Comparison of immunohistochemistry for activated caspase-3 and cleaved cytokeratin 18 with the TUNEL method for quantification of apoptosis in histological sections of PC-3 subcutaneous xenografts

The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling (TUNEL) technique has been extensively used for the detection and quantification of apoptosis in histological tissue sections. However, the interpretation and specificity of this assay have been controversial. With accumulating knowledge of the molecular mechanisms of cell death and the discovery of the caspases as key mediators of apoptosis, more direct and earlier measurements of apoptosis in tissue sections have emerged. This study, using antibodies that specifically recognize activated caspase-3 and caspase-cleaved cytokeratin (CK) 18, evaluated whether immunohistochemical stains would improve the detection and quantification of apoptosis in tissue sections, compared with the TUNEL assay. Tumour xenografts of the prostate cancer cell line PC-3 were used as an example, since these tissues contain large numbers of cells undergoing apoptosis. Apoptotic cells were quantified and apoptotic indices were calculated by computer-assisted image analysis following identification of apoptotic cells by morphological analysis, the TUNEL assay, activated caspase-3 and cleaved CK18 immunohistochemistry. The results indicated that activated caspase-3 immunohistochemistry was an easy, sensitive, and reliable method for detecting and quantifying apoptosis in this model. An excellent correlation (R = 0.89) between the apoptotic indices obtained using activated caspase-3 and cleaved CK18 immunostaining was observed. A good correlation (R = 0.75) between the apoptotic indices obtained using activated caspase-3 immunostaining and the TUNEL assay was also found. Activated caspase-3 immunohistochemistry is therefore recommended for the detection and quantification of apoptosis in tissue sections.     

异氟醚对膀胱癌 5637 细胞恶性生物学行为及化疗敏感性的影响

目的 探究异氟醚对膀胱癌 5637 细胞恶性生物学行为及化疗敏感性的影响。 方法 单独或联合 给予异氟醚及顺铂处理膀胱癌 5637 细胞, 将细胞随机分为 4 组: 对照组、 异氟醚组、 顺铂组和异氟醚 + 顺 铂联合处理组。 BrdU 染色检测 BrdU 阳性细胞数; 流式细胞仪检测细胞凋亡率和细胞周期分布; Transwell 检测侵袭细胞数; 划痕愈合实验检测划痕愈合率; Western 印迹检测 Ki67、 PCNA、 Bax、 Bcl-2、 cleaved caspase-3 和 caspase-3 蛋白表达; 结果 与顺铂组相比较, 异氟醚 + 顺铂联合处理组 BrdU 阳性细胞数和 Ki67、 PCNA 蛋白表达降低, G0 / G1期比率和 S 期细胞数降低, G2 / M 期比率升高, 细胞凋亡率和 Bax / Bcl-2、 cleaved caspase-3 / caspase-3 比值升高, 侵袭细胞数、 划痕愈合率降低, 均有显著性差异 (P< 0. 05)。 结论 异氟醚可通过抑制细胞增殖、 侵袭、 迁移, 诱导细胞凋亡, 提高 5637 细胞对顺铂的敏感性。     

Expression of tumor necrosis factor alpha converting enzyme in endocrine cancers

Tumor necrosis factor alpha converting enzyme (TACE) mediates shedding of human epidermal growth factor receptor-4 (HER4). Recent data suggest that released HER4 intracellular domain (4ICD) induces apoptosis in breast cancer.TACE expression, as measured by immunohistochemical analysis, was observed in 183 of 383 breast carcinomas, 39 of 217 ovarian carcinomas, and 16 of 24 and 17 of 24 hormonesensitive and hormone-insensitive prostate carcinomas, respectively. HER4 expression was detected in breast carcinomas by using 2 antibodies recognizing an extracellular or intracellular epitope. TACE expression was predominantly seen in tumors with high levels of 4ICD and membranous HER4. Apoptotic activity was measured by the terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) assay and cleaved caspase-3 staining in breast carcinomas. There was no significant association between cleaved caspase-3 or TUNEL positivity and 4ICD, whereas TUNEL positivity was seen predominantly in tumors with high levels of internalized HER4. The data presented herein show TACE expression in endocrine cancers and further support a role for TACE in breast cancer apoptosis.     

Apoptosis in acute shigellosis is associated with increased production of Fas/Fas ligand,perforin, caspase-1, and caspase-3 but reduced production of Bcl-2 and interleukin-2

Shigella dysenteriae type 1-induced apoptotic cell death in rectal tissues from patients infected with Shigella dysenteriae type 1 was studied by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) technique and annexin V staining. Expression of proteins and cytokines participating in the apoptotic process (caspase-1, caspase-3, Fas [CD95], Fas ligand [Fas-L], perforin, granzyme A, Bax, WAF-1, Bcl-2, interleukin-2 [IL-2], IL-18, and granulocyte-macrophage colony-stimulating factor) in tissue in the acute and convalescent stages of dysentery was quantified at the single-cell level by in situ immunostaining. Apoptotic cell death in the lamina propria was markedly up-regulated at the acute stage (P < 0.05), where an increased number of necrotic cells were also seen. Phenotypic analysis of apoptotic cells revealed that 43% of T cells (CD3), 10% of granulocytes (CD15), and 5% of macrophages (CD56) underwent apoptosis. Increased activity of caspase-1 persisted in the rectum up to 1 month after onset. More-extensive expression of Fas, Fas-L, perforin, caspase-3, and IL-18, but not IL-2, at the acute stage than at the convalescent stage was observed. Increased expression of caspase-3 and IL-18 in tissues with severe inflammation compared to expression in those with mild inflammation was evident, implying a possible role in the perpetuation of inflammation. Significantly reduced cell death during convalescence was associated with a significant up-regulation of Bcl-2, Bax, and WAF-1 expression in the rectum compared to that in the acute phase of infection. Thus, induction of apoptosis at the local site in the early phase of S. dysenteriae type 1 infection was associated with a significant up-regulation of Fas/Fas-L and perforin and granzyme A expression and a down-regulation of Bcl-2 and IL-2, which promote cell survival.     

The imbalanced mixed lymphocyte reaction between maternal and fetal lymphocytes as well as between the lymphocytes of adult children and their parents is mediated by activated CD8+ T cells

PROBLEM: The mixed lymphocyte reaction (MLR) between maternal and fetal lymphocytes as well as between adult children and their parents is imbalanced in bidirectional mixed lymphocyte cultures. The present investigation was aimed at examining which type of T cells has primary responsibility for this phenomenon. METHODS: Two-way mixed lymphocyte cultures between lymphocytes of five female and five male adults and five male and five female newborns against lymphocytes of their parents were performed. Unrelated adults served as the controls. Before and after 72 hr incubation the mixed leukocytes cultures (MLCs) were sorted by flow cytometry according to the surface markers CD4 and CD69 or CD8 and CD69. The single cell populations within the four resulting fractions were discriminated by fluoresence in situ hybridization (FiSH) using xy-DNA probes. RESULTS: Before incubation the share of CD8(+) CD69(+) cells in the MLCs between mothers and their newborn infants was significantly shifted toward the newborn cells (P < 0.01). After 72 hr incubation the CD8(+) CD69(+) ratio in the MLCs between mother and newborn cells and between adult children (male as well female) and their parents showed a significant shift of the CD8(+) CD69(+) cells toward the children's direction. All other MLCs showed a balanced cell population for all investigated cell types, both before and after incubation. CONCLUSION: The imbalanced MLR between maternal and fetal lymphocytes and lymphocytes of adult children against their parents is mediated by a specific imbalance of the activation of CD8(+) but not of CD4(+) T cells.     

The accumulation of inflammatory cells in synovial sheath and epitenon during adhesion formation in healing rat flexor tendons.

The accumulation of inflammatory cells in synovial tissue was studied using indirect immunofluorescence assays on cell cultures and frozen tissue sections of healing rat digital flexor tendons. Flexor tendons were collected from rats 3, 7 and 14 days after crush injury. Tendon sheath and epithenon cells were isolated by sequential enzymic digestion and cultured for 2 days. Subpopulations of synovial and inflammatory cells were identified with MoAbs against cell surface glycoproteins present on B lymphocytes (CD45), T lymphocytes (CD2, CD4, CD8), macrophages (CD14) and endothelial cells. A phagocytosis assay was also used to identify macrophages. We report a substantial increase in the number of T lymphocytes (mainly helper/inducer) and phagocytotic cells with monocyte/macrophage surface markers in tendon sheath and epitenon 3 days after crush injury. The infiltration of inflammatory cells into synovial sheath and epitenon preceded an increase in fibronectin production by tendon cells which was seen 7 days after injury. To study the interaction between T lymphocytes and synovial cells in vitro, we established synovial fibroblast-like type B cell cultures and used stimulated and non-stimulated T lymphocytes in cell binding assays. We observed increased adhesiveness between unstimulated synovial cells and synovial cells previously cultured with activated and non-activated T lymphocytes. ELISA inhibition studies have shown an increase in fibronectin production by synovial fibroblasts co-cultured with stimulated CD4+ T lymphocytes. We suggest that the presence of inflammatory cells in synovial sheath and epitenon during tendon healing induces synovial fibroblasts and epitenon cells to increase their production of fibronectin, which provides a scaffold for subsequent adhesion formation.     

Structural changes in hair follicles and sebaceous glands of hairless mice following exposure to sulfur mustard

Sulfur mustard (SM) is a bifunctional alkylating agent causing skin inflammation, edema and blistering. A hallmark of SM-induced toxicity is follicular and interfollicular epithelial damage. In the present studies we determined if SM-induced structural alterations in hair follicles and sebaceous glands were correlated with cell damage, inflammation and wound healing. The dorsal skin of hairless mice was treated with saturated SM vapor. One to seven days later, epithelial cell karyolysis within the hair root sheath, infundibulum and isthmus was apparent, along with reduced numbers of sebocytes. Increased numbers of utriculi, some with connections to the skin surface, and engorged dermal cysts were also evident. This was associated with marked changes in expression of markers of DNA damage (phospho-H2A.X), apoptosis (cleaved caspase-3), and wound healing (FGFR2 and galectin-3) throughout pilosebaceous units. Conversely, fatty acid synthase and galectin-3 were down-regulated in sebocytes after SM. Decreased numbers of hair follicles and increased numbers of inflammatory cells surrounding the utriculi and follicular cysts were noted within the wound 3–7 days post-SM exposure. Expression of phospho-H2A.X, cleaved caspase-3, FGFR2 and galectin-3 was decreased in dysplastic follicular epidermis. Fourteen days after SM, engorged follicular cysts which expressed galectin-3 were noted within hyperplastic epidermis. Galectin-3 was also expressed in basal keratinocytes and in the first few layers of suprabasal keratinocytes in neoepidermis formed during wound healing indicating that this lectin is important in the early stages of keratinocyte differentiation. These data indicate that hair follicles and sebaceous glands are targets for SM in the skin.     

Immuno-inflammatory cell dynamics during cutaneous wound healing

The process of wound healing begins with an inflammatory reaction that is principally dependent on cellular immune elements. Although the involvement in wound healing of leucocytes that mediate nonspecific immunity (e.g. neutrophils and macrophages) is well known, the participation of cells which prime the immune reaction, i.e. the lymphocytes, requires further investigation. This study was performed to examine the temporal sequence and kinetics of these cells during cutaneous wound repair. The model selected was a full-thickness skin excisional wound made on the flanks of female Wistar rats. At time points ranging from 3 h to 2 wk wound samples were processed for polyester wax-embedding. Target antigens were identified and monitored quantitatively in sections stained immunohistochemically. Monoclonal antibodies against neutrophils, macrophages, pan T cells and cytotoxic populations of lymphocytes were used. The results showed that these cells are involved in the process of wound healing in a distinctive dynamic pattern. The accumulation of CD3⁺ T lymphocytes in the wound bed was mainly associated with the phase of granulation tissue formation. Intraepithelial CD3⁺ T lymphocytes were detected in considerable numbers within the regenerating epidermis. The cytotoxic cell populations (OX8⁺) were classified morphologically into the cytotoxic/suppressor subset of T cells and NK cells. The OX8⁺ T cells were shown to have a kinetic pattern similar to CD3⁺ T lymphocytes but of a lower magnitude. The accumulation of OX8⁺ NK cells was confined to the early inflammatory phase of repair. It is concluded that CD3⁺ T lymphocytes as well as OX8⁺ cytotoxic populations of the immune system are involved in the process of cutaneous wound healing in temporal sequences which suggest that they may be involved in its modulation.     

Apoptosis of macrophages and T cells in tuberculosis associated caseous necrosis

Immunity against mycobacteria is almost exclusively confined to epithelioid cell granulomas, where a long-lasting but labile balance exists between host and bacilli. The relationship between immunity and mycobacteria results in regression, growth, or caseation of granulomas. To prove whether caseation is associated with apoptosis, biopsy specimens of patients with tuberculosis were analysed by electron microscopy and by in situ end-labelling combined with immunofluorescence. Apoptotic cells were not detected in regressive granulomas. Whereas productive granulomas without histologically recognizable caseous necrosis revealed only single apoptotic cells, large numbers of apoptotic CD68+ macrophages and apoptotic CD3+, CD45RO+ T cells were observed within caseous foci. As prime candidates undergoing and/or eliciting apoptosis, vital cells surrounding caseous foci were characterized. Immunohistochemistry showed that the majority of vital CD68+ macrophages surrounding caseous foci are negative for the anti-apoptotic protein bcl2, but positive for the pro-apoptotic protein bax. In situ hybridization combined with immunofluorescence demonstrated that the majority of the adjacent lymphocytes are activated CD3+, CD45RO+ cells expressing the pro-inflammatory cytokine interferon gamma (IFN gamma) and the death ligand FasL. These results suggest that caseation is strongly associated with apoptosis of macrophages and T lymphocytes; that the onset of apoptosis in macrophages may be promoted by the lack of bcl2 and the abundance of bax; and that activation-induced cell death (AICD) may be responsible for the apoptosis of T cells.     

Superoxide dismutase inactivation in pathophysiology of asthmatic airway remodeling and reactivity

Airway hyperresponsiveness and remodeling are defining features of asthma. We hypothesized that impaired superoxide dismutase (SOD) antioxidant defense is a primary event in the pathophysiology of hyperresponsiveness and remodeling that induces apoptosis and shedding of airway epithelial cells. Mechanisms leading to apoptosis were studied in vivo and in vitro. Asthmatic lungs had increased apoptotic epithelial cells compared to controls as determined by terminal dUTP nick-end labeling-positive cells. Apoptosis was confirmed by the finding that caspase-9 and -3 and poly (ADP-ribose) polymerase were cleaved. On the basis that SOD inactivation triggers cell death and low SOD levels occur in asthma, we tested whether SOD inactivation plays a role in airway epithelial cell death. SOD inhibition increased cell death and cleavage/activation of caspases in bronchial epithelial cells in vitro. Furthermore, oxidation and nitration of MnSOD were identified in the asthmatic airway, correlating with physiological parameters of asthma severity. These findings link oxidative and nitrative stress to loss of SOD activity and downstream events that typify asthma, including apoptosis and shedding of the airway epithelium and hyperresponsiveness.     

Role of macrophage and smooth muscle cell apoptosis in association with oxidized low-density lipoprotein in the atherosclerotic development.

To examine the role of the apoptosis of macrophages and smooth muscle cells in the development of atherosclerosis, human aortic tissues with intimal lesions were immunostained with antibodies against terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL), single-stranded DNA (clone F7-26), and active caspase-3. Apoptotic cells were detected in the intima using both TUNEL and single-stranded DNA, however, the latter method was the more sensitive one for detecting apoptotic cells in the early stages of atherosclerosis. The number of apoptotic cells increased as the disease progressed. It implies that the apoptosis of intimal cells is involved in the formation of atherosclerotic lesions. In addition, quantitative analyses of the cell types undergoing apoptosis using double-immunostaining revealed that the susceptibility of macrophages and smooth muscle cells to apoptosis was greater specifically in atheroma than in the other atherosclerotic lesions, and macrophages were more susceptible to apoptosis than smooth muscle cells. The frequency and spatial distribution of oxidized low-density lipoprotein (oxLDL) (FOH1a/DLH3)-positive cells were examined by immunohistochemistry, and the results resembled those of apoptotic cells. The number of oxLDL-positive cells in the intima significantly correlated with the susceptibility of smooth muscle cells, but not with that of macrophages, to apoptosis. These results suggest that oxLDL affects the apoptosis of smooth muscle cells during the atherosclerotic development.     

Immunohistochemical analysis of cleaved caspase-3 detects high level of apoptosis frequently in diffuse large B-cell lymphomas of the central nervous system

The purpose of the present paper was to examine the level of apoptosis and the relationships among apoptosis, apoptosis-associated proteins, and proliferating potential in lymphoma tissues to clarify the characteristics of apoptosis in diffuse large B-cell lymphomas (DLBCL) of the central nervous system (CNS). The formalin-fixed, paraffin-embedded tissues of CNS and non-CNS DLBCL (20 cases each) were studied by terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) and immunohistochemistry, using antibodies against single-stranded DNA (ssDNA), cleaved caspase-3, bcl-2, bax, p53, Fas and Ki-67. The cleaved caspase-3 immunohistochemistry detected apoptosis of the lymphoma cells most sensitively compared to TUNEL and ssDNA immunohistochemistry. High expression (grade + + or + + +) of cleaved caspase-3 was found more frequently in CNS DLBCL (11 cases, 55%) than non-CNS DLBCL (three cases, 15%; P = 0.009). Bax-positivity of lymphoma cells was increased in six cases of CNS DLBCL, which also showed high positivity of cleaved caspase-3. There was no significant correlation between the cleaved caspase-3-positivity and the Ki-67 positivity. The present study indicates that the number of apoptotic cells and expression level of cleaved caspase-3 were significantly higher in CNS DLBCL than non-CNS DLBCL, and that the correlation of bax and cleaved caspase-3 expression was often present in CNS DLBCL.     

Extrinsic and intrinsic pathways of apoptosis in aseptic loosening after total hip replacement

Particle-induced osteolysis is a major cause of aseptic loosening after total joint replacement. The purpose of the current study was to identify various apoptosis-related pathways in the cellular response to wear debris. Fas receptor, BAK and caspase-3 cleaved were evaluated immunohistochemically in capsules and interface membranes from patients with aseptic hip implant loosening. Moreover, we investigated local cellular proliferation, documented by the presence of Ki-67, to evaluate the proportion of apoptosis in relation to the proliferation in the different cells. We detected a strong expression of caspase-3 cleaved, Fas and BAK in macrophages, giant cells and T-lymphocytes. The fibroblasts showed caspase-3 cleaved and BAK, but no Fas staining. Demonstrated by Ki-67 staining, we found increased proliferation of macrophages and fibroblasts. Statistical analysis showed a significant positive correlation (p<0.001) between the above mentioned results and the presence of wear debris. The intensity of apoptosis and proliferation differed, depending on the extent of osteolysis. Overall, four different patterns of immunoreactivity were identified. We think, however, that in particle-induced osteolysis apoptosis is pathologically increased - a phenomenon also seen in other diseases. In these instances, the number and degree of apoptotic reactions are so great that the resulting cell remains cannot be completely removed. This leads to an increased excretion of fibrogenic mediators that could be responsible for increased proliferation of fibroblasts in spite of the increased apoptosis. Moreover, it leads to an increased excretion of cytokines which could be responsible for the activation of osteoclasts.     

糖尿病足伤口皮肤细胞凋亡情况及AGEs对人皮肤成纤维细胞凋亡的影响

 目的：检测糖尿病足伤口皮肤细胞凋亡情况,探讨晚期糖基化终产物（AGEs）对人皮肤成纤维细胞凋亡的影响。方法：选择36例足部伤口患者,糖尿病组18例,非糖尿病组18例。对2组临床和生化数据进行统计分析。采用免疫组化和TUNEL法检测伤口皮肤细胞凋亡情况。体外培养人原代皮肤成纤维细胞,分别给予正常浓度糖、持续高糖、波动高糖和AGEs干预72 h,采用蛋白质印迹法和流式细胞术测定细胞凋亡情况。结果：与非糖尿病组相比,糖尿病组血糖水平明显增高,伤口持续时间长（P<0.05）。Cleaved caspase-3在非糖尿病组和糖尿病组的免疫反应评分分别为1.04±0.23和3.04±0.31（P<0.05）;TUNEL检测非糖尿病组和糖尿病组的细胞凋亡指数分别为（3.8±0.8）%和（8.4±1.5）%（P<0.05）。人原代皮肤成纤维细胞在正常浓度糖、持续高糖、高糖波动、AGEs干预下,cleaved caspase-3蛋白表达水平分别为0.80±0.13、1.22±0.18、1.46±0.32和1.83±025,凋亡率分别为（2.43±0.19）%、（2.89±0.51）%、（3.99±0.24）%和（6.83±0.36）%。 AGEs组cleaved caspase-3蛋白表达水平和凋亡率均明显高于正常浓度糖组和持续高糖组（P<0.05）。结论：糖尿病皮肤创面细胞凋亡增加,可能是糖尿病皮肤伤口难愈的重要原因之一。与持续高糖、高糖波动组相比,AGEs促进人皮肤成纤维细胞凋亡的作用更强。     

达沙替尼对人骨髓间充质干细胞生物学特性的影响

目的:在体外探索达沙替尼对人骨髓来源间充质干细胞(hBMSCs)的活力、迁移、细胞周期和凋亡的影响以及潜在的信号通路,以评估达沙替尼在临床应用中对骨髓造血微环境的影响。方法:CCK-8法检测细胞活力;划痕实验检测细胞迁移;流式细胞术检测细胞周期和凋亡;同时采用吖啶橙/溴化乙啶法检测细胞凋亡;酶联免疫吸附实验检测细胞转化生长因子β1(TGF-β1)和肿瘤坏死因子α(TNF-α)的分泌情况;Western blot检测蛋白激酶B(Akt)蛋白的表达和磷酸化以及cleaved caspase-3的蛋白水平。结果:与对照组相比,达沙替尼(1~10nmol/L)抑制hBMSCs的活力和迁移;在随后的实验中使用的浓度为7 nmol/L。达沙替尼促进细胞凋亡,并使更多细胞的周期阻滞在G_1期。此外,hBMSCs TGF-β1和TNF-α的分泌量显著增加。7 nmol/L达沙替尼组cleaved caspase-3的蛋白水平增加,胞内Akt的蛋白量下调且其磷酸化受到抑制。结论:达沙替尼以浓度依赖性的方式抑制hBMSCs的活力和迁移,并促进TGF-β1和TNF-α的分泌,诱导细胞G_1期阻滞和凋亡;达沙替尼可能通过影响胞内Akt蛋白的表达和磷酸化调控上述细胞学行为。     

Protection of the Peyer's patch-associated crypt and villus epithelium against methotrexate-induced damage is based on its distinct regulation of proliferation

The crypt and villus epithelium associated with Peyer's patches (PPs) is largely spared from methotrexate (MTX)-induced damage, compared with the non-patch (NP) epithelium. To assess the mechanism(s) preventing damage to the PP epithelium after MTX treatment, epithelial proliferation, apoptosis, and cell functions were studied in a rat-MTX model. Small intestinal segments containing PPs were excised after MTX treatment. Epithelial proliferation and apoptosis were assessed by detection of incorporated BrdU and cleaved caspase-3, respectively. Epithelial functions were determined by the expression of cell type-specific gene products at mRNA and protein level. Before and after MTX treatment, the number of BrdU-positive cells was higher in PP crypts than in NP crypts. BrdU incorporation was diminished in NP crypts, while in PP crypts incorporation was hardly affected. In PP and NP crypts, similar and increased levels of cleaved caspase-3-positive cells were observed after MTX. The enterocyte markers, sucrase-isomaltase, sodium-glucose co-transporter 1, glucose transporters 2 and 5, and intestinal and liver fatty acid binding protein, were down-regulated after MTX in NP epithelium but not in PP epithelium. In contrast, expression of the goblet cell markers, Muc2 and trefoil factor 3, and the Paneth cell marker, lysozyme, was maintained after MTX in both PP and NP epithelium. In conclusion, as MTX-induced apoptosis was similar in PP and NP crypts, the protection of the PP epithelium seems to be based on differences in the regulation of epithelial proliferation. Enterocyte function in the PP epithelium was unaffected by MTX treatment. Goblet and Paneth cell function was maintained in both NP and PP epithelium.     

Survivin and heat shock protein 25/27 colocalize with cleaved caspase-3 in surviving reactive astrocytes following excitotoxicity to the immature brain

Following immature excitotoxic brain damage, distinct patterns of caspase activation have been described in neurons and glial cells. Neuronal cells show activation of the mitochondrial apoptosis pathway, caspase-3 cleavage and apoptotic cell death, while reactive astrocytes show caspase-3 cleavage that is not always correlated with enzymatic protease activity and does not generally terminate in cell death. Accordingly, the aim of the present study was to evaluate the astrocytic colocalization of cleaved caspase-3 and several anti-apoptotic proteins of the inhibitor of apoptosis proteins family (IAPs), such as survivin and cellular inhibitor of apoptosis-2 (cIAP-2), and the heat shock proteins (HSPs) family, Hsp25/27 and Hsc70/Hsp70, which can all prevent caspases from cleaving their substrates. At several survival times ranging from 4 h to 14 days after cortical excitotoxic damage induced by N-methyl-d-aspartate (NMDA) injection at postnatal day 9 in rat pups, single and double immunohistochemical techniques were performed in free floating cryostat sections and sections were analyzed by confocal microscopy. Our results show that survivin and Hsp25/27 are primarily expressed in reactive astrocytes of the damaged cortex and the adjacent white matter. In addition, both molecules strongly colocalize with cleaved caspase-3. Survivin is primarily located in the nucleus, like cleaved caspase-3; while Hsp25/27 is cytoplasmic but very frequently found in cells showing nuclear caspase-3. cIAP-2 was mostly found in damaged neurons but also in some glial scar reactive astrocytes and showed fewer correlation with caspase-3. Hsc70/Hsp70 was only expressed in injured neurons and did not correlate with caspase-3. Thus, we conclude that primarily survivin and Hsp25/27 may participate in the inhibition of cleaved caspase-3 in reactive astrocytes and may be involved in protecting astrocytes after injury.     

Molecular and metabolic evidence for the restricted expression of inducible nitric oxide synthase in healing wounds

Tissue injury initiates a temporally ordered sequence of local cellular and metabolic responses presumably necessary for successful repair. Previous investigations demonstrated that metabolic evidence for nitric oxide synthase (NOS) activity is detectable in wounds only during the initial 48 to 72 hours of the repair process. Present results identify the cell types contributing inducible NOS (iNOS) to experimental wounds in rats. iNOS antigen was expressed in most macrophages present in wounds 6 to 24 hours after injury, and these cells exhibited NAPDH diaphorase and NOS activity. Polymorphonuclear leukocytes contained little iNOS antigen and no NADPH diaphorase activity and were minimally able to convert L-arginine to L-citrulline. The frequency of iNOS-positive macrophages declined on days 3 and 5 after wounding. By day 10, most macrophages in the wound were negative for iNOS. These cells, however, acquired iNOS antigen and activity in culture. Wound fluids, but not normal rat serum, suppressed the induction of iNOS during culture. Findings indicate that the expression of iNOS in healing wounds is restricted to macrophages present during the early phases of repair and that components of wound fluid suppress the induction of iNOS in macrophages in late wounds. Polymorphonuclear leukocytes contribute little iNOS activity to the healing wound.     

CD163 overexpression using a macrophage-directed gene therapy approach improves wound healing in ex vivo and in vivo human skin models

Large tissue damage or wounds cause serious comorbidities and represent a major burden for patients, families, and health systems. Due to the pivotal role of immune cells in the proper resolution of inflammation and tissue repair, we focus our current study on the interaction of macrophages with skin cells, and specifically on the effects of CD163 gene induction in macrophages in wound healing. We hypothesize that the over-expression of the scavenger receptor gene CD163 in human macrophages would result in a more efficient wound healing process. Using 3D human wounded skin organotypic tissues, we observed that CD163 overexpression in THP-1 and human primary macrophages induced a more efficient re-epithelization when compared to control cells. Using human primary skin cells and an in vitro scratch assay we observed that CD163 overexpression in THP-1 macrophages promoted a more rapid and efficient wound healing process through a unique interaction with fibroblasts. The addition of CD163-blocking antibody, but not isotype control, blocked the efficient wound healing process induced by CD163 overexpression in macrophages. We found that the co-culture of skin cells and CD163 overexpressing macrophages reduced monocyte chemoattractant protein (MCP)-1 and enhanced tumor growth factor (TGF)-α, without altering interleukin (IL)-6 or TGF-β. Our findings show that CD163 induces a more efficient wound healing and seems to promote a wound milieu with a pro-resolution molecular profile. Our studies set the foundation to study this approach in in vivo clinically relevant settings to test its effects in wound healing processes such as acute major injuries, large surgeries, or chronic ulcers.