Journal articles on the topic 'Tissue specific knock out'
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Rupec, Rudolf A., Stephanie Rämisch, Gerd Plewig, Klaus Pfeffer, and Gerald Messer. "Construction of a tissue-specific IκB-α knock-out mouse." Journal of Dermatological Science 16 (March 1998): S133. http://dx.doi.org/10.1016/s0923-1811(98)83792-4.
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Yoon, Donghoon, Bumjun Kim, Myunghi Kwon, and Josef T. Prchal. "Hematopoietic Specific GATA-1-Improved Cre Mouse for Erythroid-Specific Gene Modification." Blood 108, no. 11 (November 16, 2006): 1291. http://dx.doi.org/10.1182/blood.v108.11.1291.1291.
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Abstract Animal models of erythropoiesis related genes have been limited by the fact that some of these genes have non-erythroid expression and other functions in addition to erythropoiesis and thus their knock-out may be embryonic lethal. Tissue specific knock-out or knock-in mice models employing GATA-1-Cre and other constructs showed that these promoters are also active in non-hematopoietic tissues, i.e. GATA-1 has activity in early embryonic development and in neuronal tissue. Suzuki et al (Blood, 2002, 100; 2279) isolated the GATA-1 locus hematopoietic regulatory domain (GATA-1-HRD) and demonstrated that the expression of a transgene under its control is limited to the hematopoietic tissue. We generated a transgenic mouse expressing an improved Cre (iCre) under GATA-1-HRD promoter control. This mouse was crossbred with ROSA 26 mouse and the progeny was examined for tissue specificity of iCre expression using beta-galactosidase staining. Brain, spleen, kidney, heart, thymus, liver, lung and ovary were examined for whole organ LacZ staining. All tested organs were negative except kidney and spleen where some positivity was observed. Subsequently, we prepared tissue sections from kidney, spleen and bone marrow and stained with LacZ and anti-beta-galactosidase antibody. Only the bone marrow EpoR expressing cells were positive; the kidney and the spleen cells were negative. Although Suzuki et al previously showed expression of the GATA-1-HRD driven erythropoietin receptor in spleen using RT-PCR, we were not able to find iCre expression in the splenic cells using these approaches. We demonstrate that our transgenic mouse (GATA-1-HRD-iCre) showed a restricted iCre expression in hematopoietic tissue that differs from previous studies of other hematopoiesis specific cre mouse. We conclude that this mouse model should be useful in studies of function of erythroid specific genes.
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Dubyak, George R. "Knock-Out Mice Reveal Tissue-Specific Roles of P2Y Receptor Subtypes in Different Epithelia." Molecular Pharmacology 63, no. 4 (April 1, 2003): 773–76. http://dx.doi.org/10.1124/mol.63.4.773.
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Li, Mira, Sameer Agnihotri, Mark Wilson, Julie Metcalf, Olivia Singh, Shirin Karimi, Kelly Burrell, et al. "TMOD-02. DIFFUSE GLIOMATOSIS IN MOUSE MODEL OF GFAP TISSUE SPECIFIC KNOCK IN OF EGFRvIII AND KNOCK OUT OF p19 ARF." Neuro-Oncology 20, suppl_6 (November 2018): vi268. http://dx.doi.org/10.1093/neuonc/noy148.1115.
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Garcia-Arcos, Itsaso, Yaeko Hiyama, Konstantinos Drosatos, Kalyani G. Bharadwaj, Yunying Hu, Ni Huiping Son, Sheila M. O'Byrne, et al. "Adipose-specific Lipoprotein Lipase Deficiency More Profoundly Affects Brown than White Fat Biology." Journal of Biological Chemistry 288, no. 20 (March 31, 2013): 14046–58. http://dx.doi.org/10.1074/jbc.m113.469270.
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Adipose fat storage is thought to require uptake of circulating triglyceride (TG)-derived fatty acids via lipoprotein lipase (LpL). To determine how LpL affects the biology of adipose tissue, we created adipose-specific LpL knock-out (ATLO) mice, and we compared them with whole body LpL knock-out mice rescued with muscle LpL expression (MCK/L0) and wild type (WT) mice. ATLO LpL mRNA and activity were reduced, respectively, 75 and 70% in gonadal adipose tissue (GAT), 90 and 80% in subcutaneous tissue, and 84 and 85% in brown adipose tissue (BAT). ATLO mice had increased plasma TG levels associated with reduced chylomicron TG uptake into BAT and lung. ATLO BAT, but not GAT, had altered TG composition. GAT from MCK/L0 was smaller and contained less polyunsaturated fatty acids in TG, although GAT from ATLO was normal unless LpL was overexpressed in muscle. High fat diet feeding led to less adipose in MCK/L0 mice but TG acyl composition in subcutaneous tissue and BAT reverted to that of WT. Therefore, adipocyte LpL in BAT modulates plasma lipoprotein clearance, and the greater metabolic activity of this depot makes its lipid composition more dependent on LpL-mediated uptake. Loss of adipose LpL reduces fat accumulation only if accompanied by greater LpL activity in muscle. These data support the role of LpL as the “gatekeeper” for tissue lipid distribution.
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Shim, Kye Shik. "The Growth and Pubertal Development in Female Mice with Tissue-specific Knock out of Estrogen Receptor." Journal of Korean Society of Pediatric Endocrinology 16, no. 2 (2011): 67. http://dx.doi.org/10.6065/jkspe.2011.16.2.67.
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Chesney, Kari L., Hongsheng Men, Miriam A. Hankins, and Elizabeth C. Bryda. "The Atg16l1 gene: characterization of wild type, knock-in, and knock-out phenotypes in rats." Physiological Genomics 53, no. 6 (June 1, 2021): 269–81. http://dx.doi.org/10.1152/physiolgenomics.00114.2020.
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ATG16L1 is a ubiquitous autophagy gene responsible, in part, for formation of the double-membrane bound autophagosome that delivers unwanted cellular debris and intracellular pathogens to the lysosome for degradation. A single, nonsynonymous adenine to guanine polymorphism resulting in a threonine to alanine amino acid substitution (T300A) directly preceded by a caspase cleavage site (DxxD) causes an increased susceptibility to Crohn’s disease (CD) in humans. The mechanism behind this increased susceptibility is still being elucidated, however, the amino acid change caused by this point mutation results in increased ATG16L1 protein sensitivity to caspase 3-mediated cleavage. To generate novel rat strains carrying genetic alterations in the rat Atg16l1 gene, we first characterized the wild-type rat gene. We identified four alternative splice variants with tissue-specific expression. Using CRISPR-Cas9 genome editing technology, we developed a knock-in rat model for the human ATG16L1 T300A CD risk polymorphism, as well as a knock-out rat model to evaluate the role of Atg16l1 in autophagy as well as its potential effect on CD susceptibility. These are the first reported rat strains with alterations of the Atg16l1 gene. Consistent with studies of the effects of human ATG16L1 polymorphisms, models exhibit morphological abnormalities in both Paneth and goblet cells, but do not develop spontaneous intestinal permeability or inflammatory bowel disease. Analysis of the gut microbiota does not show inherent differences in bacterial composition between wild-type and genetically modified animals. These Atg16l1 strains are valuable new animal models for the study of both autophagy and CD susceptibility.
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Man Law, Ivy Ka, Carl Rankin, and Charalabos Pothoulakis. "14 COLONIC EPITHELIAL CELL-SPECIFIC AFTIPHILIN KNOCKDOWN REGULATES EPITHELIAL BARRIER FUNCTION THROUGH MYOSIN LIGHT CHAIN-ASSOCIATED ACTIN ORGANIZATION IN VITRO AND INTESTINAL LENGTH IN VIVO." Inflammatory Bowel Diseases 26, Supplement_1 (January 2020): S28. http://dx.doi.org/10.1093/ibd/zaa010.067.
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Abstract Background and Aims Colonic epithelial integrity is often compromised during colonic inflammation and Inflammatory Bowel Disease. Aftiphilin (AFTPH) is a downstream target of microRNA-133a and its expression is reduced in colonic tissues of wild type mice from experimental colitis models and colonic biopsies from patients with ulcerative colitis. We have previously shown that AFTPH is involved in regulating intestinal epithelial barrier function and actin organization in human colonic epithelial cells in vitro (DDW 2016). On the other hand, our results suggested that global aftiphilin knock-out is embryonic lethal in mouse models (DDW 2019). Here, we further examined the role of AFTPH in regulating actin organization in vitro and characterize the colonic epithelial cell-specific aftiphilin knock-out mice. Methods Human colonic epithelial NCM460 cells were transfected with si-RNA against AFTPH to achieve transient AFTPH gene-silencing. Stable AFTPH knock-down clones were generated by transducing Caco2-BBE cells with recombinant lentivirus carrying sh-AFTPH or control sh-RNA. To create intestinal epithelial cell-specific aftiphilin knock-out mice, Aftph flox/flox mice were cross-bred with B6.Cg-Tg(Vil1-cre)997Gum/J mice, which express Villin-driven Cre recombinase (Vil-Cre), to generate intestinal epithelial cell-specific aftiphilin knock-out mice (Aftph Vil-/Vil-). Protein expression of F- and G-actin and p70S6K were detected using Western blot. Tissues from various organs were collected with Aftph Vil-/Vil- and its wildtype counterparts at 12 weeks. Results Results from western blot analysis showed that F-/G-actin ratio in AFTPH gene-silenced NCM460 cells were 0.6±0.17 fold, when compared to the treatment control. In addition, AFTPH gene-silencing in human colonic epithelial cells activated p70S6K, a kinase that is involved in actin organization, when compared to treatment control (1.2±0.15 vs. 2.0±0.15, p=0.0354). Furthermore, transepithelial electric resistance (TER) of Caco2-BBE cells deficient in AFTPH is significantly lower than that of control cells (0.5±0.07 fold). Lastly, in vivo intestinal epithelial cell-specific Aftph knock-out increased the length of small intestine, when compared to that of wild type mice (30.7±0.33 vs. 34.8±0.97, p=0.02), while the tissue weight of spleen to body weight was reduced (0.30±0.011 vs. 0.26±0.006, p=0.0169). Summary and Conclusions Our results indicate that AFTPH directly regulates epithelial barrier function and actin organization through mediating F-/G-actin ratio in human colonic epithelial cells, possibly through p70S6K. Importantly, intestinal epithelial cell-specific knock-out in vivo increased intestinal length and reduced size of the spleen. Our results suggested that AFTPH is crucial in regulating colonic epithelial barrier function in vitro and intestinal homeostasis.
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Petrova, Tsvetana, Kyle Bennett, Sambit Nanda, Sam Strickson, Cheryl L. Scudamore, Alan R. Prescott, and Philip Cohen. "Why are the phenotypes of TRAF6 knock-in and TRAF6 knock-out mice so different?" PLOS ONE 17, no. 2 (February 14, 2022): e0263151. http://dx.doi.org/10.1371/journal.pone.0263151.
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The expression of TNF-Receptor Associated Factor 6 (TRAF6) is essential for many physiological processes. Here we studied the phenotype of TRAF6[L74H] knock-in mice which are devoid of TRAF6 E3 ligase activity in every cell of the body, but express normal levels of the TRAF6 protein. Remarkably, TRAF6[L74H] mice have none of the phenotypes seen in TRAF6 KO mice. Instead TRAF6[L74H] mice display an entirely different phenotype, exhibiting autoimmunity, and severe inflammation of the skin and modest inflammation of the liver and lungs. Similar to mice with a Treg-specific knockout of TRAF6, or mice devoid of TRAF6 in all T cells, the CD4+ and CD8+ T cells in the spleen and lymph nodes displayed an activated effector memory phenotype with CD44high/CD62Llow expression on the cell surface. In contrast, T cells from WT mice exhibited the CD44low/CD62Lhigh phenotype characteristic of naïve T cells. The onset of autoimmunity and autoinflammation in TRAF6[L74H] mice (two weeks) was much faster than in mice with a Treg-specific knockout of TRAF6 or lacking TRAF6 expression in all T cells (2–3 months) and we discuss whether this may be caused by secondary inflammation of other tissues. The distinct phenotypes of mice lacking TRAF6 expression in all cells appears to be explained by their inability to signal via TNF Receptor Superfamily members, which does not seem to be impaired significantly in TRAF6[L74H] mice.
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Salveridou, Eva, Steffen Mayerl, Sivaraj Mohana Sundaram, Boyka Markova, and Heike Heuer. "Tissue-Specific Function of Thyroid Hormone Transporters: New Insights from Mouse Models." Experimental and Clinical Endocrinology & Diabetes 128, no. 06/07 (November 13, 2019): 423–27. http://dx.doi.org/10.1055/a-1032-8328.
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AbstractThyroid hormone (TH) transporters are required for cellular transmembrane passage of TH and are thus mandatory for proper TH metabolism and action. Consequently, inactivating mutations in TH transporters such as MCT8 or OATP1C1 can cause tissue- specific changes in TH homeostasis. As the most prominent example, patients with MCT8 mutations exhibit elevated serum T3 levels, whereas their CNS appear to be in a TH deficient state. Here, we will briefly summarize recent studies of mice lacking Mct8 alone or in combination with the TH transporters Mct10 or Oatp1c1 that shed light on many aspects and pathogenic events underlying global MCT8 deficiency and also underscore the contribution of Mct10 and Oatp1c1 in tissue-specific TH transport processes. Moreover, development of conditional knock-out mice that allow a cell-specific inactivation of TH transporters in distinct tissues, disclosed cell-specific changes in TH signaling, thereby highlighting the pathophysiological significance of local control of TH action.
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Li, Gang, Xin Li, Li Yang, Shuyue Wang, Yulin Dai, Baharan Fekry, Lucas Veillon, et al. "Adipose tissue–specific ablation of Ces1d causes metabolic dysregulation in mice." Life Science Alliance 5, no. 8 (April 22, 2022): e202101209. http://dx.doi.org/10.26508/lsa.202101209.
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Carboxylesterase 1d (Ces1d) is a crucial enzyme with a wide range of activities in multiple tissues. It has been reported to localize predominantly in ER. Here, we found that Ces1d levels are significantly increased in obese patients with type 2 diabetes. Intriguingly, a high level of Ces1d translocates onto lipid droplets where it digests the lipids to produce a unique set of fatty acids. We further revealed that adipose tissue–specific Ces1d knock-out (FKO) mice gained more body weight with increased fat mass during a high fat-diet challenge. The FKO mice exhibited impaired glucose and lipid metabolism and developed exacerbated liver steatosis. Mechanistically, deficiency of Ces1d induced abnormally large lipid droplet deposition in the adipocytes, causing ectopic accumulation of triglycerides in other peripheral tissues. Furthermore, loss of Ces1d diminished the circulating free fatty acids serving as signaling molecules to trigger the epigenetic regulations of energy metabolism via lipid-sensing transcriptional factors, such as HNF4α. The metabolic disorders induced an unhealthy microenvironment in the metabolically active tissues, ultimately leading to systemic insulin resistance.
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Hong, G., B. Park, J. Park, S. Kim, and J. Ro. "Transglutaminase 2 Knock-out Protects Against Airway Inflammation And Tissue Remodeling In Ova-specific Allergic Asthma In Mice." Journal of Allergy and Clinical Immunology 129, no. 2 (February 2012): AB53. http://dx.doi.org/10.1016/j.jaci.2011.12.695.
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Kim, Matthew, Richard Howlett, Thomas Moore, Peter Wagner, and Randall Johnson. "533 Phenotypic expression in murine skeletal muscle: The impact of a tissue specific conditional knock-out of HIF-1α." American Journal of Obstetrics and Gynecology 185, no. 6 (December 2001): S226. http://dx.doi.org/10.1016/s0002-9378(01)80565-2.
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Bader, Cameron S., Henry Barreras, Casey O. Lightbourn, Sabrina Copsel, Jeonghyun Ahn, Glen N. Barber, Lei Jin, and Robert B. Levy. "The Innate Immune Sensor Sting Regulates Intestinal Inflammation and GVHD after Allogeneic Hematopoietic Stem Cell Transplantation in Knock-out and Human Allele Knock-in Recipient Mice." Blood 132, Supplement 1 (November 29, 2018): 65. http://dx.doi.org/10.1182/blood-2018-99-109999.
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Abstract Graft-versus-host disease (GVHD) remains a significant cause of morbidity and mortality in patients receiving allogeneic hematopoietic stem cell transplants (allo-HSCTs). Pre-HSCT conditioning typically consists of irradiation and drug administration resulting in the death of rapidly dividing cells. Damage to host tissues initiates a cytokine storm, promoting activation and expansion of donor anti-host alloreactive T cells. Cell death following conditioning has promoted the hypothesis that sensors of cytoplasmic DNA damage in GVHD target tissues contribute to cytokine production. We identified a role for Stimulator of Interferon Genes (STING), an innate immune sensor, in GVHD using pre-clinical MHC-matched (MUD) allo-HSCT models. Our studies show that the STING pathway rapidly regulates cytokine production in the intestinal tract and non-hematopoietic cells can contribute to these responses. Using mice expressing a human STING allele associated with decreased STING activity (Patel S, et al, J Immunol. 2016), we demonstrate its potential clinical importance. To assess STING involvement immediately post-HSCT, cytokine mRNA expression was examined 48 hrs after transplant of C3H.SW bone marrow (BM) + T cells into irradiated B6-WT or STING-/- recipients. Colonic tissue from STING-/- recipients had >2x reduction in IFNβ, TNFα and IL-6 mRNA vs. WT. On day 10 post-transplant, colons from STING-/- recipients exhibited reduced inflammation and overall pathology scores than WT. MHC-matched STING-/- HSCT recipients also experienced decreased weight loss, GVHD scores and skin pathology 6 weeks post-HSCT vs. WT. Chimeric studies demonstrated that the absence of STING in non-hematopoietic cells was responsible for the amelioration of GVHD. Therefore, to test STING signaling in non-hematopoietic intestinal cells, we generated intestinal organoid cultures. Intestinal organoids upregulated IFNβ, TNFα, IL-6 and CXCL10 mRNA 6hrs after stimulation with the highly specific STING agonist DMXAA, supporting the notion that STING in intestinal tissues can contribute to inflammation in vivo. Interestingly, expression of these cytokines returned to baseline levels 24 hrs after stimulation (Fig. 1A). Next, we posited that if the absence of the STING pathway in recipients ameliorated GVHD after MHC-matched HSCT, pathway stimulation would exacerbate GVHD. B6-WT mice were injected with DMXAA immediately prior to HSCT with donor C3H.SW BM + T cells. Administration of a single dose of DMXAA increased expression of IFNβ, TNFα and IL-6 mRNA in colon tissue 48 hrs after transplant (Fig. 1B). Importantly, DMXAA treatment of WT - but not STING-/- - recipients significantly increased GVHD scores and lethality post-HSCT. To evaluate the potential impact of STING in the clinical setting, we evaluated recipients after transplant of C3H.SW BM + T cells into mice homozygous for a human allele associated with diminished STING activity (HAQ-MPYS knock-in mice, termed B6N-STINGHAQ/HAQ here) and found that STINGHAQ/HAQ mice contained a lower frequency of donor T cells expressing an activated phenotype (CD44hiCD62Llo) vs. WT recipients and the former also exhibited diminished GVHD (Fig. 1C,D). In contrast to STING knock-out recipients completely lacking protein, these results indicate that reduced STING activity can also affect GVHD. Interestingly, our findings that STING deficiency ameliorates GVHD in MHC-matched allo-HSCT contrast reported observations that STING activation can exacerbate GVHD after MHC-mismatched HSCT (Fischer J, et al, Sci. Transl. Med. 2017). We are currently investigating how the STING pathway regulates CD4+ and CD8+ T cell mediated GVHD and initial findings may provide insight into understanding the pathway's involvement in MHC-matched vs. mismatched allo-HSCT. In total, our studies demonstrate that STING plays an important role in regulating allo-HSCT and suggest this pathway can provide a target for new therapeutic strategies to ameliorate GVHD. Disclosures Levy: Allergan: Consultancy; Capricor Therapeutics: Consultancy; HEAT Biologics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pelican Therapeutics: Consultancy; OccuRx: Research Funding; Shire: Research Funding.
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Blüher, Matthias, Mary-Elizabeth Patti, Stephane Gesta, Barbara B. Kahn, and C. Ronald Kahn. "Intrinsic Heterogeneity in Adipose Tissue of Fat-specific Insulin Receptor Knock-out Mice Is Associated with Differences in Patterns of Gene Expression." Journal of Biological Chemistry 279, no. 30 (May 6, 2004): 31891–901. http://dx.doi.org/10.1074/jbc.m404569200.
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Brosnihan, K. Bridget, Jeffrey B. Hodgin, Oliver Smithies, Nobuyo Maeda, and Patricia Gallagher. "Tissue-specific regulation of ACE/ACE2 and AT1/AT2receptor gene expression by oestrogen in apolipoprotein E/oestrogen receptor-α knock-out mice." Experimental Physiology 93, no. 5 (February 18, 2008): 658–64. http://dx.doi.org/10.1113/expphysiol.2007.041806.
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Susanna H. M. Sluka, Simon F. Stämpfli, Alexander Akhmedov, Tanja Klein-Rodewald, Adrián Sanz-Moreno, Marion Horsch, Paula Grest, et al. "Murine tissue factor disulfide mutation causes a bleeding phenotype with sex specific organ pathology and lethality." Haematologica 105, no. 10 (September 5, 2019): 2484–95. http://dx.doi.org/10.3324/haematol.2019.218818.
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Tissue factor is highly expressed in sub-endothelial tissue. The extracellular allosteric disulfide bond Cys186-Cys209 of human tissue factor shows high evolutionary conservation and in vitro evidence suggests that it significantly contributes to tissue factor procoagulant activity. To investigate the role of this allosteric disulfide bond in vivo, we generated a C213G mutant tissue factor mouse by replacing Cys213 of the corresponding disulfide Cys190-Cys213 in murine tissue factor. A bleeding phenotype was prominent in homozygous C213G tissue factor mice. Pre-natal lethality of 1/3rd of homozygous offspring was observed between E9.5 and E14.5 associated with placental hemorrhages. After birth, homozygous mice suffered from bleedings in different organs and reduced survival. Homozygous C213G tissue factor male mice showed higher incidence of lung bleedings and lower survival rates than females. In both sexes, C213G mutation evoked a reduced protein expression (about 10-fold) and severely reduced pro-coagulant activity (about 1000-fold). Protein glycosylation was impaired and cell membrane exposure decreased in macrophages in vivo. Single housing of homozygous C213G tissue factor males reduced the occurrence of severe bleeding and significantly improved survival, suggesting that inter-male aggressiveness might significantly account for the sex differences. These experiments show that the tissue factor allosteric disulfide bond is of crucial importance for normal in vivo expression, post-translational processing and activity of murine tissue factor. Although C213G tissue factor mice do not display the severe embryonic lethality of tissue factor knock-out mice, their postnatal bleeding phenotype emphasizes the importance of fully functional tissue factor for hemostasis.
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Zieseniss, Anke, Amke R. Hesse, Aline Jatho, Sabine Krull, Marion Hölscher, Sabine Vogel, and Dörthe M. Katschinski. "Cardiomyocyte-Specific Transgenic Expression of Prolyl-4-Hydroxylase Domain 3 Impairs the Myocardial Response to Ischemia." Cellular Physiology and Biochemistry 36, no. 3 (2015): 843–51. http://dx.doi.org/10.1159/000430260.
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Aims: The prolyl-4-hydroxylase domain (PHD) enzymes are representing novel therapeutic targets for ischemic tissue protection. Whereas the consequences of a knock out of the PHDs have been analyzed in the context of cardioprotection, the implications of PHD overexpression is unknown so far. Methods and Results: We generated cardiomyocyte-specific PHD3transgenic mice (cPhd3tg). Resting cPhd3tg mice did not show constitutive accumulation of HIF-1α or HIF-2α or changes in HIF target gene expression in the heart. Cardiac function was followed up for 14 months in these mice and found to be unchanged. After challenging the cPhd3tg mice with ligation of the left anterior descending artery, HIF-1α/-2α accumulation in the left ventricles was blunted. This was associated with a significantly increased infarct size of the cPhd3tg compared to wild type mice. Conclusion: Whereas overexpression of PHD3 in the resting state does not significantly influence cardiac function, it is crucial for the cardiac response to ischemia by affecting HIFα accumulation in the ischemic tissue.
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den Boer, B. G., S. Sookhareea, P. Dufourcq, and M. Labouesse. "A tissue-specific knock-out strategy reveals that lin-26 is required for the formation of the somatic gonad epithelium in Caenorhabditis elegans." Development 125, no. 16 (August 15, 1998): 3213–24. http://dx.doi.org/10.1242/dev.125.16.3213.
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The Caenorhabditis elegans LIN-26 protein is required to specify and/or maintain the fates of all non-neuronal ectodermal cells. Here we show that lin-26 is expressed until the somatic gonad primordium stage in all cells of the somatic gonad, except in distal tip cells, and later in all uterine cells. To determine if lin-26 functions in the somatic gonad, we have generated gonad-specific lin-26 alleles obtained by integration of lin-26 promoter deletion derivatives into a lin-26 null mutant background. In this way, we rescued the lethal phenotype imparted by lin-26 null mutations and uncovered a highly penetrant sterile phenotype. Specifically, the strongest of these new alleles was characterized by the absence of lin-26 expression in the somatic gonad, the presence of endomitotic oocytes, decreased germline proliferation, a protruding vulva and a less penetrant absence of gonad arms. Lineage analysis of mutant somatic gonads and examination of several markers expressed in the spermatheca, sheath cells, distal tip cells and the uterus, suggest that LIN-26 is required in sheath, spermatheca and uterine precursors, and in uterine cells. We conclude that lin-26 performs a similar function in the non-neuronal ectoderm and the somatic gonad, a mesoderm derivative, and we speculate that lin-26 is required to express epithelial characteristics.
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Choi, Wonsuk, Joon Ho Moon, and Hail Kim. "Serotonergic regulation of energy metabolism in peripheral tissues." Journal of Endocrinology 245, no. 1 (April 2020): R1—R10. http://dx.doi.org/10.1530/joe-19-0546.
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Serotonin is a biogenic amine synthesized from the essential amino acid tryptophan. Because serotonin cannot cross the blood-brain barrier, it functions differently in neuronal and non-neuronal tissues. In the CNS, serotonin regulates mood, behavior, appetite, and energy expenditure. Although most serotonin in the body is synthesized at the periphery, its biological roles have not been well elucidated. Older studies using chemical agonists and antagonists yielded conflicting results, because the complexity of serotonin receptors and the low selectivity of agonists and antagonists were not known. Several recent studies using specific knock-out of serotonin receptors have been performed to assess the role of peripheral serotonin in regulating energy metabolism. This review discusses (1) the tissue-specific roles of peripheral serotonin in regulating energy metabolism, (2) the mechanism by which dysfunctional peripheral serotonin signaling can progress to metabolic diseases, and (3) how peripheral serotonin signaling could be a therapeutic target for metabolic diseases.
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Copland, Ian B., Jessica Cuerquis, Eugenia Wang, and Jacques Galipeau. "Genomic, Proteomic and Functional Screens Identify PAI-1 as an Important Factor Influencing Mesenchymal Stromal Cell Survival Post-Transplantation In Vivo." Blood 110, no. 11 (November 16, 2007): 180. http://dx.doi.org/10.1182/blood.v110.11.180.180.
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Abstract MSCs have robust reparative properties through their ability to limit apoptosis, enhance angiogenesis and direct positive tissue remodelling. However, a major limitation on the widespread application of MSC transplantation is that transplanted cells have low survival rates in vivo. Thus, enhancing MSCs survival post-transplantation is critical for the successful implementation of cellular therapy. One explanation for their low survival is that MSCs are often transplanted into ischemic tissue - such as infarcted myocardium - where there is poor blood supply and low oxygen tension. How these factors influence MSC survival is still unclear, therefore the objective of this project was to better understand how hypoxia under low nutrient conditions affects the behaviour and survival of MSCs. To answer this question, we performed the following series of experiments: Isolate and immunophenotype murine MSCs. Assess impact of in vitro ischemic conditions (hypoxia and serum deprivation) on MSCs by microarray analysis and ITRAQ proteomics for secreted proteins. Confirm changes of specific factors in vitro. Isolate and immunophenotype MSCs from knock-out mice for specific factors. Determine whether knock-out MSCs have altered survival in vitro and in vivo. As confirmed by PCR and Western blotting, microarray and proteomic screens, identified plasminogen activator inhibitor-1 (PAI-1) as one factor consistently upregulated in hypoxia (3% oxygen) treated MSCs. In vitro migration studies demonstrated that PAI-1 blocks migration of MSCs towards a chemotactic gradient, while isolation of PAI-1 knock-out MSCs, revealed an enhanced survival of PAI-1null MSCs over wild-type MSCs. In vivo subcutaneous transplantation of beta-galactosidase engineered wild-type and PAI-1null MSCs, recapitulated our in vitro results, such that 14 days post-implantation PAI-1null MSCs had a five fold increase in survival compared to wild-type MSCs. In conclusion, PAI-1 is a multifunctional protein. As an inhibitor, PAI-1 acts as ‘bait’ regulating the conversion of plasminogen to plasmin thus functioning as a major control point in the regulation of systemic fibrinolysis and local growth factor availability. In addition, PAI-1 can also act as a ligand for vitronectin to influence cellular migration/adhesion. Up-regulation of PAI-1 by MSCs after transplantation impacts MSC survival. Determining how to block PAI-1 following transplantation of MSCs is currently under investigation.
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Kaut, Oliver, Ina Schmitt, Fabian Stahl, Holger Fröhlich, Per Hoffmann, Frank J. Gonzalez, and Ullrich Wüllner. "Epigenome-Wide Analysis of DNA Methylation in Parkinson’s Disease Cortex." Life 12, no. 4 (March 29, 2022): 502. http://dx.doi.org/10.3390/life12040502.
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Background: Epigenetic factors including DNA methylation contribute to specific patterns of gene expression. Gene–environment interactions can change the methylation status in the brain, and accumulation of these epigenetic changes over a lifespan may be co-responsible for a neurodegenerative disease like Parkinson’s disease, which that is characterised by a late onset in life. Aims: To determine epigenetic modifications in the brains of Parkinson’s disease patients. Patients and Methods: DNA methylation patterns were compared in the cortex tissue of 14 male PD patients and 10 male healthy individuals using the Illumina Methylation 450 K chip. Subsequently, DNA methylation of candidate genes was evaluated using bisulphite pyrosequencing, and DNA methylation of cytochrome P450 2E1 (CYP2E1) was characterized in DNA from blood mononuclear cells (259 PD patients and 182 healthy controls) and skin fibroblasts (10 PD patients and 5 healthy controls). Protein levels of CYP2E1 were analysed using Western blot in human cortex and knock-out mice brain samples. Results: We found 35 hypomethylated and 22 hypermethylated genes with a methylation M-value difference >0.5. Decreased methylation of cytochrome P450 2E1 (CYP2E1) was associated with increased protein levels in PD brains, but in peripheral tissues, i.e., in blood cells and skin fibroblasts, DNA methylation of CYP2E1 was unchanged. In CYP2E1 knock-out mice brain alpha-synuclein (SNCA) protein levels were down-regulated compared to wild-type mice, whereas treatment with trichloroethylene (TCE) up-regulated CYP2E1 protein in a dose-dependent manner in cultured cells. We further identified an interconnected group of genes associated with oxidative stress, such as Methionine sulfoxide reductase A (MSRA) and tumour protein 73 (TP73) in the brain, which again were not paralleled in other tissues and appeared to indicate brain-specific changes. Conclusions: Our study revealed surprisingly few dysmethylated genes in a brain region less affected in PD. We confirmed hypomethylation of CYP2E1.
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Takaluoma, Kati, Marjo Hyry, Juha Lantto, Raija Sormunen, Ruud A. Bank, Kari I. Kivirikko, Johanna Myllyharju, and Raija Soininen. "Tissue-specific Changes in the Hydroxylysine Content and Cross-links of Collagens and Alterations in Fibril Morphology in Lysyl Hydroxylase 1 Knock-out Mice." Journal of Biological Chemistry 282, no. 9 (December 29, 2006): 6588–96. http://dx.doi.org/10.1074/jbc.m608830200.
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Lin, Feng, Yoshihiro Fukuoka, Andrew Spicer, Rieko Ohta, Noriko Okada, Claire L. Harris, Steven N. Emancipator, and M. Edward Medof. "Tissue distribution of products of the mouse decay-accelerating factor (DAF) genes. Exploitation of a Daf1 knock-out mouse and site-specific monoclonal antibodies." Immunology 104, no. 2 (October 2001): 215–25. http://dx.doi.org/10.1046/j.1365-2567.2001.01287.x.
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Gazit, Roi, Pankaj K. Mandal, Wataru Ebina, Ayal Ben-Zvi, César Nombela-Arrieta, Leslie E. Silberstein, and Derrick J. Rossi. "Fgd5 identifies hematopoietic stem cells in the murine bone marrow." Journal of Experimental Medicine 211, no. 7 (June 23, 2014): 1315–31. http://dx.doi.org/10.1084/jem.20130428.
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Hematopoietic stem cells (HSCs) are the best-characterized tissue-specific stem cells, yet experimental study of HSCs remains challenging, as they are exceedingly rare and methods to purify them are cumbersome. Moreover, genetic tools for specifically investigating HSC biology are lacking. To address this we sought to identify genes uniquely expressed in HSCs within the hematopoietic system and to develop a reporter strain that specifically labels them. Using microarray profiling we identified several genes with HSC-restricted expression. Generation of mice with targeted reporter knock-in/knock-out alleles of one such gene, Fgd5, revealed that though Fgd5 was required for embryonic development, it was not required for definitive hematopoiesis or HSC function. Fgd5 reporter expression near exclusively labeled cells that expressed markers consistent with HSCs. Bone marrow cells isolated based solely on Fgd5 reporter signal showed potent HSC activity that was comparable to stringently purified HSCs. The labeled fraction of the Fgd5 reporter mice contained all HSC activity, and HSC-specific labeling was retained after transplantation. Derivation of next generation mice bearing an Fgd5-CreERT2 allele allowed tamoxifen-inducible deletion of a conditional allele specifically in HSCs. In summary, reporter expression from the Fgd5 locus permits identification and purification of HSCs based on single-color fluorescence.
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Virtanen, Mari A., Pavel Uvarov, Christian A. Hübner, and Kai Kaila. "NKCC1, an Elusive Molecular Target in Brain Development: Making Sense of the Existing Data." Cells 9, no. 12 (December 4, 2020): 2607. http://dx.doi.org/10.3390/cells9122607.
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Ionotropic GABA transmission is mediated by anion (mainly Cl−)-permeable GABAA receptors (GABAARs). In immature neurons, GABA exerts depolarizing and sometimes functionally excitatory actions, based on active uptake of Cl− by the Na-K-2Cl cotransporter NKCC1. While functional evidence firmly shows NKCC1-mediated ion transport in immature and diseased neurons, molecular detection of NKCC1 in the brain has turned out to be extremely difficult. In this review, we describe the highly inconsistent data that are available on the cell type-specific expression patterns of the NKCC1 mRNA and protein in the CNS. We discuss the major technical caveats, including a lack of knock-out-controlled immunohistochemistry in the forebrain, possible effects of alternative splicing on the binding of antibodies and RNA probes, and the wide expression of NKCC1 in different cell types, which make whole-tissue analyses of NKCC1 useless for studying its neuronal expression. We also review novel single-cell RNAseq data showing that most of the NKCC1 in the adult CNS may, in fact, be expressed in non-neuronal cells, especially in glia. As future directions, we suggest single-cell NKCC1 mRNA and protein analyses and the use of genetically tagged endogenous proteins or systematically designed novel antibodies, together with proper knock-out controls, for the visualization of endogenous NKCC1 in distinct brain cell types and their subcellular compartments.
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Hui, Xiaoyan Hannah, and Tianshi Feng. "Adipocyte-Derived Lactate Potentiates Obesity-Evoked Adipose Macrophage Inflammation." Journal of the Endocrine Society 5, Supplement_1 (May 1, 2021): A40—A41. http://dx.doi.org/10.1210/jendso/bvab048.079.
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Abstract Introduction: Obesity is characterized by mobilization of macrophage inflammation, which represents the major events of obesity-associated adipose tissue inflammation. . On the other hand, lactate accumulation in adipose tissue long been observed. However, whether elevation of lactate plays an essential role in adipose inflammation is not known. In this study, we sought to examine the intermediary role of lactate in macrophage polarization and adipose inflammation upon obesity. Method: Lactate level and activity of lactate dehydrogense (LDH), the key enzyme of lactate production, were measured by biochemical assays. Adipocyte- and macrophage- specific Ldha knock out mice were constructed by cre-LoxP system to study the physiological role of lactate in diet induced obesity. Macrophage polarization and inflammation were examined by western blotting and Q-PCR. Results: Lactate and LDH activity were selectively upregulated in adipose tissues of obese mice. Adipocyte-, but not macrophage-selective deletion of LDHA, led to a significant improvement of adipose inflammation and metabolic dysfunctions. In vitro experiments showed that the lactate promoted M1 polarization through direct interation and inhibition of the PHD2, which subsequently stabilizes HIF-1alpha. In addition, a positive correlation between adipose lactate level and adipose tissue inflammation was found in obese patients. Conclusion: In obese condition, increased production of lactate from adipocytes enhances adipose tissue inflammation by promoting the proinflammatory polarization of adipose macrophages.
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Lensch, M. William, and George Q. Daley. "Scientific and clinical opportunities for modeling blood disorders with embryonic stem cells." Blood 107, no. 7 (April 1, 2006): 2605–12. http://dx.doi.org/10.1182/blood-2005-07-2991.
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AbstractOur considerable wealth of data concerning hematologic processes has come despite difficulties working with stem and progenitor cells in vitro and their propensity to differentiate. Key methodologies that have sought to overcome such limitations include transgenic/knock-out animals and in vitro studies using murine embryonic stem cells, because both permit investigation of the formation of hematopoietic tissue from nonhematopoietic precursors. Although there have been many successful studies in model animals for understanding hematopoietic-cell development, differences between lower vertebrates and humans have left gaps in our understanding. Clearly, human-specific strategies to study the onset of hematopoiesis, particularly the earliest events leading to the specification of both normal and abnormal hematopoietic tissue, could bring an investigational renaissance. The recent availability of human embryonic stem (hES) cells suggests that such a system is now at hand. This review highlights the potential of hES cells to model human hematologic processes in vitro with an emphasis on disease targets.
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Sabo, Peter, Vahagn Makaryan, Tanoya Poulsen, Lital Povodovski, Yosef Dicken, Asael Herman, Rafi Emmanuel, and David C. Dale. "CRISPR Mediated ELANE Single-Allele Knock-out Restores Proliferation and Myeloid Differentiation of Neutropenia Patient Derived BM HSCs." Blood 136, Supplement 1 (November 5, 2020): 23. http://dx.doi.org/10.1182/blood-2020-137215.
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Background: Mutations in ELANE are a cause of cyclic and severe congenital neutropenia (SCN), predominantly autosomal dominant disorders. ELANE encodes neutrophil elastase (NE), a tissue specific serine protease. Neutropenia occurs because mutant NE impairs survival and maturation of myeloid cells. More than 130 disease causing mutations in ELANE have been identified and cell permeable inhibitors of NE can correct the defect in cell survival and maturation in cellular models. (Makaryan et al. J Leukoc Biol. 2017;102:1143). CRISPR/Cas9 knock-out (KO) of ELANE in patients' hematopoietic stem cells (HSCs) corrects myeloid cell differentiation, (Nasri et al Haematologica 2020:105:598), but this approach targets both alleles, eliminates normal NE and may impair innate immunity. Hypothesis: Selective, single allele editing is a preferred strategy for correcting ELANE associated neutropenia and other autosomal dominant disorders. Methods: Emendo Biotherapeutics developed CRISPR-associated allele specific KO based on single nucleotide polymorphisms (SNPs) located in the vicinity of ELANE. Three SNPs cover ~80% of the population. In the current study, editing of the mutated allele was achieved by targeting SNP rs1683564 located downstream to the 3'UTR of ELANE and mediating a biallelic break in intron 4, with excision of the 3'UTR, destabilized the mutated allele transcript. For editing we utilized RNPs assembled from Emendo's proprietary nuclease, OMNI-50, a guide targeting the SNP and a guide targeting a region in intron 4. OMNI-50 has low off-target activity and high allele specific editing when targeting either the reference or the alternative form of the SNPs. The efficacy of this composition was tested on normal and SCN patient-derived HSCs harboring the relevant SNP. (Figure 1) A patient's bone marrow CD34+ cells with the S126L mutation and rs1683564 were enriched using RosetteSep™ Progenitor Cell Enrichment Cocktail (Stemcell Technologies), purified by density gradient centrifugation using Lymphoprep (Stemcell Technologies). The purified CD34+ cells were expanded 4 days in StemSpan™ SFEM II media, supplemented with StemSpan™ CD34+ Expansion Supplement. Expanded CD34+ cells were further purified using StemSep™ Human CD34 Positive Cocktail and purity verified by FACS analysis using CD34 and CD45 antibodies. Purified CD34+ cells were cryopreserved using serum free CryoStor® CS10 media (Stemcell Technologies) and stored in Liquid Nitrogen Vapor Phase. Normal human blood CD34+ progenitor cells, harboring rs1683564 were similarly treated as the control. Allele specific excision was determined by ddPCR. To determine excision efficiency for the patient and control, we amplified two regions in ELANE, Exon 1 and Exon 5 (excised region), using two different probes labeled with FAM and HEX, respectively. We used OMNI-50 nuclease (Emendo Bio, Ness Ziona, Israel), electroporation (Lonza 4D nucleofector) and StemCell media (Stemcell Technologies, Vancouver, BC) to modify, grow and differentiate the CD34+ cells. We assessed myeloid cell proliferation and differentiation using daily cell counts, cytospins stained with Diff-Quik, and CD14, CD66b, CD15, CD16, CD11b labeling and flow cytometry. Results: Unedited patient cells demonstrated significant abnormalities in proliferation and differentiation. Cytospins from unedited patient CD34+ cells at 14 days showed block of myeloid differentiation and 4-fold greater monocytes compared to control, consistent with the hematopoietic defect in SCN patients. Single allele ELANE KO significantly improved these cellular abnormalities: total cell proliferation increased 41% and CD14/CD66b and CD15/CD11b positive cells increased by 107% and 65.5%, respectively. Studies are in progress for additional patients harboring different ELANE mutations. Conclusions: Single allele KO may resolve concerns about unwanted effects of total elimination of the normal gene products with gene editing for autosomal dominant diseases and be an effective strategy for treating ELANE associate neutropenia. Disclosures Sabo: Emendo BioTherapeutics: Research Funding. Makaryan:Emendo BioTherapeutics: Research Funding. Poulsen:Emendo BioTherapeutics: Research Funding. Povodovski:Emendo BioTherapeutics: Current Employment, Current equity holder in private company. Dicken:Emendo BioTherapeutics: Current Employment, Current equity holder in private company. Herman:Emendo BioTherapeutics: Current Employment, Current equity holder in private company. Emmanuel:Emendo BioTherapeutics: Current Employment, Current equity holder in private company. Dale:X4 Pharmaceuticals: Research Funding; Emendo BioTherapeutics: Consultancy; X4 Pharmaceuticals: Honoraria.
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Ghigo, Clement, Aude de Gassard, Patrick Brune, Caroline Imbert, Clemence Demerle, ROUVIERE Marie-Sarah, René Hoet, Daniel Olive, and Emmanuel Valentin. "3 Butyrophilin-3a is expressed in multiple solid tumors: translational research supporting the EVICTION study with ICT01, an anti-BTN3A mAb activating Vg9Vd2 T-Cells." Journal for ImmunoTherapy of Cancer 8, Suppl 3 (November 2020): A3. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0003.
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BackgroundButyrophillin-3A (BTN3A) three isoforms (3A1/3A2/3A3) are widely expressed on a variety of tumors.1 BTN3A1 plays a key role in phosphoantigen activation of Vg9Vd2 T-cells, key mediators of innate and adaptive anti-tumor immunity.2 Vg9Vd2 T-cell infiltration into tumor tissues is associated with a positive prognosis across multiple cancers,3 which makes BTN3A an interesting target for enhancing anti-tumor immunity. ImCheck Therapeutics is developing ICT01, an anti-BTN3A mAb that specifically activates Vg9Vd2 T-cells. ICT01 is currently in an international, multi-center Phase 1/2a clinical trial (NCT04243499, EVICTION Study). The level of BTN3A expression required for a clinical response to ICT01 is not known. Therefore, we developed novel immunohistochemistry (IHC) methods to enable a precision-medicine based approach to target population selection for dose escalation and potentially guiding patient selection in the expansion cohorts of the ongoing EVICTION study.MethodsA panBTN3A IHC staining that recognizes the three isoforms was developed on Fresh frozen (FF) tissues, while BTN3A2- and BTN3A3-specific IHC methods were developed on formalin-fixed paraffin embedded (FFPE) tissues. BTN3A1-specific staining is still under development. Transfected knock-out/knock-in cell lines and positive tissues were used to assess antibody specificity. BTN3A expression was then analyzed on both normal and associated tumor tissue using tissue microarrays (TMA) and selected frozen blocks from tumor biopsies. FACS analyses were also performed on dissociated lung and pancreatic cancer biopsies to determine BTN3A (3 isoforms) membrane expression on tumor-infiltrating immune cells and cancer/stromal cells.ResultsIn normal tissues, BTN3A2 and BTN3A3 specific IHC signals were granular cytoplasmic in epithelial cells, with positive mononuclear and endothelial cells. Higher expression in lung, colon, and small intestine tissues was observed. Regarding panBTN3A expression, inter-indication and inter-patient heterogeneity was observed among head and neck, lung, melanoma, bladder, colon, pancreas, breast, and prostate cancer tissues, with both cytoplasmic and membranous localizations. The major finding was higher expression of BTN3A2 on malignant cells in melanoma, lung, colon, and prostate cancers, as compared to normal tissue. Finally, FACS analyses of lung and pancreatic cancer tissues revealed stronger expression of all BTN3A isoforms at the cell surface of infiltrated immune cells compared to its expression on stromal cells.ConclusionsThese validated IHC methods supported the selection of cancer indications for the EVICTION trial and will potentially help identify specific tumor subtypes and patients that will most likely benefit from ICT01 treatment.ReferencesCompte E, Pontarotti P, Collette Y, Lopez M, Olive D. Frontline: characterization of BT3 molecules belonging to the B7 family expressed on immune cells. Eur J Immunol 2004;34:2089–99.Juan-Luis Blazquez, Audrey Benyamine, Christine Pasero, and Daniel Olive. New Insights into the Regulation of ?d T Cells by BTN3A and Other BTN/BTNL in Tumor Immunity. Front Immunol 2018;9:1601.Andrew J. Gentles, Aaron M. Newman, Chih Long Liu, Scott V. Bratman, Weiguo Feng, Dongkyoon Kim, Viswam S. Nair, Yue Xu, Amanda Khuong, Chuong D. Hoang, Maximilian Diehn, Robert B. West, Sylvia K. Plevritis, Ash A. Alizadeh. The prognostic landscape of genes and infiltrating immune cells across human cancers. Nat Med 2015 Aug; 21(8):938–945.
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Gifre-Renom, Laia, Estefania Ugarte-Berzal, Erik Martens, Lise Boon, Olivia Cano-Garrido, Esther Martínez-Núñez, Teresa Luque, et al. "Recombinant Protein-Based Nanoparticles: Elucidating Their Inflammatory Effects In Vivo and Their Potential as a New Therapeutic Format." Pharmaceutics 12, no. 5 (May 13, 2020): 450. http://dx.doi.org/10.3390/pharmaceutics12050450.
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Bacterial inclusion bodies (IBs) are protein-based nanoparticles of a few hundred nanometers formed during recombinant protein production processes in different bacterial hosts. IBs contain active protein in a mechanically stable nanostructured format that has been broadly characterized, showing promising potential in different fields such as tissue engineering, protein replacement therapies, cancer, and biotechnology. For immunomodulatory purposes, however, the interference of the format immunogenic properties—intrinsic to IBs—with the specific effects of the therapeutic protein is still an uncovered gap. For that, active and inactive forms of the catalytic domain of a matrix metalloproteinase-9 (MMP-9 and mutMMP-9, respectively) have been produced as IBs and compared with the soluble form for dermal inflammatory effects in mmp9 knock-out mice. After protein injections in air-pouches in the mouse model, MMP-9 IBs induce local neutrophil recruitment and increase pro-inflammatory chemokine levels, lasting for at least two days, whereas the effects triggered by the soluble MMP-9 format fade out after 3 h. Interestingly, the IB intrinsic effects (mutMMP-9 IBs) do not last more than 24 h. Therefore, it may be concluded that IBs could be used for the delivery of therapeutic proteins, such as immunomodulating proteins while preserving their stability in the specific tissue and without triggering important unspecific inflammatory responses due to the protein format.
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Laoharawee, Kanut, Matthew J. Johnson, Walker S. Lahr, Christopher J. Sipe, Evan Kleinboehl, Joseph J. Peterson, Cara-lin Lonetree, et al. "A Pan-RNase Inhibitor Enabling CRISPR-mRNA Platforms for Engineering of Primary Human Monocytes." International Journal of Molecular Sciences 23, no. 17 (August 28, 2022): 9749. http://dx.doi.org/10.3390/ijms23179749.
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Monocytes and their downstream effectors are critical components of the innate immune system. Monocytes are equipped with chemokine receptors, allowing them to migrate to various tissues, where they can differentiate into macrophage and dendritic cell subsets and participate in tissue homeostasis, infection, autoimmune disease, and cancer. Enabling genome engineering in monocytes and their effector cells will facilitate a myriad of applications for basic and translational research. Here, we demonstrate that CRISPR-Cas9 RNPs can be used for efficient gene knockout in primary human monocytes. In addition, we demonstrate that intracellular RNases are likely responsible for poor and heterogenous mRNA expression as incorporation of pan-RNase inhibitor allows efficient genome engineering following mRNA-based delivery of Cas9 and base editor enzymes. Moreover, we demonstrate that CRISPR-Cas9 combined with an rAAV vector DNA donor template mediates site-specific insertion and expression of a transgene in primary human monocytes. Finally, we demonstrate that SIRPa knock-out monocyte-derived macrophages have enhanced activity against cancer cells, highlighting the potential for application in cellular immunotherapies.
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Melicoff, Ernestina, Shakeel M. Thakurdas, Tanya Siddiqi, Jayasimha Murthy, Leticia Sansores-Garcia, and Roberto Adachi. "Mast Cell-Specific Gene Targeting." Blood 106, no. 11 (November 16, 2005): 3876. http://dx.doi.org/10.1182/blood.v106.11.3876.3876.
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Abstract For years mast cell (MC) studies have relied on MC lines or bone marrow-derived MC. Regular knockout mice have some disadvantages such as early lethality and developmental defects. Also, the phenotype in MCs from these animals could not be a primary defect but secondary to other cell lines affected by the mutation. The Cre/loxP system allows us to target genetic deletions to specific tissues or cells. Cre, a recombinase from bacteriophage P1, recognizes a DNA sequence motif of 34 bases (loxP). If a DNA segment is flanked by two loxP sites in the same orientation, Cre excises that segment activating or silencing a gene. Many genes have been “floxed” (flanked by two loxP sites), but no MC-specific Cre mouse has been created. We are using promoter regions of MC-specific proteins to drive expression of Cre-recombinase. We produced transgenic mice using the promoter for human alpha-subunit and murine beta-subunit of the high-affinity receptor for IgE (hFcεRIα and mFcεRIβ), both expressed in MCs and basophils. We also used the promoter of the murine mast cell protease mMCP-5, a member of a family of cargo proteins expressed only in MCs. For even more controlled expression, we knocked-in Cre cDNA into the mMCP-5, mMCP-6 and Ras guanine releasing protein 4 (RasGRP4) loci, all of them proteins expressed exclusively in MCs, while knocking-out the respective gene. Heterozygotes will express Cre only in MCs, and homozygotes for mMCP-5, mMCP-6 or RasGRP4 deletions will be regular knockouts that will allow us to study the role of these proteins in mast cells. These three transgenic and three knock-out/knock-in lines have been created and are being crossed with reporter mice. We decided to use a double reporter mouse, the Bgeo/GFP (Jackson Laboratory). This mouse has “floxed” lacZ that is constitutively expressed under the control of the CMV enhancer/chicken actin promoter, and when crossed with our Cre recombinase-expressing mouse, lacZ is excised and enhanced green fluorescent protein (EGFP) is expressed in cells expressing Cre. EGFP can be easily detected in MCs from peritoneal lavage by flow-cytometry, using antibodies against c-kit (CD117) and FcεRI to label the MCs. Results of this screening will be presented at the meeting. After documenting the presence of functional Cre-recombinase in our reporter mice, we will cross them with our “floxed” mice to obtain conditional mast cell-specific knockouts.
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Zhang, Yujian, Defeng Tian, Hironori Matsuyama, Takashi Hamazaki, Takayuki Shiratsuchi, Naohiro Terada, Derek J. Hook, Michael A. Walters, Gunda I. Georg, and Jon E. Hawkinson. "Human Adenine Nucleotide Translocase (ANT) Modulators Identified by High-Throughput Screening of Transgenic Yeast." Journal of Biomolecular Screening 21, no. 4 (January 8, 2016): 381–90. http://dx.doi.org/10.1177/1087057115624637.
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Transport of ADP and ATP across mitochondria is one of the primary points of regulation to maintain cellular energy homeostasis. This process is mainly mediated by adenine nucleotide translocase (ANT) located on the mitochondrial inner membrane. There are four human ANT isoforms, each having a unique tissue-specific expression pattern and biological function, highlighting their potential as drug targets for diverse clinical indications, including male contraception and cancer. In this study, we present a novel yeast-based high-throughput screening (HTS) strategy to identify compounds inhibiting the function of ANT. Yeast strains generated by deletion of endogenous proteins with ANT activity followed by insertion of individual human ANT isoforms are sensitive to cell-permeable ANT inhibitors, which reduce proliferation. Screening hits identified in the yeast proliferation assay were characterized in ADP/ATP exchange assays employing recombinant ANT isoforms expressed in isolated yeast mitochondria and Lactococcus lactis as well as by oxygen consumption rate in mammalian cells. Using this approach, closantel and CD437 were identified as broad-spectrum ANT inhibitors, whereas leelamine was found to be a modulator of ANT function. This yeast “knock-out/knock-in” screening strategy is applicable to a broad range of essential molecular targets that are required for yeast survival.
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Bradley, Madelyn, Joseph Arballo, Molly Black, John Erdman, and Jaume Amengual. "Tissue Lycopene Accumulation in Transgenic Mice Lacking One or Both Carotenoid Cleaving Enzymes." Current Developments in Nutrition 6, Supplement_1 (June 2022): 55. http://dx.doi.org/10.1093/cdn/nzac049.001.
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Abstract Objectives To evaluate the impact of gender and ablation of β-carotene oxygenase 1 (BCO1), β-carotene oxygenase 2(BCO2) or both on tissue accumulation of lycopene dosed transgenic mice. Previous work from our laboratories suggests that BCO2 appears to be the primary carotenoid cleavage enzyme for lycopene in vivo. Methods Three-week old C57BL/6 male and female mice (wild type [WT], Bco1-/-, Bco2-/-, Bco1-/- X Bco2-/- double knock out [DKO]) were divided into groups based on genotype (n = 8 per group/per gender) and fed a powdered AIN 93G diet for 2 weeks. The mice were gavaged daily for 2 weeks with 1 mg of lycopene dissolved in cottonseed oil after which they were fasted and sacrificed. Liver, serum, and extra-hepatic tissues were harvested. Lycopene concentration was measured with high-performance liquid chromatography. Data analyses were performed using one-way ANOVA. Results On a concentration basis, liver, duodenum and adrenal lycopene were higher than other tissues. Serum and tissues of DKO mice accumulated the highest lycopene. DKO mice had significantly higher lycopene than Bco1-/- mice in the liver (P < .002), heart (P < .004), adipose (P < .03), and the testes (P < .004). Compared to Bco2-/- mice, DKO mice had greater accumulation in the serum (P < .001), intestine (P < .04), heart (P < .0001), kidneys (P < .0001), adipose (P < .04), and testes (P < .0001). Liver (P < .007) and adrenal (ns) tissues in Bco2-/- mice had higher levels of lycopene than Bco1-/- mice whereas Bco1-/- mice had significantly higher levels in the kidneys (P < .001) and tended to have greater accumulation in other tissues. Conclusions Accumulation of lycopene in tissues depended upon gender, genotype and tissue type. The abolition of both enzymes and mice of a female sex generated a higher accumulation. We are probing whether tissue-specific expression levels of BCO1, BCO2 or carotenoid transport proteins explain differential tissue accumulation across genotypes. Funding Sources N/A.
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Kondaiah, Palsa, Puneeta Singh Yaduvanshi, Paul A. Sharp, and Raghu Pullakhandam. "Iron and Zinc Homeostasis and Interactions: Does Enteric Zinc Excretion Cross-Talk with Intestinal Iron Absorption?" Nutrients 11, no. 8 (August 13, 2019): 1885. http://dx.doi.org/10.3390/nu11081885.
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Iron and zinc are essential micronutrients required for growth and health. Deficiencies of these nutrients are highly prevalent among populations, but can be alleviated by supplementation and food fortification. Cross-sectional studies in humans showed positive association of serum zinc levels with hemoglobin and markers of iron status. Dietary restriction of zinc or intestinal specific conditional knock out of ZIP4 (SLC39A4), an intestinal zinc transporter, in experimental animals demonstrated iron deficiency anemia and tissue iron accumulation. Similarly, increased iron accumulation has been observed in cultured cells exposed to zinc deficient media. These results together suggest a potential role of zinc in modulating intestinal iron absorption and mobilization from tissues. Studies in intestinal cell culture models demonstrate that zinc induces iron uptake and transcellular transport via induction of divalent metal iron transporter-1 (DMT1) and ferroportin (FPN1) expression, respectively. It is interesting to note that intestinal cells are exposed to very high levels of zinc through pancreatic secretions, which is a major route of zinc excretion from the body. Therefore, zinc appears to be modulating the iron metabolism possibly via regulating the DMT1 and FPN1 levels. Herein we critically reviewed the available evidence to hypothesize novel mechanism of Zinc-DMT1/FPN1 axis in regulating intestinal iron absorption and tissue iron accumulation to facilitate future research aimed at understanding the yet elusive mechanisms of iron and zinc interactions.
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Tripathi, Ankita, Khushboo Bhardwaj, Sapna Chaudhar, Radhika Chaurasia, Ravindra Kumar, and Shiva Mishra. "RECENT TRENDS IN TREATMENT AND VALIDITY OF SCREENING MODELS USED IN TYPE 2 DIABETES MELLITUS: A REVIEW." International Research Journal of Pharmacy 12, no. 1 (January 31, 2021): 10–16. http://dx.doi.org/10.7897/2230-8407.1201110.
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Type II Diabetes mellitus (NIDDM) is a complex and metabolic disease that affects and presents a serious health problem for more than 100 million people around the world. Genetics and the environment are the variables which affect NIDDM. Reasonable screening models of animals will easily understand the pharmacological screening of different therapeutic agents. NIDDM animal models are acquired either naturally or caused by chemical reagents or by dietary, surgical or combination manipulation. A significant number of new genetically engineered animal models, including knock-out, transgenic, and tissue-specific knockout mice, have been designed for the Diabetes mellitus (D.M.) research in the past year. With regard to their characteristic characteristics and processes by which they arise, this analysis focuses on the animal models of NIDDM. In addition, this analysis explicitly discusses the optimal selection and use of various animal screening models for the treatment of NIDDM in preclinical research.
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Pegoli, Gloria, Marika Milan, Pierluigi Giuseppe Manti, Andrea Bianchi, Federica Lucini, Philina Santarelli, Claudia Bearzi, Roberto Rizzi, and Chiara Lanzuolo. "Role of Cdkn2a in the Emery–Dreifuss Muscular Dystrophy Cardiac Phenotype." Biomolecules 11, no. 4 (April 6, 2021): 538. http://dx.doi.org/10.3390/biom11040538.
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The Cdkn2a locus is one of the most studied tumor suppressor loci in the context of several cancer types. However, in the last years, its expression has also been linked to terminal differentiation and the activation of the senescence program in different cellular subtypes. Knock-out (KO) of the entire locus enhances the capability of stem cells to proliferate in some tissues and respond to severe physiological and non-physiological damages in different organs, including the heart. Emery–Dreifuss muscular dystrophy (EDMD) is characterized by severe contractures and muscle loss at the level of skeletal muscles of the elbows, ankles and neck, and by dilated cardiomyopathy. We have recently demonstrated, using the LMNA Δ8–11 murine model of Emery–Dreifuss muscular dystrophy (EDMD), that dystrophic muscle stem cells prematurely express non-lineage-specific genes early on during postnatal growth, leading to rapid exhaustion of the muscle stem cell pool. Knock-out of the Cdkn2a locus in EDMD dystrophic mice partially restores muscle stem cell properties. In the present study, we describe the cardiac phenotype of the LMNA Δ8–11 mouse model and functionally characterize the effects of KO of the Cdkn2a locus on heart functions and life expectancy.
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Li, Jiajia, Renee Yin Yu, Farida Emran, Brian E. Chen, and Michael E. Hughes. "Achilles-Mediated and Sex-Specific Regulation of Circadian mRNA Rhythms in Drosophila." Journal of Biological Rhythms 34, no. 2 (February 25, 2019): 131–43. http://dx.doi.org/10.1177/0748730419830845.
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The circadian clock is an evolutionarily conserved mechanism that generates the rhythmic expression of downstream genes. The core circadian clock drives the expression of clock-controlled genes, which in turn play critical roles in carrying out many rhythmic physiological processes. Nevertheless, the molecular mechanisms by which clock output genes orchestrate rhythmic signals from the brain to peripheral tissues are largely unknown. Here we explored the role of one rhythmic gene, Achilles, in regulating the rhythmic transcriptome in the fly head. Achilles is a clock-controlled gene in Drosophila that encodes a putative RNA-binding protein. Achilles expression is found in neurons throughout the fly brain using fluorescence in situ hybridization (FISH), and legacy data suggest it is not expressed in core clock neurons. Together, these observations argue against a role for Achilles in regulating the core clock. To assess its impact on circadian mRNA rhythms, we performed RNA sequencing (RNAseq) to compare the rhythmic transcriptomes of control flies and those with diminished Achilles expression in all neurons. Consistent with previous studies, we observe dramatic upregulation of immune response genes upon knock-down of Achilles. Furthermore, many circadian mRNAs lose their rhythmicity in Achilles knock-down flies, suggesting that a subset of the rhythmic transcriptome is regulated either directly or indirectly by Achilles. These Achilles-mediated rhythms are observed in genes involved in immune function and in neuronal signaling, including Prosap, Nemy and Jhl-21. A comparison of RNAseq data from control flies reveals that only 42.7% of clock-controlled genes in the fly brain are rhythmic in both males and females. As mRNA rhythms of core clock genes are largely invariant between the sexes, this observation suggests that sex-specific mechanisms are an important, and heretofore under-appreciated, regulator of the rhythmic transcriptome.
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Dockal, Michael, Rudolf Hartmann, Thomas Polakowski, Erwin Panholzer, Willibald Kammlander, Frank Osterkamp, Ulrich Reineke, Alexandra Schiviz, Werner Hoellriegl, and Friedrich Scheiflinger. "A TFPI Inhibitory Half Life Extended Fusion Peptide Proves Efficacy in FVIII Knock out Mice and Marmoset Monkeys." Blood 124, no. 21 (December 6, 2014): 1469. http://dx.doi.org/10.1182/blood.v124.21.1469.1469.
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Abstract Introduction Tissue factor pathway inhibitor (TFPI) is an important inhibitor of the extrinsic coagulation pathway as it inhibits factor Xa (FXa) and the tissue factor (TF) – FVIIa complex. Inhibition of TFPI with blocking antibodies, aptamers, or peptide inhibitors improves hemostasis and may become an option to treat patients with hemophilia including those with inhibitors. We developed a TFPI-antagonistic fusion peptide (FP) consisting of a linear and a cyclic peptide connected by a linker. The two peptide entities bind to different epitopes on TFPI and together synergistically inhibit TFPI. The FP was further improved by half-life extending (HL) non-covalent albumin binding. HL-FP was characterized for in vitroinhibition of TFPI, pharmacokinetics, and improvement of coagulation in animal models of hemophilia. Methods HL-FP was characterized in a set of in vitro assays for binding to and inhibition of TFPI. Interaction with immobilized TFPI was studied by BiaCore. Functional inhibition was analyzed in model assay systems such as inhibition of FXa and FX activation by TF/FVIIa and plasma assays according to the calibrated automated thrombography (CAT) protocol at low TF in hemophilia plasma. Addition of TFPI simulated conditions of potentially elevated TFPI plasma concentrations. In a single dose PK study, mice (n=6 per time point) received 400 nmol/kg of the HL-FP intravenously (i.v.) or subcutaneously (s.c.). Plasma was sampled up to 38 h after dosing and HL-FP level quantified by a compound specific LC-MS protocol. To provide an ex vivo activity measure, FVIII inhibitory antibodies were added to mouse plasma to mimic a hemophilic condition and then analyzed by calibrated automated thrombography (CAT). A 2-week repeated i.v. dose study in mice investigated TFPI accumulation due to HL-FP. HL-FP was dosed at 40, 400, and 2000 nmol/kg and mouse plasma TFPI levels determined by ELISA. The efficacy of the HL-FP was studied in a hemophilia A mouse tail cut model and in a marmoset monkey model of ex vivoimprovement of coagulation. FVIII knockout mice (n=16 per group) were dosed i.v. with 12-400 nmol/kg HL-FP in the presence of a sub-therapeutic level of recombinant FVIII (10 U/kg) and blood loss (mg) was assessed. Marmoset monkeys (N=4) received 400 nmol/kg HL-FP i.v. and plasma samples obtained 1 h after dosing were analyzed by CAT in the presence of FVIII inhibitory antibodies. Results HL-FP bound to and efficiently inhibited TFPI as demonstrated in several in vitro test systems. Binding affinity of < 1nM correlated well with functional inhibition of TFPI in model assays, resulting in IC50s of ~0.7nM. The HL-fusion peptide (HL-FP) efficiently inhibited plasma TFPI, which resulted in an improvement of all thrombin generation parameters in plasma of hemophilia A and B patients, with EC50s ranging from 6 to 20nM. HL-FP increased peak thrombin levels of hemophilia plasma to or slightly above a range established for individual normal plasma. Non-covalent binding to albumin substantially increased the half-life to ~4 h with ~ 50% s.c. bioavailability in mice. The ex vivo procoagulant activity determined by CAT correlated well with HL-FP plasma concentrations. In the repeated dose study, the HL-FP was well tolerated and did not accumulate TFPI, which strongly indicates that HL-FP did not interfere with TFPI clearance receptor interactions. HL-FP significantly reduced bleeding in the hemophilia mouse tail cut bleeding model at a dose as low as 40 nmol/kg. In marmoset monkeys, HL-FP efficiently improved ex vivo plasma thrombin generation, even at low peptide plasma concentrations (25- 55 nM). Summary We developed a TFPI inhibitor composed of two TFPI antagonistic peptides that completely inhibits TFPI. Introduction of an entity non-covalently bound to albumin provides intermediate half-life extension and s.c. bioavailability. This HL-FP improved coagulation and hemostasis in animal models of hemophilia and did not interfere with TFPI clearance receptor interactions. TFPI-antagonistic peptides with a prolonged half-life, resistance to elevated TFPI, and minimal interference with TFPI clearance. Our HL-FP appears to be useful in preventing bleeding in hemophilia and provides a FVIII and FIX independent approach for non-i.v. treatment. Disclosures Dockal: Baxter Innovations GmbH, Vienna, Austria: Employment. Hartmann:Baxter Innovations GmbH, Vienna, Austria: Employment. Polakowski:3B Pharmaceuticals GmbH, Berlin, Germany: Employment. Panholzer:Baxter Innovations GmbH, Vienna, Austria: Employment. Kammlander:Baxter Innovations GmbH, Vienna, Austria: Employment. Osterkamp:3B Pharmaceuticals, Berlin, Germany: Employment. Reineke:3B Pharmaceuticals GmbH, Berlin, Germany: Employment. Schiviz:Baxter Innovations GmbH, Vienna, Austria: Employment. Hoellriegl:Baxter Innovations GmbH, Vienna, Austria: Employment. Scheiflinger:Baxter Innovations GmbH, Vienna, Austria: Employment.
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Rokvic, Ljiljana, Jens Titze, Wolfgang Schuh, and Hans-Martin Jäck. "The importance of the NFAT5/TonEBP-mediated osmotic stress response in B cells." Journal of Immunology 198, no. 1_Supplement (May 1, 2017): 152.5. http://dx.doi.org/10.4049/jimmunol.198.supp.152.5.
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Abstract Introduction Hyperosmotic stress is well investigated within renal borders, many non-renal tissues are facing fluctuations of osmolality as part of normal, physiological behavior. For example, compared to blood, osmolality of lymphoid tissues, like spleen and thymus, is substantially hypertonic. Therefore, the ability of immune cells to respond and adapt to osmotic stress is crucial for normal immune responses. The osmotic stress response is driven by nuclear factor of activated T cells (NFAT5)/tonicity enhancer binding protein (TonEBP), the only known mammalian osmosensitive transcription factor. It has been shown that mice with a heterozygous deletion of NFAT5 display impaired adaptive immune responses. To specifically analyze the functions of NFAT5 in B lymphocytes, we generated conditional B cell specific knock-out mice. Objectives Using a conditional B cell-specific NFAT5 knock-out mouse model, we want to assess the importance of NFAT5 for B cell development, B cell activation and antibody production. Results NFAT5-deficient mice fed with high salt diet (HSD; 4% NaCl in food and 0.9% NaCl in water) and immunized with TNP-KLH showed impaired IgM and IgG responses compared to wildtype controls. Accordingly, frequencies and numbers of PC and GC B cells were reduced in immunized NFAT5-deficient mice. In support of these findings, we found lower cell numbers and diminished viability of LPS-stimulated NFAT5-deficient B cells cultured in hypertonic medium (+40mM NaCl). Conclusion NFAT5 in B lymphocytes controls antibody responses by controlling B cell activation and survival. Gene expression profiling will now elucidate transcriptional programs and signaling pathways involved in NFAT5-controlled B cell responses.
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Makaryan, Vahagn, Isabella N. Archibald, Merideth L. Kelley, Breanna Fletcher, and David C. Dale. "CRISPR/Cas9 Mediated ELANE Knock-out Restores Survival and Granulocytic Differentiation of HL60 Cells Expressing Mutant Neutrophil Elastase: Is Neutrophil Elastase a Dispensible Granulocyte Protease?" Blood 134, Supplement_1 (November 13, 2019): 435. http://dx.doi.org/10.1182/blood-2019-124988.
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Background : Mutations in ELANE are the most common cause of both cyclic and severe congenital neutropenia. ELANE encodes neutrophil elastase (NE), a tissue specific serine protease expressed primarily in neutrophils. Expression of the mutant protein impairs survival and maturation of myeloid precursors in bone marrow. More than 130 different ELANE mutations have been found in patients with cyclic and congenital neutropenia, and genotype-phenotype studies suggest that specific mutations cause more severe disease. (Curr Op Hematol. 2015;22:3-11) Mutant NE is also implicated as the primary cause of ELANE associated neutropenia by studies showing that cell permeable inhibitors of NE correct the defect in cell survival and maturation in cellular models. (Makaryan et al. J Leukoc Biol. 2017;102:1143). Hypothesis: If inhibition of NE can correct the cellular defect, deletion of ELANE will have similar effects and permit studies to see if NE is an essential neutrophil protease. Methods: We used CRISPR/Cas9 mediated gene editing to create a cellular model of congenital neutropenia in the commercially available human promyelocytic cell line, HL60. We used CRISPR/Cas9 editing technology to create engineered HL60 cell lines with knock-in (KI) ELANE P139L, C151Y and G214R single point heterozygous mutations. Using the same technology, we then performed ELANE gene complete knock out (KO) of ELANE in each mutant cell line. All engineered cell lines as well as wild-type HL60 cells were cultured for 5-7 days in complete RPMI supplemented with 2uM all-trans retinoic acid (ATRA) to trigger myeloid differentiation. Survival of these cell lines was investigated using Annexin V-PE staining and flow cytometric analysis. Granulocytic differentiation was evaluated using CD11b surface marker staining and flow cytometry and by performing manual differential cell counts. We measured the unfolded protein response (UPR) by western blotting using UPR specific antibodies, phagocytosis with E. coli particles using fluorescence detection, IL-8 stimulated chemotaxis in trans-well system and PMA activated respiratory burst by flow cytometry. Results: Cells expressing the P139L and C151Y mutant ELANE had increased Annexin V staining more than a 2-fold increase in apoptotic cells at 7 days in culture. Granulocytic differentiation, measured by surface CD11b expression, was significantly impaired. (p<0.0001). Cytospins stained with Diff-Quik showed a typical block of myeloid differentiation and a significant deficiency of mature neutrophils in 7-day cultures. Western blot analysis using antibodies to GRP78/BiP and ATF6 showed a typical UPR signature in both ELANE mutant cell lines compared to wild type. Chemotaxis to the IL8 chemo-attractant was severely impaired in cells expressing mutant NE. Respiratory burst and phagocytosis was also altered in cells expressing mutant NE. ELANE KO corrected all these cellular and functional abnormalities, reverting these functions toward the wild phenotype. Studies are in progress for the third cell line expressing the G214R mutation. Conclusions: CRISPR/Cas9 engineered HL60 cell lines expressing mutant NE are a highly reproducible and reliable cellular model for investigating ELANE associated neutropenia. The presence of mutant NE severely impairs neutrophil functions, and its deletion through KO of ELANE corrects the cellular abnormalities and reverses the induced abnormalities. These results suggest that CRISPR/Cas9 mediated genetic knock down of NE is a novel therapeutic approach for treatment of this disorder. This data also suggests that total absence of NE does not alter basic functions of granulocytes. Disclosures Dale: Sanofi Aventis: Consultancy, Honoraria; Athelas: Equity Ownership; Amgen: Consultancy, Research Funding; Cellerant: Other: Scientific Advisory Board; Hospira: Consultancy; Prolong: Consultancy; Beheringer/Ingelheim: Consultancy; Coherus: Consultancy; x4pharma: Consultancy, Honoraria, Research Funding.
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Addeo, Martina, Silvia Buonaiuto, Ilaria Guerriero, Elena Amendola, Feliciano Visconte, Antonio Marino, Maria Teresa De Angelis, et al. "Insight into Nephrocan Function in Mouse Endoderm Patterning." International Journal of Molecular Sciences 21, no. 1 (December 18, 2019): 8. http://dx.doi.org/10.3390/ijms21010008.
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Endoderm-derived organs as liver and pancreas are potential targets for regenerative therapies, and thus, there is great interest in understanding the pathways that regulate the induction and specification of this germ layer. Currently, the knowledge of molecular mechanisms that guide the in vivo endoderm specification is restricted by the lack of early endoderm specific markers. Nephrocan (Nepn) is a gene whose expression characterizes the early stages of murine endoderm specification (E7.5–11.5) and encodes a secreted N-glycosylated protein. In the present study, we report the identification of a new transcript variant that is generated through alternative splicing. The new variant was found to have differential and tissue specific expression in the adult mouse. In order to better understand Nepn role during endoderm specification, we generated Nepn knock-out (KO) mice. Nepn−/− mice were born at Mendelian ratios and displayed no evident phenotype compared to WT mice. In addition, we produced nullizygous mouse embryonic stem cell (mESC) line lacking Nepn by applying (CRISPR)/CRISPR-associated systems 9 (Cas9) and employed a differentiation protocol toward endoderm lineage. Our in vitro results revealed that Nepn loss affects the endoderm differentiation impairing the expression of posterior foregut-associated markers.
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Frascoli, Michela, Enxhi Ferraj, Bing Miu, Kelsey Esposito, Nicholas Spidale, Justin Malin, Jennifer Cowan, Avinash Bhandoola, Joonsoo Kang, and Andrea Reboldi. "Crosstalk between skin homing innate T cells and epithelial cells via cholesterol byproduct messengers is required for tissue immunity." Journal of Immunology 208, no. 1_Supplement (May 1, 2022): 171.02. http://dx.doi.org/10.4049/jimmunol.208.supp.171.02.
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Abstract Innate T cells and innate lymphoid cells in barrier tissues are essential for both tissue fitness and immunity against pathogens. γδTCR+ lymphocytes that secrete IL-17 (Tγδ17) localize to dermis and are critical regulators of skin immune responses. Despite their functional importance, how Tγδ17 cells mediate their unique function in the skin is largely unknown. Tγδ17 cells express the two prototypic G-protein coupled receptors (GPCRs) CCR6 and GPR183 (EBI2): mice lacking both receptors had significantly diminished dermal Tγδ17 cells and were resistant to psoriasis induction. This was due to a block in Tγδ17 cell maturation in the thymus of Ccr6−/−Gpr183−/− double knock-out mice. EBI2 recognizes oxysterols generated by the cholesterol hydroxylase CH25H and functional oxysterols were detected in both the thymus and skin. Analysis of Ch25h-reporter mice showed that interfollicular epithelial cells (IFEs) and a subset of medullary thymic epithelial cells (mTEC) are the depot for oxysterols in skin and thymus respectively. Ch25h+ TECs were also identified by unbiased single cell transcriptome analyses. These results demonstrate that developing Tγδ17 cells must be conditioned by sensing cholesterol byproducts synthesized by a discrete subset of mTECs to position in specific oxysterol rich niches in the skin. Moreover, in the skin, dietary cholesterol increased tissue oxysterol concentration and modulated homeostatic and pathogenic effector function of Tγδ17 cells in a EBI2 dependent fashion. Given that oxysterols can be regulated by infection and inflammation, this sensory circuit directed by cholesterol is predicted to program skin innate T cells’ ability to rapidly survey for perturbations in the skin. Supported by grant from NIH R21AI143225.
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Freiin von Hövel, Friederike, Ekaterini Kefalakes, and Claudia Grothe. "What Can We Learn from FGF-2 Isoform-Specific Mouse Mutants? Differential Insights into FGF-2 Physiology In Vivo." International Journal of Molecular Sciences 22, no. 1 (December 31, 2020): 390. http://dx.doi.org/10.3390/ijms22010390.
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Fibroblast growth factor 2 (FGF-2), ubiquitously expressed in humans and mice, is functionally involved in cell growth, migration and maturation in vitro and in vivo. Based on the same mRNA, an 18-kilo Dalton (kDa) FGF-2 isoform named FGF-2 low molecular weight (FGF-2LMW) isoform is translated in humans and rodents. Additionally, two larger isoforms weighing 21 and 22 kDa also exist, summarized as the FGF-2 high molecular weight (FGF-2HMW) isoform. Meanwhile, the human FGF-2HMW comprises a 22, 23, 24 and 34 kDa protein. Independent studies verified a specific intracellular localization, mode of action and tissue-specific spatiotemporal expression of the FGF-2 isoforms, increasing the complexity of their physiological and pathophysiological roles. In order to analyze their spectrum of effects, FGF-2LMW knock out (ko) and FGF-2HMWko mice have been generated, as well as mice specifically overexpressing either FGF-2LMW or FGF-2HMW. So far, the development and functionality of the cardiovascular system, bone formation and regeneration as well as their impact on the central nervous system including disease models of neurodegeneration, have been examined. This review provides a summary of the studies characterizing the in vivo effects modulated by the FGF-2 isoforms and, thus, offers a comprehensive overview of its actions in the aforementioned organ systems.
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Vick, Philipp, Birgit Eberle, Daniela Choukair, Birgit Weiss, Ralph Roeth, Isabelle Schneider, Nagarajan Paramasivam, Markus Bettendorf, and Gudrun A. Rappold. "Identification of ZBTB26 as a Novel Risk Factor for Congenital Hypothyroidism." Genes 12, no. 12 (November 24, 2021): 1862. http://dx.doi.org/10.3390/genes12121862.
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Congenital primary hypothyroidism (CH; OMIM 218700) is characterized by an impaired thyroid development, or dyshormonogenesis, and can lead to intellectual disability and growth retardation if untreated. Most of the children with congenital hypothyroidism present thyroid dysgenesis, a developmental anomaly of the thyroid. Various genes have been associated with thyroid dysgenesis, but all known genes together can only explain a small number of cases. To identify novel genetic causes for congenital hypothyroidism, we performed trio whole-exome sequencing in an affected newborn and his unaffected parents. A predicted damaging de novo missense mutation was identified in the ZBTB26 gene (Zinc Finger A and BTB Domain containing 26). An additional cohort screening of 156 individuals with congenital thyroid dysgenesis identified two additional ZBTB26 gene variants of unknown significance. To study the underlying disease mechanism, morpholino knock-down of zbtb26 in Xenopus laevis was carried out, which demonstrated significantly smaller thyroid anlagen in knock-down animals at tadpole stage. Marker genes expressed in thyroid tissue precursors also indicated a specific reduction in the Xenopus ortholog of human Paired-Box-Protein PAX8, a transcription factor required for thyroid development, which could be rescued by adding zbtb26. Pathway and network analysis indicated network links of ZBTB26 to PAX8 and other genes involved in thyroid genesis and function. GWAS associations of ZBTB26 were found with height. Together, our study added a novel genetic risk factor to the list of genes underlying congenital primary hypothyroidism and provides additional support that de novo mutations, together with inherited variants, might contribute to the genetic susceptibility to CH.
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Atanackovic, Djordje, York Hildebrandt, Adam Jadczak, Tim Luetkens, Sabrina Meyer, Katrin Bartels, Yanran Cao, Axel R. Zander, Carsten Bokemeyer, and Nicolaus Kroeger. "Expression of Cancer-Testis Antigens MAGE-C1/CT7 and MAGE-A3 Is Central to the Survival of Myeloma Cells and Their Resistance to Chemotherapy." Blood 112, no. 11 (November 16, 2008): 3668. http://dx.doi.org/10.1182/blood.v112.11.3668.3668.
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Abstract The cancer-testis (CT) class of tumor antigens is a group of proteins, the expression of which is characteristically restricted to cancer and the human germline. Based on their immunogenicity and restricted tissue expression, CT antigens seem ideal targets for immunotherapeutic approaches. This is particularly the case in multiple myeloma (MM) where these tumor antigens are frequently and strongly expressed. Unfortunately, very little is known about the biological function of CT antigens which seems surprising given the strong impact of CT antigen expression on the prognosis of a variety of malignancies including MM. We examined for the first time the impact of the expression of two CT antigens frequently found in MM, MAGE-C1/CT7 and MAGE-A3, on the biology of the disease conducting knock-down experiments using RNA interference (RNAi) technology. As read-out assays, Western Blots as well as a large variety of tests measuring proliferation, cytotoxicity, cell adhesion, and clonogenic growth were performed. Transfecting myeloma cell line MOLP-8 with RNAi specific for MAGE-C1/CT7 and MAGE-A3 we observed a down-regulation of CT antigen protein expression by 80% and 70%, respectively. Importantly, transfection with MAGE-A3 RNA stealth led to a specific decrease in MAGE-A3 expression while the protein expression of other MAGE genes (MAGE-A4, MAGE-C1/CT7, MAGE-C2/CT10) and non-MAGE CT antigens such as Ropporin-1 was not affected. The same was true for knock-down experiments targeting MAGE-C1/CT7 with the exception of a slight decrease in expression of the highly homologous gene MAGE-C2/CT10. Comparing myeloma cell lines treated with gene-specific versus nonsense RNA stealth, we found that knocking down MAGE-C1/CT7 and MAGE-A3 led to the induction of apoptosis in in about 70% of cells at 72h post transfection as indicated by MTT assays, LDH release assays and Annexin V expression analyzed by flow cytometry. No such effect was observed when transfection was performed with nonsense RNAi (p<0.01). Importantly, CD138-negative myeloma precursors, which also expressed the CT antigens investigated, were also affected by downregulation of CT antigen expression as indicated by an 48% decrease in clonogenic growth (p<0.01). Applying chemotherapy with melphalan to the cell cultures we observed that knock-down of MAGE-C1/CT7 or MAGE-A3 increased the chemosensitivity of MM cell lines by 67% (p<0.01). Importantly, knock-down of MAGE-C1/CT7 or MAGE-A3 specifically affected cell survival and did not alter the proliferative capacity of myeloma cells or cell adhesion. In conclusion, we show here for the first time that CT antigens MAGE-C1/CT7 and MAGE-A3, which are also expressed in the majority of patient myeloma samples, are important for the survival of myeloma cell lines and clonogenic myeloma precursors. In addition, the expression of these CT antigens most likely contributes to promoting resistance to chemotherapy in multiple myeloma. Targeting CT antigens expressed by myeloma cells, either by applying CT antigen-specifric immunotherapy or other targeted therapies, might significantly improve myeloma therapy, i.e. by eliminating chemotherapy-resistant clones.
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Zhang, H., X. Chen, and M. R. Sairam. "Novel Genes of Visceral Adiposity: Identification of Mouse and Human Mesenteric Estrogen-Dependent Adipose (MEDA)-4 Gene and Its Adipogenic Function." Endocrinology 153, no. 6 (April 17, 2012): 2665–76. http://dx.doi.org/10.1210/en.2011-2008.
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Visceral adiposity represents a high risk factor for type 2 diabetes, metabolic syndrome, and cardiovascular disease as well as various cancers. While studying sex hormone imbalance-induced early obesity and late onset of insulin resistance in FSH receptor knock out female mice, we identified a novel mesenteric estrogen-dependent adipose gene (MEDA-4) selectively up-regulated in a depot-specific manner in mesenteric adipose tissue. Meda-4 cloned from both mouse and human adipose tissue codes for a 34-kDa cytosolic protein with 91% homology. Mouse Meda-4 mRNA is expressed highest in visceral adipose tissue and localizes predominantly in the adipocyte fraction. Human MEDA-4 is also more abundant in omental fat than sc depot in obese patients. In 3T3-L1 cells endogenous Meda-4 expression increases early during differentiation, and its overexpression promotes differentiation of preadipocytes into adipocytes and enhances glucose uptake. Conversely, short hairpin RNA-mediated knockdown of Meda-4 reduces both adipogenic and glucose uptake potential. In promoting adipogenesis, Meda-4 up-regulates transcription factor peroxisome proliferator-activated receptor-γ2. Meda-4 promotes lipid accumulation in adipocytes, regulating adipocyte fatty acid-binding protein 2, CD36, lipoprotein lipase, hormone-sensitive lipase, acyl-Coenzyme A oxidase-1, perilipin-1, and fatty acid synthase expression. 17β-Estradiol reduced Meda-4 expression in mesenteric adipose tissue of ovariectomized mice and in 3T3-L1 adipocytes. Thus our study identifies Meda-4 as a novel adipogenic gene, capable of promoting differentiation of preadipocytes into adipocytes, increasing lipid content and glucose uptake in adipocytes. Therefore it might play an important role in adipose tissue expansion in normal and aberrant hormonal conditions and pathophysiological states.
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Cuesta, Natalia, Sonia Fernández-Veledo, Carmen Punzón, Cristóbal Moreno, Beatriz Barrocal, Vinatha Sreeramkumar, Manuel Desco, and Manuel Fresno. "Opposing Actions of TLR2 and TLR4 in Adipocyte Differentiation and Mature-Onset Obesity." International Journal of Molecular Sciences 23, no. 24 (December 10, 2022): 15682. http://dx.doi.org/10.3390/ijms232415682.
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Understanding the signaling cascades that govern adipocyte metabolism and differentiation is necessary for the development of therapies for obesity. Toll-like receptors (TLRs) are key mediators in adipogenesis, but their specific role is not completely understood. In this study, siRNA knockdown of Tlr2 in 3T3-L1 cells allowed them to differentiate more efficiently into adipocytes, whereas the opposite was observed for the knockdown of Tlr4. At the same time, we show that TLR2 knock-out mice spontaneously developed mature-onset obesity and insulin resistance. Besides a higher incidence of hyperplasia and hypertrophy in white adipose tissue (WAT), we found a significantly increased number of adipocyte precursor cells in TLR2−/− mice compared to TLR4−/− mice. Interestingly, genetic inactivation of Tlr4 in TLR2−/− mice reverted their increased adiposity, insulin resistance, and restored normal levels of adipocyte precursor cells. These findings provide evidence that TLR2 and TLR4 play opposing roles in WAT homeostasis and point to the existence of cross-regulation among TLR2 and TLR4 during adipocyte differentiation both in vitro and in vivo.
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Oh, Sun-Young, Stephanie Brandal, Axel Roers, Zhou Zhu, and Clifford Takemoto. "Global microRNA expression is essential for mast cell development in vivo (177.18)." Journal of Immunology 188, no. 1_Supplement (May 1, 2012): 177.18. http://dx.doi.org/10.4049/jimmunol.188.supp.177.18.
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Abstract microRNAs (miRNAs) are small, non-coding RNAs that have been shown to play a critical role in both normal physiology and disease, such as hematopoietic development and cancer. However, their role in mast cell function and development is unexplored. The RNaseIII endonuclease, Dicer, is required for the processing of pre-miRNAs into mature miRNAs. To investigate the role of global miRNA depletion on mast cells in vivo, we generated a mast cell-specific knock out of Dicer in mice. Transgenic mice (Mcpt5-Cre) that express Cre selectively in mast cells were crossed with mice containing the floxed conditional Dicer allele (Dicer f/f). Mice with both homozygous (Dicer -/-) and heterozygous (Dicer f/-) mast cell-specific deletion of Dicer were viable and healthy. However, Dicer -/- mice were found to have a profound mast cell deficiency with near complete loss of peritoneal, gastrointestinal, and skin mast cells. We examined the in vivo functional consequence of mast cell-specific Dicer deletion using an IgE-dependent passive systemic anaphylaxis (PSA) murine model. IgE sensitized wild type (Dicer f/f) and heterozygous (Dicer f/-) mice show marked hypothermia with antigen; however, homozygous (Dicer -/-) mice were completely unreactive to antigen challenge. These studies suggest a critical role for Dicer and miRNA expression for establishment of mast cell tissue compartments in vivo.