Epithelial Apoptosis and Loss in Airways of Children with Asthma

Objective. To examine loss and apoptosis of bronchial epithelial cells in children with asthma. Methods. We examined endobronchial biopsies from 13 asthmatic children and 11 non-asthmatic control subjects with other respiratory diseases. Postmortem samples were obtained from six children who died from non-respiratory diseases. We examined bronchial epithelial shedding by morphology; expression of caspase-3 and terminal deoxynucleotidyl-mediated dUTP nick end labeling (TUNEL) were used to study bronchial epithelial apoptosis. Results. We found epithelial loss to be increased in asthmatic children compared with non-asthmatic control subjects (p = .001) and postmortem children (p = .001). Caspase-3⁺ epithelial cells were significantly greater in children with asthma compared with both non-asthmatic control subjects (p = .001) and the postmortem group (p = .002); TUNEL⁺ epithelial cells were also increased in columnar cells in the asthmatic children compared with the non-asthmatic control subjects (p = .002) and the postmortem group (p = .001). Eosinophilia was absent in 11 of 13 asthmatic children, although they tended to have submucosal lymphocyte infiltration. Smooth muscle and mucus gland hyperplasia were seen in some asthmatic children whose biopsy specimens included these structures. Basement membranes of childhood asthmatics were thicker than in non-asthmatic controls (p = .002) and postmortem subjects (p = .001). Conclusion. Generally, apoptosis and loss of bronchial epithelial cells were increased in childhood asthma; increased apoptosis might be related to epithelial loss.     

Caspase-3 activation in the guinea pig cochlea exposed to salicylate

In the current study, we explored whether chronic salicylate exposure could induce apoptosis in outer hair cells (OHCs) and spiral ganglion neurons (SGNs) of the cochlea. Guinea pig received sodium salicylate (400 mg/kg/d) or saline vehicle for 10 consecutive days. Programmed cell death (PCD) executioner was evaluated with immunohistochemistry detection of activated caspase-3. Apoptosis was examined with a terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method. Repeated salicylate administration activated caspase-3 and caused apoptosis in OHCs and SGNs (p < 0.01 vs. saline control for both measures and in both cell types). Cell counting showed a significant loss in OHCs (p < 0.01 vs. saline control), but not in inner hair cells (IHCs). Transmission electron microscopy (TEM) revealed chromatin condensation and nucleus margination in salicylate-treated cochlea. Scanning electron microscopy (SEM) demonstrated stereociliary bundles breakdown and fusion at the apical of OHCs, villous matter was discovered to attach on the surface of SGNs. These findings suggest that long-term administration of high-dose salicylate can activate caspase-3 pathway to induce OHC and SGN apoptosis.     

Blocking tumor necrosis factor-alpha inhibits folic acid-induced acute renal failure

Systemic administration of mice with folic acid (FA) has been used for studying the pathogenesis of acute renal failure. However, the molecular mechanisms by which FA induces acute renal failure remain poorly understood. We found that CD-1 mice treated with FA developed acute renal failure characterized by increased blood urea nitrogen, necrosis, and apoptosis of tubular epithelial cells. Compared to control mice, tumor necrosis factor-alpha (TNF-alpha) was markedly elevated in blood and kidneys of these FA-treated mice, accompanied by markedly reduced expression of anti-apoptotic protein BclxL in their kidneys. In vivo administration of FA-treated CD-1 mice with neutralizing anti-TNF-alpha antibody restored the expression of BclxL in kidneys and inhibited the necrosis and apoptosis of renal tubular epithelial cells, leading to the amelioration of acute renal failure. In ex vivo cultures, we found that FA enhanced production of TNF-alpha, decreased expression of BclxL protein, and induced apoptosis of mouse cortical tubule (MCT) cells. Addition of neutralizing anti-TNF-alpha antibody, but not control IgG, in the cultures markedly blocked the apoptotic death of FA-treated MCT cells and restored expression of BclxL to the same levels as those MCT cells cultured in the absence of FA. All these results suggest that TNF-alpha is a critical inflammatory cytokine responsible for FA-mediated acute renal failure. Furthermore, in vivo administration of anti-TNF-alpha antibody may be proved as an effective approach for acute renal failure prevention and treatment.     

Expression of Mcl-1 in mantle cell lymphoma is associated with high-grade morphology,a high proliferative state,and p53 overexpression

Mantle cell lymphoma (MCL) is a distinct type of B-cell non-Hodgkin's lymphoma characterized by the t(11;14)(q13;q32) and cyclin D1 overexpression. Defects in apoptosis may contribute to pathogenesis. This study evaluated the expression of the anti-apoptotic protein Mcl-1 in two MCL cell lines and five frozen MCL tumours (four small-cell, one blastoid/large-cell) using western blot analysis. Mcl-1 expression was also assessed in 36 formalin-fixed, paraffin wax-embedded MCL tumours (24 small-cell, 12 blastoid/large-cell) by immunohistochemistry. Western blot analysis revealed the expected 37 kD protein product in both MCL cell lines and in five frozen tumours, with the blastoid case having the highest expression level. Using a cut-off of >10% immunolabelled cells for Mcl-1, it was found that 12 of 36 MCL tumours were positive. Mcl-1-positive tumours had a higher frequency of blastoid/large-cell morphology (8/12 versus 4/24, p = 0.009), p53 overexpression (3/10 versus 1/23, p = 0.04), and higher Ki67 immuno-labelling (p = 0.002). It is concluded that expression of Mcl-1 in MCL is heterogeneous. A relatively high level of Mcl-1 expression correlates with high-grade morphology, a high proliferative state, and p53 overexpression.     

PPARs激动剂罗格列酮对大鼠癫痫持续状态海马神经元的保护作用

目的探讨罗格列酮(RSG)对大鼠癫痫持续状态(SE)后海马神经元凋亡的影响及其可能作用机制。方法采用免疫组化法对氯化锂-匹罗卡品诱发癫痫大鼠模型脑内Bcl-2、Bax及TUNEL阳性细胞的表达进行测定,并研究RSG对癫痫影响及可能机制。结果模型组(M组)海马区TUNEL、Bcl-2、Bax较对照组(N组)明显增多(P0.05);RSG组Bax、TUNEL较M组明显减少(P0.05),Bcl-2阳性细胞、Bcl-2/Bax比率较M组明显增多(P0.05)。结论SE后脑内TUNEL、Bcl-2、Bax表达明显增加;RSG能够减轻海马TUNEL、Bax的表达,增加海马Bcl-2的表达从而增加Bcl-2/Bax比率。RSG可能具有减轻癫痫发作和保护神经元的作用。     

Absence of age-related changes in nigral dopaminergic neurons of Asian Indians: Relevance to lower incidence of Parkinson's disease

Age-related loss of melanized nigral neurons reported in the British Caucasians is not observed in Asian Indian, American and French adults. In the Americans, loss of dopaminergic phenotype occurs from midlife, without frank neurodegeneration. Here, we investigated whether nigral dopaminergic neurons in Asian Indians are lost with age or undergo morphological or biochemical dysfunction. Using unbiased stereology we estimated volume, number of melanized, borderline/non-melanized (n=34, 28 gestational weeks to 80 years) and tyrosine hydroxylase (TH)–Nurr1 co-labeled neurons (n=32, 28 gestational weeks to 80 years) in substantia nigra pars compacta. We quantified Nurr1 and TH proteins by immunoblotting (n=18, 28 gestational weeks to 69 years) and apoptotic neurons by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) staining. Nuclear and soma size was estimated by morphometry. There was no age-related decline in volume, neuronal density, neuronal numbers and TH-Nurr1 co-labeled neurons. TH and Nurr1 protein expression remained stable. Lack of TUNEL-TH co-labeled cells confirmed absence of neuronal apoptosis. The neuronal size remained unaltered. Our findings of preserved nigral dopaminergic neurons suggest no age-related loss of nigral function in Asian Indians, unlike the Americans. This may explain the lower incidence of Parkinson's disease in Asian Indians.     

EGCG inhibits activation of the insulin-like growth factor (IGF)/IGF-1 receptor axis in human hepatocellular carcinoma cells

The receptor tyrosine kinase (RTK) insulin like growth factor-1 (IGF-1)/IGF-1 receptor (IGF-1R) axis plays an important role in the development of hepatocellular carcinoma (HCC). EGCG inhibits activation of the various types of RTKs and that this is associated with inhibition of multiple downstream signaling pathways. In this study we examined the effects of EGCG on activity of the IGF/IGF-1R axis in HepG2 human HCC cells which express constitutive activation of this axis. The level of phosphorylated (i.e. activated) form of the IGF-1R protein (p-IGF-1R) was increased in a series of human HCC cell lines when compared with the Hc normal human hepatocytes. EGCG preferentially inhibited growth of HepG2 cells when compared with Hc cells. Treatment of HepG2 cells with EGCG induced apoptosis and caused a decrease in the p-IGF-1R protein and its downstream signaling molecules including the p-ERK, p-Akt, p-Stat-3, and p-GSK-3β proteins, both in the absence or presence of ligand stimulation. EGCG also decreased the levels of both IGF-1 and IGF-2 proteins and mRNAs, but increased the levels of the IGFBP-3 protein. These findings suggest that EGCG can overcome the stimulatory effects of IGFs on the IGF-1R dependent signaling pathway, thus expanding the roles of EGCG as an inhibitor of critical RTKs involved in HCC cell proliferation. These results provide further evidence that EGCG may be useful in the chemoprevention or treatment of liver cancer.     

Chronic cerebral hypoperfusion induces white matter lesions and loss of oligodendroglia with DNA fragmentation in the rat

Cerebrovascular white matter lesions represent an age-related neurodegenerative condition that appears as a hyperintense signal on magnetic resonance images. These lesions are frequently observed in aging, hypertension and cerebrovascular disease, and are responsible for cognitive decline and gait disorders in the elderly population. In humans, cerebrovascular white matter lesions are accompanied by apoptosis of oligodendroglia, and have been thought to be caused by chronic cerebral ischemia. In the present study, we tested whether chronic cerebral hypoperfusion induces white matter lesions and apoptosis of oligodendroglia in the rat. Doppler flow meter analysis revealed an immediate reduction of cerebral blood flow ranging from 30% to 40% of that before operation; this remained at 52–64% between 7 and 30 days after operation. Transferrin-immunoreactive oligodendroglia decreased in number and the myelin became degenerated in the medial corpus callosum at 7 days and thereafter. Using the TUNEL method, the number of cells showing DNA fragmentation increased three- to eightfold between 3 and 30 days post-surgery compared to sham-operated animals. Double labeling with TUNEL and immunohistochemistry for markers of either astroglia or oligodendroglia showed that DNA fragmentation occurred in both of these glia. Messenger RNA for caspase-3 increased approximately twofold versus the sham-operated rats between 1 and 30 days post-surgery. Immunohistochemistry revealed up-regulation of caspase-3 in the oligodendroglia of the white matter, and also in the astroglia and neurons of the gray matter. Molecules involved in apoptotic signaling such as TNF- and Bax were also up-regulated in glial cells. These results indicate that chronic cerebral hypoperfusion induces white matter degeneration in association with DNA fragmentation in oligodendroglia.     

Trans-neuronal regulation of cortical apoptosis in the adult rat olfactory system

Previous work has identified a population of neurons within the anterior piriform cortex that undergo rapid apoptosis following de-afferentation by olfactory bulbectomy in adult rats. The specific initiation signal for apoptosis in this paradigm is unknown, but may include an activity-dependent trans-neuronal cascade. The present report examined the effect of adult-onset unilateral naris occlusion, which reduces olfactory bulb afferent excitation of piriform cortex, on apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling [TUNEL]) in the rat anterior piriform cortex. Adult Long-Evans hooded rats received unilateral naris occlusion or a control manipulation and were sacrificed after 1, 5, 7, 10 or 20 days later. For comparison, a second group of rats received a unilateral bulbectomy and were sacrificed 24 h later. Counts of TUNEL-stained cell profiles were performed for layers I/II and layer III of the anterior piriform cortex ipsilateral and contralateral to the manipulation. The results confirmed that unilateral bulbectomy produced a dramatic increase in TUNEL labeling in layers I/II of the ipsilateral piriform cortex 24 h after bulbectomy. Unilateral naris closure also produced enhanced TUNEL labeling, although the magnitude of the effect was less than that produced by bulbectomy, and enhanced TUNEL labeling was apparent both ipsilateral and contralateral to the sealed naris compared to controls. Deprivation-induced TUNEL labeling was detectable by 24 h post-closure, peaked at 5 days and was no different from controls by 20 days post-closure. Neither bulbectomy nor naris closure affected TUNEL labeling in layer III. Together, these results suggest that there is a population of superficial cells in piriform cortex whose survival is tightly regulated by sensory input.