Terminal Deoxyuridine Nick-end Labeling Research Articles

Effect of parenteral glutamine supplementation combined with enteral nutrition on Hsp90 expression and Peyer's patch apoptosis in severely burned rats.

The aim of this study was to investigate the effects of parenteral glutamine (GLN) supplementation combined with enteral nutrition (EN) on heat shock protein (Hsp) 90 expression and Peyer's patch (PP) apoptosis in severely burned rats. Male Sprague-Dawley (SD) rats were randomly assigned to four groups: Sham burn+EN+GLN-free amino acid (AA; n=10), sham burn+EN+GLN (n=10), burn+EN+AA (n=10), and burn+EN+GLN (n=10). Two hours after a 30% total body surface area (TBSA), full-thickness scald burn injury on the back, burned rats in two of the experimental groups (burn+EN+AA and burn+EN+GLN groups) were fed with a conventional EN solution by oral gavage for 7d. Simultaneously, rats in the burn+EN+GLN group were given 0.35g GLN/kg body weight/d once via a tail vein injection for 7d and rats in the burn+EN+AA group were administered isocaloric/isonitrogenous GLN-free amino acid solution (Tyrosine) for comparison. Rats in two sham burn control groups (sham burn+EN+AA and sham burn+EN+GLN groups) were treated in the same manner except for the burn injury. All rats in the four groups were given 175kcal/kg body wt/d. There was isonitrogenous, isovolumic, and isocaloric intake among the four groups. At the end of the seventh day after completion of the nutritional program, all rats were anesthetized and samples were collected for further analysis. PP apoptosis was measured by terminal deoxyuridine nick-end labeling (TUNEL). The expression of Hsp90 in PPs was analyzed by western blotting. Caspase-3 activity of PPs was also assessed. Levels of proinflammatory cytokines of gut tissues were evaluated by enzyme-linked immunosorbent assay (ELISA). The intestinal immunoglobulin A (IgA) content was also determined by ELISA. The results revealed that intestinal IgA content in rats of the burn+EN+GLN group were significantly increased compared with those in the burn+EN+AA group (P<0.05). The expression of Hsp90 of PPs in rats in the burn+EN+GLN group was significantly upregulated compared with those in the burn+EN+AA group (P<0.05). On the other hand, levels of proinflammatory cytokines of gut tissues, caspase-3 activity, and the number of TUNEL-stained cells of PPs in rats of the burn+EN+GLN group were markedly decreased compared with those of the burn+EN+AA group (P<0.05). The results of this study show that parenteral glutamine supplementation combined with EN may upregulate the expression of Hsp90, reduce caspase-3 activity, lessen the release of proinflammatory cytokines, attenuate PP apoptosis, and improve intestinal IgA response in burned rats. Clinically, therapeutic efforts to improve intestinal immunity may contribute to a favorable outcome in severely burned patients.

Lawsonia intracellularis in Pigs: Progression of Lesions and Involvement of Apoptosis.

The purpose of this study was to follow the progression of gross and histologic lesions and apoptosis events in Lawsonia intracellularis-infected enterocytes through the course of the disease, proliferative enteropathy (PE). Thirty 5-week-old pigs were divided into 2 groups: 20 challenged and 10 control animals. Groups of 3 pigs, 2 challenged and 1 control, were euthanized at 1, 3, 5, 8, 11, 15, 19, 24, 29, and 35 days after inoculation. Complete necropsies were performed with gross evaluation. Tissue samples from different sites of the gastrointestinal tract and other visceral organs were collected for routine histologic staining and for immunohistochemistry (IHC) for L. intracellularis. In addition, caspase-3, terminal deoxyuridine nick-end labeling assay, and electron microscopy were performed in ileum samples. Macroscopic and histologic lesions suggestive of PE were first detected 11 days after infection and continued through day 24. L. intracellularis antigen was first detected in the intestine by IHC on day 5 after inoculation, and the bacterium was first detected by transmission electron microscopy on day 15. Positive IHC staining for [L. intracellularis] and enterocyte proliferation, but no gross lesion, were detected on day 29. All 3 pigs euthanized on day 35 were grossly and histologically normal and IHC negative. Hyperplastic crypts in challenge pigs had more apoptotic cells on days 15, 19, and 24 postinfection ( P < .05) compared to control pigs. Our results demonstrated the progression of lesions and infection by L. intracellularis and that inhibition of enterocyte apoptosis is not involved in the pathogenesis of proliferative enteropathy.

Parenteral glutamine supplementation in combination with enteral nutrition improves intestinal immunity in septic rats

ObjectivesThe gut-associated lymphoid tissue is continuously exposed to antigens in the gut lumen and becomes the first line of defense against enteric bacteria and associated toxin. The aim of this study was to investigate the effects of parenteral glutamine (GLN) supplementation in combination with enteral nutrition (EN) on intestinal mucosal immunity in septic rats by cecal ligation and puncture (CLP). MethodsMale Sprague-Dawley rats were randomly assigned into four groups: A sham CLP + EN + saline group (n = 10), a sham CLP + EN + GLN group (n = 10), a CLP + EN + saline group (n = 10), and a CLP + EN + GLN group (n = 10). At 2 h after CLP or sham CLP, all rats in each of the four groups received an identical enteral nutrition solution as their base formula. Then, the rats in the sham CLP + EN + GLN group and CLP + EN + GLN group were given 0.35 g GLN/kg body weight daily for 7 d, all at the same time, via a tail vein injection; whereas those in the sham CLP + EN + saline group and CLP + EN + saline group were daily administered isovolumic sterile 0.9% saline for comparison. All rats in each of the four groups were given 290 kcal/kg body wt/d for 7 d. At the end of the seventh day after the nutritional program was finished, all rats were euthanized and the entire intestine was collected. Total Peyer's patches (PP) cell yield was counted by a hemocytometer. The percentage of PP lymphocyte subsets was analyzed by flow cytometry. The number of intestinal lamina propria IgA plasma cells was determined by the immunohistochemistry technique. The intestinal immunoglobulin A (IgA) levels were assessed by ELISA. PP apoptosis was evaluated by terminal deoxyuridine nick-end labeling. ResultsThe results revealed total PP cell yield, the numbers of PP lymphocyte subsets, intestinal lamina propria IgA plasma cells, and intestinal IgA levels in the CLP + EN + GLN group were significantly increased when compared with the CLP + EN + saline group (P < 0.05). On the other hand, the number of TUNEL-stained cells within PPs in the CLP + EN + GLN group was markedly decreased as compared with the CLP + EN + saline group (P < 0.05). ConclusionThe results of this study show that parenteral glutamine supplementation in combination with enteral nutrition may attenuate PP apoptosis, increase PP cell yield and intestinal lamina propria IgA plasma cells, and subsequently improve intestinal mucosal immunity. Clinically, these results suggest therapeutic efforts at improving intestinal immunity may contribute to the prevention and treatment of sepsis.

Hepatic autophagy after severe burn in response to endoplasmic reticulum stress

BackgroundPrevious studies showed that liver dysfunction develops soon after severe burn, and this is associated with activation of endoplasmic reticulum (ER) stress. Autophagy is a catabolic process to maintain cellular organelle balance; ER stress is associated with autophagy signaling cascades. We thus sought to determine whether autophagy signals were associated with damage in the liver after burn, and further whether burn-associated ER stress activates autophagy signals in hepatocytes. MethodsC57BL/6 male mice received a 25% total body surface area full-thickness scald burn, and liver was harvested at 24 h after burn. HepG2 cells were stimulated with an ER stress inducer thapsigargin (TG) for 24 h to mimic ER stress in vitro. Terminal deoxyuridine nick-end labeling staining was performed on histologic sections of liver. Autophagy was assessed by immunoblotting. Statistical analysis was performed using the Student t-test and significance was accepted at P < 0.05. ResultsTerminal deoxyuridine nick-end labeling positive–stained hepatocytes increased in burned animals with a significant elevation of caspase 3 activity (P < 0.05). Hepatic autophagy-related (ATG) protein 3, ATG5 and light chain (LC) 3B elevated significantly in burn animals as well (P < 0.05). Expression of Beclin-1, LC3A, and LC3B increased in HepG2 cells in response to TG, similar to the response seen in vivo. Cytosolic adenosine triphosphate dropped significantly, and adenosine monophosphate–activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) were phosphorylated as well in response to TG (P < 0.05). ConclusionsER stress, which occurs in hepatocytes after severe injury, is associated with autophagy and liver damage after severe burn. In response to ER stress, activated autophagy is associated with adenosine monophosphate–activated protein kinase and mammalian target target of rapamycin pathway.

Role of Cytotoxic Protease Granzyme‐b in Neuronal Degeneration During Human Stroke

Infiltration of leukocytes into post-ischemic cerebrum is a well-described phenomenon in stroke injury. Because CD-8(+) T-lymphocytes secrete cytotoxic proteases, including granzyme-b (Gra-b) that exacerbates post-ischemic brain damage, we investigated roles of Gra-b in human stroke. To study the role of Gra-b in stroke, ischemic and non-ischemic tissues (from post-mortem stroke patients) were analyzed using immunoblotting, co-immunoprecipitation, terminal deoxy uridine nick end labeling (TUNEL) and Annexin-V immunostaining, and in vitro neuron survival assays. Activated CG-SH cells and supernatants were used to model leukocyte-dependent injury. Non-ischemic brain tissues were used as non-pathological controls. Non-activated CG-SH cells and supernatants were used as controls for in vitro experiments. Human stroke (ischemic) samples contained significantly higher levels of Gra-b and interferon-gamma inducible protein-10 (IP-10/CXCL10) than non-ischemic controls. In stroke, poly (ADP-ribose) polymerase-1 and heat shock protein-70 were cleaved to canonical proteolytic "signature" fragments by Gra-b. Gra-b was also found to bind to Bid and caspase-3. Gra-b also co-localized with Annexin-V(+) /TUNEL(+) in degenerating neurons. Importantly, Gra-b inhibition protected both normal and ischemia-reperfused neurons against in vitro neurotoxicity mediated by activated CG-SH cells and supernatants. These results suggest that increased leukocyte infiltration and elevated Gra-b levels in the post-stroke brain can induce contact-dependent and independent post-ischemic neuronal death to aggravate stroke injury.

Proximal Gut Mucosal Epithelial Homeostasis in Aged IL-1 Type I Receptor Knockout Mice after Starvation

Previous studies have shown that starvation induces small bowel atrophy, and that atrophy diminishes with aging. In this experiment, we assessed whether starvation-induced atrophy of proximal gut mucosa is associated with the Interleukin-1 receptor (IL-1R) signaling pathway in aged mice. Thirty 26-month-old IL-1R knockout mice and age-matched wild-type C57BL/6 mice were randomly divided into two groups: ad libitum fed and fasted. Mice were euthanized 12 or 48 hours after starvation. The proximal small bowel was harvested for morphologic analysis. Gut epithelial cell proliferation was detected using immunohistochemical staining for proliferating cell nuclear antigen (PCNA), and apoptosis was identified using terminal deoxyuridine nick-end labeling (TUNEL) staining. Aged IL-1R knockout mice were larger than aged-matched wild-type mice (P < 0.05). Proximal gut mucosal height and mucosal cell number were not different between aged IL-1R knockout and wild-type groups. The apoptosis index in gut epithelial cells was higher in fed IL-1R knockout versus wild-type mice (P < 0.05), while there was no significant difference in cell proliferation between both groups. Mucosal atrophy was induced in both aged IL-1R knockout and wild-type groups by starvation (P < 0.05), however, aged IL-1R knockout mice experienced greater loss in proximal gut weight, mucosal length, and corresponding cell number than did wild-type mice at the 12-h time point (P < 0.05). The apoptosis index in gut epithelial cells significantly increased in both groups after starvation (P < 0.05). Starvation decreased cell proliferation in IL-1R knockout mice (P < 0.05), but not in wild-type mice. The response in aged IL-1R knockout mice differs from wild-type mice in that starvation increases atrophy and is associated with decreased cell proliferation rather than increased apoptosis.

Antitumor Effect of Docetaxel-Loaded Lipid Microbubbles Combined With Ultrasound-Targeted Microbubble Activation on VX2 Rabbit Liver Tumors

The purpose of the study was to explore the antitumor effect of docetaxel-loaded lipid microbubbles combined with ultrasound-targeted microbubble activation (UTMA) on VX2 rabbit liver tumors. Docetaxel-loaded lipid microbubbles were made by a mechanical vibration technique. VX2 liver tumor models were established in 90 rabbits, which were randomly divided into 6 groups, including control, docetaxal-loaded lipid microbubbles alone, docetaxal alone, docetaxal combined with ultrasound, pure lipid microbubbles combined with ultrasound, and docetaxel-loaded lipid microbubbles combined with ultrasound (DOC+MB/US). The tumor volume and inhibition rate (IR) of tumor growth were calculated and compared. Apoptosis was detected by terminal deoxyuridine nick end labeling. Proliferating cell nuclear antigen and matrix metalloproteinase 2 (MMP2) protein expression was detected by immunohistochemistry. Caspase 3 and MMP2 messenger RNA (mRNA) expression was detected by in situ hybridization histochemistry. The tumor metastasis rate and survival time of the animals were compared. The IR and apoptotic index of the DOC+MB/US group were the highest among all groups, and the proliferating labeling index was the lowest. Matrix metalloproteinase 2 protein and mRNA expression in the DOC+MB/US group was the lowest among all groups, and caspase 3 mRNA expression in the DOC+MB/US group was the highest. The extensive metastasis rate in the DOC+MB/US group was the lowest, and the survival time of the animals in the DOC+MB/US group was the longest. Docetaxel-loaded lipid microbubbles combined with UTMA could inhibit the growth of VX2 rabbit liver tumors by deferring proliferation and promoting apoptosis, which may provide a novel targeted strategy for chemotherapy of liver carcinoma.

Hematoporphyrin Monomethyl Ether Enhances the Killing Action of Ultrasound on Osteosarcoma In Vivo

Osteosarcoma is one of the most common malignant cancers afflicting young adults. Ultrasound is a new therapeutic modality for controlling malignant cancers. Enhancing the efficacy of ultrasound treatment will improve clinical outcomes. The aim of this study was to investigate the killing action of ultrasound on osteosarcoma enhanced by hematoporphyrin monomethyl ether (HMME) in vivo. An animal model of an osteosarcoma xenograft was set up to investigate the inhibitory effect of sonoactivating HMME on osteosarcoma. High-performance liquid chromatography was used to analyze the time course of HMME in the osteosarcoma xenograft. Three hours after intravenous (IV) administration of HMME, ultrasound radiation was administered in the osteosarcoma xenograft. On day 7 after ultrasound radiation, the tumor volume and weight were measured and calculated for effect assessment, hematoxylin-eosin staining for histopathologic examination, immunohistochemical staining for proliferating cell nuclear antigen (PCNA) expression, and terminal deoxyuridine nick end-labeling (TUNEL) staining for apoptosis examinations. The peak value of HMME in osteosarcoma tissues increased with time after IV administration of HMME and reached a plateau at 3 hours. The increasing rates of the tumor volume and weight in the control group were very fast, but the increasing rates in the ultrasound-alone group were slower, and those in the ultrasound-HMME group were the slowest throughout the observation period. There was a significant difference between the HMME-ultrasound, ultrasound-alone, and control groups (P < .01). Hematoxylin-eosin staining showed that some cells had typical cell death such as pyknosis and nuclear fragmentation after ultrasound radiation alone. More cells with pyknosis, nuclear fragmentation, and even karyolysis were found after HMME-ultrasound treatment. Immunohistochemical staining showed that the percentage of PCNA-positive cells decreased and that of TUNEL-positive cells increased after ultrasound treatment alone, and the changes in the PCNA- and TUNEL-positive percentages were significantly enhanced by pretreatment with HMME (20 mg/kg, IV) for 3 hours and ultrasound radiation (10.5 MHz) for 120 seconds at an intensity of 0.8 W/cm(2) (P < .05). Hematoporphyrin monomethyl ether pretreatment could substantially enhance the growth inhibition of ultrasound on osteosarcoma, which suggests that HMME is an efficient sonosensitizer, and ultrasound radiation with HMME could be developed as a new modality for treating osteosarcoma.

Loss of Cyclin G1 Expression in Human Uterine Leiomyoma Cells Induces Apoptosis

Observations from the authors' laboratory suggest a physiological role for increased cyclin G1 protein levels in human uterine leiomyoma. The hypothesis of the present study is that the strategic modulation of cyclin G1 by antisense technology will inhibit the survival of in vitro-grown uterine leiomyoma cells. Cultured uterine leiomyoma cells were transfected with cyclin G1 ribbon-type antisense oligonucleotide (cyclin G1 RiAS) to effectively reduce cyclin G1 expression. Cell viability, in situ terminal deoxyuridine nick end-labeling (TUNEL) assay, flow cytometry, DNA fragmentation, and expression of cell cycle regulatory-related proteins were evaluated by Western blot. Antisense oligonucleotides compromised uterine leiomyoma cell viability and inducted apoptosis in a caspase-independent mechanism. In situ TUNEL and DNA fragmentation revealed apoptosis induction, and fluorescent-activated cell sorting analysis showed increased sub-G1-phase cells. Furthermore, abrogation of cyclin G1 enhanced p53 accumulation, phosphorylation of p53 at Ser-15 residue, and increased expression of cyclin-dependent kinase inhibitors p21 and p27. These data imply that cyclin G1 expression is associated with growth promotion and the potential utility and novelty of using ribbon-type antisense oligonucleotides as a gene therapy strategy to treat human uterine leiomyoma.

Cyclin D1 Overexpression and Response to Bortezomib Treatment in a Breast Cancer Model

Cyclin D1 is frequently overexpressed in breast cancer, and its overexpression is, surprisingly, associated with improved survival. One potential mechanism for this association involves signal transducer and activator of transcription 3 (STAT3). Cyclin D1 and STAT3 expression were assessed in human tumors using microarray analysis and in breast cancer cell lines HBL100, T47D, MCF7, MDA-MB-453, and BT20 and in HBL100 and T47D cells stably overexpressing cyclin D1 using immunoblot analysis. Cyclin D1 protein was stabilized by treatment with the proteasome inhibitor bortezomib, and the effects on STAT3 expression in vitro was determined by using immunoblotting and on xenograft tumor growth and apoptosis in vivo was determined by using terminal deoxyuridine nick-end labeling assays. All statistical tests were two-sided. Tumors with high cyclin D1 expression (n = 17) had low STAT3 expression (mean = 274 arbitrary units), and those with low cyclin D1 expression (n = 31) had high STAT3 expression (mean = 882 arbitrary units) (P<.001). In HBL100 and T47D parental and cyclin D1-overexpressing cells, cyclin D1 overexpression was also inversely associated with STAT3 expression, and cyclin D1 directly reduced the expression of STAT3. Stabilization of cyclin D1 protein by bortezomib treatment further amplified the cyclin D1-dependent repression of STAT3 in vitro and slowed tumor growth in vivo (week 7: untreated mean = 185.7 mm3 versus treated mean = 136.2 mm3, difference = 49.5 mm3, 95% confidence interval [CI] = 18 to 81 mm3, P = .007; week 8: untreated mean = 240.2 mm3 versus treated mean = 157.3 mm3, difference = 82.9 mm3, 95% CI = 9.1 to 156.7 mm3, P = .0014; and week 9: untreated mean = 256.4 mm3 versus treated mean = 170.2 mm3, difference = 86.2 mm3, 95% CI = 22.8 to 149.6 mm3, P = .006) and increased apoptosis (untreated mean = 19% versus treated mean = 54%, difference = 35%, 95% CI = 24.7% to 45.4%; P = .013) of xenograft tumors. Cyclin D1 repression of STAT3 expression may explain the association between cyclin D1 overexpression and improved outcome in breast cancer. In addition, bortezomib can amplify the proapoptotic function of cyclin D1, raising the possibility that cyclin D1 levels may be a marker for predicting the response to this novel drug.

In vivo evidence for apoptosis, but not inflammation in the hindlimb muscle of neuropathic rats

Loose ligation of the rat sciatic nerve (chronic constriction injury (CCI) model) provokes signs and symptoms like those observed in complex regional pain syndrome (CRPS) patients. Neurogenic inflammation is a purported cause of neuropathic pain despite inconsistent evidence to support this hypothesis. To clarify this issue, we examined effects of CCI on microcirculation, inflammatory cell–cell interaction and cell integrity in muscle tissue using intravital fluorescence microscopic, molecular and immunohistochemical techniques. CCI-rats, but not sham-operated animals developed symptoms of neuropathic pain and oedema on the ipsilateral hindpaw. Despite signs of neuropathic pain, high resolution in vivo multifluorescence microscopy revealed physiological values for functional capillary density, leukocyte–endothelial cell interaction and microvascular permeability in muscle tissue of CCI-animals, similarly as found in controls, indicating absence of perfusion failure and inflammatory cell reaction. However, CCI-animals represented with marked apoptosis of bisbenzimide-stained muscle tissue cells, as given by in vivo fluorescence microscopic assessment of cell death-associated condensation, fragmentation and/or crescent-shaped formation of their nuclear chromatin. Apoptosis was further confirmed by increased caspase 3 protein levels and positive terminal deoxyuridine nick end labeling histochemistry. The present study demonstrates that sciatic nerve ligation-induced neuropathy causes cell apoptosis without concomitant inflammation-associated microcirculatory dysfunction in muscle tissue. Beside the well-known pattern of neuropathic pain, the CCI-model has now additionally been shown to reflect the response of muscle tissue to impaired innervation, i.e. prompting muscle cells to undergo non-inflammatory apoptotic cell death. This finding deserves further investigation in that apoptosis may contribute to neuropathic pain conditions like CRPS.

Burn-induced gut mucosal homeostasis in TCR delta receptor-deficient mice.

Gammadelta T lymphocytes make up approximately 50% of lymphocytes in the intestine. These cells have been shown to prime macrophages for TNF-alpha production after burn. We previously showed that neutralizing anti-TNF-alpha antibodies reduce mucosal atrophy by decreasing gut epithelial apoptosis after severe burn. We hypothesized that burn-induced mucosal turnover is diminished in T cell receptor delta gene knockout (TCR delta-/-) mice through diminished TNF-alpha activity. Forty-two wild-type and 42 TCR delta-/- mice (C57-BL6) were randomly assigned to burn and sham burn groups. The burn group underwent a 25% total body surface area (TBSA) scald burn. The proximal small intestine was harvested at 2, 12, and 48 h. To assess mucosal atrophy, mucosal height and cell numbers in the villi and crypts were determined on hematoxylin and eosin-stained tissue sections. Apoptotic gut epithelium was identified by terminal deoxyuridine nick-end labeling (TUNEL) staining, and cell proliferation was detected by immunostaining for proliferative cell nuclear antigen (PCNA). TNF-alpha mRNA expression was measured by RT-PCR. Caspase-8 activity was measured by colorimetric assay. Statistical analysis was performed with two-way analysis of variance and t testing. Significance was accepted at P < 0.05. Data are expressed as means +/- SEM. TNF-alpha mRNA expression was significantly decreased in TCR delta-/- mice at 2 h after burn. Gut epithelial apoptosis and proliferation in both wild-type and TCR delta-/- mice were significantly increased after burn, but TCR delta-/- mice had a significantly lower levels of apoptosis (P < 0.01) and proliferation (P < 0.05) when compared with wild-type mice. Burn-induced mucosal atrophy was identified in groups by decreasing villus height, crypt depth, and villus and crypt cell number (P < 0.001) compared with sham, but no difference was found between wild-type and TCR delta-/- mice. Caspase-8 activity was significantly diminished in TCR delta-/- mice compared with wild-type mice. Gammadelta T cells are associated with increased TNF-alpha expression and gut epithelial turnover in the small bowel after severe burn. However, absence of delta T cell receptor did not inhibit mucosal atrophy after severe burn. This study suggests that gut mucosal atrophy after severe burn is a multifactorial process associated with increased TNF-alpha activity.

Decreased lymphocyte apoptosis by anti-tumor necrosis factor antibody in Peyer's patches after severe burn.

Severe burn results in immunosuppression, with increased lymphocyte apoptosis in both the central and peripheral immune system. As atrophy of the small intestine has been described in mouse models and intestinal lymphocytes have been implicated in the burn inflammatory response, we examined the effects of burn and tumor necrosis factor (TNF)-alpha on lymphocytes in intestinal Peyer's patches. Anesthetized C57BL6 mice received a 30% full-thickness scald burn on the upper back. Sham-burned animals served as controls. Anti-TNF or control immunoglobulin (Ig) G antibody (200 microg) was given immediately after the burn. The animals were initially resuscitated with 2 mL of normal saline, and were then sacrificed 12 h postburn. Terminal deoxyuridine nick-end labeling (TUNEL) and proliferative cell nuclear antigen (PCNA) staining was performed. Apoptosis was quantified as apoptotic lymphocytes/high-powered field (hpf). Results, expressed as mean +/- SEM, were compared using analysis of variance (ANOVA) and the Student-Newman-Keuls test. All mice survived the burn. An initial time-course experiment demonstrated maximal Peyer's patch apoptosis 12 h after the burn. Sham mice had 25 +/- 7 TUNEL-stained cells/hpf in Peyer's patches, whereas burned mice had 93 +/- 18 cells/hpf (P < 0.05). In contrast, burned mice receiving anti-TNF antibody had 28 +/- 8 TUNEL-stained cells/hpf (P < 0.05 vs. burn), whereas sham mice receiving anti-TNF antibody had 20 +/- 4 cells/hpf. There were no significant differences in PCNA staining between the groups. Scald burn results in lymphocyte apoptosis in Peyer's patches. This apoptosis can be abrogated by the addition of anti-TNF antibody. Apoptotic changes may lead to the failure of the intestinal immunological barrier and increased risk of sepsis.

Role of TNF-alpha in gut mucosal changes after severe burn.

Gut epithelial cell death by apoptosis is increased in the gut epithelium after severe burn associated with mucosal atrophy. We hypothesized that tumor necrosis factor (TNF)-alpha-TNF receptor (TNFR) interaction activates apoptosis in small bowel mucosal cells after severe burn. C57BL6 mice received a 30% total body surface area scald burn and were treated with neutralizing anti-TNF-alpha. The proximal small bowel was assessed for mucosal atrophy. Proliferation and apoptosis of mucosal cells were assessed by proliferative cell nuclear antigen-immunostaining and terminal deoxyuridine nick-end labeling assay, respectively. Mucosal height and mucosal cell number decreased after burn. Anti-TNF-alpha-treated mice showed significantly less mucosal atrophy. Proliferation of intestinal cells was not changed with burn or anti-TNF-alpha treatment. An over threefold increase in apoptotic cell number was seen after burn, which was diminished by anti-TNF-alpha treatment. Changes in gut mucosal homeostasis after severe burn are affected, in part, by the activation of apoptosis by TNF-alpha-TNFR interaction.

Expression of p53 and Ki-67 antigen in bone marrow giant proerythroblasts associated with human parvovirus B19 infection.

Giant proerythroblasts are hallmarks of human parvovirus B19 infection. We attempted to characterize these cells in 5 patients with parvovirus B19-induced pure red cell aplasia using immunostaining of paraffin-embedded bone marrow sections with antibodies against erythroid-lineage-specific proteins, viral capsid antigen VP-1, and apoptosis- and cell-cycle-related proteins. Giant proerythroblasts are immunohistochemically consistent with early erythroid precursors of cells in the differentiation stage of CD34-, cytoplasmic spectrin+, glycophorin A-, and band-3-. VP-1 was expressed in the nucleus and cytoplasm of small- to medium-sized spectrin+ erythroid cells but not in giant proerythroblasts. The giant proerythroblasts displayed nuclear staining for p53 (41%+/-16%) and Ki-67 antigen (100%+/-0%) and cytoplasmic staining for Bax (65%+/-11%) and procaspase-3 (78%+/-10%), whereas they were not stained for p21Wafl/Cip1, active form of caspase-3, or terminal deoxynucleotidyltransferase-mediated deoxyuridine nick-end labeling (TUNEL). Antiapoptotic proteins, Bcl-2 and Mcl-1, were not expressed in the giant cells, and Bcl-x was infrequently expressed in these cells (11%+/-4%). These immunohistochemical findings suggest that giant proerythroblasts are proliferating erythroid precursors with accumulation of nonfunctional p53.

Cell proliferation, apoptosis, NF-kappaB expression, enzyme, protein, and weight changes in livers of burned rats.

Thermal injury has been shown to alter gut epithelium and heart myocyte homeostasis by inducing programmed cell death. The effect of thermal injury on hepatocyte apoptosis and proliferation, however, has not been established. The purpose of this study was to determine whether a large thermal injury increases liver cell apoptosis and proliferation and whether these changes were associated with alterations in hepatic nuclear factor kappaB (NF-kappaB) expression and changes in liver enzymes and amount of protein. Sprague-Dawley rats received a 40% total body surface area scald burn or sham burn. Rats were killed and livers were harvested at 1, 2, 5, and 7 days after burn. Liver cell apoptosis was determined by terminal deoxyuridine nick end labeling (TUNEL) assay and cell proliferation by immunohistochemistry for proliferating cell nuclear antigen. Hepatic NF-kappaB expression was determined by Western blot, and total hepatic protein content was determined by protein assay. Protein concentration decreased after burn compared with sham controls (P < 0.05). Liver cell apoptosis, proliferation, and NF-kappaB expression in hepatocytes increased in burned rats compared with controls (P < 0.05). It was concluded that thermal injury induces hepatic cell apoptosis and proliferation associated with an increase in hepatic NF-kappaB expression and a decrease in hepatic protein concentration.

Increased small bowel epithelial turnover in interleukin-1 receptor knockout mice.

To determine whether interleukin-1 (IL-1) affects the cellular homeostasis of small bowel mucosa, the authors studied apoptosis and proliferation in small bowel epithelium in two groups of C57 mice: an IL-1 receptor knockout group, and a control wild-type group. Gut mucosal integrity is maintained by a balance of cell proliferation and cell death. Recent reports suggest that IL-1, a proinflammatory cytokine, increases cell death by apoptosis in some epithelial cells. Twenty-four male C57BL6 IL-1 receptor (type I) knockout mice were killed, and small bowel was removed for study. Twenty-four wild-type mice (C57-BL6) served as controls. Body weights, bowel length, and mucosal morphology were examined for phenotypic differences. Apoptosis was quantified by terminal deoxyuridine nick-end labeling (TUNEL) immunohistochemical staining and cellular proliferation by proliferation cell nuclear antigen staining. Whole mucosal protein was analyzed for nuclear factor-kappaB expression. Groups were analyzed by t test. The absence of IL-1 type I receptor in knockout mice was verified by reverse transcriptase-polymerase chain reaction. IL-1 receptor null mice were larger than wild-type controls, with a longer small bowel; however, the index of small bowel length to total body weight did not differ between groups. The percentage of apoptotic cells was higher in IL-1 receptor null mice than in wild-type mice; the proliferation index also increased. Mucosal height and other measures of mucosal morphology were not different. Genotypic absence of IL-1 receptors was associated with decreased expression of nuclear factor-kappaB in whole mucosal protein extracts. Both apoptosis and proliferation increased in gut epithelial cells of mice without IL-1 receptors, suggesting increased cell turnover with no change in net balance. This model represents an opportunity to examine potential mechanisms of gut epithelial turnover in vivo, under both normal conditions and in conditions of mucosal proliferation and atrophy.

Subarachnoid hemolysate produces DNA fragmentation in a pattern similar to apoptosis in mouse brain

Stroke and traumatic brain/spinal cord injuries are often associated with hemorrhage. Despite the relative frequency of hemorrhage in the central nervous system (CNS), little is known about what role blood and hemoglobin (Hb) play in mediating cellular injury. Since Hb and hemolysate have been associated with generation of oxidative stress and cell injury, we examined whether apoptosis was present after cortical exposure to subarachnoid hemolysate. Subarachnoid hemorrhage (SAH) was induced in CD-1 mice ( n=25) by injection of 50 μl of autologous hemolysate over the right parietal cortex. Saline-injected mice ( n=13) were used as controls. Subjects were sacrificed at 24 h. Transcardiac perfusion fixation was performed on a subgroup of hemolysate- ( n=15) and saline-injected ( n=9) animals. Sections were stained for DNA fragmentation using the terminal deoxyuridine nick end-labeling (TUNEL) method and also immunostained for the hemeoxygenase-1 (HO-1) protein to assess blood distribution. In the remaining animals ( n=6 SAH, n=4 saline), DNA was extracted and precipitated from 40 mg of tissue and subjected to electrophoresis on a 1.5% agarose gel. DNA fragmentation was evident on TUNEL staining in 10/15 subjects injected with hemolysate as compared to 0/9 subjects injected with saline ( p<0.01, Fisher exact test). TUNEL-positive cells were most abundant closest to the site of cortical SAH, as evidenced by HO-1 immunoreactivity. TUNEL-positive cells were also seen remotely in the hippocampus and basal forebrain. The presence of apoptosis was suggested by DNA laddering on electrophoresis in the hemolysate-injected subjects (4/6 animals). No laddering was evident in saline-injected subjects ( n=4). These results provide evidence that the presence of subarachnoid blood products is associated with DNA fragmentation and apoptotic cell death.

Role of Nuclear Factor‐KB in Colon Cancer Cell Apoptosis Mediated by Aminopyropheophorbide Photosensitization

Aminopyropheophorbide (APP) is a second generation of photosensitizer for photodynamic therapy (PDT). We demonstrated that APP strongly absorbed red light and, after being taken up by colon cancer cells (HCT-116 cells), was localized in cytoplasmic and internal membranes but not in mitochondria. The APP-mediated photosensitization was cytotoxic for HCT-116 cells through an induction of apoptosis. Indeed, DNA fragmentation (DNA laddering and terminal deoxyuridine nick-end labeling) and chromatin condensation (4',6-diamidine-2'-phenylindole staining) could be visualized soon after photosensitization. Because nuclear factor (NF)-kappa B is involved in the response to many photosensitizers, we also demonstrated its nuclear translocation in two waves: a rapid and transient one, followed by a slow and sustained phase. The NF-kappa B turned out to be involved in an antiapoptotic response to APP-mediated photosensitization because the HCT-116 cell line expressing the dominant negative mutant of inhibitor-kappa B alpha was more sensitive to apoptosis as measured by DNA fragmentation and caspase activation. These data unambiguously show that a membrane-located photosensitizer can lead to effective apoptosis, reinforcing the idea that PDT can be an effective means to eradicate colon cancer cells.

Cutaneous burn increases apoptosis in the gut epithelium of mice

Background: Gut mucosal homeostasis depends on a balance between cell proliferation and cell death. After cutaneous burn injury, gut mucosal weight has been shown to decrease. This decrease in weight was paradoxically associated with an increase in gut proliferative factors. For mucosal weight to decrease in the presence of increased proliferation, there must be an even greater increase in cell death. We postulate that cutaneous burn injury causes an increase in gut epithelial cell death primarily by apoptosis. Study Design: We produced a 30% full-thickness scald burn in the dorsum of anesthetized male C57BL6 mice and collected the proximal small bowel at 12, 24, 36, 48, and 60 hours after injury. Sham burned animals served as controls. Apoptosis and proliferation were measured by immunohistochemical assays (terminal deoxyuridine nick-end labeling for apoptosis and proliferative cell nuclear antigen assay for proliferation). Apoptosis was also measured by ELISA for cytoplasmic histone-associated DNA fragments. Mucosal height was determined on histologic sections. The two groups were compared at each time point using Wilcoxon two-sample test and t-test with Bonferroni’s correction where appropriate. Results: The percentage of apoptotic cells (number of cells stained by terminal deoxyuridine nick-end labeling per 100 villus cells) was significantly higher at 12, 24, and 48 hours after injury. This increase was corroborated by an increase in the ELISA at 12 hours. Proliferation as measured by immunostaining for proliferative cell nuclear antigen significantly increased at 12, 24, 48, and 60 hours. Mucosal height as a gross measure of mucosal atrophy was not different between the groups. Conclusions: We have shown an increase in apoptosis coupled with an increase in proliferation after a burn injury. These results imply an increase in cell turnover in the gut epithelial cells after a burn. Impaired bowel function has been demonstrated repeatedly after burn injury, and this increase in cell turnover may be related.