CD10 as a novel marker of therapeutic resistance and cancer stem cells in head and neck squamous cell carcinoma.

BACKGROUND: Cancer stem cells (CSCs) are responsible for treatment failure. However, their identification and roles in resistance are not well established in head and neck squamous cell carcinoma (HNSCC). METHODS: Three HNSCC cell lines (FaDu, Detroit562 and BICR6) were treated with cisplatin or radiation. Cell surface antigens were analysed by LyoPlate, a novel cell surface antigen array. The expression levels of antigens highly expressed after treatments were further compared between cisplatin-resistant Detroit562 cells and its parental line. Association of the candidate antigen with CSCs properties, namely sphere formation and in vivo tumourigenicity, was also examined. RESULTS: CD10, CD15s, CD146 and CD282 were upregulated across the treated cell lines, while the increased expression of CD10 was prominent in the cisplatin-resistant cell line. Isolation mediated by FACS revealed that the CD10-positive subpopulation was more refractory to cisplatin, fluorouracil and radiation than the CD10-negative subpopulation. It also showed an increased ability to form spheres in vitro and tumours in vivo. Moreover, the CD10-positive subpopulation expressed the CSC marker OCT3/4 at a higher level than that in the CD10-negative subpopulation. CONCLUSIONS: CD10 is associated with therapeutic resistance and CSC-like properties of HNSCC. CD10 may serve as a target molecule in the treatment of refractory HNSCC.

Blockade of cysteinyl leukotriene-1 receptors suppresses airway remodelling in mice overexpressing GATA-3.

BACKGROUND: We demonstrated previously that GATA-3 overexpression markedly enhanced allergen-induced airway inflammation and airway remodelling, including subepithelial fibrosis, and smooth muscle cell hyperplasia, in transgenic mice. OBJECTIVE: Because cysteinyl leukotrienes (cysLTs) have been shown to be involved in such structural changes, the effects of a specific cysLT1 receptor antagonist, montelukast, were evaluated in a mouse model of chronic asthma. METHODS: GATA-3-overexpressing mice and wild-type Balb/c mice were sensitized and repeatedly challenged by ovalbumin (OVA) or saline. The effects of montelukast on the development of airway remodelling were compared between the two mouse genotypes. RESULTS: CysLTs in the lung were increased after repeated allergen challenges, and significantly enhanced in GATA-3-overexpressing mice. The enhanced cysLT levels were accompanied by the development of eosinophilia, smooth muscle cell hyperplasia, and increased stromal cell-derived factor-1 gene expression with a small increase in pro-collagen gene expression in OVA-challenged GATA-3-overexpressing mice, but not in wild-type mice. Montelukast significantly decreased lung cysLT levels and inhibited the GATA-3-overexpression-related airway remodelling, potently preventing smooth muscle cell hyperplasia, but partially suppressed the increased pro-collagen gene expression and eosinophilic inflammation. Increases in the levels of IL-4, IL-5, IL-13, and eotaxin in bronchial lavage and TGF-ß gene expression in the lungs were induced by OVA in both mouse genotypes. Montelukast treatment also significantly reduced these levels to the levels seen after saline challenges in GATA-3-overexpressing mice. CONCLUSION: Montelukast efficaciously prevented airway inflammation and remodelling in a GATA-3-overexpression antigen challenge mouse model by decreasing the cysLT-driven Th2 cytokine cycle of amplification of airway pathologies.

Clinical significance of LAMB3 and COL7A1 mRNA in esophageal squamous cell carcinoma.

AIMS: LAMB3 and COL7A1 genes code for the laminin-5beta3 chain and type VII collagen, respectively. They constitute the major components of the basement membrane zone. The aim of the current study was to clarify the clinical significance of LAMB3 and COL7A1 mRNA expression in esophageal squamous cell carcinoma (ESC). METHODS: We quantitated the expression of LAMB3 mRNA and COL7A1 mRNA in malignant esophageal tissues (T) and corresponding normal tissues (N) by real-time quantitative reverse transcription-polymerase chain reaction assays. The clinicopathologic significance of LAMB3 and COL7A1 expression was also determined. Paired T and N tissues were obtained from 66 patients who underwent curative esophagectomy. RESULTS: The expression levels of LAMB3 and COL7A1 mRNAs were higher in malignant tissues than in the corresponding normal tissues. The level of LAMB3 expression was significantly correlated with the depth of invasion and venous invasion (p=0.007 and 0.001, respectively). COL7A1 expression was significantly correlated with depth of tumor invasion and lymphatic invasion (p=0.046, 0.013, respectively). The five-year survival rate was better in the 22 patients with relatively low expression of both LAMB3 and COL7A1 in comparison with the other 44 cases (p<0.05). CONCLUSION: The evaluation of LAMB3 and COL7A1 mRNA expression is useful for predicting the malignant properties of ESC and may prove valuable in predicting the future course of the disease.

Mitotic centromere-associated kinesin is a novel marker for prognosis and lymph node metastasis in colorectal cancer.

Mitotic centromere-associated kinesin (MCAK) is a microtubule depolymerase that is essential for proper kinetochore-microtubule attachment during spindle formation. Overexpression of MCAK has been correlated with aggressive forms of carcinoma, resulting in poor prognosis of colorectal cancer. The purpose of this study was to quantify MCAK expression in malignant and benign colorectal tissues and to determine if MCAK expression levels correlate with clinicopathologic factors and prognosis in colorectal cancer patients. Paired colorectal tissue samples from tumours and the corresponding normal tissues were obtained from 120 patients with colorectal cancer who underwent surgical resection. The real-time reverse transcriptase-PCR and immunohistochemistry were used to analyse mRNA and protein expression status with respect to various clinicopathological factors. MCAK expression was higher in colorectal cancer tissue (P<0.01) than in corresponding normal tissue, and this elevated expression level was markedly associated with factors such as lymph node metastasis (P=0.0023), venous invasion (P=0.019), peritoneal dissemination (P=0.021) and Dukes' classification (P=0.0023). Patients with high MCAK mRNA expression also showed a far poorer survival rate than those with low MCAK mRNA expression (P<0.01). Elevated MCAK expression was an independent predictor of overall survival and lymph node metastasis. These data suggest that MCAK expression may serve as a good marker of prognosis and lymph node metastasis in colorectal cancer.

Clinicopathological and biological significance of mitotic centromere-associated kinesin overexpression in human gastric cancer.

Mitotic centromere-associated kinesin (MCAK) is a microtubule (MT) depolymerase necessary for ensuring proper kinetochore MT attachment during spindle formation. To determine MCAK expression status and its clinicopathological significance, real-time reverse transcriptase-polymerase chain reaction was used in 65 cases of gastric cancer. MCAK gene expression in cancer tissue was significantly higher than expression in non-malignant tissue (P<0.05). Elevated MCAK expression was significantly associated with lymphatic invasion (P=0.01) and lymph node metastasis (P=0.04). Furthermore, patients with high MCAK expression had a significantly poorer survival rate than those with low MCAK expression (P=0.008). Immunohistochemical study revealed that expression of MCAK was primarily observed in cancer cells. Additionally, a gastric cancer cell line (AZ521) that stably expressed MCAK was established and used to investigate the biological effects of the MCAK gene. In vitro results showed that cells transfected with MCAK had a high rate of proliferation (P<0.001) and increased migratory ability (P<0.001) compared to mock-transfected cells. This study demonstrated that elevated expression of MCAK may be associated with lymphatic invasion, lymph node metastasis, and poor prognosis. These characteristics may be due in part to the increased proliferative and migratory ability of cells expressing MCAK.

Renal clearance of glycolaldehyde- and methylglyoxal-modified proteins in mice is mediated by mesangial cells through a class A scavenger receptor (SR-A).

AIMS/HYPOTHESIS: Glomerular mesangial expansion is a characteristic feature of diabetic nephropathy, and the accumulation of AGE in the mesangial lesion has been implicated as one of its potential causes. However, the route for the AGE accumulation in mesangial lesions in diabetic patients is poorly established. METHODS: Glycolaldehyde-modified BSA (GA-BSA) and methylglyoxal-modified BSA (MG-BSA) were prepared as model AGE proteins, and their in vivo plasma clearance was examined in mice, and renal uptake by in vitro studies with isolated renal mesangial cells. RESULTS: Both (111)In-GA-BSA and (111)In-MG-BSA were rapidly cleared from the circulation mainly by both the liver and kidney. Immunohistochemical studies with an anti-GA-BSA antibody demonstrated that intravenously injected GA-BSA accumulated in mesangial cells, suggesting that such cells play an important role in the renal clearance of circulating AGE proteins. Binding experiments at 4 degrees C using mesangial cells isolated from mice showed that (125)I-GA-BSA and (125)I-MG-BSA exhibited specific and saturable binding. Upon incubation at 37 degrees C, (125)I-GA-BSA and (125)I-MG-BSA underwent endocytic degradation by these cells. The binding of the ligands to these cells was inhibited by several ligands for scavenger receptors. The endocytic degradation of GA-BSA by mesangial cells from class A scavenger receptor (SR-A) knock-out mice was reduced by 80% when compared with that of wild-type cells. The glomerular accumulation of GA-BSA after its intravenous administration was attenuated in SR-A knock-out mice, as evidenced by immunohistochemical observations. CONCLUSIONS/INTERPRETATION: These results raise the possibility that circulating AGE-modified proteins are subjected to renal clearance by mesangial cells, mainly via SR-A. This pathway may contribute to the pathogenesis of AGE-induced diabetic nephropathy.

Nitroglycerin as a nitric oxide donor accelerates lipid peroxidation but preserves ventricular function in a canine model of orthotopic heart transplantation.

BACKGROUND: Nitric oxide has cardioprotective effects through several mechanisms. However, nitric oxide may have deleterious effects in the presence of superoxide because it is converted to peroxynitrite, which then initiates lipid peroxidation. Using a canine model of orthotopic heart transplantation, we examined whether adding an organic nitric oxide donor, nitroglycerin, to preservation solution elicits lipid peroxidation after reperfusion and causes deleterious effects on coronary endothelial function and left ventricular function. METHODS AND RESULTS: The donor heart was preserved for 24 hours in cold University of Wisconsin solution with nitroglycerin (0.1 mg/mL) supplementation (group NTG, n = 8) or in standard University of Wisconsin solution (group C, n = 8). After reperfusion, changes of coronary resistance were measured during the infusion of acetylcholine (0.1 mg/min) and of sodium nitroprusside (1 mg/min), and percent coronary relaxation was calculated. Left ventricular function was evaluated by pressure-volume relations with the use of a conductance catheter, thereby deriving the slopes of end-systolic pressure-volume relation, stroke work-end-diastolic volume relation, and maximum rate of change of left ventricular pressure-end-diastolic volume relation. Serum lipid peroxide level was measured. Percent coronary relaxation was similar for the 2 groups. The slopes of end-systolic pressure-volume relation, stroke work-end-diastolic volume relation, and maximum rate of change of left ventricular pressure-end-diastolic volume relation in group NTG were significantly higher than those in group C. On the other side, serum lipid peroxide level in group NTG was significantly higher than that in group C. CONCLUSIONS: Nitroglycerin may have detrimental effects evidenced by the increase in lipid peroxidation, which implied peroxynitrite formation. However, the overall effect of nitroglycerin was cardioprotective. Although the exact mechanism is yet to be clarified, the superb cardioprotective effect of nitroglycerin overwhelms the exaggeration of lipid peroxidation.

Antifibrinolytic therapy with tranexamic acid in cardiac operations.

To demonstrate its antifibrinolytic effects and establish an effective regimen of tranexamic acid for hemostasis, the authors measured alpha2-plasmin inhibitor-plasmin complexes, thrombin-antithrombin III complexes and postoperative blood loss in three groups undergoing different regimens during cardiac operations. Forty-six patients undergoing coronary artery bypass grafting or valve replacement were enrolled in this study. They were divided into three groups of drug administration. A bolus infusion of 50 mg/kg tranexamic acid was given to 17 patients at the end of cardiopulmonary bypass (control group) and to 14 patients at the beginning of cardiopulmonary bypass (group A). In addition to the same bolus infusion at the beginning of cardiopulmonary bypass as group A, a continuous infusion of 10 mg/kg per h, starting at the time of skin incision and maintained for 6 h after cardiopulmonary bypass was given to 15 patients (group B). The marked increase in alpha2-plasmin inhibitor-plasmin complexes at the end of cardiopulmonary bypass in the control group was significantly reduced in group A (P < 0.01) and a further reduction was observed in group B (P < 0.001). The difference in postoperative blood loss only reached significant levels between the control group and group B (P < 0.05). Although a significant increase in thrombin-antithrombin III complexes during cardiopulmonary bypass was similarly observed in all groups, no thromboembolic events occurred in any group, nor was any difference seen in graft patency. From the tranexamic acid therapy regimens tested in this study, a continuous infusion of 10 mg/kg per h starting at the time of skin incision to 6 h after cardiopulmonary bypass, with a bolus infusion of 50 mg/kg at the beginning of cardiopulmonary bypass, proved to be the most effective.

Ischemic heart attacks following cessation of aspirin before coronary artery bypass surgery: A report of two cases.

We present herein two patients who suffered life-threatening ischemic heart attacks following the preoperative discontinuation of aspirin. Since their cardiac conditions were stable in the long-term while under medication, the cessation of aspirin was highly suspected to have caused or at least to have contributed to their ischemic events. We thus discuss the appropriate timing for discontinuation of aspirin before surgery in patients with ischemic heart disease.

New radiographic projections for avulsion fractures of the lateral malleolus.

We report two new radiographic projections for evaluating avulsion fractures at the lateral malleolus. We used seven freshly amputated legs with simulated avulsion fractures and radiopaque markers to assess their value. The projections allow accurate assessment of the displacement of fragments without superimposition, and also show whether they affect the anterior talofibular or the calcaneofibular ligament or both.

[A prospective study on the timing of discontinuation of aspirin before coronary artery bypass grafting].

The effects of the timing of discontinuation of aspirin before coronary artery bypass grafting (CABG) on postoperative blood loss and blood requirements were examined in 22 patients undergoing elective CABG, who were randomly assigned into two groups. In Group I (11 patients), aspirin was discontinued two days before the operation and in Group II (11 patients), aspirin was continued up to the operation. The other 40 patients, who did not take aspirin for at least seven days before the operation, served as a control Group. There were no differences in preoperative data including the platelet count and the hemoglobin concentration, nor in operative variables such as operation time, cardiopulmonary bypass duration and aortic crossclamp time among the groups. Although postoperative blood loss (six hours' loss; Group I 218 ml, Group II 183 ml and control Group 172 ml) and red blood cells transfusion requirements were not different among the groups, platelet concentrates transfusion was more frequently required in Group II (54.5%) as compared with control Group (7.5%) and Group I (9.1%). The difference between Group II and the control Group reached statistical significance (p < 0.01), but there was no significant difference between Group I and control Group. This fact suggests that preoperative two days' discontinuation of aspirin works as effectively as seven days' discontinuation.

Cloning and expression of the cDNA encoding prolyl oligopeptidase (prolyl endopeptidase) from bovine brain.

Prolyl oligopeptidase (EC 3.4.21.26, prolyl endopeptidase) cDNA from bovine brain was cloned by PCR, and the amplified fragment was used as a probe to screen the cDNA library from bovine brain. The obtained clone contained a 2.7 kb DNA fragment with an open reading frame of 2130 nucleotides, and encoded a protein of 710 amino acids with a deduced molecular weight of 80640 Da. The deduced amino acid sequence is 95, 94, 51 and 48% homologous to those of human T-cell, porcine brain, Aeromonas hydrophila, and Flavobacterium meningosepticum prolyl endopeptidases, respectively. The bovine brain prolyl endopeptidase-encoding cDNA was expressed using an expression vector bearing a tac promoter, with an approximate yield of 20 micrograms/ml of cell culture.

Active hypothalamic-pituitary-gonadal axis in an infant with X-linked adrenal hypoplasia congenita.

To evaluate the hypothalamic-pituitary-gonadal axis in an infant with adrenal hypoplasia congenita, we measured the serum levels of testosterone and performed a luteinizing hormone-releasing hormone stimulation test. The diagnosis was made because of the presence of a mutation, A300V, in the DAX-1 gene. The results demonstrated an active hypothalamic-pituitary-gonadal axis, with adult-level testosterone of 266 ng/dl on day 0, and maintenance of testosterone concentration in the 100 to 250 ng/dl range for 140 days as expected. The luteinizing hormone-releasing hormone lest was compatible with an active pituitary gland with a luteinizing hormone peak of 13.1 IU/L and a follicle-stimulating hormone of 5.0 IU/L We conclude that the DAX-1 mutation does allow a normal reproductive axis at birth. We speculate that sometime between infancy and puberty this mutation in the DAX-1 gene leads to an inability to activate the reproductive axis from its childhood suppression; thus puberty will not develop in this infant.

Inhalation sedation with sevoflurane: a comparative study with nitrous oxide.

PURPOSE: To evaluate the possibility of using sevoflurane for inhalation sedation. PATIENTS AND METHODS: Thirty-five volunteers were divided randomly into two groups: sevoflurane group (n = 20) and nitrous oxide (N2O) group (n = 15). At the beginning of the sedation they all inhaled 100% O2, then a 0.1 minimum alveolar concentration (MAC) of sevoflurane or 10% N2O with oxygen, a 0.2 MAC of sevoflurane or 20% N2O with oxygen, and 0.3 MAC of sevoflurane or 30% N2O with oxygen for 10 minutes each. RESULTS: The respiratory and cardiovascular functions were stable during inhalation of the gas mixtures. There were five negative comments about breathing N2O, but none about breathing sevoflurane. CONCLUSION: All subjects in the sevoflurane group stated they would be willing to submit to the same inhalation procedure again.

Reflex changes in the masticatory muscles with load perturbations during chewing hard and soft food.

Reflex changes in masticatory muscles were investigated in naturally behaving rabbits while chewing soft food (bread) and hard food (raw rice). To study peripheral control mechanisms of mastication, reflex changes in masticatory muscles were correlated with the jaw movement trajectories. When the hard food was tested, the closing muscle was activated isometrically and the antagonist activity was evident during closure. Under such conditions, the so-called masseteric inhibitory periods (MIPs) and digastric short bursts (DSBs) were found in the closing phase, which was not the case with soft food. The reflex changes in the masticatory muscles were similar to those in an unloading reflex or in a reflex after tooth tap. Precise comparison of the EMG and the movement orbit showed that DSB with MIP was preceded by a trough in the closing velocity which bottomed at 8 +/- 1 ms (mean +/- S.D.: n = 9) before the DSB onset. These results suggest that the DSB with MIP may be a reflex change generated by periodontal mechanoreceptor stimulation after the upper and lower teeth come together with hard food, which have a role in regulatory mechanisms of mastication when the teeth are suddenly loaded by hard food.

Variation of the jaw-opening reflex during spontaneous mastication in rabbits.

To understand the modulation of periodontal mechanoreceptor activity on mastication, the jaw-opening reflex (JOR) evoked by electrical stimulation of the inferior alveolar nerve (IAN) was studied during chewing in freely behaving rabbits. Spontaneous chewing movements were intact from the IAN stimulation below two times the threshold (T) which was measured at rest. As the stimulus intensity was increased to more than 2.5 T, the amplitude of the masseter activity decreased or it was abolished; however, the chewing rhythm was still maintained up to 3.0 T. When the low-threshold primary afferents were tested while the rabbit chewed pellets rhythmically, the amplitude in the JOR was inhibited below the prior control level. The inhibitory effect on the JOR was larger in the opening phase than that in the closing phase. In conclusion, the JOR was tonically depressed during the masticatory cycle and the inhibition of the JOR was rhythmically modulated in a phase-linked manner that was larger in the opening phase than in the closing phase.

Jaw movements and EMG activities of limb-licking behavior during grooming in rabbits.

Jaw movements and masticatory muscle activity were recorded in freely moving rabbits during limb-licking, chewing, and drinking. The limb-licking movement was characterized by rhythmical jaw movement and digastric burst and relatively small masseter activity. The mean cycle time was 292 ms +/- 11.0 (mean +/- SE: n = 7) and was longer than that in chewing (267 ms +/- 20.8, p < 0.01) or drinking (208 ms +/- 15.2, p < 0.01). Jaw movements in the frontal plane of the ingestive behaviors coincided with those reported in previous studies; however, that of the licking movements was featured here. The basic cycle included; a) an opening stroke in which the mandible moved downward and laterally, b) a lateral excursion stroke in which the mandible moved medially and either upward or downward, and c) a closing stroke in which the mandible moved upward to the closing position. During opening, the digastric activity on the working side was determined to be larger than that on the other side (p < 0.001).