Measurement of serum hepcidin-25 by latex agglutination in healthy volunteers and patients with hematologic disorders.

Iron is an essential trace metal, vital for various physiologic processes, but excess levels can harm health. Maintaining iron homeostasis is critical, with hepcidin playing a key role. The isoform hepcidin-25 exerts the most significant influence on iron metabolism, making its serum levels a valuable diagnostic tool. However, mass-spectrometry and other conventional measurement methods can be difficult to perform, and some immunoassays lack reliability. In this study, we employed a recently developed latex agglutination method integrated with a readily available automated analyzer to quantify serum hepcidin-25 levels in both volunteers recruited from personnel of our hospital (n = 93) and patients with various hematological disorders (n = 112). Our findings unveiled a robust positive correlation between serum hepcidin-25 and ferritin, as well as C-reactive protein levels, in both volunteers and patients. Among the patients with hematological disorders, there was a noteworthy negative correlation between hepcidin-25 levels and hemoglobin concentrations, as well as reticulocyte counts. Interestingly, the hepcidin-25/ferritin ratio was remarkably low in patients with hemolytic anemia and myelodysplastic syndromes with ring sideroblasts. Our findings suggest that quantifying serum hepcidin-25 and the hepcidin-25/ferritin ratio using this method may be valuable for screening of hematopoietic diseases and other iron metabolism disorders.

Clinical impact and characteristics of erythroid dysplasia in adult aplastic anaemia: Results from a multicentre registry.

Morphological dysplasia in haematopoietic cells, defined by a 10% threshold in each lineage, is one of the diagnostic criteria for myelodysplastic neoplasms. Dysplasia limited to the erythroid lineage has also been reported in some cases of aplastic anaemia (AA); however, its significance remains unclear. We herein examined the impact of erythroid dysplasia on immunosuppressive therapy responses and survival in AA patients. The present study included 100 eligible AA patients without ring sideroblasts. Among them, 32 had dysplasia in the erythroid lineage (AA with minimal dysplasia [mini-D]). No significant sex or age differences were observed between AA groups with and without erythroid dysplasia. In severe/very severe AA and non-severe AA patients, a response to anti-thymocyte globulin + ciclosporin within 12 months was observed in 80.0% and 60.0% of AA with mini-D and 42.9% and 90.0% of those without dysplasia, with no significant difference (p = 0.29 and p = 0.24 respectively). Overall survival and leukaemia-free survival did not significantly differ between the groups. Collectively, the present results indicate that the presence of erythroid dysplasia did not significantly affect clinical characteristics or outcomes in AA patients, suggesting that its presence in AA is acceptable. Therefore, erythroid dysplasia should not exclude an AA diagnosis.

Weak response of bovine hepcidin induction to iron through decreased expression of Smad4.

Hepcidin negatively regulates systemic iron levels by inhibiting iron entry into the circulation. Hepcidin production is increased in response to an increase in systemic iron via the activation of the bone morphogenetic protein (BMP) pathway. Regulation of hepcidin expression by iron status has been proposed on the basis of evidence mainly from rodents and humans. We evaluated the effect of iron administration on plasma hepcidin concentrations in calves and the expression of bovine hepcidin by the BMP pathway in a cell culture study. Hematocrit as well as levels of blood hemoglobin and plasma iron were lower than the reference level in calves aged 1-4 weeks. Although intramuscular administration of iron increased iron-related parameters, plasma hepcidin concentrations were unaffected. Treatment with BMP6 increased hepcidin expression in human liver-derived cells but not in bovine liver-derived cells. A luciferase-based reporter assay revealed that Smad4 was required for hepcidin reporter transcription induced by Smad1. The reporter activity of hepcidin was lower in the cells transfected with bovine Smad4 than in those transfected with murine Smad4. The lower expression levels of bovine Smad4 were responsible for the lower activity of the hepcidin reporter, which might be due to the instability of bovine Smad4 mRNA. In fact, the endogenous Smad4 protein levels were lower in bovine cells than in human and murine cells. Smad4 also confers TGF-ß/activin-mediated signaling. Induction of TGF-ß-responsive genes was also lower after treatment with TGF-ß1 in bovine hepatocytes than in human hepatoma cells. We revealed the unique regulation of bovine hepcidin expression and the characteristic TGF-ß family signaling mediated by bovine Smad4. The present study suggests that knowledge of the regulatory expression of hepcidin as well as TGF-ß family signaling obtained in murine and human cells is not always applicable to bovine cells.

The Usefulness of Machine Learning-Based Evaluation of Clinical and Pretreatment [18F]-FDG-PET/CT Radiomic Features for Predicting Prognosis in Hypopharyngeal Cancer.

PURPOSE: To examine whether the machine learning (ML) analyses using clinical and pretreatment 2-deoxy-2-[18F]fluoro-D-glucose positron emission tomography ([18F]-FDG-PET)-based radiomic features were useful for predicting prognosis in patients with hypopharyngeal cancer. PROCEDURES: This retrospective study included 100 patients with hypopharyngeal cancer who underwent [18F]-FDG-PET/X-ray computed tomography (CT) before treatment, and these patients were allocated to the training (n=80) and validation (n=20) cohorts. Eight clinical (age, sex, histology, T stage, N stage, M stage, UICC stage, and treatment) and 40 [18F]-FDG-PET-based radiomic features were used to predict disease progression. A feature reduction procedure based on the decrease of the Gini impurity was applied. Six ML algorithms (random forest, neural network, k-nearest neighbors, naïve Bayes, logistic regression, and support vector machine) were compared using the area under the receiver operating characteristic curve (AUC). Progression-free survival (PFS) was assessed using Cox regression analysis. RESULTS: The five most important features for predicting disease progression were UICC stage, N stage, gray level co-occurrence matrix entropy (GLCM_Entropy), gray level run length matrix run length non-uniformity (GLRLM_RLNU), and T stage. Patients who experienced disease progression displayed significantly higher UICC stage, N stage, GLCM_Entropy, GLRLM_RLNU, and T stage than those without progression (each, p<0.001). In both cohorts, the logistic regression model constructed by these 5 features was the best performing classifier (training: AUC=0.860, accuracy=0.800; validation: AUC=0.803, accuracy=0.700). In the logistic regression model, 5-year PFS was significantly higher in patients with predicted non-progression than those with predicted progression (75.8% vs. 8.3%, p<0.001), and this model was only the independent factor for PFS in multivariate analysis (hazard ratio = 3.22; 95% confidence interval = 1.03-10.11; p=0.045). CONCLUSIONS: The logistic regression model constructed by UICC, T and N stages and pretreatment [18F]-FDG-PET-based radiomic features, GLCM_Entropy, and GLRLM_RLNU may be the most important predictor of prognosis in patients with hypopharyngeal cancer.

TAFRO Syndrome: A Disease Requiring Immediate Medical Attention.

TAFRO syndrome was first described in 2010, standing for thrombocytopenia, anasarca, fever, reticulin fibrosis and organomegaly. Because the lymph node histopathology of TAFRO syndrome mimics idiopathic multicentric Castleman disease (iMCD), some researchers consider TAFRO syndrome to be a subtype of iMCD. However, the clinical features of TAFRO syndrome considerably differ from those of iMCD without TAFRO. The clinical features of patients with TAFRO syndrome with or without iMCD-histopathology are similar, and these patients require an accurate diagnosis and urgent treatment. Although a histological diagnosis, including a differential diagnosis, is important, lymph node involvement in patients with TAFRO syndrome is usually modest or sometimes absent. Furthermore, a bleeding tendency due to thrombocytopenia and severe anasarca hampers performing a biopsy. Nonetheless, patients with various other disorders may manifest TAFRO syndrome-like symptoms, making the differential diagnosis in borderline cases difficult. Therefore, the establishment of precise and specific biomarkers is important.

[Evaluation of oral medications in the geriatric medical care ward based on the Guidelines for Appropriate Use of Medications for the Elderly (2018), and an analysis of the number of oral medicines in relation to the nutritional route and activities of daily living (ADL) categories].

AIM: This study aimed to evaluate the use of potentially inappropriate medications (PIMs) and to examine the number of oral medicines based on the swallowing function and activities of daily living (ADL) categories in a geriatric medical care ward. METHODS: A prospective investigation of oral medication use of 124 consecutive patients (male, n=58; female, n=66) admitted to a geriatric medical care ward was conducted from November 2019 to October 2020. Nutritional routes and ADL categories were quantitatively assessed, and the respective medication quantities were subjected to a statistical analysis. RESULTS: The average number of oral medications was 5.8 at acute care admission, 4.4 upon transfer to the geriatric medical care ward and 4.8 at discharge. Approximately 30% of oral medications were classified as PIMs, including antithrombotic agents, diuretics, antidiabetic drugs, magnesium oxide, sleep and anxiolytic medications, and antipsychotic drugs. Magnesium oxide, antipsychotic drugs, sleep and anxiolytic medications were frequently discontinued during the patient's stay at the geriatric medical care ward. The proportion of PIMs significantly decreased from 35.1% at admission, to 28.8% at ward transfer, and 24.3% at discharge (P<0.01). The number of oral medicines at discharge varied based on the nutritional route, with averages of 5.5 for oral intake, 3.6 for enteral nutrition, and 0.7 for venous nutrition. It also varied based on ADL categories, with averages of 6.0 for ADL 1, 5.8 for ADL 2, and 3.8 for ADL 3. CONCLUSION: The use of PIMs decreased in the geriatric medical care ward. A reduced swallowing function and lower ADL were associated with a decrease in the quantity of oral medicines.

A tetravalent peptide that binds to the RANK-binding region of TRAF6 via a multivalent interaction efficiently inhibits osteoclast differentiation.

Inhibition of osteoclast differentiation is a promising approach for the treatment of osteoporosis and rheumatoid arthritis. Receptor activator of nuclear factor kappa B (NF-κB) (RANK), which is an essential molecule for osteoclast differentiation, interacts with tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) to transduce downstream signals. Both RANK and TRAF6 have homo-trimeric structures, forming a multivalent interaction between the Pro-X-Glu-X-X-(aromatic/acidic) motif of RANK and the C-terminal domain of TRAF6 (TRAF-C), that markedly increases the binding affinity. Here, we designed a tetravalent peptide, RANK-tet, containing the TRAF-C-binding motif of RANK and found that RANK-tet binds to TRAF-C with high affinity. In contrast, a monomeric form of RANK-tet (RANK-mono) with the same TRAF-C-binding motif did not bind to TRAF-C, clearly indicating the multivalent interaction is strictly required for the high-affinity binding to TRAF-C. RANK-tet did not bind to a series of TRAF-C-mutants with an amino acid substitution in the RANK-binding region, indicating that RANK-tet specifically targets the RANK-binding region of TRAF-C. A cell-permeable form of RANK-tet that has poly-Arg residues at each C-terminal of the TRAF-C-binding motif efficiently inhibited the RANK ligand (RANKL)-induced differentiation of bone marrow cells to osteoclasts. Thus, this compound can be an effective anti-osteoclastogenic agent.

Intravenous ferric derisomaltose versus saccharated ferric oxide for iron deficiency anemia associated with menorrhagia: a randomized, open-label, active-controlled, noninferiority study.

A multicenter, randomized, open-label, phase III study was conducted to compare the efficacy and safety of intravenous ferric derisomaltose (FDI) versus saccharated ferric oxide (SFO) in Japanese patients with iron deficiency anemia associated with menorrhagia. FDI can be administered as a single dose up to 1000 mg, whereas SFO has a maximum single dose of 120 mg. The primary endpoint, which was the maximum change in hemoglobin concentration from baseline, was noninferior for the FDI group compared with the SFO group. The incidence of treatment-emergent adverse events was lower in the FDI group (66.2%) than in the SFO group (90.8%). Notably, the incidence of serum phosphorus level < 2.0 mg/dL was significantly lower in the FDI group (8.4%) than in the SFO group (83.2%), and severe hypophosphatemia (≤ 1.0 mg/dL) occurred in 6.7% of SFO­treated patients compared with none in the FDI group. The percentage of patients who achieved the cumulative total iron dose during the 8-week treatment period was higher in the FDI group (92.8%) than in the SFO group (43.2%). The study met its primary endpoint, and also demonstrated the tolerability of a high dose of FDI per infusion, with a lower incidence of hypophosphatemia.

ASXL1 mutations with serum EPO levels predict poor response to darbepoetin alfa in lower-risk MDS: W-JHS MDS01 trial.

Darbepoetin alfa (DA) is used to treat anemia in lower-risk (IPSS low or int-1) myelodysplastic syndromes (MDS). However, whether mutations can predict the effectiveness of DA has not been examined. The present study aimed to determine predictive gene mutations. The primary endpoint was a correlation between the presence of highly frequent (≥ 10%) mutations and hematological improvement-erythroid according to IWG criteria 2006 by DA (240 µg/week) until week 16. The study included 79 patients (age 29-90, median 77.0 years; 52 [65.8%] male). Frequently (≥ 10%) mutated genes were SF3B1 (24 cases, 30.4%), TET2 (20, 25.3%), SRSF2 (10, 12.7%), ASXL1 (9, 11.4%), and DNMT3A (8, 10.1%). Overall response rate to DA was 70.9%. Multivariable analysis including baseline erythropoietin levels and red blood cell transfusion volumes as variables revealed that erythropoietin levels and mutations of ASXL1 gene were significantly associated with worse response (odds ratio 0.146, 95% confidence interval 0.042-0.503; p = 0.0023, odds ratio 0.175, 95% confidence interval 0.033-0.928; p = 0.0406, respectively). This study indicated that anemic patients who have higher erythropoietin levels and harbor ASXL1 gene mutations may respond poorly to DA. Alternative strategies are needed for the treatment of anemia in this population. Trial registration number and date of registration: UMIN000022185 and 09/05/2016.

Intravenous ferric derisomaltose for iron-deficiency anemia associated with gastrointestinal diseases: a single-arm, randomized, uncontrolled, open-label study.

Iron-deficiency anemia (IDA) associated with gastrointestinal diseases is the second most common etiology of IDA in Japan, and is most often caused by gastrointestinal bleeding. A multicenter, single-arm (2 groups), open-label, phase III study was conducted to assess the efficacy and safety of ferric derisomaltose (FDI) when administered by intravenous (IV) bolus injection (n = 30) or drip infusion (n = 10) in Japanese patients with IDA associated with gastrointestinal diseases. The primary endpoint, which was the mean maximum change in hemoglobin (Hb) concentration from baseline, was 4.33 (95% confidence interval, 3.82-4.83) g/dL in the overall population (4.27 [3.83-4.71] g/dL in the bolus injection group and 4.49 [2.69-6.29] g/dL in the drip infusion group). Treatment-emergent adverse events (TEAEs) were reported in 24 patients (60.0%) in the overall population (18 patients [60.0%] in the bolus injection group and 6 patients [60.0%] in the drip infusion group). No serious treatment-related TEAEs or unexpected safety findings were reported during the study. These findings reveal a favorable efficacy and safety profile for FDI when administered by IV bolus injection or drip infusion in Japanese patients with IDA associated with gastrointestinal diseases.

Prognostic impacts of serum levels of C-reactive protein, albumin, and total cholesterol in patients with myelodysplastic syndromes.

Various systems for predicting the prognosis of patients with myelodysplastic syndromes (MDS) have been developed. However, associations between performance status (PS) and prognosis of MDS require further investigation. To objectively assess the impact of PS on survival, we examined laboratory findings associated with PS, including serum levels of C-reactive protein (CRP), albumin (ALB), and total cholesterol (CHOL). Patients (n = 123; male 86, female 37; median age 74 yrs.) diagnosed with MDS or myelodysplastic/myeloproliferative neoplasms at Kanazawa Medical University Hospital between 2010 and 2020 were enrolled and grouped by cutoff values determined by receiver operating characteristic analysis: 0.44 mg/dL for CRP, 4.0 g/dL for ALB, and 120 mg/dL for CHOL. The median follow-up period was 17.6 months. Kaplan-Meier analysis revealed that overall survival (OS) in the high CRP, low ALB, and low CHOL groups was significantly shorter than in the low CRP, high ALB, and high CHOL groups, respectively. Multivariable analysis revealed that elevated serum CRP was an independent prognostic risk factor independent of gender, bone marrow blast percentage, and cytogenetics.

Castleman disease and TAFRO syndrome.

Although Castleman disease was first described in 1956, this disease includes various conditions, including unicentric Castleman disease with hyaline vascular histology, human herpesvirus-8 (HHV-8) related multicentric Castleman disease, idiopathic multicentric Castleman disease, and mimics of Castleman disease associated with other conditions. To date, Castleman disease remains incompletely understood due to its rareness and difficulties in clinical and pathological diagnosis. TAFRO syndrome was reported in Japan in 2010. Because lymph node histology is similar in patients with TAFRO syndrome and Castleman disease, TAFRO syndrome is described as a related disorder of Castleman disease. Clinically, however, these conditions differ markedly. Although elevated interleukin-6 (IL-6) expression is characteristic of Castleman disease, increased expression of IL-6 may occur in patients with other diseases, making elevated IL-6 unsuitable for differential diagnosis. Further understanding of these disorders requires the identification of novel disease-specific biomarkers. This review article therefore outlines the characteristics of Castleman disease and TAFRO syndrome.

Azacitidine is a potential therapeutic drug for pyridoxine-refractory female X-linked sideroblastic anemia.

X-linked sideroblastic anemia (XLSA) is associated with mutations in the erythroid-specific δ-aminolevulinic acid synthase (ALAS2) gene. Treatment of XLSA is mainly supportive, except in patients who are pyridoxine responsive. Female XLSA often represents a late onset of severe anemia, mostly related to the acquired skewing of X chromosome inactivation. In this study, we successfully generated active wild-type and mutant ALAS2-induced pluripotent stem cell (iPSC) lines from the peripheral blood cells of an affected mother and 2 daughters in a family with pyridoxine-resistant XLSA related to a heterozygous ALAS2 missense mutation (R227C). The erythroid differentiation potential was severely impaired in active mutant iPSC lines compared with that in active wild-type iPSC lines. Most of the active mutant iPSC-derived erythroblasts revealed an immature morphological phenotype, and some showed dysplasia and perinuclear iron deposits. In addition, globin and HO-1 expression and heme biosynthesis in active mutant erythroblasts were severely impaired compared with that in active wild-type erythroblasts. Furthermore, genes associated with erythroblast maturation and karyopyknosis showed significantly reduced expression in active mutant erythroblasts, recapitulating the maturation defects. Notably, the erythroid differentiation ability and hemoglobin expression of active mutant iPSC-derived hematopoietic progenitor cells (HPCs) were improved by the administration of δ-aminolevulinic acid, verifying the suitability of the cells for drug testing. Administration of a DNA demethylating agent, azacitidine, reactivated the silent, wild-type ALAS2 allele in active mutant HPCs and ameliorated the erythroid differentiation defects, suggesting that azacitidine is a potential novel therapeutic drug for female XLSA. Our patient-specific iPSC platform provides novel biological and therapeutic insights for XLSA.

Comprehensive analysis of protein-expression changes specific to immunoglobulin G4-related disease.

BACKGROUND AND AIMS: Immunoglobulin 4 (IgG4)-related disease (IgG4-RD) is a lymphoproliferative disorder characterized by elevated serum IgG4 levels and tissue infiltration of IgG4-positive plasma cells. We analyzed the serum proteins, whose levels varied based on the disease state and treatment. MATERIALS AND METHODS: Serum proteins from patients with IgG4-related disease and healthy subjects were resolved using two-dimensional electrophoresis, silver-stained, and scanned. Alternatively, the proteins were labeled with Cy2, Cy3, and Cy5 before electrophoresis. The proteins, whose expression differed significantly between patients and healthy individuals, and between before and after steroid treatment, were identified and validated using enzyme-linked immunosorbent assays. RESULTS: Pre-treatment sera from patients with IgG4-related disease was characterized by increased levels of immunoglobulins such as IgG1, IgG4; inflammatory factors such as α-1 antitrypsin (A1AT); and proteins associated with immune system regulation such as clusterin and leucine-rich α-2-glycoprotein (LRG-1). The serum levels of A1AT, LRG-1 and clusterin, during treatment with prednisolone for up to 12 months revealed that LRG-1 levels were halved after 1 month of treatment, comparable to those in healthy subjects; LRG-1 levels remained normal until the end of treatment. CONCLUSION: LRG-1 could serve as a novel biomarker of IgG4-related diseases.

Patient's age and D-dimer levels predict the prognosis in patients with TAFRO syndrome.

OBJECTIVES: To identify prognostic factors for TAFRO syndrome, a rare inflammatory disorder of unknown etiology characterized by thrombocytopenia, anasarca, fever, reticulin myelofibrosis, renal dysfunction, and organomegaly. METHODS: Data of patients with TAFRO syndrome were extracted from a Japanese patient registry. Patients were divided into groups according to the clinical and laboratory parameters at initial presentation. Cut-off values for the laboratory parameters were determined using receiver operating characteristic curve analysis and by clinical relevance. Patient survival was analyzed by the Kaplan-Meier method. Univariable analysis was performed using log-rank tests. Multivariable analyses were performed with the logistic regression model and the Cox proportional hazards model. RESULTS: We extracted the data of 83 patients with TAFRO syndrome from the registry. Univariable analysis identified several potential prognostic factors. Of these factors, age ≥60 years and D-dimer ≥18 µg/dL remained significant predictors of poor overall survival in the multivariable Cox proportional hazards model. Based on these results, we developed a simple prognostic scoring system for TAFRO syndrome (TS-PSS). CONCLUSION: Patients in our cohort were stratified into low, intermediate, and high-risk groups by the TS-PSS. This system should be verified with independent patient cohorts in future studies.

Optimal treatments for TAFRO syndrome: a retrospective surveillance study in Japan.

TAFRO syndrome is a systemic inflammatory disorder of unknown etiology characterized by thrombocytopenia, anasarca, fever, reticulin myelofibrosis, renal dysfunction, and organomegaly. Mortality in patients with this syndrome is high; however, an optimal treatment strategy has not been established. To explore the strategy, we retrospectively analyzed 81 patients with TAFRO syndrome registered in the Multicenter Collaborative Retrospective Study for Establishing the Concept of TAFRO Syndrome in Japan by December 2019. Sixty-eight patients received corticosteroid therapy as the first-line treatment, and as the second-line treatment, 21 received tocilizumab (Toc), 14 received cyclosporine A (CsA), and 8 received rituximab (Rit) in addition to corticosteroids. We compared these second-line treatment groups by setting the primary endpoint as time to next treatment or death (TTNT). Kaplan-Meier analysis showed that the median TTNT in the Toc, CsA, and Rit groups were 2.8 months, 9.2 months, and not reached, respectively. The TTNT of the Rit group was significantly longer than that of the Toc group. In contrast, there were no significant differences in overall survival between groups, indicating that subsequent salvage therapies rescued a large proportion of patients who failed the second-line treatments. Further studies are warranted to establish the optimal treatment strategies for this syndrome.

[The pathogenesis of anemia in inflammation].

Inflammation is a physiological process that primarily occurs as a way to help protect the host against tissue damage and invasion by pathogens. During inflammation, erythropoiesis is suppressed and, if it lasts, anemia develops. The mechanisms underlying this are complex and not fully understood, but various cytokines, such as tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-1ß (IL-1ß), and IL-6, are involved. TNF-α upregulates PU.1, which is a crucial transcription factor in granulocytic differentiation, and downregulates GATA-1, a master transcription factor for erythroid differentiation, in hematopoietic stem cells. TNF-α and IL-1ß suppress erythropoietin production in the kidney, whereas IFN-γ downregulates the expression of erythropoietin receptors in erythroid precursor cells. Moreover, IL-6 upregulates the production of hepcidin, the master regulator of systemic iron metabolism, in the liver. Hepcidin reduces the iron available for erythropoiesis by downregulating the rate of iron release from macrophages. Activated macrophages may also contribute to the development of anemia by shortening the erythrocyte lifespan. Proper management of the underlining conditions is necessary in treating anemia associated with inflammation. Erythropoiesis-stimulating agents may be administered to patients with chronic kidney disease, whereas anti-IL-6 agents may be beneficial for anemic patients with rheumatoid arthritis and idiopathic multicentric Castleman disease.