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Imaging, myeloid precursor immortalization, and genome editing for defining mechanisms of leukocyte recruitment in vivo.

ABSTRACT: Recruitment of leukocytes from the blood to sites of inflammation poses a promising target for new diagnostic and therapeutic approaches. We aimed to develop a novel method to non-invasively analyze molecular mechanisms of leukocyte migration in pre-clinical models of inflammation in vivo. Methods: We used the ER-HoxB8 system to transiently immortalize murine myeloid precursors from wildtype and CD18- as well as MRP14-deficient mice. A VLA4α-/- cell line was generated by CRISPR/Cas9-mediated gene editing. We analyzed the migration of wildtype and knockout leukocytes in vivo by optical and nuclear imaging in mice with irritant contact dermatitis, cutaneous granuloma, experimental arthritis and myocardial infarction. Results: Transient immortalization, gene editing and in vivo imaging can be combined to analyze migratory mechanisms of murine leukocytes, even for gene deletions resulting in lethal phenotypes in mice. We reliably confirmed known migratory defects of leukocytes deficient for the adhesion molecules CD18 or VLA4α. Also, using our new method we identified a new role of the most abundant calcium-binding proteins in phagocytes and major alarmins in many inflammatory diseases, MRP8 and MRP14, for transmigration in vivo. Conclusion: We provide a combinatorial approach to rapidly characterize molecular mechanisms of leukocyte recruitment in vivo, with the potential to aid in identification of diagnostic and therapeutic targets in inflammatory pathologies.

SUBMITTER: Gran S

PROVIDER: S-EPMC5928898 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Imaging, myeloid precursor immortalization, and genome editing for defining mechanisms of leukocyte recruitment in vivo.

Gran Sandra S Honold Lisa L Fehler Olesja O Zenker Stefanie S Eligehausen Sarah S Kuhlmann Michael T MT Geven Edwin E van den Bosch Martijn M van Lent Peter P Spiekermann Christoph C Hermann Sven S Vogl Thomas T Schäfers Michael M Roth Johannes J

Theranostics 20180323 9

Recruitment of leukocytes from the blood to sites of inflammation poses a promising target for new diagnostic and therapeutic approaches. We aimed to develop a novel method to non-invasively analyze molecular mechanisms of leukocyte migration in pre-clinical models of inflammation in vivo. Methods: We used the ER-HoxB8 system to transiently immortalize murine myeloid precursors from wildtype and CD18- as well as MRP14-deficient mice. A VLA4α-/- cell line was generated ...[more]

PMID: 29721088

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Project description:BackgroundProlapse recurrence after transvaginal surgical repair is common; however, its mechanisms are ill-defined. A thorough understanding of how and why prolapse repairs fail is needed to address their high rate of anatomic recurrence and to develop novel therapies to overcome defined deficiencies.ObjectiveThis study aimed to identify mechanisms and contributors of anatomic recurrence after vaginal hysterectomy with uterosacral ligament suspension (native tissue repair) vs transvaginal mesh (VM) hysteropexy surgery for uterovaginal prolapse.Study designThis multicenter study was conducted in a subset of participants in a randomized clinical trial by the Eunice Kennedy Shriver National Institute of Child Health and Human Development Pelvic Floor Disorders Network. Overall, 94 women with uterovaginal prolapse treated via native tissue repair (n=48) or VM hysteropexy (n=46) underwent pelvic magnetic resonance imaging at rest, maximal strain, and poststrain rest (recovery) 30 to 42 months after surgery. Participants who desired reoperation before 30 to 42 months were imaged earlier to assess the impact of the index surgery. Using a novel 3-dimensional pelvic coordinate system, coregistered midsagittal images were obtained to assess study outcomes. Magnetic resonance imaging-based anatomic recurrence (failure) was defined as prolapse beyond the hymen. The primary outcome was the mechanism of failure (apical descent vs anterior vaginal wall elongation), including the frequency and site of failure. Secondary outcomes included displacement of the vaginal apex and perineal body and change in the length of the anterior wall, posterior wall, vaginal perimeter, and introitus of the vagina from rest to strain and rest to recovery. Group differences in the mechanism, frequency, and site of failure were assessed using the Fisher exact tests, and secondary outcomes were compared using Wilcoxon rank-sum tests.ResultsOf the 88 participants analyzed, 37 (42%) had recurrent prolapse (VM hysteropexy, 13 of 45 [29%]; native tissue repair, 24 of 43 [56%]). The most common site of failure was the anterior compartment (VM hysteropexy, 38%; native tissue repair, 92%). The primary mechanism of recurrence was apical descent (VM hysteropexy, 85%; native tissue repair, 67%). From rest to strain, failures (vs successes) had greater inferior displacement of the vaginal apex (difference, -12 mm; 95% confidence interval, -19 to -6) and perineal body (difference, -7 mm; 95% confidence interval, -11 to -4) and elongation of the anterior vaginal wall (difference, 12 mm; 95% confidence interval, 8-16) and vaginal introitus (difference, 11 mm; 95% confidence interval, 7-15).ConclusionThe primary mechanism of prolapse recurrence following vaginal hysterectomy with uterosacral ligament suspension or VM hysteropexy was apical descent. In addition, greater inferior descent of the vaginal apex and perineal body, lengthening of the anterior vaginal wall, and increased size of the vaginal introitus with strain were associated with anatomic failure. Further studies are needed to provide additional insight into the mechanism by which these factors contribute to anatomic failure.

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Imaging, myeloid precursor immortalization, and genome editing for defining mechanisms of leukocyte recruitment in vivo.

Publications

Imaging, myeloid precursor immortalization, and genome editing for defining mechanisms of leukocyte recruitment <i>in vivo</i>.

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