Transferase-mediated Deoxyuridine Triphosphate-biotin Nick End Labeling Research Articles

Non-malignant migration of B16 mouse melanoma cells in the neural crest and invasive growth in the eye cup of the chick embryo

Melanocytes originate from the neural crest. In a previous study, we observed that human SK-Mel 28 human melanoma cells resumed neural crest cell migration after transplantation into the chick embryo neural tube. Here, we used transgenic mouse B16-F1 melanoma cells transfected with green fluorescent protein-vasodilator-stimulated phosphoprotein construct to extend these observations. After the injection of a cell suspension into the trunk neural tube of E2 chick embryos, the migration of melanoma cells was followed by live fluorescence microscopy. Within 12 h, the melanoma cells formed clusters in the neural tube at the levels of the intersegmental clefts between somites. After 24 h, a segmental pattern of emigration was visible. Emigrated melanoma cells were identified in serial paraffin sections by immunohistochemistry with ab732 as a marker for melanoma cells and by in-situ hybridization of mouse-specific repetitive genomic sequence mL1. After 24 h, melanoma cells were found along the medial neural crest pathway and in the sympathetic trunk ganglia and, after 48 h, also in the lateral melanocytic pathway. During migration along the neural crest pathways, mouse melanoma cells underwent apoptosis, which was assessed by anti-caspase 3 and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling staining. To prove the ablation of malignant behavior after back-transplantation into the original embryonic neural crest environment, we injected the same cell suspension into the eye cup of the E3 embryo. In this location, invasive melanomas formed.

Histological changes and apoptosis of cartilage layer in human anterior cruciate ligament tibial insertion after rupture

The purpose of this study is to investigate the histological changes and apoptosis of cartilaginous layers in human anterior cruciate ligament (ACL) tibial insertion at different time periods after rupture. By using a core reamer, 35 tibial insertions of ruptured ACLs were obtained during primary ACL reconstructions (number of days after injury: 19-206 days). A histological examination was performed and a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining assay was carried out to detect apoptosis. The average thickness of the cartilage layer, the glycosaminoglycan-stained area and the number of chondrocytes per millimeter decreased with time. The percentage average of TUNEL-positive chondrocytes was 42.0 +/- 16.2. The histological degenerative changes of the cartilage layer in the ruptured ACL tibial insertion progressed with time, especially in the first 2 months. Moreover, chondrocyte apoptosis continued from 19 to 206 days after rupture. The results may help elucidate the etiology of the histological changes of the insertion, and may help in devising optimal treatment protocols for ACL injuries if apoptosis is controlled. Moreover, we consider that using a surviving ligament and minimizing a debridement of ACL remnant during ACL reconstruction may be important for ACL reconstruction to maintain cartilage layers in ACL insertions.

Ras Activation Modulates Methylglyoxal-Induced Mesangial Cell Apoptosis Through Superoxide Production

Aims. While previous studies have demonstrated that diabetic nephropathy is attributable to glucose-derived dicarbonyl compounds, methylglyoxal (MGO)-inducing apoptosis in renal mesangial cells, the molecular mechanism of upper stream redox signaling modulation, has not been fully elucidated. Methods: Rat mesangial cells pretreated with or without superoxide dismutase, diphenyloniodium, SB203580, and manumycin A were cultured in methylglyoxal stress-induced apoptosis. Signaling protein expression, flow cytometry, and morphological features of apoptotic cell death were assessed. Results: Methylglyoxal decreased cell viability in mesangial cells. Superoxide mediated methylglyoxal-induced caspase 3 cleavage. Pretreatment with diphenyloniodium, SB203580, and manumycin A reduced methylglyoxal augmentation of superoxide synthesis and caspase-3 activation. Methylglyoxal rapidly enhanced Ras activation and progressively increased cytosolic P38 and nuclear c-Jun activation. Scavenging of superoxide by superoxide dismutase or diphenyloniodium, inhibiting P38 by SB203580, and inhibiting Ras with manumycin A successfully reduced the promoting effect of methylglyoxal on P38 and c-Jun phosphorylation (activation). Furthermore, pretreatment with superoxide dismutase, diphenyloniodium, SB203580, and manumycin A significantly attenuated methylglyoxal induction of apoptosis on the basis of Annexin-V assay and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labelling (TUNEL) staining. Conclusions. This study has shown that methylglyoxal increased Ras modulation of superoxide-mediated P38 activation and c-Jun activation, which resulted in increased apoptosis.

Mechanisms underlying the radioprotective effect of histamine on small intestine

Purpose: To examine the protective effects of histamine on intestinal damage produced by gamma-radiation.Materials and methods: 56 mice were divided into 4 groups. Histamine and Histamine-10 Gy groups received a daily subcutaneous histamine injection (0.1 mg/kg) starting 20 hours before irradiation and continued until the end of the experimental period; the untreated group received saline. Histamine-10 Gy and untreated-10 Gy groups were irradiated with a single dose on whole-body using Cesium-137 source (7 Gy/min) and were sacrificed 3 days after irradiation. Small intestine was removed, fixed and stained with hematoxylin and eosin. The number of intestinal crypts per circumference, and other histological characteristics of intestinal cells were evaluated. We further determined by immunohistochemistry the expression of proliferating cell nuclear antigen (PCNA), Bax, Bcl-2 (pro- and anti-apoptotic protein, respectively), antioxidant enzymes (Superoxide dismutase (SOD), Catalase and Glutathione peroxidase), histamine content and apoptosis by terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate biotin nick end labeling (TUNEL) assay. Cells in the S phase of the cell cycle were identified by immunohistochemical detection of 5-bromo-2′-deoxyuridine (BrdU) incorporation.Results: Histamine treatment reduced mucosal atrophy, edema and preserved villi, crypts and nuclear and cytoplasmic characteristics of small intestine after radiation exposure. Additionally, histamine treatment increased PCNA expression and the BrdU-positive cell number, histamine content, decreased the number of apoptotic cells and significantly increased Catalase and copper-zinc-containing SOD of irradiated mice.Conclusions: Histamine prevents radiation-induced toxicity by increasing proliferation of damaged intestinal mucosa and suppressing apoptosis that was associated with an increase in SOD and Catalase levels. This effect might be of clinical value in patients undergoing radiotherapy.

Chondrocyte Apoptosis and Decrease of Glycosaminoglycan in Cranial Cruciate Ligament Insertion after Resection in Rabbits

We examined time-dependent histological changes of the calcified fibrocartilage area in a tibial cranial cruciate ligament (CCL) insertion after ligament resection in rabbits. The animals were divided into two groups: those undergoing CCL substance resection in the right stifle (resected group) and those receiving the same operation without CCL resection in the left stifle (sham operated group). Five animals were euthanized with deep anaesthesia at four time periods (1, 2, 4 and 6 weeks), and Haematoxylin-eosin and Safranin-O stainings and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining were performed. The average percentage of TUNEL-positive chondrocytes and the average thickness of the glycosaminoglycan (GAG)-stained area in the calcified fibrocartilage area were measured. Two and 4 weeks after the surgery, the average percentages of TUNEL-positive chondrocytes in the resected group (23.8 +/- 10.3% and 15.9 +/- 6.7%, respectively) were significantly higher than those in the sham operated group (8.9 +/- 3.8% and 7.4 +/- 1.6%, P<0.05, respectively). Six weeks after the surgery, the average thickness of the GAG-stained area in the resected group (7.7 +/- 13. 5 microm) was significantly smaller than that in the sham operated group (69.4 +/- 39.9 microm, P<0.05). Our results suggest that the average percentage of TUNEL-positive chondrocytes became a peak in 2 weeks and that histological changes occurred in 6 weeks. The chondrocyte apoptosis can induce decrease of GAG-stained area after resection of CCL. Therefore, chondrocyte apoptosis in the calcified cartilage area in the CCL tibial insertion might lead to histological changes.

Staining Ability and Biocompatibility of Brilliant Blue G

To evaluate the effectiveness and biocompatibility of brilliant blue G (BBG) for capsular visualization for continuous curvilinear capsulorrhexis. The capsular staining ability of BBG was evaluated at graded concentrations of 10.0, 1.0, 0.5, 0.25, 0.1, and 0.01 mg/mL in enucleated pig's eyes. The biocompatibility of BBG was assessed in rat's eyes for 2 months. The eyes were analyzed using light, fluorescence, transmission electron, and scanning electron microscopy. TUNEL (terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridine triphosphate nick-end labeling) was used to detect apoptotic cells, and endothelial cell counts were analyzed using scanning electron microscopy. The results were compared using indocyanine green and trypan blue. The BBG improved capsular visualization, and a complete capsulorrhexis could be performed. In the rat model, no apparent toxic effect was observed using biomicroscopy during 2 months. Histologically, BBG showed satisfactory biocompatibility. Apoptotic cell death of the endothelial cells was detected in only the trypan blue group. In contrast to BBG, indocyanine green and trypan blue showed degeneration of corneal endothelial cells using transmission and scanning electron microscopy. The BBG contributed to better capsular visualization and caused no apparent complications to the corneal endothelium.Clinical Relevance The BBG is effective and safe capsular staining for continuous curvilinear capsulorrhexis.

Mesna Protects Intestinal Mucosa from Ischemia/Reperfusion Injury

Mesna is a thiol used for the prevention of oxazaphosphorine-induced hemorrhagic cystitis. However, its antioxidant properties on renal and hepatorenal oxidative damage, as well as its mucoprotective effect on the intestinal epithelium have also been shown. The aim of this study was to investigate the potential beneficial effect of mesna on ischemia/reperfusion (I/R)-induced oxidant damage of the intestinal mucosa. Wistar rats were subjected to intestinal I/R for 30 min, induced by occlusion of the superior mesenteric artery, followed by 60 min reperfusion. Mesna was administered at 3 time points relative to ischemia; 60 min before ischemia, at the onset of ischemia or at the onset of reperfusion. At the end of the study period, jejunal segments were excised and assessed for histopathologic score, apoptotic index using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling (TUNEL) assay and glutathione/glutathione disulfide (GSH/GSSG) ratio, as a marker of oxidative stress. I/R caused deterioration of histological characteristics and induction of apoptosis and oxidative stress in the intestinal mucosa. Changes regarding histology and apoptosis were prevented when mesna was administered 60 min before ischemia, but were attenuated when mesna was administered at the onset of ischemia or reperfusion. In all mesna groups, oxidative stress was reduced. Mesna can ameliorate or even prevent intestinal I/R injury by reducing oxidative stress.

Relationship between TNF‐α and TUNEL‐positive chondrocytes in antigen‐induced arthritis of the rabbit temporomandibular joint

Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end-labeling (TUNEL) staining is a widely accepted method for the detection of DNA fragmentation in nuclei of apoptotic cells. Tumor necrosis factor (TNF)-alpha is closely associated with changes in condylar cartilage and modulates apoptosis in various tissues including cartilage. The aim of this study was to investigate the relationship between apoptotic chondrocytes and TNF-alpha in a rabbit model of arthritis. Unilateral temporomandibular joint (TMJ) arthritis was induced in 20 adult New Zealand White rabbits. From 1 day to 6 weeks after the induction of arthritis, immunohistochemical analysis for TNF-alpha and TUNEL was performed. In condylar cartilage, TNF-alpha-positive cells and TUNEL-positive cells were localized together. TNF-alpha-positive chondrocytes seemed to precede TUNEL-positive cells. The results of the present study suggest that TNF-alpha may be involved in apoptosis and/or apoptotic necrosis of chondrocytes as TMJ arthritis progresses from the acute to chronic stage.

ICSI in Cases of Sperm DNA Damage: Beneficial Effect of Oral Antioxidant Treatment

referral clinic. Patient(s): We collected 303 semen samples from patients undergoing IVF with or without ICSI. Intervention(s): Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assay, measurement of fertilization rates, good embryo rates, and pregnancy rates for the IVF/ICSI program. Main Outcome Measure(s): The percentage of sperm with DNA fragmentation, correlated with semen analysis parameters and IVF/ICSI outcomes. Result(s): Sperm DNA fragmentation rates were significantly higher in patients with abnormal sperm parameters than in those with normal sperm parameters. When sperm DNA fragmentation was 10%, fertilization rates were affected. Sperm DNA fragmentation rates were negatively correlated with sperm velocity parameters but did not affect pregnancy outcomes. Conclusion(s): The results indicated that sperm DNA fragmentation affects fertilization rates and sperm motility but might not affect pregnancy rates. Editorial Comment: Since ICSI allows a single immotile sperm, even a dead one, to fertilize an ovum, an assay for sperm DNA integrity would prove useful in counseling couples undergoing the procedure. These investigators assessed sperm DNA integrity as measured by the TUNEL assay, and correlated outcomes in 303 women undergoing in vitro fertilization (86 with ICSI.) DNA fragmentation correlated poorly with bulk seminal parameters, including morphology, supporting the concept that the look of a sperm and its DNA are different things, and drawing into question the ability of a technician to select a good sperm for ICSI based on appearance alone. DNA fragmentation did affect fertilization rates and embryo quality, suggesting a possible prognostic value for sperm TUNEL assay.

Basic Fibroblast Growth Factor Suppresses Retinal Neuronal Apoptosis in Form-Deprivation Myopia in Chicks

Purpose: Chorioretinal atrophy including retinal neuronal apoptosis occurred in chronic form-deprivation myopia induced by lid suture in chicks. We investigated whether exogenous basic fibroblast growth factor (bFGF) could simultaneously inhibit the excessive axial elongation and retinal neuronal loss in the myopic chick eyes. Methods: Unilateral form deprivation was produced in neonatal male Hyline chicks by lid suture 24 hr after hatching. Ten microliters of solution containing 5 ng bFGF or PBS (vehicle) was injected into the vitreal chamber at 10 weeks of age, once every 3 days until week 12, when the animals were sacrificed. Ocular refraction and axial length were assessed by retinoscopy and calipers at the age of 12 weeks. Retinal apoptotic neurons and their caspase-3-like protease activity were analyzed by transmission electron microscopy, the terminal-deoxynucleotidyl transferase-mediated biotin-deoxyuridine triphosphate nick-end labeling (TUNEL) staining, and a colorimetric method using Ac-DEVD-pNA as a substrate. The number of TUNEL-positive cells was counted to analyize the survival activity of bFGF on retina. Results: After 12 weeks of lid suture, the control eyes were either emmetropic or hyperopic, whereas the deprived eyes became myopic with axial length increased. TUNEL-positive nuclei, condensed nuclear chromatin, and apoptotic bodies were observed in posterior retinal outer nuclear layer (ONL) and inner nuclear layer (INL) in the myopic chick eyes. Activity of retinal caspase-3-like protease in these eyes was elevated. In addition to ameliorating myopic ocular growth, intravitreal bFGF therapy significantly reduced the number of retinal apoptotic neurons and downregulated caspase-3 activity. Conclusions: Exogenous bFGF effectively ameliorates the excessive axial elongation and retinal neuron apoptosis in chronic form-deprivation myopia in chicks. It is possible that bFGF will be a promising therapeutic agent for high human myopia.

Interleukin-1 beta induction of neuron apoptosis depends on p38 mitogen-activated protein kinase activity after spinal cord injury1

Interleukin-1 beta (IL-1beta) has been implicated as an extracellular signal in the initiation of apoptosis in neurons and oligodendrocytes after spinal cord injury (SCI). To further characterize the apoptotic cascade initiated by IL-1beta after SCI, we examined the expression of IL-1beta, p38 mitogen-activated protein kinase (p38 MAPK) and caspase-3 after SCI, and further investigated whether p38 MAPK was involved in neuron apoptosis induced by IL-1beta. Adult rats were given contusion SCI at the T-10 vertebrae level with a weight-drop impactor (10 g weight dropped 25.0 mm). The expression levels of IL-1beta, p38 MAPK and caspase-3 after SCI were assessed with Western blots, immunohistochemistry staining, and real time reverse transcription polymerase chain reactions (RT-PCR). Neuron apoptosis was assessed with the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) method. Increased levels of IL-1beta and p38 MAPK were observed soon after injury, with a peak in expression levels within 6 h of injury. By 24 h after injury, caspase-3 expression was markedly increased in the injured spinal cord. TUNEL-positive cells were first observed in the lesioned area 6 h after SCI. The largest number of TUNEL-positive cells was observed at 24 h post-SCI. Intrathecal injection of the IL-1 receptor antagonist IL-1Ra significantly reduced expression of p38 MAPK and caspase-3, and reduced the number of TUNEL-positive cells. Moreover, intrathecal injection of an inhibitor of p38 MAPK, SB203580, also significantly reduced the expression of caspase-3, and reduced the number of TUNEL-positive cells in the injured spinal cord. The p38MAPK signaling pathway plays an important role in IL-1beta mediated induction of neuron apoptosis following SCI in rats.

Sperm DNA fragmentation negatively correlates with velocity and fertilization rates but might not affect pregnancy rates

To evaluate sperm DNA fragmentation in correlation with sperm parameters and IVF/intracytoplasmic sperm injection (ICSI) outcomes. Retrospective review. A tertiary infertility referral clinic. We collected 303 semen samples from patients undergoing IVF with or without ICSI. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assay, measurement of fertilization rates, good embryo rates, and pregnancy rates for the IVF/ICSI program. The percentage of sperm with DNA fragmentation, correlated with semen analysis parameters and IVF/ICSI outcomes. Sperm DNA fragmentation rates were significantly higher in patients with abnormal sperm parameters than in those with normal sperm parameters. When sperm DNA fragmentation was >10%, fertilization rates were affected. Sperm DNA fragmentation rates were negatively correlated with sperm velocity parameters but did not affect pregnancy outcomes. The results indicated that sperm DNA fragmentation affects fertilization rates and sperm motility but might not affect pregnancy rates.

Different Susceptibility of Cells of Porcine Skin and Internal Organs to Ultraviolet A‐Induced Breaking of Nuclear DNA¶

ABSTRACTThere is a continuously growing interest in medical applications of ultraviolet radiation (UV‐A and long‐wavelength UV‐B) especially for laser surgery, phototherapy and photodiagnostics of human internal organs. UV‐B and UV‐A radiation is potentially mutagenic, however, there has been very little information published to date concerning the significance of possible deleterious action of such photons on cells of internal tissues. The aim of this study is to compare the sensitivities of skin cells to those of internal organs upon exposure to UV‐A. To assess this sensitivity we have determined the UV‐A dose‐dependent frequency of nuclear DNA breaks detected with the terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate‐biotin nick end‐labeling (TUNEL) technique. The materials for the study were macroscopic samples of porcine skin, colon and esophagus. The UV‐A dose ranged from 0.1 to 1000 mJ/cm2, which is similar to doses received by cells in regions examined with laser‐induced fluorescence or by cells surrounding areas subject to a laser ablation. To reduce the influence of DNA repair processes the tissue samples were kept at a low temperature during the irradiation and were deep frozen immediately after completing the irradiation procedure. The cells of the internal organs are much more susceptible to UV‐A‐induced breaking of DNA than the skin cells. The percentage fractions and the spatial distributions of the damaged cells and the characteristics of the UV‐A dose dependence seem to vary by type of internal organ.

Expression of Apoptotic and Antiapoptotic Markers in Epithelial Cells in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic, usually fatal lung disease of unknown etiology. A common feature is the presence of microscopic areas of epithelial cell dropout. Increased apoptosis of these cells could elucidate the speculative pathogenesis of the disease. Therefore, the aim of our study was to examine the expression of p53, p21, bcl-2, bax, and caspase-3 in association with DNA strand breaks in bronchial and alveolar epithelial cells in lung specimens from IPF patients and control subjects. We examined by immunohistochemistry the expression of p53, p21, bax, bcl-2, and caspase-3 in association with DNA strand breaks detected by terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) in bronchial and alveolar epithelial cells in lung specimens taken by biopsy in 12 IPF patients and 10 control subjects. An independent tissue evaluation by two pathologists graded semiquantatively the degree of staining present. TUNEL was positive in epithelial cells in all IPF patients and only in one control subject. The expression of p53, p21, bax, and caspase-3 was up-regulated in IPF patients compared to control subjects. Bcl-2 was expressed less in IPF patients than in control subjects. These results confirm that apoptotic hyperplastic epithelial cells are present in patients with IPF and that the expression of p53, p21, bax, and caspase-3 appears to be up-regulated and that of bcl-2 down-regulated in these cells. The increased expression of proapoptotic molecules in epithelial cells in IPF may be involved in the inadequate and delayed reepithelialization, which in turn contributes to fibroblast proliferation.

Inducible nitric oxide synthase, nitrotyrosine and apoptosis in gastric adenocarcinomas and their correlation with a poor survival

To detect the presence of inducible nitric oxide synthase (iNOS), nitrotyrosine (NT) and apoptosis in gastric adenocarcinomas and their possible correlations with the clinicopathological characteristics and prognosis of gastric adenocarcinoma. Sixty-six specimens of gastric adenocarcinoma and corresponding adjacent normal gastric tissues were studied. Immunohistochemistry was employed to localize iNOS and NT protein and an immunohistochemical scoring system was used. The occurrence of apoptotic cell death (apoptotic index (AI)) was analyzed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labeling (TUNEL) method. Results showed that iNOS expression was detected at an intermediate or high level in 41 of 66 (62%) specimens of gastric adenocarcinoma. NT expression was 58%. Neither of them was found in the normal gastric tissues; there were significant positive correlations among iNOS expression, NT expression and AI. Many clinicopathologic characteristics of gastric adenocarcinoma, such as tumor size, depth of invasion, lymph node metastasis and TNM staging, were related to iNOS and NT expressions (P<0.05). In 66 surviving patients, the 5-year survival rate of 41 patients who had tumors with intermediate or high iNOS expressions and high AIs (4.09%; 19.96%) was significantly lower than that of 25 patients who had tumors with negative or low iNOS expressions and low AIs (0.79%; 47.14%) (P = 0.001). COX's multivariate analysis revealed that the iNOS expression was identified as one of the significant independent prognostic factors predictive of a poor survival (relative risk (RR) = 2.69). NO produced by iNOS may play a stronger role in promoting gastric adenocarcinoma growth than in suppressing its growth. iNOS and NT expressions by gastric adenocarcinoma may correlate with a poor survival.

Associations among beta-TrCP, an E3 ubiquitin ligase receptor, beta-catenin, and NF-kappaB in colorectal cancer.

The ubiquitin-proteasome pathway is important in regulating protein signaling pathways that are involved in tumorigenesis. beta-transducin repeat-containing proteins (beta-TrCP) are components of the ubiquitin ligase complex targeting beta-catenin and IkappaBalpha for proteasomal degradation and are thus a negative regulator of Wnt/beta-catenin signaling and a positive regulator of NF-kappaB signaling. We analyzed expression of beta-TrCP in colorectal cancers and its association with types of beta-catenin subcellular localization, an indirect measure of activation. Levels of beta-TrCP1 mRNA and protein were measured by quantitative reverse transcription-polymerase chain reaction and immunoblotting, respectively, in samples of tumor and normal tissues from 45 patients with colorectal cancer. Types of beta-catenin activation (diffuse or invasion edge) and NF-kappaB activation were examined by immunohistochemistry. Apoptosis was determined by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling (TUNEL) assay. All statistical tests were two-sided. Compared with the beta-TrCP1 levels in normal tissues, 25 (56%) of 45 tumors had increased beta-TrCP1 mRNA and protein levels. Of the 22 (49%) tumors with beta-catenin activation, 12 had the diffuse type (i.e., nuclear accumulation throughout the tumor) and 10 had the invasion edge type (i.e., nuclear accumulation predominantly in the tumor cells that formed the invasion edge). Increased beta-TrCP1 levels were statistically significantly associated with beta-catenin activation (P =.023) and decreased apoptosis (P =.035). beta-TrCP accumulated in the nuclei of tumor cells that contained increased levels of beta-TrCP1 mRNA and the active form of NF-kappaB. Higher levels of beta-TrCP1 mRNA were detected in primary tumors of patients who had metastases (0.960 arbitrary units, 95% confidence interval = 0.878 to 1.042) than in the tumors of patients who did not (0.722 arbitrary units, 95% confidence interval = 0.600 to 0.844; P =.016). In colorectal cancer, increased expression of beta-TrCP1 is associated with activation of both beta-catenin and NF-kappaB, suggesting that the integration of these signaling pathways by increased beta-TrCP expression may contribute to an inhibition of apoptosis and tumor metastasis.

Mesna ameliorates intestinal mucosa damage after ifosfamide administration in the rabbit at a dose-Related manner

Background Cancer chemotherapy may lead to mucositis, a serious dose-limiting side effect. The alkylating agent ifosfamide is used in the treatment of various forms of cancer in combination with the uroprotective thiol mesna (2-mercaptoethane-sulfonate). The aims of this study were to assess the dose response intestinal mucosa damage of ifosfamide and to investigate the potential protective effect of mesna on rabbit intestinal epithelium. Materials and methods Fifty New Zealand White rabbits were randomly assigned to 10 groups of five animals each and received intravenously every week for 10 weeks either normal saline, ifosfamide, mesna, or ifosfamide plus mesna at three escalating dose levels (ifosfamide: 30, 45, or 60 mg/kg; mesna: 12, 18, or 24 mg/kg divided into two equal doses administered 4 h apart). Intestinal mucosa damage was assessed on the basis of crypt cell apoptosis and proliferation as well as intestinal morphometry. Apoptosis was detected by agarose gel electrophoresis and quantified by the DNA fragmentation assay and a standard terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling (TUNEL) method by which the percentage of fragmented DNA and the apoptotic index were determined, respectively. The mitotic index and the crypt-villus (c/v) unit height were also measured in histological sections. Results Ifosfamide caused a dose-related increase of crypt cell apoptosis and shortening of c/v unit, while it had a steady antimitotic effect. Mesna as a sole agent had no apoptotic or trophic effect on intestinal mucosa and hence no effect on intestinal morphometry. However, mesna, when administered concurrently with ifosfamide, ameliorated apoptosis, hypoproliferation, and mucosal atrophy at a dose-related manner. Conclusions Ifosfamide causes intestinal mucosa damage, which may be ameliorated in a dose-related manner by coadministration of mesna.

Influence of biologic markers on the outcome of Hodgkin's lymphoma: a study by the Spanish Hodgkin's Lymphoma Study Group.

Current therapies fail to cure a significant proportion of patients with Hodgkin's lymphoma (HL). Predictive systems for stratification of the disease and selection of treatment based on sets of clinical variables, such as the international prognostic score (IPS), are of relatively small practical value. The predictive use of biologic parameters has so far provided limited and inconsistent results. Here we explore the influence of a set of molecular markers on the outcome of HL. Forty molecular markers involved in B-cell differentiation and activation, signal transduction, cell cycle, and apoptosis control were analyzed in 259 classic HL patient cases by using tissue microarrays. Univariate analysis was performed to evaluate the influence of markers on favorable outcome (complete remission of > 12 months). Significant variables were included in a multivariate logistic regression analysis, and the probability of favorable outcome was estimated. Univariate analysis revealed four molecular markers that predicted outcome, and the multivariate analysis showed p53, Bcl-X(L), and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling (TUNEL) to have independent significance. The combination of these factors determined two groups of patients (group I, zero to one factor; group II, two to three factors) with a probability of a favorable outcome of.948 and.687, respectively. A multivariate Cox's model shows that these biologic risk groups have special predictive power in low-IPS patients. The data from this exploratory study suggest that the accumulation of molecular events seems to influence the outcome of HL, particularly in the low-IPS group.

Regional low-flow perfusion improves neurologic outcome compared with deep hypothermic circulatory arrest in neonatal piglets

BackgroundRegional low-flow perfusion is an alternative to deep hypothermic circulatory arrest, but whether regional low-flow perfusion improves neurologic outcome after deep hypothermic circulatory arrest in neonates remains unknown. We tested neurologic recovery after regional low-flow perfusion compared with deep hypothermic circulatory arrest in a neonatal piglet model. MethodsSixteen neonatal piglets underwent cardiopulmonary bypass, were randomized to 90 minutes of deep hypothermic circulatory arrest or regional low-flow perfusion (10 mL · kg−1 · min−1) at 18°C, and survived for 1 week. Standardized neurobehavioral scores were obtained on postoperative days 1, 3, and 7 (0 = no deficit to 90 = brain death). Histopathologic scores were determined on the basis of the percentage of injured and apoptotic neurons in the neocortex and hippocampus by hematoxylin and eosin and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate–biotin nick-end labeling (0 = no injury to 4 = diffuse injury). Differences between groups were tested by using the Wilcoxon rank sum test, and results are listed as medians within a range. ResultsThere were no significant differences between groups during cardiopulmonary bypass. Postoperative neurobehavioral scores were abnormal in 25% (2/8) of the regional low-flow perfusion animals versus 88% (7/8) of controls. Regional low-flow perfusion animals had significantly less neurologic injury compared with controls on postoperative day 1 (0.00 [range, 0-5] vs 12.5 [range, 0-52]; P < .008). There was a trend for less severe injury in the regional low-flow perfusion group (2.0 [range, 1-4] vs 0.0 [range, 0-50]; P = .08) on hematoxylin and eosin. The degree of apoptosis was significantly less in the regional low-flow perfusion group (0.0 [range, 0-1] vs 2.5 [range, 0-4]; P = .03). ConclusionsRegional low-flow perfusion decreases neuronal injury and improves early postoperative neurologic function after deep hypothermic circulatory arrest in neonatal piglets.

Different Susceptibility of Cells of Porcine Skin and Internal Organs to Ultraviolet A - Induced Breaking of Nuclear DNA

There is a continuously growing interest in medical applications of ultraviolet radiation (UV-A and long-wavelength UV-B) especially for laser surgery, phototherapy and photodiagnostics of human internal organs. UV-B and UV-A radiation is potentially mutagenic, however, there has been very little information published to date concerning the significance of possible deleterious action of such photons on cells of internal tissues. The aim of this study is to compare the sensitivities of skin cells to those of internal organs upon exposure to UV-A. To assess this sensitivity we have determined the UV-A dose-dependent frequency of nuclear DNA breaks detected with the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) technique. The materials for the study were macroscopic samples of porcine skin, colon and esophagus. The UV-A dose ranged from 0.1 to 1000 mJ/cm2, which is similar to doses received by cells in regions examined with laser-induced fluorescence or by cells surrounding areas subject to a laser ablation. To reduce the influence of DNA repair processes the tissue samples were kept at a low temperature during the irradiation and were deep frozen immediately after completing the irradiation procedure. The cells of the internal organs are much more susceptible to UV-A-induced breaking of DNA than the skin cells. The percentage fractions and the spatial distributions of the damaged cells and the characteristics of the UV-A dose dependence seem to vary by type of internal organ.