Nationwide surveillance of the antimicrobial susceptibility of Neisseria gonorrhoeae from male urethritis in Japan

Neisseria gonorrhoeae is one of the most important pathogens causing sexually transmitted infection, and strains that are resistant to several antimicrobials are increasing. To investigate the trends of antimicrobial susceptibility among N. gonorrhoeae strains isolated from male patients with urethritis, a Japanese surveillance committee conducted the first nationwide surveillance. The urethral discharge was collected from male patients with urethritis at 51 medical facilities from April 2009 to October 2010. Of the 156 specimens, 83 N. gonorrhoeae strains were tested for susceptibility to 18 antimicrobial agents. The prevalence of β-lactamase-producing strains and chromosomally mediated resistant strains were 7.2 % and 16.5 %, respectively. None of the strains was resistant to ceftriaxone, but the minimum inhibitory concentration (MIC) of ceftriaxone for 7 strains (8.4 %) was 0.125 μg/ml. One strain was resistant to cefixime (MIC 0.5 μg/ml). The MICs of fluoroquinolones, such as ciprofloxacin, levofloxacin, and tosufloxacin, showed a bimodal distribution. The MIC of sitafloxacin was lower than those of the three fluoroquinolones listed here, and it was found that the antimicrobial activity of sitafloxacin was stronger than that of the fluoroquinolones. The MIC of azithromycin in 2 strains was 2 μg/ml, but no high-level resistance to macrolides was detected.     

Hypericin- and mTHPC-mediated photodynamic therapy for the treatment of cariogenic bacteria

ObjectiveDental caries is one of the most common diseases in Western countries. Its pathoetiology is multifactorial, however, bacteria including Streptococcus mutans and the closely related Streptococcus sobrinus are regarded as key factors involved in this process. The fact that therapeutic approaches to eradicate these microorganisms are still limited prompted us to investigate the treatment potential of photodynamic therapy with the photoactive compounds hypericin (HYP) and meso-tetra(hydroxyphenyl)chlorin (mTHPC) in vitro.Material and methodsS. mutans and S. sobrinus were cultivated under standard conditions and incubated with HYP (Invitrogen, Basel, Switzerland), the liposomal mTHPC derivative Foslipos (FOS, Biolitec, Jena, Germany), or a mixture of both at concentrations ranging between 0.625 and 10 μg/ml for various time points. Following a thorough washing step, bacteria were irradiated with a dental polymerization instrument (400–505 nm). All samples were subjected to serial dilutions and spiral plating on blood agar plates. Viable colony counts were determined after 48 h in culture. Photosensitizer fluorescence of bacteria was visualized by confocal microscopic techniques.ResultsOne hundred percent of S. sobrinus could be killed by a 15 min incubation with as little as 2.5 μg/ml HYP, 5 μg/ml FOS or a mixture of 1.25 μg/ml of each photosensitizer followed by light activation of 120 s. In contrast to S. sobrinus, S. mutans displayed a significant dark toxicity for FOS (10–1.25 μg/ml) and no relevant PDT effects using HYP (10–0.625 μg/ml) under these conditions. HYP-mediated PDT effects (10 μg/ml) could be enhanced to more than 99.9% by prolonging photosensitizer incubation to 30 min and fractional illumination (2×120 s). Complete eradication of S. mutans was achieved by incubation for 15 min with a mixture of 0.625 μg/ml each of FOS and HYP and illumination for 120 s.ConclusionFor both S. mutans and S. sobrinus, short PDT protocols with FOS and/or HYP could be established that completely eradicated these cariogenic bacteria in suspension. Our study, however, indicated that careful optimization of PDT conditions may be necessary for successful treatment of even closely related bacterial species. In multispecies microbial populations, the application of photosensitizer combinations for PDT may be useful.     

Evaluation of anti-diabetic and anti-tumoral activities of bioactive compounds from Phoenix dactylifera L’s leaf: In vitro and in vivo approach

Among various chronic disorders, cancer and diabetes mellitus are the most common disorders. This study was designed to evaluate the effectiveness of hydroalcoholic extract of Phoenix dactylifera L. leaves (HEPdL) in animal models of type II diabetes in vitro/in vivo and in a human melanoma-derived cell line (IGR-39). A liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis was also performed to determine the amount of phenolic and flavonoid compounds in this plant. The physicochemical results by LC–MS/MS analysis of HEPdL showed the presence of 10 phenolic compounds. The in vitro study showed that the extract exhibited a more specific and potent inhibitor of α-glucosidase than α-amylase with an IC₅₀ value of 20 ± 1 μg/mL and 30 ± 0.8 μg/mL, respectively. More importantly, the in vivo study of the postprandial hyperglycemia activity with (20 mg/kg) of HEPdL showed a decrease in plasma glucose levels after 60 min in resemblance to the glucor (acarbose) (50 mg/kg) effect. The oral administration of HEPdL (20 mg/kg) in alloxan-induced diabetic mices for 28 days showed a more significant anti-diabetic activity than that of the drug (50 mg/kg). Moreover, cytotoxicity effects of HEPdL in IGR-39 cancer cell lines were tested by MTT assay. This extract was effective in inhibiting cancer cells growth (IGR-39) at dose 35 and 75 μg/mL. These results confirm ethnopharmacological significance of the plant and could be taken further for the development of an effective pharmaceutical drug against diabetes and cancer.     

Bactericidal activity of garenoxacin against in vitro biofilm formed by nontypeable Haemophilus influenzae

Using β-lactamase-negative ampicillin (ABPC)-susceptible (BLNAS) and β-lactamase-negative ABPC-resistant (BLNAR) nontypeable Haemophilus influenzae (NTHi) strains isolated from otological patients, colony biofilm was prepared on membrane filter substrates. Bactericidal activities of garenoxacin (GRNX), levofloxacin (LVFX), cefditoren (CDTR), and clavulanic acid/amoxicillin (CVA/AMPC) were examined by counting viable cells after drug exposure to biofilm cells for 6 and 24 h and by observation under a scanning electron microscope (SEM). After exposure of biofilm to the 100-fold MIC of GRNX or LVFX for 24 h, GRNX and LVFX showed potent bactericidal activity (?log₁₀ CFU/ml, ≥5.1). In this case, the drug-exposure AUC, exposure concentration × 24 μg h/ml, was 64–128 % for GRNX and 121 % for LVFX of free AUC at the clinical dosage in humans, respectively. CDTR and CVA/AMPC at 100-fold MIC exhibited little bactericidal activity against biofilm cells. Under an SEM, after exposure of BLNAS and BLNAR biofilms to GRNX or LVFX, most of the biofilm matrices were transformed. Quinolones such as GRNX show potent bactericidal activity against biofilm-forming NTHi at the usual clinical dosage.     

Anti-proliferation effects,efficacy of cyasterone in vitro and in vivo and its mechanism

Cyasterone was demonstrated potential inhibition effect in mouse skin carcinoma cells in published report. However, the molecular mechanisms of the cyasterone on cells remain unknown. Herein, we investigated the effects of cyasterone-induced apoptosis in A549 and MGC823 cells in vitro. MTT assay showed that cyasterone caused a significantly decreasing of the proliferation of A549 and MGC823 cells in a time-and dose-dependent manner with IC₅₀ values of 38.50 ± 3.73 μg/mL on A549 cells and 32.96 ± 1.24 μg/mL on MGC823 cells at 48 h, respectively. Hoechst staining and TUNEL staining results indicated the quintessential apoptosis features in immunofluorescence image. Apoptosis and cell cycle were determined by flow cytometry. Cyasterone treatment triggered inhibition of epidermal growth factor receptor- phosphatidylinositol 3 kinase/protein kinase B (EGFR-AKT) signaling pathways and activation of P38 pathways. Furthermore, cyasterone inhibited MGC823 cells xenografted tumor growth in vivo with few changes in body weights. In conclusion, our findings provide the evidence that cyasterone inhibits growth of A549 and MGC823 cells, via regulating EGFR signaling pathway. Our results indicated that cyasterone, a natural EGFR inhibitor, maybe a promising anti-cancer agent.     

Automated cerebrospinal fluid cell count — New reference ranges and evaluation of its clinical use in central nervous system infections

ObjectivesThe purposes of this study were to establish new reference ranges for leukocytes in the CSF and to examine if the separation of mononuclear cells into lymphocytes and monocytes could be used to differentiate between various CNS infections that present with a similar picture in manual CSF cell counts.Design and methodsThe automated cell counter Siemens ADVIA 2120i was used. For the reference range section, we analyzed CSF from 80 neurologically healthy volunteers. For the differential diagnosis section we analyzed cell counts and hospital records from 175 patients with CSF mononuclear pleocytosis.ResultsCorrelation was good between automated and manual leukocyte counts for samples with erythrocyte counts < 250 cells/μL. For the neurologically healthy volunteers studied in the reference range section, the 95th percentile was 3.0 cells/μL for lymphocytes, 1.0 cell/μL for monocytes and 1.0 cell/μL for granulocytes. In the differential diagnosis section, comparisons were done between the groups Lyme neuroborreliosis and viral CNS infection. There were no significant differences between these two groups regarding cell counts; neither for lymphocytes, median 58 cells/μL vs. 72 cells/μL (P = n.s.); nor for monocytes, median 13 cells/μL vs. 16 cells/μL (P = n.s.); nor for granulocytes, median 1 cell/μL vs. 2 cells/μL (P = n.s.)ConclusionsWe suggest new CSF cell count reference ranges of < 4 cells/μL for lymphocytes, < 3 cells/μL for monocytes and < 3 cells/μL for granulocytes. The separation of mononuclear cells into lymphocytes and monocytes did not facilitate the discrimination between Lyme neuroborreliosis and viral CNS infection.     

Superiority of postmortem liver fentanyl concentrations over peripheral blood influenced by postmortem interval for determination of fentanyl toxicity

ObjectiveThe current study was undertaken to determine the relationship between postmortem (PM) peripheral blood (PB) and liver fentanyl concentrations and the role of measuring liver fentanyl concentrations in cause of death investigations in medical examiner cases in which fentanyl was identified.Design and methodsFB and liver tissue were routinely collected at autopsy from 4 Minnesota medical examiners' offices in 2010–2011. Samples were analyzed by gas chromatography–mass spectrometry (GC–MS).ResultsPB fentanyl ranged from < 2–15 μg/L in non-drug related deaths (n = 5), < 2–22 μg/L from mixed drug toxicity (n = 26) and 3.7–56 μg/L from fentanyl toxicity (n = 33). Liver fentanyl ranged from 11 to 104 μg/kg, 6 to 235 μg/kg, and 18 to 365 μg/kg, respectively. PB and liver fentanyl showed a modest correlation (r = 0.67). PM interval to the liver/blood ratio showed a decreasing ratio over increasing PM interval in cases from fentanyl and mixed drug toxicity. Liver fentanyl concentrations best define therapeutic use at < 23 μg/kg and fatal toxicity at > 56 μg/kg, without substantial overlap as found in blood fentanyl concentrations.ConclusionDiscriminatory liver fentanyl concentrations suggestive of therapeutic or toxic drug levels may better assist cause of death determination in cases of suspected fentanyl toxicity than postmortem PB concentrations. Peripheral blood fentanyl concentrations appear to undergo postmortem redistribution, associated with an increasing PM interval.     

Mild mental retardation and low levels of urinary heparan sulfate in a patient with the attenuated phenotype of mucopolysaccharidosis type IIIA

ObjectivesWe report the case of a 28-year-old female subject affected by the attenuated phenotype of mucopolysaccharidosis type IIIA characterized by moderate slowly evolving mental retardation in which the urinary content of heparan sulfate was demonstrated as being substantially low compared to that found in patients with the severe phenotype.Design and methodsThe specific evaluation of macromolecular heparan sulfate by electrophoresis and the determination of related glucosamine in the urine were performed.ResultsIn our patient, the urinary macromolecular heparan sulfate content (4.2 μg/mg creatinine) was ~ 7.5-times higher than in healthy subjects (0.56 μg/mg creatinine ± 0.9 SD) while it was ~ 28-times lower compared to the severe mucopolysaccharidosis IIIA group (117 μg/mg creatinine ± 44.8 SD). Furthermore, the urinary glucosamine (86.4 μg/mg creatinine) was ~ 2.4-times greater than in healthy subjects (36.0 μg/mg creatinine ± 18.2 SD) but ~ 2.4-times lower than in severe subjects (208.1 μg/mg creatinine ± 55.0 SD).ConclusionsThe above data could reflect the reduced heparan sulfate storage in her tissues and organs, and in particular in the brain, consequently explaining her moderate mental retardation. Furthermore, the clinical presentation of patients with an attenuated form of MPS III confirms the need for a specific evaluation of urinary GAGs in all young and adult subjects showing a not well-defined or not particularly severe mental retardation, along with an early MPS diagnosis. Such investigation should also be associated with a more specific characterization of heparan sulfate.     

Particle-enhanced turbidimetric immunoassay for determination of serum neutrophil gelatinase-associated lipocalin on the Roche Cobas c501 analyzer

ObjectiveNeutrophil gelatinase-associated lipocalin (NGAL) has been reported to be a good marker for tubular damage and acute kidney injury. The aim of this study was to develop a high throughput assay for the quantification of serum NGAL (sNGAL).MethodsImprecision, interference, linearity, recovery, and reference values were evaluated on Cobas c501.ResultsThe assay was linear over the dynamic range of the study (R² = 0.9988). The total assay imprecision was below 5%. The assay recovery was estimated at 98.89%–102.61%. The assay displayed a good linearity over the range from 35 μg/L to 4250 μg/L. A typical high-dose hook effect was observed for the assay at NGAL concentration > 28,800 μg/L. No interference was observed with hemoglobin ≤ 5 g/L, bilirubin ≤ 0.3 g/L, vitamin C ≤ 0.5 g/L, sodium heparin ≤ 5 g/L and intralipid ≤ 1%. The 95th centile for serum NGAL was < 122.57 μg/L from 454 healthy donors. There were no gender-related differences for serum NGAL. There were significant age-related differences between the 21–44 and 45–75 year categories for serum NGAL. The reference value for sNGAL was < 116.52 μg/L in the 21–44 year group and < 126.9 μg/L in the 45–75 year group.ConclusionsThe NGAL assay verified to be a reliable assay with convenient performance characteristics. The assay improves and simplifies the laboratory workload.     

Vitamin E (α-tocopherol) enhances the PDT action of hematoporphyrin derivatives on cervical cancer cells

ObjectiveThe effect of antioxidant vitamin E (VE) on PDT (a mainly reactive oxygen species-driven process) has in the past shown contradicting results. Hence, we studied the effect of different concentrations and different incubation periods of VE on the PDT cytotoxicity of the cervical adenocarcinoma HeLa cell line.Materials and methodsHeLa cells were incubated with 25 μg/ml of hematoporphyrin derivatives (HpD) for 25 min (1st PDT regimen) or 24 h (2nd PDT regimen), then irradiated with visible light (total light dose of 10 J/cm²) either with or without different concentrations of VE (1–1000 μM) which had been incubated with the cells for 1 h or 24 h prior to PDT. After irradiation, viability was measured using MTT assay.ResultsThe results obtained showed that PDT is effective against cervical cancer cells. Incubation of HpD for 24 h leads to improved PDT action. Higher concentrations of VE incubated in HeLa cells for 1 h before the 2nd PDT regimen significantly enhanced cytotoxicity of PDT and the maximal enhancement was at 1000 μM of VE. The cytotoxic effect of VE on HeLa cells after incubation for 24 h before PDT is enhanced by the PDT action.ConclusionIn conclusion 1000 μM of VE can be used 1 h before PDT to enhance its effect on cervical adenocarcinoma, a disease which is steadily increasing in young women. It is well-known that the cervical adenocarcinoma is resistant to anticancer agents and radiotherapy and it was previously considered to be HpD-PDT resistant.     

Nanomedicine-based combination of gambogic acid and retinoic acid chlorochalcone for enhanced anticancer efficacy in osteosarcoma

In this study, gambogic acid (GA) and retinoic acid chlorochalcone (RACC) co-loaded glycol chitosan nanoparticle was successfully developed and studied for its therapeutic efficacy against osteosarcoma cancer cells. The GA/RACC loaded glycol chitosan nanoparticles (RGNP) was nanosized and exhibited a controlled release of drug in either pH 7.4 and pH 5.0. Owing to the strong positive charge on the RGNP surface, efficiency cellular uptake was observed in cancer cells. Moreover, a synergistic combination of GA and RACC were effectively suppressed the tumor growth progression. The half maximal inhibitory concentration (IC50) values in MG63 cells were 0.89 μg/ml and 0.35 μg/ml for GA and RGNP after 24 h. The results clearly suggest the synergist effect of GA and RACC in effectively inhibiting the cancer cell proliferation. The RGNP as expected induced a remarkably higher apoptosis of cancer cells with ∼28%. Overall, combination of GA and RACC encapsulated in a nanocarrier could be an effective strategy to treat osteosarcoma. Future studies will focus on the in vivo evaluation of GA/RACC-loaded polymeric nanoparticles.     

Verification of an immunoturbidimetric assay for heart-type fatty acid-binding protein (H-FABP) on a clinical chemistry platform and establishment of the upper reference limit

BackgroundHeart-type fatty acid-binding protein (H-FABP) is an early biomarker of cardiac injury. Randox Laboratories developed an immunoturbidimetric H-FABP assay for non-proprietary automated clinical chemistry analysers that could be useful in the emergency department. We verified the analytical performances claimed by Randox Laboratories on Roche Cobas 6000 clinical chemistry platform in use in our laboratory, and we defined our own 99th percentile upper reference limit for H-FABP.MethodsFor the verification of method performances, we used pools of spared patient samples from routine and two levels of quality control material, while samples for the reference value study were collected from 545 blood donors. Following CLSI guidelines we verified limit of blank (LOB), limit of detection (LOD), limit of quantitation (LOQ), repeatability and within-laboratory precision, trueness, linearity, and the stability of H-FABP in EDTA over 24 h.Results and discussionThe LOQ (3.19 μg/L) was verified with a CV% of 10.4. The precision was verified for the low (mean 5.88 μg/L, CV = 6.7%), the medium (mean 45.28 μg/L, CV = 3.0%), and the high concentration (mean 88.81 μg/L, CV = 4.0%). The trueness was verified as well as the linearity over the indicated measurement interval of 0.747–120 μg/L. The H-FABP in EDTA samples is stable throughout 24 h both at room temperature and at 4 °C. The H-FABP 99th percentile upper reference limit for all subjects (3.60 μg/L, 95% CI 3.51–3.77) is more appropriate than gender-specific ones that are not statistically different.     

Synthesis and photodynamic activity of unsymmetrical A3B tetraarylporphyrins functionalized with l-glutamate and their Zn(II) and Cu(II) metal complex derivatives

Four novel unsymmetrical A₃B porphyrins 1, 2, 3 and 4 were synthesized following Lindsey procedure. Porphyrins 3 and 4 include one and three l-glutamate groups, respectively, and all porphyrins were metallated with Zn(II) (1a–4a) or Cu(II) (1b–4b). Porphyrins and metalloporphyrins presented values of singlet oxygen quantum yields (ΦD) ranging from 0.21 to 0.67. The tetraaryl derivatives in this study showed phototoxicity in SiHa cells with IC₅₀ values ranging from <0.01 to 6.56 ± 0.11 μM, the metalloporphyrin 4a showed the lowest IC₅₀ value. Comparing the phototoxic activity between all porphyrins, functionalization of porphyrins with glutamate increased 100 times phototoxic activity (1 (IC₅₀ 4.81 ± 0.34 μM) vs. 3 (IC₅₀ 0.04 ± 0.02 μM) and 2 (IC₅₀ 5.19 ± 0.42 μM) vs. 4 (IC₅₀ 0.05 ± 0.01 μM)). This increased activity could be attributed to reduced hydrophobicity and increased ΦΔ, given by functionalization with l-glutamate. Metalloporphyrins 3a (IC₅₀ 0.04 ± 0.01 μM) and 4a (IC₅₀ <0.01 μM) presented the best values ​​of phototoxic activity. Therefore, functionalization and zinc metalation increased the phototoxic activity. SiHa cells treated with porphyrins 3, 4, 3a and 4a at a final concentration of 10 μM, showed increased activity of caspase-3 enzyme compared to the negative control; indicating the induction of apoptosis. Differential gene expression pattern in SiHa cells was determined; treatments with metalloporphyrins 4a and 4b were performed, respectively, comparing the expression with untreated control. Treatments in both cases showed similar gene expression pattern in upregulated genes, since they share about 25 biological pathways and a large number of genes. According to the new photophysical properties related to the structural improvement and phototoxic activity, these molecules may have the potential application as photosensitizers in the photodynamic therapy.     

Activity of omadacycline tested against Streptococcus pneumoniae from a global surveillance program (2014)

The activity of omadacycline and comparators when tested against a subset of Streptococcus pneumoniae from US and European regions of a 2014 global surveillance program (304 isolates) are reported. These MIC results were compared to those obtained when testing S. pneumoniae from 2010 surveillance (1,834 isolates). The omadacycline MIC_50/90 for S. pneumoniae (2014) was 0.06/0.06 μg/mL, similar to 2010 (MIC_50/90, 0.06/0.12 μg/mL). The omadacycline MIC₉₀ (0.06–0.12 μg/mL) was similar for the penicillin-susceptible, -intermediate, -resistant, multidrug-resistance (MDR; ≥3 classes), and ceftriaxone nonsusceptible subgroups. Omadacycline MIC₉₀ values were 0.06–0.12 μg/mL for S. pneumoniae from the US and Europe. There was a high degree of resistance with doxycycline, erythromycin and trimethoprim-sulfamethoxazole in both US and EU. For penicillin-resistant S. pneumoniae, resistance to doxycycline and tetracycline in US/Europe was 64.2/61.0% and 63.8/60.5%, respectively, erythromycin 91.2/75.1, and ceftriaxone 7.3/4.0%. The potent activity of omadacycline against S. pneumoniae indicates that omadacycline merits further study in bacterial pneumonia, especially where MDR may be a concern.     

Antileishmanial,antioxidant, and cytotoxic activities of Quercus infectoria Olivier extract

Currently, there is no effective vaccine available, and chemotherapy is the main approach for treatment of cutaneous leishmaniasis (CL). During recent decades, studies have demonstrated that a number of plant-derived compounds may act as new therapeutic tools against leishmaniasis. This study was evaluated the antileishmanial, antioxidant, and cytotoxic activities of Quercus infectoria Olivier (oak) extract. The total amount of phenolic and flavonoid compounds was measured in oak extract. High performance liquid chromatography (HPLC) analysis was also performed to determine the amount of quercetin and gallic acid in this plant. This extract (0–80 g/mL) was evaluated in vitro against promastigote and intracellular amastigote forms of Leishmania major (MRHO/IR/75/ER) using MTT assay and in a macro-phage model, respectively. Then oak extract was tested on CL in infected male BALB/c mice with L. major in order to evaluate the antileishmanial activity topically. Moreover, cytotoxicity effects of oak in murine macrophage cells were tested by MTT assay. Antioxidative activity of oak was also determined by the 2,2-diphenyl-1,1-picrylhydrazyl (DPPH) scavenging test. The amount of phenolic and flavonoid compounds in the oak extract was 57.50 and 1.86%, respectively. The amount of quercetin and gallic acid in the oak extract was 0.0064 and 0.22%, respectively. The findings revealed that oak significantly (P < 0.05) inhibited the growth rate of promastigote of (IC₅₀ 12.65 μg/mL) and amastigotes (IC₅₀ 10.31 μg/mL) as a dose-dependent response. In the in vivo assay, after 4 weeks of treatment, 91.6, 66.66, and 50% recovery was observed in the infected mice treated with 20, 10, and 5 mg/kg of oak extract, respectively. After treatment of the infected mice with the concentration of 10 and 20 mg/kg of oak, the mean diameter of lesions, parasite load and mean number of parasites was significantly (P < 0.05) reduced. Selectivity index of greater than 10 for oak revealed that oak extract had no cytotoxic effects on macrophage cells. Moreover, DPPH test demonstrated that radical inhibition occurred at greater power with increasing the concentration of oak. To conclude, the present study showed potent antileishmanial and antioxidant activity of oak extract; whereas this plant had no toxic effect on mammalian cells.     

Validating urinary measurement of beta-2-microglobulin with a Roche reagent kit designed for serum measurements

ObjectiveTo validate the use of a Roche serum beta-2-microglobulin (B2MG) kit for urinary B2MG measurements, and to establish reference limits for urinary B2MG/creatinine ratio from healthy individuals.Design and methodsThe Roche B2MG Tina-Quant serum kit was used to measure urinary B2MG immunoturbidimetrically.ResultsUsing human urine as a diluent, the B2MG method was linear from 73–2156 μg/L. The imprecision on a commercially available urine QC was 4.4% at a concentration of 380 μg/L. Limit of quantification at < 20% CV was 40 μg/L. Method comparison with Immulite 2000 (Siemens) yielded slope = 1.180 (95% CI 1.14–1.22), intercept = 11.5 (95% CI ? 3.6–26.6), SEE = 27.6 and r = 0.99 (n = 26) by the Deming regression analysis. The upper reference limit of B2MG/creatinine ratio determined from 195 healthy adults was 29 μg/mmol (97.5th centile).ConclusionsThe serum B2MG Tina Quant reagent kit is acceptable to measure urinary B2MG.     

Bactericidal effects of antimicrobial agents on epithelial cell-associated Pseudomonas aeruginosa

It is not clear whether antipseudomonal agents can kill cell-associated bacteria within a short time. Madin–Darby canine kidney (MDCK) and A549 cells were infected with Pseudomonas aeruginosa ATCC 27853 and PAO1 and the bactericidal activity of ceftazidime, imipenem, meropenem, gentamicin, and ciprofloxacin against the organisms was investigated. In both MDCK and A549 cells, β-lactams could not kill epithelial cell-associated bacteria within 2 h. Gentamicin at concentrations ≤32 μg/ml killed more than 99% of epithelial cell-associated bacteria. Ciprofloxacin at 0.5 μg/ml killed more than 99.9% of MDCK cell-associated bacteria. Ciprofloxacin has the strongest and most rapid bactericidal activity against epithelial cell-associated bacteria, which may be explained by the combination of potent in-vitro bactericidal activity and high penetration ability into epithelial cells.     

Involvement of l-arginine-nitric oxide pathway in anxiolytic-like effects of zinc chloride in rats

PurposeZinc is crucial for normal development of the brain, and Zinc deficiency has been shown to associate with neurological disorders (e.g. anxiety) through interactions with several neurotransmitter systems such as nitric oxide (NO). In this regard, our study aimed to evaluate the possible involvement of l-arginine NO pathway on anxiolytic effects of zinc in adult male rats.MethodsZinc chloride at doses of 2.5 and 10 mg/kg (intraperitoneal or ip) or saline (1 ml/kg, ip) were injected 30 min before the anxiety test. Zinc administrated rats (10 mg/kg) were pre-treated with intra-CA1 microinjection of l-arginine in sub-effective dose of 1 μg/rat (dorsal hippocampus, vehicle: saline1 μl/rat). In addition, zinc chloride and NG-nitro-l-arginine methyl ester (l-NAME) were intraperitoneally co-administrated in sub-effective doses of 2.5 mg/kg and 80 mg/kg, respectively. The percentage of open arm time (OAT%), percentage of open arm entry (OAE%), as measures of anxiety, and total number of arm entries, as measures of locomotor activity, were recorded.ResultsTreatment with zinc (10 mg/kg) markedly produced an increase in OAT% and OAE% in the Elevated plus maze test (EPM). A decrease of OAT% and OAE% was shown in groups which received zinc (10 mg/kg) and l-arginine (1 μg/rat) concomitantly as compared to the control group. Moreover, an increase of OAE% was revealed in the group exposed to Zinc (2.5 mg/kg) and l-NAME (80 mg/kg) co-administration. Although, Two-way ANOVA showed no significant differences of anxiety indices in rats received drug + zinc chloride in compare to the zinc pretreated with saline group.ConclusionAnxiolytic- like effect of zinc reversed by nitric oxide precursor l-arginine. Additionally, the synergistic effects of l-NAME and ZnCl₂ were shown in the EPM. Thus our findings suggest that at least in part the anxiolytic effects of zinc can be mediated through the nitric oxide system.     

Soluble transferrin receptor in urine,a new biomarker for IgA nephropathy and Henoch–Schönlein purpura nephritis

ObjectivesIgA nephropathy (IgAN) and Henoch–Schönlein purpura nephritis (HSPN) might represent different ends of a continuous spectrum of glomerular disease. In both conditions, upregulated soluble transferrin receptor (sTfR) might be excreted in urine, which could be a potential biomarker to monitor disease activity and therapeutic response.MethodsIn this pilot study, 132 Caucasian patients consulting the Nephrology Department at the Ghent University Hospital because of a glomerulopathy and 50 normal controls were included. Urinary sTfR concentrations were determined in concentrated urine using a newly developed latex-enhanced immunonephelometric assay.ResultsMedian urinary sTfR concentration was higher in patients with a primary glomerulopathy than in healthy subjects (p < 0.0001). More importantly, absolute median levels of urinary sTfR were markedly higher in patients with active IgAN or HSPN [10 μg/L, 95% confidence interval (CI): 6–18 μg/L] in comparison with those with other morphological types of glomerulopathy (2 μg/L, 95%CI: 1–4 μg/L) (p < 0.0001). A statistically significant difference in urinary sTfR concentration was observed between patients with active IgAN or HSPN and patients who had achieved partial or complete remission (p < 0.0001). Multiple regression analysis with urinary sTfR as dependent variable revealed that proteinuria was the main predictor of urinary sTfR concentration (r² = 0.52, p < 0.001).ConclusionDetermination of sTfR in urine is a new and sensitive method for a potential biomarker of IgAN and HSPN.     

Inter-method variability in bone alkaline phosphatase measurement: Clinical impact on the management of dialysis patients

BackgroundBone-specific alkaline phosphatase (BAP) is now recommended to assess bone turnover in hemodialysis (HD) patients. However, little is known about potential variability between methods available to measure BAP.MethodsWe measured BAP in 76 HD patients with six different assays (Beckman-Coulter Ostase IRMA, Beckman-Coulter Ostase Access, IDS iSYS Ostase, IDS Ostase enzyme immunoassay, DiaSorin Liaison Ostase and Quidel MicroVue BAP).ResultsWe observed a high correlation between all the assays ranging from 0.9948 (IDS iSYS vs. IDS EIA) to 0.9215 (DiaSorin Liaison vs. Quidel MicroVue). However, using the regression equations, the equivalent concentration of a Beckman-Coulter Access value of 10 μg/L can range from 7.7 to 14.4 μg/L and of 20 μg/L can range from 16.9 to 27.9 μg/L with other assays. According to Beckman-Coulter Access, 13%, 50% and 37% of the patients presented BAP values ≤ 10, between 10 and 20 and ≥ 20 μg/L, respectively. Discrepancies are observed when other assays are used (concordance from 10 to 100%).ConclusionsAnalytical problems leading to inter-method variation should be overcome to improve the usefulness of this marker in clinical practice. According to correlation results, recalibration of BAP assays is necessary but should not be a major issue.