BackgroundChronic inflammation is a characteristic feature of diabetic cutaneous wounds. We sought to delineate novel mechanisms involved in the impairment of resolution of inflammation in diabetic cutaneous wounds. At the wound-site, efficient dead cell clearance (efferocytosis) is a pre-requisite for the timely resolution of inflammation and successful healing.Methodology/Principal FindingsMacrophages isolated from wounds of diabetic mice showed significant impairment in efferocytosis. Impaired efferocytosis was associated with significantly higher burden of apoptotic cells in wound tissue as well as higher expression of pro-inflammatory and lower expression of anti-inflammatory cytokines. Observations related to apoptotic cell load at the wound site in mice were validated in the wound tissue of diabetic and non-diabetic patients. Forced Fas ligand driven elevation of apoptotic cell burden at the wound site augmented pro-inflammatory and attenuated anti-inflammatory cytokine response. Furthermore, successful efferocytosis switched wound macrophages from pro-inflammatory to an anti-inflammatory mode.Conclusions/SignificanceTaken together, this study presents first evidence demonstrating that diabetic wounds suffer from dysfunctional macrophage efferocytosis resulting in increased apoptotic cell burden at the wound site. This burden, in turn, prolongs the inflammatory phase and complicates wound healing.

Carica papaya Linn is widely known as a medicinal fruit. We sought to study a standardized fermented papaya preparation (FPP) for its effects on wound healing in adult obese diabetic (db=db) mice. FPP blunted the gain in blood glucose and improved the lipid profile after 8 weeks of oral supplementation. However, FPP did not influence weight gain during the supplementation period. FPP (0.2 g=kg body weight) supplementation for 8 weeks before wounding was effective in correcting wound closure. Studies on viable macrophages isolated from the wound site demonstrated that FPP supplementation improved respiratory-burst function as well as inducible NO production. Reactive oxygen species support numerous aspects of wound healing; NO availability in diabetic wounds is known to be compromised. Diabetic mice supplemented with FPP showed a higher abundance of CD68 as well as CD31 at the wound site, suggesting effective recruitment of monocytes and an improved proangiogenic response. This work provides the first evidence that diabetic-wound outcomes may benefit from FPP supplementation by specifically influencing the response of wound-site macrophages and the subsequent angiogenic response. Given that FPP has a long track record of safe human consumption, testing of the beneficial effects of FPP on diabetic wound-related outcomes in a clinical setting is warranted. Antioxid. Redox Signal. 13, 599-606.

Summary. Inclusion of the BCR±ABL ES probe in routine cytogenetics led to the identification of a subgroup of Philadelphia positive (Ph1) chronic myeloid leukaemia patients characterized by a 5 H -ABL deletion. This anomaly was observed in 5/51 cases (9´8%). Cytological and clinical data suggest that the 5 H -ABL deletion may be associated with dysplastic features of polymorphonuclear cells and metamyelocytes and a short chronic phase duration.

Endogenous nitric oxide (NO) plays important physiological roles in the body. As a small diatomic molecule, NO has been assumed to freely diffuse in tissues with a diffusion rate similar to that in water. However, this assumption has not been tested experimentally. In this study, a modified Clark-type NO electrode attached with a customized aorta holder was used to directly measure the flux of NO diffusion across the aortic wall at 37 degrees C. Experiments were carefully designed for accurate measurements of the apparent NO diffusion coefficient D and the partition coefficient alpha in the aortic wall. A mathematical model was presented for analyzing experimental data. It was determined that alpha = 1.15 +/- 0.11 and D = 848 +/- 45 mum(2)/s (n = 12). The NO diffusion coefficient in the aortic wall is nearly fourfold smaller than the reported diffusion coefficient in solution at 37 degrees C, indicating that NO diffusion in the vascular wall is no longer free, but markedly dependent on the environment in the tissue where these NO molecules are. These results imply that the NO diffusion rate in the vascular wall may be upregulated and downregulated by certain physiological and/or pathophysiological processes affecting the composition of tissues.

Endothelium-derived nitric oxide (NO) is critical in maintaining vascular tone. Accumulating evidence shows that NO bioavailability is regulated by oxygen concentration. However, it is unclear to what extent the oxygen concentration regulates NO bioavailability in the vascular wall. In this study, a recently developed experimental setup was used to measure the NO diffusion fluxes across the aortic wall at different oxygen concentrations. It was observed that for a constant NO concentration at the endothelial surface, the measured NO diffusion flux out of the adventitial surface at [O2]=0 μM is around 5-fold greater than at [O2]=150 μM, indicating that NO is consumed in the aortic wall in an oxygen-dependent manner. Analysis of experimental data shows that the rate of NO consumption in the aortic wall is first order with respect to [NO] and first order with respect to [O2], and the rate constant k1 was determined as (4.0 ± 0.3)×103 M-1s-1. Computer simulations demonstrate that NO concentration distribution significantly changes with oxygen concentration and the effective NO diffusion distance at low oxygen level ([O2]≤25 μM) is significantly longer than that at high oxygen level ([O2]=200 μM). These results suggest that the oxygen-dependent NO consumption may play an important role in dilating blood vessels during hypoxia by increasing the effective NO diffusion distance.

Glucans are known to promote wound repair. Non-cellulosic β-glucans are recognized as potent immunological activators. β-Glucans are generally safe and are known to attenuate the rate of postoperative infection. Glyc101 is a particulate β-glucan isolated from Saccharomyces cerevisiae. In this study, the hypothesis that Glyc101 regulates wound macrophage function was tested. Glyc101 induced TNFα transcription in macrophages isolated from murine wound site. Multiplex assay identified IL-10 and TNFα as two cytokines that are induced by Glyc101 in human blood monocyte derived macrophages. Glyc101-induced TNFα production was observed to be mediated via the TLR-2 and dectin-1 receptors, receptor tyrosine kinases and NFκB activation. In murine wound macrophages, Glyc101 potentiated PMA-induced respiratory burst. In vivo, implantation of Glyc101 enriched PVA-sponges at the wound-site induced TNFα expression in macrophages. Consistently, Glyc101 induced TNFα expression in wound-site macrophages isolated from two patients with chronic wounds. These observations establish the translational significance of the net findings of this study. Activation of wound macrophages by Glyc101 represents one of the potential mechanisms by which this beta-glucan may benefit chronic wounds where inefficient inflammatory response is one of the underlying causes of impaired healing.

Carbon fiber microelectrodes and carbon fiber composite minielectrodes (CFM/CFCM) have been generally used for measurements of nitric oxide (NO) concentration in chemical and biological systems. The response time of a CFM/CFCM is usually from milliseconds to seconds depending on the electrode size, the thickness of coating layers on the electrode, and NO diffusion coefficients of the coating layers. As a result, the time course of recoded current changes (I-t curves) by the CFM/CFCM may be different from the actual time course of NO concentration changes (c-t curves) if the half-life of NO decay is close to or shorter than the response time of the electrode used. This adds complexity to the process for determining rate constants of NO decay kinetics from the recorded current curves (I-t curves). By computer simulations based on a mathematical model, an approximation method was developed for determining rate constants of NO decay from the recorded current curves. This method was first tested and valuated using a commercial CFCM in several simple reaction systems with known rate constants. The response time of the CFCM was measured as 4.7±0.7 seconds (n=5). The determined rate constants of NO volatilization and NO autoxidation in our measurement system at 37 °C are (1.9±0.1)×10 −3 s −1 (n=4) and (2.0±0.3)×10 3 M −1 s −1 (n=7), which are close to the reported rate constants. The method was then applied to determine the rate of NO decay in blood samples from control and smoking exposed mice. It was observed that the NO decay rate in the smoking group is >20% higher than that in control group, and the increased NO decay rate in the smoking group was reversed by 10 μM diphenyleneiodonium chloride (DPI), an inhibitor of flavin enzymes such as leukocyte NADPH oxidase.

Endothelium-derived nitric oxide (NO) plays an important role in maintaining vascular tone. It is known that NO may be consumed by heme proteins, superoxide and oxygen during diffusion from the endothelium to smooth muscle cells in the vascular wall. Due to the limitation of available techniques, it is unclear to what extent these consumptions can affect the diffusion distance of NO, and if the vascular NO consumption could serve as a “sensor” of oxygen concentrations in the blood vessels. In this study, rat aortas were used as an experimental model for studying NO diffusion process in the vascular system. A Clark-type NO electrode was used to directly measure the flux of NO diffusion across the vascular wall at 37 °C. A segment of aorta was isolated from a 12-week old WKY rat. After the aorta was cleaned and surrounding tissue was removed, it was longitudinally opened. A specifically-designed aorta holder was attached on the tip of the Clark-type NO electrode. The aorta holder surface and the electrode tip surface were aligned in the same plane so that the opened aorta segment could be placed flat on the electrode tip surface and pinned to the aorta holder. Using this technique, we measured the flux of NO diffusion across the aortic wall at different oxygen concentration. It was observed that the NO flux increased 6 to 10 fold when oxygen concentrations dropped from 200 μM to zero. A mathematical model describing the steady-state diffusion-reaction was used in analyzing the experimental data. It was found that the rate of NO decay is first order with respect to [O 2 ] and first order with respect to [NO], and hence of the form k[O 2 ][NO]. The rate constant k was determined as (3.8±0.4)x10 −3 μM −1 s −1 (n=6). With this rate constant, the half-life of NO in the aortic wall in the presence of 200 μM O 2 (equilibrium with room air) will be 0.9 seconds. Our results show that the flux and diffusion distance of NO in the aortic wall is largely regulated by oxygen concentration. When oxygen concentrations drop, NO diffusion distance will significantly increase. As a result, the blood vessel will dilate to a larger extent to allow more blood to be delivered to the hypoxic tissues. Therefore this vascular NO consumption appears to play the role of an oxygen sensor in the regulation of blood flow in the body.