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The mammalian cornea contains an extensive network of resident macrophages and dendritic cells. To determine the role of these cells in LPS-induced corneal inflammation, TLR4−/− mice were sublethally irradiated and reconstituted with bone marrow cells from either enhanced GFP (eGFP)+/C57BL/6 or eGFP+/TLR4−/− mice. The corneal epithelium was abraded, LPS was added topically, and cellular infiltration to the corneal stroma and development of corneal haze were examined after 24 h. TLR4−/− mice reconstituted with C57BL/6, but not TLR4−/− bone marrow cells donor cells were found to cause infiltration of eGFP+ cells to the cornea, including neutrophils, and also increased corneal haze compared with saline-treated corneas. In a second experimental approach, corneas of transgenic macrophage Fas induced apoptosis (Mafia) mice were stimulated with LPS. These mice express eGFP and a suicide gene under control of the c-fms promoter, and systemic treatment with the FK506 dimerizer (AP20187) causes Fas-mediated apoptosis of monocytic cells. AP20187-treated mice had significantly fewer eGFP+ cells in the cornea than untreated mice. After stimulation with LPS neutrophil recruitment and development of corneal haze were impaired in AP20187-treated mice compared with untreated controls. Furthermore, LPS induced CXCL1/KC and IL-1α production within 4 h in corneas of untreated Mafia mice, which is before cellular infiltration; however, cytokine production was impaired after AP20187 treatment. Together, results from both experimental approaches demonstrate an essential role for resident corneal monocytic lineage cells (macrophages and dendritic cells) in development of corneal inflammation.

Background We present a new mouse model for the study of peritoneal adhesions using macrophage Fasinduced apoptosis (Mafia) transgenic mice expressing a Fas-FKBP construct under control of the murine c-fms promoter. Mafia mice allow systemic macrophage depletion by dimerization of Fas with a synthetic dimerizer, AP20187. Results demonstrate that macrophage depletion in Mafia mice induces peritoneal adhesion formation when the peritoneal cavity is also exposed to an irritant. The Mafia mouse model presents a reproducible, non-surgical approach for research in adhesion formation and prevention. Materials and methods Mafia mice were treated with AP20187 using an intravenous (i.v.) or intraperitoneal (i.p.) injection. Control groups included mock-treated Mafia mice and both AP20187 and mock-treated wild type mice. Seven days after treatment, mice were observed for the presence of adhesions. Results After i.p. injection with AP20187, 76% of Mafia mice developed adhesions whereas none of the mock-treated Mafia or wild-type mice developed adhesions, and only one AP20187-treated wild-type mouse (5.8%) developed a mild adhesion. Mafia mice treated with AP20187 i.v. exhibited macrophage depletion not significantly different than i.p. treated mice, but did not develop adhesions. In contrast, Mafia mice treated with AP20187 i.v. developed adhesions when diluent was also injected into the peritoneal cavity, whereas i.p diluent alone had no effect. Conclusion Macrophage depletion, combined with a peritoneal irritant, results in peritoneal adhesion formation in transgenic Mafia mice. Macrophages appear to play a protective role in the development and/or repair of peritoneal adhesions.

Transgenic mice expressing an inducible suicide gene, which allows systemic and reversible elimination of macrophages, were developed. A macrophage-specific c-fms promoter was used to express enhanced green fluorescent protein and a drug-inducible suicide gene that leads to Fas-mediated apoptosis in resting and cycling cells of the macrophage lineage. Transgenic mice were fertile, of normal weight, and showed no abnormal phenotype before drug exposure. The transgene was expressed constitutively in macrophages and dendritic cells (DC) but not significantly in T cells or B cells. Induction of the suicide gene led to depletion of 70–95% of macrophages and DC in nearly all tissues examined. Depletion reduced the ability to clear bacteria from the blood and led to increased bacterial growth in the liver. Depleted mice displayed several abnormalities, including splenomegaly, lymphadenopathy, thymic atrophy, extramedullary hematopoiesis, and development of peritoneal adhesions. This new, transgenic line will be useful in investigating the role of macrophages and DC.

Being able to deliver molecular loads to the intracellular space of mammalian cells is a key initial step of genetic engineering. In the following work, experimentation with nanoinjection, a non-viral molecular load delivery technique, was examined in regards to transmembrane delivery of propidium iodide (PI), a dye that cannot penetrate the cell membrane and fluoresces when bound to genetic material. Investigation includes two environmental factors: peak pulse amplitude (1.5 to 3, 5, 7, or 9 V) and saline type (HBSS, PBS with potassium, and PBS without potassium). Results indicate that PBS with potassium has significantly higher PI uptake efficiency than the other two saline solutions for pulsed voltages of 3V, 5V, and 7V (with the peak value being 3.352 times greater than the positive control). Also, cell viability analysis indicates that there is a measureable reduction in cell viability for voltage protocol samples in comparison to non-voltage protocol samples. Cell viabilities range from 74.5% to 89.4% for voltage protocol samples. Findings suggest that a possible combination of physical/electrical variables work in concert with biological mechanisms to contribute to overall cell survival and PI uptake efficiency in nanoinjection.

Nanoinjection lance arrays have been used to inject propidium iodide, a dye, into living cells. The lance arrays consist of approximately four million needle-like structures on a 2 × 2 cm silicon chip, fabricated using standard methods for silicon wafers. A culture of HeLa cancer cells, commonly used in genetic research, has been employed for testing the nanoinjection process. Propidium iodide at several concentrations has been used as the dye for cell injection. The dye binds to nucleic acids after injection, and does not fluoresce when not bound, allowing accurate flow cytometry measurement of successful injections. Over 200 wells were tested to gather reliable testing data, consisting of negative controls (no treatment), positive controls (dye added with no injection), and samples (dye added and injection performed). Tests were performed for dye concentrations of 20, 40, 60, and 80 μL per 1 mL of cell media solution, for two different lance array geometries. The multi-cell nanoinjection process presented in this paper has proven to produce positive dye uptake results in injected cells while maintaining high cell viability, with an average uptake rate of about 52% for cells injected with the highest concentration of dye. Reviewing all data, the average sample success rate is 8 times higher than the rate of dye uptake in the positive controls. Cell viability in these tests is on average 96.2%. The process may be used in a variety of research areas.Copyright © 2013 by ASME

Intranasal application of vesicular stomatitis virus (VSV) induces acute encephalitis characterized by a pronounced myeloid and T cell infiltrate. The role of distinct phagocytic populations on VSV encephalitis was therefore examined in this study. Ablation of peripheral macrophages did not impair VSV encephalitis or viral clearance from the brain, whereas, depletion of splenic marginal dendritic cells impaired this response and enhanced morbidity/mortality. Selective depletion of brain perivascular macrophages also suppressed this response without altering viral clearance. Thus, two anatomically distinct phagocytic populations regulate VSV encephalitis in a non-redundant fashion although neither population is essential for viral clearance in the CNS.

The tumor microenvironment, which includes inflammatory cells, vasculature, extracellular matrix, and fibroblasts, is a critical mediator of neoplastic progression and metastasis. Using an inducible transgenic mouse model of preneoplastic progression in the mammary gland, we discovered that activation of inducible fibroblast growth factor receptor-1 (iFGFR1) in the mammary epithelium rapidly increased the expression of several genes involved in the inflammatory response. Further analysis revealed that iFGFR1 activation induced recruitment of macrophages to the epithelium and continued association with the alveolar hyperplasias that developed following long-term activation. Studies using HC-11 mammary epithelial cells showed that iFGFR1-induced expression of the macrophage chemoattractant osteopontin was required for macrophage recruitment in vitro. Finally, conditional depletion of macrophages inhibited iFGFR1-mediated epithelial cell proliferation and lateral budding. These findings show that inflammatory cells, specifically macrophages, are critical for mediating early events in an inducible transgenic mouse model of preneoplastic progression. (Cancer Res 2006; 66(11): 5676-85)