No abstract

Hepcidin, the iron regulatory hormone, has three isoforms; -20, -22 and -25. While hepcidin-25 has been studied extensively, the physiological significance of other isoforms remains poorly understood. Using a quantitative method based on liquid chromatography-tandem mass spectrometry (LC-tandem MS) developed by our group, we quantified hepcidin isoforms in human serum to elucidate their characteristics, and investigated the role of hepatocytes in isoform processing. Hepcidin isoforms in serum obtained from 40 healthy volunteers were quantified. Synthetic hepcidin peptides were added to healthy serum, and to HepG2 culture media, and hepcidin isoform concentrations determined. All three hepcidin isoforms were detected in human serum; however, hepcidin-25 concentrations were highest. The three hepcidin isoforms showed a strong positive correlation with each other and with serum ferritin. Additionally, while hepcidin-20 was strongly correlated with serum creatinine, the other isoforms were not. Hepcidin-20 and -25 levels were also increased in chronic kidney disease (CKD) serum. Hepcidin-22 rapidly degraded into hepcidin-20, whereas hepcidin-25 remained relatively stable. Finally, hepcidin-22 degradation into hepcidin-20 was accelerated in the presence of HepG2. This method has enabled us to reveal fundamental characteristics of the three hepcidin isoforms in serum and may be a powerful tool for quantifying hepcidin isoform expression and processing.

Our novel method for NTBI measurement is high-throughput and may be a useful and powerful tool in the study of the physiological and clinical importance of NTBI.

Transfusion is believed to be the main cause of iron overload in Japan. A nationwide survey on post-transfusional iron overload subsequently led to the establishment of guidelines for iron chelation therapy in this country. To date, however, detailed clinical information on the entire iron overload population in Japan has not been fully investigated. In the present study, we obtained and studied detailed clinical information on the iron overload patient population in Japan. Of 1109 iron overload cases, 93.1% were considered to have occurred post-transfusion. There were, however, 76 cases of iron overload of unknown origin, which suggest that many clinicians in Japan may encounter some difficulty in correctly diagnosing and treating iron overload. Further clinical data were obtained for 32 cases of iron overload of unknown origin; median of serum ferritin was 1860.5 ng/mL. As occurs in post-transfusional iron overload, liver dysfunction was found to be as high as 95.7% when serum ferritin levels exceeded 1000 ng/mL in these patients. Gene mutation analysis of the iron metabolism-related genes in 27 cases of iron overload with unknown etiology revealed mutations in the gene coding hemojuvelin, transferrin receptor 2, and ferroportin; this indicates that although rare, hereditary hemochromatosis does occur in Japan.

Non-Tf-bound iron (NTBI), which appears in serum in iron overload, is thought to contribute to organ damage; the monitoring of serum NTBI levels may therefore be clinically useful in iron-overloaded patients. However, NTBI quantification methods remain complex, limiting their use in clinical practice. To overcome the technical difficulties often encountered, we recently developed a novel automated NTBI quantification system capable of measuring large numbers of samples. In the present study, we investigated the in vivo behavior of NTBI in human and animal serum using this newly established automated system. Average NTBI in healthy volunteers was 0.44 ± 0.076 μM (median 0.45 μM, range 0.28-0.66 μM), with no significant difference between sexes. Additionally, serum NTBI rapidly increased after iron loading, followed by a sudden disappearance. NTBI levels also decreased in inflammation. The results indicate that NTBI is a unique marker of iron metabolism, unlike other markers of iron metabolism, such as serum ferritin. Our new automated NTBI quantification method may help to reveal the clinical significance of NTBI and contribute to our understanding of iron overload.

Recent studies have revealed that tumor cells decrease their immunogenicity by epigenetically repressing the expression of highly immunogenic antigens to survive in immunocompetent hosts. We hypothesized that these epigenetically hidden “stealth” antigens should be favorable targets for cancer immunotherapy due to their high immunogenicity. To identify these stealth antigens, we treated human lung cell line A549 with DNA methyltransferase inhibitor 5‐aza‐2′‐deoxycytidine (5Aza) and its prodrug guadecitabine for 3 d in vitro and screened it using cDNA microarray analysis. We found that the gene encoding sperm equatorial segment protein 1 (SPESP1) was re‐expressed in cell lines including solid tumors and leukemias treated with 5Aza, although SPESP1 was not detected in untreated tumor cell lines. Using normal human tissue cDNA panels, we demonstrated that SPESP1 was not detected in normal human tissue except for testis and placenta. Moreover, we found using immunohistochemistry SPESP1 re‐expression in xenografts in BALB/c‐nu/nu mice that received 5Aza treatment. To assess the antigenicity of SPESP1, we stimulated human CD4+ T‐cells with a SPESP1‐derived peptide designed using a computer algorithm. After repetitive stimulation, SPESP1‐specific helper T‐cells were obtained; these cells produced interferon‐γ against HLA‐matched tumor cell lines treated with 5Aza. We also detected SPESP1 expression in freshly collected tumor cells derived from patients with acute myeloid leukemia or lung cancer. In conclusion, SPESP1 can be classified as a stealth antigen, a molecule encoded by a gene that is epigenetically silenced in tumor cells but serves as a highly immunogenic antigen suitable for cancer immunotherapy.

The role of histopathological characteristics in distinguishing amalgam-associated oral lichenoid reactions and oral lichen planus.

Background: Anemia is a significant worldwide health problem, and approximately 30% of world people suffer from anemia, the half of which is iron deficiency (ID). The diagnosis of anemia requires the confirmation of a decrease in hemoglobin (Hb) concentration. For the diagnosis of iron deficiency anemia (IDA), the determinations of serum ferritin and iron related parameters must be necessary even if microcytic hypochromic anemia is confirmed. With recent technological advances, the Hb content of reticulocytes can be quantified by flow cytometry. Reticulocytes exist for 1-2 days in the peripheral blood and its Hb levels might be a good index of ID.There are several markers for the assessment of Hb content in reticulocytes, including reticulocyte Hb equivalent (RET-He) and reticulocyte Hb content (CHr). RET-He, which can be measured in the same sample used for complete blood count tests by the latest automated hematology analyzers, is considered to reflect iron content in reticulocytes. If RET-He is capable of evaluating ID, it must be useful for immediate diagnosis of IDA. Therefore, we evaluated the usefulness of RET-He for determining of ID. Methods: This prospective study was approved by the ethics committee of Asahikawa Medical University (authorization numbers 1356, 1679, and 1356-3). Blood samples were obtained from 211 patients (63 males and 148 females) from 14 to 91 years old. RET-He levels were determined using an automated hematology analyzer (XN-3000® or XE-5000®, Sysmex, Kobe, Japan). Serum iron, total iron binding capacity (TIBC), serum ferritin, and biochemical data were measured using an automated chemical analyzer. Soluble transferrin receptor (sTfR) was measured by an enzyme-linked immunosorbent assay. Anemia was defined as Hb level of <12 g/dL. ID state was defined as serum ferritin level of <12 ng/mL. Patients were classified into four groups which are IDA, ID, control, and anemia without ID groups according to their Hb and serum ferritin levels (Table 1). Laboratory parameters were compared among four groups. The changes of RET-He during oral iron administration were also determined for 21 IDA patients. Results: There were 72 (14 males and 58 females), 28 (12 males and 16 females), 67 (23 males and 44 females), and 44 (14 males and 30 females) patients in the IDA, ID, control, and anemia without ID groups, respectively. As shown in Table 1, The median RET-He levels were 22.3 pg (15.1-35.6 pg), 29.7 pg (19.2-34.9 pg), 34.0 pg (25.9-38.0 pg), and 32.5 pg (19.1-46.3 pg) in the IDA, ID, control, and anemia without ID groups, respectively. Patients in not only IDA but ID groups had significantly lower RET-He levels than those in control group (p < 0.001) while there was no significant difference in RET-He levels between anemia without ID and control. RET-He correlated positively with serum iron (r = 0.654) and transferrin saturation (TSAT) (r = 0.666), and correlated negatively with TIBC (r = -0.617) and sTfR (r = -0.655). There was no correlation between RET-He and serum ferritin when all patients were included in the analysis (r = 0.287); however, analysis of groups according to their iron status revealed a positive correlation between RET-He and serum ferritin in the IDA and ID groups (r = 0.604). The area under the ROC curve (AUC) detecting ID for RET-He was 0.902, whereas AUC for serum iron, TIBC, TSAT, and sTfR were 0.889, 0.879, 0.922 and 0.821, respectively. The cutoff value of RET-He with maximal sensitivity and specificity was 30.9 pg, and the cutoff RET-He value of 28.5 pg had a specificity of >90% (sensitivity, 68%; specificity 91%). Among patients receiving iron treatments, the Hb levels increased in 14 patients, whereas Hb values decreased or did not change in 7 patients. Serum ferritin and RET-He values seemed to change in parallel with changes in Hb levels. Conclusions: In the present study, our data showed the efficacy of RET-He for diagnosis of IDA and the usefulness for monitoring drug iron administration. Because other parameters related to ID such as iron and ferritin should be measured biochemically in serum, it takes a longer time to measure serum iron and ferritin levels when compared with complete blood count tests. We would therefore suggest that measurement of RET-He might be useful to diagnose IDA because its assessment is rapid, fully automated, and can be measured in same sample used for complete blood count test. Disclosures Toki: Sysmex Corporation: Research Funding. Ikuta:Sysmex Corporation: Research Funding. Yamamoto:Sysmex Corporation: Research Funding. Hatayama:Sysmex Corporation: Research Funding. Shindo:Sysmex Corporation: Research Funding. Fujiya:Sysmex Corporation: Research Funding. Okumura:Sysmex Corporation: Research Funding.