Comparison of interleukin 10 homologs on dermal wound healing using a novel human skin ex vivo organ culture model.

BACKGROUND: Anti-inflammatory cytokine interleukin (IL)-10 has been shown to induce regenerative healing in postnatal wounds. A viral homolog of IL-10 produced by human cytomegalovirus (CMV IL-10) similarly generates potent immunoregulatory effects, but its effects on wound healing have not been investigated. Currently, there are limited cost-effective methods of screening vulnerary therapeutics. Taken together, we aim to develop and validate a novel human ex vivo dermal wound model and hypothesize that CMV IL-10 will enhance dermal wound healing. METHODS: Full-thickness circular (6-mm) explants were taken from surgical skin samples and 3-mm full-thickness wounds were created. Explants were embedded in collagen I matrix and maintained in specially formulated media with the epidermis at air-liquid interface, and treated with human IL-10 or CMV IL-10 (200 ng/mL). The viability of cultured explants was validated by histology and lactate dehydrogenase (LDH) activity. Epithelial gap, epithelial height, basal keratinocyte migration, vascular endothelial growth factor levels, and neovascularization were measured at days 3 and 7 to determine IL-10 effects on wound healing. RESULTS: Culture explants at day 7 appeared similar to fresh skin in morphology, cell, and vessel density. By day 14, the epidermis separated from the dermis and the cell density diminished. Day 7 wounds appeared viable with advancing epithelial and basal keratinocyte migration with no evidence of necrosis. Cytotoxicity analysis via the quantification of LDH revealed no differences between controls and treated groups. There was a slight increase in the quantity of LDH in media at day 3; however, this decreased at day 5 and continued to decline up to day 21. CMV IL-10 treatment resulted in a significant decrease in the epithelial gap and an increase in epithelial height. There were no differences in the rates of basal keratinocyte migration at day 7 between treated and control groups. Interestingly, human IL-10 increased vascular endothelial growth factor expression and neovascularization compared with controls. CONCLUSIONS: The human ex vivo wound model provides a simple and viable design to study dermal wound healing. Both IL-10 homologs demonstrate vulnerary effects. The viral homolog demonstrates enhanced effects on wound closure compared with human IL-10. These data represent a novel tool that can be used to screen therapeutics, such as CMV IL-10, before preclinical studies.

Adenoviral-mediated gene transfer of insulin-like growth factor 1 enhances wound healing and induces angiogenesis.

BACKGROUND: Chronic wounds are characterized by a wound healing and neovascularization deficit. Strategies to increase neovascularization can significantly improve chronic wound healing. Insulin-like growth factor (IGF)-1 is reported to be a keratinocyte mitogen and is believed to induce angiogenesis via a vascular endothelial growth factor (VEGF)-dependent pathway. Using a novel ex vivo human dermal wound model and a diabetic-impaired wound healing murine model, we hypothesized that adenoviral overexpression of IGF-1 (Ad-IGF-1) will enhance wound healing and induce angiogenesis through a VEGF-dependent pathway. METHODS: Ex vivo: 6-mm full-thickness punch biopsies were obtained from normal human skin, and 3-mm full-thickness wounds were created at the center. Skin explants were maintained at air liquid interface. Db/db murine model: 8-mm full-thickness dorsal wounds in diabetic (db/db) mice were created. Treatment groups in both human ex vivo and in vivo db/db wound models include 1×10(8) particle forming units of Ad-IGF-1 or Ad-LacZ, and phosphate buffered saline (n=4-5/group). Cytotoxicity (lactate dehydrogenase) was quantified at days 3, 5, and 7 for the human ex vivo wound model. Epithelial gap closure (hematoxylin and eosin; Trichrome), VEGF expression (enzyme-linked immunosorbent assay), and capillary density (CD 31+CAPS/HPF) were analyzed at day 7. RESULTS: In the human ex vivo organ culture, the adenoviral vectors did not demonstrate any significant difference in cytotoxicity compared with phosphate buffered saline. Ad-IGF-1 overexpression significantly increases basal keratinocyte migration, with no significant effect on epithelial gap closure. There was a significant increase in capillary density in the Ad-IGF-1 wounds. However, there was no effect on VEGF levels in Ad-IGF-1 samples compared with controls. In db/db wounds, Ad-IGF-1 overexpression significantly improves epithelial gap closure and granulation tissue with a dense cellular infiltrate compared with controls. Ad-IGF-1 also increases capillary density, again with no significant difference in VEGF levels in the wounds compared with control treatments. CONCLUSIONS: In two different models, our data demonstrate that adenoviral-mediated gene transfer of IGF-1 results in enhanced wound healing and induces angiogenesis via a VEGF-independent pathway. Understanding the underlying mechanisms of IGF-1 effects on angiogenesis may help produce novel therapeutics for chronic wounds or diseases characterized by a deficit in neovascularization.

Pseudotyped adeno-associated viral vectors for gene transfer in dermal fibroblasts: implications for wound-healing applications.

BACKGROUND: Cell-specific gene transfer and sustained transgene expression are goals of cutaneous gene therapy. Pseudotyping strategy with adeno-associated viral (AAV) vectors has the potential to confer unique cellular tropism and transduction efficiency. We hypothesize that pseudotyped AAV vectors have differential tropism and transduction efficiency under normal and wound conditions in dermal fibroblasts. MATERIALS AND METHODS: We packaged AAV2 genome with green fluorescent protein reporter in capsids of other serotypes, AAV5, AAV7, and AAV8, producing pseudotyped vectors AAV2/5, AAV2/7, and AAV2/8, respectively. Murine and human dermal fibroblasts were transduced by the different pseudotypes for 24 h at multiplicities of infection 10(2), 10(3), 10(4), and 10(5). We assessed transduction efficiency at days 3 and 7. Experiments were repeated in a simulated wound environment by adding 10 ng/mL platelet-derived growth factor-B to culture media. RESULTS: Transduction efficiency of the pseudotyped AAV vectors was dose dependent. Multiplicity of infection 10(5) resulted in significantly higher gene transfer. Under normal culture conditions, the pseudotyping strategy conferred differential transduction of dermal fibroblasts, with significantly enhanced transduction of murine cells by AAV2/5 and AAV2/8 compared with AAV2/2. Adeno-associated virus 2/8 was more efficacious in transducing human cells. Under wound conditions, transduction efficiency of AAV2/2, 2/5, and 2/8 was significantly lower in murine fibroblasts. At day 3 under wound conditions, all vectors demonstrated similar transduction efficiency, but by day 7, the three pseudotyped vectors transduced significantly more murine cells compared with AAV2/2. However, in human cells, there was no significant difference in the transduction efficiency of each pseudotype between normal and wound conditions at both 3 and 7 d. CONCLUSIONS: The AAV pseudotyping strategy represents a gene transfer technology that can result in differential transduction of dermal fibroblasts. The differences in transduction efficiency in murine and human dermal fibroblasts in both the normal and wound environment highlight issues with translatability of gene transfer techniques. These data provide a template for using pseudotyped AAV vectors in cutaneous applications.

Systemic Allergic Reactions and Anaphylaxis Associated with Allergen Immunotherapy.

Subcutaneous allergen immunotherapy (SCIT) is a proven treatment of allergic rhinitis, asthma, atopic dermatitis, and prevention of Hymenoptera venom anaphylaxis. The known benefit of SCIT, however, must be considered in each patient relative to the potential risks of systemic allergic reactions (SRs). A mean of 1 SR per 1000 injection visits (0.1%) was estimated to occur between 2008 and 2018. Life-threatening anaphylactic events are estimated to occur in 1/160,000 injection visits. The factors that contribute to SRs and fatal reactions (FRs) are reviewed. Risk management strategies are proposed to prevent and decrease future SCIT associated with SRs, anaphylaxis, and FR.

Demographic Undertones for Sepsis Mortality in a Community-Based Hospital.

BACKGROUND: Sepsis continues to take main stage in healthcare. Therefore, it remains crucial to elucidate contributors to sepsis mortality. The aim of this study is to determine the impact of race, insurance type, and code status on sepsis mortality in a community health system. METHODS: We conducted a retrospective cohort study of inpatient adults of any sex, race, and insurance type with a diagnosis of sepsis, severe sepsis, septic shock, or pneumonia. RESULTS: We included 913 patients, with an average age of 69 years for expired patients and 62 years for non-expiring patients (P < 0.0001). After controlling for other variables, patients who presented as comfort care arrest were 4.3 (95% confidence interval (CI): 1.8 to 9.9, P = 0.0007) times more likely to have died than full code patients. Those who were comfort care only were 10.6 (95% CI: 0.8 to 140.6, P = 0.0741) times more likely to have died than the full code, although this was not statistically significant. CONCLUSIONS: The results suggest that patients who are comfort care arrest have an increased risk of sepsis mortality. The results show no impact of insurance type or race on sepsis mortality, which is in contrast to some existing literature. The study suggests that institutions may need to investigate internal variables related to sepsis mortality.

The role of interleukin-10 and hyaluronan in murine fetal fibroblast function in vitro: implications for recapitulating fetal regenerative wound healing.

BACKGROUND: Mid-gestation fetal cutaneous wounds heal scarlessly and this has been attributed in part to abundant hyaluronan (HA) in the extracellular matrix (ECM) and a unique fibroblast phenotype. We recently reported a novel role for interleukin 10 (IL-10) as a regulator of HA synthesis in the fetal ECM, as well as the ability of the fetal fibroblast to produce an HA-rich pericellular matrix (PCM). We hypothesized that IL-10-mediated HA synthesis was essential to the fetal fibroblast functional phenotype and, moreover, that this phenotype could be recapitulated in adult fibroblasts via supplementation with IL-10 via an HA dependent process. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the differences in functional profile, we compared metabolism (MTS assay), apoptosis (caspase-3 staining), migration (scratch wound assay) and invasion (transwell assay) between C57Bl/6J murine fetal (E14.5) and adult (8 weeks) fibroblasts. We found that fetal fibroblasts have lower rates of metabolism and apoptosis, and an increased ability to migrate and invade compared to adult fibroblasts, and that these effects were dependent on IL-10 and HA synthase activity. Further, addition of IL-10 to adult fibroblasts resulted in increased fibroblast migration and invasion and recapitulated the fetal phenotype in an HA-dependent manner. CONCLUSIONS/SIGNIFICANCE: Our data demonstrates the functional differences between fetal and adult fibroblasts, and that IL-10 mediated HA synthesis is essential for the fetal fibroblasts' enhanced invasion and migration properties. Moreover, IL-10 via an HA-dependent mechanism can recapitulate this aspect of the fetal phenotype in adult fibroblasts, suggesting a novel mechanism of IL-10 in regenerative wound healing.