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Ikuo Igarashi - One of the best experts on this subject based on the ideXlab platform.

  • Dose-response curves of CH against Babesia and Theileria parasites in vitro.
    2019
    Co-Authors: Gaber El-saber Batiha, Amany Magdy Beshbishy, Dickson Stuart Tayebwa, Oluyomi Stephen Adeyemi, Hazem Shaheen, Naoaki Yokoyama, Ikuo Igarashi
    Abstract:

    The curves show the Relative Fluorescence Units of B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi treated with increasing concentrations of CH. The results were determined via Fluorescence assay after 96 h of incubation in three separate trials. The values obtained from three separate trials were used to determine the IC50 values using the non-linear regression (curve fit analysis) in GraphPad Prism software (GraphPad Software Inc., USA).

  • Performance and consistency of a Fluorescence-based high-throughput screening assay for use in Babesia drug screening in mice
    Nature Publishing Group, 2017
    Co-Authors: Mohamed Abdo Rizk, Naoaki Yokoyama, Shimaa Abd El-salam El-sayed, Mahmoud Aboulaila, Rasha Eltaysh, Ikuo Igarashi
    Abstract:

    Abstract In this study, we evaluated the validity of a Fluorescence-based assay using SYBR Green I (SG I) stain for screening antibabesial compounds against B. microti in mice. Two different hematocrits (HCTs; 2.5% and 5%) were used. Correlating Relative Fluorescence Units (RFUs) with parasitemia showed significant linear relationships with R2 values of 0.97 and 0.99 at HCTs of 2.5% and 5%, respectively. Meanwhile, the Z′ factors in a high-throughput screening (HTS) assay were within the permissible limit (≥0.5) at 2.5% HCT and lower than this value at 5% HCT. Taken together, the highest signal-to-noise (S/N) ratios were obtained at 2.5% HCT; therefore, we concluded that 2.5% was the best HCT for applying Fluorescence assay in antibabesial drug screening in mice. Additionally, positive control mice and those treated with diminazene aceturate, pyronaridine tetraphosphate, and an allicin/diminazene aceturate combination showed peak parasitemia and Fluorescence values on the same day post-inoculation. Moreover, using different concentrations of SG I revealed that the optimal concentration was 2x. In summary, considering that all experiments were applied under optimal laboratory conditions, Fluorescence assay at 2.5% HCT using 2x SG I for B. microti parasite offers a novel approach for drug screening in mice

  • Evaluation of a Fluorescence-Based Method for Antibabesial Drug Screening
    Antimicrobial agents and chemotherapy, 2014
    Co-Authors: Azirwan Guswanto, Naoaki Yokoyama, Mohamed Abdo Rizk, Shimaa Abd El-salam El-sayed, Thillaiampalam Sivakumar, Mohamed Youssef, Elsaid El Shirbini Elsaid, Ikuo Igarashi
    Abstract:

    In vitro evaluation of chemotherapeutic agents against Babesia and Theileria parasites has become routine, and the effectiveness of these chemicals is usually determined by comparing the parasitemia dynamics of untreated and treated parasites. Although microscopy is widely used to calculate parasitemia, several disadvantages are associated with this technique. The present study evaluated a Fluorescence-based method using SYBR green I stain (SG I) to screen antibabesial agents in in vitro cultures of Babesia bovis. The linearity between Relative Fluorescence Units (RFU) and parasitemia was found to be well correlated with a 0.9944 goodness-of-fit (r2) value. Subsequently, 50% inhibitory concentration (IC50) values were calculated for 3 antiprotozoan agents, diminazene aceturate, nimbolide, and gedunin, by this method. For diminazene aceturate and nimbolide, the IC50s determined by the Fluorescence-based method (408 nM and 8.13 μM, respectively) and microscopy (400.3 nM and 9.4 μM, respectively) were in agreement. Furthermore, the IC50 of gedunin determined by the Fluorescence-based method (19 μM) was similar to the recently described microscopy-based value (21.7 μM) for B. bovis. Additionally, the Z′ factor (0.80 to 0.90), signal-to-noise (S/N) ratio (44.15 to 87.64), coefficient of variation at the maximum signal (%CVmax) (0.50 to 2.85), and coefficient of variation at the minimum signal (%CVmin) (1.23 to 2.21) calculated for the Fluorescence method using diminazene aceturate were comparable to those previously determined in malaria research for this assay. These findings suggest that the Fluorescence-based method might be useful for antibabesial drug screening and may have potential to be developed into a high-throughput screening (HTS) assay.

Heikki Repo - One of the best experts on this subject based on the ideXlab platform.

  • Constitutive STAT3 Phosphorylation in Circulating CD4(+) T Lymphocytes Associates with Disease Activity and Treatment Response in Recent-Onset Rheumatoid Arthritis
    PloS one, 2015
    Co-Authors: Krista Kuuliala, Hannu Kautiainen, Antti Kuuliala, Riitta Koivuniemi, Suvi Oksanen, Mari Hämäläinen, Eeva Moilanen, Marjatta Leirisalo-repo, Heikki Repo
    Abstract:

    The aim of the present study was to examine constitutive signal transducer and activator of transcription 3 (STAT3) phosphorylation in circulating leukocytes as a candidate biomarker in rheumatoid arthritis (RA). 25 patients with recent-onset, untreated RA provided samples for whole blood flow cytometric determination of intracellular STAT3 phosphorylation, expressed as Relative Fluorescence Units. The occurrence of constitutive STAT3 phosphorylation was evaluated by determining proportion of STAT3-phosphorylated cells among different leukocyte subtypes. Plasma levels of interleukin (IL)-6, IL-17 and IL-21 were measured by immunoassay, radiographs of hands and feet were examined and disease activity score (DAS28) was determined. Biomarkers were restudied and treatment response (according to European League Against Rheumatism) was determined after 12 months of treatment with disease-modifying antirheumatic drugs. At baseline, constitutive phosphorylation of STAT3 occurred in CD4+ T cells of 14 (56%) patients, CD8+ T cells of 13 (52%) patients, in CD19+ B cells of 7 (28%) patients, and in CD14+ monocytes of 12 (48%) patients. STAT3 phosphorylation levels of CD4+ T cells associated with DAS28, and those of all leukocyte subtypes studied associated with erosive disease. The presence of constitutive STAT3 phosphorylation in CD4+ T lymphocytes, pSTAT3 Fluorescence intensity of CD4+ and CD8+ T cells and C-reactive protein (CRP) levels at baseline associated with good treatment response. In conclusion, constitutive STAT3 phosphorylation in circulating CD4+ T cells is common in recent-onset untreated RA and associates with good treatment response in patients characterized by high disease activity and the presence of systemic inflammation.

  • Neutrophil activation in preterm infants who have respiratory distress syndrome.
    PEDIATRICS, 2002
    Co-Authors: Irmeli Nupponen, Eero Pesonen, Sture Andersson, Aila Makela, Riikka Turunen, Hannu Kautiainen, Heikki Repo
    Abstract:

    Objective To study neutrophil activation in circulation as a sign of systemic inflammation in preterm infants with respiratory distress syndrome. Methods The study comprised very low birth weight preterm infants who had respiratory distress syndrome and required intubation and mechanical ventilation (n = 51), 1-day-old preterm infants who had no need for mechanical ventilation (n = 12), term infants (n = 47), and adult volunteers (n = 25). Neutrophil surface expression of CD11b was quantified with flow cytometry. Results In preterm infants with respiratory distress syndrome, neutrophil CD11b expression during the first day of life was higher than in cord blood (mean: 165 Relative Fluorescence Units [RFU] [standard deviation [SD]: 53], n = 29 vs 83 RFU [SD: 21], n = 11; 95% confidence interval [CI] for difference: 59-106) or in preterm infants without mechanical ventilation (106 RFU [SD: 33], n = 12; 95% CI for difference: 17-90). CD11b expression decreased by age of 10 days. CD11b expression was lower in preterm cord than in term cord blood (95% CI for difference: 5-53). However, in preterm infants with respiratory distress syndrome aged 2 to 5 days, it was higher than in term infants of that age. Conclusions The observations demonstrate an early transient postnatal neutrophil activation indicative of systemic inflammation that may contribute to the tissue injury in preterm infants with respiratory distress syndrome.

  • Neutrophil activation in preterm infants who have respiratory distress syndrome.
    Pediatrics, 2002
    Co-Authors: Irmeli Nupponen, Eero Pesonen, Sture Andersson, Aila Makela, Riikka Turunen, Hannu Kautiainen, Heikki Repo
    Abstract:

    To study neutrophil activation in circulation as a sign of systemic inflammation in preterm infants with respiratory distress syndrome. The study comprised very low birth weight preterm infants who had respiratory distress syndrome and required intubation and mechanical ventilation (n = 51), 1-day-old preterm infants who had no need for mechanical ventilation (n = 12), term infants (n = 47), and adult volunteers (n = 25). Neutrophil surface expression of CD11b was quantified with flow cytometry. In preterm infants with respiratory distress syndrome, neutrophil CD11b expression during the first day of life was higher than in cord blood (mean: 165 Relative Fluorescence Units [RFU] [standard deviation [SD]: 53], n = 29 vs 83 RFU [SD: 21], n = 11; 95% confidence interval [CI] for difference: 59-106) or in preterm infants without mechanical ventilation (106 RFU [SD: 33], n = 12; 95% CI for difference: 17-90). CD11b expression decreased by age of 10 days. CD11b expression was lower in preterm cord than in term cord blood (95% CI for difference: 5-53). However, in preterm infants with respiratory distress syndrome aged 2 to 5 days, it was higher than in term infants of that age. The observations demonstrate an early transient postnatal neutrophil activation indicative of systemic inflammation that may contribute to the tissue injury in preterm infants with respiratory distress syndrome.

Steven B. Abramson - One of the best experts on this subject based on the ideXlab platform.

  • Nitric oxide stimulates ADP ribosylation of actin in association with the inhibition of actin polymerization in human neutrophils.
    Journal of leukocyte biology, 1995
    Co-Authors: Robert R. Clancy, Joanna Leszczynska, Ashok R. Amin, Levartovsky D, Steven B. Abramson
    Abstract:

    In these studies we provide conclusive evidence that (beta/gamma) actin present in human neutrophils is a substrate for nitric oxide (NO)-dependent ADP ribosylation and that this modification is associated with the inhibition of actin polymerization. A 43-kDa substrate for NO-dependent ADP ribosylation was identified as actin by four methods: (1) comigration with the botulinum C2 toxin substrate by two-dimensional gel electrophoresis (pI 5.2), (2) identity between the peptide map generated by V8 protease digestion of the NO and botulinum C2 substrates, (3) immunoprecipitation with antiactin antibodies, and (4) the ability of NO to ADP ribosylate purified neutrophil G-actin in the presence of plasma membrane cofactors. Because the ADP ribosylation of actin by the botulinum C2 toxin is known to inhibit F-actin polymerization, we examined the effect of NO on actin assembly. Flow cytometry revealed that NO inhibited formyl-methionine-leucine-phenylalanine (fMLP)-dependent (30 s at 37 degrees C) F-actin formation (108 +/- 8 vs. 89 +/- 6 Relative Fluorescence Units, P < .02). These results were confirmed by quantification of F-actin formation by gel scanning (10% sodium dodecyl sulfate gel, Coomassie, and densitometry): pretreatment of polymorphonuclear leukocytes with NO resulted in a reduction of fMLP-induced, cytoskeletal-associated F-actin, which was accompanied by an increase of Triton-soluble G-actin. NO also inhibited F-actin formation, as observed by means of rhodamine phalloidin staining of neutrophils adherent to a fibronectin-coated surface. This effect was accompanied by a dose-dependent inhibition of neutrophil adherence in NO-treated cells. The data indicate that NO inhibits cytoskeletal assembly and adherence in human neutrophils in association with the ADP ribosylation of actin.

  • Intravascular neutrophil activation in systemic lupus erythematosus (SLE): dissociation between increased expression of CD11b/CD18 and diminished expression of L-selectin on neutrophils from patients with active SLE.
    Clinical immunology and immunopathology, 1994
    Co-Authors: Yair Molad, Jill P. Buyon, Donald C. Anderson, Steven B. Abramson, Bruce N. Cronstein
    Abstract:

    Abstract Previous studies have shown that neutrophils in the circulation of patients with active systemic lupus erythematosus (SLE) are activated as judged by their increased surface expression of the β2-integrin CD11b/CD18. Since activation of neutrophils leads to altered expression of another adhesion molecule, L-selectin (LS), we examined neutrophils from patients with SLE for changes in the expression of CD11b/CD18 and LS by cytofluorographic analysis of immunofluorescent-labeled cells. Overall there was no difference between surface expression of CD11b/CD18 on neutrophils from SLE patients or controls [mean Fluorescence 225 ± 26 vs 225 ± 13 Relative Fluorescence Units (RFU), respectively]. However, as previously reported, neutrophils from patients with more active disease (activity score ⩾3, UCH Middlesex activity score) expressed greater CD11b/CD18 than neutrophiIs from controls (319 ± 40 RFU, P

Mark R. Bowlby - One of the best experts on this subject based on the ideXlab platform.

  • a novel high throughput screening assay for hcn channel blocker using membrane potential sensitive dye and flipr
    Journal of Biomolecular Screening, 2009
    Co-Authors: Dmitry V Vasilyev, Qin J Shan, Stanley P Nawoschik, Edward J Kaftan, Scott Christian Mayer, John Dunlop, Veronica Soloveva, Mark R. Bowlby
    Abstract:

    Hyperpolarization-activated cation nonselective (HCN) channels represent an interesting group of targets for drug development. In this study, the authors report the development of a novel membrane potential–sensitive dye (MPSD) assay for HCN channel modulators that has been miniaturized into 384-well fluorescent imaging plate reader (FLIPR) high-throughput screening (HTS) format. When optimized (by cell plating density, plate type, cell recovery from cryopreservation), the wellto-well signal variability was low, with a Z′ = 0.73 and coefficient of variation = 6.4%, whereas the MPSD Fluorescence signal amplitude was –23,700 ± 1500 FLIPR 3 Relative Fluorescence Units (a linear relationship was found between HCN1 MPSD Fluorescence signal and the cell plating density) and was completely blocked by 30 µM ZD7288. The assay tolerated up to 1% DMSO, inclusion of which did not significantly change the signal kinetics or amplitude. A single-concentration screening of an ion channel–focused library composed of 4855 compounds resulted in 89 HCN1 blocker hits, 51 of which were subsequently analyzed with an 8-point concentration-response analysis on the IonWorks HT electrophysiology platform. The correlation between MPSD and the electrophysiology assay was moderate, as shown by the linear regression analysis (r 2 = 0.56) between the respective IC 50 s obtained using these 2 assays. The reported HTS-compatible HCN channel blocker assay can serve as a tool in drug discovery in the pursuit of HCN channel isoform-selective small molecules that could be used in the development of clinically relevant compounds. (Journal of Biomolecular Screening 2009:1119-1128)

Andrew D. Berti - One of the best experts on this subject based on the ideXlab platform.

  • Reduced Production of Bacterial Membrane Vesicles Predicts Mortality in ST45/USA600 Methicillin-Resistant Staphylococcus aureus Bacteremia.
    Antibiotics (Basel Switzerland), 2019
    Co-Authors: Somrita Dey, George Sakoulas, Smitha Gudipati, Christopher Giuliano, Marcus J. Zervos, Jonathan M. Monk, Richard Szubin, Sarah C. J. Jorgensen, Andrew D. Berti
    Abstract:

    Immune biomarkers can stratify mortality risk in staphylococcal bacteremia. Microbial biomarkers may provide more consistent signals during early infection. We demonstrate that in ST45/USA600 bacteremia, bacterial membrane vesicle production in vitro predicts clinical mortality (773 vs. 116 RFU, survivors vs. decedents, p < 0.0001). Using a threshold of 301 Relative Fluorescence Units (RFU), the sensitivity and specificity of the membrane vesicles to predict mortality are 78% and 90%, respectively. This platform is facile, scalable and can be integrated into clinical microbiology lab workflows.