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Objective: To assess the response of cellular infiltration in wounds treated with Exendin-4., Method: In this study, 16 mice were used. On each mouse, two wounds were produced, one above the other, in order to study the effects of the various treatments carried out. The wounds then received an intradermal injection of either saline (20μl; Group 1) or Exendin-4 (Exe4, 62ng; Group 2) in the upper and lower wounds, respectively. The mice were euthanised in order to collect the wounds at time of abrasion (T0), at 48 hours (T1), 96 hours (T2) and 144 hours (T3). The expression of the glucagon-like peptide-1 receptor (GLP-1R) was evaluated by Western blot in wound lysates. Histological and histochemistry methods were applied in cryosections., Results: In T2 and T3 treated wounds, the mast cells degranulation index increased while GLP-1R expression, tumour necrosis factor (TNF )-α, or heat shock protein (HSP)47 antigens were detected in their cytoplasm. These cells interacted with dendritic cells, vessels or granulocytes. The density of dendritic cells increased progressively, and intercellular connections were found between these cells and vessels. Among the dendritic cells at T2, only M2 macrophages increased. However, M1 cells expressed transforming growth factor (TGF )-β and both interacted with either fibroblasts or with vessels. The number of plasmacytoid dendritic cells increased and established close contacts with regulatory T cells., Conclusion: We propose that after treatment with Exe4, early activation of mast cells is critical in wound healing acceleration. This is crucial in understanding the potential effect of this drug for viable clinical therapies., Declaration of Interest: No potential conflict of interest was reported by the authors.

Mast cells (MCs) are bone marrow-derived cells capable of secreting many active molecules, ranging from the mediators stored in specific granules, some of which have been known about for several decades (histamine, heparin), to small molecules produced immediately upon stimulation (membrane lipid derivatives, nitric oxide), to a host of constitutively secreted, multifunctional cytokines. With the aid of a wide array of mediators, the activated MCs control the key events of inflammation and therefore participate in the regulation of local immune response. On the basis of the structure, origin, principal subtypes, localization and function of these cells, their involvement in injury repair is therefore to be considered in acute and chronic conditions, respectively. The importance of MCs in regulating the healing processes is underscored by the proposed roles of a surplus or a deficit of their mediators in the formation of exuberant granulation tissue (such as keloids and hypertrophic scars), the delayed closure or dehiscence of wounds and the transition of acute to chronic inflammation.

Mast cell histochemistry has been proposed in addition to classic histological methods to estimate the course of traumatic events before and after death. We have addressed the utility of this approach on nine victims of different types of trauma. Sections of wounded skin were stained with hematoxylin and eosin and with fluorescent avidin to tag mast cells. Mast cell numbers were evaluated by both direct and digitalized counts. Intact skin was used as control. The results on mast cells implemented the findings upon hematoxylin and eosin stain and helped to put the wounds and death in chronological sequence. Digitalized morphometry allowed to reduce intra- and inter-observer variation. We conclude that combined histological and histochemical analyses can be of practical use in forensic pathology, that a preliminary setting of the reference values is needed for each laboratory, and that image analysis can be of help for the quantification of the results., (© 2011 American Academy of Forensic Sciences.)

Mast cells are bone marrow derived cells capable of secreting many active molecules: mediators stored in specific granules, such as histamine and heparin; small molecules produced immediately upon stimulation, such as lipid derivatives and nitric oxide; and many constitutively secreted, pleiotropic cytokines. Thanks to these secretion products and perhaps direct cell-cell interactions, mast cells play roles in inflammation and tissue repair, angiogenesis and fibrosis. Mast cells themselves respond to many mediators of their own, giving rise to autocrine loops. Successful anti-allergic therapies have typically targeted the receptors for mast cell secretory products, particularly those for histamine. Among agents directly affecting mast cells, disodium chromoglycate and glucocorticoids are known since some time, while new pharmacological approaches may stem from the recognition of an interference with mast cell growth and differentiation by cyclosporine A, monoclonal antibodies, interferons, and JAK3 inhibitors. The action of agents that affect mast cell differentiation and function is considered here from a cell and tissue biological perspective as a premise to the application of these agents to the clinics, therefore special attention has been paid to references pertaining to humans.

Langerhans cells are a dendritic cell type characteristic of the epidermis. Since mast cells secrete molecules potentially influent on dendritic cell differentiation, we have addressed the degree of proximity between these two cell types in biopsies of skin diseases characterized by massive influx of dendritic cell precursors. By fluorescence microscopy, avidin labeled mast cells were found in contact with CD1a+ dendritic cells. By electron microscopy, contacts between mast cells and blunty dendritic cells were found in areas corresponding to those where CD1a+ cells were localized by immunohistochemistry. We propose that mast cells can induce the differentiation of precursors into Langerhans cells through both the release of short range acting soluble factors and contact-mediating plasma membrane molecules.

We have evaluated the numbers and immunohistochemical positivity for tumor necrosis factor (TNF) alpha of the mast cells in (a) 40 skin samples collected at autopsy from subjects who had survived for a few seconds to 1 h, (b) 10 samples of post-mortem skin lesions and (c) 10 surgical biopsies of healthy skin. Sections were treated with fluoresceinated avidin, to tag mast cell granules, followed by indirect immunohistochemistry for TNF-alpha with polyclonal primary and rhodaminated secondary antibodies. We could confirm a progressive increase in mast cell numbers, which became significant 1 h after trauma. More important, the fraction of mast cells positive for TNF-alpha increased progressively in the same time period and became significantly higher than controls in specimens collected more than 15 min after trauma. Samples from post-mortem lesions had significantly fewer mast cells and fewer TNF-alpha-positive cells than any other group of samples. The results suggest that mast cells are quickly recruited to an injured site in response to trauma and upregulate their TNF-alpha content, which can play an early role in directing tissue response to injury. The forensic pathologist can take advantage from this behavior of mast cells for the evaluation of the timing of early vital lesions.

Human nasal mucosal mast cells contain and secrete tumour necrosis factor (TNF)-alpha, which in turn can stimulate histamine secretion by these cells. Interferon (IFN)-alpha can inhibit TNF-alpha secretion by mast cells in vitro. We have addressed the interrelationships between IFN-alpha and the content in TNF-alpha and number of mast cells in vivo, in the human nasal mucosa. Biopsies were taken from two healthy control patients, two allergic patients and two more allergic patients treated topically with IFN-alpha for two weeks; biopsies from the last two patients were taken both before and after stimulation with the specific allergen. Mast cells were counted upon tagging with rhodaminated avidin and by indirect immunofluorescence for TNF-alpha. Data were subjected to analysis of variance. Mast cell numbers were significantly lower in all allergic patients than in controls (P<0.001). Upon IFN-alpha treatment, TNF-alpha positive mast cells were less than in allergic, untreated patients and the opposite was true for TNF-alpha negative mast cells (p<0.05). Allergen challenge caused selective, significant decrease only in the number of TNF-alpha negative mast cells (p<0.05). The results suggest that upon topical IFN-alpha treatment: (1) mast cells stores of TNF-alpha in the nasal mucosa of allergic patients are decreased; and (2) only TNF-alpha negative cells degranulate in response to allergen challenge. Therefore, one may expect that such a treatment reduces the TNF-alpha burden to the mucosa in these patients.

The histamine content in vital wounds is known to increase, with a zenith after 3 h, and then decrease until 24 h after wounding. We addressed whether this biochemical alteration has a morphological counterpart. Since the main source of skin histamine are mast cells, the distribution and number of these cells was assessed upon labeling with fluorescent avidin and with antibodies to the mast cell specific enzymes, chymase and tryptase. Analyses were performed on skin from 15 healthy controls (from surgical biopsies), from 15 post-mortem lesions and 75 vital lesions, obtained at autopsy from subjects who had survived from a few seconds to 24 h. The number of mast cells per unit area of section surface increased progressively with survival time, up to a maximum in subjects who survived 1-3 h ( p<0.01), and decreased thereafter becoming less than in the controls if lesions had occurred earlier than 6 h before death ( p<0.01). Samples from post-mortem lesions had significantly fewer mast cells than those of any other groups of samples ( p<0.01). We suggest that in association to other histological and circumstantial evidence the analysis of mast cells by affinity cytochemistry can help to discriminate vital from post-mortem lesions and to estimate survival time after lesions.

In wounds that are inflicted at least 60 min before death, histamine levels can increase up to 100%. This functional effect might have a morphometric counterpart. Mast cells play a crucial role in acute inflammatory reactions and in the healing process of wounds. Therefore, the density of these cells was immunohistochemically assessed in tissue from 20 healthy controls (Group 1), 20 vital skin lesions (Group 2) (age range: a few seconds to 1 h), and 20 postmortem lesions (Group 3). A piece of skin close to the vital lesion was also obtained from the homolateral part of the body (Group 4). Mast cell density was significantly higher at the level of the vital lesions (11.28+/-2.44) than elsewhere (healthy controls 7.66+/-1.27, postmortem lesions 4.13+/-1.46, skin close to the vital lesions 4.88+/-1.59). No differences were found between the values assessed in the skin samples close to the vital lesions and in those in the postmortem lesions. Therefore, mast cell richness in the vital lesions exhibited a proportional morphological correlation with previously detected histamine values in cutaneous vital lesions. These results suggest that the detection of mast cells with immunohistochemical techniques can lead to a high level of discrimination (based on statistical data) between antemortem and postmortem lesions. This method could also be used to ascertain the vitality of lesions.

We have demonstrated, with immunohistochemical techniques, the colocalization of tumour necrosis factor-alpha (TNF alpha) with a constitutive neuronal isoform of nitric oxide-synthase (NOS) in granules of the majority (52.77%) of the mast cells (MCs) of healthy human nasal mucosa. Very few cells were positive for NOS alone (2.54%). Some cells were positive for TNF alpha alone (16.73%) or negative for both antigens (18%). Since dim degranulation occurs in MCs of healthy nasal mucosa at any time, we propose that low concentrations of TNF alpha and NOS secreted by these cells are involved not only in the regulation of homeostasis of normal human nasal mucosa, but also in the survival and function of MCs themselves.

Gastric mucosal histamine content, enterochromaffin-like cell density, and mast cell density were studied in 13 subjects under omeprazole therapy, 13 partially gastrectomized subjects with a Billroth II reconstruction, 10 partially gastrectomized subjects with a Roux-en-Y reconstruction, and 9 control subjects. Histamine content was significantly greater both in the subjects with higher gastrinemic levels (omeprazole-treated subjects) and those with more abundant enterogastric reflux (Billroth II subjects) than in controls. Enterochromaffin-like cell density was significantly greater in the omeprazole subjects than in each of the other groups. Mast cell density was significantly greater in Billroth II subjects than in controls. Serum gastrin levels, mucosal histamine content, and enterochromaffin-like cell density were positively correlated. Gastrin was not correlated to mast cell density. These results support the existence of different control pathways for enterochromaffin-like and mast cells. Moreover, they suggest that enterochromaffin-like cells and mast cells are involved in the regulation of gastric secretion and in gastric mucosal injury-repair mechanisms, respectively, due to histamine release.

The number and histochemistry of mast cells were analyzed in surgical specimens of the ileocecal junction and neighboring intestinal segments. All the basophilic cells contained tryptase and some were immunoreactive for chymase, vasoactive intestinal polypeptide, or nitric oxide synthase. The medium density of mast cells per square millimeter was 31.90, 110.38, 72.83, 29.80, and 32.70, in the mucosa, submucosa, inner circular, outer circular, and longitudinal muscle layers, respectively. Mast cell density was higher at the ileocecal junction (for all layers together, 79.29 mast cells/mm2) than elsewhere (mast cells/mm2: ileum, 52.29; cecum, 59.22; cecocolonic junction, 54.65; ascending colon, 48.63). The differences among layers and among segments were significant and might be due to layer- and region-specific mast cell roles. Mast cell richness in the muscle coat, especially in the inner circular muscle layer, might be important in regulating its motility.

Nitric oxide (NO)-synthase immunoreactivity has been detected for the first time in mast cells of human normal nasal mucosa, with an antibody specific for neuronal NO-synthase. Intense immunoreactivity was revealed in secretion granules of mast cells but was found in mast cell granules free in the extracellular matrix only in some instances; no reactivity was found in the cytoplasm of this or other cell types. These findings suggest that human nasal mast cells contain a particulate isoform of NO-synthase, which shares epitopes with neuronal NO-synthase and is rapidly removed from granules upon exocytosis.

The response to wounds until healing requires the activity of many cell types coordinate in space and time, so that the types of cells in a wound and their localization may be of help to date lesions with respect to death, which would be useful in forensic pathology. Cells reacting to injury include dendritic cells; the early reaction of these cells to skin wounding has not yet been investigated in humans, which was the aim of this study. Samples of wounded and control skin were taken at autopsy and analyzed by affinity histochemistry. Both epidermal and dermal MHC-II+ cells increased transiently in number within the first hour after wounding, then decreased. In the epidermis the increase affected also CD1a+ cells, i.e. well differentiated Langherhans cells, which however increased less, earlier and for a shorter time period than MHC-II+ cells. Dermal MHC-II+ cells became part of a perivascular mononuclear cell infiltrate visible in the subpapillary dermis by 60 min after wounding, which contained also mast cells. The immediately perivascular MHC-II+ cells were DC-SIGN- and CD11c-, while MHC-II+, DC-SIGN+, CD11c+ dendritic cells were predominantly located at the periphery of infiltrates and some were near the epidermis. Mast cells underwent degranulation, besides increase in number, in the first hours after wounding. The results suggest that skin dendritic cells, including Langerhans cells, participate to the early response to wounding in concert with mast cells, and that subpapillary blood vessels are primary sites of cell infiltration during that response in humans. The results show that the ratio between CD1a positive and MHC-II positive cells in the epidermis, the degranulation index of mast cells and the relative volume of MHC-II positive cells in the dermis can be added to the tools useful to distinguish vital from post mortem lesions and, the first two of them, to estimate the interval between a lesion and death. [ABSTRACT FROM AUTHOR]