Various methodologies for the genetic analysis of longitudinal data have been proposed and applied to data from large-scale genome-wide association studies (GWAS) to identify single nucleotide polymorphisms (SNPs) associated with traits of interest and to detect SNP-time interactions. We recently proposed a grid-based Bayesian mixed model for longitudinal genetic data and showed that our Bayesian method increased the statistical power compared to the corresponding univariate method and well detected SNP-time interactions. In this paper, we further analyze longitudinal obesity-related traits such as body mass index, hip circumference, waist circumference, and waist-hip ratio from Korea Association Resource data to evaluate the proposed Bayesian method. We first conducted GWAS analyses of cross-sectional traits and combined the results of GWAS analyses through a meta-analysis based on a trajectory model and a random-effects model. We then applied our Bayesian method to a subset of SNPs selected by meta-analysis to further discover SNPs associated with traits of interest and SNP-time interactions. The proposed Bayesian method identified several novel SNPs associated with longitudinal obesity-related traits, and almost 25% of the identified SNPs had significant p-values for SNP-time interactions.

Background: We sought to compare the performance of three commercially available automated urine sediment analyzers that represent the current urine sediment analysis technology. Methods: A total of 232 patient samples were analyzed using manual microscopy and three automated analyzers: IRIS Iq200 (Beckman Coulter, USA), UF-1000i (Sysmex, Japan), and Cobas u701 (Roche, Switzerland). We analyzed precision, linearity, carry-over, concordance rate, and agreement between the three analyzers and manual microscopy. Results: The repeatability and within-laboratory precision showed results similar to those of previous studies. All analyzers showed excellent linearity. The carry-over rates were within 1%. The correlation coefficient (r) between the three analyzers and manual microscopy was good. Regarding red blood cell (RBC), the UF-1000i showed a better concordance rate (90.52%) with manual microscopy than the other two analyzers and the agreement was substantial for UF-1000i (κ=0.63) and IRIS Iq200 (κ=0.61). Regarding white blood cell (WBC), Cobas u701 showed the best concordance rate (96.55%) and the agreement was moderate for IRIS Iq200 (κ=0.57) and Cobas u701 (κ=0.56), and fair for UF-1000i (κ=0.47). Regarding epithelial cell (EPI), IRIS Iq200 showed the highest concordance rate (99.2%) and the agreement was moderate for IRIS Iq200 (κ=0.59) and Cobas u701 (κ=0.54), and fair for UF-1000i (κ=0.40). Conclusions IRIS Iq200 offered the best agreement with manual microscopy for WBC and EPI count, while UF-1000i showed a better agreement for RBC count. The agreement is insufficient for fully replacing the manual microscopy.

Background: We sought to compare the performance of three commercially available automated urine sediment analyzers that represent the current urine sediment analysis technology. Methods: A total of 232 patient samples were analyzed using manual microscopy and three automated analyzers: IRIS Iq200 (Beckman Coulter, USA), UF-1000i (Sysmex, Japan), and Cobas u701 (Roche, Switzerland). We analyzed precision, linearity, carry-over, concordance rate, and agreement between the three analyzers and manual microscopy. Results: The repeatability and within-laboratory precision showed results similar to those of previous studies. All analyzers showed excellent linearity. The carry-over rates were within 1%. The correlation coefficient (r) between the three analyzers and manual microscopy was good. Regarding red blood cell (RBC), the UF-1000i showed a better concordance rate (90.52%) with manual microscopy than the other two analyzers and the agreement was substantial for UF-1000i (κ=0.63) and IRIS Iq200 (κ=0.61). Regarding white blood cell (WBC), Cobas u701 showed the best concordance rate (96.55%) and the agreement was moderate for IRIS Iq200 (κ=0.57) and Cobas u701 (κ=0.56), and fair for UF-1000i (κ=0.47). Regarding epithelial cell (EPI), IRIS Iq200 showed the highest concordance rate (99.2%) and the agreement was moderate for IRIS Iq200 (κ=0.59) and Cobas u701 (κ=0.54), and fair for UF-1000i (κ=0.40). Conclusions IRIS Iq200 offered the best agreement with manual microscopy for WBC and EPI count, while UF-1000i showed a better agreement for RBC count. The agreement is insufficient for fully replacing the manual microscopy.

Phthalates widely used in the manufacture of plastics have deeply penetrated into our everyday lives. Recently, a concern over the toxicity of phthalates on thyroid, has been raised but in most of cases, the doses employed were unrealistically high. To investigate the effects of phthalates on thyroid, we investigated the effects of the repeated oral exposure to low to high doses (0.3, 3, 30 and 150 mg/kg) di-2-ethylhexylphthalate (DEHP) from weaning to maturity for 90 days in juvenile rats on the thyroid. The histological examination revealed that DEHP significantly induced hyperplasia in the thyroid from the doses of 30 mg/kg, which was confirmed with Ki67 staining. In line with this finding, increased mRNA expression of thyrotropin releasing hormone (Trh) was observed in the thyroid of female at 0.3 mg/kg and 150 mg/kg as determined by RNAseq analysis. Moreover, significantly increased expression of parathyroid hormone (Pth) in the female at 0.3 mg/kg, and thyroglobulin (Tg) and thyroid hormone responsive (Thrsp) in the male at 0.3 mg/kg were noted in the blood, of which changes were substantially attenuated at 150 m/kg, alluding the meaningful effects of low dose DEHP on the thyroid hormone regulation. Urinary excretion of mono-2-ethylhexyl-phthalate (MEHP), a major metabolite of DEHP was determined to be 4.10 and 12.26 ppb in male, 6.65 and 324 ppb in female at 0.3 and 30 mg/kg DEHP, respectively, which fell within reported human urine levels. Collectively, these results suggest a potential adverse effects of low dose phthalates on the thyroid.
Animals ; Diethylhexyl Phthalate* ; Female ; Humans ; Hyperplasia ; Male ; Parathyroid Hormone ; Plastics ; Rats* ; RNA, Messenger ; Thyroglobulin ; Thyroid Gland* ; Thyrotropin-Releasing Hormone ; Weaning

The adoption of high-sensitivity flow cytometry (HSFC) in routine laboratory settings has been slow owing to concerns regarding the reliability and reproducibility of results. Validation is an essential prerequisite for conducting assays, and implementing the CLSI guidelines has been confusing, primarily because many aspects are not yet established. We aimed to validate an HSFC protocol for detecting follicular helper T (Tfh) cells in a real-world laboratory environment. The analytical validity of the Tfh cell panel was ensured through rigorous testing, including evaluations of precision, stability, carryover, and sensitivity, following the CLSI H62 guidelines. We found that Tfh cells, present in very small numbers in the blood, could be sufficiently detected through HSFC, and concerns about the reliability and reproducibility of the results in real-world laboratories could be solved through systematic validation. Establishing the lower limit of quantification (LLOQ) is a critical step in HSFC evaluations. By selecting an appropriate sample, for example, collecting residual cells from CD4 isolation in our experiment and using them as low-level samples, the LLOQ could be accurately established. The strategic validation of flow cytometry panels can facilitate the adoption of HSFC in clinical laboratories, even with limited resources.

BACKGROUND: A co-inhibitory molecule, B7-H4, is believed to negatively regulate T cell immunity by suppressing T cell proliferation and inhibiting cytokine production. However, the mechanism behind B7-H4-mediated tolerance remains unclear. METHODS: Balb/c (H-2(d)) mice were fed with dendritic cell line, DC2.4 (H-2(b)) every day for 10 days. Meantime, mice were hydrodynamically injected with recombinant plasmid expressing B7-H4 fusion protein (B7-H4.hFc) or hFc via tail vein. One day after last feeding, mice were immunized with allogeneic B6 spleen cells. 14 days following immunization, mice were challenged with B6 spleen cells to ear back and the ear swelling was determined the next day. Subsequently, a mixed lymphocyte reaction (MLR) was also performed and cytokines profiles from the reaction were examined by sandwich ELISA. Frequency of immunosuppressive cell population was assayed with flow cytometry and mRNA for FoxP3 was determined by RT-PCR. RESULTS: Tolerant mice given plasmid expressing B7-H4.hFc showed a significant reduction in ear swelling compared to control mice. In addition, T cells from mice given B7-H4.hFc plasmid revealed a significant hyporesponsiveness of T cells against allogeneic spleen cells and showed a significant decrease in Th1 and Th2 cytokines such as IFN-gamma, IL-5, and TNF-alpha. Interestingly, flow cytometric analysis showed that the frequency of CD4+CD25+FoxP3+ Tregs in spleen was increased in tolerant mice given recombinant B7-H4.hFc plasmid compared to control group. CONCLUSION: Our results demonstrate that B7-H4 synergistically potentiates oral tolerance induced by allogeneic cells by increasing the frequency of FoxP3+ CD4+CD25+ Treg and reducing Th1 and Th2 cytokine production.
Animals ; Cell Proliferation ; Cytokines ; Dendritic Cells ; Ear ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Immunization ; Interleukin-5 ; Lymphocyte Culture Test, Mixed ; Mice ; Plasmids ; RNA, Messenger ; Spleen ; T-Lymphocytes ; Tumor Necrosis Factor-alpha ; Veins

BACKGROUND: A co-inhibitory molecule, B7-H4, is believed to negatively regulate T cell immunity by suppressing T cell proliferation and inhibiting cytokine production. However, the mechanism behind B7-H4-mediated tolerance remains unclear. METHODS: Balb/c (H-2(d)) mice were fed with dendritic cell line, DC2.4 (H-2(b)) every day for 10 days. Meantime, mice were hydrodynamically injected with recombinant plasmid expressing B7-H4 fusion protein (B7-H4.hFc) or hFc via tail vein. One day after last feeding, mice were immunized with allogeneic B6 spleen cells. 14 days following immunization, mice were challenged with B6 spleen cells to ear back and the ear swelling was determined the next day. Subsequently, a mixed lymphocyte reaction (MLR) was also performed and cytokines profiles from the reaction were examined by sandwich ELISA. Frequency of immunosuppressive cell population was assayed with flow cytometry and mRNA for FoxP3 was determined by RT-PCR. RESULTS: Tolerant mice given plasmid expressing B7-H4.hFc showed a significant reduction in ear swelling compared to control mice. In addition, T cells from mice given B7-H4.hFc plasmid revealed a significant hyporesponsiveness of T cells against allogeneic spleen cells and showed a significant decrease in Th1 and Th2 cytokines such as IFN-gamma, IL-5, and TNF-alpha. Interestingly, flow cytometric analysis showed that the frequency of CD4+CD25+FoxP3+ Tregs in spleen was increased in tolerant mice given recombinant B7-H4.hFc plasmid compared to control group. CONCLUSION: Our results demonstrate that B7-H4 synergistically potentiates oral tolerance induced by allogeneic cells by increasing the frequency of FoxP3+ CD4+CD25+ Treg and reducing Th1 and Th2 cytokine production.
Animals ; Cell Proliferation ; Cytokines ; Dendritic Cells ; Ear ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Immunization ; Interleukin-5 ; Lymphocyte Culture Test, Mixed ; Mice ; Plasmids ; RNA, Messenger ; Spleen ; T-Lymphocytes ; Tumor Necrosis Factor-alpha ; Veins

Colorectal cancer (CRC) is one of the leading causes of cancer death in western countries or in the developed countries. Zinc intake has been associated with decreased risk of CRC. We investigated the effect of zinc on the formation of colonic aberrant crypt foci (ACF) induced by azoxymethane followed by dextran sodium sulfate in mice. Five-week old ICR mice were fed with the different zinc levels (0.01, 0.1, 1 ppm) for 12 weeks. The numbers of ACF were measured in the colonic mucosa. The ACF number of HZn group was significantly low compared with LZn group or MZn group. Cytosolic superoxide dismutase activity was the highest in HZn group, while thiobarbituric acid reactive substance level for lipid peroxidation was the highest in LZn group. There was no difference in number of PCNA-positive proliferative cells among the groups. TUNEL-positive apoptotic cells were increased in HZn group compared with LZn group. The HZn group exhibited a decrease of beta-catenin immunostaining areas compared with the LZn or MZn group. These findings indicate that dietary zinc might exert a protecting effect against colon carcinogenesis by inhibiting the development of ACF in the mice.
Aberrant Crypt Foci ; Animals ; Azoxymethane ; beta Catenin ; Colon ; Colorectal Neoplasms ; Cytosol ; Developed Countries ; Dextrans ; Lipid Peroxidation ; Mice ; Mice, Inbred ICR ; Mucous Membrane ; Sodium ; Sulfates ; Superoxide Dismutase ; Thiobarbiturates ; Zinc