer, Dietmar; Veelken, Hendrik; Warnatz, Klaus; Tahami, Fariba; Jamal, Sarah; Manguiat, Annabelle; Rezaei, Nima; Amirzargar, Ali Akbar; Plebani, Alessandro; Hannesschläger, Nicole; Gross, Olaf; Ruland, Jürgen; Grimbacher, Bodo

2009-10-29

Chronic mucocutaneous candidiasis may be manifested as a primary immunodeficiency characterized by persistent or recurrent infections of the mucosa or the skin with candida species. Most cases are sporadic, but both autosomal dominant inheritance and autosomal recessive inheritance have been described. We performed genetic studies in 36 members of a large, consanguineous five-generation family, in which 4 members had recurrent fungal infections and an additional 3 members died during adolescence, 2 after invasive infection of the brain with candida species. All 36 family members were enrolled in the study, and 22 had blood samples taken for DNA analysis. Homozygosity mapping was used to locate the mutated gene. In the 4 affected family members (patients) and the 18 unaffected members we sequenced CARD9, the gene encoding the caspase recruitment domain-containing protein 9, carried out T-cell phenotyping, and performed functional studies, with the use of either leukocytes from the patients or a reconstituted murine model of the genetic defect. We found linkage (lod score, 3.6) to a genomic interval on chromosome 9q, including CARD9. All four patients had a homozygous point mutation in CARD9, resulting in a premature termination codon (Q295X). Healthy family members had wild-type expression of the CARD9 protein; the four patients lacked wild-type expression, which was associated with low numbers of Th17 cells (helper T cells producing interleukin-17). Functional studies based on genetic reconstitution of myeloid cells from Card9(-/-) mice showed that the Q295X mutation impairs innate signaling from the antifungal pattern-recognition receptor dectin-1. An autosomal recessive form of susceptibility to chronic mucocutaneous candidiasis is associated with homozygous mutations in CARD9. 2009 Massachusetts Medical Society

Preliminary characterization of a murine model for 1-bromopropane neurotoxicity: Role of cytochrome P450.

PubMed

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

2016-09-06

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. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Deficits in serum amyloid A contribute to increased neonatal mortality during murine listeriosis.

PubMed

Hawkins, J Seth; Wu, Qingqing; Wang, Yanxia; Lu, Christopher Y

2013-12-01

To understand the increased susceptibility of preterm neonates to infection. A murine listeriosis model using immunohistochemistry, microarray technology, and real-time polymerase chain reaction (PCR). We report that recombinant serum amyloid A (SAA) administered prophylactically 18 h before intraperitoneal (i.p.) inoculation with Listeria monocytogenes conferred a dramatic survival benefit compared with administration of only vehicle in neonatal mice. Neonates that received the recombinant SAA protein had significantly fewer Listeria colony counts on plating of infected liver and showed significantly more activated macrophages, but SAA did not affect postnatal growth. Real-time PCR was used to confirm the microarray findings that gene expression levels for the SAA proteins 1 (Saa1) and 2 (Saa2), in addition to that for orosomucoid-2 (Orm2), were strikingly elevated in the adult compared with those in the neonate. Real-time PCR analysis showed that of the acute phase cytokines, tumor necrosis factor (TNF) gene expression increased exponentially with time in the infected adult, whereas neonates did not show similar increases. The increased susceptibility of neonatal mice to listeriosis is in part mediated by a deficiency in the acute phase response, specifically expression of SAA, and that prophylactic SAA protein before neonatal murine listeriosis results in more macrophage activation, lower Listeria counts, and greater survival.

The role of adrenergic activation on murine luteal cell viability and progesterone production.

PubMed

Wang, Jing; Tang, Min; Jiang, Huaide; Wu, Bing; Cai, Wei; Hu, Chuan; Bao, Riqiang; Dong, Qiming; Xiao, Li; Li, Gang; Zhang, Chunping

2016-09-15

Sympathetic innervations exist in mammalian CL. The action of catecholaminergic system on luteal cells has been the focus of a variety of studies. Norepinephrine (NE) increased progesterone secretion of cattle luteal cells by activating β-adrenoceptors. In this study, murine luteal cells were treated with NE and isoprenaline (ISO). We found that NE increased the viability of murine luteal cells and ISO decreased the viability of luteal cells. Both NE and ISO promoted the progesterone production. Nonselective β-adrenergic antagonist, propranolol reversed the effect of ISO on cell viability but did not reverse the effect of NE on cell viability. Propranolol blocked the influence of NE and ISO on progesterone production. These results reveal that the increase of luteal cell viability induced by NE is not dependent on β-adrenergic activation. α-Adrenergic activation possibly contributes to it. Both NE and ISO increased progesterone production through activating β-adrenergic receptor. Further study showed that CyclinD2 is involved in the increase of luteal cell induced by NE. 3β-Hydroxysteroid dehydrogenase, LHR, steroidogenic acute regulatory protein (StAR), and PGF2α contribute to the progesterone production induced by NE and ISO. Copyright © 2016 Elsevier Inc. All rights reserved.

Baicalin maintains late-stage functional cardiomyocytes in embryoid bodies derived from murine embryonic stem cells.

PubMed

Tang, Meilin; Yin, Mengmeng; Tang, Ming; Liang, Huamin; Yu, Chong; Hu, Xinwu; Luo, Hongyan; Baudis, Birte; Haustein, Moritz; Khalil, Markus; Sarić, Tomo; Hescheler, Jürgen; Xi, Jiaoya

2013-01-01

Low efficiency of cardiomyocyte (CM) differentiation from embryonic stem (ES) cells limits their therapeutic use. The objective of this study was to investigate the effect of baicalin, a natural flavonoid compound, on the in vitro cardiac differentiation of murine ES cells. The induction of ES cells into cardiac-like cells was performed by embryoid body (EB)-based differentiation method. The electrophysiological properties of the ES cell-derived CMs (ES-CMs) were measured by patch-clamp. The biomarkers of ES-CMs were determined by quantitative RT-PCR and immunofluorescence. Continuous baicalin treatment decreased the size of EBs, and increased the proportion of α-actinin-positive CMs and transcript level of cardiac specific markers in beating EBs by inducing cell death of non-CMs. Baicalin increased the percentage of working ES-CMs which had typical responses to β-adrenergic and muscarinic stimulations. Baicalin maintains the late-stage functional CMs in EBs derived from murine ES cells. This study describes a new insight into the various biological effects of baicalin on cardiac differentiation of pluripotent stem cells. Copyright © 2013 S. Karger AG, Basel.

NMR structure of the 101-nucleotide core encapsidation signal of the Moloney murine leukemia virus.

PubMed

D'Souza, Victoria; Dey, Anwesha; Habib, Dina; Summers, Michael F

2004-03-19

The full length, positive-strand genome of the Moloney Murine Leukemia Virus contains a "core encapsidation signal" that is essential for efficient genome packaging during virus assembly. We have determined the structure of a 101-nucleotide RNA that contains this signal (called mPsi) using a novel isotope-edited NMR approach. The method is robust and should be generally applicable to larger RNAs. mPsi folds into three stem loops, two of which (SL-C and SL-D) co-stack to form an extended helix. The third stem loop (SL-B) is connected to SL-C by a flexible, four-nucleotide linker. The structure contains five mismatched base-pairs, an unusual C.CG base-triple platform, and a novel "A-minor K-turn," in which unpaired adenosine bases A340 and A341 of a GGAA bulge pack in the minor groove of a proximal stem, and a bulged distal uridine (U319) forms a hydrogen bond with the phosphodiester of A341. Phylogenetic analyses indicate that these essential structural elements are conserved among the murine C-type retroviruses.

Characteristics of scrub typhus, murine typhus, and Q fever among elderly patients: Prolonged prothrombin time as a predictor for severity.

PubMed

Chang, Ko; Lee, Nan-Yao; Ko, Wen-Chien; Lin, Wei-Ru; Chen, Yen-Hsu; Tsai, Jih-Jin; Chen, Tun-Chieh; Lin, Chun-Yu; Chang, Ya-Ting; Lu, Po-Liang

2017-06-22

The clinical manifestations of scrub typhus, murine typhus and acute Q fever in the elderly are not clear. We conducted a retrospective study to identify the characteristics of the elderly aged ≥65 years with a comparison group aged 18-64 years among patients with scrub typhus, murine typhus, or acute Q fever who were serologically confirmed at three hospitals in Taiwan during 2002-2011. Among 441 cases, including 187 cases of scrub typhus, 166 acute Q fever, and 88 murine typhus, 68 (15.4%) cases were elderly patients. The elderly had a higher severe complication rate (10.3% vs. 3.5%, p = 0.022), but did not have a significantly higher mortality rate (1.47% vs. 0.54%, p = 0.396). Compared with those without severe complications, we found the elderly (p = 0.022), dyspnea (p = 0.006), less relative bradycardia (p = 0.004), less febrile illness (p = 0.004), prolonged prothrombin time (PT) (p = 0.002), higher levels of initial C-reactive protein (p = 0.039), blood leukocyte counts (p = 0.01), and lower platelet counts (p = 0.012) are significantly associated with severe complications. Only prolonged prothrombin time was associated with severe complications in multivariate analysis (p = 0.018, CI 95% 0.01-0.66). Among clinical symptoms and laboratory data, multivariate analysis revealed chills was less frequently occurred in the elderly (p = 0.012, 95% confidence interval [CI]: 1.33-9.99). The elderly cases with scrub typhus, murine typhus, or acute Q fever would be more likely to have severe complications, for which prothrombin time prolongation is an important predictor for severe complications. Copyright © 2017. Published by Elsevier B.V.

[Effects of interferon-gamma on cytotoxicity of murine activated macrophages against murine glioma cells].

PubMed

Ohyama, K; Kikuchi, H; Oda, Y; Moritake, K; Yamasaki, T

1993-06-01

We studied the effects of mouse IFN-gamma on the cytotoxic activity of murine activated macrophages (M phi) against mouse VM-Glioma cells (H-2b). Activated M phi were obtained from peritoneal exudate cells of mice from four strains, C57BL/6 (H-2b), C3H/He(H-2k), DBA/2 (H-2d), and BALB/c (H-2d), following intraperitoneal injection of (1) LPS 200 micrograms, (2) BCG 200 micrograms, (3) C. parvum 200 micrograms, or (4) MDP 350 micrograms 7 days prior to 20-hr 51Cr release-assay. Of the various combination of mouse strains and activating agents tested, that of activated M phi of the C3H/He mouse with induction by LPS had the most tumoricidal effect against the glioma cells, which was not MHC restricted. Although LPS-activated M phi underwent marked loss of cytotoxicity following initiation of in vitro culture, this 24 hr pretreatment with IFN-gamma inhibited this reduction in tumoricidal effects in a dose-dependent fashion. On the other hand, 24 hr pretreatment of target cells with IFN-gamma did not increase their susceptibility to lysis by activated M phi. These findings suggest that IFN-gamma augments the in vitro tumoricidal activation of M phi; This effect appears to be unrelated to any influence of IFN-gamma on target sensitivity to lysis by macrophages.

Viability and functional assessment of murine pancreatic islets after transportation between Korea and Japan.

PubMed

Lee, S; Takahashi, Y; Lee, K M; Mizuno, M; Nemeno, J G; Takebe, T; Lee, J I

2015-04-01

Organ donor scarcity remains a restricting factor for pancreatic islet transplantation. To date, limited information is available on the impact of long-distance transportation on transplantable pancreatic islets. The objective of this study was to assess the effects of transportation on the viability and function of murine pancreatic islet cells. The isolated murine pancreatic islets were transported from Japan to Korea with the use of commercial modes of transportation: subway and commercial airplane. After transportation, the islets were assessed by performing a viability assay and by evaluating the islets' insulin secretion in response to glucose stimulation. A comparative study was performed for evaluating the insulin secretory responses of transported and control islets (not transported). There was no evidence of contamination in the transported pancreatic islets. No significant differences were observed in the viability and functionality of the transported and control islet cells. These findings show the feasibility of pancreatic islet transportation from Japan to Korea. Our data could be used not only for the inter-Asian but also for global advancement of animal and human islet transportation methods and transplantation research. Copyright © 2015 Elsevier Inc. All rights reserved.

A Real Time Chemotaxis Assay Unveils Unique Migratory Profiles amongst Different Primary Murine Macrophages

PubMed Central

Iqbal, Asif J.; Regan-Komito, Daniel; Christou, Ivy; White, Gemma E.; McNeill, Eileen; Kenyon, Amy; Taylor, Lewis; Kapellos, Theodore S.; Fisher, Edward A.; Channon, Keith M.; Greaves, David R.

2013-01-01

Chemotaxis assays are an invaluable tool for studying the biological activity of inflammatory mediators such as CC chemokines, which have been implicated in a wide range of chronic inflammatory diseases. Conventional chemotaxis systems such as the modified Boyden chamber are limited in terms of the data captured given that the assays are analysed at a single time-point. We report the optimisation and validation of a label-free, real-time cell migration assay based on electrical cell impedance to measure chemotaxis of different primary murine macrophage populations in response to a range of CC chemokines and other chemoattractant signalling molecules. We clearly demonstrate key differences in the migratory behavior of different murine macrophage populations and show that this dynamic system measures true macrophage chemotaxis rather than chemokinesis or fugetaxis. We highlight an absolute requirement for Gαi signaling and actin cytoskeletal rearrangement as demonstrated by Pertussis toxin and cytochalasin D inhibition. We also studied the chemotaxis of CD14+ human monocytes and demonstrate distinct chemotactic profiles amongst different monocyte donors to CCL2. This real-time chemotaxis assay will allow a detailed analysis of factors that regulate macrophage responses to chemoattractant cytokines and inflammatory mediators. PMID:23516549

Interleukin 37 limits monosodium urate crystal-induced innate immune responses in human and murine models of gout.

PubMed

Liu, Lei; Xue, Yu; Zhu, Yingfeng; Xuan, Dandan; Yang, Xue; Liang, Minrui; Wang, Juan; Zhu, Xiaoxia; Zhang, Jiong; Zou, Hejian

2016-11-18

Interleukin (IL)-37 has emerged as a fundamental inhibitor of innate immunity. Acute gout is a self-limiting inflammatory response to monosodium urate (MSU) crystals. In the current study, we assessed the preventive and therapeutic effect of recombinant human IL-37 (rhIL-37) in human and murine gout models. We investigated the expression of IL-37 in patients with active and inactive gouty arthritis and assessed the effect of rhIL-37 in human and murine gout models: a human monocyte cell line (THP-1) and human synovial cells (containing macrophage-like and fibroblast-like synoviocytes) exposed to MSU crystals, a peritoneal murine model of gout and a murine gouty arthritis model. After inhibition of Mer receptor tyrosine kinase (Mertk), levels of IL-1β, IL-8 and chemokine (C-C motif) ligand 2 (CCL-2) were detected by ELISA and expression of mammalian homologs of the drosophila Mad gene 3 (Smad), suppressor of cytokine signaling 3 (SOCS3), NACHT-LRR-PYD-containing protein 3 (NLRP3), and IL-8R of THP-1 were assessed by qPCR and western blot to explore the molecular mechanisms. Our studies strongly indicated that rhIL-37 played a potent immunosuppressive role in the pathogenesis of experimental gout models both in vitro and in vivo, by downregulating proinflammatory cytokines and chemokines, markedly reducing neutrophil and monocyte recruitment, and mitigating pathological joint inflammation. In our studies, rhIL-37 suppressed MSU-induced innate immune responses by enhancing expression of Smad3 and IL-1R8 to trigger multiple intracellular switches to block inflammation, including inhibition of NLRP3 and activation of SOCS3. Mertk signaling participated in rhIL-37 inhibitory pathways in gout models. By inhibition of Mertk, the anti-inflammatory effect of rhIL-37 was partly abrogated, and IL-1R8, Smad3 and S​OCS3 expression were suppressed, whereas NLRP3 expression was reactivated. Our studies reveal that IL-37 limits runaway inflammation initiated by MSU crystal

Pannexin1 channels act downstream of P2X7 receptors in ATP-induced murine T-cell death

PubMed Central

Shoji, Kenji F; Sáez, Pablo J; Harcha, Paloma A; Aguila, Hector L; Sáez, Juan C

2014-01-01

Death of murine T cells induced by extracellular ATP is mainly triggered by activation of purinergic P2X7 receptors (P2X7Rs). However, a link between P2X7Rs and pannexin1 (Panx1) channels, which are non-selective, has been recently demonstrated in other cell types. In this work, we characterized the expression and cellular distribution of pannexin family members (Panxs 1, 2 and 3) in isolated T cells. Panx1 was the main pannexin family member clearly detected in both helper (CD4+) and cytotoxic (CD8+) T cells, whereas low levels of Panx2 were found in both T-cell subsets. Using pharmacological and genetic approaches, Panx1 channels were found to mediate most ATP-induced ethidium uptake since this was drastically reduced by Panx1 channel blockers (10Panx1, Probenecid and low carbenoxolone concentration) and absent in T cells derived from Panx1−/− mice. Moreover, electrophysiological measurements in wild-type CD4+ cells treated with ATP unitary current events and pharmacological sensitivity compatible with Panx1 channels were found. In addition, ATP release from T cells treated with 4Br-A23187, a calcium ionophore, was completely blocked with inhibitors of both connexin hemichannels and Panx1 channels. Panx1 channel blockers drastically reduced the ATP-induced T-cell mortality, indicating that Panx1 channels mediate the ATP-induced T-cell death. However, mortality was not reduced in T cells of Panx1−/− mice, in which levels of P2X7Rs and ATP-induced intracellular free Ca2+ responses were enhanced suggesting that P2X7Rs take over Panx1 channels lose-function in mediating the onset of cell death induced by extracellular ATP. PMID:24590064

Family ties: constructing family time in low-income families.

PubMed

Tubbs, Carolyn Y; Roy, Kevin M; Burton, Linda M

2005-03-01

"Family time" is reflected in the process of building and fortifying family relationships. Whereas such time, free of obligatory work, school, and family maintenance activities, is purchased by many families using discretionary income, we explore how low-income mothers make time for and give meaning to focused engagement and relationship development with their children within time constraints idiosyncratic to being poor and relying on welfare. Longitudinal ethnographic data from 61 low-income African American, European American, and Latina American mothers were analyzed to understand how mothers construct family time during daily activities such as talking, play, and meals. We also identify unique cultural factors that shape family time for low-income families, such as changing temporal orientations, centrality of television time, and emotional burdens due to poverty. Implications for family therapy are also discussed.

A Minimal Chimera of Human Cyclin T1 and Tat Binds TAR and Activates Human Immunodeficiency Virus Transcription in Murine Cells

PubMed Central

Fujinaga, Koh; Irwin, Dan; Taube, Ran; Zhang, Fan; Geyer, Matthias; Peterlin, B. Matija

2002-01-01

The transcriptional elongation of human immunodeficiency virus type 1 (HIV-1) is mediated by the virally encoded transactivator Tat and its cellular cofactor, positive transcription elongation factor b (P-TEFb). The human cyclin T1 (hCycT1) subunit of P-TEFb forms a stable complex with Tat and the transactivation response element (TAR) RNA located at the 5′ end of all viral transcripts. Previous studies have demonstrated that hCycT1 binds Tat in a Zn2+-dependent manner via the cysteine at position 261, which is a tyrosine in murine cyclin T1. In the present study, we mutated all other cysteines and histidines that could be involved in this Zn2+-dependent interaction. Because all of these mutant proteins except hCycT1(C261Y) activated viral transcription in murine cells, no other cysteine or histidine in hCycT1 is responsible for this interaction. Next, we fused the N-terminal 280 residues in hCycT1 with Tat. Not only the full-length chimera but also the mutant hCycT1 with an N-terminal deletion to position 249, which retained the Tat-TAR recognition motif, activated HIV-1 transcription in murine cells. This minimal hybrid mutant hCycT1-Tat protein bound TAR RNA as well as human and murine P-TEFb in vitro. We conclude that this minimal chimera not only reproduces the high-affinity binding among P-TEFb, Tat, and TAR but also will be invaluable for determining the three-dimensional structure of this RNA-protein complex. PMID:12438619

The localisation of the heparin binding sites of human and murine interleukin-12 within the carboxyterminal domain of the P40 subunit.

PubMed

Garnier, Pascale; Mummery, Rosemary; Forster, Mark J; Mulloy, Barbara; Gibbs, Roslyn V; Rider, Christopher C

2018-05-09

We have previously shown that the heterodimeric cytokine interleukin-12, and the homodimer of its larger subunit p40, both bind to heparin and heparan sulfate with relatively high affinity. In the present study we characterised these interactions using a series of chemically modified heparins as competitive inhibitors. Human interleukin-12 and p40 homodimer show indistinguishable binding profiles with a panel of heparin derivatives, but that of murine interleukin-12 is distinct. Heparin markedly protects the human and murine p40 subunits, but not the p35 subunits, from cleavage by the bacterial endoprotease LysC, further implicating the larger subunit as the location of the heparin binding site. Moreover the essential role of the carboxyterminal D3 domain in heparin binding is established by the failure of a truncated construct of the p40 subunit lacking this domain to bind. Predictive docking calculations indicate that a cluster of basic residues at the tip of the exposed C'D' loop within D3 is important in heparin binding. However since the human and murine C'D' loops differ considerably in length, the mode and three dimensional orientation of heparin binding are likely to differ substantially between the human and murine p40s. Thus overall the binding of IL-12 via its p40 subunit to heparin-related polysaccharides of the extracellular matrix appears to be functionally important since it has been conserved across mammalian species despite this structural divergence. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Lessons from the Murine Models of West Nile Virus Infection.

PubMed

McGruder, Brenna; Saxena, Vandana; Wang, Tian

2016-01-01

West Nile virus (WNV), a mosquito-borne, single positive-stranded RNA virus, has been the leading cause of arboviral encephalitis in the U.S. and other parts of the world over the past decade. Up to 50 % of WNV convalescent patients were reported to have long-term neurological sequelae or chronic kidney diseases. However, there are neither antiviral drugs nor vaccines available for humans. The underlying mechanism of the long-term sequelae is not clearly understood either. Animal models have been an effective tool to investigate viral pathogenesis and host immunity in humans. Here, we will review several commonly used murine models of WNV infection.

Spatial distribution of endogenous retinoids in the murine embryonic mandible.

PubMed

Kronmiller, J E; Beeman, C S

1994-12-01

Retinoids play an important part in pattern formation during embryonic development. Exogenous retinoids alter the pattern of skeletal, neural and odontogenic tissues. Endogenous retinoids have been demonstrated previously in the murine embryonic mandible, reaching a concentration peak during the initiation of odontogenesis. It was now found that endogenous retinoids are present in a concentration gradient in the embryonic mouse mandible at the time of the initiation of the dental lamina. All-trans-retinoic acid was more concentrated in the incisor region and retinol in the molar region. These results, and the fact that exogenous retinoids produce supernumerary incisors and missing molars, suggest that all-trans-retinoic acid may instruct incisor morphology.

Citrus hallabong [(Citrus unshiu × C. sinensis) × C. reticulata)] exerts potent anti-inflammatory properties in murine splenocytes and TPA-induced murine ear oedema model.

PubMed

Herath, Kalahe Hewage Iresha Nadeeka Madushani; Bing, So Jin; Cho, Jinhee; Kim, Areum; Kim, Gi-Ok; Lee, Jong-Chul; Jee, Youngheun

2016-12-01

Hallabong [(Citrus unshiu × C. sinensis) X C. reticulata)] (Rutaceae) is a hybrid citrus cultivated in temperate regions of South Korea. Its fruit is well-known for pharmacological properties. This study examined the anti-inflammatory effect of 80% ethanol extract of Hallabong (HE) on concanavalin A (Con A)-stimulated splenocytes and mouse oedema model induced by 12-O-tetradecanoylphorbal acetate (TPA). Murine splenocytes treated with HE were stimulated with Con A (10 μg/mL, for 24 h) were evaluated for T-cell population and production of inflammatory cytokines IL-2, IL-4 and IFN-γ. Anti-inflammatory effect of topically applied HE (100 μg/20 μL) on TPA (4 μg/20 μL/ear)-induced ear oedema was investigated in mouse model. HE-treated Con A-stimulated murine splenocytes showed a marked decrease in CD44/CD62L + memory T-cell population, an important marker for anti-inflammatory activity, and a significant inhibition in the production of IL-2 and IFN-γ. HE treatment had reduced the mouse skin oedema (47%) and myeloperoxidase (MPO) activity significantly (40%) in TPA-challenged tissues. More importantly, immunohistochemical localization revealed the suppressed (p < 0.05) expression of inducible nitric oxide (iNOS), cyclooxygenase-2 (COX2). HE decreased the infiltration of CD3 + T cells and F4/80 + macrophages to the site of inflammation and a topical application of HE significantly suppressed the expression of TNF-α (20.2%). A topical application of HE can exert a potential anti-inflammatory effect and HE can be explored further as a putative alternative therapeutic agent for inflammatory oedema.

A MURINE MODEL FOR LOW MOLECULAR WEIGHT CHEMICALS: DIFFERENTIATION OF RESPIRATORY SENSITIZERS (TMA) FROM CONTACT SENSITIZERS (DNFB)

EPA Science Inventory

Exposure to low molecular weight (LMW) chemicals contributes to both dermal and respiratory sensitization and is an important occupational health problem. Our goal was to establish an in vivo murine model for hazard identification of LMW chemicals that have the potential to indu...

Markers of murine embryonic and neural stem cells, neurons and astrocytes: reference points for developmental neurotoxicity testing

EPA Science Inventory

Developmental neurotoxicity (DNT) is a significant concern for environmental chemicals, as well as for food and drug constituents. The sensitivity of animal-based DNT models is unclear, and they are expensive and time consuming. Murine embryonic stem cells (mESC) recapitulate sev...

Lin28b is sufficient to drive liver cancer and necessary for its maintenance in murine models

PubMed Central

Nguyen, Liem H.; Robinton, Daisy A.; Seligson, Marc; Wu, Linwei; Li, Lin; Rakheja, Dinesh; Comerford, Sarah; Ramezani, Saleh; Sun, Xiankai; Parikh, Monisha; Yang, Erin; Powers, John T.; Shinoda, Gen; Shah, Samar; Hammer, Robert; Daley, George Q.; Zhu, Hao

2014-01-01

SUMMARY Lin28a/b are RNA-binding proteins that influence stem cell maintenance, metabolism, and oncogenesis. Poorly differentiated, aggressive cancers often overexpress Lin28, but its role in tumor initiation or maintenance has not been definitively addressed. We report that LIN28B overexpression is sufficient to initiate hepatoblastoma and hepatocellular carcinoma in murine models. We also detected Lin28b overexpression in MYC-driven hepatoblastomas, and liver-specific deletion of Lin28a/b reduced tumor burden, extended latency, and prolonged survival. Both intravenous siRNA against Lin28b and conditional Lin28b deletion reduced tumor burden and prolonged survival. Igf2bp proteins are upregulated and Igf2bp3 is required in the context of LIN28B overexpression to promote growth. Thus, multiple murine models demonstrate that Lin28b is both sufficient to initiate liver cancer and necessary for its maintenance. PMID:25117712

Importance of basophils in eosinophilic asthma: the murine counterpart.

PubMed

Poddighe, D; Mathias, C B; Brambilla, I; Marseglia, G L; Oettgen, H C

2018-01-01

Several experimental studies in mice showed that basophils participate in the initiation of Th2 adaptive immune response, in addition to the effector phase. However, the role of basophils in allergic airway inflammation is less clear. The aim of this experiment was to assess the importance of basophils in recruiting inflammatory cells and, in particular, eosinophils in a murine model of asthma induced by Aspergillus fumigatus allergens. Additionally, bronchial reactivity was evaluated. Basophil depletion resulted in a reduction of inflammatory cells in the airways and eosinophil recruitment was significantly impaired. Also bronchial reactivity seemed to be impaired in basophil-depleted mice, but the result was not statistically significant. According to these preliminary data, basophils seem to influence the local eosinophilic response of allergic asthma.

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

PubMed

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

2015-08-01

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. Copyright © 2015 Elsevier B.V. All rights reserved.

Murine Models of Sepsis and Trauma: Can We Bridge the Gap?

PubMed

Stortz, Julie A; Raymond, Steven L; Mira, Juan C; Moldawer, Lyle L; Mohr, Alicia M; Efron, Philip A

2017-07-01

Sepsis and trauma are both leading causes of death in the United States and represent major public health challenges. Murine models have largely been used in sepsis and trauma research to better understand the pathophysiological changes that occur after an insult and to develop potential life-saving therapeutic agents. Mice are favorable subjects for this type of research given the variety of readily available strains including inbred, outbred, and transgenic strains. In addition, they are relatively easy to maintain and have a high fecundity. However, pharmacological therapies demonstrating promise in preclinical mouse models of sepsis and trauma often fail to demonstrate similar efficacy in human clinical trials, prompting considerable criticism surrounding the capacity of murine models to recapitulate complex human diseases like sepsis and traumatic injury. Fundamental differences between the two species include, but are not limited to, the divergence of the transcriptomic response, the mismatch of temporal response patterns, differences in both innate and adaptive immunity, and heterogeneity within the human population in comparison to the homogeneity of highly inbred mouse strains. Given the ongoing controversy, this narrative review aims to not only highlight the historical importance of the mouse as an animal research model but also highlight the current benefits and limitations of the model as it pertains to sepsis and trauma. Lastly, this review will propose future directions that may promote further use of the model. Published by Oxford University Press on behalf of the Institute for Laboratory Animal Research 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Comparisons between Murine Polyomavirus and Simian Virus 40 Show Significant Differences in Small T Antigen Function ▿

PubMed Central

Andrabi, Shaida; Hwang, Justin H.; Choe, Jennifer Kean; Roberts, Thomas M.; Schaffhausen, Brian S.

2011-01-01

Although members of a virus family produce similar gene products, those products may have quite different functions. Simian virus 40 (SV40) large T antigen (LT), for example, targets p53 directly, but murine polyomavirus LT does not. SV40 small T antigen (SVST) has received considerable attention because of its ability to contribute to transformation of human cells. Here, we show that there are major differences between SVST and polyomavirus small T antigen (POLST) in their effects on differentiation, transformation, and cell survival. Both SVST and POLST induce cell cycle progression. However, POLST also inhibits differentiation of 3T3-L1 preadipocytes and C2C12 myoblasts. Additionally, POLST induces apoptosis of mouse embryo fibroblasts. SVST reduces the proapoptotic transcriptional activity of FOXO1 through phosphorylation. On the other hand, SVST complements large T antigen and Ras for the transformation of human mammary epithelial cells (HMECs), but POLST does not. Mechanistically, the differences between SVST and POLST may lie in utilization of protein phosphatase 2A (PP2A). POLST binds both Aα and Aβ scaffolding subunits of PP2A while SVST binds only Aα. Knockdown of Aβ could mimic POLST-induced apoptosis. The two small T antigens can target different proteins for dephosphorylation. POLST binds and dephosphorylates substrates, such as lipins, that SVST does not. PMID:21835797

Development of a broadly reactive nested reverse transcription-PCR assay to detect murine noroviruses, and investigation of the prevalence of murine noroviruses in laboratory mice in Japan.

PubMed

Kitajima, Masaaki; Oka, Tomoichiro; Tohya, Yukinobu; Katayama, Hiroyuki; Takeda, Naokazu; Katayama, Kazuhiko

2009-09-01

A broadly reactive nested RT-PCR assay to detect MNV was developed and subsequently used to investigate the prevalence of MNV in laboratory mice in Japan. MNV were detected in 8 (22%) of 37 murine stool specimens by second-round PCR, although no positive band was obtained from any specimen by first-round PCR. Genetic analysis of the second round PCR products showed that MNV sequences detected in this study were closely matched (97.2 approximately 99.1%) to that of MNV-3 (DQ223042). This is the first report demonstrating the prevalence of MNV in Japan.

CD11c identifies a subset of murine liver natural killer cells that responds to adenoviral hepatitis

PubMed Central

Burt, Bryan M.; Plitas, George; Stableford, Jennifer A.; Nguyen, Hoang M.; Bamboat, Zubin M.; Pillarisetty, Venu G.; DeMatteo, Ronald P.

2008-01-01

The liver contains a unique repertoire of immune cells and a particular abundance of NK cells. We have found that CD11c defines a distinct subset of NK cells (NK1.1+CD3−) in the murine liver whose function was currently unknown. In naïve animals, CD11c+ liver NK cells displayed an activated phenotype and possessed enhanced effector functions when compared with CD11c− liver NK cells. During the innate response to adenovirus infection, CD11c+ NK cells were the more common IFN-γ-producing NK cells in the liver, demonstrated enhanced lytic capability, and gained a modest degree of APC function. The mechanism of IFN-γ production in vivo depended on TLR9 ligation as well as IL-12 and -18. Taken together, our findings demonstrate that CD11c+ NK cells are a unique subset of NK cells in the murine liver that contribute to the defense against adenoviral hepatitis. PMID:18664530

Identification of factors for physicians to facilitate early differential diagnosis of scrub typhus, murine typhus, and Q fever from dengue fever in Taiwan.

PubMed

Chang, Ko; Lee, Nan-Yao; Ko, Wen-Chien; Tsai, Jih-Jin; Lin, Wei-Ru; Chen, Tun-Chieh; Lu, Po-Liang; Chen, Yen-Hsu

2017-02-01

Dengue fever, rickettsial diseases, and Q fever are acute febrile illnesses with similar manifestations in tropical areas. Early differential diagnosis of scrub typhus, murine typhus, and Q fever from dengue fever may be made by understanding the distinguishing clinical characteristics and the significance of demographic and weather factors. We conducted a retrospective study to identify clinical, demographic, and meteorological characteristics of 454 dengue fever, 178 scrub typhus, 143 Q fever, and 81 murine typhus cases in three Taiwan hospitals. Case numbers of murine typhus and Q fever correlated significantly with temperature and rainfall; the scrub typhus case number was only significantly related with temperature. Neither temperature nor rainfall correlated with the case number of dengue fever. The rarity of dengue fever cases from January to June in Taiwan may be a helpful clue for diagnosis in the area. A male predominance was observed, as the male-to-female rate was 2.1 for murine typhus and 7.4 for Q fever. Multivariate analysis revealed the following six important factors for differentiating the rickettsial diseases and Q fever group from the dengue fever group: fever ≥8 days, alanine aminotransferase > aspartate aminotransferase, platelets >63,000/mL, C-reactive protein >31.9 mg/L, absence of bone pain, and absence of a bleeding syndrome. Understanding the rarity of dengue in the first half of a year in Taiwan and the six differentiating factors may help facilitate the early differential diagnosis of rickettsial diseases and Q fever from dengue fever, permitting early antibiotic treatment. Copyright © 2015. Published by Elsevier B.V.

Assessment of carbon nanoparticle exposure on murine macrophage function

NASA Astrophysics Data System (ADS)

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

Protopine reduces the inflammatory activity of lipopolysaccharide-stimulated murine macrophages.

PubMed

Bae, Deok Sung; Kim, Young Hoon; Pan, Cheol-Ho; Nho, Chu Won; Samdan, Javzan; Yansan, Jamyansan; Lee, Jae Kown

2012-02-01

Protopine is an isoquinoline alkaloid contained in plants in northeast Asia. In this study, we investigated whether protopine derived from Hypecoum erectum L could suppress lipopolysaccharide (LPS)-induced inflammatory responses in murine macrophages (Raw 264.7 cells). Protopine was found to reduce nitric oxide (NO), cyclooxygenase-2 (COX-2), and prostaglandin E(2) (PGE(2)) production by LPS-stimulated Raw 264.7 cells, without a cytotoxic effect. Pre-treatment of Raw 264.7 cells with protopine reduced the production of pro-inflammatory cytokines. These inhibitory effects were caused by blocking phosphorylation of mitogen-activated protein kinases (MAP kinases) and also blocking activation of a nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB).

Expression of human factor IX gene in murine plasma through lentiviral vector-infected haematopoietic stem cells.

PubMed

Chen, Haoming; Yao, Hengmei; Huang, Lu; Shen, Qi; Jia, William; Xue, Jinglun

2006-12-01

1. Haematopoietic stem cells (HSC) are an attractive target for gene therapy. Gene transfer to HSC can provide a potential cure for many inherited diseases. Moreover, recombinant lentiviral vectors can transfer genes efficiently to HSC. In the present study, we used the recombinant lentiviruses FUGW (Flip, ubiquitin promoter, GFP and WRE vector) and FUXW (Flip, ubiquitin promoter, F IX and WRE vector), which carry the enhanced green fluorescent protein (EGFP) and human factor IX (hFIX) gene, respectively, to infect HSC. 2. High titres of recombinant lentivirus were prepared from 293T cells by calcium phosphate-mediated transient cotransfection. Murine mononuclear cells (MNC) separated from murine bone marrow and HSC separated by magnetic cell sorting were cultured in vitro. Cells they were infected by the recombinant lentiviruses FUGW and FUXW. The expression of EGFP was observed under a fluorescent microscope and was analysed by fluorescence-activated cell sorting, whereas the expression of hFIX was detected by ELISA. 3. The results show that the lentiviral vectors can efficiently infect murine HSC in vitro and that transduction was more efficient following cytokine treatment with interleukin (IL)-3, IL-6 and stem cell factor. 4. Haematopoietic stem cells infected with lentivirus FUXW were transplanted into [(60)Co]-irradiated non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mice. The expression of hFIX in the blood plasma of the transplanted mice reached a peak of 44.9 +/- 7.6 ng/mL on Day 7. An assay of transaminase levels and a histological study of the liver showed that there was no significant damage following HSC transplantation to mice. 5. The results of the present study suggest that transplantation of HSC results in the persistant expression of hFIX in mice, which may be useful in haemophilia B gene therapy.

In Vivo Pharmacodynamic Target Assessment of Eravacycline against Escherichia coli in a Murine Thigh Infection Model.

PubMed

Zhao, Miao; Lepak, Alexander J; Marchillo, Karen; VanHecker, Jamie; Andes, David R

2017-07-01

Eravacycline is a novel fluorocycline antibiotic with potent activity against a broad range of pathogens, including strains with tetracycline and other drug resistance phenotypes. The goal of the studies was to determine which pharmacokinetic/pharmacodynamic (PK/PD) parameter and magnitude best correlated with efficacy in the murine thigh infection model. Six Escherichia coli isolates were utilized for the studies. MICs were determined using CLSI methods and ranged from 0.125 to 0.25 mg/liter. A neutropenic murine thigh infection model was utilized for all treatment studies. Single-dose plasma pharmacokinetics were determined in mice after administration of 2.5, 5, 10, 20, 40, and 80 mg/kg of body weight. Pharmacokinetic studies exhibited maximum plasma concentration ( C max ) values of 0.34 to 2.58 mg/liter, area under the concentration-time curve (AUC) from time zero to infinity (AUC 0-∞ ) values of 2.44 to 57.6 mg · h/liter, and elimination half-lives of 3.9 to 17.6 h. Dose fractionation studies were performed using total drug doses of 6.25 mg/kg to 100 mg/kg fractionated into 6-, 8-, 12-, or 24-h regimens. Nonlinear regression analysis demonstrated that the 24-h free drug AUC/MIC ( f AUC/MIC) was the PK/PD parameter that best correlated with efficacy ( R 2 = 0.80). In subsequent studies, we used the neutropenic murine thigh infection model to determine if the magnitude of the AUC/MIC needed for the efficacy of eravacycline varied among pathogens. Mice were treated with 2-fold increasing doses (range, 3.125 to 50 mg/kg) of eravacycline every 12 h. The mean f AUC/MIC magnitudes associated with the net stasis and the 1-log-kill endpoints were 27.97 ± 8.29 and 32.60 ± 10.85, respectively. Copyright © 2017 American Society for Microbiology.

A Novel Subgenomic Murine Leukemia Virus RNA Transcript Results from Alternative Splicing

PubMed Central

Déjardin, Jérôme; Bompard-Maréchal, Guillaume; Audit, Muriel; Hope, Thomas J.; Sitbon, Marc; Mougel, Marylène

2000-01-01

Here we show the existence of a novel subgenomic 4.4-kb RNA in cells infected with the prototypic replication-competent Friend or Moloney murine leukemia viruses (MuLV). This RNA derives by splicing from an alternative donor site (SD′) within the capsid-coding region to the canonical envelope splice acceptor site. The position and the sequence of SD′ was highly conserved among mammalian type C and D oncoviruses. Point mutations used to inactivate SD′ without changing the capsid-coding ability affected viral RNA splicing and reduced viral replication in infected cells. PMID:10729146

Characterization of tumor cell lines derived from murine gammaherpesvirus-68-infected mice.

PubMed Central

Usherwood, E J; Stewart, J P; Nash, A A

1996-01-01

Cell lines were derived from mice with murine gammaherpesvirus-68 (MHV-68)-associated lymphoproliferative disease. Four were of an ambiguous phenotype and were MHV-68 negative. One, S11, was a B lymphocyte that contained MHV-68 genomes in both linear and episomal forms and released virus. The line was clonable and grew into tumors in nude mice. This is the first naturally occurring MHV-68-positive B-cell line to be generated, and it will be an invaluable tool for the study of MHV-68 latency. PMID:8709292

Induction of Protective CTL Responses in Newborn Mice by a Murine Retrovirus

NASA Astrophysics Data System (ADS)

Sarzotti, Marcella; Robbins, Deanna S.; Hoffman, Paul M.

1996-03-01

The susceptibility of neonates to virus-induced disease is thought to reflect, in part, the immaturity of their immune systems. However, inoculation of newborn mice with low doses of Cas-Br-M murine leukemia virus induced a protective cytotoxic T lymphocyte (CTL) response. The inability of neonates to develop a CTL response to high doses of virus was not the result of immunological immaturity but correlated with the induction of a nonprotective type 2 cytokine response. Thus, the initial viral dose is critical in the development of protective immunity in newborns.

DNA methyltransferase inhibition increases efficacy of adoptive cellular immunotherapy of murine breast cancer.

PubMed

Terracina, Krista P; Graham, Laura J; Payne, Kyle K; Manjili, Masoud H; Baek, Annabel; Damle, Sheela R; Bear, Harry D

2016-09-01

Adoptive T cell immunotherapy is a promising approach to cancer treatment that currently has limited clinical applications. DNA methyltransferase inhibitors (DNAMTi) have known potential to affect the immune system through multiple mechanisms that could enhance the cytotoxic T cell responses, including: upregulation of tumor antigen expression, increased MHC class I expression, and blunting of myeloid derived suppressor cells (MDSCs) expansion. In this study, we have investigated the effect of combining the DNAMTi, decitabine, with adoptive T cell immunotherapy in the murine 4T1 mammary carcinoma model. We found that expression of neu, MHC class I molecules, and several murine cancer testis antigens (CTA) was increased by decitabine treatment of 4T1 cells in vitro. Decitabine also increased expression of multiple CTA in two human breast cancer cell lines. Decitabine-treated 4T1 cells stimulated greater IFN-gamma release from tumor-sensitized lymphocytes, implying increased immunogenicity. Expansion of CD11b + Gr1 + MDSC in 4T1 tumor-bearing mice was significantly diminished by decitabine treatment. Decitabine treatment improved the efficacy of adoptive T cell immunotherapy in mice with established 4T1 tumors, with greater inhibition of tumor growth and an increased cure rate. Decitabine may have a role in combination with existing and emerging immunotherapies for breast cancer.

Immunotoxicity and allergic potential induced by topical application of dimethyl carbonate (DMC) in a murine model

PubMed Central

Anderson, Stacey E.; Franko, Jennifer; Anderson, Katie L.; Munson, Albert E.; Lukomska, Ewa; Meade, B. Jean

2015-01-01

Dimethyl carbonate (DMC) is an industrial chemical, used as a paint and adhesive solvent, with the potential for significant increases in production. Using select immune function assays, the purpose of these studies was to evaluate the immunotoxicity of DMC following dermal exposure using a murine model. Following a 28-day exposure, DMC produced a significant decrease in thymus weight at concentrations of 75% and greater. No effects on body weight, hematological parameters (erythrocytes, leukocytes, and their differentials), or immune cell phenotyping (B-cells, T-cells, and T-cell sub-sets) were identified. The IgM antibody response to sheep red blood cell (SRBC) was significantly reduced in the spleen but not the serum. DMC was not identified to be an irritant and evaluation of the sensitization potential, conducted using the local lymph node assay (LLNA) at concentrations ranging from 50–100%, did not identify increases in lymphocyte proliferation. These results demonstrate that dermal exposure to DMC induces immune suppression in a murine model and raise concern about potential human exposure and the need for occupational exposure regulations. PMID:22953780

Peptidylarginine deiminase inhibition reduces vascular damage and modulates innate immune responses in murine models of atherosclerosis.

PubMed

Knight, Jason S; Luo, Wei; O'Dell, Alexander A; Yalavarthi, Srilakshmi; Zhao, Wenpu; Subramanian, Venkataraman; Guo, Chiao; Grenn, Robert C; Thompson, Paul R; Eitzman, Daniel T; Kaplan, Mariana J

2014-03-14

Neutrophil extracellular trap (NET) formation promotes vascular damage, thrombosis, and activation of interferon-α-producing plasmacytoid dendritic cells in diseased arteries. Peptidylarginine deiminase inhibition is a strategy that can decrease in vivo NET formation. To test whether peptidylarginine deiminase inhibition, a novel approach to targeting arterial disease, can reduce vascular damage and inhibit innate immune responses in murine models of atherosclerosis. Apolipoprotein-E (Apoe)(-/-) mice demonstrated enhanced NET formation, developed autoantibodies to NETs, and expressed high levels of interferon-α in diseased arteries. Apoe(-/-) mice were treated for 11 weeks with daily injections of Cl-amidine, a peptidylarginine deiminase inhibitor. Peptidylarginine deiminase inhibition blocked NET formation, reduced atherosclerotic lesion area, and delayed time to carotid artery thrombosis in a photochemical injury model. Decreases in atherosclerosis burden were accompanied by reduced recruitment of netting neutrophils and macrophages to arteries, as well as by reduced arterial interferon-α expression. Pharmacological interventions that block NET formation can reduce atherosclerosis burden and arterial thrombosis in murine systems. These results support a role for aberrant NET formation in the pathogenesis of atherosclerosis through modulation of innate immune responses.

The Effects of Simulated Weightlessness on Susceptibility to Viral and Bacterial Infections Using a Murine Model

NASA Technical Reports Server (NTRS)

Gould, C. L.

1985-01-01

Certain immunological responses may be compromised as a result of changes in environmental conditions, such as the physiological adaptation to and from the weightlessness which occurs during space flight and recovery. A murine antiorthostatic model was developed to simulate weightlessness. Using this model, the proposed study will determine if differences in susceptibility to viral and bacterial infections exist among mice suspended in an antiorthostatic orientation to simulate weightlessness, mice suspended in an orthostatic orientation to provide a stressful situation without the condition of weightlessness simulation, and non-suspended control mice. Inbred mouse strains which are resistant to the diabetogenic effects of the D variant of encephalomyocarditis virus (EMC-D) and the lethal effects of Salmonella typhimurium will be evaluated. Glucose tolerance tests will be performed on all EMC-D-infected and non-infected control groups. The incidence of EMC-D-induced diabetes and the percentage survival of S. typhimurium-infected animals will be determined in each group. An additional study will determine the effects of simulated weightlessness on murine responses to exogenous interferon.

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

PubMed

Li, Mi; Gustchina, Alla; Matúz, Krisztina; Tözsér, Jozsef; Namwong, Sirilak; Goldfarb, Nathan E; Dunn, Ben M; Wlodawer, Alexander

2011-11-01

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(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. Journal compilation © 2011 FEBS. No claim to original US government works.

Maternal methyl-donor supplementation induces prolonged murine offspring colitis susceptibility in association with mucosal epigenetic and microbiomic changes

USDA-ARS?s Scientific Manuscript database

Developmental epigenetic changes, such as DNA methylation, have been recognized as potential pathogenic factors in inflammatory bowel diseases, the hallmark of which is an exaggerated immune response against luminal microbes. A methyl-donor (MD) diet can modify DNA methylation at select murine genom...

Who counts as family? Family typologies, family support, and family undermining among young adult gay and bisexual men.

PubMed

Soler, Jorge H; Caldwell, Cleopatra H; Córdova, David; Harper, Gary; Bauermeister, José A

2018-06-01

Gay and bisexual men may form chosen families in addition to or in place of families of origin. However, the characteristics of these diverse families remain largely unexamined in the quantitative literature. The purpose of this study was to develop a family typology based on responses from a racially and ethnically diverse sample of young adult gay and bisexual men (YGBM) recruited from the Detroit Metropolitan Area (N=350; 18-29 years old). To explore the role of family, we then examined family social support and social undermining in relation to YGBM psychological distress within different family types. A series of multivariate regressions were used to examine associations between family social support and social undermining with depression and anxiety outcomes. The majority (88%) of YGBM included family of origin in their definitions of family and 63% indicated having chosen families. Associations between family social processes and psychological outcomes varied by type of family, suggesting that family composition shapes how perceptions of support and undermining relate to experiencing symptoms of depression and anxiety. Chosen families play a prominent role in the lives of YGBM and should not be overlooked in family research. Findings also highlight the importance of examining co-occurring family social support and social stress processes to further address psychological distress symptoms among YGBM.

Temporal profile of inflammatory response to fracture and hemorrhagic shock: Proposal of a novel long-term survival murine multiple trauma model.

PubMed

Kleber, Christian; Becker, Christopher A; Malysch, Tom; Reinhold, Jens M; Tsitsilonis, Serafeim; Duda, Georg N; Schmidt-Bleek, Katharina; Schaser, Klaus D

2015-07-01

Hemorrhagic shock (hS) interacts with the posttraumatic immune response and fracture healing in multiple trauma. Due to the lack of a long-term survival multiple trauma animal models, no standardized analysis of fracture healing referring the impact of multiple trauma on fracture healing was performed. We propose a new long-term survival (21 days) murine multiple trauma model combining hS (microsurgical cannulation of carotid artery, withdrawl of blood and continuously blood pressure measurement), femoral (osteotomy/external fixation) and tibial fracture (3-point bending technique/antegrade nail). The posttraumatic immune response was measured via IL-6, sIL-6R ELISA. The hS was investigated via macrohemodynamics, blood gas analysis, wet-dry lung ration and histologic analysis of the shock organs. We proposed a new murine long-term survival (21 days) multiple trauma model mimicking clinical relevant injury patterns and previously published human posttraumatic immune response. Based on blood gas analysis and histologic analysis of shock organs we characterized and standardized our murine multiple trauma model. Furthermore, we revealed hemorrhagic shock as a causative factor that triggers sIL-6R formation underscoring the fundamental pathophysiologic role of the transsignaling mechanism in multiple trauma. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

[Expression of homeobox gene Msx-1, Msx-2 and Dlx-2 during murine mandibular first molar development].

PubMed

Ma, Li; Chen, Zhi; Song, Guang-tai; Fan, Ming-wen; Zhang, Qi; Wang, Zhi-feng

2003-11-01

To observe the expression of homeobox gene Msx-1, Msx-2 and Dlx-2 during murine mandibular first molar development. The murine heads or mandibles on embryonic days 11-18 (E11-18) and postnatal day 1-3 (P1-3) were removed, fixed and embedded, 5 micro m serial sections were cut in the coronal plane. Msx-1, Msx-2 and Dlx-2 RNA probes were synthesized by in vitro transcription and labeled with digoxigenin. Msx-1, Msx-2 and Dlx-2 mRNA expression was observed after in situ hybridization. During molar development Msx-1 transcripts appeared only in mesenchymal cells, not in epithelial cells. Msx-2 and Dlx-2 both expressed in the epithelial and mesenchymal cells. At the initiation stage of the molar development Msx-2 and Dlx-2 had similar expression. At the bud stage (E13-14) Msx-2 mRNA signaling was intensive in the enamel organ and slight in the dental mesenchyme; Dlx-2 signaling was stronger in the dental papilla. At cap stage (E15-16) Msx-2 showed prominent mRNA signaling in enamel knot and Dlx-2 was maximal in the dental papilla. At the late bell stage (P2-3) Msx-2 transcripts were observed in odontoblasts but not labeled in ameloblasts, and Dlx-2 transcripts appeared in ameloblasts but no labeling was seen in odontoblasts. Msx-1, Msx-2 and Dlx-2 are expressed in various patterns during murine mandibular first molar development, suggesting they possibly play a role in the interaction between the epithelium and mesenchyme during the molar development.

Modulation of phenotypic and functional maturation of murine dendritic cells (DCs) by purified Achyranthes bidentata polysaccharide (ABP).

PubMed

Zou, Yaxuan; Meng, Jingjuan; Chen, Wenna; Liu, Jingling; Li, Xuan; Li, Weiwei; Lu, Changlong; Shan, Fengping

2011-08-01

There are a large number of interactions at molecular and cellular levels between the plant polysaccharides and immune system. Plant polysaccharides present an interesting effects as immunomodulators, particularly in the induction of the cells both in innate and adaptive immune systems. Activation of DCs could improve antitumoral responses usually diminished in cancer patients, and natural adjuvants provide a possibility of inducing this activation. ABP is a purified polysaccharide isolated from Achyranthes bidentata, a traditional Chinese medicine (TCM). The aim of this study is to investigate modulation of phenotypic and functional maturation of murine DCs by ABP. Both phenotypic and functional activities were assessed with use of conventional scanning electronic microscopy (SEM) for the morphology of the DC, transmitted electron microscopy (TEM) for intracellular lysosomes inside the DC, cellular immunohistochemistry for phagocytosis by the DCs, flow cytometry (FCM) for the changes in key surface molecules, bio-assay for the activity of acidic phosphatases (ACP), and ELISA for the production of pro-inflammatory cytokine IL-12. In fact, we found that purified ABP induced phenotypic maturation revealed by increased expression of CD86, CD40, and MHC II. Functional experiments showed the down-regulation of ACP inside DCs (which occurs when phagocytosis of DCs is decreased, and antigen presentation increased with maturation). Finally, ABP increased the production of IL-12. These data reveal that ABP promotes effective activation of murine DCs. This adjuvant-like activity may have therapeutic applications in clinical settings where immune responses need boosting. It is therefore concluded that ABP can exert positive modulation to murine DCs. Copyright © 2011 Elsevier B.V. All rights reserved.

Generation of murine induced pluripotent stem cells by using high-density distributed electrodes network.

PubMed

Lu, Ming-Yu; Li, Zhihong; Hwang, Shiaw-Min; Linju Yen, B; Lee, Gwo-Bin

2015-09-01

This study reports a robust method of gene transfection in a murine primary cell model by using a high-density electrodes network (HDEN). By demonstrating high cell viability after gene transfection and successful expression of transgenes including fluorescent proteins, the HDEN device shows great promise as a solution in which reprogramming efficiency using non-viral induction for generation of murine induced pluripotent stem cells (iPSCs) is optimized. High and steady transgene expression levels in host cells of iPSCs can be demonstrated using this method. Moreover, the HDEN device achieved successful gene transfection with a low voltage of less than 180 V while requiring relatively low cell numbers (less than 1.5 × 10(4) cells). The results are comparable to current conventional methods, demonstrating a reasonable fluorescent-plasmid transfection rate (42.4% in single transfection and 24.5% in triple transfection) and high cell viability of over 95%. The gene expression levels of each iPSC factor was measured to be over 10-fold higher than that reported in previous studies using a single mouse embryonic fibroblast cell. Our results demonstrate that the generation of iPSCs using HDEN transfection of plasmid DNA may be a feasible and safe alternative to using viral transfection methods in the near future.

[Combined effect of cisplatin and caffeine on murine B16-BL6 melanoma cells].

PubMed

Yasutake, H; Tsuchiya, H; Sugihara, M; Tomita, K; Ueda, Y; Tanaka, M; Sasaki, T

1989-05-01

Combined effect of cisplatin and caffeine on murine B16-BL6 melanoma cells was studied. Synergistic inhibition of the cell growth was observed when caffeine (2 mM) was added continuously after one hour exposure of cisplatin. On the other hand, when caffeine was added before one hour exposure of cisplatin or one hour simultaneous exposure with cisplatin, synergistic effect was not shown. In the analysis of DNA histogram obtained from flow cytometry, S and G2/M accumulation was observed by the treatment of cisplatin and that accumulation was reduced by the combination of cisplatin and caffeine. From this findings, it was suggested that caffeine would inhibit DNA repair process. Furthermore, according to morphological studies with hematoxylin-eosin stain and Fontana-Masson stain, the addition of caffeine alone resulted in mild swelling of melanoma cells and the decrease of nuclear-cytoplasmic ratio. The combination of cisplatin and caffeine caused marked swelling of melanoma cells and remarkable increase of dendrite-like processes. Melanogenesis was also enhanced by the addition of these two drugs. Many matured melanosomes, increases of mitochondria, Golgi's apparatus and endoplasmic reticula were observed by the use of electron microscope. These findings implied that the combination of cisplatin and caffeine induced a differentiation of murine melanoma cells.

Detailed methodology for high resolution scanning electron microscopy (SEM) of murine malaria parasitized-erythrocytes.

PubMed

Hayakawa, Eri H; Matsuoka, Hiroyuki

2016-10-01

Scanning electron microscopy (SEM) is a powerful tool used to investigate object surfaces and has been widely applied in both material science and biology. With respect to the study of malaria, SEM revealed that erythrocytes infected with Plasmodium falciparum, a human parasite, display 'knob-like' structures on their surface comprising parasitized proteins. However, detailed methodology for SEM studies of malaria parasites is lacking in the literature making such studies challenging. Here, we provide a step-by-step guide to preparing Plasmodium-infected erythrocytes from two mouse strains for SEM analysis with minimal structural deterioration. We tested three species of murine malaria parasites, P. berghei, P. yoelii, and P. chabaudi, as well as non-parasitized human erythrocytes and P. falciparum-infected erythrocytes for comparisons. Our data demonstrated that the surface structures of parasitized erythrocytes between the three species of murine parasites in the two different strains of mice were indistinguishable and no surface alterations were observed in P. falciparum-erythrocytes. Our SEM observations contribute towards an understanding of the molecular mechanisms of parasite maturation in the erythrocyte cytoplasm and, along with future studies using our detailed methodology, may help to gain insight into the clinical phenomena of human malaria. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Effects of murine leukemia virus env gene proteins on macrophage-mediated cytotoxicity in vitro

NASA Technical Reports Server (NTRS)

Chapes, S. K.; Takemoto, L. J.; Spooner, B. S. (Principal Investigator)

1991-01-01

F5b Tumor cells were incubated with concentrated culture supernatants taken from cells resistant (F5m) or sensitive (F5b) to contact-dependent macrophage cytotoxicity. Macrophage cell line B6MP102 and murine peritoneal macrophages killed targets incubated with supernatants taken from sensitive cells but poorly killed cells incubated in supernatants isolated from resistant cells. Membranes from cells resistant to macrophage killing, F5m, were fused into F5b cells. The fused F5b cells were killed significantly less than F5b cells fused with F5b cell membranes or untreated F5b cells. The decreased killing of F5b cells corresponded to increased concentrations of gp70(a) molecules on F5b cells. Affinity purified gp70(a) was added to cytotoxicity assays but failed to inhibit macrophage cytotoxicity. P15E molecules were detectable on both F5b and F5m cells. In addition, a synthetic peptide found to exhibit the inhibitory properties of p15E was added to cytotoxicity assays. P15E synthetic peptide also did not inhibit macrophage cytotoxicity. Therefore, env gene proteins of murine leukemia virus do not appear responsible for inducing tumor cell resistance to activated macrophage contact-dependent cytotoxicity.

Advances in the development of enterohemorrhagic Escherichia coli vaccines using murine models of infection

PubMed Central

Garcia-Angulo, Victor A.; Kalita, Anjana; Torres, Alfredo G.

2013-01-01

Enterohemorrhagic Escherichia coli (EHEC) strains are food borne pathogens with importance in public health. EHEC colonizes the large intestine and causes diarrhea, hemorrhagic colitis and in some cases, life-threatening hemolytic-uremic syndrome (HUS) due to the production of Shiga toxins (Stx). The lack of effective clinical treatment, sequelae after infection and mortality rate in humans supports the urgent need of prophylactic approaches, such as development of vaccines. Shedding from cattle, the main EHEC reservoir and considered the principal food contamination source, has prompted the development of licensed vaccines that reduce EHEC colonization in ruminants. Although murine models do not fully recapitulate human infection, they are commonly used to evaluate EHEC vaccines and the immune/protective responses elicited in the host. Mice susceptibility differs depending of the EHEC inoculums; therefore, displaying different mortality rates and Stx-mediated renal damage. Therefore, several experimental protocols have being pursued in this model to develop EHEC-specific vaccines. Recent candidate vaccines evaluated include those composed of virulence factors alone or as fused-subunits, DNA-based, attenuated bacteria and bacterial ghosts. In this review, we summarize progress in the design and testing of EHEC vaccines and the use of different strategies for the evaluation of novel EHEC vaccines in the murine model. PMID:23707170

Family Violence and Family Physicians

PubMed Central

Herbert, Carol P.

1991-01-01

The acronym IDEALS summarizes family physicians' obligations when violence is suspected: to identify family violence; document injuries; educate families and ensure safety for victims; access resources and coordinate care; co-operate in the legal process; and provide support for families. Failure to respond reflects personal and professional experience and attitudes, fear of legal involvement, and lack of knowledge. Risks of intervention include physician burnout, physician overfunctioning, escalation of violence, and family disruption. PMID:21228987

Immunomodulatory activity of a novel, synthetic beta-glucan (β-glu6) in murine macrophages and human peripheral blood mononuclear cells.

PubMed

Li, Xiaofei; Wang, Jing; Wang, Wei; Liu, Chunhong; Sun, Shuhui; Gu, Jianxin; Wang, Xun; Boraschi, Diana; Huang, Yuxian; Qu, Di

2013-01-01

Natural β-glucans extracted from plants and fungi have been used in clinical therapies since the late 20th century. However, the heterogeneity of natural β-glucans limits their clinical applicability. We have synthesized β-glu6, which is an analog of the lentinan basic unit, β-(1→6)-branched β-(1→3) glucohexaose, that contains an α-(1→3)-linked bond. We have demonstrated the stimulatory effect of this molecule on the immune response, but the mechanisms by which β-glu6 activates innate immunity have not been elucidated. In this study, murine macrophages and human PBMCs were used to evaluate the immunomodulatory effects of β-glu6. We showed that β-glu6 activated ERK and c-Raf phosphorylation but suppressed the AKT signaling pathway in murine macrophages. Additionally, β-glu6 enhanced the secretion of large levels of cytokines and chemokines, including CD54, IL-1α, IL-1β, IL-16, IL-17, IL-23, IFN-γ, CCL1, CCL3, CCL4, CCL12, CXCL10, tissue inhibitor of metalloproteinase-1 (TIMP-1) and G-CSF in murine macrophages as well as IL-6, CCL2, CCL3, CCL5, CXCL1 and macrophage migration inhibitory factor (MIF) in human PBMCs. In summary, it demonstrates the immunomodulatory activity of β-glu6 in innate immunity.

Comparative genomic analysis of Acinetobacter strains isolated from murine colonic crypts.

PubMed

Saffarian, Azadeh; Touchon, Marie; Mulet, Céline; Tournebize, Régis; Passet, Virginie; Brisse, Sylvain; Rocha, Eduardo P C; Sansonetti, Philippe J; Pédron, Thierry

2017-07-11

A restricted set of aerobic bacteria dominated by the Acinetobacter genus was identified in murine intestinal colonic crypts. The vicinity of such bacteria with intestinal stem cells could indicate that they protect the crypt against cytotoxic and genotoxic signals. Genome analyses of these bacteria were performed to better appreciate their biodegradative capacities. Two taxonomically different clusters of Acinetobacter were isolated from murine proximal colonic crypts, one was identified as A. modestus and the other as A. radioresistens. Their identification was performed through biochemical parameters and housekeeping gene sequencing. After selection of one strain of each cluster (A. modestus CM11G and A. radioresistens CM38.2), comparative genomic analysis was performed on whole-genome sequencing data. The antibiotic resistance pattern of these two strains is different, in line with the many genes involved in resistance to heavy metals identified in both genomes. Moreover whereas the operon benABCDE involved in benzoate metabolism is encoded by the two genomes, the operon antABC encoding the anthranilate dioxygenase, and the phenol hydroxylase gene cluster are absent in the A. modestus genomic sequence, indicating that the two strains have different capacities to metabolize xenobiotics. A common feature of the two strains is the presence of a type IV pili system, and the presence of genes encoding proteins pertaining to secretion systems such as Type I and Type II secretion systems. Our comparative genomic analysis revealed that different Acinetobacter isolated from the same biological niche, even if they share a large majority of genes, possess unique features that could play a specific role in the protection of the intestinal crypt.

Desipramine decreases expression of human and murine indoleamine-2,3-dioxygenases

PubMed Central

Brooks, Alexandra K.; Janda, Tiffany M.; Lawson, Marcus A.; Rytych, Jennifer L.; Smith, Robin A.; Ocampo-Solis, Cecilia; McCusker, Robert H.

2017-01-01

Abundant evidence connects depression symptomology with immune system activation, stress and subsequently elevated levels of kynurenine. Anti-depressants, such as the tricyclic norepinephrine/serotonin reuptake inhibitor desipramine (Desip), were developed under the premise that increasing extracellular neurotransmitter level was the sole mechanism by which they alleviate depressive symptomologies. However, evidence suggests that anti-depressants have additional actions that contribute to their therapeutic potential. The Kynurenine Pathway produces tryptophan metabolites that modulate neurotransmitter activity. This recognition identified another putative pathway for anti-depressant targeting. Considering a recognized role of the Kynurenine Pathway in depression, we investigated the potential for Desip to alter expression of rate-limiting enzymes of this pathway: indoleamine-2,3-dioxygenases (Ido1 and Ido2). Mice were administered lipopolysaccharide (LPS) or synthetic glucocorticoid dexamethasone (Dex) with Desip to determine if Desip alters indoleamine-dioxygenase (DO) expression in vivo following a modeled immune and stress response. This work was followed by treating murine and human peripheral blood mononuclear cells (PBMCs) with interferon-gamma (IFNγ) and Desip. In vivo: Desip blocked LPS-induced Ido1 expression in hippocampi, astrocytes, microglia and PBMCs and Ido2 expression by PBMCs. Ex vivo: Desip decreased IFNγ-induced Ido1 and Ido2 expression in murine PBMCs. This effect was directly translatable to the human system as Desip decreased IDO1 and IDO2 expression by human PBMCs. These data demonstrate for the first time that an anti-depressant alters expression of Ido1 and Ido2, identifying a possible new mechanism behind anti-depressant activity. Furthermore, we propose the assessment of PBMCs for anti-depressant responsiveness using IDO expression as a biomarker. PMID:28212884

Splenomegaly, myeloid lineage expansion and increased osteoclastogenesis in osteogenesis imperfecta murine.

PubMed

Matthews, Brya G; Roeder, Emilie; Wang, Xi; Aguila, Hector Leonardo; Lee, Sun-Kyeong; Grcevic, Danka; Kalajzic, Ivo

2017-10-01

Osteogenesis imperfecta (OI) is a disease caused by defects in type I collagen production that results in brittle bones. While the pathology is mainly caused by defects in the osteoblast lineage, there is also elevated bone resorption by osteoclasts resulting in high bone turnover in severe forms of the disease. Osteoclasts originate from hematopoietic myeloid cells, however changes in hematopoiesis have not been previously documented in OI. In this study, we evaluated hematopoietic lineage distribution and osteoclast progenitor cell frequency in bone marrow, spleen and peripheral blood of osteogenesis imperfecta murine (OIM) mice, a model of severe OI. We found splenomegaly in all ages examined, and expansion of myeloid lineage cells (CD11b + ) in bone marrow and spleen of 7-9week old male OIM animals. OIM spleens also showed an increased frequency of purified osteoclast progenitors. This phenotype is suggestive of chronic inflammation. Isolated osteoclast precursors from both spleen and bone marrow formed osteoclasts more rapidly than wild-type controls. We found that serum TNFα levels were increased in OIM, as was IL1α in OIM females. We targeted inflammation therapeutically by treating growing animals with murine TNFR2:Fc, a compound that blocks TNFα activity. Anti-TNFα treatment marginally decreased spleen mass in OIM females, but failed to reduce bone resorption, or improve bone parameters or fracture rate in OIM animals. We have demonstrated that OIM mice have changes in their hematopoietic system, and form osteoclasts more rapidly even in the absence of OI osteoblast signals, however therapy targeting TNFα did not improve disease parameters. Copyright © 2017 Elsevier Inc. All rights reserved.

Role of airway epithelial injury in murine orthotopic tracheal allograft rejection.

PubMed

Kuo, Elbert; Bharat, Ankit; Shih, Jennifer; Street, Tyler; Norris, Jenyi; Liu, Wei; Parks, William; Walter, Michael; Patterson, G Alexander; Mohanakumar, T

2006-10-01

Murine tracheal transplantation is a model used to study bronchiolitis obliterans syndrome, a major cause of morbidity and mortality after lung transplantation. Unlike murine heterotopic tracheal transplants, orthotopic transplantation does not cause luminal obliteration despite major histocompatibility antigen mismatch. Repopulation of the tracheal allografts with recipient-derived epithelium confers protection against luminal obliteration. The purpose of this study was to determine whether (1) orthotopic tracheal transplantation showed signs of allograft rejection, and (2) airway epithelial cell injury promoted orthotopic tracheal allograft rejection. Forty isogeneic (C57BL/6 to C57BL/6) and 40 allogeneic (BALB/c to C57BL/6) orthotopic tracheal transplants were performed. Damage to airway epithelial cells was induced by Sendai viral (SdV) infection and tracheal transplantation into non-reepithelializing matrix metalloproteinase-7 knockout (MMP7-KO) recipient mice. Percent fibrosis and lamina propria to cartilage ratio were calculated with computer assistance on harvested allografts. Allografts showed significantly more intramural fibrosis compared with isografts at 30, 60, and 180 days after transplant without luminal occlusion. Tracheal allografts infected with SdV showed an increase in fibrosis and lamina propria to cartilage ratio compared with noninfected controls. Allografts retrieved from MMP7-KO recipients also showed a significant increase in fibrosis and lamina propria to cartilage ratio. Although orthotopic tracheal transplantation does not cause luminal obliteration, it results in increased fibrosis in allografts. Damage to the respiratory epithelium by viral infection or defective reepithelialization after transplant as seen in MMP7-KO recipient mice leads to changes consistent with chronic allograft rejection, suggesting a role for epithelial injury in bronchiolitis obliterans syndrome development.

STRAIN-DEPENDENT SUSCEPTIBILITY TO TRANSPLACENTALLY-INDUCED MURINE LUNG TUMORS AND DNA ADDUCTS OF 3-METHYLCHOLANTHRENE

EPA Science Inventory

Strain-dependent susceptibility to transplacentally-induced murine lung tumors and DNA adducts of 3methylcholanthrene G B Nelson, J A Ross, J E Moore, M Xu, N D Kock, M S Miller Wake Forest University, Winston-Salem, NC and USEPA, Research Triangle Park, NC.

It has been de...

Gene transfer into hematopoietic stem cells reduces HLH manifestations in a murine model of Munc13-4 deficiency.

PubMed

Soheili, Tayebeh; Durand, Amandine; Sepulveda, Fernando E; Rivière, Julie; Lagresle-Peyrou, Chantal; Sadek, Hanem; de Saint Basile, Geneviève; Martin, Samia; Mavilio, Fulvio; Cavazzana, Marina; André-Schmutz, Isabelle

2017-12-26

Patients with mutations in the UNC13D gene (coding for Munc13-4 protein) suffer from familial hemophagocytic lymphohistiocytosis type 3 (FHL3), a life-threatening immune and hyperinflammatory disorder. The only curative treatment is allogeneic hematopoietic stem cell (HSC) transplantation, although the posttreatment survival rate is not satisfactory. Here, we demonstrate the curative potential of UNC13D gene correction of HSCs in a murine model of FHL3. We generated a self-inactivating lentiviral vector, used it to complement HSCs from Unc13d -deficient (Jinx) mice, and transplanted the cells back into the irradiated Jinx recipients. This procedure led to complete reconstitution of the immune system (ie, to wild-type levels). The recipients were then challenged with lymphocytic choriomeningitis virus to induce hemophagocytic lymphohistiocytosis (HLH)-like manifestations. All the clinical and biological signs of HLH were significantly reduced in mice having undergone HSC UNC13D gene correction than in nontreated animals. This beneficial effect was evidenced by the correction of blood cytopenia, body weight gain, normalization of the body temperature, decreased serum interferon-γ level, recovery of liver damage, and decreased viral load. These improvements can be explained by the restoration of the CD8 + T lymphocytes' cytotoxic function (as demonstrated here in an in vitro degranulation assay). Overall, our results demonstrate the efficacy of HSC gene therapy in an FHL-like setting of immune dysregulation.

The role of indoleamine 2,3-dioxygenase in LP-BPM5 murine retroviral disease progression.

PubMed

O'Connor, Megan A; Green, William R

2013-05-17

Indoleamine 2,3-dioxygenase (IDO) is an immunomodulatory intracellular enzyme involved in tryptophan degradation. IDO is induced during cancer and microbial infections by cytokines, ligation of co-stimulatory molecules and/or activation of pattern recognition receptors, ultimately leading to modulation of the immune response. LP-BM5 murine retroviral infection induces murine AIDS (MAIDS), which is characterized by profound and broad immunosuppression of T- and B-cell responses. Our lab has previously described multiple mechanisms regulating the development of immunodeficiency of LP-BM5-induced disease, including Programmed Death 1 (PD-1), IL-10, and T-regulatory (Treg) cells. Immunosuppressive roles of IDO have been demonstrated in other retroviral models, suggesting a possible role for IDO during LP-BM5-induced retroviral disease progression and/or development of viral load. Mice deficient in IDO (B6.IDO-/-) and wildtype C57BL/6 (B6) mice were infected with LP-BM5 murine retrovirus. MAIDS and LP-BM5 viral load were assessed at termination. As expected, IDO was un-inducible in B6.IDO-/- during LP-BM5 infection. B6.IDO-/- mice infected with LP-BM5 retrovirus succumbed to MAIDS as indicated by splenomegaly, serum hyper IgG2a and IgM, decreased responsiveness to B- and T-cell mitogens, conversion of a proportion of CD4+ T cells from Thy1.2+ to Thy1.2-, and increased percentages of CD11b+Gr-1+ cells. LP-BM5 infected B6.IDO-/- mice also demonstrated the development of roughly equivalent disease kinetics as compared to infected B6 mice. Splenic viral loads of B6 and B6.IDO-/- mice were also equivalent after infection as measured by LP-BM5-specific Def Gag and Eco Gag viral mRNA, determined by qRT-PCR. Collectively, these results demonstrate IDO neither plays an essential role, nor is required, in LP-BM5-induced disease progression or LP-BM5 viral load.

The role of indoleamine 2,3-dioxygenase in LP-BPM5 murine retroviral disease progression

PubMed Central

2013-01-01

Background Indoleamine 2,3-dioxygenase (IDO) is an immunomodulatory intracellular enzyme involved in tryptophan degradation. IDO is induced during cancer and microbial infections by cytokines, ligation of co-stimulatory molecules and/or activation of pattern recognition receptors, ultimately leading to modulation of the immune response. LP-BM5 murine retroviral infection induces murine AIDS (MAIDS), which is characterized by profound and broad immunosuppression of T- and B-cell responses. Our lab has previously described multiple mechanisms regulating the development of immunodeficiency of LP-BM5-induced disease, including Programmed Death 1 (PD-1), IL-10, and T-regulatory (Treg) cells. Immunosuppressive roles of IDO have been demonstrated in other retroviral models, suggesting a possible role for IDO during LP-BM5-induced retroviral disease progression and/or development of viral load. Methods Mice deficient in IDO (B6.IDO−/−) and wildtype C57BL/6 (B6) mice were infected with LP-BM5 murine retrovirus. MAIDS and LP-BM5 viral load were assessed at termination. Results As expected, IDO was un-inducible in B6.IDO−/− during LP-BM5 infection. B6.IDO−/− mice infected with LP-BM5 retrovirus succumbed to MAIDS as indicated by splenomegaly, serum hyper IgG2a and IgM, decreased responsiveness to B- and T-cell mitogens, conversion of a proportion of CD4+ T cells from Thy1.2+ to Thy1.2-, and increased percentages of CD11b+Gr-1+ cells. LP-BM5 infected B6.IDO−/− mice also demonstrated the development of roughly equivalent disease kinetics as compared to infected B6 mice. Splenic viral loads of B6 and B6.IDO−/− mice were also equivalent after infection as measured by LP-BM5-specific Def Gag and Eco Gag viral mRNA, determined by qRT-PCR. Conclusions Collectively, these results demonstrate IDO neither plays an essential role, nor is required, in LP-BM5-induced disease progression or LP-BM5 viral load. PMID:23680027

Acute Inhibition of MEK Suppresses Congenital Melanocytic Nevus Syndrome in a Murine Model Driven by Activated NRAS and Wnt Signaling.

PubMed

Pawlikowski, Jeffrey S; Brock, Claire; Chen, Sheau-Chiann; Al-Olabi, Lara; Nixon, Colin; McGregor, Fiona; Paine, Simon; Chanudet, Estelle; Lambie, Wendy; Holmes, William M; Mullin, James M; Richmond, Ann; Wu, Hong; Blyth, Karen; King, Ayala; Kinsler, Veronica A; Adams, Peter D

2015-08-01

Congenital melanocytic nevus (CMN) syndrome is the association of pigmented melanocytic nevi with extra-cutaneous features, classically melanotic cells within the central nervous system, most frequently caused by a mutation of NRAS codon 61. This condition is currently untreatable and carries a significant risk of melanoma within the skin, brain, or leptomeninges. We have previously proposed a key role for Wnt signaling in the formation of melanocytic nevi, suggesting that activated Wnt signaling may be synergistic with activated NRAS in the pathogenesis of CMN syndrome. Some familial pre-disposition suggests a germ-line contribution to CMN syndrome, as does variability of neurological phenotypes in individuals with similar cutaneous phenotypes. Accordingly, we performed exome sequencing of germ-line DNA from patients with CMN to reveal rare or undescribed Wnt-signaling alterations. A murine model harboring activated NRAS(Q61K) and Wnt signaling in melanocytes exhibited striking features of CMN syndrome, in particular neurological involvement. In the first model of treatment for this condition, these congenital, and previously assumed permanent, features were profoundly suppressed by acute post-natal treatment with a MEK inhibitor. These data suggest that activated NRAS and aberrant Wnt signaling conspire to drive CMN syndrome. Post-natal MEK inhibition is a potential candidate therapy for patients with this debilitating condition.

Purification and growth of melanocortin 1 receptor (Mc1r)-defective primary murine melanocytes is dependent on stem cell factor from keratinocyte-conditioned media

PubMed Central

Scott, Timothy L.; Wakamatsu, Kazumasa; Ito, Shosuke; D’Orazio, John A.

2015-01-01

Summary The melanocortin 1 receptor (MC1R) is a transmembrane Gs-coupled surface protein found on melanocytes that binds melanocyte stimulating hormone (MSH) and mediates activation of adenylyl cyclase and generation of the second messenger cAMP. MC1R regulates growth and differentiation of melanocytes and protects against carcinogenesis. Persons with loss-of-function polymorphisms of MC1R tend to be UV-sensitive (fair-skinned and with a poor tanning response) and are at high risk for melanoma. Mechanistic studies of the role of MC1R in melanocytic UV responses, however, have been hindered in part because Mc1r-defective primary murine melanocytes have been difficult to culture in vitro. Until now, effective growth of murine melanocytes has depended on cAMP stimulation with adenylyl cyclase activating or phosphodiesterase inhibiting agents. However, rescuing cAMP in the setting of defective MC1R signaling would be expected to confound experiments directly testing MC1R function on melanocytic UV responses. Here we report a novel method of culturing primary murine melanocytes in the absence of pharmacologic cAMP stimulation by incorporating conditioned supernatants containing stem cell factor (SCF) derived from primary keratinocytes. Importantly, this method seems to permit similar pigment expression by cultured melanocytes as that found in the skin of their parental murine strains. This novel approach will allow mechanistic investigation into MC1R’s role in protection against UV-mediated carcinogenesis and determination of the role of melanin pigment subtypes on UV-mediated melanocyte responses. PMID:19633898

WrpA Is an Atypical Flavodoxin Family Protein under Regulatory Control of the Brucella abortus General Stress Response System

DOE Office of Scientific and Technical Information (OSTI.GOV)

Herrou, Julien; Czyż, Daniel M.; Willett, Jonathan W.

ABSTRACT The general stress response (GSR) system of the intracellular pathogenBrucella abortuscontrols the transcription of approximately 100 genes in response to a range of stress cues. The core genetic regulatory components of the GSR are required forB. abortussurvival under nonoptimal growth conditionsin vitroand for maintenance of chronic infection in anin vivomouse model. The functions of the majority of the genes in the GSR transcriptional regulon remain undefined.bab1_1070is among the most highly regulated genes in this regulon: its transcription is activated 20- to 30-fold by the GSR system under oxidative conditionsin vitro. We have solved crystal structures of Bab1_1070 and demonstratemore » that it forms a homotetrameric complex that resembles those of WrbA-type NADH:quinone oxidoreductases, which are members of the flavodoxin protein family. However,B. abortusWrbA-relatedprotein (WrpA) does not bind flavin cofactors with a high affinity and does not function as an NADH:quinone oxidoreductasein vitro. Soaking crystals with flavin mononucleotide (FMN) revealed a likely low-affinity binding site adjacent to the canonical WrbA flavin binding site. Deletion ofwrpA(ΔwrpA) does not compromise cell survival under acute oxidative stressin vitroor attenuate infection in cell-based or mouse models. However, a ΔwrpAstrain does elicit increased splenomegaly in a mouse model, suggesting that WrpA modulatesB. abortusinteraction with its mammalian host. Despite high structural homology with canonical WrbA proteins, we propose thatB. abortusWrpA represents a functionally distinct member of the diverse flavodoxin family. IMPORTANCEBrucella abortusis an etiological agent of brucellosis, which is among the most common zoonotic diseases worldwide. The general stress response (GSR) regulatory system ofB. abortuscontrols the transcription of approximately 100 genes and is required for maintenance of chronic infection in a murine model; the majority of GSR-regulated genes

Family functioning in the families of psychiatric patients: a comparison with nonclinical families.

PubMed

Trangkasombat, Umaporn

2006-11-01

To examine family functioning in the families of psychiatric patients. Families of psychiatric patients and nonclinical families were compared. There were 60 families in each group. The instrument included a semistructured interview of family functioning and the Chulalongkorn Family Inventory (CFI), a self-report questionnaire designed to assess the perception of one's family. From the assessment by semistructured interview, 83.3% of psychiatric families and 45.0% of nonclinical families were found to be dysfunctional in at least one dimension. The difference was statistically significant (p < 0.001). The average number of dysfunctional dimensions in the psychiatric families was significantly higher than in the nonclinical control group, 3.5 +/- 1.9 and 0.98 +/- 1.5 respectively, p < 0.0001. The CFI scores of the psychiatric families were significantly lower than the control group, reflecting poor family functioning. The dysfunctions were mostly in the following dimensions: problem-solving, communication, affective responsiveness, affective involvement, and behavior control. Psychiatric families faced more psychosocial stressors and the average number of stressors was higher than the control families, 88.3% vs. 56.7% and 4.2 +/- 2.7 vs. 1.3 +/- 1.47 stressors respectively, p < 0.0001. Family functioning of psychiatric patients was less healthy than the nonclinical control. The present study underlined the significance of family assessment and family intervention in the comprehensive care of psychiatric patients.

Myosin-driven rescue of contractile reserve and energetics in mouse hearts bearing familial hypertrophic cardiomyopathy-associated mutant troponin T is mutation-specific

PubMed Central

He, Huamei; Hoyer, Kirsten; Tao, Hai; Rice, Ronald; Jimenez, Jesus; Tardiff, Jil C; Ingwall, Joanne S

2012-01-01

The thin filament protein troponin T (TnT) is a regulator of sarcomere function. Whole heart energetics and contractile reserve are compromised in transgenic mice bearing missense mutations at R92 within the tropomyosin-binding domain of cTnT, despite being distal to the ATP hydrolysis domain of myosin. These mutations are associated with familial hypertrophic cardiomyopathy (FHC). Here we test the hypothesis that genetically replacing murine αα-MyHC with murine ββ-MyHC in hearts bearing the R92Q cTnT mutation, a particularly lethal FHC-associated mutation, leads to sufficiently large perturbations in sarcomere function to rescue whole heart energetics and decrease the cost of contraction. By comparing R92Q cTnT and R92L cTnT mutant hearts, we also test whether any rescue is mutation-specific. We defined the energetic state of the isolated perfused heart using 31P-NMR spectroscopy while simultaneously measuring contractile performance at four work states. We found that the cost of increasing contraction in intact mouse hearts with R92Q cTnT depends on the type of myosin present in the thick filament. We also found that the salutary effect of this manoeuvre is mutation-specific, demonstrating the major regulatory role of cTnT on sarcomere function at the whole heart level. PMID:22907055

Myosin-driven rescue of contractile reserve and energetics in mouse hearts bearing familial hypertrophic cardiomyopathy-associated mutant troponin T is mutation-specific.

PubMed

He, Huamei; Hoyer, Kirsten; Tao, Hai; Rice, Ronald; Jimenez, Jesus; Tardiff, Jil C; Ingwall, Joanne S

2012-11-01

The thin filament protein troponin T (TnT) is a regulator of sarcomere function. Whole heart energetics and contractile reserve are compromised in transgenic mice bearing missense mutations at R92 within the tropomyosin-binding domain of cTnT, despite being distal to the ATP hydrolysis domain of myosin. These mutations are associated with familial hypertrophic cardiomyopathy (FHC). Here we test the hypothesis that genetically replacing murine αα-MyHC with murine ββ-MyHC in hearts bearing the R92Q cTnT mutation, a particularly lethal FHC-associated mutation, leads to sufficiently large perturbations in sarcomere function to rescue whole heart energetics and decrease the cost of contraction. By comparing R92Q cTnT and R92L cTnT mutant hearts, we also test whether any rescue is mutation-specific. We defined the energetic state of the isolated perfused heart using (31)P-NMR spectroscopy while simultaneously measuring contractile performance at four work states. We found that the cost of increasing contraction in intact mouse hearts with R92Q cTnT depends on the type of myosin present in the thick filament. We also found that the salutary effect of this manoeuvre is mutation-specific, demonstrating the major regulatory role of cTnT on sarcomere function at the whole heart level.

[The role of wild murine rodents in the selection of different strains of tick-borne encephalitis and Powassan viruses].

PubMed

Leonova, G N; Krugliak, S P; Lozovskaia, S A; Rybachuk, V N

1987-01-01

The study demonstrated the role of wild murine rodents in selection of different strains of tick-borne encephalitis and Powassan viruses, determined their ecological characteristics reflected in the epidemiological features of tick-borne encephalitis and Powassan encephalitis in southern Far East.

Vaccination with Irradiated Tumor Cells Engineered to Secrete Murine Granulocyte-Macrophage Colony-Stimulating Factor Stimulates Potent, Specific, and Long-Lasting Anti-Tumor Immunity

NASA Astrophysics Data System (ADS)

Dranoff, Glenn; Jaffee, Elizabeth; Lazenby, Audrey; Golumbek, Paul; Levitsky, Hyam; Brose, Katja; Jackson, Valerie; Hamada, Hirofumi; Pardoll, Drew; Mulligan, Richard C.

1993-04-01

To compare the ability of different cytokines and other molecules to enhance the immunogenicity of tumor cells, we generated 10 retroviruses encoding potential immunomodulators and studied the vaccination properties of murine tumor cells transduced by the viruses. Using a B16 melanoma model, in which irradiated tumor cells alone do not stimulate significant anti-tumor immunity, we found that irradiated tumor cells expressing murine granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulated potent, long-lasting, and specific anti-tumor immunity, requiring both CD4^+ and CD8^+ cells. Irradiated cells expressing interleukins 4 and 6 also stimulated detectable, but weaker, activity. In contrast to the B16 system, we found that in a number of other tumor models, the levels of anti-tumor immunity reported previously in cytokine gene transfer studies involving live, transduced cells could be achieved through the use of irradiated cells alone. Nevertheless, manipulation of the vaccine or challenge doses made it possible to demonstrate the activity of murine GM-CSF in those systems as well. Overall, our results have important implications for the clinical use of genetically modified tumor cells as therapeutic cancer vaccines.

Elastic force restricts growth of the murine utricle

PubMed Central

Gnedeva, Ksenia; Jacobo, Adrian; Salvi, Joshua D; Petelski, Aleksandra A; Hudspeth, A J

2017-01-01

Dysfunctions of hearing and balance are often irreversible in mammals owing to the inability of cells in the inner ear to proliferate and replace lost sensory receptors. To determine the molecular basis of this deficiency we have investigated the dynamics of growth and cellular proliferation in a murine vestibular organ, the utricle. Based on this analysis, we have created a theoretical model that captures the key features of the organ’s morphogenesis. Our experimental data and model demonstrate that an elastic force opposes growth of the utricular sensory epithelium during development, confines cellular proliferation to the organ’s periphery, and eventually arrests its growth. We find that an increase in cellular density and the subsequent degradation of the transcriptional cofactor Yap underlie this process. A reduction in mechanical constraints results in accumulation and nuclear translocation of Yap, which triggers proliferation and restores the utricle’s growth; interfering with Yap’s activity reverses this effect. DOI: http://dx.doi.org/10.7554/eLife.25681.001 PMID:28742024

Immunostaining to visualize murine enteric nervous system development.

PubMed

Barlow-Anacker, Amanda J; Erickson, Christopher S; Epstein, Miles L; Gosain, Ankush

2015-04-29

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must then differentiate into neurons with markers specific for their neurotransmitter phenotype. Cholinergic neurons, a major neurotransmitter phenotype in the enteric nervous system, are identified by staining for choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine. Historical efforts to visualize cholinergic neurons have been hampered by antibodies with differing specificities to central nervous system versus peripheral nervous system ChAT. We and others have overcome this limitation by using an antibody against placental ChAT, which recognizes both central and peripheral ChAT, to successfully visualize embryonic enteric cholinergic neurons. Additionally, we have compared this antibody to genetic reporters for ChAT and shown that the antibody is more reliable during embryogenesis. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

Clindamycin in a murine model of toxoplasmic encephalitis.

PubMed Central

Hofflin, J M; Remington, J S

1987-01-01

We investigated the efficacy of clindamycin in a murine model of toxoplasmic encephalitis using direct intracerebral inoculation. Clindamycin reduced mortality from 40% in normal mice and 100% in cortisone-treated mice to 0% in both groups. Although we were unable to document appreciable levels of clindamycin in the brains of infected mice, the histological features of cerebral infection were markedly altered. The formation of large numbers of cysts and the intense inflammatory response seen in the brains of normal mice and the unchecked infection and tissue necrosis in the brains of cortisone-treated mice were absent in the brains of clindamycin-treated mice. Enumeration of cysts in the brains of mice 10 weeks after infection revealed a significantly lower number in the clindamycin-treated mice. Spread of infection to other organs was also decreased during clindamycin administration. These observations suggest that clindamycin may have a role in the therapy of toxoplasmic encephalitis. Images PMID:3606059

Heterogeneity across the murine small and large intestine

PubMed Central

Bowcutt, Rowann; Forman, Ruth; Glymenaki, Maria; Carding, Simon Richard; Else, Kathryn Jane; Cruickshank, Sheena Margaret

2014-01-01

The small and large intestine of the gastrointestinal tract (GIT) have evolved to have discrete functions with distinct anatomies and immune cell composition. The importance of these differences is underlined when considering that different pathogens have uniquely adapted to live in each region of the gut. Furthermore, different regions of the GIT are also associated with differences in susceptibility to diseases such as cancer and chronic inflammation. The large and small intestine, given their anatomical and functional differences, should be seen as two separate immunological sites. However, this distinction is often ignored with findings from one area of the GIT being inappropriately extrapolated to the other. Focussing largely on the murine small and large intestine, this review addresses the literature relating to the immunology and biology of the two sites, drawing comparisons between them and clarifying similarities and differences. We also highlight the gaps in our understanding and where further research is needed. PMID:25386070

Heterogeneity across the murine small and large intestine.

PubMed

Bowcutt, Rowann; Forman, Ruth; Glymenaki, Maria; Carding, Simon Richard; Else, Kathryn Jane; Cruickshank, Sheena Margaret

2014-11-07

The small and large intestine of the gastrointestinal tract (GIT) have evolved to have discrete functions with distinct anatomies and immune cell composition. The importance of these differences is underlined when considering that different pathogens have uniquely adapted to live in each region of the gut. Furthermore, different regions of the GIT are also associated with differences in susceptibility to diseases such as cancer and chronic inflammation. The large and small intestine, given their anatomical and functional differences, should be seen as two separate immunological sites. However, this distinction is often ignored with findings from one area of the GIT being inappropriately extrapolated to the other. Focussing largely on the murine small and large intestine, this review addresses the literature relating to the immunology and biology of the two sites, drawing comparisons between them and clarifying similarities and differences. We also highlight the gaps in our understanding and where further research is needed.

Tracing the fate of limbal epithelial progenitor cells in the murine cornea.

PubMed

Di Girolamo, N; Bobba, S; Raviraj, V; Delic, N C; Slapetova, I; Nicovich, P R; Halliday, G M; Wakefield, D; Whan, R; Lyons, J G

2015-01-01

Stem cell (SC) division, deployment, and differentiation are processes that contribute to corneal epithelial renewal. Until now studying the destiny of these cells in a living mammal has not been possible. However, the advent of inducible multicolor genetic tagging and powerful imaging technologies has rendered this achievable in the translucent and readily accessible murine cornea. K14CreER(T2)-Confetti mice that harbor two copies of the Brainbow 2.1 cassette, yielding up to 10 colors from the stochastic recombination of fluorescent proteins, were used to monitor K-14(+) progenitor cell dynamics within the corneal epithelium in live animals. Multicolored columns of cells emerged from the basal limbal epithelium as they expanded and migrated linearly at a rate of 10.8 µm/day toward the central cornea. Moreover, the permanent expression of fluorophores, passed on from progenitor to progeny, assisted in discriminating individual clones as spectrally distinct streaks containing more than 1,000 cells within the illuminated area. The centripetal clonal expansion is suggestive that a single progenitor cell is responsible for maintaining a narrow corridor of corneal epithelial cells. Our data are in agreement with the limbus as the repository for SC as opposed to SC being distributed throughout the central cornea. This is the first report describing stem/progenitor cell fate determination in the murine cornea using multicolor genetic tracing. This model represents a powerful new resource to monitor SC kinetics and fate choice under homeostatic conditions, and may assist in assessing clonal evolution during corneal development, aging, wound-healing, disease, and following transplantation. © 2014 AlphaMed Press.

Dynamic Tumor Growth Patterns in a Novel Murine Model of Colorectal Cancer

PubMed Central

Olson, Terrah J. Paul; Hadac, Jamie N.; Sievers, Chelsie K.; Leystra, Alyssa A.; Deming, Dustin A.; Zahm, Christopher D.; Albrecht, Dawn M.; Nomura, Alice; Nettekoven, Laura A.; Plesh, Lauren K.; Clipson, Linda; Sullivan, Ruth; Newton, Michael A.; Schelman, William R.; Halberg, Richard B.

2014-01-01

Colorectal cancer (CRC) often arises from adenomatous colonic polyps. Polyps can grow and progress to cancer, but may also remain static in size, regress, or resolve. Predicting which progress and which remain benign is difficult. We developed a novel long-lived murine model of CRC with tumors that can be followed by colonoscopy. Our aim was to assess whether these tumors have similar growth patterns and histologic fates to human colorectal polyps to identify features to aid in risk-stratification of colonic tumors. Long-lived ApcMin/+ mice were treated with dextran sodium sulfate to promote colonic tumorigenesis. Tumor growth patterns were characterized by serial colonoscopy with biopsies obtained for immunohistochemistry and gene expression profiling. Tumors grew, remained static, regressed, or resolved over time with different relative frequencies. Newly developed tumors demonstrated higher rates of growth and resolution than more established tumors that tended to remain static in size. Colonic tumors were hyperplastic lesions (3%), adenomas (73%), intramucosal carcinomas (20%), or adenocarcinomas (3%). Interestingly, the level of β-catenin was higher in adenomas that became intratumoral carcinomas as compared to those that failed to progress. In addition, differentially expressed genes between adenomas and intramucosal carcinomas were identified. This novel murine model of intestinal tumorigenesis develops colonic tumors that can be monitored by serial colonoscopy, mirror growth patterns seen in human colorectal polyps, and progress to CRC. Further characterization of cellular and molecular features are needed to determine which features can be used to risk-stratify polyps for progression to CRC and potentially guide prevention strategies. PMID:24196829

MHC class II molecules control murine B cell responsiveness to lipopolysaccharide stimulation.

PubMed

Rodo, Joana; Gonçalves, Lígia A; Demengeot, Jocelyne; Coutinho, António; Penha-Gonçalves, Carlos

2006-10-01

LPS is a strong stimulator of the innate immune system and inducer of B lymphocyte activation. Two TLRs, TLR4 and RP105 (CD180), have been identified as mediators of LPS signaling in murine B cells, but little is known about genetic factors that are able to control LPS-induced cell activation. We performed a mouse genome-wide screen that aside from identifying a controlling locus mapping in the TLR4 region (logarithm of odds score, 2.77), also revealed that a locus closely linked to the MHC region (logarithm of odds score, 3.4) governed B cell responsiveness to LPS stimulation. Using purified B cells obtained from MHC congenic strains, we demonstrated that the MHC(b) haplotype is accountable for higher cell activation, cell proliferation, and IgM secretion, after LPS stimulation, when compared with the MHC(d) haplotype. Furthermore, B cells from MHC class II(-/-) mice displayed enhanced activation and proliferation in response to LPS. In addition, we showed that the MHC haplotype partially controls expression of RP105 (a LPS receptor molecule), following a pattern that resembles the LPS responsiveness phenotype. Together, our results strongly suggest that murine MHC class II molecules play a role in constraining the B cell response to LPS and that genetic variation at the MHC locus is an important component in controlling B cell responsiveness to LPS stimulation. This work raises the possibility that constraining of B cell responsiveness by MHC class II molecules may represent a functional interaction between adaptive and innate immune systems.

St. John’s Wort inhibits insulin signaling in murine and human adipocytes

PubMed Central

Richard, Allison J.; Amini, Zhaleh J.; Ribnicky, David M.; Stephens, Jacqueline M.

2012-01-01

Adipocytes are insulin-sensitive cells that play a major role in energy homeostasis. Obesity is the primary disease of fat cells and a major risk factor for the development of Type 2 diabetes, cardiovascular disease, and metabolic syndrome. The use of botanicals in the treatment of metabolic diseases is an emerging area of research. In previous studies, we screened over 425 botanical extracts for their ability to modulate adipogenesis and insulin sensitivity. We identified St. John’s Wort (SJW) extracts as inhibitors of adipogenesis of 3T3-L1 cells and demonstrated that these extracts also inhibited insulin-sensitive glucose uptake in mature fat cells. In these follow-up studies we have further characterized the effects of SJW on insulin action in both murine and human fat cells. We have shown that SJW also attenuates insulin-sensitive glucose uptake in human adipocytes. Moreover, SJW inhibits IRS-1 tyrosine phosphorylation in both murine and human fat cells. Botanical extracts are complex mixtures. Many bioactive compounds have been identified in SJW, including hypericin (HI) and hyperforin (HF). We have examined the ability of HI and HF, purified from SJW, to modulate adipocyte development and insulin action in mature adipocytes. Our novel studies indicate that the profound effects of SJW on adipogenesis, IRS-1 activation, and insulin-stimulated glucose uptake are not mediated by HI and/or HF. Nonetheless, we propose that extracts of SJW may contribute to adipocyte related diseases by limiting differentiation of preadipocytes and significantly inducing insulin resistance in mature fat cells. PMID:22198320

Automated segmentation of murine lung tumors in x-ray micro-CT images

NASA Astrophysics Data System (ADS)

Swee, Joshua K. Y.; Sheridan, Clare; de Bruin, Elza; Downward, Julian; Lassailly, Francois; Pizarro, Luis

2014-03-01

Recent years have seen micro-CT emerge as a means of providing imaging analysis in pre-clinical study, with in-vivo micro-CT having been shown to be particularly applicable to the examination of murine lung tumors. Despite this, existing studies have involved substantial human intervention during the image analysis process, with the use of fully-automated aids found to be almost non-existent. We present a new approach to automate the segmentation of murine lung tumors designed specifically for in-vivo micro-CT-based pre-clinical lung cancer studies that addresses the specific requirements of such study, as well as the limitations human-centric segmentation approaches experience when applied to such micro-CT data. Our approach consists of three distinct stages, and begins by utilizing edge enhancing and vessel enhancing non-linear anisotropic diffusion filters to extract anatomy masks (lung/vessel structure) in a pre-processing stage. Initial candidate detection is then performed through ROI reduction utilizing obtained masks and a two-step automated segmentation approach that aims to extract all disconnected objects within the ROI, and consists of Otsu thresholding, mathematical morphology and marker-driven watershed. False positive reduction is finally performed on initial candidates through random-forest-driven classification using the shape, intensity, and spatial features of candidates. We provide validation of our approach using data from an associated lung cancer study, showing favorable results both in terms of detection (sensitivity=86%, specificity=89%) and structural recovery (Dice Similarity=0.88) when compared against manual specialist annotation.

Anatomy and nomenclature of murine lymph nodes: Descriptive study and nomenclatory standardization in BALB/cAnNCrl mice.

PubMed

Van den Broeck, Wim; Derore, Annie; Simoens, Paul

2006-05-30

Murine lymph nodes are intensively studied but often assigned incorrectly in scientific papers. In BALB/cAnNCrl mice, we characterized a total of 22 different lymph nodes. Peripheral nodes were situated in the head and neck region (mandibular, accessory mandibular, superficial parotid, cranial deep cervical nodes), and at the forelimb (proper axillary, accessory axillary nodes) and hindlimb (subiliac, sciatic, popliteal nodes). Intrathoracic lymph nodes included the cranial mediastinal, tracheobronchal and caudal mediastinal nodes. Abdominal lymph nodes were associated with the gastrointestinal tract (gastric, pancreaticoduodenal, jejunal, colic, caudal mesenteric nodes) or were located along the major intra-abdominal blood vessels (renal, lumbar aortic, lateral iliac, medial iliac and external iliac nodes). Comparative and nomenclative aspects of murine lymph nodes are discussed. The position of the lymph nodes of BALB/cAnNCrl mice is summarized and illustrated in an anatomical chart containing proposals for both an official nomenclature according to the Nomina Anatomica Veterinaria and English terms.

Strengthening Family Practices for Latino Families.

PubMed

Chartier, Karen G; Negroni, Lirio K; Hesselbrock, Michie N

2010-01-01

The study examined the effectiveness of a culturally-adapted Strengthening Families Program (SFP) for Latinos to reduce risks for alcohol and drug use in children. Latino families, predominantly Puerto Rican, with a 9-12 year old child and a parent(s) with a substance abuse problem participated in the study. Pre- and post-tests were conducted with each family. Parental stress, parent-child dysfunctional relations, and child behavior problems were reduced in the families receiving the intervention; family hardiness and family attachment were improved. Findings contribute to the validation of the SFP with Latinos, and can be used to inform social work practice with Puerto Rican families.

Isolation, Purification, and Culture of Primary Murine Sensory Neurons.

PubMed

Katzenell, Sarah; Cabrera, Jorge R; North, Brian J; Leib, David A

2017-01-01

Cultured primary neurons have been of extraordinary value for the study of neuronal anatomy, cell biology, and physiology. While use of neuronal cell lines has ease and utility, there are often caveats that arise due to their mitotic nature. This methods article presents detailed methodology for the preparation, purification, and culture of adult murine sensory neurons for the study of herpes simplex virus lytic and latent infections. While virology is the application for our laboratory, these cultures also have broad utility for neurobiologists and cell biologists. While these primary cultures have been highly informative, the methodology is challenging to many investigators. Through publication of this highly detailed protocol, it is our hope that the use of this culture system can spread in the field to allow more rapid progress in furthering our understanding of neurotropic virus infection.

Oviductal extracellular vesicles (oviductosomes, OVS) are conserved in humans: murine OVS play a pivotal role in sperm capacitation and fertility.

PubMed

Bathala, Pradeepthi; Fereshteh, Zeinab; Li, Kun; Al-Dossary, Amal A; Galileo, Deni S; Martin-DeLeon, Patricia A

2018-03-01

Are extracellular vesicles (EVs) in the murine oviduct (oviductosomes, OVS) conserved in humans and do they play a role in the fertility of Pmca4-/- females? OVS and their fertility-modulating proteins are conserved in humans, arise via the apocrine pathway, and mediate a compensatory upregulation of PMCA1 (plasma membrane Ca2+-ATPase 1) in Pmca4-/- female mice during proestrus/estrus, to account for their fertility. Recently murine OVS were identified and shown during proestrus/estrus to express elevated levels of PMCA4 which they can deliver to sperm. PMCA4 is the major Ca2+ efflux pump in murine sperm and Pmca4 deletion leads to loss of sperm motility and male infertility as there is no compensatory upregulation of the remaining Ca2+ pump, PMCA1. Of the four family members of PMCAs (PMCA1-4), PMCA1 and PMCA4 are ubiquitous, and to date there have been no reports of one isoform being upregulated to compensate for another in any organ/tissue. Since Pmca4-/- females are fertile, despite the abundant expression of PMCA4 in wild-type (WT) OVS, we propose that OVS serve a role of packaging and delivering to sperm elevated levels of PMCA1 in Pmca4-/- during proestrus/estrus to compensate for PMCA4's absence. Fallopian tubes from pre-menopausal women undergoing hysterectomy were used to study EVs in the luminal fluid. Oviducts from sexually mature WT mice were sectioned after perfusion fixation to detect EVs in situ. Oviducts were recovered from WT and Pmca4-/- after hormonally induced estrus and sectioned for PMCA1 immunofluorescence (IF) (detected with confocal microscopy) and hematoxylin and eosin staining. Reproductive tissues, luminal fluids and EVs were recovered after induced estrus and after natural cycling for western blot analysis of PMCA1 and qRT-PCR of Pmca1 to compare expression levels in WT and Pmca4-/-. OVS, uterosomes, and epididymal luminal fluid were included in the comparisons. WT and Pmca4-/- OVS were analyzed for the presence of known PMCA4 partners

Quality control of murine monoclonal antibodies using isoelectric focusing affinity immunoblot analysis

NASA Technical Reports Server (NTRS)

Hamilton, Robert G.; Rodkey, L. Scott; Reimer, Charles B.

1987-01-01

The quality control of murine hybridoma secretory products has been performed using two approaches for isoelectric focusing affinity immunoblot analysis: (1) a method in which antigen-coated nitrocellulose is placed on top of an acrylamide gel containing isoelectrically focused ascites to bind the antigen specific monoclonal antibody; and (2) a method in which focused ascite proteins were passively blotted onto nitrocellulose and specific monoclonal antibodies were detected with enzyme-conjugated antigen. Analysis by both methods of batches of ascites containing antihuman IgG antibodies that were produced by six hybridomas permitted effective monitoring of immunoreactive antibodies for pI microheterogeneity.

Borrelia burgdorferi basic membrane protein A could induce chemokine production in murine microglia cell line BV2.

PubMed

Zhao, Hua; Liu, Aihua; Cui, Yuhui; Liang, Zhang; Li, Bingxue; Bao, Fukai

2017-10-01

Lyme neuroborreliosis is a nervous system infectious disease caused by Borrelia burgdorferi (B. burgdorferi). It has been demonstrated that cytokines induced by B. burgdorferi are related to Lyme neuroborreliosis. Microglia is known as a key player in the immune responses that occur within the central nervous system. In response to inflammation, it will be activated and generate cytokines and chemokines. Experiments in vitro cells have showed that B. Burgdorferi membrane protein A (BmpA), a major immunogen of B. Burgdorferi, could induce Lyme arthritis and stimulate human and murine lymphocytes to produce inflammatory cytokines. In our study, the murine microglia BV2 cell line was used as a cell model to explore the stimulating effects of recombinant BmpA (rBmpA); Chemokine chip, ELISA and QPCR technology were used to measure the production of chemokines from microglial cells stimulated by rBmpA. Compared with the negative control group, CXCL2, CCL22, and CCL5 concentrations in the cell supernatant increased significantly after the rBmpA stimulation; the concentration of these chemokines increased with rBmpA concentration increasing; the mRNA expression levels of chemokines (CXCL2, CCL22, and CCL5) in murine BV2 cells increased significantly with 10 μg/mL and 20 μg/mL rBmpA stimulation; CXCL13 was not change after the rBmpA stimulation. Our study shows that chemokines, such as CXCL2, CCL22, and CCL5 were up-regulated by the rBmpA in the BV2 cells. The production of chemokines in Lyme neuroborreliosis may be mainly from microglia cells and the rBmpA may be closely related with the development of Lyme neuroborreliosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Housing Temperature-Induced Stress Is Suppressing Murine Graft-versus-Host Disease through β2-Adrenergic Receptor Signaling.

PubMed

Leigh, Nicholas D; Kokolus, Kathleen M; O'Neill, Rachel E; Du, Wei; Eng, Jason W-L; Qiu, Jingxin; Chen, George L; McCarthy, Philip L; Farrar, J David; Cao, Xuefang; Repasky, Elizabeth A

2015-11-15

Graft-versus-host disease (GVHD) is the major complication of allogeneic hematopoietic cell transplantation, a potentially curative therapy for hematologic diseases. It has long been thought that murine bone marrow-derived T cells do not mediate severe GVHD because of their quantity and/or phenotype. During the course of experiments testing the impact of housing temperatures on GVHD, we discovered that this apparent resistance is a function of the relatively cool ambient housing temperature. Murine bone marrow-derived T cells have the ability to mediate severe GVHD in mice housed at a thermoneutral temperature. Specifically, mice housed at Institutional Animal Care and Use Committee-mandated, cool standard temperatures (∼ 22°C) are more resistant to developing GVHD than are mice housed at thermoneutral temperatures (∼ 30°C). We learned that the mechanism underlying this housing-dependent immunosuppression is associated with increased norepinephrine production and excessive signaling through β-adrenergic receptor signaling, which is increased when mice are cold stressed. Treatment of mice housed at 22°C with a β2-adrenergic antagonist reverses the norepinephrine-driven suppression of GVHD and yields similar disease to mice housed at 30°C. Conversely, administering a β2-adrenergic agonist decreases GVHD in mice housed at 30°C. In further mechanistic studies using β2-adrenergic receptor-deficient (β2-AR(-/-)) mice, we found that it is host cell β2-AR signaling that is essential for decreasing GVHD. These data reveal how baseline levels of β-adrenergic receptor signaling can influence murine GVHD and point to the feasibility of manipulation of β2-AR signaling to ameliorate GVHD in the clinical setting. Copyright © 2015 by The American Association of Immunologists, Inc.

Rescue of murine F508del CFTR activity in native intestine by low temperature and proteasome inhibitors.

PubMed

Wilke, Martina; Bot, Alice; Jorna, Huub; Scholte, Bob J; de Jonge, Hugo R

2012-01-01

Most patients with Cystic Fibrosis (CF) carry at least one allele with the F508del mutation, resulting in a CFTR chloride channel protein with a processing, gating and stability defect, but with substantial residual activity when correctly sorted to the apical membranes of epithelial cells. New therapies are therefore aimed at improving the folding and trafficking of F508del CFTR, (CFTR correctors) or at enhancing the open probability of the CFTR chloride channel (CFTR potentiators). Preventing premature breakdown of F508del CFTR is an alternative or additional strategy, which is investigated in this study. We established an ex vivo assay for murine F508del CFTR rescue in native intestinal epithelium that can be used as a pre-clinical test for candidate therapeutics. Overnight incubation of muscle stripped ileum in modified William's E medium at low temperature (26°C), and 4 h or 6 h incubation at 37°C with different proteasome inhibitors (PI: ALLN, MG-132, epoxomicin, PS341/bortezomib) resulted in fifty to hundred percent respectively of the wild type CFTR mediated chloride secretion (forskolin induced short-circuit current). The functional rescue was accompanied by enhanced expression of the murine F508del CFTR protein at the apical surface of intestinal crypts and a gain in the amount of complex-glycosylated CFTR (band C) up to 20% of WT levels. Sustained rescue in the presence of brefeldin A shows the involvement of a post-Golgi compartment in murine F508del CFTR degradation, as was shown earlier for its human counterpart. Our data show that proteasome inhibitors are promising candidate compounds for improving rescue of human F508del CFTR function, in combination with available correctors and potentiators.

Binding Affinity, Specificity and Comparative Biodistribution of the Parental Murine Monoclonal Antibody MX35 (Anti-NaPi2b) and Its Humanized Version Rebmab200.

PubMed

Lindegren, Sture; Andrade, Luciana N S; Bäck, Tom; Machado, Camila Maria L; Horta, Bruno Brasil; Buchpiguel, Carlos; Moro, Ana Maria; Okamoto, Oswaldo Keith; Jacobsson, Lars; Cederkrantz, Elin; Washiyama, Kohshin; Aneheim, Emma; Palm, Stig; Jensen, Holger; Tuma, Maria Carolina B; Chammas, Roger; Hultborn, Ragnar; Albertsson, Per

2015-01-01

The aim of this preclinical study was to evaluate the characteristics of the monoclonal antibody Rebmab200, which is a humanized version of the ovarian-specific murine antibody MX35. This investigation contributes to the foundation for future clinical α-radioimmunotherapy of minimal residual ovarian cancer with 211At-Rebmab200. Here, the biodistribution of 211At-Rebmab200 was evaluated, as was the utility of 99mTc-Rebmab200 for bioimaging. Rebmab200 was directly compared with its murine counterpart MX35 in terms of its in-vitro capacity for binding the immobilized NaPi2B epitope and live cells; we also assessed its biodistribution in nude mice carrying subcutaneous OVCAR-3 tumors. Tumor antigen and cell binding were similar between Rebmab200 and murine MX35, as was biodistribution, including normal tissue uptake and in-vivo tumor binding. We also demonstrated that 99mTc-Rebmab200 can be used for single-photon emission computed tomography of subcutaneous ovarian carcinomas in tumor-bearing mice. Taken together, our data support the further development of Rebmab200 for radioimmunotherapy and diagnostics.

The kunitz protease inhibitor domain of protease nexin-2 inhibits factor XIa and murine carotid artery and middle cerebral artery thrombosis

PubMed Central

Wu, Wenman; Li, Hongbo; Navaneetham, Duraiswamy; Reichenbach, Zachary W.; Tuma, Ronald F.

2012-01-01

Coagulation factor XI (FXI) plays an important part in both venous and arterial thrombosis, rendering FXIa a potential target for the development of antithrombotic therapy. The kunitz protease inhibitor (KPI) domain of protease nexin-2 (PN2) is a potent, highly specific inhibitor of FXIa, suggesting its possible role in the inhibition of FXI-dependent thrombosis in vivo. Therefore, we examined the effect of PN2KPI on thrombosis in the murine carotid artery and the middle cerebral artery. Intravenous administration of PN2KPI prolonged the clotting time of both human and murine plasma, and PN2KPI inhibited FXIa activity in both human and murine plasma in vitro. The intravenous administration of PN2KPI into WT mice dramatically decreased the progress of FeCl3-induced thrombus formation in the carotid artery. After a similar initial rate of thrombus formation with and without PN2KPI treatment, the propagation of thrombus formation after 10 minutes and the amount of thrombus formed were significantly decreased in mice treated with PN2KPI injection compared with untreated mice. In the middle cerebral artery occlusion model, the volume and fraction of ischemic brain tissue were significantly decreased in PN2KPI-treated compared with untreated mice. Thus, inhibition of FXIa by PN2KPI is a promising approach to antithrombotic therapy. PMID:22674803

Stage-specific apoptosis, developmental delay, and embryonic lethality in mice homozygous for a targeted disruption in the murine Bloom's syndrome gene.

PubMed

Chester, N; Kuo, F; Kozak, C; O'Hara, C D; Leder, P

1998-11-01

Bloom's syndrome is a human autosomal genetic disorder characterized at the cellular level by genome instability and increased sister chomatid exchanges (SCEs). Clinical features of the disease include proportional dwarfism and a predisposition to develop a wide variety of malignancies. The human BLM gene has been cloned recently and encodes a DNA helicase. Mouse embryos homozygous for a targeted mutation in the murine Bloom's syndrome gene (Blm) are developmentally delayed and die by embryonic day 13.5. The fact that the interrupted gene is the homolog of the human BLM gene was confirmed by its homologous sequence, its chromosomal location, and by demonstrating high numbers of SCEs in cultured murine Blm-/- fibroblasts. The proportional dwarfism seen in the human is consistent with the small size and developmental delay (12-24 hr) seen during mid-gestation in murine Blm-/- embryos. Interestingly, the growth retardation in mutant embryos can be accounted for by a wave of increased apoptosis in the epiblast restricted to early post-implantation embryogenesis. Mutant embryos do not survive past day 13.5, and at this time exhibit severe anemia. Red blood cells and their precursors from Blm-/- embryos are heterogeneous in appearance and have increased numbers of macrocytes and micronuclei. Both the apoptotic wave and the appearance of micronuclei in red blood cells are likely cellular consequences of damaged DNA caused by effects on replicating or segregating chromosomes.

Endogenous biosynthesis of thromboxane and prostacyclin in 2 distinct murine models of atherosclerosis.

PubMed

Praticò, D; Cyrus, T; Li, H; FitzGerald, G A

2000-12-01

Thromboxane A(2) is a potent vasoconstrictor and platelet agonist; prostacyclin is a potent platelet inhibitor and vasodilator. Altered biosynthesis of these eicosanoids is a feature of human hypercholesterolemia and atherosclerosis. This study examined whether in 2 murine models of atherosclerosis their levels are increased and correlated with the evolution of the disease. Urinary 2,3-dinor thromboxane B(2) and 2,3-dinor-6-keto prostaglandin F(1 alpha), metabolites of thromboxane and prostacyclin, respectively, were assayed in apoliprotein E (apoE)-deficient mice on chow and low-density lipoprotein receptor (LDLR)-deficient mice on chow and a Western-type diet. Atherosclerosis lesion area was measured by en face method. Both eicosanoids increased in apoE-deficient mice on chow and in LDLR-deficient mice on a high-fat diet, but not in LDLR-deficient mice on chow by the end of the study. Aspirin suppressed ex vivo platelet aggregation, serum thromboxane B(2), and 2,3-dinor thromboxane B(2), and significantly reduced the excretion of 2,3-dinor-6-keto prostaglandin F(1 alpha) in these animals. This study demonstrates that thromboxane as well as prostacyclin biosynthesis is increased in 2 murine models of atherogenesis and is secondary to increased in vivo platelet activation. Assessment of their generation in these models may afford the basis for future studies on the functional role of these eicosanoids in the evolution and progression of atherosclerosis. (Blood. 2000;96:3823-3826)

Induction of heme oxygenase-1 by chamomile protects murine macrophages against oxidative stress.

PubMed

Bhaskaran, Natarajan; Shukla, Sanjeev; Kanwal, Rajnee; Srivastava, Janmejai K; Gupta, Sanjay

2012-06-27

Protection of cells from oxidative insult may be possible through direct scavenging of reactive oxygen species, or through stimulation of intracellular antioxidant defense mechanisms by induction of antioxidant gene expression. In this study we investigated the cytoprotective effect of chamomile and elucidated the underlying mechanisms. The cytoprotective effect of chamomile was examined on H(2)O(2)-induced cellular stress in RAW 264.7 murine macrophages. RAW 264.7 murine macrophages treated with chamomile were protected from cell death caused by H(2)O(2). Treatment with 50μM H(2)O(2) for 6h caused significant increase in cellular stress accompanied by cell death in RAW 264.7 macrophages. Pretreatment with chamomile at 10-20μg/mL for 16h followed by H(2)O(2) treatment protected the macrophages against cell death. Chamomile exposure significantly increased the expression of antioxidant enzymes viz. heme oxygenase-1 (HO-1), peroxiredoxin-1 (Prx-1), and thioredoxin-1 (Trx-1) in a dose-dependent manner, compared with their respective controls. Chamomile increased nuclear translocation of Nrf2 with increased phosphorylated Nrf2 levels, and binding to the antioxidant response element in the nucleus. These molecular findings for the first time provide insights into the mechanisms underlying the induction of phase 2 enzymes through the Keap1-Nrf2 signaling pathway by chamomile, and provide evidence that chamomile possesses antioxidant and cytoprotective properties. Copyright © 2012 Elsevier Inc. All rights reserved.

Induction of heme oxygenase-1 by chamomile protects murine macrophages against oxidative stress

PubMed Central

Bhaskaran, Natarajan; Shukla, Sanjeev; Kanwal, Rajnee; Srivastava, Janmejai K; Gupta, Sanjay

2012-01-01

Aims Protection of cells from oxidative insult may be possible through direct scavenging of reactive oxygen species, or through stimulation of intracellular antioxidant defense mechanisms by induction of antioxidant gene expression. In this study we investigated the cytoprotective effect of chamomile and elucidated the underlying mechanisms. Main Methods The cytoprotective effect of chamomile was examined on H2O2-induced cellular stress in RAW 264.7 murine macrophages. Key Findings RAW 264.7 murine macrophages treated with chamomile were protected from cell death caused by H2O2. Treatment with 50 μM H2O2 for 6 h caused significant increase in cellular stress accompanied by cell death in RAW 264.7 macrophages. Pretreatment with chamomile at 10-20 μg/mL for 16 h followed by H2O2 treatment protected the macrophages against cell death. Chamomile exposure significantly increased the expression of antioxidant enzymes viz. heme oxygenase-1 (HO-1), peroxiredoxin-1 (Prx-1), and thioredoxin-1 (Trx-1) in a dose-dependent manner, compared with their respective controls. Chamomile increased nuclear translocation of Nrf2 with increased phosphorylated Nrf2 levels, and binding to the antioxidant response element in the nucleus. Significance These molecular findings for the first time provide insights into the mechanisms underlying the induction of phase 2 enzymes through the Keap1-Nrf2 signaling pathway by chamomile, and provide evidence that chamomile possesses antioxidant and cytoprotective properties. PMID:22683429

Protective effect of kombucha tea against tertiary butyl hydroperoxide induced cytotoxicity and cell death in murine hepatocytes.

PubMed

Bhattacharya, Semantee; Manna, Prasenjit; Gachhui, Ratan; Sil, Parames C

2011-07-01

Kombucha (KT), a fermented black tea (BT), is known to have many beneficial properties. In the present study, antioxidant property of KT has been investigated against tertiary butyl hydroperoxide (TBHP) induced cytotoxicity using murine hepatocytes. TBHP, a reactive oxygen species inducer, causes oxidative stress resulting in organ pathophysiology. Exposure to TBHP caused a reduction in cell viability, increased membrane leakage and disturbed the intra-cellular antioxidant machineries in hepatocytes. TBHP exposure disrupted mitochondrial membrane potential and induced apoptosis as evidenced by flow cytometric analyses. KT treatment, however, counteracted the changes in mitochondrial membrane potential and prevented apoptotic cell death of the hepatocytes. BT treatment also reverted TBHP induced hepatotoxicity, however KT was found to be more efficient. This may be due to the formation of antioxidant molecules like D-saccharic acid-1,4-lactone (DSL) during fermentation process and are absent in BT. Moreover, the radical scavenging activities of KT were found to be higher than BT. Results of the study showed that KT has the potential to ameliorate TBHP induced oxidative insult and cell death in murine hepatocytes more effectively than BT.

Murine CMV-Induced Hearing Loss Is Associated with Inner Ear Inflammation and Loss of Spiral Ganglia Neurons

PubMed Central

Golemac, Mijo; Pugel, Ester Pernjak; Jonjic, Stipan; Britt, William J.

2015-01-01

Congenital human cytomegalovirus (HCMV) occurs in 0.5–1% of live births and approximately 10% of infected infants develop hearing loss. The mechanism(s) of hearing loss remain unknown. We developed a murine model of CMV induced hearing loss in which murine cytomegalovirus (MCMV) infection of newborn mice leads to hematogenous spread of virus to the inner ear, induction of inflammatory responses, and hearing loss. Characteristics of the hearing loss described in infants with congenital HCMV infection were observed including, delayed onset, progressive hearing loss, and unilateral hearing loss in this model and, these characteristics were viral inoculum dependent. Viral antigens were present in the inner ear as were CD3+ mononuclear cells in the spiral ganglion and stria vascularis. Spiral ganglion neuron density was decreased after infection, thus providing a mechanism for hearing loss. The lack of significant inner ear histopathology and persistence of inflammation in cochlea of mice with hearing loss raised the possibility that inflammation was a major component of the mechanism(s) of hearing loss in MCMV infected mice. PMID:25875183

Characterization of Short Range DNA Looping in Endotoxin-mediated Transcription of the Murine Inducible Nitric-oxide Synthase (iNOS) Gene*

PubMed Central

Guo, Hongtao; Mi, Zhiyong; Kuo, Paul C.

2008-01-01

The local structural properties and spatial conformations of chromosomes are intimately associated with gene expression. The spatial associations of critical genomic elements in inducible nitric-oxide synthase (iNOS) transcription have not been previously examined. In this regard, the murine iNOS promoter contains 2 NF-κB binding sites (nt –86 and nt –972) that are essential for maximal transactivation of iNOS by LPS. Although AP-1 is commonly listed as an essential transcription factor for LPS-mediated iNOS transactivation, the relationship between AP-1 and NF-κB in this setting is not well studied. In this study using a model of LPS-stimulated ANA-1 murine macrophages, we demonstrate that short range DNA looping occurs at the iNOS promoter. This looping requires the presence of AP-1, c-Jun, NF-κB p65, and p300-associated acetyltransferase activity. The distal AP-1 binding site interacts via p300 with the proximal NF-κB binding site to create this DNA loop to participate in iNOS transcription. Other geographically distant AP-1 and NF-κB sites are certainly occupied, but selected sites are critical for iNOS transcription and the formation of the c-Jun, p65, and p300 transcriptional complex. In this “simplified” model of murine iNOS promoter, numerous transcription factors recognize and bind to various response elements, but these locales do not equally contribute to iNOS gene transcription. PMID:18596035

Characterization of intravitreally delivered capsid mutant AAV2-Cre vector to induce tissue-specific mutations in murine retinal ganglion cells.

PubMed

Langouet-Astrie, Christophe J; Yang, Zhiyong; Polisetti, Sraavya M; Welsbie, Derek S; Hauswirth, William W; Zack, Donald J; Merbs, Shannath L; Enke, Raymond A

2016-10-01

Targeted expression of Cre recombinase in murine retinal ganglion cells (RGCs) by viral vector is an effective strategy for creating tissue-specific gene knockouts for investigation of genetic contribution to RGC degeneration associated with optic neuropathies. Here we characterize dosage, efficacy and toxicity for sufficient intravitreal delivery of a capsid mutant Adeno-associated virus 2 (AAV2) vector encoding Cre recombinase. Wild type and Rosa26 (R26) LacZ mice were intravitreally injected with capsid mutant AAV2 viral vectors. Murine eyes were harvested at intervals ranging from 2 weeks to 15 weeks post-injection and were assayed for viral transduction, transgene expression and RGC survival. 10(9) vector genomes (vg) were sufficient for effective in vivo targeting of murine ganglion cell layer (GCL) retinal neurons. Transgene expression was observed as early as 2 weeks post-injection of viral vectors and persisted to 11 weeks. Early expression of Cre had no significant effect on RGC survival, while significant RGC loss was detected beginning 5 weeks post-injection. Early expression of viral Cre recombinase was robust, well-tolerated and predominantly found in GCL neurons suggesting this strategy can be effective in short-term RGC-specific mutation studies in experimental glaucoma models such as optic nerve crush and transection experiments. RGC degeneration with Cre expression for more than 4 weeks suggests that Cre toxicity is a limiting factor for targeted mutation strategies in RGCs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Antigen-Specific Gut Inflammation and Systemic Immune Responses Induced by Prolonging Wheat Gluten Sensitization in BALB/c Murine Model.

PubMed

Vijaykrishnaraj, M; Mohan Kumar, B V; Muthukumar, S P; Kurrey, Nawneet K; Prabhasankar, P

2017-10-06

Gluten-related diseases such as wheat allergy, celiac disease, and gluten intolerance are widespread around the globe to genetically predisposed individuals. The present study aims to develop a wheat-gluten induced BALB/c murine model for addressing wheat-gluten related disorders by sensitizing the wheat gluten through the route of intraperitoneal and oral challenge in prolonged days. During the sensitization, the sera were collected for specific antigliadin antibodies response and proinflammatory markers quantification. Ex vivo primary cells and organs were collected for subsequent analysis of inflammatory profile. Prolonging sensitization of gluten can moderate the antigen-specific inflammatory markers such as IL-1β, IL-4, IL-15, IL-6, IFN-γ and TNF-α levels in mice sera. However, ex vivo primary cells of splenocytes (SPLs) and intestinal epithelial lymphocytes (IELs) significantly increased the IL-6, IL-15, IL-1β, and IL-4 levels in G+ (gliadin and gluten) treated cells. Histopathology staining of jejunum sections indicates enterocyte degeneration in the apical part of villi and damage of tight junctions in G+ (gliadin and gluten) sensitized murine model. Immunohistochemistry of embedded jejunum sections showed significant expression of positive cells of IL-15, tTG and IL-4 in G+ sensitized murine model. In contrast, all markers of gluten-related disorders are expressed exclusively such as tTG, ZO-1, IL-15, IL-6, IL-4, and intestinal inflammation was mediated by iNOS, COX-2, TLR-4 and NF- k Bp50 signaling mechanism in G+ sensitized mice.

3-D QSAR ANALYSIS OF INHIBITION OF MURINE SOLUBLE EPOXIDE HYDROLASE (MSEH) BY BENZOYLUREAS, ARYLUREAS, AND THEIR ANALOGUES. (R825433)

EPA Science Inventory

Two hundred and seventy-one compounds including benzoylureas, arylureas and related compounds were assayed using recombinant murine soluble epoxide hydrolase (MsEH) produced from a baculovirus expression system. Among all the insect growth regulators assayed, 18 benzoylphenylu...

Family and family therapy in the Netherlands.

PubMed

Wagenaar, Karin; Baars, Jan

2012-04-01

This article describes how families are functioning in the Netherlands, and how family therapy is used in mental healthcare. In the open Dutch society, new ideas are easily incorporated, as exemplified by the rapid introduction and growth of family therapy in the 1980s. In recent decades, however, family therapy has lost ground to other treatment models that are more individually orientated, and adhere to stricter protocols. This decline of family therapy has been exacerbated by recent budget cuts in mental healthcare. In regular healthcare institutes family therapy now has a marginal position at best, although family treatment models are used in specific areas such as forensic treatments. In addition, the higher trained family therapists have found their own niches to work with couples and families. We argue that a stronger position of family therapy would be beneficial for patients and for families, in order to counteract the strong individualization of Dutch society.

Methanol extract of Ocimum gratissimum protects murine peritoneal macrophages from nicotine toxicity by decreasing free radical generation, lipid and protein damage and enhances antioxidant protection

PubMed Central

Mahapatra, Santanu Kar; Chakraborty, Subhankari Prasad; Das, Subhasis

2009-01-01

In the present study, methanol extract of Ocimum gratissimum Linn (ME-Og) was tested against nicotine-induced murine peritoneal macrophage in vitro. Phytochemical analysis of ME-Og shown high amount of flavonoid and phenolic compound present in it. The cytotoxic effect of ME-Og was studied in murine peritoneal macrophages at different concentrations (0.1 to 100 µg/ml) using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide (MTT) method. To establish the protective role of ME-Og against nicotine toxicity, peritoneal macrophages from mice were treated with nicotine (10 mM), nicotine + ME-Og (1 to 25 µg/ml) for 12 h in culture media. The significantly (p < 0.05) increased super oxide anion generation, reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, myeloperoxidase (MPO) activity, lipid peroxidation, protein carbonyls, oxidized glutathione levels were observed in nicotine-treated group as compared to control group; those were significantly (p < 0.05) reduced in ME-Og supplemented groups in concentration dependent manner. More over, significantly (p < 0.05) reduced antioxidant status due to nicotine exposure was effectively ameliorated by ME-Og supplementation in murine peritoneal macrophages. Among the different concentration of ME-Og, maximum protective effect was observed by 25 µg/ml, which does not produce significant cell cytotoxicity in murine peritoneal macrophages. These findings suggest the potential use and beneficial role of O. gratissimum as a modulator of nicotine-induced free radical generation, lipid-protein damage and antioxidant status in important immune cell, peritoneal macrophages. PMID:20716908

Handling stress may confound murine gut microbiota studies.

PubMed

Allen-Blevins, Cary R; You, Xiaomeng; Hinde, Katie; Sela, David A

2017-01-01

Accumulating evidence indicates interactions between human milk composition, particularly sugars (human milk oligosaccharides or HMO), the gut microbiota of human infants, and behavioral effects. Some HMO secreted in human milk are unable to be endogenously digested by the human infant but are able to be metabolized by certain species of gut microbiota, including Bifidobacterium longum subsp. infantis (B. infantis) , a species sensitive to host stress (Bailey & Coe, 2004). Exposure to gut bacteria like B. infantis during critical neurodevelopment windows in early life appears to have behavioral consequences; however, environmental, physical, and social stress during this period can also have behavioral and microbial consequences. While rodent models are a useful method for determining causal relationships between HMO, gut microbiota, and behavior, murine studies of gut microbiota usually employ oral gavage, a technique stressful to the mouse. Our aim was to develop a less-invasive technique for HMO administration to remove the potential confound of gavage stress. Under the hypothesis that stress affects gut microbiota, particularly B. infantis , we predicted the pups receiving a prebiotic solution in a less-invasive manner would have the highest amount of Bifidobacteria in their gut. This study was designed to test two methods, active and passive, of solution administration to mice and the effects on their gut microbiome. Neonatal C57BL/6J mice housed in a specific-pathogen free facility received increasing doses of fructooligosaccharide (FOS) solution or deionized, distilled water. Gastrointestinal (GI) tracts were collected from five dams, six sires, and 41 pups over four time points. Seven fecal pellets from unhandled pups and two pellets from unhandled dams were also collected. Qualitative real-time polymerase chain reaction (qRT-PCR) was used to quantify and compare the amount of Bifidobacterium , Bacteroides , Bacteroidetes, and Firmicutes. Our results

Fibroblast drug scavenging increases intratumoural gemcitabine accumulation in murine pancreas cancer

PubMed Central

Hessmann, E; Patzak, M S; Klein, L; Chen, N; Kari, V; Ramu, I; Bapiro, T E; Frese, K K; Gopinathan, A; Richards, F M; Jodrell, D I; Verbeke, C; Li, X; Heuchel, R; Löhr, J M; Johnsen, S A; Gress, T M; Ellenrieder, V; Neesse, A

2018-01-01

Objective Desmoplasia and hypovascularity are thought to impede drug delivery in pancreatic ductal adenocarcinoma (PDAC). However, stromal depletion approaches have failed to show clinical responses in patients. Here, we aimed to revisit the role of the tumour microenvironment as a physical barrier for gemcitabine delivery. Design Gemcitabine metabolites were analysed in LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) murine tumours and matched liver metastases, primary tumour cell lines, cancer-associated fibroblasts (CAFs) and pancreatic stellate cells (PSCs) by liquid chromatography-mass spectrometry/mass spectrometry. Functional and preclinical experiments, as well as expression analysis of stromal markers and gemcitabine metabolism pathways were performed in murine and human specimen to investigate the preclinical implications and the mechanism of gemcitabine accumulation. Results Gemcitabine accumulation was significantly enhanced in fibroblast-rich tumours compared with liver metastases and normal liver. In vitro, significantly increased concentrations of activated 2′,2′-difluorodeoxycytidine-5′-triphosphate (dFdCTP) and greatly reduced amounts of the inactive gemcitabine metabolite 2′,2′-difluorodeoxyuridine were detected in PSCs and CAFs. Mechanistically, key metabolic enzymes involved in gemcitabine inactivation such as hydrolytic cytosolic 5′-nucleotidases (Nt5c1A, Nt5c3) were expressed at low levels in CAFs in vitro and in vivo, and recombinant expression of Nt5c1A resulted in decreased intracellular dFdCTP concentrations in vitro. Moreover, gemcitabine treatment in KPC mice reduced the number of liver metastases by >50%. Conclusions Our findings suggest that fibroblast drug scavenging may contribute to the clinical failure of gemcitabine in desmoplastic PDAC. Metabolic targeting of CAFs may thus be a promising strategy to enhance the antiproliferative effects of gemcitabine. PMID:28077438

Immunoproteomic profiling of Saccharomyces cerevisiae systemic infection in a murine model.

PubMed

Hernández-Haro, Carolina; Llopis, Silvia; Molina, María; Monteoliva, Lucía; Gil, Concha

2015-01-01

Saccharomyces cerevisiae is considered a safe microorganism widely used as a dietary supplement. However, in the latest decades several cases of S. cerevisiae infections have been reported. Recent studies in a murine model of systemic infection have also revealed the virulence of some S. cerevisiae dietary strains. Here we use an immunoproteomic approach based on protein separation by 2D-PAGE followed by Western-blotting to compare the serological response against a virulent dietary and a non-virulent laboratory strains leading to the identification of highly different patterns of antigenic proteins. Thirty-six proteins that elicit a serological response in mice have been identified. Most of them are involved in stress responses and metabolic pathways. Their selectivity as putative biomarkers for S. cerevisiae infections was assessed by testing sera from S. cerevisiae-infected mice against Candida albicans and C. glabrata proteins. Some chaperones and metabolic proteins showed cross-reactivity. We also compare the S. cerevisiae immunodetected proteins with previously described C. albicans antigens. The results point to the stress-related proteins Ahp1, Yhb1 and Oye2, as well as the glutamine synthetase Gln1 and the oxysosterol binding protein Kes1 as putative candidates for being evaluated as biomarkers for diagnostic assays of S. cerevisiae infections. S. cerevisiae can cause opportunistic infections, and therefore, a precise diagnosis of fungal infections is necessary. This immunoproteomic analysis of sera from a model murine infection with a virulent dietary S. cerevisiae strain has been shown to be a source of candidate proteins for being evaluated as biomarkers to develop assays for diagnosis of S. cerevisiae infections. To our knowledge, this is the first study devoted to the identification of S. cerevisiae immunogenic proteins and the results allowed the proposal of five antigens to be further investigated. Copyright © 2014 Elsevier B.V. All rights reserved.

Fibroblast drug scavenging increases intratumoural gemcitabine accumulation in murine pancreas cancer.

PubMed

Hessmann, E; Patzak, M S; Klein, L; Chen, N; Kari, V; Ramu, I; Bapiro, T E; Frese, K K; Gopinathan, A; Richards, F M; Jodrell, D I; Verbeke, C; Li, X; Heuchel, R; Löhr, J M; Johnsen, S A; Gress, T M; Ellenrieder, V; Neesse, A

2018-03-01

Desmoplasia and hypovascularity are thought to impede drug delivery in pancreatic ductal adenocarcinoma (PDAC). However, stromal depletion approaches have failed to show clinical responses in patients. Here, we aimed to revisit the role of the tumour microenvironment as a physical barrier for gemcitabine delivery. Gemcitabine metabolites were analysed in LSL-Kras G12D/+ ; LSL-Trp53 R172H/+ ; Pdx-1-Cre (KPC) murine tumours and matched liver metastases, primary tumour cell lines, cancer-associated fibroblasts (CAFs) and pancreatic stellate cells (PSCs) by liquid chromatography-mass spectrometry/mass spectrometry. Functional and preclinical experiments, as well as expression analysis of stromal markers and gemcitabine metabolism pathways were performed in murine and human specimen to investigate the preclinical implications and the mechanism of gemcitabine accumulation. Gemcitabine accumulation was significantly enhanced in fibroblast-rich tumours compared with liver metastases and normal liver. In vitro, significantly increased concentrations of activated 2',2'-difluorodeoxycytidine-5'-triphosphate (dFdCTP) and greatly reduced amounts of the inactive gemcitabine metabolite 2',2'-difluorodeoxyuridine were detected in PSCs and CAFs. Mechanistically, key metabolic enzymes involved in gemcitabine inactivation such as hydrolytic cytosolic 5'-nucleotidases (Nt5c1A, Nt5c3) were expressed at low levels in CAFs in vitro and in vivo, and recombinant expression of Nt5c1A resulted in decreased intracellular dFdCTP concentrations in vitro. Moreover, gemcitabine treatment in KPC mice reduced the number of liver metastases by >50%. Our findings suggest that fibroblast drug scavenging may contribute to the clinical failure of gemcitabine in desmoplastic PDAC. Metabolic targeting of CAFs may thus be a promising strategy to enhance the antiproliferative effects of gemcitabine. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a

Early Molecular Events in Murine Gastric Epithelial Cells Mediated by Helicobacter pylori CagA.

PubMed

Banerjee, Aditi; Basu, Malini; Blanchard, Thomas G; Chintalacharuvu, Subba R; Guang, Wei; Lillehoj, Erik P; Czinn, Steven J

2016-10-01

Murine models of Helicobacter pylori infection are used to study host-pathogen interactions, but lack of severe gastritis in this model has limited its usefulness in studying pathogenesis. We compared the murine gastric epithelial cell line GSM06 to the human gastric epithelial AGS cell line to determine whether similar events occur when cultured with H. pylori. The lysates of cells infected with H. pylori isolates or an isogenic cagA-deficient mutant were assessed for translocation and phosphorylation of CagA and for activation of stress pathway kinases by immunoblot. Phosphorylated CagA was detected in both cell lines within 60 minutes. Phospho-ERK 1/2 was present within several minutes and distinctly present in GSM06 cells at 60 minutes. Similar results were obtained for phospho-JNK, although the 54 kDa phosphoprotein signal was dominant in AGS, whereas the lower molecular weight band was dominant in GSM06 cells. These results demonstrate that early events in H. pylori pathogenesis occur within mouse epithelial cells similar to human cells and therefore support the use of the mouse model for the study of acute CagA-associated host cell responses. These results also indicate that reduced disease in H. pylori-infected mice may be due to lack of the Cag PAI, or by differences in the mouse response downstream of the initial activation events. © 2016 John Wiley & Sons Ltd.

Secondary Metabolites in Allergic Plant Pollen Samples Modulate Afferent Neurons and Murine Tracheal Rings.

PubMed

Božičević, Alen; De Mieri, Maria; Nassenstein, Christina; Wiegand, Silke; Hamburger, Matthias

2017-11-22

Plant pollens are strong airborne elicitors of asthma. Their proteinaceous allergens have been studied intensively, but little is known about a possible contribution of pollen secondary metabolites to the nonallergic exacerbation of asthma. Pollen samples originating from 30 plant species were analyzed by HPLC coupled to PDA, ESIMS, and ELSD detectors and off-line NMR spectroscopy. Polyamine conjugates, flavonoids, and sesquiterpene lactones were identified. Polyamine conjugates were characteristic of all Asteraceae species. The presence of sesquiterpene lactones in Asteraceae pollen varied between species and pollen lots. All plant pollen, including those from non-Asteraceae species, contained to some extent electrophiles as determined by their reaction with N-acetyl-l-cysteine. Selected pollen extracts and pure compounds were tested in murine afferent neurons and in murine tracheal preparations. Tetrahydrofuran extracts of Ambrosia artemisiifolia and Ambrosia psilostachya pollen and a mixture of sesquiterpene lactones coronopilin/parthenin increased the intracellular Ca 2+ concentration in 15%, 32%, and 37% of cinnamaldehyde-responsive neurons, respectively. In organ bath experiments, only the sesquiterpene lactones tested induced a weak dilatation of naïve tracheas and strongly lowered the maximal methacholine-induced tracheal constriction. A tetrahydrofuran extract of A. psilostachya and coronopilin/parthenin led to a time-dependent relaxation of the methacholine-preconstricted trachea. These results provide the first evidence for a potential role of pollen secondary metabolites in the modulation of the tracheal tone.

Effect of Different Titanium Surfaces on Maturation of Murine Bone Marrow-Derived Dendritic Cells

NASA Astrophysics Data System (ADS)

Zheng, Xiaofei; Zhou, Fengjuan; Gu, Yifei; Duan, Xiaobo; Mo, Anchun

2017-02-01

Dendritic cells (DCs) play a pivotal role in the host response to implanted biomaterials. Osseointegration of titanium (Ti) implant is an immunological and inflammatory-driven process. However, the role of DCs in this complex process is largely unknown. This study aimed to investigate the effect of different Ti surfaces on DC maturation, and evaluate its subsequent potential on osteogenic differentiation of preosteoblasts. Murine bone marrow-derived DCs were seeded on Ti disks with different surface treatments, including pretreatment (PT), sandblasted/acid-etched (SLA) and modified SLA (modSLA) surface. Compared with DCs cultured on PT and SLA surfaces, the cells seeded on modSLA surface demonstrated a more round morphology with lower expression of CD86 and MHC-II, the DC maturation markers. Those cells also secreted high levels of anti-inflammatory cytokine IL-10 and TGF-β. Notably, addition of conditioned medium (CM) from modSLA-induced DCs significantly increased the mRNA expression of Runx2 and ALP as well as ALP activity by murine preosteoblast MC3T3-E1 cells. Our data demonstrated that Ti disks with different surfaces lead to differential DCs responses. PT and SLA surfaces induce DCs mature, while DCs seeded on modSLA-Ti surface maintain an immature phenotype and exhibit a potential of promoting osteogenic differentiation of MC3T3-E1 cells.

Ube3a reinstatement identifies distinct developmental windows in a murine Angelman syndrome model

PubMed Central

Silva-Santos, Sara; van Woerden, Geeske M.; Bruinsma, Caroline F.; Mientjes, Edwin; Jolfaei, Mehrnoush Aghadavoud; Distel, Ben; Kushner, Steven A.; Elgersma, Ype

2015-01-01

Angelman syndrome (AS) is a severe neurodevelopmental disorder that results from loss of function of the maternal ubiquitin protein ligase E3A (UBE3A) allele. Due to neuron-specific imprinting, the paternal UBE3A copy is silenced. Previous studies in murine models have demonstrated that strategies to activate the paternal Ube3a allele are feasible; however, a recent study showed that pharmacological Ube3a gene reactivation in adulthood failed to rescue the majority of neurocognitive phenotypes in a murine AS model. Here, we performed a systematic study to investigate the possibility that neurocognitive rescue can be achieved by reinstating Ube3a during earlier neurodevelopmental windows. We developed an AS model that allows for temporally controlled Cre-dependent induction of the maternal Ube3a allele and determined that there are distinct neurodevelopmental windows during which Ube3a restoration can rescue AS-relevant phenotypes. Motor deficits were rescued by Ube3a reinstatement in adolescent mice, whereas anxiety, repetitive behavior, and epilepsy were only rescued when Ube3a was reinstated during early development. In contrast, hippocampal synaptic plasticity could be restored at any age. Together, these findings suggest that Ube3a reinstatement early in development may be necessary to prevent or rescue most AS-associated phenotypes and should be considered in future clinical trial design. PMID:25866966

Cytomegalovirus infection of the BS-1 human stroma cell line: effect on murine hemopoiesis.

PubMed

Steinberg, H N; Anderson, J; Lim, B; Chatis, P A

1993-10-01

BS-1, a stromal cell line derived from human bone marrow, can support the growth of murine erythroid (BFU-E), granulocyte-macrophage (CFU-GM), and megakaryocyte (CFU-M) progenitor cells in a short term in vitro coculture system. Exposure of BS-1 cells to cytomegalovirus (CMV) for 3 hr prior to coculture results in a marked reduction in the stroma cell's ability to support murine hemopoiesis. CMV's effect on the BS-1 cell's hematopoietic support function is dependent on the multiplicity of infection with total suppression of BFU-E observed at a 1:1 ratio of virus to bone marrow cells. A 50% loss in the ability of BS-1 cells to support BFU-E is observed at a 0.1:1 ratio. No effect of CMV is observed with further log dilutions of virus. CMV infection of BS-1 cells affects its support of erythroid progenitor cell growth to a greater extent than its influence on the development of granulocyte-macrophage colonies. Antibody to CMV or heat inactivation of the virus reverses the inhibitory affect on BS-1 cells. The results suggest that CMV can infect a cell that constitutes one of the cellular elements of the normal bone marrow microenvironment causing a decrease in the stroma's ability to support the growth and development of normal progenitor cells.

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

NASA Astrophysics Data System (ADS)

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

2015-11-01

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.

Peptidylarginine Deiminase Inhibition Reduces Vascular Damage and Modulates Innate Immune Responses in Murine Models of Atherosclerosis

PubMed Central

Knight, Jason S.; Luo, Wei; O’Dell, Alexander A.; Yalavarthi, Srilakshmi; Zhao, Wenpu; Subramanian, Venkataraman; Guo, Chiao; Grenn, Robert C.; Thompson, Paul R.; Eitzman, Daniel T.; Kaplan, Mariana J.

2014-01-01

Rationale Neutrophil extracellular trap (NET) formation promotes vascular damage, thrombosis, and activation of interferon-α-producing plasmacytoid dendritic cells in diseased arteries. Peptidylarginine deiminase inhibition is a strategy that can decrease in vivo NET formation. Objective To test whether peptidylarginine deiminase inhibition, a novel approach to targeting arterial disease, can reduce vascular damage and inhibit innate immune responses in murine models of atherosclerosis. Methods and Results Apolipoprotein-E (Apoe)−/− mice demonstrated enhanced NET formation, developed autoantibodies to NETs, and expressed high levels of interferon-α in diseased arteries. Apoe−/− mice were treated for 11 weeks with daily injections of Cl-amidine, a peptidylarginine deiminase inhibitor. Peptidylarginine deiminase inhibition blocked NET formation, reduced atherosclerotic lesion area, and delayed time to carotid artery thrombosis in a photochemical injury model. Decreases in atherosclerosis burden were accompanied by reduced recruitment of netting neutrophils and macrophages to arteries, as well as by reduced arterial interferon-α expression. Conclusions Pharmacological interventions that block NET formation can reduce atherosclerosis burden and arterial thrombosis in murine systems. These results support a role for aberrant NET formation in the pathogenesis of atherosclerosis through modulation of innate immune responses. PMID:24425713

Growth inhibition and differentiation of murine melanoma B16-BL6 cells caused by the combination of cisplatin and caffeine.

PubMed

Tsuchiya, H; Tomita, K; Yasutake, H; Ueda, Y; Tanaka, M; Sasaki, T

1989-12-01

We preliminarily investigated the combined effects of cisplatin and caffeine on murine melanoma B16-BL6 cells in vitro. When caffeine was added before or simultaneously with cisplatin, there was little growth inhibition. The addition of 2.0 mM caffeine after 1 h of exposure to cisplatin inhibited growth and induced cell differentiation. This treatment resulted in fewer cells, and the numbers of melanosomes and mitochondria and the amount of Golgi's complex and endoplasmic reticulum were increased. DNA histograms obtained by flow cytometry showed that cells treated with cisplatin alone accumulated in the G2/M phase, with a partial G2 block. The addition of 2.0 mM caffeine after 1 h of treatment with cisplatin reduced this block. Caffeine caused murine melanoma B16-BL6 cells treated with cisplatin to differentiate, and this inhibited growth.

Local effect of zoledronic acid on new bone formation in posterolateral spinal fusion with demineralized bone matrix in a murine model.

PubMed

Zwolak, Pawel; Farei-Campagna, Jan; Jentzsch, Thorsten; von Rechenberg, Brigitte; Werner, Clément M

2018-01-01

Posterolateral spinal fusion is a common orthopaedic surgery performed to treat degenerative and traumatic deformities of the spinal column. In posteriolateral spinal fusion, different osteoinductive demineralized bone matrix products have been previously investigated. We evaluated the effect of locally applied zoledronic acid in combination with commercially available demineralized bone matrix putty on new bone formation in posterolateral spinal fusion in a murine in vivo model. A posterolateral sacral spine fusion in murine model was used to evaluate the new bone formation. We used the sacral spine fusion model to model the clinical situation in which a bone graft or demineralized bone matrix is applied after dorsal instrumentation of the spine. In our study, group 1 received decortications only (n = 10), group 2 received decortication, and absorbable collagen sponge carrier, group 3 received decortication and absorbable collagen sponge carrier with zoledronic acid in dose 10 µg, group 4 received demineralized bone matrix putty (DBM putty) plus decortication (n = 10), and group 5 received DBM putty, decortication and locally applied zoledronic acid in dose 10 µg. Imaging was performed using MicroCT for new bone formation assessment. Also, murine spines were harvested for histopathological analysis 10 weeks after surgery. The surgery performed through midline posterior approach was reproducible. In group with decortication alone there was no new bone formation. Application of demineralized bone matrix putty alone produced new bone formation which bridged the S1-S4 laminae. Local application of zoledronic acid to demineralized bone matrix putty resulted in significant increase of new bone formation as compared to demineralized bone matrix putty group alone. A single local application of zoledronic acid with DBM putty during posterolateral fusion in sacral murine spine model increased significantly new bone formation in situ in our model. Therefore, our

The Aminopeptidase Inhibitor CHR-2863 Is an Orally Bioavailable Inhibitor of Murine Malaria

PubMed Central

Skinner-Adams, Tina S.; Peatey, Christopher L.; Anderson, Karen; Trenholme, Katharine R.; Krige, David; Brown, Christopher L.; Stack, Colin; Nsangou, Desire M. M.; Mathews, Rency T.; Thivierge, Karine; Dalton, John P.

2012-01-01

Malaria remains a significant risk in many areas of the world, with resistance to the current antimalarial pharmacopeia an ever-increasing problem. The M1 alanine aminopeptidase (PfM1AAP) and M17 leucine aminopeptidase (PfM17LAP) are believed to play a role in the terminal stages of digestion of host hemoglobin and thereby generate a pool of free amino acids that are essential for parasite growth and development. Here, we show that an orally bioavailable aminopeptidase inhibitor, CHR-2863, is efficacious against murine malaria. PMID:22450967

Cell-delivered magnetic nanoparticles caused hyperthermia-mediated increased survival in a murine pancreatic cancer model.

PubMed

Basel, Matthew T; Balivada, Sivasai; Wang, Hongwang; Shrestha, Tej B; Seo, Gwi Moon; Pyle, Marla; Abayaweera, Gayani; Dani, Raj; Koper, Olga B; Tamura, Masaaki; Chikan, Viktor; Bossmann, Stefan H; Troyer, Deryl L

2012-01-01

Using magnetic nanoparticles to absorb alternating magnetic field energy as a method of generating localized hyperthermia has been shown to be a potential cancer treatment. This report demonstrates a system that uses tumor homing cells to actively carry iron/iron oxide nanoparticles into tumor tissue for alternating magnetic field treatment. Paramagnetic iron/ iron oxide nanoparticles were synthesized and loaded into RAW264.7 cells (mouse monocyte/ macrophage-like cells), which have been shown to be tumor homing cells. A murine model of disseminated peritoneal pancreatic cancer was then generated by intraperitoneal injection of Pan02 cells. After tumor development, monocyte/macrophage-like cells loaded with iron/ iron oxide nanoparticles were injected intraperitoneally and allowed to migrate into the tumor. Three days after injection, mice were exposed to an alternating magnetic field for 20 minutes to cause the cell-delivered nanoparticles to generate heat. This treatment regimen was repeated three times. A survival study demonstrated that this system can significantly increase survival in a murine pancreatic cancer model, with an average post-tumor insertion life expectancy increase of 31%. This system has the potential to become a useful method for specifically and actively delivering nanoparticles for local hyperthermia treatment of cancer.

Cell-delivered magnetic nanoparticles caused hyperthermia-mediated increased survival in a murine pancreatic cancer model

PubMed Central

Basel, Matthew T; Balivada, Sivasai; Wang, Hongwang; Shrestha, Tej B; Seo, Gwi Moon; Pyle, Marla; Abayaweera, Gayani; Dani, Raj; Koper, Olga B; Tamura, Masaaki; Chikan, Viktor; Bossmann, Stefan H; Troyer, Deryl L

2012-01-01

Using magnetic nanoparticles to absorb alternating magnetic field energy as a method of generating localized hyperthermia has been shown to be a potential cancer treatment. This report demonstrates a system that uses tumor homing cells to actively carry iron/iron oxide nanoparticles into tumor tissue for alternating magnetic field treatment. Paramagnetic iron/ iron oxide nanoparticles were synthesized and loaded into RAW264.7 cells (mouse monocyte/ macrophage-like cells), which have been shown to be tumor homing cells. A murine model of disseminated peritoneal pancreatic cancer was then generated by intraperitoneal injection of Pan02 cells. After tumor development, monocyte/macrophage-like cells loaded with iron/ iron oxide nanoparticles were injected intraperitoneally and allowed to migrate into the tumor. Three days after injection, mice were exposed to an alternating magnetic field for 20 minutes to cause the cell-delivered nanoparticles to generate heat. This treatment regimen was repeated three times. A survival study demonstrated that this system can significantly increase survival in a murine pancreatic cancer model, with an average post-tumor insertion life expectancy increase of 31%. This system has the potential to become a useful method for specifically and actively delivering nanoparticles for local hyperthermia treatment of cancer. PMID:22287840

cis elements and trans-acting factors involved in dimer formation of murine leukemia virus RNA.

PubMed

Prats, A C; Roy, C; Wang, P A; Erard, M; Housset, V; Gabus, C; Paoletti, C; Darlix, J L

1990-02-01

The genetic material of all retroviruses examined so far consists of two identical RNA molecules joined at their 5' ends by the dimer linkage structure (DLS). Since the precise location of the DLS as well as the mechanism and role(s) of RNA dimerization remain unclear, we analyzed the dimerization process of Moloney murine leukemia virus (MoMuLV) genomic RNA. For this purpose we derived an in vitro model for RNA dimerization. By using this model, murine leukemia virus RNA was shown to form dimeric molecules. Deletion mutagenesis in the 620-nucleotide leader of MoMuLV RNA showed that the dimer promoting sequences are located within the encapsidation element Psi between positions 215 and 420. Furthermore, hybridization assays in which DNA oligomers were used to probe monomer and dimer forms of MoMuLV RNA indicated that the DLS probably maps between positions 280 and 330 from the RNA 5' end. Also, retroviral nucleocapsid protein was shown to catalyze dimerization of MoMuLV RNA and to be tightly bound to genomic dimer RNA in virions. These results suggest that MoMuLV RNA dimerization and encapsidation are probably controlled by the same cis element, Psi, and trans-acting factor, nucleocapsid protein, and thus might be linked during virion formation.

cis elements and trans-acting factors involved in dimer formation of murine leukemia virus RNA.

PubMed Central

Prats, A C; Roy, C; Wang, P A; Erard, M; Housset, V; Gabus, C; Paoletti, C; Darlix, J L

1990-01-01

The genetic material of all retroviruses examined so far consists of two identical RNA molecules joined at their 5' ends by the dimer linkage structure (DLS). Since the precise location of the DLS as well as the mechanism and role(s) of RNA dimerization remain unclear, we analyzed the dimerization process of Moloney murine leukemia virus (MoMuLV) genomic RNA. For this purpose we derived an in vitro model for RNA dimerization. By using this model, murine leukemia virus RNA was shown to form dimeric molecules. Deletion mutagenesis in the 620-nucleotide leader of MoMuLV RNA showed that the dimer promoting sequences are located within the encapsidation element Psi between positions 215 and 420. Furthermore, hybridization assays in which DNA oligomers were used to probe monomer and dimer forms of MoMuLV RNA indicated that the DLS probably maps between positions 280 and 330 from the RNA 5' end. Also, retroviral nucleocapsid protein was shown to catalyze dimerization of MoMuLV RNA and to be tightly bound to genomic dimer RNA in virions. These results suggest that MoMuLV RNA dimerization and encapsidation are probably controlled by the same cis element, Psi, and trans-acting factor, nucleocapsid protein, and thus might be linked during virion formation. Images PMID:2153242

CD8+ T Cells Contribute to the Development of Coronary Arteritis in the Lactobacillus casei Cell Wall Extract-Induced Murine Model of Kawasaki Disease.

PubMed

Noval Rivas, Magali; Lee, Youngho; Wakita, Daiko; Chiba, Norika; Dagvadorj, Jargalsaikhan; Shimada, Kenichi; Chen, Shuang; Fishbein, Michael C; Lehman, Thomas J A; Crother, Timothy R; Arditi, Moshe

2017-02-01

Kawasaki disease (KD) is the leading cause of acquired heart disease among children in developed countries. Coronary lesions in KD in humans are characterized by an increased presence of infiltrating CD3+ T cells; however, the specific contributions of the different T cell subpopulations in coronary arteritis development remain unknown. Therefore, we sought to investigate the function of CD4+ and CD8+ T cells, Treg cells, and natural killer (NK) T cells in the pathogenesis of KD. We addressed the function of T cell subsets in KD development by using a well-established murine model of Lactobacillus casei cell wall extract (LCWE)-induced KD vasculitis. We determined which T cell subsets were required for development of KD vasculitis by using several knockout murine strains and depleting monoclonal antibodies. LCWE-injected mice developed coronary lesions characterized by the presence of inflammatory cell infiltrates. Frequently, this chronic inflammation resulted in complete occlusion of the coronary arteries due to luminal myofibroblast proliferation (LMP) as well as the development of coronary arteritis and aortitis. We found that CD8+ T cells, but not CD4+ T cells, NK T cells, or Treg cells, were required for development of KD vasculitis. The LCWE-induced murine model of KD vasculitis mimics many histologic features of the disease in humans, such as the presence of CD8+ T cells and LMP in coronary artery lesions as well as epicardial coronary arteritis. Moreover, CD8+ T cells functionally contribute to the development of KD vasculitis in this murine model. Therapeutic strategies targeting infiltrating CD8+ T cells might be useful in the management of KD in humans. © 2016, American College of Rheumatology.

Growth differentiation factor‑5 induces tenomodulin expression via phosphorylation of p38 and promotes viability of murine mesenchymal stem cells from compact bone.

PubMed

Qu, Yanlong; Zhou, Li; Lv, Bing; Wang, Chunlei; Li, Pengwei

2018-03-01

Growth differentiation factor (GDF)‑5 serves a role in tissue development and tenomodulin serves an important role in the development of tendons. The effects of GDF‑5 on mesenchymal stem cells (MSCs), particularly with regards to tendon bioengineering, are poorly understood. The present study aimed to investigate the effects of GDF‑5 on cell viability and tenomodulin expression in MSCs from murine compact bone. MSCs were isolated from murine compact bones and confirmed by flow cytometric analysis. In addition, the adipogenic, osteoblastic and chondrocyte differentiation capabilities of the MSCs were determined. MSCs were treated with GDF‑5 and the effects of GDF‑5 on MSC viability were determined. The mRNA and protein expression levels of tenomodulin were detected by reverse transcription‑quantitative polymerase chain reaction and western blotting, respectively. MSCs from murine compact bone were successfully isolated. GDF‑5 had optimal effects on cell viability at 100 ng/ml (+36.9% of control group without GDF‑5 treatment, P<0.01) and its effects peaked after 6 days of treatment (+56.6% of control group, P<0.001). Compared with the control group, treatment with 100 ng/ml GDF‑5 for 4 days enhanced the mRNA expression levels of tenomodulin (3.56±0.94 vs. 1.02±0.25; P<0.05). In addition, p38 was activated by GDF‑5, as determined by enhanced expression levels of phosphorylated p38 (p‑p38). The GDF‑5‑induced protein expression levels of p‑p38 and tenomodulin were markedly inhibited following treatment with SB203580, an inhibitor of p38 mitogen‑activated protein kinase. These results suggested that GDF‑5 treatment may increase tenomodulin protein expression via phosphorylation of p38 in MSCs from murine compact bone. These findings may aid the future development of tendon bioengineering.

Family demands, social support and family functioning in Taiwanese families rearing children with Down syndrome.

PubMed

Hsiao, C-Y

2014-06-01

Down syndrome (DS) affects not only children but also their families. Much remains to be learned about factors that influence how families of children with DS function, especially families in non-Western populations. The purpose of this cross-sectional, correlational study was to examine how family demographics, family demands and social support relate to family functioning as well as the potential mediating effect of social support on the relationship between family demands and family functioning in Taiwanese families of children with DS. One hundred and fifty-five parents (80 mothers and 75 fathers) from 83 families independently completed mailed questionnaires. Data were analysed using a principal component analysis and mixed linear modelling. Families having older children with DS, greater parental education, higher family income, fewer family demands and greater social support contributed to healthier family functioning. Social support partially mediated the effects of family demands on family functioning. Family demographics, family demands and social support appear to be important factors that may play a critical role in how Taiwanese families respond to the birth of a child with DS. Care of children with DS and their families is likely to be more effective if professionals working with these families are aware of factors that contribute to healthy family functioning. © 2013 MENCAP and International Association of the Scientific Study of Intellectual and Developmental Disabilities and John Wiley & Sons Ltd.

Phagocytosis of Candida albicans Enhances Malignant Behavior of Murine Tumor Cells

NASA Astrophysics Data System (ADS)

Ginsburg, Isaac; Fligiel, Suzanne E. G.; Kunkel, Robin G.; Riser, Bruce L.; Varani, James

1987-12-01

Murine tumor cells were induced to phagocytize either Candida albicans or group A streptococcal cells. The presence of microbial particles within the tumor cell cytoplasm had no effect on in vitro tumor cell growth. However, when Candida albicans-infected tumor cells were injected into syngeneic mice, they formed tumors that grew faster, invaded the surrounding normal tissue more rapidly and metastasized more rapidly than control tumor cells. Tumor cells infected with group A streptococcal particles did not grow faster or show increased malignant behavior. These data indicate that the in vivo behavior of malignant tumor cells can be modulated by microbial particles, which are often present in the microenvironment of the growing tumor.

cDNA cloning and characterization of mouse DTEF-1 and ETF, members of the TEA/ATTS family of transcription factors.

PubMed

Yockey, C E; Shimizu, N

1998-02-01

Members of the TEA/ATTS family of transcription factors have been found in most representative eukaryotic organisms. In vertebrates, the TEA family contains at least four members, which share overlapping DNA-binding specificity and have similar transcriptional activation properties. In this article, we describe the cDNA cloning and characterization of the murine TEA proteins DTEF-1 (mDTEF-1) and ETF. Using in situ hybridization analysis of mouse embryos, we found that mDTEF-1 and ETF transcript distributions substantially overlap. ETF is expressed throughout the embryo except in the myocardium early in development, whereas late in development, it is enriched in lung and neuroectoderm. Mouse DTEF-1 is expressed at a much lower level throughout development and is substantially enriched in ectoderm and skin, as well as in the developing pituitary at midgestation. Northern blot analysis of adult mouse tissue total RNA showed that both ETF and mDTEF-1 are abundant in uterus and lung relative to other tissues. Using gel mobility shift assays and GAL4-fusion protein analysis, we demonstrated that the full coding sequences of ETF and mDTEF-1 encode M-CAT/GT-IIC-binding proteins containing activation domains.

Ibuprofen Potentiates the In Vivo Antifungal Activity of Fluconazole against Candida albicans Murine Infection

PubMed Central

Miranda, Isabel M.; Silva-Dias, Ana; Silva, Ana P.; Rodrigues, Acácio G.; Pina-Vaz, Cidália

2015-01-01

Candida albicans is the most prevalent cause of fungemia worldwide. Its ability to develop resistance in patients receiving azole antifungal therapy is well documented. In a murine model of systemic infection, we show that ibuprofen potentiates fluconazole antifungal activity against a fluconazole-resistant strain, drastically reducing the fungal burden and morbidity. The therapeutic combination of fluconazole with ibuprofen may constitute a new approach for the management of antifungal therapeutics to reverse the resistance conferred by efflux pump overexpression. PMID:25845879

Dynein Regulators Are Important for Ecotropic Murine Leukemia Virus Infection

PubMed Central

Valle-Tenney, Roger; Opazo, Tatiana; Cancino, Jorge; Goff, Stephen P.

2016-01-01

ABSTRACT During the early steps of infection, retroviruses must direct the movement of the viral genome into the nucleus to complete their replication cycle. This process is mediated by cellular proteins that interact first with the reverse transcription complex and later with the preintegration complex (PIC), allowing it to reach and enter the nucleus. For simple retroviruses, such as murine leukemia virus (MLV), the identities of the cellular proteins involved in trafficking of the PIC in infection are unknown. To identify cellular proteins that interact with the MLV PIC, we developed a replication-competent MLV in which the integrase protein was tagged with a FLAG epitope. Using a combination of immunoprecipitation and mass spectrometry, we established that the microtubule motor dynein regulator DCTN2/p50/dynamitin interacts with the MLV preintegration complex early in infection, suggesting a direct interaction between the incoming viral particles and the dynein complex regulators. Further experiments showed that RNA interference (RNAi)-mediated silencing of either DCTN2/p50/dynamitin or another dynein regulator, NudEL, profoundly reduced the efficiency of infection by ecotropic, but not amphotropic, MLV reporters. We propose that the cytoplasmic dynein regulators are a critical component of the host machinery needed for infection by the retroviruses entering the cell via the ecotropic envelope pathway. IMPORTANCE Retroviruses must access the chromatin of host cells to integrate the viral DNA, but before this crucial event, they must reach the nucleus. The movement through the cytoplasm—a crowded environment where diffusion is slow—is thought to utilize retrograde transport along the microtubule network by the dynein complex. Different viruses use different components of this multisubunit complex. We found that the preintegration complex of murine leukemia virus (MLV) interacts with the dynein complex and that regulators of this complex are essential for

Regulatory Elements Associated with Paternally-Expressed Genes in the Imprinted Murine Angelman/Prader-Willi Syndrome Domain

PubMed Central

Khadake, Jyoti; Heggestad, Arnold D.; Ma, Xiaojie; Johnstone, Karen A.; Resnick, James L.; Yang, Thomas P.

2013-01-01

The Angelman/Prader-Willi syndrome (AS/PWS) domain contains at least 8 imprinted genes regulated by a bipartite imprinting center (IC) associated with the SNRPN gene. One component of the IC, the PWS-IC, governs the paternal epigenotype and expression of paternal genes. The mechanisms by which imprinting and expression of paternal genes within the AS/PWS domain – such as MKRN3 and NDN – are regulated by the PWS-IC are unclear. The syntenic region in the mouse is organized and imprinted similarly to the human domain with the murine PWS-IC defined by a 6 kb interval within the Snrpn locus that includes the promoter. To identify regulatory elements that may mediate PWS-IC function, we mapped the location and allele-specificity of DNase I hypersensitive (DH) sites within the PWS-IC in brain cells, then identified transcription factor binding sites within a subset of these DH sites. Six major paternal-specific DH sites were detected in the Snrpn gene, five of which map within the 6 kb PWS-IC. We postulate these five DH sites represent functional components of the murine PWS-IC. Analysis of transcription factor binding within multiple DH sites detected nuclear respiratory factors (NRF's) and YY1 specifically on the paternal allele. NRF's and YY1 were also detected in the paternal promoter region of the murine Mrkn3 and Ndn genes. These results suggest that NRF's and YY1 may facilitate PWS-IC function and coordinately regulate expression of paternal genes. The presence of NRF's also suggests a link between transcriptional regulation within the AS/PWS domain and regulation of respiration. 3C analyses indicated Mkrn3 lies in close proximity to the PWS-IC on the paternal chromosome, evidence that the PWS-IC functions by allele-specific interaction with its distal target genes. This could occur by allele-specific co-localization of the PWS-IC and its target genes to transcription factories containing NRF's and YY1. PMID:23390487

Corn silk induces nitric oxide synthase in murine macrophages.

PubMed

Kim, Kyung A; Choi, Sang Kyu; Choi, Hye Seon

2004-12-31

Corn silk has been purified as an anticoagulant previously and the active component is a polysaccharide with a molecular mass of 135 kDa. It activates murine macrophages to induce nitric oxide synthase (NOS) and generate substantial amounts of NO in time and dose-dependent manners. It was detectable first at 15 h after stimulation by corn silk, peaked at 24 h, and undetectable by 48 h. Induction of NOS is inhibited by pyrolidine dithiocarbamate (PDTC) and genistein, an inhibitor of nuclear factor kappa B (NF-kappaB) and tyrosine kinase, respectively, indicating that iNOS stimulated by corn silk is associated with tyrosine kinase and NF-kappaB signaling pathways. IkappaB-alpha degradation was detectible at 10 min, and the level was restored at 120 min after treatment of corn silk. Corn silk induced nuclear translocation of NF-kappaB by phosphorylation and degradation of IkappaB-alpha.

Angelica acutiloba Kitagawa Extract Attenuates DSS-Induced Murine Colitis

PubMed Central

Jang, Jong-Chan; Lee, Kang Min

2016-01-01

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. PMID:27293323

High hydrostatic pressure processing of murine norovirus 1-contaminated oysters inhibits oral infection in STAT-1 -/- deficient female mice

USDA-ARS?s Scientific Manuscript database

We have previously demonstrated that high pressure processing (HPP) is effective in preventing in vitro replication of murine norovirus strain 1 (MNV-1), a human norovirus surrogate, in a monocyte cell line following extraction from MNV-1-contaminated oysters. In the present study, the efficacy of ...

Family functioning of child-rearing Japanese families on family-accompanied work assignments in Hong Kong.

PubMed

Hohashi, Naohiro; Honda, Junko

2011-11-01

Although the number of employees on overseas assignments accompanied by their families has increased steadily, little is known about the effects of this experience on family functioning. Japanese families on family-accompanied assignments living in Hong Kong were compared with families living in Japan (consisting of 135 and 248 paired partners, respectively). Applying an ecological framework, family functioning was examined using the Feetham Family Functioning Survey-Japanese (FFFS-J). Japanese wives living in Hong Kong rated family functioning lower, particularly in the area of "relationship between family and family members." Between paired marital partners living in Hong Kong, the level of satisfaction in the area of "relationship between family and society" was significantly lower for wives than for husbands. This study provides application of the family ecological framework in families in a multicultural environment and identifies potential areas for family assessment and intervention that may of interest to health care professionals who care for families living away from their home countries.

Beta-catenin regulates vitamin C biosynthesis and cell survival in murine liver.

PubMed

Nejak-Bowen, Kari N; Zeng, Gang; Tan, Xinping; Cieply, Benjamin; Monga, Satdarshan P

2009-10-09

Because the Wnt/beta-catenin pathway plays multiple roles in liver pathobiology, it is critical to identify gene targets that mediate such diverse effects. Here we report a novel role of beta-catenin in controlling ascorbic acid biosynthesis in murine liver through regulation of expression of regucalcin or senescence marker protein 30 and L-gulonolactone oxidase. Reverse transcription-PCR, Western blotting, and immunohistochemistry demonstrate decreased regucalcin expression in beta-catenin-null livers and greater expression in beta-catenin overexpressing transgenic livers, HepG2 hepatoma cells (contain constitutively active beta-catenin), regenerating livers, and in hepatocellular cancer tissues that exhibit beta-catenin activation. Interestingly, coprecipitation and immunofluorescence studies also demonstrate an association of beta-catenin and regucalcin. Luciferase reporter and chromatin immunoprecipitation assays verified a functional TCF-4-binding site located between -163 and -157 (CTTTGCA) on the regucalcin promoter to be critical for regulation by beta-catenin. Significantly lower serum ascorbate levels were observed in beta-catenin knock-out mice secondary to decreased expression of regucalcin and also of L-gulonolactone oxidase, the penultimate and last (also rate-limiting) steps in the synthesis of ascorbic acid, respectively. These mice also show enhanced basal hepatocyte apoptosis. To test if ascorbate deficiency secondary to beta-catenin loss and regucalcin decrease was contributing to apoptosis, beta-catenin-null hepatocytes or regucalcin small interfering RNA-transfected HepG2 cells were cultured, which exhibited significant apoptosis that was alleviated by the addition of ascorbic acid. Thus, through regucalcin and L-gulonolactone oxidase expression, beta-catenin regulates vitamin C biosynthesis in murine liver, which in turn may be one of the mechanisms contributing to the role of beta-catenin in cell survival.

PAR-2 agonists induce contraction of murine small intestine through neurokinin receptors.

PubMed

Zhao, Aiping; Shea-Donohue, Terez

2003-10-01

Protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor and is expressed throughout the gut. It is well known that PAR-2 participates in the regulation of gastrointestinal motility; however, the results are inconsistent. The present study investigated the effect and mechanism of PAR-2 activation on murine small intestinal smooth muscle function in vitro. Both trypsin and PAR-2-activating peptide SLIGRL induced a small relaxation followed by a concentration-dependent contraction. The sensitivity to trypsin was greater than that to SLIGRL (EC50 = 0.03 vs. 40 microM), but maximal responses were similar (12.3 +/- 1.6 vs. 13.7 +/- 1.3 N/cm2). Trypsin-evoked contraction (1 microM) exhibited a rapid desensitization, whereas the desensitization of response to SLIGRL was less even at high concentration (50 microM). Atropine had no effect on PAR-2 agonist-induced contractions. In contrast, TTX and capsaicin significantly attenuated those contractions, implicating a neurogenic mechanism that may involve capsaicin-sensitive sensory nerves. Furthermore, contractions induced by trypsin and SLIGRL were reduced by neurokinin receptor NK1 antagonist SR-140333 or NK2 antagonist SR-48968 alone or were further reduced by combined application of SR-140333 and SR-48968, indicating the involvement of neurokinin receptors. In addition, desensitizing neurokinin receptors with substance P and/or neurokinin A decreased the PAR-2 agonist-evoked contraction. We concluded that PAR-2 agonists induced a contraction of murine intestinal smooth muscle that was mediated by nerves. The excitatory effect is also dependent on sensory neural pathways and requires both NK1 and NK2 receptors.

Acute exercises induce disorders of the gastrointestinal integrity in a murine model.

PubMed

Gutekunst, Katrin; Krüger, Karsten; August, Christian; Diener, Martin; Mooren, Frank-Christoph

2014-03-01

Many endurance athletes complain about gastrointestinal (GI) symptoms. It is assumed that exercise-induced shift of perfusion with consecutive hypoperfusion of the enteral vascular system leads to an increased GI permeability and tissue damage. Therefore, the aim of the study was to investigate permeability, apoptosis, electrogenic ion transport (Isc), and tissue conductance (Gt) of the small intestine in a murine exercise model. After spirometry, male Swiss CD-1 mice were subjected to an intensive treadmill exercise (80% VO2max). Sedentary mice served as controls. The small intestine was removed at several time intervals post-exercise. Apoptotic cells were determined by the TUNEL method, while fluorescein isothiocyanate dextran permeation indicated intestinal permeability. The Gt and Isc measurements were carried out in a modified Ussing chamber. Apoptosis of epithelial cells increased continuously until 24 h post exercise (0.8 ± 0.42 versus 39.2 ± 26.0%; p < 0.05). Compared with the control group the permeability increased 2 h after exercise (0.47 ± 0.07 versus 0.67 ± 0.14 FU/min; p < 0.05). Isc measurements of the ileum were augmented after 24 h (3.33 ± 0.56 versus 5.77 ± 1.16 μEq/h/cm(2); p < 0.05). At this time the Gt increased as well (28.8 ± 3.37 versus 32.5 ± 2.59 mS/cm(2); p < 0.05). In the murine exercise model there is evidence that after intense endurance exercise repair processes occur in small intestinal epithelial cells, which affect permeability, Gt, and Isc. The formation of lamellipodia to close the "leaky" tight junctions caused by apoptosis might be an underlying mechanism.

Immunomodulatory properties of silver nanoparticles contribute to anticancer strategy for murine fibrosarcoma

PubMed Central

Chakraborty, Biswajit; Pal, Ramkrishna; Ali, Mohammed; Singh, Leichombam Mohindro; Shahidur Rahman, Dewan; Kumar Ghosh, Sujit; Sengupta, Mahuya

2016-01-01

The use of nanotechnology in nanoparticle-based cancer therapeutics is gaining impetus due to the unique biophysical properties of nanoparticles at the quantum level. Silver nanoparticles (AgNPs) have been reported as one type of potent therapeutic nanoparticles. The present study is aimed to determine the effect of AgNPs in arresting the growth of a murine fibrosarcoma by a reductive mechanism. Initially, a bioavailability study showed that mouse serum albumin (MSA)-coated AgNPs have enhanced uptake; therefore, toxicity studies of AgNP-MSA at 10 different doses (1–10 mg/kg b.w.) were performed in LACA mice by measuring the complete blood count, lipid profile and histological parameters. The complete blood count, lipid profile and histological parameter results showed that the doses from 2 to 8 mg (IC50: 6.15 mg/kg b.w.) sequentially increased the count of leukocytes, lymphocytes and granulocytes, whereas the 9- and 10-mg doses showed conclusive toxicity. In an antitumor study, the incidence and size of fibrosarcoma were reduced or delayed when murine fibrosarcoma groups were treated by AgNP-MSA. Transmission electron micrographs showed that considerable uptake of AgNP-MSA by the sentinel immune cells associated with tumor tissue and a morphologically buckled structure of the immune cells containing AgNP-MSA. Because the toxicity studies revealed a relationship between AgNPs and immune function, the protumorigenic cytokines TNF-α, IL-6 and IL-1β were also assayed in AgNP-MSA-treated and non-treated fibrosarcoma groups, and these cytokines were found to be downregulated after treatment with AgNP-MSA. PMID:25938978

Families and family therapy in Hong Kong.

PubMed

Tse, Samson; Ng, Roger M K; Tonsing, Kareen N; Ran, Maosheng

2012-04-01

Family therapy views humans not as separate entities, but as embedded in a network of relationships, highlighting the reciprocal influences of one's behaviours on one another. This article gives an overview of family demographics and the implementation of family therapy in Hong Kong. We start with a review of the family demographics in Hong Kong and brief notes on families in mainland China. Demographics show that the landscape has changed markedly in the past decade, with more cross-border marriages, an increased divorce rate, and an ageing overall population - all of which could mean that there is increasing demand for professional family therapy interventions. However, only a limited number of professionals are practising the systems-based approach in Hong Kong. Some possible reasons as to why family therapy is not well disseminated and practised are discussed. These reasons include a lack of mental health policy to support family therapy, a lack of systematic family therapy training, and a shortage of skilled professionals. Furthermore, challenges in applying the western model in Chinese culture are also outlined. We conclude that more future research is warranted to investigate how family therapy can be adapted for Chinese families.

Proteolysis of the Matricellular Protein Hevin by Matrix Metalloproteinase-3 Produces a SPARC-like Fragment (SLF) Associated with Neovasculature in a Murine Glioma Model

PubMed Central

Weaver, Matt; Workman, Gail; Schultz, Chad R.; Lemke, Nancy; Rempel, Sandra A.; Sage, E. Helene

2011-01-01

The matricellular SPARC-family member hevin (Sparc-like 1/SPARCL-1/SC1/Mast9) contributes to neural development and alters tumor progression in a range of mammalian models. Based on sequence similarity, we hypothesized that proteolytic digestion of hevin would result in SPARC-like fragments (SLF) that affect the activity and/or location of these proteins. Incubation of hevin with matrix metalloproteinase-3 (MMP-3), a protease known to cleave SPARC, produced a limited number of peptides. Sequencing revealed the major proteolytic products to be SPARC-like in primary structure. In gliomas implanted into murine brain, a SLF was associated with SPARC in the neovasculature but not with hevin, the latter prominent in the astrocytes encompassed by infiltrating tumor. In this model of invasive glioma that involves MMP-3 activity, host-derived SLF was not observed in the extracellular matrix adjacent to tumor cells. In contrast, it occurred with its homolog SPARC in the angiogenic response to the tumor. We conclude that MMP-3-derived SLF is a marker of neovessels in glioma, where it could influence the activity of SPARC. PMID:21688302

Methodological issues in interviewing families in family nursing research.

PubMed

Astedt-Kurki, P; Paavilainen, E; Lehti, K

2001-07-01

The aim of this study is to discuss what methodological problems can be met in family research with one family member as an interviewee speaking on behalf of the whole family and, vice versa, what is the meaning of having multiple family members or the whole family unit as informants. Family nursing research is part of multidisciplinary research with families. It is a basis for family nursing and contributes to research, especially from the perspective of family welfare and its promotion. Family nursing research generates knowledge concerning families' and family members' wellbeing and experiences and expectations of nursing and health care. The examination of methodological problems while pursuing family research is based on two studies conducted in Finland. Quantitative methods add to the general knowledge of families. Qualitative methods are well suited to the study of family experiences. Family interviews performed for research purposes differ from interviews aiming at caring for families. They aim at obtaining knowledge of families on a general level so as to improve family nursing. Family research has to be looked at as a whole. It faces many challenges such as the definition of the family, gaining access, methods of data collection and data management. A family is a complex system and research with families need flexible, sensitive and practical methods. Family research should also aim at developing new methods for data collection and analysis.

Strengthening Families: Exploring the Impacts of Family Camp Experiences on Family Functioning and Parenting

ERIC Educational Resources Information Center

Garst, Barry A.; Baughman, Sarah; Franz, Nancy K.; Seidel, Richard W.

2013-01-01

Research suggests that family camp experiences can enhance family relationships. Families often participate in family camp experiences for a vacation, as part of a therapeutic and/or intervention strategy, or to gain general enrichment or engagement. To better understand the impacts of family camp experiences on family functioning, a mixed-methods…

Family interactions in adoptive compared to nonadoptive families.

PubMed

Rueter, Martha A; Keyes, Margaret A; Iacono, William G; McGue, Matt

2009-02-01

Despite the large and growing numbers of adoptive families, little research describes interactions in families with adopted adolescents. Yet, adopted adolescents' increased risk for adjustment problems, combined with the association between family interactions and adolescent adjustment in nonadoptive families, raises questions about differences in adoptive and nonadoptive family interactions. We compared observed and self-reported family interactions between 284 adoptive and 208 nonadoptive families and within 123 families with 1 adopted and 1 nonadopted adolescent. Adolescents averaged 14.9 years of age. Comparisons were made using analysis of variance incorporating hierarchical linear methods in SAS PROC MIXED to control family-related correlations in the data. Parents and children reported more conflict in adoptive families when compared with nonadoptive families. Families with 1 adopted and 1 nonadopted adolescent reported more conflict between parents and adopted adolescents. Observed parental behavior was similar across adoptive and nonadoptive children although adopted adolescents were less warm and, in families with 2 adopted children, more conflictual than nonadopted adolescents. These findings suggest a need for further investigation of the association between family interactions and adopted adolescent problem behavior. Copyright 2009 APA, all rights reserved.

Family Interactions in Adoptive Compared to Nonadoptive Families

PubMed Central

Rueter, Martha A.; Keyes, Margaret A.; Iacono, William G.; McGue, Matt

2009-01-01

Despite the large and growing numbers of adoptive families, little research describes interactions in families with adopted adolescents. Yet, adopted adolescents’ increased risk for adjustment problems, combined with the association between family interactions and adolescent adjustment in nonadoptive families, raises questions about differences in adoptive and nonadoptive family interactions. We compared observed and self-reported family interactions between 284 adoptive and 208 nonadoptive families and within 123 families with 1 adopted and 1 nonadopted adolescent. Adolescents averaged 14.9 years of age. Comparisons were made using analysis of variance incorporating hierarchical linear methods in SAS PROC MIXED to control family-related correlations in the data. Parents and children reported more conflict in adoptive families when compared with nonadoptive families. Families with 1 adopted and 1 nonadopted adolescent reported more conflict between parents and adopted adolescents. Observed parental behavior was similar across adoptive and nonadoptive children although adopted adolescents were less warm and, in families with 2 adopted children, more conflictual than nonadopted adolescents. These findings suggest a need for further investigation of the association between family interactions and adopted adolescent problem behavior. PMID:19203160

Editor's Highlight: Hydroxyurea Exposure Activates the P53 Signaling Pathway in Murine Organogenesis-Stage Embryos

PubMed Central

El Husseini, Nazem; Schlisser, Ava E.; Hales, Barbara F.

2016-01-01

Hydroxyurea, an anticancer agent and potent teratogen, induces oxidative stress and activates a DNA damage response pathway in the gestation day (GD) 9 mouse embryo. To delineate the stress response pathways activated by this drug, we investigated the effect of hydroxyurea exposure on the transcriptome of GD 9 embryos. Timed pregnant CD-1 mice were treated with saline or hydroxyurea (400 mg/kg or 600 mg/kg) on GD 9; embryonic gene and protein expression were examined 3 h later. Microarray analysis revealed that the expression of 1346 probe sets changed significantly in embryos exposed to hydroxyurea compared with controls; the P53 signaling pathway was highly affected. In addition, P53 related family members, P63 and P73, were predicted to be activated and had common and unique downstream targets. Western blot analysis revealed that active phospho-P53 was significantly increased in drug-exposed embryos; confocal microscopy showed that the translocation of phospho-P53 to the nucleus was widespread in the embryo. Furthermore, qRT-PCR showed that the expression of P53-regulated genes (Cdkn1A, Fas, and Trp53inp1) was significantly upregulated in hydroxyurea-exposed embryos; the concentration of the redox sensitive P53INP1 protein was also increased in a hydroxyurea dose-dependent fashion. Thus, hydroxyurea elicits a significant effect on the transcriptome of the organogenesis stage murine embryo, activating several key developmental signaling pathways related to DNA damage and oxidative stress. We propose that the P53 pathway plays a central role in the embryonic stress response and the developmental outcome after teratogen exposure. PMID:27208086

Enteric serotonin and oxytocin: endogenous regulation of severity in a murine model of necrotizing enterocolitis.

PubMed

Gross Margolis, Kara; Vittorio, Jennifer; Talavera, Maria; Gluck, Karen; Li, Zhishan; Iuga, Alina; Stevanovic, Korey; Saurman, Virginia; Israelyan, Narek; Welch, Martha G; Gershon, Michael D

2017-11-01

Necrotizing enterocolitis (NEC), a gastrointestinal inflammatory disease of unknown etiology that may also affect the liver, causes a great deal of morbidity and mortality in premature infants. We tested the hypothesis that signaling molecules, which are endogenous to the bowel, regulate the severity of intestinal and hepatic damage in an established murine NEC model. Specifically, we postulated that mucosal serotonin (5-HT), which is proinflammatory, would exacerbate experimental NEC and that oxytocin (OT), which is present in enteric neurons and is anti-inflammatory, would oppose it. Genetic deletion of the 5-HT transporter (SERT), which increases and prolongs effects of 5-HT, was found to increase the severity of systemic manifestations, intestinal inflammation, and associated hepatotoxicity of experimental NEC. In contrast, genetic deletion of tryptophan hydroxylase 1 (TPH1), which is responsible for 5-HT biosynthesis in enterochromaffin (EC) cells of the intestinal mucosa, and TPH inhibition with LP-920540 both decrease the severity of experimental NEC in the small intestine and liver. These observations suggest that 5-HT from EC cells helps to drive the inflammatory damage to the gut and liver that occurs in the murine NEC model. Administration of OT decreased, while the OT receptor antagonist atosiban exacerbated, the intestinal inflammation of experimental NEC. Data from the current investigation are consistent with the tested hypotheses-that the enteric signaling molecules, 5-HT (positively) and OT (negatively) regulate severity of inflammation in a mouse model of NEC. Moreover, we suggest that mucosally restricted inhibition of 5-HT biosynthesis and/or administration of OT may be useful in the treatment of NEC. NEW & NOTEWORTHY Serotonin (5-HT) and oxytocin reciprocally regulate the severity of intestinal inflammation and hepatotoxicity in a murine model of necrotizing enterocolitis (NEC). Selective depletion of mucosal 5-HT through genetic deletion or

mCLCA3 Modulates IL-17 and CXCL-1 Induction and Leukocyte Recruitment in Murine Staphylococcus aureus Pneumonia

PubMed Central

Dietert, Kristina; Reppe, Katrin; Mundhenk, Lars; Witzenrath, Martin; Gruber, Achim D.

2014-01-01

The human hCLCA1 and its murine ortholog mCLCA3 (calcium-activated chloride channel regulators) are exclusively expressed in mucus cells and linked to inflammatory airway diseases with increased mucus production, such as asthma, cystic fibrosis and chronic obstructive pulmonary disease. Both proteins have a known impact on the mucus cell metaplasia trait in these diseases. However, growing evidence points towards an additional role in innate immune responses. In the current study, we analyzed Staphylococcus aureus pneumonia, an established model to study pulmonary innate immunity, in mCLCA3-deficient and wild-type mice, focusing on the cellular and cytokine-driven innate inflammatory response. We compared clinical signs, bacterial clearance, leukocyte immigration and cytokine responses in the bronchoalveolar compartment, as well as pulmonary vascular permeability, histopathology, mucus cell number and mRNA expression levels of selected genes (mClca1 to 7, Muc5ac, Muc5b, Muc2, Cxcl-1, Cxcl-2, Il-17). Deficiency of mCLCA3 resulted in decreased neutrophilic infiltration into the bronchoalveolar space during bacterial infection. Only the cytokines IL-17 and the murine CXCL-8 homolog CXCL-1 were decreased on mRNA and protein levels during bacterial infection in mCLCA3-deficient mice compared to wild-type controls. However, no differences in clinical outcome, histopathology or mucus cell metaplasia were observed. We did not find evidence for regulation of any other CLCA homolog that would putatively compensate for the lack of mCLCA3. In conclusion, mCLCA3 appears to modulate leukocyte response via IL-17 and murine CXCL-8 homologs in acute Staphylococcus aureus pneumonia which is well in line with the proposed function of hCLCA1 as a signaling molecule acting on alveolar macrophages. PMID:25033194

Proteoglycan 4 regulates macrophage function without altering atherosclerotic lesion formation in a murine bone marrow-specific deletion model.

PubMed

Nahon, Joya E; Hoekstra, Menno; Havik, Stefan R; Van Santbrink, Peter J; Dallinga-Thie, Geesje M; Kuivenhoven, Jan-Albert; Geerling, Janine J; Van Eck, Miranda

2018-05-05

Proteoglycan 4 (Prg4) has a high structural similarity with the established atherosclerosis-modulating proteoglycan versican, but its role in atherogenesis is still unknown. Therefore, the impact of Prg4 deficiency on macrophage function in vitro and atherosclerosis susceptibility in vivo was investigated. The presence and localization of Prg4 was studied in atherosclerotic lesions. Furthermore, the effect of Prg4 deficiency on macrophage foam cell formation, cholesterol efflux and lipopolysaccharide (LPS) response was determined. Finally, susceptibility for atherosclerotic lesion formation was investigated in bone marrow-specific Prg4 knockout (KO) mice. Prg4 mRNA expression was induced 91-fold (p<0.001) in murine initial atherosclerotic lesions and Prg4 protein co-localized with human lesional macrophages. Murine Prg4 KO macrophages showed increased foam cell formation (+2.1-fold, p<0.01). In parallel, the expression of the cholesterol efflux genes ATP-binding cassette transporter A1 and scavenger receptor type B1 was lower (-35%, p<0.05;-40%, p<0.05) in Prg4 KO macrophages. This translated into an impaired cholesterol efflux to high-density lipoprotein (-13%, p<0.001) and apolipoprotein A1 (-8%, p<0.05). Furthermore, Prg4 KO macrophages showed an impaired LPS-induced rise in TNFα secretion as compared to wild-type controls (-31%, p<0.001), indicating a reduced inflammatory response. Combined, these pro- and anti-atherogenic effects did not translate into a significant difference in atherosclerotic lesion formation upon bone marrow-specific deletion of Prg4 in low-density lipoprotein receptor KO mice. Prg4 is present in macrophages in both murine and human atherosclerotic lesions and critically influences macrophage function, but deletion of Prg4 in bone marrow-derived cells does not affect atherosclerotic lesion development. Copyright © 2018 Elsevier B.V. All rights reserved.