Comparing the Role of ROS and RNS in the Thermal Stress Response of Two Cnidarian Models, Exaiptasia diaphana and Galaxea fascicularis.

Coral reefs are threatened by climate change, because it causes increasingly frequent and severe summer heatwaves, resulting in mass coral bleaching and mortality. Coral bleaching is believed to be driven by an excess production of reactive oxygen (ROS) and nitrogen species (RNS), yet their relative roles during thermal stress remain understudied. Here, we measured ROS and RNS net production, as well as activities of key enzymes involved in ROS scavenging (superoxide dismutase and catalase) and RNS synthesis (nitric oxide synthase) and linked these metrics to physiological measurements of cnidarian holobiont health during thermal stress. We did this for both an established cnidarian model, the sea anemone Exaiptasia diaphana, and an emerging scleractinian model, the coral Galaxea fascicularis, both from the Great Barrier Reef (GBR). Increased ROS production was observed during thermal stress in both species, but it was more apparent in G. fascicularis, which also showed higher levels of physiological stress. RNS did not change in thermally stressed G. fascicularis and decreased in E. diaphana. Our findings in combination with variable ROS levels in previous studies on GBR-sourced E. diaphana suggest G. fascicularis is a more suitable model to study the cellular mechanisms of coral bleaching.

Minor impact of patient alloantibodies against human platelet antigen (HPA)-15 in the effectiveness of platelet transfusion: A pilot study.

BACKGROUND: Alloantibodies against human platelet antigen (HPA)-15 are sometimes detected in patients with platelet transfusion refractoriness (PTR); however, little is known about their impact on PTR. STUDY DESIGN AND METHODS: Two patients who possessed HPA-15 alloantibodies (Patient 1, anti-HPA-15b; Patient 2, anti-HPA-15a) and human leukocyte antigen (HLA) antibodies were enrolled. The efficacy of HPA-15-compatible vs -incompatible platelet transfusion was compared by focusing on ABO- and HLA-matched transfusions on the basis of the 24-hour corrected count increment (CCI-24 hours) for platelets. The titers of HPA-15 antibodies in the patients' sera were also monitored. RESULTS: The patients received 71 and 12 ABO-compatible, HLA-matched platelet transfusions, respectively, during the monitoring periods. Among these transfusions, CCI-24 hours could be calculated in 27 and 10 transfusions, respectively, and the HPA-15 genotype of the donors was determined. There were no significant differences in the CCI-24 hours between the HPA-15 compatible and incompatible transfusions in both patients (P = .30 and .56, respectively, Mann-Whitney U test). There was no significant change in the HPA-15b antibody titer in Patient 1 during the monitoring period, while the HPA-15a antibody level in Patient 2 was undetectable at the end of the monitoring period, although the titer was low at the beginning. CONCLUSION: The efficacy of HPA-15-incompatible platelet transfusions was not necessarily inferior to that of HPA-15 compatible ones. Although the case number was limited, our results suggest that HPA-15 antibodies do not have a significant impact on the effects of platelet transfusion.

A novel simple assay system for the detection of human platelet antigen 15 (HPA-15) alloantibodies based on three techniques: an HPA-15 expressing cell line, a monoclonal antibody-specific antigen-capture method and mixed-passive haemagglutination.

BACKGROUND AND OBJECTIVES: To detect HPA-15 alloantibodies, we previously developed a human platelet antigen 15 (HPA-15)-expressing cell line-based modified rapid monoclonal antibody immobilization of platelet antigen (CL-MR-MAIPA) assay. In this study, the protocol was modified for easier performance by introducing the mixed-passive haemagglutination (MPHA) principle. MATERIAL AND METHODS: In total, 20 samples that tested negative for HPA alloantibodies and eight that tested positive for HPA-15 alloantibodies (two and six positive for HPA-15a and HPA-15b antibodies, respectively) by CL-MR-MAIPA assay were used in this study. HPA-15 cell lines were incubated with serum/plasma and then solubilized. The lysate was transferred to a round-bottom well, which was coated with anti-human CD109 monoclonal antibodies. After incubation and repeated washings, sheep red blood cells, coated with anti-human IgG, were added to the wells. Haemagglutination was assessed the next day. RESULTS: The proposed cell line-based immune complex capture-dependent mixed-passive haemagglutination (CL-IC-MPHA) assay consisted of four steps, but required only 2 h to perform, except for overnight incubation for haemagglutination. Two HPA-15a alloantibody samples were reactive only for HPA-15a cells, and six HPA-15b alloantibody samples were reactive only for HPA-15b cells with the CL-IC-MPHA assay. The 20 samples that tested negative for HPA alloantibodies did not react with HPA-15a or HPA-15b cells. These data indicated that the CL-IC-MPHA assay was highly specific and sensitive. Unfortunately, the CL-IC-MPHA assay's analytic sensitivity was twofold to eightfold lower than that of the CL-MR-MAIPA assay. CONCLUSION: A novel, easy-to-perform protocol was successfully developed to detect HPA-15 alloantibodies with high specificity and sensitivity.

Quantitative comparison of cancer and normal cell adhesion using organosilane monolayer templates: an experimental study on the anti-adhesion effect of green-tea catechins.

The main constituent of green tea, (-)-Epigallocatechin-3-O-gallate (EGCG), is known to have cancer-specific chemopreventive effects. In the present work, we investigated how EGCG suppresses cell adhesion by comparing the adhesion of human pancreatic cancer cells (AsPC-1 and BxPC-3) and their counterpart, normal human embryonic pancreas-derived cells (1C3D3), in catechin-containing media using organosilane monolayer templates (OMTs). The purpose of this work is (1) to evaluate the quantitativeness in the measurement of cell adhesion with the OMT and (2) to show how green-tea catechins suppress cell adhesion in a cancer-specific manner. For the first purpose, the adhesion of cancer and normal cells was compared using the OMT. The cell adhesion in different type of catechins such as EGCG, (-)-Epicatechin-3-O-gallate (ECG) and (-)-Epicatechin (EC) was also evaluated. The measurements revealed that the anti-adhesion effect of green-tea catechins is cancer-specific, and the order is EGCG≫ECG>EC. The results agree well with the data reported to date, showing the quantitativeness of the new method. For the second purpose, the contact area of cells on the OMT was measured by reflection interference contrast microscopy. The cell-OMT contact area of cancer cells decreases with increasing EGCG concentration, whereas that of normal cells remains constant. The results reveal a twofold action of EGCG on cancer cell adhesion-suppressing cell attachment to a candidate adhesion site and decreasing the contact area of the cells-and validates the use of OMT as a tool for screening cancer cell adhesion.

Quantitative Evaluation of Cancer Cell Adhesion to Self-Assembled Monolayer-Patterned Substrates by Reflection Interference Contrast Microscopy.

Adhesion of cancer cells with different metastatic potential and anticancer drug resistance has been quantitatively evaluated by using self-assembled monolayer (SAM)-patterned substrates and reflection interference contrast microscopy (RICM). Cell-adhesive SAM spots with optimized diameter could prevent cell-cell adhesion and thus allowed the systematic evaluation of statistically reliable numbers of contact area between single cancer cells and substrates by RICM. The statistical image analysis revealed that highly metastatic mouse melanoma cells showed larger contact area than lowly metastatic cells. We also found that both cancer cell types exhibited distinct transition from the "strong" to "weak" adhesion states with increase in the concentration of (-)-epigallocatechin gallate (EGCG), which is known to exhibit cancer preventive activity. Mathematical analysis of the adhesion transition revealed that adhesion of the highly metastatic mouse melanoma cells showed more EGCG tolerance than that of lowly metastatic cells. Moreover, time-lapse RICM observation revealed that EGCG weakened cancer cell adhesion in a stepwise manner, probably via focal adhesion complex. These results clearly indicate that contact area can be used as a quantitative measure for the determination of cancer phenotypes and their drug resistance, which will provide physical insights into the mechanism of cancer metastasis and cancer prevention.