Suellen Greco

In vivo developmental stages in murine natural killer cell maturation

Natural killer (NK) cells develop in the bone marrow, but their in vivo stages of maturation, expansion and acquisition of receptors that guide target cell specificity are not well defined. We describe here such stages of development. We also show that developing NK cells actively proliferate at a phenotypically distinguishable immature stage after they have acquired expression of Ly49 and CD94-NKG2 receptors. These studies provide a developmental framework for NK cell maturation in vivo and suggest the possible involvement of the Ly49 and CD94-NKG2 receptors themselves in modulating expansion of NK cell populations with a given NK cell receptor repertoire.

Gr1+ Inflammatory Monocytes Are Required for Mucosal Resistance to the Pathogen Toxoplasma gondii

The enteric pathogen Toxoplasma gondii is controlled by a vigorous innate T helper 1 (Th1) cell response in the murine model. We demonstrated that after oral infection, the parasite rapidly recruited inflammatory monocytes [Gr1(+) (Ly6C(+), Ly6G(-)) F4/80(+)CD11b(+)CD11c(-)], which established a vital defensive perimeter within the villi of the ileum in the small intestine. Mice deficient of the chemokine receptor CCR2 or the ligand CCL2 failed to recruit Gr1(+) inflammatory monocytes, whereas dendritic cells and resident tissue macrophages remained unaltered. The selective lack of Gr1(+) inflammatory monocytes resulted in an inability of mice to control replication of the parasite, high influx of neutrophils, extensive intestinal necrosis, and rapid death. Adoptive transfer of sorted Gr1(+) inflammatory monocytes demonstrated their ability to home to the ileum in infected animals and protect Ccr2(-/-) mice, which were otherwise highly susceptible to oral toxoplasmosis. Collectively, these findings illustrate the critical importance of inflammatory monocytes as a first line of defense in controlling intestinal pathogens.

Histologic and Biomechanical Evaluation of Crosslinked and Non-Crosslinked Biologic Meshes in a Porcine Model of Ventral Incisional Hernia Repair

BACKGROUND The objective of this study was to evaluate the biomechanical characteristics and histologic remodeling of crosslinked (Peri-Guard, Permacol) and non-crosslinked (AlloDerm, Veritas) biologic meshes over a 12 month period using a porcine model of incisional hernia repair. STUDY DESIGN Bilateral incisional hernias were created in 48 Yucatan minipigs and repaired after 21 days using an underlay technique. Samples were harvested at 1, 6, and 12 months and analyzed for biomechanical and histologic properties. The same biomechanical tests were conducted with de novo (time 0) meshes as well as samples of native abdominal wall. Statistical significance (p < 0.05) was determined using 1-way analysis of variance with a Fishers least significant difference post-test. RESULTS All repair sites demonstrated similar tensile strengths at 1, 6, and 12 months and no significant differences were observed between mesh materials (p > 0.05 in all cases). The strength of the native porcine abdominal wall was not augmented by the presence of the mesh at any of the time points, regardless of de novo tensile strength of the mesh. Histologically, non-crosslinked materials showed earlier cell infiltration (p < 0.01), extracellular matrix deposition (p < 0.02), scaffold degradation (p < 0.05), and neovascularization (p < 0.02) compared with crosslinked materials. However, by 12 months, crosslinked materials showed similar results compared with the non-crosslinked materials for many of the features evaluated. CONCLUSIONS The tensile strengths of sites repaired with biologic mesh were not impacted by very high de novo tensile strength/stiffness or mesh-specific variables such as crosslinking. Although crosslinking distinguishes biologic meshes in the short-term for histologic features, such as cellular infiltration and neovascularization, many differences diminish during longer periods of time. Characteristics other than crosslinking, such as tissue type and processing conditions, are likely responsible for these differences.

Intrawound vancomycin powder eradicates surgical wound contamination: An in vivo rabbit study

BACKGROUND Surgical site infection remains a complication of spine surgery despite routine use of prophylactic antibiotics. Retrospective clinical studies of intrawound vancomycin use have documented a decreased prevalence of surgical site infection after spine surgery. The purpose of the present study was to assess the efficacy of intrawound vancomycin powder in terms of eradicating a known bacterial surgical site contamination in a rabbit spine surgery model. METHODS Twenty New Zealand White rabbits underwent lumbar partial laminectomy and wire implantation. The surgical sites were inoculated, prior to closure, by injecting 100 μL of cefazolin-sensitive and vancomycin-sensitive Staphylococcus aureus (S. aureus) (1 × 10⁸ colony-forming units [CFU]/mL) into the wound. Preoperative cefazolin was administered to all rabbits, and vancomycin powder (100 mg) was placed into the wound of ten rabbits prior to closure. The rabbits were killed on postoperative day four, and tissue and wire samples were obtained for bacteriologic assessment. An independent samples t test was used to assess mean group differences, and a Fisher exact test was used to assess differences in categorical variables. RESULTS The vancomycin-treated and the control rabbits were similar in weight (mean [and standard deviation], 4.1 ± 0.5 kg and 4.0 ± 0.4 kg, respectively; p = 0.60) and sex distribution and had similar durations of surgery (21.7 ± 7.7 minutes and 16.9 ± 6.7 minutes; p = 0.15). The bacterial cultures of the surgical site tissues were negative for all ten vancomycin-treated rabbits and positive for all ten control rabbits (p < 0.0001). Bacterial growth occurred in thirty-nine of forty samples from the control group but in zero of forty samples from the vancomycin group (p < 0.0001). All blood and liver samples were sterile. No rabbit had evidence of sepsis or vancomycin toxicity. Gross examination of the surgical sites showed no differences between the groups. CONCLUSIONS In a rabbit spine-infection model, intrawound vancomycin powder in combination with preoperative cefazolin eliminated S. aureus surgical site contamination. All rabbits that were managed with only prophylactic cefazolin had persistent S. aureus contamination. CLINICAL RELEVANCE This animal study supports the findings in prior clinical reports that intrawound vancomycin powder helps reduce the risk of surgical site infections.

Biomechanical and Histologic Evaluation of Fenestrated and Nonfenestrated Biologic Mesh in a Porcine Model of Ventral Hernia Repair

BACKGROUND The purpose of this study was to compare tissue incorporation and adhesion characteristics of a novel fenestrated versus nonfenestrated crosslinked porcine dermal matrix (CPDM) (Bard CollaMend) in a porcine model of ventral hernia repair. STUDY DESIGN Bilateral abdominal wall defects were created in 24 Yucatan minipigs, resulting in 48 defects, which were allowed to mature for 21 days. Twelve defects were repaired with fenestrated CPDM using a preperitoneal technique, 12 with fenestrated CPDM using an intraperitoneal technique, 12 with nonfenestrated CPDM using a preperitoneal technique, and 12 with nonfenestrated CPDM using an intraperitoneal technique. Half of the animals in the intraperitoneal group were euthanized after 1 month, and the other half after 3 months. Similarly, half of the animals in the preperitoneal group were euthanized after 1 month, and the other half after 6 months. Biomechanical testing and histologic evaluation were performed. RESULTS Intraperitoneal placement of the CPDM products resulted in significantly greater adhesed area compared with preperitoneal placement (p < 0.05). Tissue ingrowth into preperitoneal fenestrated and nonfenestrated CPDM resulted in significantly greater incorporation strengths after 6 months compared with 1 month (p = 0.03 and p < 0.0001). Histologic analysis showed significantly greater cellular infiltration, extracellular matrix deposition, and neovascularization, with less fibrous encapsulation through the center of the fenestrations compared with all other sites evaluated, including nonfenestrated grafts. CONCLUSIONS Histologic findings revealed increased tissue incorporation at fenestration sites compared with nonfenestrated grafts regardless of implant location or time in vivo. However, preperitoneal placement resulted in greater incorporation strength, less adhesed area, and lower adhesion scores compared with intraperitoneal placement for both fenestrated and nonfenestrated CPDM.

Indolyl-quinuclidinols inhibit ENOX activity and endothelial cell morphogenesis while enhancing radiation-mediated control of tumor vasculature

There is a need for novel strategies that target tumor vasculature, specifically those that synergize with cytotoxic therapy, in order to overcome resistance that can develop with current therapeutics. A chemistry‐driven drug discovery screen was employed to identify novel compounds that inhibit endothelial cell tubule formation. Cell‐based phenotypic screening revealed that noncytotoxic concentrations of (Z)‐(±)‐2– (1‐benzenesulfonylindol‐3–ylmethylene)‐1‐azabicyclo[2. 2.2]octan‐3‐ol (analog I) and (Z)‐(±)‐2‐(l‐benzylindol‐3‐ylmethylene)‐1‐azabicyclo[2.2.2]octan‐3‐ol (analog II) inhibited endothelial cell migration and the ability to form capillary‐like structures in Matrigel by ≥70%. The ability to undergo neoangiogenesis, as measured in a window‐chamber model, was also inhibited by 70%. Screening of biochemical pathways revealed that analog II inhibited the enzyme ENOX1 (EC50 = 10 µM). Retroviral‐mediated shRNA suppression of endothelial ENOX1 expression inhibited cell migration and tubule formation, recapitulating the effects observed with the small‐molecule analogs. Genetic or chemical suppression of ENOX1 significantly increased radiation‐mediated Caspase3‐activated apoptosis, coincident with suppression of p70S6K1 phosphorylation. Administration of analog II prior to fractionated X‐irradiation significantly diminished the number and density of tumor microvessels, as well as delayed syngeneic and xenograft tumor growth compared to results obtained with radiation alone. Analysis of necropsies suggests that the analog was well tolerated. These results suggest that targeting ENOX1 activity represents a novel therapeutic strategy for enhancing the radiation response of tumors.—Geng, L., Rachakonda, G., Morre, D. J., Morre, D. M., Crooks, P. A., Sonar, V. N., Roti Roti, J. L., Rogers, B. E., Greco, S., Ye, F., Salleng, K. J., Sasi, S., Freeman, M. L., Sekhar, K. R. Indolyl‐quinuclidinols inhibit ENOX activity and endothelial cell morphogenesis while enhancing radiation‐mediated control of tumor vasculature. FASEB J. 23, 2986–2995 (2009). www.fasebj.org

Maternal obesogenic diet induces endometrial hyperplasia, an early hallmark of endometrial cancer, in a diethylstilbestrol mouse model

Thirty-eight percent of US adult women are obese, meaning that more children are now born of overweight and obese mothers, leading to an increase in predisposition to several adult onset diseases. To explore this phenomenon, we developed a maternal obesity animal model by feeding mice a diet composed of high fat/ high sugar (HF/HS) and assessed both maternal diet and offspring diet on the development of endometrial cancer (ECa). We show that maternal diet by itself did not lead to ECa initiation in wildtype offspring of the C57Bl/6J mouse strain. While offspring fed a HF/HS post-weaning diet resulted in poor metabolic health and decreased uterine weight (regardless of maternal diet), it did not lead to ECa. We also investigated the effects of the maternal obesogenic diet on ECa development in a Diethylstilbestrol (DES) carcinogenesis mouse model. All mice injected with DES had reproductive tract lesions including decreased number of glands, condensed and hyalinized endometrial stroma, and fibrosis and increased collagen deposition that in some mice extended into the myometrium resulting in extensive disruption and loss of the inner and outer muscular layers. Fifty percent of DES mice that were exposed to maternal HF/HS diet developed several features indicative of the initial stages of carcinogenesis including focal glandular and atypical endometrial hyperplasia versus 0% of their Chow counterparts. There was an increase in phospho-Akt expression in DES mice exposed to maternal HF/HS diet, a regulator of persistent proliferation in the endometrium, and no difference in total Akt, phospho-PTEN and total PTEN expression. In summary, maternal HF/HS diet exposure induces endometrial hyperplasia and other precancerous phenotypes in mice treated with DES. This study suggests that maternal obesity alone is not sufficient for the development of ECa, but has an additive effect in the presence of a secondary insult such as DES.