Cholest-4-en-3-one attenuates TGF-ß responsiveness by inducing TGF-ß receptors degradation in Mv1Lu cells and colorectal adenocarcinoma cells.

PURPOSE: The transforming growth factor-beta (TGF-ß) pathway is an important in the initiation and progression of cancer. Due to a strong association between an elevated colorectal cancer risk and increase fecal excretion of cholest-4-en-3-one, we aim to determine the effects of cholest-4-en-3-one on TGF-ß signaling in the mink lung epithelial cells (Mv1Lu) and colorectal cancer cells (HT29) in vitro. METHODS: The inhibitory effects of cholest-4-en-3-one on TGF-ß-induced Smad signaling, cell growth inhibition, and the subcellular localization of TGF-ß receptors were investigated in epithelial cells using a Western blot analysis, luciferase reporter assays, DNA synthesis assay, confocal microscopy, and subcellular fractionation. RESULTS: Cholest-4-en-3-one attenuated TGF-ß signaling in Mv1Lu cells and HT29 cells, as judged by a TGF-ß-specific reporter gene assay of plasminogen activator inhibitor-1 (PAI-1), Smad2/3 phosphorylation and nuclear translocation. We also discovered that cholest-4-en-3-one suppresses TGF-ß responsiveness by increasing lipid raft and/or caveolae accumulation of TGF-ß receptors and facilitating rapid degradation of TGF-ß and thus suppressing TGF-ß-induced signaling. CONCLUSIONS: Our results suggest that cholest-4-en-3-one inhibits TGF-ß signaling may be due, in part to the translocation of TGF-ß receptor from non-lipid raft to lipid raft microdomain in plasma membranes. Our findings also implicate that cholest-4-en-3-one may be further explored for its potential role in colorectal cancer correlate to TGF-ß deficiency.

Inhaled corticosteroids can reduce osteoporosis in female patients with COPD.

BACKGROUND: Whether the use of inhaled corticosteroids (ICSs) in patients with COPD can protect from osteoporosis remains undetermined. The aim of this study is to assess the incidence of osteoporosis in patients with COPD with ICS use and without. PATIENTS AND METHODS: This is a retrospective cohort and population-based study in which we extracted newly diagnosed female patients with COPD between 1997 and 2009 from Taiwan's National Health Insurance (TNHI) database between 1996 and 2011 (International Classification of Diseases, Ninth Revision - Clinical Modification [ICD-9-CM] 491, 492, 496). The patients with COPD were defined by the presence of two or more diagnostic codes for COPD within 12 months on either inpatient or outpatient service claims submitted to TNHI. Patients were excluded if they were younger than 40 years or if osteoporosis had been diagnosed prior to the diagnosis of COPD and cases of asthma (ICD-9 CM code 493.X) before the index date. These enrolled patients were followed up till 2011, and the incidence of osteoporosis was determined. The Cox proportional hazards regression model was also used to estimate hazard ratios (HRs) for incidences of lung cancer. RESULTS: Totally, 10,723 patients with COPD, including ICS users (n=812) and nonusers (n=9,911), were enrolled. The incidence rate of osteoporosis per 100,000 person years is 4,395 in nonusers and 2,709 in ICS users (HR: 0.73, 95% confidence interval [CI]: 0.63-084). The higher ICS dose is associated with lower risk of osteoporosis (0 mg to ≤20 mg, HR: 0.84, 95% CI: 0.69-1.04; >20 mg to ≤60 mg, HR: 0.78, 95% CI: 0.59-1.04; and >60 mg, HR: 0.72, 95% CI: 0.55-0.96; P for trend =0.0023) after adjusting for age, income, and medications. The cumulative osteoporosis probability significantly decreased among the ICS users when compared with the nonusers (P<0.001). CONCLUSION: Female patients with COPD using ICS have a dose-response protective effect for osteoporosis.

Theophylline-Based KMUP-1 Improves Steatohepatitis via MMP-9/IL-10 and Lipolysis via HSL/p-HSL in Obese Mice.

KMUP-1 (7-[2-[4-(2-chlorobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine) has been reported to cause hepatic fat loss. However, the action mechanisms of KMUP-1 in obesity-induced steatohepatitis remains unclear. This study elucidated the steatohepatitis via matrix metallopeptidase 9 (MMP-9) and tumor necrosis factor α (TNFα), and related lipolysis via hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) by KMUP-1. KMUP-1 on steatohepatitis-associated HSL/p-HSL/ATGL/MMP-9/TNFα/interleukin-10 (IL-10) and infiltration of M1/M2 macrophages in obese mice were examined. KMUP-1 was administered by oral gavage from weeks 1-14 in high-fat diet (HFD)-supplemented C57BL/6J male mice (protection group) and from weeks 8-14, for 6 weeks, in HFD-induced obese mice (treatment group). Immunohistochemistry (IHC) and hematoxylin and eosin (H&E) staining of tissues, oil globules number and size, infiltration and switching of M1/M2 macrophages were measured to determine the effects on livers. IL-10 and MMP-9 proteins were explored to determine the effects of KMUP-1 on M1/M2 macrophage polarization in HFD-induced steatohepatitis. Long-term administration of KMUP-1 reversed HFD-fed mice increased in body weight, sGOT/sGPT, triglyceride (TG) and glucose. Additionally, KMUP-1 decreased MMP-9 and reactive oxygen species (ROS), and increased HSL/p-HSL and IL-10 in HFD mice livers. In conclusion, KMUP-1, a phosphodiesterase inhibitor (PDEI), was shown to reduce lipid accumulation in liver tissues, suggesting that it could be able to prevent or treat steatohepatitis induced by HFD.

Altered mitochondrial dynamics and response to insulin in cybrid cells harboring a diabetes-susceptible mitochondrial DNA haplogroup.

The advantage of using a cytoplasmic hybrid (cybrid) model to study the genetic effects of mitochondria is that the cells have the same nuclear genomic background. We previously demonstrated the independent role of mitochondria in the pathogenesis of insulin resistance (IR) and pro-inflammation in type 2 diabetes. In this study, we compared mitochondrial dynamics and related physiological functions between cybrid cells harboring diabetes-susceptible (B4) and diabetes-protective (D4) mitochondrial haplogroups, especially the responses before and after insulin stimulation. Cybrid B4 showed a more fragmented mitochondrial network, impaired mitochondrial biogenesis and bioenergetics, increased apoptosis and ineffective mitophagy and a low expression of fusion-related molecules. Upon insulin stimulation, increases in network formation, mitochondrial DNA (mtDNA) content, and ATP production were observed only in cybrid D4. Insulin promoted a pro-fusion dynamic status in both cybrids, but the trend was greater in cybrid D4. In cybrid B4, the imbalance of mitochondrial dynamics and impaired biogenesis and bioenergetics, and increased apoptosis were significantly improved in response to antioxidant treatment. We concluded that diabetes-susceptible mtDNA variants are themselves resistant to insulin.

Leukocyte Mitochondrial DNA Copy Number Is Associated with Chronic Obstructive Pulmonary Disease.

BACKGROUND: Oxidative stress is known to be involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). Evidence suggests that leukocytes mitochondria DNA (mtDNA) is susceptible to undergo mutations, insertions, or depletion in response to reactive oxidative stress (ROS). We hypothesize that mtDNA copy number is associated with the development of COPD. METHODOLOGY/PRINCIPAL FINDINGS: Relative mtDNA copy number was measured by a quantitative real-time PCR assay using DNA extracted from peripheral leukocytes. MtDNA copy number of peripheral leukocytes in the COPD group (n = 86) is significantly decreased compared with non-smoker group (n = 77) (250.3± 21.5 VS. 464.2± 49.9, P<0.001). MtDNA copy number in the COPD group was less than that in the healthy smoking group, but P value nearly achieved significance (250.3± 21.5 VS. 404.0± 76.7, P = 0.08) MtDNA copy number has no significance with age, gender, body mass index, current smoking, and pack-years in COPD group, healthy smoker group and no smoker group, respectively. Serum glutathione level in the COPD group is significantly decreased compared with healthy smoker and non-smoker groups (4.5± 1.3 VS. 6.2± 1.9 and 4.5± 1.3 VS. 7.1±1.1 mU/mL; P<0.001 respectively). Pearson correlation test shows a significant liner correlation between mtDNA copy number and serum glutathione level (R = 0.2, P = 0.009). CONCLUSIONS/SIGNIFICANCE: COPD is associated with decreased leukocyte mtDNA copy number and serum glutathione. COPD is a regulatory disorder of leukocytes mitochondria. However, further studies are needed to determine the real mechanisms about the gene and the function of mitochondria.

Pretreatment with Evans blue, a stimulator of BK(Ca) channels, inhibits compound 48/80-induced shock, systemic inflammation, and mast cell degranulation in the rat.

The present study demonstrated that intravenous injection of a high dose of compound 48/80 to the rat induced 50% drop, within a few min, in the mean arterial pressure and pulse pressure as well as systemic inflammatory plasma leakage that might lead to circulatory and respiratory failure. We also investigated whether pretreatment with Evans blue, a stimulator of BK(Ca) channels, could exert inhibitory effect against compound C48/80-induced allergic circulatory shock and systemic inflammation. Different groups of Sprague-Dawley rats received an intravenous injection of a dose of Evans blue (0, 5, 10, or 50 mg/kg) just 20 s prior to injection of compound 48/80 (200 µg/kg, over 2 min). The present study found that pretreatment with Evans blue in a dose of 10 or 50 mg/kg exerted acute inhibitory effect on compound 48/80-induced sudden drop in mean arterial and pulse pressures. We also showed that pretreatment with Evans blue in a dose of 5, 10, or 50 mg/kg significantly inhibited compound 48/80-induced extensive plasma extravasation, mast cell degranulation, and edema formation in various organs including the airways, esophagus, and skin. Pretreatment with Evans blue 50 mg/kg 1 h earlier exhibited longer-term inhibitory effect on compound 48/80-induced arterial hypotension and systemic inflammation. We concluded that Evans blue pretreatment prevented rats from compound 48/80-triggered allergic shock and systemic inflammation, possibly mainly through inhibition of mast cell degranulation. Evans blue might be potentially useful in elucidating the mechanism and acting as a therapeutic agent of allergic shock and systemic inflammation.

Biphasic response of mitochondrial biogenesis to oxidative stress in visceral fat of diet-induced obesity mice.

AIMS: Studies in skeletal muscle demonstrate a strong association of mitochondrial dysfunction with insulin resistance (IR). However, there is still a paucity of knowledge regarding the alteration of mitochondria in adipose tissue (AT) in the pathogenesis of IR in obesity. We investigated the mitochondrial biogenesis in visceral fat (VF) and subcutaneous fat (SF) in C57BL/6J mice fed a high-fat high-sucrose diet for 12 months. RESULTS: Impairment of glucose tolerance and insulin sensitivity developed after 1 month of the diet and was associated with a prompt increase of VF. The VF adipocytes were larger than those in the SF and had increased expressions of HIF-1α and p-NFκB p65. However, the alteration of mitochondrial biogenesis did not occur in the early stage when increased intracellular reactive oxygen species (ROS), mitochondrial oxygen consumption rate, and mitochondrial ROS emerged at the 1st, 2nd and 2nd month, respectively. Until the 6th month, the VF had markedly increased mitochondrial DNA content and expression of PGC-1α, Tfam, ATP5A, and MnSOD. This increase of mitochondrial biogenesis was followed by a generalized decrease at the 12th month and the mitochondrial morphology altered markedly. In the late stage, although mitochondrial ROS decreased, the increased expression of 8-OHdG in VF continued. INNOVATION AND CONCLUSION: These data suggest that IR and ROS production occur before the biphasic changes of mitochondrial biogenesis in AT, and the VF plays a more crucial role.

NF-κB activation in myeloid cells mediates ventilator-induced lung injury.

BACKGROUND: Although use of the mechanical ventilator is a life-saving intervention, excessive tidal volumes will activate NF-κB in the lung with subsequent induction of lung edema formation, neutrophil infiltration and proinflammatory cytokine/chemokine release. The roles of NF-κB and IL-6 in ventilator-induced lung injury (VILI) remain widely debated. METHODS: To study the molecular mechanisms of the pathogenesis of VILI, mice with a deletion of IкB kinase in the myeloid cells (IKKß(Δmye)), IL-6(-/-) to WT chimeric mice, and C57BL/6 mice (WT) were placed on a ventilator for 6 hr.WT mice were also given an IL-6-blocking antibody to examine the role of IL-6 in VILI. RESULTS: Our results revealed that high tidal volume ventilation induced pulmonary capillary permeability, neutrophil sequestration, macrophage drifting as well as increased protein in bronchoalveolar lavage fluid (BALF). IL-6 production and IL-1ß, CXCR2, and MIP2 expression were also increased in WT lungs but not in those pretreated with IL-6-blocking antibodies. Further, ventilator-induced protein concentrations and total cells in BALF, as well as lung permeability, were all significantly decreased in IKKß(Δmye) mice as well as in IL6(-/-) to WT chimeric mice. CONCLUSION: Given that IKKß(Δmye) mice demonstrated a significant decrease in ventilator-induced IL-6 production, we conclude that NF-κB-IL-6 signaling pathways induce inflammation, contributing to VILI, and IкB kinase in the myeloid cells mediates ventilator-induced IL-6 production, inflammation, and lung injury.

Blocking TNF-α enhances Pseudomonas aeruginosa-induced mortality in burn mice through induction of IL-1ß.

PURPOSE: Tumor necrosis factor (TNFα) is a proinflammatory cytokine and has been a target for intervention in human sepsis. However, inhibition of TNF-α with a high dose of a TNF-receptor fusion protein in patients with septic shock worsened patient survival. This study was designed to investigate whether blocking TNF-α enhances mortality in infected burn mice through the induction of IL-1ß. METHODS: WT or Tnfrsf1a(-/-) mice received Pseudomonas aeruginosa injection in the back at 8h after burn injury. The animals were sacrificed at 24h after burn and lung tissues were harvested and examined for determining myeloperoxidase (MPO) activity, pulmonary microvascular dysfunction, NF-κB DNA binding activity, and IL-1ß expression. Also, the lung and blood were harvested for bacterial count assay. RESULT: Thermal injury alone induced NF-κB DNA binding activity and neutrophil infiltration in the lung in WT but not in Tnfrsf1a(-/-) mice. A 50% total body surface area (TBSA) burn induced a significant increase of mortality in WT compared with Tnfrsf1a(-/-) mice. In contrast, P. aeruginosa injection with a 30% TBSA burn pretreatment enhanced IL-1ß expression, bacterial counts in lung and blood, pulmonary microvascular dysfunction, and mortality in Tnfrsf1a(-/-) mice compared with WT mice. Injection of the IL-1 receptor antagonist, Anakinra, reduced P. aeruginosa infection with burn pretreatment-induced blood bacterial counts, IL-1ß levels as well as permeability of lung, and mortality in Tnfrsf1a(-/-) mice. CONCLUSIONS: Our findings suggest that thermal injury induces lung NF-κB activation and neutrophil sequestration through TNFα signaling. However, blocking TNF-α enhances P. aeruginosa infection-induced lung damage in burn mice via induction of IL-1ß. Using an IL-1 receptor antagonist combined with the neutralization of TNF-α could be a useful strategy for decreasing P. aeruginosa infection-induced mortality in burn patients.

Histamine-induced changes in rat tracheal goblet cell mucin store and mucosal edema.

The pathology of chronic asthma in human and mouse is characterized by inflammation and remodeling of airway tissues. As a result of repeated inflammatory insults to the lower airways, smooth muscle thickening, mucin secretion and airway hyperreactivity may develop. In ovalbumin (OVA)-sensitized mice with repeated challenges with OVA to the lower airways, the trachea and bronchi are characterized by goblet cell hyperplasia and mucus hypersecretion from goblet cells. Previous study reports that intravenous (i.v.) application of a high dose of capsaicin releases tachykinin from capsaicin-sensitive nerves, producing acute plasma leakage and mucosal edema formation and causing depletion of mucin granules in goblet cells that results in a reduction in the number and size of Alcian blue (AB)-positive goblet cells in the rat trachea within a few minute after capsaicin application. Histamine is an important non-neural mediator of asthma from mast cells. The present study investigated whether i.v. application of a high dose of histamine (18 µmol/ml/kg) could result in these acute changes and the similar time-course changes in rat trachea. The tracheal whole mounts stained with chloroacetate esterase reagent and AB and tracheal methacrylate sections stained with AB and periodic acid-Schiff reagent were used for evaluation of histological and cellular changes. At 5 min after histamine application, mucosal leaky venules were numerous and subepithelial edema ratio (% of length of edema along the mucosal epithelial circumference of tracheal cross section) was found to be 48.2 ± 4.9, which was greater (P < 0.01) than saline-treated rats. But, the number of AB-positive goblet cells, 2,030 ± 170/mm(2) of mucosal surface epithelium, was similar to saline-treated group (P > 0.05). One day later, edema ratio remained large and the number of AB-positive goblet cells was 1,140 ± 150/mm(2) epithelium, reduced to half the number of the group at 5 min after histamine (P < 0.01). It is suggested that mucus hypersecretion occurred at this time point. At 3 or 5 days after histamine, edema ratio gradually decreased. The number of AB-positive goblet cells continued to remain small on day 3. On day 5 after histamine, the number of AB-positive goblet cells restored to the level of rat group at 5 min after histamine application. At 7 days after histamine, edema ratio returned to the level of saline-treated group. It is concluded that degranulation and thinning of tracheal goblet cells and mucus hypersecretion lagged behind histamine-induced acute plasma leakage and edema, and restoration of mucin store in goblet cells was associated with remission of mucosal edema.

Glial cell line-derived neurotrophic factor gene transfer exerts protective effect on axons in sciatic nerve following constriction-induced peripheral nerve injury.

Damage to peripheral nerves following trauma or neurodegenerative diseases often results in various sensory and motor abnormalities and chronic neuropathic pain. The loss of neurotrophic factor support has been proposed to contribute to the development of peripheral neuropathy. The main objective of this study was to investigate the protective effect of glial cell line-derived neurotrophic factor (GDNF) using peripheral gene delivery in a rat model of constriction-induced peripheral nerve injury. In this study, it was shown that mechanical and thermal hypersensitivity increased on the injured limb at day 7 after chronic constrictive injury (CCI) was induced. The neurological changes were correlated with the structural changes and loss of GDNF/Akt signaling, particularly in the distal stump of the injured sciatic nerve. Subsequently, recombinant adenovirus was employed to evaluate the potential of intramuscular GDNF gene delivery to alleviate the CCI-induced nerve degeneration ad neuropathic pain. After CCI for 3 days, intramuscular injection of adenovirus encoding GDNF (Ad-GDNF) restored the protein level and activity of GDNF/Akt signaling pathway in the sciatic nerve. This was associated with an improved myelination profile and behavioral outcomes in animals with CCI. In conclusion, the present study demonstrates the involvement of GDNF loss in the pathogenesis of CCI-induced neuropathic pain and the therapeutic potential of intramuscular GDNF gene delivery for the treatment of peripheral nerve degeneration.

Warm SPA-induced hyperthermia confers protection to rats against airway inflammation evoked by capsaicin and substance P.

Solus par aqua (SPA) is a traditional health care therapy. Warm SPA may enhance immunity and cellular defense to protect body against diseases. The present study investigated whether the warm SPA could confer protection to neurogenic inflammation in rats. The rats were immersed in water where the body core temperatures were maintained at hyperthermia (41.5 degrees C) or normothermia (37 degrees C) for a period of 15min. After SPA for 1 or 6 days, neurogenic inflammation was induced by intravenous injection of capsaicin (90microg/kg) or substance P (SP; 3microg/kg). The plasma leakage and arterial pressures in rats after neurogenic inflammation were monitored. The extent of capsaicin- or SP-induced plasma leakage and hypotension was significantly attenuated in rats on day 1 after SPA hyperthermia. However, such resistance to neurogenic inflammation was not found on day 6 after hyperthermia. Western blotting analysis showed that the expression of heat shock protein 72 (HSP 72) in the trachea on days 1 and 2 after hyperthermia was 9.61-fold and 6.66-fold, respectively, of that in normothermia. Afterwards, the hyperthermia-induced HSP 72 upregulation gradually declined in a time-dependent manner. Thus, SPA hyperthermia may protect rats against neurogenic inflammation through modulation of HSP expression.

Dimethylthiourea pretreatment inhibits endotoxin-induced compound exocytosis in goblet cells and plasma leakage of rat small intestine.

Intravenous application of a high dose of endotoxin, also called lipopoly-saccharide (LPS), results in endotoxemia in animals, that induces production of cytokines and free radicals, systemic inflammation and mucin discharge from mucous tissues. The present study was to investigate (1) whether LPS application increased goblet cell secretion by compound exocytotic activity in mucosal villi and crypts of rat small intestine, and (2) whether hydroxyl radicals were involved in LPS-induced compound exocytosis in goblet cells and plasma leakage. Scanning electron microscopy showed that the numbers of goblet cells undergoing compound exocytosis (cavitated goblet cells) per mm(2) of ileal villus epithelium in rats 5 and 30 min after LPS (15 mg kg(-1)) were 693 +/- 196 (N = 6) and 547 +/- 213 (N = 6), respectively, which were 5.1 and 8.4 times (P < 0.05) the number of saline control. The percentage of villus cavitated goblet cell numbers, in both duodenum and ileum 5 min after LPS and in the ileum 30 min after LPS, increased significantly (P < 0.05). Pretreatment with dimethylthiourea (DMTU), a hydroxyl radical scavenger, decreased the number of cavitated goblet cells to saline control (P > 0.05). Morphometric analysis showed that the percentage of crypt epithelial area in the duodenum and ileum occupied by goblet cell mucin stores in the duodenum and ileum 30 min after LPS were 3.8 +/- 0.2% (N = 6) and 6.9 +/- 0.5 (N = 6), respectively reducing to one half the amount of control (P < 0.01). When DMTU was given prior to LPS the crypt goblet cell mucin stores and the amount of plasma leakage returned to the level of control. It is concluded that hydroxyl radicals were involved in the LPS-induced increase in compound exocytotic activity of goblet cells and the increase in plasma leakage during acute phases of inflammatory response in rat small intestine.

Prophylactic proopiomelanocortin expression alleviates capsaicin-induced neurogenic inflammation in rat trachea.

Neurogenic inflammation frequently causes acute plasma leakage in airways and life-threatening pulmonary edema. However, limited strategies are available to alleviate neurogenic inflammation. Proopiomelanocortin (POMC) is the precursor of anti-inflammatory melanocortins, which have been proposed of therapeutic potential for various inflammatory diseases. The present study aimed to evaluate whether peripheral POMC expression ameliorated capsaicin-induced acute neurogenic inflammation in rat trachea. Prophylactic POMC expression was achieved by intravenous injection of adenovirus encoding POMC (Ad-POMC), which led to POMC expression in livers and elevated plasma adrenocorticotropin levels for approximately 60 days. After gene delivery for 7 days, neurogenic inflammation was induced in rats by capsaicin injection. The extent of capsaicin-evoked plasma leakage in trachea was alleviated in Ad-POMC-treated rats compared with animals of control groups (P < 0.01). Moreover, the number of endothelial gaps in tracheal venules was also significantly decreased in Ad-POMC-treated animals (P < 0.01). Prophylactic POMC expression, however, did not alter the basal substance P (SP) expression or the capsaicin-induced SP elevation in trachea and circulation. Instead, cell cultures studies revealed that POMC overexpression or application of POMC-derived melanocortins potently inhibited the SP-induced migration of endothelial cells (P < 0.01), thereby possibly contributing to the attenuation of endothelial gap formation and plasma leakage. The present study indicates that the anti-inflammatory POMC gene vector or melanocortins may constitute a therapeutic alternative for neurogenic inflammation.

Peripheral gene transfer of glial cell-derived neurotrophic factor ameliorates neuropathic deficits in diabetic rats.

Deprivation of neurotrophic factors contributes to the pathogenesis of diabetic neuropathy. However, the role of glial cell-derived neurotrophic factor (GDNF) in the pathogenesis of diabetic neuropathy remains unclear. The present study evaluated the pathogenic role of GDNF deficiency and the therapeutic potential of GDNF gene transfer for diabetic neuropathy. After injection of streptozotocin (STZ) for 2 weeks, diabetic rats displayed significant alteration in electrophysiological parameters, which was associated with structural changes and defective myelination in the sciatic nerves. The early diabetic neuropathy was accompanied by attenuation of the GDNF/GFRalpha1/Akt signaling cascade and depletion of sensory neuropeptides in the peripheral nerves. After detection of neuropathy, intramuscular GDNF gene transfer reversed the deficiency of GDNF/Akt signaling in the sciatic nerve and improved the neurological functions of diabetic rats. Moreover, GDNF gene delivery alleviated the axonal demyelination and restored the sensory neuropeptide levels in the sciatic nerve of diabetic rats. In summary, peripheral GDNF gene delivery ameliorates the diabetes-induced downregulation of the GDNF signaling complex in the peripheral nervous system and holds promises for treatment of diabetic neuropathy.

Inhibitory effect of dimethylthiourea on rat urinary bladder inflammation produced by 6-hydroxydopamine application.

The present study was to investigate 6-hydroxydopamine (6-OHDA)-induced inflammatory response and underlying mechanisms in the urinary bladder in anesthetized male rats of Long-Evans strain. The magnitude of inflammation was evaluated by morphometric analysis of the relative number of leaky blood vessels expressed by the area density of India ink-labeled blood vessels in whole mount specimens. Light and scanning electron microscopies were employed to study the changes in histologic structure and endothelial ultrastructure of bladder wall. Local injection of 6-OHDA to lumen of urinary bladder induced a dose-dependent increase in plasma leakage. Following application of vehicle, 5 mg/kg 6-OHDA, and 10 mg/kg 6-OHDA, area densities of India ink-labeled leaky vessels were 5.65+/-3.72% (N=6), 22.63+/-5.12% (N=6), and 35.02+/-11.25% (N=6), respectively. Inflammatory response was completely abolished by pretreatment alone with dimethylthiourea (DMTU), a hydroxyl radical scavenger, and was also attenuated by pretreatment with L-732,138, a NK1 receptor antagonist. 6-OHDA caused edema formation and venular endothelial gap formation in bladder tissue. It is concluded that 6-OHDA induced inflammation in the rat urinary bladder, the response of which was dose-dependently increased and free radicals and tachykinins were involved in the inflammatory process.

Heat preconditioning ameliorates hepatocyte viability after cold preservation and rewarming, and modulates its immunoactivity.

BACKGROUND: Heat preconditioning significantly preserved liver graft function after cold preservation in animal experimental model. The elevation of heat shock protein 70 (HSP70) was claimed to play a critical role in protecting grafts against cold preservation-induced hepatocyte apoptosis. However, little is known about whether HSP70 also plays an immunomodulatory role in cold preserved cells. This study aimed at investigating the relationship between HSP70 protein and the immunoactivity in response to lipopolysaccharide (LPS) stimulation. METHODS AND RESULTS: A normal rat hepatocyte cell line was preserved with University of Wisconsin (UW) solution, Ringer's lactate solution (RL), and phosphate-buffered saline (PBS) at 4 degrees C. No significant morphological alteration was noted in UW-preserved cells after 24 h through phase-contrast microscopic observation and fluorescent viability stain. Western blotting showed a two-fold increase in the ratio of HSP70/Bax proteins in cells after 24 h of UW preservation. Heat preconditioning significantly enhanced the recovery of lactate dehydrogenase (LDH) activity in both RL- and UW-preserved cells that were stored for a period of 12 h or less. Moreover, heat preconditioning promoted HSP70 and NF-kappaB p50 nuclear translocation and suppressed the LPS-induced nuclear p50 accumulation in cells before UW preservation. Immunofluorescent stain revealed that the LPS-induced p50 protein redistribution to nuclear membrane might contribute to NF-kappaB activation, while heat preconditioning and UW cold preservation completely abrogated the p50 intranuclear redistribution. Thus NF-kappaB p50 might be responsible for the endotoxin tolerance induction. CONCLUSIONS: These findings strongly suggest that heat preconditioning not only preserves hepatocyte viability after cold preservation and rewarming, but also ameliorates its immunoactivity.

Scanning electron microscopic study of the nasolabial cyst: its clinical and embryological implications.

OBJECTIVE: Nasolabial cyst is an uncommon midfacial cyst. It is considered to be a developmental anomaly arising from the rest of nasal respiratory epithelium. Although the cyst is a well-recognized entity, there remains some confusion of its origin, cell types, and ultrastructures. Based on the routine light microscopic study, some authors reported the epithelial cells of the inner lining of the nasolabial cyst were ciliated; some others reported they were nonciliated. To clarify this, a scanning electron microscopic study is needed. STUDY DESIGN: This was a prospective clinical series. METHODS: A transnasal marsupialization method was used to treat 10 patients with nasolabial cyst. With patients under local anesthesia, the roof of the cyst wall and a disk of nearby nasal mucosa were excised together with a sickle knife and scissors. Surgical specimens were dissected and processed for scanning electron microscopy and histochemistry. Patients were followed up for 8 to 65 months. RESULTS: Marsupialization of cysts was successfully performed on all patients. Electron microscopically, the inner surface of the nasolabial cysts in all the cases was lined with nonciliated columnar epithelium consisting chiefly of goblet cells and basal cells. It is suggested that goblet cells contributed to clear, thin, and yellow mucus present in the cyst lumen. Instead of cilia, these epithelial cell surfaces were equipped with numerous short, globular, or irregular microvilli. Apical cytoplasm of adjacent cells did not tightly adhere to each other. Instead, microsulci of 1 to 3 microm in width formed between cells. Cytoplasmic processes from the lateral border spanned the microsulcus and contacted with those from neighboring cells. CONCLUSION: The novel study has proved that the lining epithelium on the inner surface of the nasolabial cyst is columnar epithelium that chiefly consisted of two types of cells: goblet cells and basal cells. Not present were ciliated cells that were essential in the other portion of the respiratory tract. Numerous microvilli, instead of cilia, covered the inner lining of the nasolabial cyst, probably as a result of lacking the stimulation of air in ventilation as that on the other portion of the respiratory tract. The cilia of the epithelium were ill developed.

Histological, ultrastructural, and in situ hybridization study on enlarged cells in grouper Epinephelus hybrids infected by grouper iridovirus in Taiwan (TGIV).

Grouper iridovirus in Taiwan (TGIV) infection in the Epinephelus hybrid is a major problem in the grouper industry. ATPase gene sequences indicate that this virus is closely related to cell hypertrophy iridoviruses. Histologically, the appearance of basophilic or eosinophilic enlarged cells in internal organs is the most characteristic feature of this disease. These cells are acid-phosphatase positive and are able to phagocytose injected carbon particles. In our study, TGIV infection inhibited normal phagocytic ability in these cells in vivo after 4 d post-infection (p.i.) but not before 2 d p.i. Their staining properties and phagocytic ability suggested a monocyte origin of enlarged cells, which appeared in high numbers in the trunk kidney, head kidney, spleen and gill. After infection, the enlarged cells first appeared in the spleen, with an abundance peak at 64 h p.i. (Peak 1); at 120 h p.i., a second peak (Peak 2) occurred in the spleen, head kidney, trunk kidney and gill. Lower numbers of enlarged cells were observed in the liver, muscle, heart, eye, intestine, but no enlarged cells were found in the brain. A TGIV-specific DNA probe labeled most of the basophilic but not eosinophilic enlarged cells. Nuclei of infected cells were labeled during an early stage of the infection; at later stages, both nuclei and cytoplasms were labeled. Ultrastructurally, heterochromatins of the infected cells were marginated or aggregated to one side of the nuclei during the early stages of infection. Damage and rupture of the nuclear membrane started before formation of the viromatrix. Capsids were assembled in ring-shaped or disc-shaped structures. Bullet-shaped electron-dense material was present near the incomplete virus particles, and is speculated to be inserted into the capsids later.

Spontaneous remission of edema and regranulation of goblet cells in rat tracheae after capsaicin-induced acute inflammation.

Previous studies have investigated the short-term effect of capsaicin on edema formation and goblet-cell secretion in the trachea. The present study sought to investigate the long-term effect of a high dose of capsaicin (90 micro g/ml/kg), administered intravenously, on changes in the formation of endothelial gaps among venular endothelial cells, mucosal tissue edema and the secretory activity of goblet cells, including the number and size of goblet cells, and the mucus score and secretory ratio of goblet-cell mucus secretion in the trachea of rats. The tracheal whole mounts with silver staining, those stained with chloroacetate esterase reagent and Alcian blue and tracheal tissue sections stained with Alcian blue and periodic acid-Schiff reagent were used for evaluation. Formation of endothelial gaps occurred a few min after administration of capsaicin, and gaps almost closed within 12 min after capsaicin injection. Five min after capsaicin, the leaky blood vessels were numerous and the subepithelial edema ratio (% of length of edema along the inner circumference of tracheal cross section) was found to be 57.8+/-3.0% ( n=6). The number of Alcian blue-positive goblet cells (1,090+/-220 per mm(2) of mucosal surface) was reduced to half the number of goblet cells in the vehicle-treated rats (2,200+/-230). The mucus score of goblet cell secretion was not changed. The secretory ratio was greatly increased. One day after capsaicin, the edema ratio remained large and the number of Alcian blue-positive goblet cells was also small. The mucus score was also not changed. The secretory ratio was still large. On day 3, the edema ratio remained large, but the number of Alcian blue-positive goblet cells was increased to the level of the controls. The mucus score and secretory ratio returned to the control level. On day 5, the edema ratio was greatly decreased, but it was still significantly larger than that of the controls. The mucus score and secretory ratio remained at the baseline level. Seven days after capsaicin, the edema ratio was similar to the controls. The number of goblet cells was even larger than controls. It is concluded that capsaicin-induced acute inflammation in the rat trachea involves formation of endothelial gaps, extensive plasma extravasation and edema formation, and depletion of goblet-cell secretory granules. Spontaneous gradual remission of edema was accompanied by regranulation of goblet cells with gradual mucogenesis for several days.