ICSH guidelines for the verification and performance of automated cell counters for body fluids.

One of the many challenges facing laboratories is the verification of their automated Complete Blood Count cell counters for the enumeration of body fluids. These analyzers offer improved accuracy, precision, and efficiency in performing the enumeration of cells compared with manual methods. A patterns of practice survey was distributed to laboratories that participate in proficiency testing in Ontario, Canada, the United States, the United Kingdom, and Japan to determine the number of laboratories that are testing body fluids on automated analyzers and the performance specifications that were performed. Based on the results of this questionnaire, an International Working Group for the Verification and Performance of Automated Cell Counters for Body Fluids was formed by the International Council for Standardization in Hematology (ICSH) to prepare a set of guidelines to help laboratories plan and execute the verification of their automated cell counters to provide accurate and reliable results for automated body fluid counts. These guidelines were discussed at the ICSH General Assemblies and reviewed by an international panel of experts to achieve further consensus.

Meropenem as predictive risk factor for isolation of multidrug-resistant Pseudomonas aeruginosa.

The objective of this study was to explore independent risk factors for the isolation of multidrug-resistant (MDR) Pseudomonas aeruginosa in a Japanese university hospital between January 1997 and December 2010. MDR P. aeruginosa was defined when the organism was resistant or intermediately susceptible to all five antimicrobials tested. In all, 159 patients with MDR P. aeruginosa were identified over the 14-year period. Multivariate logistic regression analysis revealed that prolonged hospital stay, prior exposure to meropenem and fluoroquinolones, and patients suffering from diabetes mellitus or receiving surgery were predictive risk factors for the isolation of MDR P. aeruginosa.

Role of stromal microenvironment in nonpharmacological resistance of CML to imatinib through Lyn/CXCR4 interactions in lipid rafts.

We and others have previously demonstrated that p210 Bcr-Abl tyrosine kinase inhibits stromal cell-derived factor-1α/CXCR4 chemokine receptor signaling, contributing to the deficient adhesion of chronic myeloid leukemia (CML) cells to bone marrow stroma. Conversely, exposure of CML cells to a tyrosine kinase inhibitor (TKI) enhances migration of CML cells towards stromal cell layers and promotes non-pharmacological resistance to imatinib. Src-related kinase Lyn is known to interact with CXCL12/CXCR4 signaling and is directly activated by p210 Bcr-Abl. In this study, we demonstrate that TKI treatment promoted CXCR4 redistribution into the lipid raft fraction, in which it co-localized with active phosphorylated form of Lyn (LynTyr396) in CML cells. Lyn inhibition or cholesterol depletion abrogated imatinib-induced migration, and dual Src/Abl kinase inhibitor dasatinib induced fewer CML cells to migrate to the stroma. These findings demonstrate the novel mechanism of microenvironment-mediated resistance through lipid raft modulation, which involves compartmental changes of the multivalent CXCR4 and Lyn complex. We propose that pharmacological targeting of lipid rafts may eliminate bone marrow-resident CML cells through interference with microenvironment-mediated resistance.

Effects of denervation at ileocecal junction and ileocecal resection in dogs.

BACKGROUND: To investigate neural regulation at the ileocecal junction (ICJ) and motility changes after ileocecal resection (ICR). Previous studies showed normal basal motility at the ICJ directly by force transducers in dogs, but these observations were limited to normal contractile activity. METHODS: Continuous strain gauge recordings of stomach, terminal ileum, ileocecal sphincter (ICS), and colon were performed in dogs. The dogs were divided into four groups, namely control (CONT), extrinsic denervation at ICJ (ED), intrinsic denervation at ICJ (ID), and ICR groups. Colonic activity was recorded 2 h before a meal, in the early postprandial period (first 2 h), and in the late postprandial period (4-6 h after a meal). The meal lasted 5 min. KEY RESULTS: Motility index was significantly increased at the ICS (P = 0.0056) and proximal colon (P = 0.0059) after feeding. However, such changes were not observed in the ED and ID groups. The amplitude of contractions at proximal colon in the interdigestive state was significantly decreased by ED. In the ID and ICR groups, the numbers of nonmigrating contractions were significantly decreased (P < 0.05), and colonic migrating motor complex (CMMC) ratio was significantly higher than that of the CONT group (P < 0.001). The dogs in these two groups had diarrhea. CONCLUSIONS & INFERENCES: Gastrocolonic response at the ICJ may require both intrinsic and extrinsic innervation. When ID was performed, CMMC ratio increased. As a result, intraluminal water absorption may have decreased. ID may be one of the causes of diarrhea after ICR.

Antiproliferative and proapoptotic activity of GUT-70 mediated through potent inhibition of Hsp90 in mantle cell lymphoma.

BACKGROUND: Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma with poor prognosis, requiring novel anticancer strategies. METHODS: Mantle cell lymphoma cell lines with known p53 status were treated with GUT-70, a tricyclic coumarin derived from Calophyllum brasiliense, and the biological and biochemical consequences of GUT-70 were studied. RESULTS: GUT-70 markedly reduced cell proliferation/viability through G(1) cell cycle arrest and increased apoptosis, with greater sensitivity in mutant (mt)-p53-expressing MCL cells than in wild-type (wt)-p53-bearing cells. Mechanistically, GUT-70 showed binding affinity to heat-shock protein 90 (Hsp90) and ubiquitin-dependent proteasomal degradation of Hsp90 client proteins, including cyclin D1, Raf-1, Akt, and mt-p53. Depletion of constitutively overexpressed cyclin D1 by GUT-70 was accompanied by p27 accumulation and decreased Rb phosphorylation. GUT-70 induced mitochondrial apoptosis with Noxa upregulation and Mcl-1 downregulation in mt-p53 cells, but Mcl-1 accumulation in wt-p53 cells. Noxa and Mcl-1 were coimmunoprecipitated, and activated BAK. Treatment with a combination of GUT-70 and bortezomib or doxorubicin had synergistic antiproliferative effects in MCL cells that were independent of p53 status. CONCLUSION: GUT-70 has pronounced antiproliferative effects in MCL with mt-p53, a known negative prognostic factor for MCL, through Hsp90 inhibition. These findings suggest that GUT-70 has potential utility for the treatment of MCL.

Molecular dynamics simulation of condensed-phase chiral molecular propellers.

Molecular dynamics simulations were performed for an axial-chiral liquid crystalline (LC) monolayer under trans-monolayer gas flow. The rotational dynamics of the monolayer chiral LC molecule along its long-molecular axis were analyzed at the molecular level. We found a precise correspondence between the flow-driven molecular rotation direction and molecular chirality as well as between the rotation direction and the trans-monolayer flow direction. The rotational direction exactly corresponded to what was expected in the proposed chiral molecular propeller model (Tabe, Y.; Yokoyama, H. Nat. Mater. 2003, 2, 806). Among the four trans-monolayer gas species we investigated, we found argon to be the most efficient at driving the chiral molecular propeller and helium the least efficient.

Selective FLT3 inhibitor FI-700 neutralizes Mcl-1 and enhances p53-mediated apoptosis in AML cells with activating mutations of FLT3 through Mcl-1/Noxa axis.

Treatment using Fms-like tyrosine kinase-3 (FLT3) inhibitors is a promising approach to overcome the dismal prognosis of acute myeloid leukemia (AML) with activating FLT3 mutations. Current trials are combining FLT3 inhibitors with p53-activating conventional chemotherapy. The mechanisms of cytotoxicity of FLT3 inhibitors are poorly understood. We investigated the interaction of FLT3 and p53 pathways after their simultaneous blockade using the selective FLT3 inhibitor FI-700 and the MDM2 inhibitor Nutlin-3 in AML. We found that FI-700 immediately reduced antiapoptotic Mcl-1 levels and enhanced Nutlin-induced p53-mediated mitochondrial apoptosis in FLT3/internal tandem duplication cells through the Mcl-1/Noxa axis. FI-700 induced proteasome-mediated degradation of Mcl-1, resulting in the reduced ability of Mcl-1 to sequester proapoptotic Bim. Nutlin-3 induced Noxa, which displaced Bim from Mcl-1. The FI-700/Nutlin-3 combination profoundly activated Bax and induced apoptosis. Our findings suggest that FI-700 actively enhances p53 signaling toward mitochondrial apoptosis and that a combination strategy aimed at inhibiting FLT3 and activating p53 signaling could potentially be effective in AML.

Gas permeation of LC films observed by smectic bubble expansion.

Gas permeation through liquid crystal (LC) films was examined using hemispherical smectic bubbles. A smectic bubble, when the inside and the outside are filled with different gases, should expand or shrink toward the quasi-equilibrium state, where the influx and efflux caused by osmotic pressure are balanced. Deriving a simple formula that directly converts the quasi-equilibrated bubble radius to the gas permeation, we determined the absolute permeability coefficients of 8 simple gases through the smectic bubble. The permeability was distributed in such a wide range that carbon-dioxide had more than 20 times larger value than nitrogen, the dependence of which on the gas species was mostly dominated by their solubility into the LCs. Dividing the measured permeability by the calculated solubility, we obtained the diffusion constants as well, yet whose magnitude and the dependence on the solute size could not be explained by either conventional continuum theories or microscopic diffusion models. In order to describe the diffusion of small solutes in the liquid solvent composed of large molecules, a new theoretical framework may be necessary.

Correlation between colonic motility and defecatory disorders after anterior resection of the rectum in canine models.

The objective of this study was to describe the correlation between changes in colonic motility and defecatory disorders in four experimental canine models, with an emphasis on denervation. Therefore, we constructed a model by dividing 20 healthy mongrel dogs into four groups, i.e. control, denervation, transection and anterior resection of the rectum (AR) (denervation plus transection), and focused on the correlation between colonic motility and defecatory disorders by counting the colonic migrating motor complexes (CMMCs) and colonic non-migrating motor complexes (CNMCs). Gastrointestinal and colonic contractile activities were continuously recorded on a computer with strain gauge force transducers. The dogs' feces were checked daily, and their consistency was recorded as normal, semisolid, or watery. Compared with the control group, the transection group showed elongation of the propagation time (P < 0.05), and the mean motility index of colonic contractile activity at C4 and C5 in the denervation group was greater than that in the control group (P < 0.05). The AR group showed three features of colonic motility: (i) elongation of the mean CMMC cycle (P < 0.05); (ii) shortening of the propagation time (P < 0.05); and (iii) increment of the number of CNMCs. Concerning fecal consistency, the AR group only showed watery diarrhoea. In conclusion, we revealed the existence of a correlation between defecatory disorders and changes in colonic motility. Increased knowledge among colorectal surgeons of the changes in colonic motility that occur following colorectal surgery is very important and could lead to the curtailment of defecatory disorders among patients.

Apoptosis-based dual molecular targeting by INNO-406, a second-generation Bcr-Abl inhibitor, and ABT-737, an inhibitor of antiapoptotic Bcl-2 proteins, against Bcr-Abl-positive leukemia.

Bcr-Abl is the cause of Philadelphia-positive (Ph(+)) leukemias and also constitutes their principal therapeutic target, as exemplified by dramatic effects of imatinib mesylate. However, mono-targeting of Bcr-Abl does not always achieve complete leukemia eradication, and additional strategies those enable complete elimination of leukemic cells are desired to develop. Here we demonstrate that INNO-406, a much more active Bcr-Abl tyrosine kinase inhibitor than imatinib, augments the activities of several proapoptotic Bcl-2 homology (BH)3-only proteins (Bim, Bad, Bmf and Bik) and induces apoptosis in Ph(+) leukemia cells via Bcl-2 family-regulated intrinsic apoptosis pathway. ABT-737, an inhibitor of antiapoptotic Bcl-2 and Bcl-X(L), greatly enhanced the apoptosis by INNO-406, even in INNO-406-less sensitive cells with Bcr-Abl point mutations except T315I mutation. In contrast, co-treatment with INNO-406 and other pharmacologic inducers of those BH3-only proteins, such as 17-allylaminogeldanamycin, an heat shock protein-90 inhibitor, or PS-341, a proteasome inhibitor, did not further increase the BH3-only protein levels or sensitize leukemic cells to INNO-406-induced apoptosis, suggesting a limit to how much expression levels of BH3-only proteins can be increased by anticancer agents. Thus, double-barrelled molecular targeting for Bcr-Abl-driven oncogenic signaling and the cell protection by antiapoptotic Bcl-2 family proteins may be the rational therapeutic approach for eradicating Ph(+) leukemic cells.

Air tube formation at the freezing transition in nematic liquid crystals.

A phenomenon is presented, which changes the shape of gas bubbles in liquid crystals and also creates long gas tubes. The system consists of air bubbles which are injected into a nematic liquid crystal host. The shape of these air bubbles changes from spherical to ellipsoidal by initiating freezing of the sample. Furthermore, long gas tubes are formed from the air which was formerly dissolved in the liquid crystal. The gas tubes are created by the progression of the crystalline-liquid interface. Their length can reach up to 40 times their diameter. The diameter of the tubes depends on the pressure applied to the system, as well as on the interface velocity.

Novel role of HDAC inhibitors in AML1/ETO AML cells: activation of apoptosis and phagocytosis through induction of annexin A1.

The chimeric fusion protein AML1-ETO, created by the t(8;21) translocation, recruits histone deacetylase (HDAC) to AML1-dependent promoters, resulting in transcriptional repression of the target genes. We analyzed the transcriptional changes in t(8;21) Kasumi-1 AML cells in response to the HDAC inhibitors, depsipeptide (FK228) and suberoylanilide hydroxamic acid (SAHA), which induced marked growth inhibition and apoptosis. Using cDNA array, annexin A1 (ANXA1) was identified as one of the FK228-induced genes. Induction of ANXA1 mRNA was associated with histone acetylation in ANXA1 promoter and reversal of the HDAC-dependent suppression of C/EBPalpha by AML1-ETO with direct recruitment of C/EBPalpha to ANXA1 promoter. This led to increase in the N-terminal cleaved isoform of ANXA1 protein and accumulation of ANXA1 on cell membrane. Neutralization with anti-ANXA1 antibody or gene silencing with ANXA1 siRNA inhibited FK228-induced apoptosis, suggesting that the upregulation of endogenous ANXA1 promotes cell death. FK228-induced ANXA1 expression was associated with massive increase in cell attachment and engulfment of Kasumi-1 cells by human THP-1-derived macrophages, which was completely abrogated with ANXA1 knockdown via siRNA transfection or ANXA1 neutralization. These findings identify a novel mechanism of action of HDAC inhibitors, which induce the expression and externalization of ANXA1 in leukemic cells, which in turn mediates the phagocytic clearance of apoptotic cells by macrophages.

Director-configurational transitions around microbubbles of hydrostatically regulated size in liquid crystals.

A high-pressure technique is introduced which allows a continuous variation of the inclusion size in liquid crystal colloids. We use a nematic liquid crystal host into which micrometer-sized gas bubbles are injected. By applying hydrostatic pressures, the diameter of these gas bubbles can be continuously decreased via compression and absorption of gas into the host liquid crystal, so that the director configurations around a single bubble can be investigated as a function of the bubble size. The theoretically predicted transition from a hyperbolic hedgehog to a Saturn-ring configuration, on reduction of the particle size below a certain threshold, is confirmed to occur at the radius of a few micrometers.

Glycopeptide susceptibility profiles of nosocomial multiresistant Staphylococcus haemolyticus isolates.

We investigated 48 Staphylococcus haemolyticus isolates from patients and medical staff in terms of susceptibility to and in-vitro selection for vancomycin and teicoplanin in regard to their antibiotypes. On comparison of multiresistant S. haemolyticus isolates with non-multiresistant isolates, the geometric mean minimum inhibitory concentration (MIC) of vancomycin for multiresistant S. haemolyticus was 2.9 microg/ml, and that of teicoplanin was 18.0 microg/ml, both of which values were significantly greater than the corresponding mean MICs of vancomycin (2.0 microg/ml) and teicoplanin (4.7 microg/ml) for nonmultiresistant isolates. After agar selection, the mean of the highest teicoplanin concentration of selected plates for multiresistant S. haemolyticus was 97.1 microg/ml, which was significantly higher than that for nonmultiresistant isolates (57.8 microg/ml). However, the means' of the highest vancomycin concentrations after agar selection for multiresistant and nonmulti-resistant isolates were the same, at 7.4 microg/ml, with no colonies capable of growing in 32 microg/ml of vancomycin. There was no significant difference in glycopeptide susceptibility between oxacillin-resistant and oxacillin-susceptible isolates among nonmultiresistant S. haemolyticus. The geometric mean MICs of vancomycin for oxacillin-resistant and oxacillin-susceptible isolates were 2.1 microg/ml and 1.6 microg/ml, and those of teicoplanin were 4.4 microg/ml and 5.6 microg/ml, while the means of the highest concentrations of the selected plates of vancomycin were 8.6 microg/ml and 3.3 microg/ml, and those of teicoplanin were 52.8 microg/ml and 74.7 microg/ml, respectively. Multiresistant isolates showed significantly greater mean MICs of vancomycin and teicoplanin and higher teicoplanin concentration of the selected plates than nonmultiresistant isolates, irrespective of oxacillin resistance. These results indicate that methicillin resistance may not be related to reduced susceptibility to glycopeptide in S. haemolyticus, and that a multiresistant profile is associated more with a decreasing susceptibility to glycopeptides then with resistance to oxacillin. In this study, antibiotypes showed good concordance with pulsed-field gel electrophoresis typing results, with a sufficiently high discriminatory ability index, of 0.912. We consider that primary screening with antimicrobial susceptibility testing and antibiotyping, with attention to the multiresistant profile, would be useful for monitoring nosocomial S. haemolyticus colonization and infection.

[Epidemiological study on Staphylococcus epidermidis isolated from bloodstream and blood vessel catheter].

An epidemiological study on 35 strains of Staphylococcus epidermidis was conducted in Juntendo University Hospital between 1994 and 1996. The strains were isolated from blood and blood vessel catheters. Three epidemiological markers; PFGE type (pulsed-field gel electrophoresis using SmaI), biotype by STAPHYOGRAM and antibiotype (antibiotic resistant pattern) were used. There were 12 types in PFGE type, 6 types in biotype and 7 types in antibiotype. 1. The predominant types were PFGE type A (57.1%), biotype 1 (62.9%), and antibiotype I (resistant for oxacillin, ampicillin and gentamicin; 34.3%) in Juntendo University Hospital. 2. The strains with antibiotic V-VII (resistant for over 6 antibiotics) showed only PFGE type A and B. All strains with PFGE type B showed biotype 4-6 (negative nitrate reduction strain). 3. The strains having PFGE type A and B were isolated from various patient wards. The strains showing PFGE type A and antibiotype I were isolated from the pediatric ward. 4. There was no strain with PFGE type C or D in 1996. 5. Three patients in whom S. epidermidis was frequently isolated for a few months had the same types of PFGE type, biotype as well as antibiotype.

Molecular characterization of epidemic multiresistant Staphylococcus haemolyticus isolates.

Fifty-five Staphylococcus haemolyticus specimens isolated from patients and neonatal intensive care unit staff were tested for susceptibility to 12 antimicrobial agents. There were 34 multidrug-resistant isolates which were resistant to oxacillin, ampicillin, cefazolin, cefmetazole, imipenem, and gentamicin. These isolates had a higher frequency of resistance to tobramicin and ofloxacin, and relatively high MICs (2 to 4 micrograms/mL) for vancomycin, although none of the isolates were vancomycin resistant. To investigate hospital-acquired colonization and infection by multiresistant S. haemolyticus, we examined all isolates by pulsed-field gel electrophoresis (PFGE) after SmaI and SstII digestion, and detected an endemic PFGE pattern in multiresistant isolates. The results suggested that local spread of multiresistant S. haemolyticus was hospital acquired, and that the hospital staffs functioned as a reservoir.

[Nationwide survey of susceptibilities of clinical isolates to antibacterial agents in 1992].

This study was conducted to investigate susceptibilities of clinical isolates to imipenem (IPM) and other antibacterial agents in 144 hospital laboratories throughout Japan from September to December of 1992. In this study, the isolates were identified and susceptibility tests were performed at individual laboratories. The susceptibility tests were performed using the disk dilution method recommended by NCCLS. S. aureus (including MRSA) strains were highly susceptible to arbekacin (ABK) and netilmicin (NTL). S. pneumoniae and H. influenzae were susceptible to most of the agents tested. E. faecalis were highly susceptible to penicillins and imipenem (IPM). P. aeruginosa showed high susceptibility to ceftazidime (CAZ), IPM and amikacin (AMK). Annual changes in antimicrobial susceptibility patterns over 5 years (1988-1992) were examined. The frequency of sensitive strains of S. aureus to methicillin (DMPPC) has slightly increased from 1991 to 1992. A moderate increases of PCG-insensitive S. pneumoniae was observed. B. fragilis group showed a slight increase in sensitivity to minocycline (MINO) but no yearly changes in IPM sensitivity was observed.

[Nationwide survey on susceptibilities of clinical isolates to antibacterial agents in 1991].

This study was conducted to investigate susceptibilities of clinical isolates to different antibacterial agents at 123 hospital laboratories throughout Japan from September to December of 1991. In this study, identifications and susceptibility testings were carried out at each hospital laboratory. The susceptibility testing were performed using the disk dilution method recommended by NCCLS. Staphylococcus aureus and CNS showed high or moderate resistance rates to methicillin (DMPPC). Streptococcus pneumoniae, Escherichia coli, Klebsiella pneumoniae, Haemophilus influenzae, Proteus mirabilis were highly susceptible to many agents including beta-lactam antibiotics. Though Enterococcus faecalis was highly susceptible to ampicillin (ABPC), piperacillin (PIPC), imipenem (IPM), sulfamethoxazole-trimethoprim (ST) compounds, Enterococcus faecium was resistant to almost all antibacterial agents but to ST compounds. High susceptibility rates were observed for strains of Enterobacter cloacae to IPM, gentamicin (GM) and ofloxacin (OFLX) and for strains of Proteus vulgaris to latamoxef (LMOX), IPM, aztreonam (AZT), GM and OFLX. Serratia marcescens and Bacteroides fragilis group were highly susceptible only to IPM. Pseudomonas aeruginosa were sensitive to ceftazidime (CAZ), IPM, amikacin (AMK) and tobramycin (TOB). Pseudomonas cepacia was relatively susceptible only to CAZ. IPM showed strong antibacterial activity to many species except for S. aureus and CNS.