A simple method for quantifying high density antigens in erythrocyte membrane by flow cytometry

RBC flow cytometric analysis is usually used to quantify antigen content. Calibration systems enable antigen content determination by relating mean fluorescence intensity with the number of bound antibody molecules (equivalent to the number of antigen molecules). For that reason, antibodies must be used at saturating concentration, which may lead to agglutination when working with high density antigens. Then, forward scattering, side scattering and fluorescence will be increased, thus obtaining wrong results. In this work, the simple Langmuir adhesion model was applied. Flow cytometry was used to quantify GPA, a transmembrane protein present at high density on RBC. The fluorescence intensity of samples at different anti-GPA sub-saturating concentrations was measured. Sometimes, agglutinates were present and two peaks of fluorescence were observed, the principal one corresponding to isolated cells and the secondary one corresponding to agglutinated cells. In those cases, the principal peak was taken into account for the analysis. The GPA antigen content obtained for nine analyzed samples ranged from 3 to 13 x 10(5) sites per cell, which is similar to those values found in literature. Therefore, the Langmuir adsorption model enables us to determine the antigen content for the anti-GPA/GPA system on RBC membrane. This model could be used to quantify high density antigens in RBC and in other cells.     

Effects of a modified dye-labeled nucleotide spacer arm on incorporation by thermophilic DNA polymerases

The ability of eight commercially available thermophilic DNA polymerases to sequentially incorporate fluorescently labeled nucleotides sequentially was analyzed by a gel based primer extension assay. Cy5-dUTP or a variant nucleotide in which the linker had been lengthened by 14 atoms between the dye and the nucleobase were compared. We found that the Cy5-dUTP with a longer linker resulted in longer primer extension lengths. Furthermore, some of the assayed polymerases are capable of extending the primer to the full or near full length of 30 nucleotides using dye-labeled nucleotides exclusively.     

Flow cytometry and spectral imaging multiphoton microscopy analysis of CD36 expression with quantum dots 605 of untreated and 7-ketocholesterol-treated human monocytic cells

OBJECTIVE: To evaluate CD36 expression with quantum dots 605 (QDs 605) on untreated and 7-ketocholesterol (7KC)-treated monocytic U937 cells by flow cytometry (FCM) and confocal and multiphoton laser scanning microscopy (CLSM). STUDY DESIGN: Cells were analyzed by CLSM, following flow cytometric quantification of CD36 expression and 7KC uptake. Image sequences were obtained by spectral analysis in monophoton and multiphoton CLSM and analyzed by the factor analysis of medical image sequences (FAMIS) algorithm to differentiate emission spectra. In CLSM analysis, cell deposits were screened in ultraviolet excitation modes to optimize the possibilities of QDs 605 and have the benefit of nuclei counterstaining by DAPI. RESULTS: FCM and CLSM reveal the expression of CD36 by means of QDs 605. FCM provides information on 7KC uptake. CLSM provides the localization of 7KC vs. DAPI. As factor curves and images show the red, narrow emission of QDs 605 vs. violet and blue emissions of 7KC and DAPI, respectively, a reliable identification of CD36 is obtained. CONCLUSION: QDs 605 are useful tools to perform antigenic expression in FCM and CLSM. Moreover, CLSM and subsequent spectral analysis provide a more specific characterization of QDs 605 fluorescent emission in the UV excitation mode and a simultaneous identification of 7KC.     

Influence of polyelectrolyte multilayer films on the ICAM-1 expression of endothelial cells

Recently, the use of polyelectrolyte films has been suggested as a new versatile technique of surface modification aimed at tissue engineering. In the present study, we evaluated the expression of intercellular adhesion molecule (ICAM)-1 of endothelial cells (ECs) seeded on two types of polyelectrolyte multilayer films either terminated by poly(D-lysine) (PDL) or poly(allylamine hydrochloride) (PAH). This work showed that chemical stimulations with tumor necrosis factor (TNF)-alpha induced the ICAM-1 expression of ECs differently depending largely on the film architecture employed. Compared with PAH-ending films, the PDL-ending ones upregulated the ICAM-1 expression of the ECs after a prolonged exposition to TNF-alpha, rendering this film type less favorable in tissue engineering. Cytochalasin D (an F-actin disrupting agent) showed the involvement of the cytoskeleton in the upregulation of ICAM-1 for cells deposited on films terminated by PDL. The PAH-ending films did not perturb the ICAM-1 expression of ECs and might thus enhance the seeding of ECs in vascular engineering.     

Influence of mechanical stress on cell viability

The cartilage is a hydrated connective tissue in joints that withstands and distributes mechanical forces. The chondrocytes utilize mechanical signals to regulate their metabolic activity through complex biological and biophysical interactions with the extracellular matrix (ECM). The aim of this work was to study the influence of mechanical stress on cells behavior cultured in 3D biosystems (alginate and alginate supplemented with hyaluronate). After mechanical stimulation, cell viability and cell death process were the main studied parameters. Our results indicated that viability and cell cycle progression were inhibited under mechanical stimulation, as far as the extracellular matrix was not yet synthesized. In contrast, on day 21, the mechanical stimulation had positive effect on these parameters.     

Effects of a Modified Dye-Labeled Nucleotide Spacer Arm on Incorporation by Thermophilic DNA Polymerases

The ability of eight commercially available thermophilic DNA polymerases to sequentially incorporate fluorescently labeled nucleotides sequentially was analyzed by a gel based primer extension assay. Cy5-dUTP or a variant nucleotide in which the linker had been lengthened by 14 atoms between the dye and the nucleobase were compared. We found that the Cy5-dUTP with a longer linker resulted in longer primer extension lengths. Furthermore, some of the assayed polymerases are capable of extending the primer to the full or near full length of 30 nucleotides using dye-labeled nucleotides exclusively.     

Cytomegalovirus Viral Load Kinetics in Patients with HIV/AIDS Admitted to a Medical Intensive Care Unit: A Case for Pre-Emptive Therapy

Background

Cytomegalovirus (CMV) infection is associated with severe diseases in immunosuppressed patients; however, there is a lack of data for pre-emptive therapy in patients with HIV/AIDS.

Method

This was a retrospective study, which enrolled patients diagnosed with HIV/AIDS (CD4<200 cells/μl), who had detectable CMV viral load (VL) during their stay in an adult medical intensive care unit between 2009–2012.

Results

After screening 82 patients’ records, 41 patients met the enrolment criteria. Their median age was 37 (interquartile range [IQR]: 31–46), and median CD4 count was 29 cells/μl (IQR: 5–55). Sixteen patients (39%) had serial measurements of CMV VL before treatment with ganciclovir. Patients whose baseline CMV VL values were between 1,000–3,000 copies/ml had significantly higher values (median of 14,650 copies/ml) on follow-up testing done 4–12 days later. Those with undetectable VLs at baseline testing had detectable VLs (median of 1,590 copies/ml) mostly within 20 days of follow-up testing. Patients who had VLs >1,000 copies/ml at baseline testing had significantly higher mortality compared to those who had <1,000 copies/ml {hazard ratio of 3.46, p = 0.003 [95% confidence interval (CI): 1.55–7.71]}. Analysis of the highest CMV VL per patient showed that patients who had VLs of >5,100 copies/ml and did not receive ganciclovir had 100% mortality compared to 58% mortality in those who received ganciclovir at VLs of >5,100 copies/ml, 50% mortality in those who were not treated and had low VLs of <5,100 copies/ml, and 44% mortality in those who had ganciclovir treatment at VLs of <5,100 copies/ml (p = 0.084, 0.046, 0.037, respectively).

Conclusion

This study showed a significantly increased mortality in patients with HIV/AIDS who had high CMV VLs, and suggests that a threshold value of 1,000 copies/ml may be appropriate for pre-emptive treatment in this group.