Potential effect of ezetimibe against Mycobacterium tuberculosis infection in type II diabetes.

BACKGROUND AND OBJECTIVE: Tuberculosis (TB) risk might be increased in patients with diabetes by factors other than hyperglycaemia, such as dyslipidaemia. Host lipids are essential energy sources used by mycobacteria to persist in a latent TB state. A potential therapy targeting cholesterol catabolism of mycobacteria has been proposed, but the potential of cholesterol-lowering drugs as anti-TB therapy is unclear. The purpose of this study was to determine the effects of ezetimibe, a 2-azetidinone cholesterol absorption inhibitor, on intracellular mycobacteria survival and dormancy. METHODS: Intracellular mycobacteria survival was determined by measurements of ATP activity and colony-formation units (CFUs). Gene expression profiles of hypoxia-induced dormant Mycobacterium tuberculosis (Mtb) were analysed by real-time PCR. Flow cytometry and microscopy analysis were used to measure the lipid loads of human macrophages with or without ezetimibe treatment. QuantiFERON-TB Gold In-Tube (QFT-G-IT) assays were performed to diagnose latent TB infection. The levels of intracellular cholesterol/ triglyceride were measured by an enzymatic fluorometric method. RESULTS: Ezetimibe was capable of effectively lowering intracellular growth of Mtb and hypoxia-induced dormant Mtb. There was a significant decrease in Mtb growth in leucocytes from ezetimibe-treated patients with diabetes in terms of ATP levels of intracellular mycobacteria and CFU formation. Also, patients receiving ezetimibe therapy had a lower prevalence of latent TB and had lower intracellular lipid contents. CONCLUSION: Ezetimibe, which is a currently marketed drug, could hold promise as an adjunctive, host-directed therapy for TB.

Stimuli-responsive and hemocompatible pseudozwitterionic interfaces.

We report a novel biomacromolecular formula for the design of hemocompatible gel interfaces of N-isopropylacrylamide (NIPAAm) and mixed-charge pairs of [2-(methacryloyloxy)ethyl]trimethylammonium (TMA) and 3-sulfopropyl methacrylate (SA) with overall electrical neutrality. The study stresses on how well-defined compositions of nonionic NIPAAm and pseudozwitterionic TMA/SA in the poly(NIPAAm-co-TMA/SA) hydrogels along with environmental conditions (temperature, ionic strength, and solution pH) affect swelling and adhesion of biofoulants on their surfaces. When challenged with plasma proteins, bacteria, recalcified platelets, or whole blood, stimuli-responsive hydrogels better resisted their adhesion as the content of mixed charges in the copolymer increased, to reach nonbiofouling for the gels made of 100% TMA/SA. The low hemolytic activity (0.5%) associated with a long plasma clotting time (10 min) suggests excellent hemocompatibility excellent hemocompatibility. Finally, hydrogels containing both NIPAAm and TMA/SA tend to exhibit preferential adhesion of leukocytes.

Aminopeptidase N facilitates entry and intracellular survival of Mycobacterium tuberculosis in monocytes.

BACKGROUND AND OBJECTIVE: Aminopeptidase N (CD13) is an ectoenzyme located in the outer membrane of a variety of cells. Proteomic profiling indicates an increased expression of CD13 in phagocytes during Mycobacterium tuberculosis infection. The purpose of this study was to investigate the role of CD13 on the internalization and intracellular survival of M. tuberculosis in monocytes. METHODS: Magnetic nanoparticles and confocal microscopy were used to observe interactions between CD13 and M. tuberculosis. Mycobacterial entry and intracellular survival in monocytes were assessed with and without anti-CD13 antibody (WM15 and WM47) using flow cytometry and colony formation assay. RESULTS: By using magnetic nanoparticles and confocal microscopy, M. tuberculosis was found to be capable of binding to either soluble CD13 or membranous CD13 on monocytes. Flow cytometry showed that pretreatment of monocytes with WM15 or WM47 reduced the number of intracellular M. tuberculosis. Collectively, the data suggest that CD13 is a binding and entry receptor for M. tuberculosis on monocytes. Treatment of infected monocytes showed a greater effect of WM47 than WM15 in reducing the intracellular colonization of M. tuberculosis, suggesting that specific epitopes of CD13 may play an important role modulating intracellular M. tuberculosis survival. CONCLUSIONS: CD13 acts as a receptor for M. tuberculosis on human monocytes. The molecule facilitates internalization, and interaction of CD13 with an anti-CD13 antibody reduces intracellular M. tuberculosis survival.

Mycobacterium tuberculosis DNA detection using surface plasmon resonance modulated by telecommunication wavelength.

A surface plasmon resonance sensor for Mycobacterium tuberculosis (MTB) deoxyribonucleic acid (DNA) is developed using repeatable telecommunication wavelength modulation based on optical fiber communications laser wavelength and stability. MTB DNA concentrations of 1 µg/mL and 10 µg/mL were successfully demonstrated to have the same spectral half-width in the dip for optimum coupling. The sensitivity was shown to be -0.087 dB/(µg/mL) at all applied telecommunication wavelengths and the highest sensitivity achieved was 115 ng/mL without thiolated DNA immobilization onto a gold plate, which is better than the sensor limit of 400 ng/mL possible with commercial biosensor equipment.

Hemocompatible control of sulfobetaine-grafted polypropylene fibrous membranes in human whole blood via plasma-induced surface zwitterionization.

In this work, the hemocompatibility of zwitterionic polypropylene (PP) fibrous membranes with varying grafting coverage of poly(sulfobetaine methacrylate) (PSBMA) via plasma-induced surface polymerization was studied. Charge neutrality of PSBMA-grafted layers on PP membrane surfaces was controlled by the low-pressure and atmospheric plasma treatment in this study. The effects of grafting composition, surface hydrophilicity, and hydration capability on blood compatibility of the membranes were determined. Protein adsorption onto the different PSBMA-grafted PP membranes from human fibrinogen solutions was measured by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. Blood platelet adhesion and plasma clotting time measurements from a recalcified platelet-rich plasma solution were used to determine if platelet activation depends on the charge bias of the grafted PSBMA layer. The charge bias of PSBMA layer deviated from the electrical balance of positively and negatively charged moieties can be well-controlled via atmospheric plasma-induced interfacial zwitterionization and was further tested with human whole blood. The optimized PSBMA surface graft layer in overall charge neutrality has a high hydration capability and keeps its original blood-inert property of antifouling, anticoagulant, and antithrmbogenic activities when it comes into contact with human blood. This work suggests that the hemocompatible nature of grafted PSBMA polymers by controlling grafting quality via atmospheric plasma treatment gives a great potential in the surface zwitterionization of hydrophobic membranes for use in human whole blood.

The characteristics of new semi-quantitative method for diagnosing proteinuria by using random urine samples.

We assessed the characteristics of the new semi-quantitative test paper (Clinitek ATLAS Pro(12)) using random urine samples. Three hundred urine samples were analyzed using either the new test paper, conventional dipsticks, quantitative (P/C ratio), or immunological quantitative methods (A/C ratio). Our study showed that the new test paper is highly sensitive and specific for the detection of urinary protein. The new test paper also detected the urine protein more accurately than the conventional test and has a lower false-positive rate. In addition, the new test paper detected 14 of the 300 patients (4.7%) as dilute urine samples needing reassessment. Seventeen of the 300 samples tested were negative with conventional dipsticks but positive with the new test paper. The new semi-quantitative test paper not only has higher sensitivity than the conventional dipstick method, but also has potential to detect dilute samples.

Fluorescence detection of single nucleotide polymorphisms using a universal molecular beacon.

We present a simple and novel assay-employing a universal molecular beacon (MB) in the presence of Hg(2+)-for the detection of single nucleotide polymorphisms (SNPs) based on Hg(2+)-DNA complexes inducing a conformational change in the MB. The MB (T(7)-MB) contains a 19-mer loop and a stem of a pair of seven thymidine (T) bases, a carboxyfluorescein (FAM) unit at the 5'-end, and a 4-([4-(dimethylamino)phenyl]azo)benzoic acid (DABCYL) unit at the 3'-end. Upon formation of Hg(2+)-T(7)-MB complexes through T-Hg(2+)-T bonding, the conformation of T(7)-MB changes from a random coil to a folded structure, leading to a decreased distance between the FAM and DABCYL units and, hence, increased efficiency of fluorescence resonance energy transfer (FRET) between the FAM and DABCYL units, resulting in decreased fluorescence intensity of the MB. In the presence of complementary DNA, double-stranded DNA complexes form (instead of the Hg(2+)-T(7)-MB complexes), with FRET between the FAM and DABCYL units occurring to a lesser extent than in the folded structure. Under the optimal conditions (20 nM T(7)-MB, 20 mM NaCl, 1.0 muM Hg(2+), 5.0 mM phosphate buffer solution, pH 7.4), the linear plot of the fluorescence intensity against the concentration of perfectly matched DNA was linear over the range 2-30 nM (R(2) = 0.991), with a limit of detection of 0.5 nM at a signal-to-noise ratio of 3. This new probe provides higher selectivity toward DNA than that exhibited by conventional MBs.

Antithrombin-III attenuates hepatocyte apoptosis in bile duct ligated rat: a striking cellular change.

BACKGROUND AND PURPOSE: Retention and accumulation of toxic hydrophobic bile salts within hepatocyte may cause hepatocyte toxicity by inducing apoptosis. This study was designed with the purpose of evaluating the possible effect of antithrombin-III on hepatocyte apoptosis in bile duct ligated rat. MATERIALS AND METHODS: The rats were randomized to 3 groups: group 1 (control, C) underwent sham operation; group 2 (obstructive jaundice, OB) underwent common bile duct ligation; and group 3 (obstructive jaundice with antithrombin-III, OBAT-III) underwent common bile duct ligation and simultaneously were treated with antithrombin-III. Liver tissues were harvested on the fifth postoperative day. RESULTS: Hepatocyte apoptosis was significantly increased in bile duct ligated group when compared with the sham operation group. The administration of antithrombin-III effectively attenuates such phenomenon in obstructive jaundice with antithrombin-III group. CONCLUSION: Bile duct ligation significantly increased hepatocyte apoptosis and the administration of antithrombin-III effectively attenuates such phenomenon.

Anti-"Mi(a)" immunization is associated with HLA-DRB1*0901.

BACKGROUND: Anti-"Mi(a)" is one of the most important irregular red blood cell antibodies found in Taiwan. The aim of this study was to investigate whether specific HLA-DRB1 alleles are associated with anti-"Mi(a)" production. STUDY DESIGN AND METHODS: A case-control retrospective study was performed on 68 patients showing presence of anti-"Mi(a)" and 219 unrelated control subjects from the Mackay Memorial Hospital. HLA-DRB1 genotyping was carried out using sequence-based typing method. Fisher's exact test using 2 x 2 contingency tables was used to analyze significance of the association between DRB1 polymorphisms and presence of anti-"Mi(a)" in patients. RESULTS: HLA-DRB1*0901 allele frequency in the anti-"Mi(a)" group (30%) was significantly higher than in the control group (16%) with an odds ratio of 2.27 (95% confidence interval, 1.44-3.55; p = 0.0005; p(c) = 0.016). CONCLUSION: HLA-DRB1*0901 is significantly more prevalent in the anti-"Mi(a)" patients group than in the control group. It is suggested that cells from DR9 individuals might present processed "Mi(a)" antigen-allospecific peptides more effectively than cells from individuals carrying other DR phenotypes. Finally, it was predicted that two epitopes, derived from the MiIII glycophorin amino acid sequence, were likely to bind preferentially with the DR9 molecule. Further work will be necessary to determine if these epitopes are responsible for anti-"Mi(a)" alloimmunization.

Primary autoimmune neutropenia in children in Taiwan.

BACKGROUND: Autoimmune neutropenia in children is caused by granulocyte-specific autoantibodies. These antibodies react to the patient's own neutrophils but disappear when the neutropenia spontaneously remits. This study reviewed our experience with autoimmune neutropenia in children and investigated possible associations with HLA-DR and HLA-DQ alleles. STUDY DESIGN AND METHODS: From 1993 to 2006, our laboratory received 155 blood samples from children with neutropenia. Of these samples, 55 had granulocyte-specific autoantibodies on the indirect granulocyte immunofluorescence test. As the children had no other disorders associated with neutropenia, they were diagnosed with primary autoimmune neutropenia. HLA-DRB1 and -DQB1 allele typing was performed in 31 cases, and the results were compared with those of 190 normal healthy unrelated Taiwanese controls. RESULTS: The mean ages of onset and resolution of neutropenia were 9.8 months (median, 9.0 months; range, 4-28 months) and 22.5 months (median, 20.0 months; range, 13-44 months), respectively. The male-to-female ratio was 1.2:1. The mean absolute neutrophil count was 190 per microL (standard deviation, 213/microL). Most patients (74%) had antibodies against HNA-1a. Autoimmune neutropenia in children in Taiwan was significantly associated with HLA-DQB1*0503 (odds ratio, 6.48; p = 0.0002; p(c) = 0.003) allele. CONCLUSION: In Taiwan, autoimmune neutropenia in children is associated with HLA-DQB1*0503. The autoantibody in autoimmune neutropenia is most commonly anti-HNA-1a.

Z-LLY-FMK attenuates intestinal apoptosis after bile duct ligation in rats.

Apoptosis is an important process in a wide variety of different biological systems. In addition to caspases, recently, calpains, another family of proteases, have been found to be involved in apoptosis of many cell systems. This study is designed with the aims to evaluate the possible effect of Z-LLY-FMK (a calpain inhibitor) on intestine apoptosis after bile duct ligation in rats. Male Sprague-Dawley rats weighing 250-300 g were randomized to five groups (n = 6 in each group). Group 1 (CONTROL: C) underwent Sham operation and were simultaneously treated with the same amount of normal saline. Group 2 (CONTROL with DMSO: CDMSO) underwent Sham operation and were simultaneously treated with the same amount of dimethylsulfoxide (DMSO). Group 3 (Obstructive jaundice: OB) underwent common bile duct ligation without any other manipulation. Group 4 (Obstructive jaundice with Z-LLY-FMK: OBZLLY) underwent common bile duct ligation and were simultaneously treated with Z-LLY-FMK (dissolved in DMSO). Group 5 (Obstructive jaundice with ZFA-FMK: OBZFA) underwent common bile duct ligation and were simultaneously treated with ZFA-FMK (dissolved in DMSO). After 3 days, intestine tissue was harvested for apoptosis measurements. There was no significant difference between Sham operation group (C) and Sham operation with DMSO group (CDMSO) either in jejunum (P = 0.924) or in ileum (P = 0.996). When compared to Sham operation group (C), increased intestine apoptosis occurred in either jejunum (P < 0.001) or in ileum (P < 0.001) after common bile duct ligation (OB). After administration of Z-LLY-FMK (OBZLLY), the increased intestine apoptosis after common bile duct ligation (OB) was significantly diminished either in jejunum or in ileum (P < 0.001 and P < 0.001). Moreover, administration of ZFA (OBZFA) failed to show the same phenomenon in either jejunum (P = 0.993) or ileum (P = 0.485). There was a significant difference in intestine apoptosis in either jejunum (P < 0.001) or in ileum (P < 0.001) between OBZLLY group and OBZFA group. Significantly increased intestine apoptosis occurred after common bile duct ligation. The administration of Z-LLY-FMK could effectively diminish the intestine apoptosis after common bile duct ligation, whereas the administration of ZFA-FMK failed to show the same effect.

The hsp65 gene patterns of less common Mycobacterium and Nocardia spp. by polymerase chain reaction-restriction fragment length polymorphism analysis with capillary electrophoresis.

To rapidly identify Mycobacterium and Nocardia spp. without costly probes, we had implemented capillary electrophoresis (CE) in polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis to analyze their 65-kDa heat shock protein (hsp65) gene. The PCR-RFLP analysis with CE (PRACE) involved only one restriction enzyme, HaeIII, and a single electrophoretic separation less than 10 min. Full-range (10-200 bp) RFLP patterns of 12 less common Mycobacterium and 7 Nocardia spp. were investigated. A good agreement was observed between the sizes of restriction fragments resolved by CE and the real sizes deduced from sequence analysis. Including hsp65 gene patterns of 12 Mycobacterium spp. published earlier, differentiation was distinct among 24 Mycobacterium and 7 Nocardia spp. Some closely related species exhibiting similar biochemical characteristics could be well discriminated by an extra HaeIII digestion site. Thus, PRACE offers a nonprobe alternative for rapid identification of various cultured Mycobacterium and Nocardia to the species level.

Surface charge-bias impact of amine-contained pseudozwitterionic biointerfaces on the human blood compatibility.

This work discusses the impact of the charge bias and the hydrophilicity on the human blood compatibility of pseudozwitterionic biomaterial gels. Four series of hydrogels were prepared, all containing negatively-charged 3-sulfopropyl methacrylate (SA), and either acrylamide, N-isopropylacrylamide, 2-dimethylaminoethyl methacrylate (DMAEMA) or [2-(methacryloyloxy)ethyl]trimethylammonium (TMA), to form SnAm, SnNm, SnDm or SnTm hydrogels, respectively. An XPS analysis proved that the polymerization was well controlled from the initial monomer ratios. All gels present high surface hydrophilicity, but varying bulk hydration, depending on the nature/content of the comonomer, and on the immersion medium. The most negative interfaces (pure SA, S7A3, S5A5) showed significant fibrinogen adsorption, ascribed to the interactions of the αC domains of the protein with the gels, then correlated to considerable platelet adhesion; but low leukocyte/erythrocyte attachments were measured. Positive gels (excess of DMAEMA or TMA) are not hemocompatible. They mediate protein adsorption and the adhesion of human blood cells, through electrostatic attractive interactions. The neutral interfaces (zeta potential between -10mV and +10mV) are blood-inert only if they present a high surface and bulk hydrophilicity. Overall, this study presents a map of the hemocompatible behavior of hydrogels as a function of their surface charge-bias, essential to the design of blood-contacting devices.

Colonization of severe acute respiratory syndrome-associated coronavirus among health-care workers screened by nasopharyngeal swab.

STUDY OBJECTIVES: To report the efficacy and findings of a large-scale preventive screening program for severe acute respiratory syndrome-associated coronavirus (SARS-CoV) using amplification of the virus from a nasopharyngeal swab (NPS) obtained from the health-care workers (HCWs). DESIGN: A prospective observational study. SETTING: A medical center in Taiwan. PARTICIPANTS: Two hundred thirty HCWs. INTERVENTION: NPS examination for the presence of SARS-CoV by two nested reverse transcription-polymerase chain reaction (RT-PCR) assays. MEASUREMENTS AND RESULTS: During the outbreak of severe acute respiratory syndrome (SARS), NPS polymerase chain reaction screening of HCWs for SARS-CoV was performed. SARS-CoV was examined by two nested RT-PCRs and a quantitative RT-PCR. Serum-specific antibodies were assessed by enzyme immunoassay and indirect immunofluorescence. We monitored 230 HCWs, including 217 first-line HCWs and 13 non-first-line HCWs. One hundred ninety first-line HCWs and 13 non-first-line HCWs had negative results in both nested RT-PCR assays. Two first-line HCWs who were positive on both nested RT-PCR assays had SARS. They had 16,900 +/- 7,920 copies (mean +/- SD) of RNA per milliliter in the NPS and had detectable anti-SARS antibodies. The remaining 25 first-line HCWs were negative for the first nested RT-PCR but positive for the second nested RT-PCR. Their corresponding titers were 338 +/- 227 copies of RNA per milliliter; antibodies developed in none of these 25 HCWs. The expression and function of angiotensin-converting enzyme-2 were not different among these HCWs. This study shows that colonization of SARS-CoV occurred in 25 of 217 well-protected first-line HCWs on a SARS-associated service, but they remained seronegative. CONCLUSION: With the second RT-PCR assay more sensitive than the first RT-PCR assay, we are able to show that approximately 11.5% of well-protected HCWs exposed to SARS patients or specimens may have colonization without seroconversion. Only those with significant clinical symptoms or disease would have active immunity. Thus, regular NPS screening for nested RT-PCR assays in conjunction with a daily recording of body temperature in all first-line HCWs may provide an effective way of early detection.

Enhancing a Blood Transfusion Platform with Clinic Decision Support Component to Better Assure Patient Safety.

Blood transfusion is an important but complex and high-risky clinical procedure. Any error could cause serious injuries to patients. To better assure the procedure safety, we enhancing our home-made blood transfusion platform with new clinic decision support components to assure patient's identity and to inform clinicians of any event in time. So far, our transfusion incidence case has been reduced to 0 from 9 before the system implemented.

Zwitterionic fibrous polypropylene assembled with amphiphatic carboxybetaine copolymers for hemocompatible blood filtration.

UNLABELLED: The present study serves three main functions. First, it presents a novel random copolymer, made of octadecyl acrylate hydrophobic blocks and 2-(dimethylamino)ethyl methacrylate hydrophilic groups, and it zwitterionic form. Second, random copolymer and zwitterionic random copolymer, OmDn and Z-OmDn, are used to modify polypropylene membranes by evaporation coating. Our investigations unveil that this method leads to sufficiently stable self-assembling provided a minimum number of hydrophobic repeat units of 77, which also corresponds to a hydrophobic degree of 74%. Third, antifouling and hemocompatible properties of membranes are thoroughly investigated using all types of blood cells separately, as well as challenging membranes against whole blood in static and dynamic conditions. Membranes modified with zwitterionic copolymer containing 26% of zwitterionic groups are shown to be highly antifouling and hemocompatible, for a coating density as low as 0.2mg/cm(2). Their application in a specially designed blood filtration module enabled to almost totally inhibit blood cells interactions with membrane material, as well as to importantly reduce platelet activation in the permeate (2.5-fold reduction). STATEMENT OF SIGNIFICANCE: The design of new zwitterionic copolymer material is proposed and demonstrated in this study. It was showed that hydrophobicoctadecyl acrylate segments can be introduced in the zwitterioniccarboxybetaine polymer chain with a well-controlled random sequence. Stable, efficient, and effective surface zwitterionization of hydrophobic polypropylene are obtained via grafting onto approach by evaporation-induced self-assembling coating. In the perspective of potential application, hemocompatible blood filtration was demonstrated with the excellent results of non-activated platelets obtained. DESIGN: New zwitterionicmaterial, amphiphatic carboxybetaine copolymers. DEVELOPMENT: Evaporation-induced self-assembling grafting. APPLICATION: Hemocompatible blood filtration.

Effect of ZVAD-fmk on hepatocyte apoptosis after bile duct ligation in rat.

AIM: Retention and accumulation of toxic hydrophobic bile salts within hepatocyte may cause hepatocyte toxicity by inducing apoptosis. Apoptosis is a pathway of cell death orchestrated by a family of proteases called caspases. Z-Val-Ala-Asp (OMe)-fluoromethyl ketone (ZVAD-fmk) is a cell-permeable irreversible inhibitor of caspase. The purpose of this study was to evaluate the possible effect of ZVAD-fmk on hepatocyte apoptosis after bile duct ligation in the rat. METHODS: Male Sprague-Dawley rats, weighing 250-300 g, were randomized to five groups of five rats each. Group 1 underwent common bile duct ligation and simultaneous treatment with ZVAD-fmk (dissolved in dimethylsulfoxide (DMSO)). Group 2 underwent common bile duct ligation and simultaneous treatment with Z-Phe-Ala-fluoromethyl ketone ( ZFA-fmk, dissolved in DMSO). Group 3 underwent sham operation and simultaneous treatment with the same amount of DMSO. Group 4 underwent sham operation and simultaneous treatment with the same amount of normal saline. Group 5 underwent common bile duct ligation without other manipulation. After three days, liver tissue was harv-ested for histopathologic analysis and measurements of apoptosis. RESULTS: When compared with sham operation, common bile duct ligation significantly increased hepatocyte apoptosis (P = 0.008) and ductular proliferation (P = 0.007). ZVAD-fmk significantly diminished the increased hepatocyte apoptosis and ductular proliferation after common bile duct ligation (P = 0.008 and P = 0.007, respectively). ZFA did not show the same effects. CONCLUSION: Hepatocyte apoptosis and ductular proliferation significantly increased after common bile duct ligation. ZVAD-fmk effectively diminished the increased hepatocyte apoptosis and ductular proliferation after common bile duct ligation, whereas ZFA-fmk did not.

Evaluation of the VITEK 2 cards for identification and antimicrobial susceptibility testing of non-glucose-fermenting Gram-negative bacilli.

We evaluated VITEK 2 cards (NGNC and AST-GN10) for the accuracy of identification (ID) and antimicrobial susceptibility testing (AST) of non-glucose-fermenting Gram-negative bacilli (NGF-GNB). In a total of 201 strains, 190 strains (94.5%) were correctly identified, seven strains (3.5%)showed low discrimination, four strains (2.0%) had discrepancies, and no strain remained unidentified.Reference AST of amikacin, aztreonam, cefepime, cefotaxime, ceftazidime, ciprofloxacin, imipenem,levofloxacin, piperacillin-tazobactam, and trimethoprim-sulfamethoxazole was performed by the agar dilution method. Approximately 82.5% of ID and 72.9% of AST were completed within 7 and 14 h,respectively. For NGF-GNB, other than Pseudomonas aeruginosa, Acinetobacter spp., Stenotrophomonas maltophilia, and the Burkholderia cepacia group, essential agreements (EAs) were 93.6-100.0%. Severe disagreements (resistant by the reference method to susceptible by AST-GN10) were observed for amikacin(0.9%), cefepime (1.8%), cefotaxime (1.8%), imipenem (0.9%), and piperacillin-tazobactam (0.9%).One major disagreement (susceptible to resistant) was observed for ceftazidime (0.1%). For P. aeruginosa,EAs were 85.7-100%, with severe disagreements observed for cefepime (4.8%) and piperacillin-tazobactam(4.8%). For Acinetobacter spp., EAs were 86.4-100% without disagreements. The VITEK 2 cards appear to be promising for rapid ID and reliable AST for most species of NGF-GNB.

A method for lactate and pyruvate determination in filter-paper dried blood spots.

Lactic acidemia is commonly associated with severe diseases in pediatric patients. Quantitation of blood lactate and pyruvate is important for the diagnosis and clinical management. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using dried blood spots (DBS) was developed and could be used for simultaneous quantification of blood lactate and pyruvate. The applicability of the developed method was tested and confirmed by the regression analysis between LC-MS/MS method and enzymatic assay. Lactate and pyruvate were extracted from DBS obtained from 580 full-term, 120 pre-term infants (gestations ranging from 24 to 36 weeks), and 65 patients with suspected lactic acidemia, with methanolic internal standard (IS) solutions of sodium L-lactate-(13)C(3) and pyruvate-(13)C(3). An API-2000 LC-MS/MS system with multiple reaction monitoring (MRM) mode was applied. The within-run and between-run precisions (CV%) were determined and the results were 1.9% and 3.9% for lactate (n=20) and 5.7% and 7.3% for pyruvate (n=20). The linearity of lactate (r=0.9986) and pyruvate (r=0.9973) based on the IS was excellent. The parameter r squared (r(2)) of linear regression between LC-MS/MS method and enzymatic assay was 0.9405 for lactate and 0.9447 for pyruvate, respectively, and the agreement between these methods was consistent and acceptable. The stability of lactate and pyruvate on DBS was also confirmed. The LC-MS/MS method we developed is a specific, sensitive, and reproducible method for measuring blood lactate and pyruvate concentrations. The use of DBS in this method makes it particularly attractive for pediatric patients.

Z-LLY-FMK can attenuate hepatocyte apoptosis after bile duct ligation in rat.

BACKGROUND: Cholestasis leading to retention and accumulation of toxic hydrophobic bile salts within hepatocytes may cause hepatocyte toxicity by inducing apoptosis. Calpains have been found to be involved in apoptosis of many cell systems. This study is designed with the aim of evaluating the possible effect of Z-LLY-FMK (a calpain inhibitor) on hepatocyte apoptosis after bile duct ligation in rat. MATERIALS AND METHODS: Male Sprague-Dawley rats were randomized to five groups. Group 1 (C) underwent sham operation. Group 2 (CDMSO) underwent Sham operation and simultaneous treatment with dimethylsulfoxide (DMSO). Group 3 (OB) underwent common bile duct ligation. Group 4 (OBZLLY) underwent common bile duct ligation and simultaneous treatment with Z-LLY-FMK. Group 5 (OBZFA) underwent common bile duct ligation and simultaneous treatment with ZFA-FMK. After 3 days, liver tissue was harvested for histopathologic analysis and apoptosis measurements. RESULTS: When compared with sham operation groups, increased hepatocyte apoptosis (P < 0.001) and ductular proliferation (P < 0.001) occurred after common bile duct ligation. Following administration of Z-LLY-FMK, the increased hepatocyte apoptosis and ductular proliferation after common bile duct ligation were significantly diminished (P < 0.001 and P < 0.001). Moreover, administration of ZFA failed to show the same phenomenon (P = 0.9 and 0.987). CONCLUSION: Significantly increased hepatocyte apoptosis and ductular proliferation occurred after common bile duct ligation. The administration of Z-LLY-FMK could effectively diminish the hepatocyte apoptosis and ductular proliferation after common bile duct ligation, whereas the administration of ZFA-FMK failed to show the same effect.