Estimation of reference interval for neutrophil activity evaluation systems: a interim report.

Neutrophils play an important role in innate immunity and produce reactive oxygen species, but they can also cause inflammation and oxidative stress that can damage their own tissues. We have developed neutrophil activity evaluation systems that simultaneously monitors superoxide radicals and hypochlorite ions secreted by stimulated neutrophils in a few microliters of whole blood and have conducted clinical studies in humans. Here, we report normal reference intervals with our systems based on the results of 3,082 persons who underwent comprehensive cancer screening between February 2020 and March 2022. A total of 344 were extracted as reference individuals based on the results of the cancer screening and the reference intervals of the two systems were interim estimated considering gender and age. Reference intervals can be used as a marker of sub-clinical inflam-mation, which is difficult to detect with other blood markers.

Effect of fat ingestion on postprandial oxidative status in healthy young women: a pilot study.

Reactive oxygen species (ROS) and highly reactive oxygen species (hROS) secreted by leukocytes are crucial to innate immunity; however, they pose a risk of oxidative stress. To monitor their balance in daily health check-ups, optical technologies for the simultaneous measurement of ROS (superoxide radicals) and hROS (hypochlorite ions) that utilize only a few microliters of whole blood have been developed. The aim of this study was to clarify whether this system could assess the effects of fat ingestion on postprandial oxidative status. Eight healthy young Japanese women ingested a beverage containing oral fat tolerance test cream. Blood samples were collected before and 0.5, 1, 2, 4, and 6 h after fat ingestion. Blood ROS and hROS levels, oxidative stress markers, and biochemical markers were monitored. Consistent with previous studies, triglyceride levels significantly increased at 4 h (p<0.01) and returned to near-baseline levels 6 h after ingestion. ROS levels peaked significantly at 2 h (p<0.05), and hROS levels peaked significantly at 1 (p<0.05) and 2 h (p<0.01) after ingestion. This study offers an insight into the acute effects of fat ingestion on leukocyte activity and provides a methodology for monitoring postprandial oxidative status.

Comparison of the oxidative profiles before and after revascularization in peripheral arterial disease: a pilot study.

Reactive and highly reactive oxygen species (ROS and hROS) produced by white blood cells are essential for innate immunity; however, they may cause oxidative stress in the host. We developed systems for simultaneously monitoring ROS and hROS, i.e., superoxide radicals (O2•-) and hypochlorite ions (OCl-) secreted from stimulated white blood cells in a few microliters of whole blood. We previously reported on the evaluation of healthy volunteers' blood using the developed system; however, whether patients' blood can be assessed remains unclear. Here, we report a pilot study of 30 cases (28 patients) with peripheral arterial disease, in whom we measured the ROS and hROS levels before and approximately one month after endovascular treatment (EVT) using the system (CFL-H2200) that we developed. At approximately the same time points, physiological indices of blood vessels, oxidative stress markers, and standard clinical parameters in the blood were also monitored. The ankle-brachial index, a diagnostic tool for peripheral arterial disease, was significantly improved after EVT (p<0.001). The ROS-hROS ratio, low-density lipoprotein cholesterol, and hematocrit levels were decreased after EVT (p<0.05), while triglyceride and lymphocyte levels were increased after EVT (p<0.05). The correlations between the study parameters were also analyzed.

Correlation between human health and reactive oxygen species produced in blood: a long-term chemiluminescence and fluorescence analysis.

The previous slide-glass type system could simultaneously detect reactive and highly reactive oxygen species, i.e., superoxide radicals (O2-·) and hypochlorite ions (OCl-) elicited from leucocytes in sample blood, but had some drawbacks, i.e., signal noise from air-flow stirring, potential biohazard risks, etc. because of open samples placed on a slide glass. We overcame these drawbacks by adopting a fluidic-chip container in a new system, which resulted in higher sensitivity and more stable measurements. Using the new system, we conducted a pilot study on nominally healthy volunteers to find whether or not the monitored activities of leukocytes can distinguish more or less unhealthy conditions from healthy ones. At first, healthy volunteers of both genders and of various ages showed that the fluctuation magnitudes (%) of O2-· and OCl- were nearly similar to each other and to that of the neutrophil count fluctuation. These parameters sometimes exceeded the healthy fluctuation range. By comparing these large fluctuations with the data of an inflammation marker C-reactive protein (CRP), the neutrophil count fluctuation and the timings/symptoms of abnormalities found in questionnaire, we could gain information suggesting the factors causing the large fluctuations. The new system could detect bodily abnormalities earlier than CRP or self-aware symptoms.

Automated single nucleotide polymorphism typing using bead array in capillary tube.

A low-cost and simple on-site technique for genotyping single nucleotide polymorphisms (SNPs) was developed. The technique is based on allele-specific primer PCR and the recently developed bead arrays in a single tip technique. The performance of the method was verified by genotyping four SNPs that correlate with cardiovascular diseases.

High relative biologic effectiveness of carbon ion radiation on induction of rat mammary carcinoma and its lack of H-ras and Tp53 mutations.

PURPOSE: The high relative biologic effectiveness (RBE) of high-linear energy transfer (LET) heavy-ion radiation has enabled powerful radiotherapy. The potential risk of later onset of secondary cancers, however, has not been adequately studied. We undertook the present study to clarify the RBE of therapeutic carbon ion radiation and molecular changes that occur in the rat mammary cancer model. METHODS AND MATERIALS: We observed 7-8-week-old rats (ACI, F344, Wistar, and Sprague-Dawley) until 1 year of age after irradiation (0.05-2 Gy) with either 290 MeV/u carbon ions with a spread out Bragg peak (LET 40-90 keV/mum) generated from the Heavy-Ion Medical Accelerator in Chiba or (137)Cs gamma-rays. RESULTS: Carbon ions significantly induced mammary carcinomas in Sprague-Dawley rats but less so in other strains. The dose-effect relationship for carcinoma incidence in the Sprague-Dawley rats was concave downward, providing an RBE of 2 at a typical therapeutic dose per fraction. In contrast, approximately 10 should be considered for radiation protection at low doses. Immunohistochemically, 14 of 18 carcinomas were positive for estrogen receptor alpha. All carcinomas examined were free of common H-ras and Tp53 mutations. Importantly, lung metastasis (7%) was characteristic of carbon ion-irradiated rats. CONCLUSIONS: We found clear genetic variability in the susceptibility to carbon ion-induced mammary carcinomas. The high RBE for carbon ion radiation further supports the importance of precise dose localization in radiotherapy. Common point mutations in H-ras and Tp53 were not involved in carbon ion induction of rat mammary carcinomas.

Development of the Handy Bio-Strand and its application to genotyping of OPRM1 (A118G).

We previously developed a three-dimensional microarray system, the Bio-Strand, which exhibits advantages in automated DNA analysis in combination with our Magtration Technology. In the current study, we have developed a compact system for the Bio-Strand, the Handy Bio-Strand, which consists of several tools for the preparation of Bio-Strand Tip, hybridization, and detection. Using the Handy Bio-Strand, we performed single nucleotide polymorphism (SNP) genotyping of OPRM1 (A118G) by allele-specific oligonucleotide competitive hybridization (ASOCH). DNA fragments containing SNP sites were amplified from genomic DNA by PCR and then were fixed on a microporous nylon thread. Thus, prepared Bio-Strand Tip was hybridized with allele-specific Cy5 probes (<15mer), on which the SNP site was designed to be located in the center. By optimizing the amount of competitors, the selectivity of Cy5 probes increased without a drastic signal decrease. OPRM1 (A118G) genotypes of 23 human genomes prepared from whole blood samples were determined by ASOCH using the Handy Bio-Strand. The results were perfectly consistent with those determined by PCR direct sequencing. ASOCH using the Handy Bio-Strand would be a very simple and reliable method for SNP genotyping for small laboratories and hospitals.