Relationship between a deep learning model and liquid-based cytological processing techniques.

OBJECTIVE: Artificial intelligence (AI)-based cytopathology studies conducted using deep learning have enabled cell detection and classification. Liquid-based cytology (LBC) has facilitated the standardisation of specimen preparation; however, cytomorphology varies according to the LBC processing technique used. In this study, we elucidated the relationship between two LBC techniques and cell detection and classification using a deep learning model. METHODS: Cytological specimens were prepared using the ThinPrep and SurePath methods. The accuracy of cell detection and cell classification was examined using the one- and five-cell models, which were trained with one and five cell types, respectively. RESULTS: When the same LBC processing techniques were used for the training and detection preparations, the cell detection and classification rates were high. The model trained on ThinPrep preparations was more accurate than that trained on SurePath. When the preparation types used for training and detection were different, the accuracy of cell detection and classification was significantly reduced (P < 0.01). The model trained on both ThinPrep and SurePath preparations exhibited slightly reduced cell detection and classification rates but was highly accurate. CONCLUSIONS: For the two LBC processing techniques, cytomorphology varied according to cell type; this difference affects the accuracy of cell detection and classification by deep learning. Therefore, for highly accurate cell detection and classification using AI, the same processing technique must be used for both training and detection. Our assessment also suggests that a deep learning model should be constructed using specimens prepared via a variety of processing techniques to construct a globally applicable AI model.

Immunocytochemical Reactivity Comparison between Formalin-Fixed and Liquid-Based Cytology-Fixed Specimens.

INTRODUCTION: Liquid-based cytology (LBC)-fixed samples can be used for preparing multiple specimens of the same quality and for immunocytochemistry (ICC); however, LBC fixing solutions affect immunoreactivity. Therefore, in this study, we examined the effect of LBC fixing solutions on immunoreactivity. METHODS: Samples were cell lines, and specimens were prepared from cell blocks of 10% neutral buffered formalin (NBF)-fixed samples and the four types of LBC-fixed samples: PreservCyt®, CytoRich™ Red, CytoRich™ Blue, and TACAS™ Ruby, which were post-fixed with NBF. ICC was performed using 24 different antibodies, and immunocytochemically stained specimens were analyzed for the percentage of positive cells. RESULTS: Immunoreactivity differed according to the type of antigen detected. For nuclear antigens, the highest percentage of positive cells of Ki-67, WT-1, ER, and p63 was observed in the NBF-fixed samples, and the highest percentage of positive cells of p53, TTF-1, and PgR was observed in the TACAS™ Ruby samples. For cytoplasmic antigens, the percentage of positive cells of CK5/6, Vimentin, and IMP3 in LBC-fixed samples was higher than or similar to that in NBF-fixed samples. The percentage of positive cells of CEA was significantly lower in CytoRich™ Red and CytoRich™ Blue samples than in the NBF-fixed sample (p < 0.01). Among the cell membrane antigens, the percentage of positive cells of Ber-EP4, CD10, and D2-40 was the highest in NBF-fixed samples and significantly lower in CytoRich™ Red and CytoRich™ Blue samples than that in NBF-fixed samples (p < 0.01). The NBF-fixed and LBC-fixed samples showed no significant differences in the percentage of positive cells of CA125 and EMA. DISCUSSION/CONCLUSION: ICC using LBC-fixed samples showed the same immunoreactivity as NBF-fixed samples when performed on cell block specimens post-fixed with NBF. The percentage of positive cells increased or decreased based on the type of fixing solution depending on the amount of antigen in the cells. Further, the detection rate of ICC with LBC-fixed samples varied according to the type of antibody and the amount of antigen in the cells. Therefore, we propose that ICC using LBC-fixed samples, including detection methods, should be carefully performed.

Characterizing the Effect of Processing Technique and Solution Type on Cytomorphology Using Liquid-Based Cytology.

INTRODUCTION: Liquid-based cytology (LBC) is increasingly used for nongynecologic applications. However, the cytological preparation of LBC specimens is influenced by the processing technique and the preservative used. In this study, the influence of the processing techniques and preservatives on cell morphology was examined mathematically and statistically. METHODS: Cytological specimens were prepared using the ThinPrep (TP), SurePath (SP), and AutoSmear methods, with 5 different preservative solutions. The cytoplasmic and nuclear areas of Papanicolaou-stained specimens were measured for all samples. RESULTS: The cytoplasmic and nuclear areas were smaller in cells prepared using the 2 LBC methods, compared to that prepared using the AutoSmear method, irrespective of the preservative used. The cytoplasmic and nuclear areas of cells prepared using the SP method were smaller than those of cells prepared using the TP method, irrespective of the preservative used. There were fewer differences among the cytoplasmic areas of cells prepared with different preservative solutions using the TP method; however, the cytoplasmic areas of cells prepared using the SP method changed with the preservative solution used. CONCLUSIONS: The most significant difference affecting the cytoplasmic and nuclear areas was the processing technique. The TP method increased the cytoplasmic and nuclear areas, while the methanol-based PreservCyt solution enabled the highest enlargement of the cell. LBC is a superior preparation technique for standardization of the specimens. Our results offer a better understanding of methods suitable for specimen preparation for developing precision AI-based diagnosis in cytology.

Helicobacter pylori protein that binds to and activates platelet specifically reacts with sera of H. pylori-associated chronic immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an acquired autoimmune disorder characterized by antiplatelet antibodies and/or CD8 + T cells, resulting in the destruction of platelets and decreased platelet counts. Helicobacter pylori that persistently colonizes the stomach causes various disorders, including extragastric diseases such as chronic ITP (cITP). Several studies have reported increased platelet counts in H. pylori-infected cITP patients with eradication treatment and also the pathophysiological pathways involving cross-reaction of antibodies against H. pylori with platelets, the modulation of Fcrγ receptors balance and others. We previously reported an immunocomplex pathway comprising H. pylori low-molecular-weight (LMW) antigens, their antibodies, and platelets, involved in the development of H. pylori-associated cITP; however, the LMW antigens were not identified. In the present study, we demonstrated that the H. pylori LMW antigen of the immunocomplex was identified as Lpp20 of outer membrane proteins. Lpp20 could bind to platelets and specifically react with sera of H. pylori-associated cITP patients.

MicroRNA-150 suppresses p27Kip1 expression and promotes cell proliferation in HeLa human cervical cancer cells.

MicroRNAs (miRNAs) exert critical roles in the majority of biological and pathological processes. Recent studies have associated miR-150 with a number of different cancer types. However, little is known about miR-150 targets in cervical cancer. In the present study, the HeLa human cervical cancer cell line was transfected with hsa-miR-150-5p mimics, hsa-miR-150-5p inhibitors or miRNA controls. miR-150 was predicted to bind the 3'untranslated region (3'UTR) of the CDKN1B gene, which encodes the cyclin-dependent kinase inhibitor 1B (p27Kip1). The direct binding between miR-150 and the 3'UTR of CDKN1B was confirmed using dual-luciferase reporter assays. The effects of miR-150 on CDKN1B mRNA expression, p27Kip1 protein expression, cell cycle and cell proliferation were determined using reverse-transcription quantitative PCR, western blot analysis, flow cytometry and WST-8 assays, respectively. miR-150 was demonstrated to directly target the 3'UTR of CDKN1B in transfected HeLa cells. The expression of CDKN1B mRNA and p27Kip1 protein was reduced by miR-150 mimics, and increased by miR-150 inhibitors. Moreover, the overexpression of miR-150 promoted cell cycle progression from the G0/G1 to the S phase and led to a significant increase in HeLa cell proliferation. The results of the present study indicated that miR-150 promotes HeLa cell cycle progression and proliferation via the suppression of p27Kip1 expression.

Drinking Refined Deep-Sea Water Improves the Gut Ecosystem with Beneficial Effects on Intestinal Health in Humans: A Randomized Double-Blind Controlled Trial.

World health trends are focusing on a balanced food and beverage intake for healthy life. Refined deep-sea water (RDSW), obtained from deep-sea water collected offshore in Muroto (Japan), is mineral-rich drinking water. We previously reported that drinking RDSW improves human gut health. Here, we analyzed the effect of drinking RDSW on the gut ecosystem to understand this effect. This was a randomized double-blind controlled trial. Ninety-eight healthy adults were divided into two groups: RDSW or mineral water (control). The participants consumed 1 L of either water type daily for 12 weeks. A self-administered questionnaire and stool and urine samples were collected through the intervention. The following were determined: fecal biomarkers of secretory immunoglobulin A (sIgA), five putrefactive products, and nine short-chain-fatty-acids (SCFAs) as the primary outcomes; and three urinary isoflavones and the questionnaire as secondary outcomes. In post-intervention in the RDSW group, we found increased concentrations of five SCFAs and decreased concentrations of phenol and sIgA (p < 0.05). The multiple logistic analysis demonstrated that RDSW significantly affected two biomarkers (acetic and 3-methylbutanoic acids) of the five SCFAs mentioned above (p < 0.05). Similarly, the concentrations of urinary isoflavones tended to increase in post-intervention in the RDSW group. Constipation was significantly alleviated in the RDSW group (94%) compared with the control group (60%). Drinking RDSW improves the intestinal environment, increasing fecal SCFAs and urinary isoflavones, which leads to broad beneficial effects in human.

ABO genotyping of various hematopoietic cell lines to select model cells for research purposes.

Various cells from humans and animals have been established as cell lines, and their features, characteristics, and origins have been reported. Many laboratories use cell lines as model cells, which are selected to suit research purposes. We attempted to identify the ABO genotypes of 31 human leukemia and lymphoma cell lines stored in our laboratory using three methods: the PCR amplification of specific alleles (PASA), PCR-restriction fragment length polymorphism (RFLP), and the direct DNA sequencing of PCR products. We distinguished 31 human leukemia and lymphoma cell lines examined into six major ABO genotypes: A/O (A101/O01: n = 1, A101/O12: n = 4, A101/O26: n = 1, A101/O49: n = 1, A102/O01: n = 3), A/A (A101/A101: n = 1, A102/A102: n = 2), B/O (Bw29/O01: n = 1), B/B (B101/B101: n = 2), O/O (O01/O01: n = 9, O01/O02: n = 1, O01/O26: n = 1, O02/O03: n = 1), and A/B (A102/B101: n = 3). To the best of our knowledge, this is the first study to identify the ABO genotypes of various cell lines. The ABO genotypes of cell lines are important when selecting an experimental model cell for an ABO blood group study, and are essential information for cell lines. These results may be employed by research and clinical laboratories as well as in the forensic field.

QPY/RAH haplotypes of the GZMB gene are associated with natural killer cell cytotoxicity.

Granzyme B (GzmB) is a component of cytolytic granules within NK cells and is involved in several pathologies. It has previously been reported that there are three non-synonymous coding SNPs (rs8192917; Q48R, rs11539752; P88A, and rs2236338; Y245H) in the GZMB gene and that the QPY/RAH allele was clustered together close to the C-terminal α-helix. However, it is unknown whether the function of GzmB produced from NK cells is influenced by QPY/RAH polymorphism. The authors investigated the distribution of QPY/RAH polymorphism of the GZMB gene in a Japanese population (n = 106), and the involvement of Q48R polymorphism in NK cell cytotoxicity, degranulation, and production of GzmB. A strong linkage disequilibrium was observed among these SNPs, and NK cell cytotoxicity was influenced by rs8192917 (Q48R). Moreover, it found that R48-GzmB is a stable protein that accumulates to similar levels in activated NK cells as Q48-GzmB. rs8192917 polymorphism may influence antitumor activity and the effect of antitumor cellular immunotherapy. The authors expect that these new informations about QPY/RAH polymorphism of the GZMB gene could help to assess the impact of NK cell cytotoxicity in several pathologies and aid their treatment.

Genetic Factors of Low-responsiveness to Hepatitis B Virus Vaccine Confirms the Importance of Human Leukocyte Antigen Class II Types in a Japanese Young Adult Population.

We investigated the genetic mechanisms underlying the association between human leukocyte antigen (HLA) types and the immune response to hepatitis B virus (HBV) vaccination in 84 healthy Japanese adults, and found that the HLA-DRB1＊04 and HLA-DQB1＊03 frequencies were higher in the low responders (<10 mIU/ml; n=9, 10.7%) compared to the responders (≥10 mIU/ml, n=75, 89.3%). The combination of DRB1＊04 and DQB1＊03 was associated with a low response to vaccination. The DRB1＊04 and DQB1＊03 haplotypes' frequencies were significantly higher in the low responders compared to responders. Novel candidate HLA types may be important in Japanese individuals.

Evaluation of effects of various drugs on platelet functions using phorbol 12-myristate 13-acetate-induced megakaryocytic human erythroid leukemia cells.

BACKGROUND: The hyperfunction and activation of platelets have been strongly implicated in the development and recurrence of arterial occlusive disease, and various antiplatelet drugs are used to treat and prevent such diseases. New antiplatelet drugs and many other drugs have been developed, but some drugs may have adverse effects on platelet functions. OBJECTIVE: The aim of this study was to establish an evaluation method for evaluating the effect and adverse effect of various drugs on platelet functions. MATERIALS AND METHODS: Human erythroid leukemia (HEL) cells were used after megakaryocytic differentiation with phorbol 12-myristate 13-acetate as an alternative to platelets. Drugs were evaluated by changes in intracellular Ca2+ concentration ([Ca2+]i) mobilization in Fura2-loaded phorbol 12-myristate 13-acetate-induced HEL cells. Aspirin and cilostazol were selected as antiplatelet drugs and ibuprofen and sodium valproate as other drugs. RESULTS: There was a positive correlation between [Ca2+]i and platelet aggregation induced by thrombin. Aspirin (5.6-560 µM) and cilostazol (5-10 µM) significantly inhibited thrombin-induced increases in [Ca2+]i in a concentration-dependent manner. On the other hand, ibuprofen (8-200 µM) and sodium valproate (50-1,000 µg/mL) also significantly inhibited thrombin-induced increases in [Ca2+]i in a concentration-dependent manner. Furthermore, the interaction effects of the simultaneous combined use of aspirin and ibuprofen or sodium valproate were evaluated. When the inhibitory effect of aspirin was higher than that of ibuprofen, the effect of aspirin was reduced, whereas when the inhibitory effect of aspirin was lower than that of ibuprofen, the effect of ibuprofen was reduced. The combination of aspirin and sodium valproate synergistically inhibited thrombin-induced [Ca2+]i. CONCLUSION: It is possible to induce HEL cells to differentiate into megakaryocytes, which are a useful model for the study of platelet functions, and the quantification of the inhibition of thrombin-induced increases in [Ca2+]i is applicable to the evaluation of the effects of various drugs on platelets.

Flow Cytometric Evaluation of Surface CD56 Expression on Activated Natural Killer Cells as Functional Marker.

Surface CD56 is the most important cell marker for defining NK cells. However, the relationship between the expression of surface CD56 and NK cell activity has not yet been elucidated in detail. Thirteen healthy volunteers were enrolled in the present study. Peripheral blood mononuclear cells (PBMCs) were stimulated with rIL-2 or rIL-12 (1, 10, 100 U/mL) for 18 h at 37°C. After incubation, surface CD56 expression on NK cells was evaluated using a flow cytometric analysis. A colorimetric-based lactate dehydrogenase (LDH) assay was used for experiments on cytotoxicity. IFN-γ mRNA gene expression was quantified by real-time PCR. The expression level of surface CD56 on NK cells, cytotoxicity, and IFN-γ mRNA gene expression were significantly increased by the rIL-2 and rIL-12 stimulations. In addition, a positive correlation was found between surface CD56 expression and cytotoxic activity or IFN-γ mRNA gene expression. We revealed that the quantification of surface CD56 expression was applicable to the evaluation of cytotoxicity and IFN-γ production in activated NK cells. These results suggest that the measurement of surface CD56 expression represent an easy and rapidly reproducible technique to evaluate the activated state of NK cells and monitor NK cell activity in immunotherapy. J. Med. Invest. 63: 199-203, August, 2016.

Influence of single nucleotide polymorphisms of cytokine genes on anti-HBs antibody production after hepatitis B vaccination in a Japanese young adult population.

Hepatitis B (HB) vaccination is one of the most efficient tools to prevent the transmission of the virus. Considerable variability exists in HB vaccine responses, with 5-10% of healthy Japanese adults demonstrating no response following a standard vaccination. Recently, polymorphisms of immune-regulatory genes, such as cytokine genes, have been reported to influence the immune response to HB vaccine. The aim of this study was to investigate the underlying mechanisms of the genetic association between several cytokine gene polymorphisms and the immune response to HB vaccination in a Japanese population. One hundred and twenty three vaccinated young adults were classified according to the level of antibody-titer (anti-HBs). Single nucleotide polymorphism typing for IFN-γ (+874, 3'-UTR), IL-10 (-591, -819, -1082), and TNF-α (-308, -857), was accomplished using the PCR-RFLP or SSP-PCR method. The TNF-α (-857) CC type and the IL-10 (-1082) AG type were present more frequently in the low titer group than in the high titer group. The TNF-α (-857) CC type was found to be significantly associated with low response of serum anti-HBs. The anti-HBs antibody was not readily produced in the IL-10 (-1082) AG and TNF-α (-857) CC haplotype. Conversely, the antibody was readily produced in the IL-10 (-1082) AA and TNF-α (-857) CC haplotype, and the IL-10 (-1082) AA and TNF-α (-857) CT haplotype, suggesting a high likelihood of the IL-10 (-1082) AG type to be included in the low anti-HBs group, and high anti-HBs antibody production in those with the TNF-α (-857) CT type. These SNPs may produce ethnically-specific differences in the immune response to HB vaccine in the Japanese population. J. Med. Invest. 63: 256-261, August, 2016.

SNPs rs4656317 and rs12071048 located within an enhancer in FCGR3A are in strong linkage disequilibrium with rs396991 and influence NK cell-mediated ADCC by transcriptional regulation.

CD16 receptors are mainly expresses on the surface of NK cells and mediate antibody-dependent cellular cytotoxicity (ADCC). The authors previously reported that NK cell-mediated ADCC is influenced by the single nucleotide polymorphism (SNP) rs396991 (T>G; F158V), and the structure and expression levels of CD16 differed among these genotypes. The authors examined haplotype frequency distributions among rs396991 and other SNPs, rs10917571 (G>T), rs4656317 (C>G), and rs12071048 (G>A), located in an enhancer of the FCGR3A gene. A total of 101 healthy Japanese were genotyped for the presence of these SNPs. The authors also measured ADCC activity, FCGR3A transcript levels, and surface CD16 expression on NK cells. We found that the regulatory SNPs (rSNPs) rs4656317 and rs12071048 were in strong linkage disequilibrium with rs396991. These two SNPs with major alleles had higher ADCC activity than those with minor alleles. In addition, FCGR3A transcript levels and surface CD16 expression levels were regulated by these SNPs. These findings suggest that NK cell-mediated ADCC could be influenced by transcriptional regulation of these rSNPs. These findings help to clarify our understanding of the linkage disequilibrium among functional SNPs in the FCGR3A gene, and provide a resource for investigating the roles of functional SNPs in NK cell-mediated ADCC.

The influence of NK cell-mediated ADCC: Structure and expression of the CD16 molecule differ among FcγRIIIa-V158F genotypes in healthy Japanese subjects.

NK cells express the CD16 (FcγRIIIa) receptor, which mediates antibody-dependent cellular cytotoxicity (ADCC), on their cell surface. Therefore, ADCC activity may be influenced by qualitative or quantitative changes in the CD16 molecule on NK cells. Responses to NK cell-mediated ADCC have been shown to depend on single nucleotide polymorphisms (SNPs) at FcγRIIIa amino acid position 158. However, a consensus has not yet been reached regarding differences in the structure and expression levels of the CD16 molecule among FcγRIIIa-V158F genotypes, which have not yet been adequately investigated in healthy Japanese individuals. We herein examined the influence of the FcγRIIIa polymorphism on ADCC, binding affinity of CD16 to the Fc region, FCGR3A gene expression, and cell-surface CD16 expression in healthy Japanese subjects. FcγRIIIa-V158F genotyping was performed for 101 subjects. The results obtained showed that all parameters analyzed increased in the order of V/V>V/F>F/F and were significantly higher in V/V subjects than in F/F subjects. Moreover, a positive correlation was observed between ADCC activity and binding affinity, FCGR3A transcript levels, and surface CD16 expression levels. These results suggest that the structure and expression of the CD16 molecule differs among FcγRIIIa-V158F genotypes, and the FcγRIIIa-V158F polymorphism may be represent a haplotype with other SNPs in regulatory regions in Japanese subjects.