Diabetic foot ulcers (DFU) are a major, debilitating complication of diabetes mellitus. Unfortunately, many DFUs are refractory to existing treatments and frequently lead to amputation. The development of more effective therapies has been hampered by the lack of predictive in vitro methods to investigate the mechanisms underlying impaired healing. To address this need for realistic wound healing models, we established patient-derived fibroblasts from DFUs and site-matched controls and used them to construct three-dimensional (3D) models of chronic wound healing. Incorporation of DFU-derived fibroblasts into these models accurately recapitulated the following key aspects of chronic ulcers: reduced stimulation of angiogenesis, increased keratinocyte proliferation, decreased re-epithelialization, and impaired extracellular matrix (ECM) deposition. In addition to reflecting clinical attributes of DFUs, the wound healing potential of DFU fibroblasts demonstrated in this suite of models correlated with in vivo wound closure in mice. Thus, the reported panel of 3D DFU models provides a more biologically-relevant platform for elucidating the cell-cell and cell-matrix related mechanisms responsible for chronic wound pathogenesis and may improve translation of in vitro findings into efficacious clinical applications.
Diabetic foot ulcers (DFUs) are characterized by an unsatisfactory inflammatory and migratory response. Skin inflammation involves the participation of many cells and particularly macrophages. Macrophage function can be modulated by neuropeptides; however, little is known regarding the role of neurotensin (NT) as a modulator of macrophages under inflammatory and hyperglycemic conditions. RAW 264.7 cells were maintained at 10/30 mM glucose, stimulated with/without LPS (1 ?g/mL), and treated with/without NT(10 nM). The results show that NT did not affect macrophage viability. However, NT reverted the hyperglycemia-induced impair in the migration of macrophages. The expression of IL-6 and IL-1? was significantly increased under 10 mM glucose in the presence of NT, while IL-1? and IL-12 expression significantly decreased under inflammatory and hyperglycemic conditions. More importantly, high glucose modulates NT and NT receptor expression under normal and inflammatory conditions. These results highlight the effect of NT on cell migration, which is strongly impaired under hyperglycemic conditions, as well as its effect in decreasing the proinflammatory status of macrophages under hyperglycemic and inflammatory conditions. These findings provide new insights into the potential therapeutic role of NT in chronic wounds, such as in DFU, characterized by a deficit in the migratory properties of cells and a chronic proinflammatory status.
Systemic inflammation is associated with impaired wound healing in diabetes mellitus (DM) patients. Using immunohistochemistry techniques, the authors investigated changes in skin inflammation and skin blood vessels in human and experimental diabetes. Comparing to the non-DM human subjects, the total number of inflammatory cells per biopsy and the number of inflammatory cells around blood vessels, a strong indication of inflammation, were higher in DM subjects irrespective of their risk for developing diabetic foot ulcer. Inflammatory cell infiltration was robustly increased in all DM animal models compared with their non-DM controls. The number and density of blood vessels and CD31 positive proliferating endothelial cells around preexisting skin vessels was also higher in the DM patients. However, there were no differences in the skin blood flow between the non-DM and DM subjects. The number of skin blood vessels was also increased in the DM animals; however, these differences were less obvious than the ones observed for inflammatory cells. We conclude that skin inflammation and skin blood vessel density is increased in diabetic human subjects and in rodent and rabbit models of diabetes.
In this study, we developed a methodology to improve the survival, vascular differentiation and regenerative potential of umbilical cord blood (UCB)-derived hematopoietic stem cells (CD34(+) cells), by co-culturing the stem cells in a 3D fibrin gel with CD34(+)-derived endothelial cells (ECs). ECs differentiated from CD34(+) cells appear to have superior angiogenic properties to fully differentiated ECs, such as human umbilical vein endothelial cells (HUVECs). Our results indicate that the pro-survival effect of CD34(+)-derived ECs on CD34(+) cells is mediated, at least in part, by bioactive factors released from ECs. This effect likely involves the secretion of novel cytokines, including interleukin-17 (IL-17) and interleukin-10 (IL-10), and the activation of the ERK 1/2 pathway in CD34(+) cells. We also show that the endothelial differentiation of CD34(+) cells in co-culture with CD34(+)-derived ECs is mediated by a combination of soluble and insoluble factors. The regenerative potential of this co-culture system was demonstrated in a chronic wound diabetic animal model. The co-transplantation of CD34(+) cells with CD34(+)-derived ECs improved the wound healing relatively to controls, by decreasing the inflammatory reaction and increasing the neovascularization of the wound.
Acute pharyngitis is a frequent pediatric consultation, being group A beta-hemolytic Streptococcus (GABHS) the main bacterial etiology. GABHS screening is controversial in children less than 5 years, because of the low frequency historically reported. Nevertheless, 24% prevalence was recently estimated in this group. The aim of this study was to estimate the GABHS frequency in symptomatic children less than 15 years, for which we performed a retrospective, cross sectional study, to evaluate its presence in the throat of 6691 patients, during 2010. The maximum frequency was observed in children from 5 to 11 years. For children aged 3 and 4, frequency was estimated at 36%, thus seeming to be mandatory to do the screening, as it is done in older children. In younger children, the epidemiology of each patient should be considered before asking for diagnostic tests.
We examined the role of vascular function and inflammation in the development and failure to heal diabetic foot ulcers (DFUs). We followed 104 diabetic patients for a period of 18.4 ± 10.8 months. At the beginning of the study, we evaluated vascular reactivity and serum inflammatory cytokines and growth factors. DFUs developed in 30 (29%) patients. DFU patients had more severe neuropathy, higher white blood cell count, and lower endothelium-dependent and -independent vasodilation in the macrocirculation. Complete ulcer healing was achieved in 16 (53%) patients, whereas 13 (47%) patients did not heal. There were no differences in the above parameters between the two groups, but patients whose ulcers failed to heal had higher tumor necrosis factor-?, monocyte chemoattractant protein-1, matrix metallopeptidase 9 (MMP-9), and fibroblast growth factor 2 serum levels when compared with those who healed. Skin biopsy analysis showed that compared with control subjects, diabetic patients had increased immune cell infiltration, expression of MMP-9, and protein tyrosine phosphatase-1B (PTP1B), which negatively regulates the signaling of insulin, leptin, and growth factors. We conclude that increased inflammation, expression of MMP-9, PTP1B, and aberrant growth factor levels are the main factors associated with failure to heal DFUs. Targeting these factors may prove helpful in the management of DFUs.
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