Novel and rapid enumeration method of peripheral blood stem cells using automated hematology analyzer.

INTRODUCTION: The number of infused CD34(+) cells is crucial to the success of peripheral blood stem cell transplantation (PBSCT). Here, we present, for the first time, a new method of enumerating hematopoietic progenitor cells (HPCs) for PBSCT. METHOD: This novel method is based on hemolysis and chemical staining, followed by flow cytometry-based optical detection, conducted using an automated hematology analyzer (XN series, Sysmex). CD34(+) cells and HPCs were compared in 76 granulocyte colony-stimulating factor (G-CSF)-mobilized blood or apheresis samples taken from healthy donors (n = 18) or patients undergoing autologous PBSCT (n = 6). RESULTS: There was a strong correlation between the numbers of HPCs and CD34(+) cells (R(2)  = 0.958). The expected total number of HPCs in the final products, which was estimated from HPCs in pre-apheresis PB or mid-apheresis products, also correlated well with the total number of CD34(+) cells in the final products. The change in HPCs in PB closely resembled that of CD34(+) cells during mobilization. Experiments using immunomagnetic beads suggested that the majority of CD34(+) cells existed in HPCs, and vice versa. CONCLUSION: Hematopoietic progenitor cells may serve as surrogates for CD34(+) cells in PBSCT. However, further investigations are required to verify this.

Non-genomic inhibitory effect of glucocorticoids on activated peripheral blood basophils through suppression of lipid raft formation.

We investigated the non-genomic effects of glucocorticoids (GCs) on inhibition of plasma membrane lipid raft formation in activated human basophils. Human basophils obtained from house dust mite (HDM)-sensitive volunteers were pretreated with hydrocortisone (CORT) or dexamethasone (Dex) for 30 min and then primed with phorbol 12-myristate 13-acetate (PMA, 10 ng/ml) or HDM (10 µg/ml). The expression of CD63, a basophil activation marker, was assessed by flow cytometry. Membrane-bound GC receptors (mGCRs) were analysed by flow cytometry and confocal laser microscopy. Lipid rafts were assessed using a GM1 ganglioside probe and visualization by confocal laser microscopy. Pretreatment of basophils with CORT (10(-4) M and 10(-5) M) and Dex (10(-7) M) significantly inhibited CD63 expression 20 min after addition of PMA or HDM. The inhibitory effects of GCs were not altered by the nuclear GC receptor (GCR) antagonist RU486 (10(-5) M) or the protein synthesis inhibitor cycloheximide (10(-4) M) (P < 0·05). CORT coupled to bovine serum albumin (BSA-CORT) mimicked the rapid inhibitory effects of CORT, suggesting the involvement of mGCRs. mGCRs were detectable on the plasma membrane of resting basophils and formed nanoclusters following treatment with PMA or HDM. Pretreatment of cells with BSA-CORT inhibited the expression of mGCRs and nanoclustering of ganglioside GM1 in lipid rafts. The study provides evidence that non-genomic mechanisms are involved in the rapid inhibitory effect of GCs on the formation of lipid raft nanoclusters, through binding to mGCRs on the plasma membrane of activated basophils.

Unexpected serum level of vancomycin after oral administration in a patient with severe colitis and renal insufficiency.

OBJECTIVE: To report a case in which the serum concentration of vancomycin (VCM) reached the supratherapeutic range following oral administration in a patient with severe pseudomembranous colitis and renal insufficiency. CASE SUMMARY: A 65-year-old, 70 kg weighing man with severe acute pancreatitis and acute renal failure was subjected to continuous hemodiafiltration (CHDF). CHDF could only be performed intermittently because of the unstable circulation dynamic of this patient. After admission, intravenous VCM therapy was initiated. Thereafter, oral VCM administration was begun (0.5 g every 6 h). Despite the discontinuation of intravenous VCM after the first 2 days of oral VCM, the serum VCM concentration increased gradually to 49.8 mg/l over a period of 2 weeks from the initiation of oral administration (34.4 mg/l). Based on pharmacokinetic analysis, the bioavailability of VCM was estimated to over 33%. Autopsy findings indicated broadly distributed necrosis on the lamina propria of the mucosa throughout all parts of the intestine below the duodenum. DISCUSSION: This case indicates necessity of the careful monitoring after oral high-dose VCM administration in a patient with a broadly distributed necrosis and renal insufficiency. CONCLUSIONS: TDM should be considered according to renal function, the severity of enteritis and the total dosage of oral VCM administration.

Interleukin-18-deficient mice exhibit diminished chronic inflammation and airway remodelling in ovalbumin-induced asthma model.

Interleukin (IL)-18, which is produced by activated monocytes/macrophages and airway epithelial cells, is suggested to contribute to the pathophysiology of asthma by modulating airway inflammation. However, the involvement of IL-18 on modulating chronic airway inflammation and airway remodelling, which are characterized in a refractory asthma model exposed to long-term antigen, has not been investigated sufficiently. We examined the role of IL-18 in chronic airway inflammation and airway remodelling by long-term antigen exposure. IL-18-deficient and C57BL/6-wild-type mice were sensitized by ovalbumin (OVA) and were then exposed to aerosolized OVA twice a week for 12 weeks. We assessed airway inflammation by assessing the infiltration of cells into the airspace and lung tissues, and airway remodelling by airway mucus expression, peribronchial fibrosis and smooth muscle thickness. In IL-18-deficient mice, when exposed to OVA, the total cells and neutrophils of the bronchoalveolar lavage fluid (BALF) were diminished, as were the number of infiltrated cells in the lung tissues. IL-18-deficient mice exposed to OVA after 12 weeks showed significantly decreased levels of interferon (IFN)-gamma, IL-13 and transforming growth factor (TGF)-beta1 in the BALF. The airway hyperresponsiveness to acetyl-beta-methacholine chloride was inhibited in IL-18-deficient mice in comparison with wild-type mice. In addition, IL-18-deficient mice exposed to OVA had fewer significant features of airway remodelling. These findings suggest that IL-18 may enhance chronic airway inflammation and airway remodelling through the production of IFN-gamma, IL-13 and TGF-beta1 in the OVA-induced asthma mouse model.

Plical resection in pre-temporal approach for basilar bifurcation aneurysms: preliminary surgical experience and cadaveric study.

OBJECT: Although a pre-temporal approach (PA) can provide a wide space for preservation of thalamoperforating atrteries in direct surgery for basilar bifurcation aneurysms (BBAs), it cannot always secure adequate proximal control. The authors described the advantages of plical resection added to PA for BBAs. METHODS: Between October 1998 and April 2000, eight consecutive patients with BBAs were treated in the neurosurgical department of Kurashiki Central Hospital. Among them, five patients received direct clipping using this method. There were four females and one male, ages ranging from 61 to 77 (mean 70.8 years). Mean aneurysmal size and distance between the in"terclinoidal line and the aneurysmal neck was 4.5 and 9.5 mm, respectively. The operative procedures consisted of the following components; 1) fronto-temporal craniotomy with translocation of orbito-zygomatico-malar bone for PA, 2) preservation of lateral branches of the superficial sylvian veins, 3) resection of plica dural folds to increase the operative field up to the oculomotor nerve (OMN). RESULTS: Complete clipping was achieved without thalamic infarction or temporal contusion in all patients. Three of the five patients suffered from transient right OMN palsy which recovered within two months after surgery. CONCLUSION: Plical resection in the pre-temporal approach might be beneficial in the surgical treatment of BBAs when proximal control seems difficult.

Characterization of carotid atherosclerosis and detection of soft plaque with use of black-blood MR imaging.

BACKGROUND AND PURPOSE: In the treatment of carotid atherosclerosis, the rate of stenosis and characteristics of plaque should be assessed to diagnose vulnerable plaques that increase the risk for cerebral infarction. We performed carotid black-blood (BB) MR imaging to diagnose plaque components and assess plaque hardness based on MR signals. MATERIALS AND METHODS: Three images of BB-MR imaging per plaque were obtained from 70 consecutive patients who underwent carotid endarterectomy (CEA) to generate T1- and T2-weighted images. To evaluate the relative signal intensity (rSI) of plaque components and the relationship between histologic findings and symptoms, we prepared sections at 2-mm intervals from 34 intact plaques. We then calculated the relative overall signal intensity (roSI) of 70 plaques to assess the relationship between MR signal intensity and plaque hardness and symptoms. RESULTS: The characteristics of rSI values on T1- and T2-weighted images of fibrous cap (FC), fibrosis, calcification, myxomatous tissue, lipid core (LC) with intraplaque hemorrhage (IPH), and LC without IPH differed. Symptomatic plaques were associated with FC disruption (P < .001) and LC with IPH (P < .05). The roSI on T1-weighted images was significantly higher for soft than nonsoft plaques. When the roSI cutoff value was set at 1.25 (mean of the roSI), soft plaques were diagnosed with 79.4% sensitivity and 84.4% specificity. The roSI was also significantly higher for symptomatic than for asymptomatic plaques. Soft and nonsoft plaques as well as symptomatic and asymptomatic plaques did not significantly differ on T2-weighted images. CONCLUSION: BB-MR imaging can diagnose plaque components and predict plaque hardness. This procedure provides useful information for planning therapeutic strategies of carotid atherosclerosis.

Transplatin, a cisplatin trans-isomer, may enhance the anticancer effect of 5-fluorouracil.

Transplatin (TDDP), a trans-isomer of cisplatin (CDDP), is well known to have faint cytotoxicity because its geometric structure allows less adduct formation with DNA than does CDDP. However, TDDP might have the potential to enhance the anticancer effect of 5-fluorouracil (5-FU) as well as CDDP. In this study, five gastric cancer cell lines were used. Cells were treated with 5-FU, TDDP, TDDP+5-FU, CDDP, and CDDP+5-FU, for 72 hrs. Synergistic effects between TDDP and 5-FU were observed in OCUM-2MD3, OCUM-2M, and OCUM-11, though they were not observed in MKN-45 or MKN-28. The cell lines in which synergistic effects were observed between TDDP and 5-FU were the same ones in which synergistic effects are shown between CDDP and 5-FU. The cell lines without synergism between 5-FU +TDDP/CDDP had lower thymidylate synthase (TS) activities than those with synergism, suggesting TS might be attributable to the synergistic mechanism. TDDP alone, compared to CDDP alone, gave rather low cytotoxicity for these cell lines. In conclusion, TDDP might be a clinically useful modulator of 5-FU.

Mucocele in an Onodi cell responsible for acute optic neuropathy.

An Onodi cell is defined as the most posterior ethmoid cell, which pneumatizes laterally and superiorly to the sphenoid sinus. A rare case of an isolated mucocele in an Onodi cell with unilateral acute visual disturbance is presented. MRI was imperative for the early and accurate diagnosis, and prompt surgical drainage resulted in dramatic visual recovery.

Gene therapy for human small-cell lung carcinoma by inactivation of Skp-2 with virally mediated RNA interference.

Increase of Skp-2, which is involved in the degradation of cell cycle regulators including p27Kip1, p21 and c-myc, is one of the important mechanisms for dysregulation of cell cycles in various cancers. We applied RNA interference (RNAi) for Skp-2 by using HIV-lentiviral or adenoviral vectors for a human small-cell lung carcinoma cell line with increased Skp-2 to evaluate RNAi strategy for cancer gene therapy. HIV-lentivirus-mediated RNAi for Skp-2 resulted in efficient inhibition of the in vitro cell growth of cancer cells with increased Skp-2 through the increase of p27Kip1 and p21, but no significant effect on the growth of cells without high Skp-2 expression. Furthermore, intratumoral administration of adenovirus siRNA vector for Skp-2 efficiently inhibited growth of established subcutaneous tumor on NOD/SCID mice. These results indicate that the Skp-2 RNAi may be a useful strategy for gene therapy of cancers with high Skp-2 expression.

[Thoracoscopic surgery using a new bronchial blocker].

We report use of a new bronchial blocker through a single-lumen endotracheal tube to achieve one-lung ventilation to perform thoracoscopic operation in patients in whom placement of the double-lumen tube failed and difficult intubation is predicted. The bronchial blocker tube was placed into the aimed bronchus under the bronchoscopic vision and the cuff of the blocker was inflated to achieve one-lung ventilation. In all of the 4 patients, the bronchial blocker could be inserted and placed safely, quickly, and exactly under the fiberoptic flexible bronchoscopic vision to perform thoracoscopic operation without any complications. The new bronchial blocker tube through the indwelling endotracheal tube may have advantages in situations where placement of double-lumen endotracheal tubes is technically impossible or inappropriate. The use of the new bronchial blocker tube will, however, require careful evaluation in larger series.

[Thoracoscopic pericardial resections for malignant pericardial effusions].

We have performed 5 thoracoscopic pericardial resections for malignant pericardial effusions. An initial trocar was placed in the seventh or eighth intercostal space posterior to the midaxillary line. Two additional trocars were placed, usually in the sixth intercostals spaces in the anterior axillary and posterior axillary lines. Using an endoscopic grasping instrument and scissors through the working ports, a pericardial resection was performed. All patients were successfully managed by thoracoscopic pericardial resections. Two of the 5 patients had associated malignant pleural effusions that were able to be managed at the same time by thoracoscopy. The average chest tube duration was 1.8 days. There has been no reaccumulation of pericardial effusions in all patients at an average follow-up of 5 months. The thoracoscopic approach could be minimally invasive and the procedure of choice in performing pericardial resections in selected patients with malignant pericardial effusions who are expected to have a reasonable life expectancy.

Static-dynamic friction transition of FRP esthetic orthodontic wires on various brackets by suspension-type friction test.

A new testing apparatus for the measurement of frictional properties was designed and the frictional coefficients were obtained and compared with each other in various combinations of brackets and orthodontic wires, including esthetic fiber-reinforced plastic (FRP) wire that was especially designed and manufactured. Three kinds of wires (stainless steel, nickel-titanium, and FRP) and four brackets (single-crystal alumina, polycrystalline alumina, polycarbonate, and stainless steel) were used. The testing was done under dry and wet conditions. The friction testing equipment was designed to attach the bracket to a C-shaped bar suspended with a variable mass, and sliding along a fixed wire. The transition between static and dynamic friction was measured as a breakaway force, with the use of a universal test machine. In addition to material properties, this testing fixture eliminates geometrical factors, such as the rotational moment at the edge of the bracket slot, deflection of the orthodontic wire, and tension of the ligature wire. Nearly ideal frictional properties between materials are obtained. The frictional properties of FRP wire were similar to those of metal wires on all brackets, except the polycrystalline alumina bracket. The frictional coefficient between the polycrystalline ceramic bracket and FRP wire was larger than that of other combinations. There was little difference in frictional coefficients between dry and wet conditions.

Xanthine oxidase inhibition reduces reactive nitrogen species production in COPD airways.

Reactive nitrogen species (RNS) have been reported to be involved in the inflammatory process in chronic obstructive pulmonary disease (COPD). However, there are no studies on the modulation of RNS in COPD. It was hypothesised that inhibition of xanthine oxidase (XO) might decrease RNS production in COPD airways through the suppression of superoxide anion production. Ten COPD and six healthy subjects participated in the study. The XO inhibitor allopurinol (300 mg x day(-1) p.o. for 4 weeks) was administered to COPD patients. RNS production in the airway was assessed by 3-nitrotyrosine immunoreactivity and enzymic activity of XO in induced sputum as well as by exhaled nitric oxide (eNO) concentration. XO activity in the airway was significantly elevated in COPD compared with healthy subjects. Allopurinol administration to COPD subjects significantly decreased XO activity and nitrotyrosine formation. In contrast, eNO concentration was significantly increased by allopurinol administration. These results suggest that oral administration of the xanthine oxidase inhibitor allopurinol reduces airway reactive nitrogen species production in chronic obstructive pulmonary disease subjects. This intervention may be useful in the future management of chronic obstructive pulmonary disease.

[A thoracoscopic technique with fibrin glue and polyglycolic acid mesh for the injured lung during thoracoscopic operation].

The injury of the lung occurred during thoracoscopic operation, especially, of the fragile, severely emphysematous lung is often difficult to treat. A fibrin glue spraying device in which an air compressor was connected to an intravenous catheter was constructed, and connected to a Dupuloject syringe. Fibrin glue was administered by spraying on the target lesion under a thoracoscopic vision. The procedure was done while the tip of the intravenous catheter was held and manipulated with forceps through a thoracoport. And then, the polyglycolic acid (PGA) mesh that had been cut into small pieces was put on the target lesion with forceps. Fibrin glue was sprayed on the lesion again in the above-mentioned manner. When necessary, the procedure was repeated. Our technique during thoracoscopic operation may be easy to manipulate and be able to apply the target lesion uniformly within a short time with a successful repair.

Room-temperature ferromagnetism in a II-VI diluted magnetic semiconductor Zn(1-x)Cr(x)Te.

The magnetic and magneto-optical properties of a Cr-doped II-VI semiconductor ZnTe were investigated. Magnetic circular dichroism measurements showed a strong interaction between the sp carriers and localized d spins, indicating that Zn(1-x)Cr(x)Te is a diluted magnetic semiconductor. The Curie temperature of the film with x=0.20 was estimated to be 300+/-10 K, which is the highest value ever reported for a diluted magnetic semiconductor in which sp-d interactions were confirmed. In spite of its high Curie temperature, Zn(1-x)Cr(x)Te film shows semiconducting electrical transport properties.

Correlation between change in pulmonary function and suppression of reactive nitrogen species production following steroid treatment in COPD.

BACKGROUND: Reactive nitrogen species (RNS) have a number of inflammatory actions and the production of these molecules has been reported to be increased in the airways of patients with chronic obstructive pulmonary disease (COPD), which suggests that they may be involved in the inflammatory and obstructive process in COPD. METHODS: The relationship between the reduction in RNS and the improvement in pulmonary function was studied in 18 patients with COPD following steroid treatment (800 micro g beclomethasone dipropionate inhalation for 4 weeks). Twelve patients were treated with inhaled steroids and the others received placebo treatment. Forced expiratory volume in 1 second (FEV(1)) and airway responsiveness to histamine were measured before and after treatment. Induced sputum cells were stained with anti-nitrotyrosine antibody, a footprint of RNS, and RNS formation was assessed by measuring nitrotyrosine immunoreactivity. The immunoreactivity of inducible nitric oxide synthase (iNOS) in induced sputum and exhaled NO levels were also measured. RESULTS: Treatment with steroids resulted in a significant reduction in both nitrotyrosine and iNOS immunoreactivity in sputum cells compared with pretreatment levels (both p<0.01). The reduction rates in both parameters were significantly related (p<0.05). The reduction in nitrotyrosine and iNOS immunoreactivity was correlated with the improvement in FEV(1) (p<0.05) and airway responsiveness to histamine (p<0.01). None of the parameters was significantly changed by placebo administration. CONCLUSIONS: These results suggest that RNS may be involved in the reversible component of inflammation in COPD that is suppressed by steroids. Further studies using specific inhibitors for RNS are needed to clarify their effects on the long term progression of COPD.

[Thoracoscopic surgery for metastatic lung tumors: computed tomography-guided localization with use of a needle with a suture].

When a metastatic lung tumor is found to be deep to the visceral surface of the pleura, or when it is found to be a small lesion, it may important to help identify the lesion by preoperative methods in order to localize it at the time of thoracoscopic operation. We performed computed tomography-guided localization of metastatic lung tumors with use of a needle with a suture in 11 cases prior to thoracoscopic resection. Placement of the needle, immediately before moving to the operation room were successfully performed in all patients. It took from 16 to 25 minutes (20.3 minutes on average). Complications included minimal pneumothorax in 8 patients, mild airway bleeding in 2, and penetration through the middle lobe to the lower lobe of the right lung in 1. However, treatment was not necessary in any of the patients. Thoracoscopic resections were successfully achieved in all patients. Our technique may be a simpler technique and advantageous for identification of small lesions and lesions deep to the visceral surface of the pleura during the thoracoscopic operation for metastatic lung tumors.

iNOS depletion completely diminishes reactive nitrogen-species formation after an allergic response.

Nitric oxide (NO) shows proinflammatory actions mainly via reactive nitrogen species (RNS) formation through superoxide- and peroxidase-dependent mechanisms. The purpose of this study was to examine the role of inducible NO synthase (iNOS) in RNS production, airway hyperresponsiveness, and inflammation after allergen challenge. Ovalbumin (OVA)-sensitised, iNOS-deficient and wild-type mice were used. RNS production was assessed by nitrotyrosine (NT) immunoreactivity in the airways. Airway inflammation and responsiveness were evaluated by eosinophil accumulation and methacholine (i.v.) challenge, respectively. In wild-type mice, OVA-inhalation challenge increased iNOS immunoreactivity in airway epithelial cells as well as iNOS protein measured by Western blotting. The total amounts of nitrite and nitrate in bronchoalveolar lavage (BAL) fluid were increased, and NT immunoreactivity was also observed abundantly in airway inflammatory cells. In iNOS-deficient mice, both iNOS expression and NT formation were completely abolished, and the total amounts of nitrite and nitrate in BAL fluid were significantly decreased. In contrast, OVA-induced airway eosinophil recruitment and hyperresponsiveness were observed almost equally in wild-type and iNOS-deficient mice. These data suggest that reactive nitrogen species production after allergic reaction occurs totally via inducible nitric oxide synthase-dependent pathways. Allergen-mediated airway eosinophil recruitment and hyperresponsiveness appear to be independent of reactive nitrogen species production.

Ganglioside GD1a inhibits HGF-induced motility and scattering of cancer cells through suppression of tyrosine phosphorylation of c-Met.

We previously reported that ganglioside GD1a, which is highly expressed in poorly metastatic FBJ-S1 cells, inhibits the serum-induced motility of FBJ-LL cells and that the metastatic potential of FBJ-LL cells is completely suppressed by enforced GD1a expression (Hyuga et al., Int J Cancer 1999;83:685-91). We recently discovered that hepatocyte growth factor (HGF) induces FBJ-LL cell motility. In the present study, the HGF-induced motility of FBJ-S1 cells was found to be one-thirtieth that of FBJ-LL cells. This motility of GD1a-expressing transfectants, which were produced by transfection of FBJ-LL cells with GM2/GD2 synthase cDNA, decreased with increases in their GD1a expression and HGF induced almost no motility in GD1a-pretreated FBJ-LL cells, indicating that GD1a inhibits the HGF-induced motility of FBJ-LL cells. The expression of the HGF receptor c-Met on FBJ-S1 cells, FBJ-LL cells, transfectants and a mock-transfectant was almost the same. The level of tyrosine phosphorylation of c-Met after HGF stimulation in FBJ-S1 cells, GD1a-pretreated FBJ-LL cells and a GD1a-expressing transfectant was significantly lower than in FBJ-LL cells and a mock-transfectant. These findings suggested that GD1a inhibits the HGF-induced motility of FBJ-LL cells through suppression of tyrosine phosphorylation of c-Met. HepG2 cells, a human hepatoma cell line, were used to investigate whether GD1a interferes with other cancer cells expressing c-Met. HepG2 cells did not express GD1a. HGF induced cell scattering of HepG2 cells and the scattering was inhibited by pretreating the cells with GD1a. The c-Met in the cells was autophosphorylated by stimulation with HGF, but after treating the cells with GD1a, the HGF-induced autophosphorylation of c-Met was suppressed. These results suggest that GD1a acts as a negative regulator of c-Met in cancer cells.