Detection of adenovirus hepatitis and acute liver failure in allogeneic hematopoietic stem cell transplant patients.

Human adenovirus (HAdV) is an important cause of the common cold and epidemic keratoconjunctivitis in immunocompetent individuals. In immunocompromised patients, HAdV can sometimes cause severe infection such as cystitis, gastroenteritis, pneumonia, encephalitis, hepatitis, or disseminated disease, resulting in significant morbidity and also mortality. In particular, severe cases have been reported in patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Indeed HAdV has been recognized as a pathogen that requires careful monitoring in allo-HSCT patients. While HAdV hepatitis leading to severe acute liver failure is rare, such liver failure progresses rapidly and is often fatal. Unfortunately, HAdV hepatitis has few characteristic symptoms and physical findings, which makes it difficult to promptly confirm and start treatment. We report here four cases of HAdV hepatitis after allo-HSCT and their autopsy findings.

Kinetics-Based Structural Requirements of Human Immunoglobulin G Binding Peptides.

Currently, antibodies are widely used not only in research but also in therapy. Hence, peptides that selectively bind to the fragment crystallizable site of an antibody have been extensively utilized in various research efforts such as the preparation of antibody-drug conjugates (ADC). Consequently, appropriate peptides that bind to immunoglobulin G (IgG) with a specific K d value and also k on and k off values will be useful in different applications, and these kinetic parameters have been perhaps overlooked but are key to development of peptide ligands with advantageous binding properties. We prepared structural derivatives of IgG-binding peptide 1 and evaluated the binding affinity and kinetic rates of the products by surface plasmon resonance assay and isothermal titration calorimetry to obtain novel peptides with beneficial antibody binding properties. In this way, 15-Lys8Leu with fast-binding and slow-release features was obtained through a shortened peptide 15-IgBP. On the other hand, we successfully obtained distinctive peptide, 15-Lys8Tle, with a similar K d value but with k on and k off values that were as much as six-fold different from those of 15-IgBP. These new peptides are useful for the elucidation of kinetic effects on the function of IgG-binding peptides and various applications of antibody or antibody-drug interactions, such as immunoliposome, ADC, or half-life extension strategy, by using a peptide with the appropriate kinetic features.

High pretransplant hepcidin levels are associated with poor overall survival and delayed platelet engraftment after allogeneic hematopoietic stem cell transplantation.

Iron overload is considered a risk factor for mortality in patients with hematopoietic malignancies. Hepcidin is a key regulator of systemic iron balance. We previously reported dynamic changes of serum hepcidin-25 levels in patients with hematologic malignancies after allogeneic hematopoietic stem cell transplantation (allo-HSCT). In this study, we retrospectively analyzed the association of pretransplant hepcidin-25 levels with overall survival (OS), engraftment, and other clinical outcomes of allo-HSCT in patients with hematologic malignancies. A total of 166 patients were divided into two groups depending on their pretransplant serum hepcidin-25 levels; their median age was 49.5 years, and the median follow-up time was 46.8 months. At 3 years, the patients in the high-hepcidin group had a significantly lower OS than those in the low-hepcidin group (49.2 vs. 69.0%, respectively; P = 0.006). Multivariate analysis revealed that pretransplant serum hepcidin-25 level, sex, and disease status were independently associated with OS. The incidence of platelet engraftment was significantly lower in the high-hepcidin group than in the low-hepcidin group, whereas no significant differences were observed in neutrophil and reticulocyte engraftments between these groups. Hence, pretransplant serum hepcidin levels can be a marker for predicting delayed platelet recovery after allo-HSCT.

Roles of transferrin receptors in erythropoiesis.

Erythropoiesis requires large amounts of iron for hemoglobin synthesis, which is mainly provided by macrophages and the intestines in a transferrin (Tf)-bound form. Bone marrow erythroblasts incorporate Tf through endocytosis, which is mediated by transferrin receptor 1 (TFR1). Recently, human TFR1, aside from its role as a Tf receptor, was also found to be a receptor for the H-subunit of ferritin (FTH). In humans, hematopoietic erythroid precursor cells express high levels of TFR1 and specifically take up the FTH homopolymer (H-ferritin). H-ferritin inhibits the formation of burst forming unit-erythroid colonies in vitro. TFR2, which is also a Tf receptor, is predominantly expressed in hepatocytes and erythroid precursor cells. In the liver, TFR2 forms a complex with HFE, a hereditary hemochromatosis-associated protein, and acts as an iron sensor. In mice, hepatocyte-specific knockout of the TFR2 gene has been shown to cause systemic iron-overload with decreased expression of hepcidin, the central regulator of iron homeostasis. In erythroid cells, TFR2 forms a complex with the erythropoietin receptor and facilitates its trafficking to the cell membrane. Moreover, hematopoietic cell-specific knockout of the TFR2 gene causes microcytic erythrocytosis in mice. This review focuses on the molecular evolution and functions of these TFRs and their ligands.

H-Ferritin Is Preferentially Incorporated by Human Erythroid Cells through Transferrin Receptor 1 in a Threshold-Dependent Manner.

Ferritin is an iron-storage protein composed of different ratios of 24 light (L) and heavy (H) subunits. The serum level of ferritin is a clinical marker of the body's iron level. Transferrin receptor (TFR)1 is the receptor not only for transferrin but also for H-ferritin, but how it binds two different ligands and the blood cell types that preferentially incorporate H-ferritin remain unknown. To address these questions, we investigated hematopoietic cell-specific ferritin uptake by flow cytometry. Alexa Fluor 488-labeled H-ferritin was preferentially incorporated by erythroid cells among various hematopoietic cell lines examined, and was almost exclusively incorporated by bone marrow erythroblasts among human primary hematopoietic cells of various lineages. H-ferritin uptake by erythroid cells was strongly inhibited by unlabeled H-ferritin but was only partially inhibited by a large excess of holo-transferrin. On the other hand, internalization of labeled holo-transferrin by these cells was not inhibited by H-ferritin. Chinese hamster ovary cells lacking functional endogenous TFR1 but expressing human TFR1 with a mutated RGD sequence, which is required for transferrin binding, efficiently incorporated H-ferritin, indicating that TFR1 has distinct binding sites for H-ferritin and holo-transferrin. H-ferritin uptake by these cells required a threshold level of cell surface TFR1 expression, whereas there was no threshold for holo-transferrin uptake. The requirement for a threshold level of TFR1 expression can explain why among primary human hematopoietic cells, only erythroblasts efficiently take up H-ferritin.

The role of growth differentiation factor 15 in the pathogenesis of primary myelofibrosis.

Growth differentiation factor 15 (GDF15) is a pleiotropic cytokine that belongs to the transforming growth factor-ß superfamily. Elevated serum concentrations of this cytokine have been reported in patients with various malignancies. To assess the potential roles of GDF15 in hematologic malignancies, we measured its serum levels in patients with these diseases. We found that serum GDF15 levels were elevated in almost all these patients, particularly in patients with primary myelofibrosis (PMF). Immunohistochemical staining of bone marrow (BM) specimens revealed that GDF15 was strongly expressed by megakaryocytes, which may be sources of increased serum GDF15 in PMF patients. Therefore, we further assessed the contribution of GDF15 to the pathogenesis of PMF. Recombinant human (rh) GDF15 enhanced the growth of human BM mesenchymal stromal cells (BM-MSCs), and it enhanced the potential of these cells to support human hematopoietic progenitor cell growth in a co-culture system. rhGDF15 enhanced the growth of human primary fibroblasts, but it did not affect their expression of profibrotic genes. rhGDF15 induced osteoblastic differentiation of BM-MSCs in vitro, and pretreatment of BM-MSCs with rGDF15 enhanced the induction of bone formation in a xenograft mouse model. These results suggest that serum levels of GDF15 in PMF are elevated, that megakaryocytes are sources of this cytokine in BM, and that GDF15 may modulate the pathogenesis of PMF by enhancing proliferation and promoting osteogenic differentiation of BM-MSCs.

A HAMP promoter bioassay system for identifying chemical compounds that modulate hepcidin expression.

Hepcidin is the central regulator of systemic iron homeostasis; dysregulation of hepcidin expression causes various iron metabolic disorders, including hereditary hemochromatosis and anemia of inflammation. To identify molecules that modulate hepcidin expression, we developed a bioassay system for hepcidin gene (HAMP) promoter activity by stable transfection of Hep3B hepatoma cells with an expression plasmid in which EGFP was linked to a 2.5-kb human HAMP promoter. Interleukin 6, bone morphogenetic protein 6 (BMP-6), and oncostatin M, well-characterized stimulators of the HAMP promoter, strongly enhanced the green fluorescence intensity of these cells. Dorsomorphin, heparin, and cobalt chloride, known inhibitors of hepcidin expression, significantly suppressed green fluorescence intensity, and these inhibitory effects were more prominent when the cells were stimulated with BMP-6. Employing this system, we screened 1,280 biologically active small molecules and found several candidate inhibitors of hepcidin expression. Apomorphine, benzamil, etoposide, CGS-15943, kenpaullone, and rutaecarpine (all at 10 µmol/L) significantly inhibited hepcidin mRNA expression by Hep3B cells without affecting cell viability. CGS-15943 was the strongest suppressor of BMP-6-induced hepcidin-25 secretion in these cells. We conclude that our newly developed hepcidin promoter bioassay system is useful for identifying and evaluating compounds that modulate hepcidin expression.

Synthesis, structure, luminescent, and magnetic properties of carbonato-bridged Zn(II)2Ln(III)2 complexes [(µ4-CO3)2{Zn(II)L(n)Ln(III)(NO3)}2] (Ln(III) = Gd(III), Tb(III), Dy(III); L(1) = N,N'-bis(3-methoxy-2-oxybenzylidene)-1,3-propanediaminato, L(2) = N,N'-bis(3-ethoxy-2-oxybenzylidene)-1,3-propanediaminato).

Carbonato-bridged Zn(II)2Ln(III)2 complexes [(µ4-CO3)2{Zn(II)L(n)Ln(III)(NO3)}2]·solvent were synthesized through atmospheric CO2 fixation reaction of [Zn(II)L(n)(H2O)2]·xH2O, Ln(III)(NO3)3·6H2O, and triethylamine, where Ln(III) = Gd(III), Tb(III), Dy(III); L(1) = N,N'-bis(3-methoxy-2-oxybenzylidene)-1,3-propanediaminato, L(2) = N,N'-bis(3-ethoxy-2-oxybenzylidene)-1,3-propanediaminato. Each Zn(II)2Ln(III)2 structure possessing an inversion center can be described as two di-µ-phenoxo-bridged {Zn(II)L(n)Ln(III)(NO3)} binuclear units bridged by two carbonato CO3(2-) ions. The Zn(II) ion has square pyramidal coordination geometry with N2O2 donor atoms of L(n) and one oxygen atom of a bridging carbonato ion at the axial site. Ln(III) ion is coordinated by nine oxygen atoms consisting of four from the deprotonated Schiff-base L(n), two from a chelating nitrate, and three from two carbonate groups. The temperature-dependent magnetic susceptibilities in the range 1.9-300 K, field-dependent magnetization from 0 to 5 T at 1.9 K, and alternating current magnetic susceptibilities under the direct current bias fields of 0 and 1000 Oe were measured. The magnetic properties of the Zn(II)2Ln(III)2 complexes are analyzed on the basis of the dicarbonato-bridged binuclear Ln(III)-Ln(III) structure, as the Zn(II) ion with d(10) electronic configuration is diamagnetic. ZnGd1 (L(1)) and ZnGd2 (L(2)) show a ferromagnetic Gd(III)-Gd(III) interaction with J(Gd-Gd) = +0.042 and +0.028 cm(-1), respectively, on the basis of the Hamiltonian H = -2J(Gd-Gd)SGd1·SGd2. The magnetic data of the Zn(II)2Ln(III)2 complexes (Ln(III) = Tb(III), Dy(III)) were analyzed by a spin Hamiltonian including the crystal field effect on the Ln(III) ions and the Ln(III)-Ln(III) magnetic interaction. The Stark splitting of the ground state was so evaluated, and the energy pattern indicates a strong easy axis (Ising type) anisotropy. Luminescence spectra of Zn(II)2Tb(III)2 complexes were observed, while those of Zn(II)2Dy(III)2 were not detected. The fine structure assignable to the (5)D4 → (7)F6 transition of ZnTb1 and ZnTb2 is in good accord with the energy pattern from the magnetic analysis. The Zn(II)2Ln(III)2 complexes (Ln(III) = Tb(III), Dy(III)) showed an out-of-phase signal with frequency-dependence in alternating current susceptibility, indicative of single molecule magnet. Under a dc bias field of 1000 Oe, the signals become significantly more intense and the energy barrier, Δ/kB, for the magnetic relaxation was estimated from the Arrhenius plot to be 39(1) and 42(8) K for ZnTb1 and ZnTb2, and 52(2) and 67(2) K for ZnDy1 and ZnDy2, respectively.

Serum neutrophil extracellular trap levels predict thrombotic microangiopathy after allogeneic stem cell transplantation.

Transplantation-associated thrombotic microangiopathy (TA-TMA) is a devastating complication of hematopoietic stem cell transplantation. TA-TMA likely represents the final stage of vascular endothelial injury; however, its pathophysiology is largely unknown, making clinical management difficult. Recently, the association of neutrophil extracellular traps (NETs) with the development of thrombotic thrombocytopenic purpura and hemolytic uremic syndrome has been reported. Thus, we explored whether NETs are also relevant to the occurrence of TA-TMA. We retrospectively analyzed post-transplant trends of serum NET levels in 90 patients, 11 of whom developed TA-TMA. Relative to baseline (before the conditioning regimen), elevated serum NET levels either at 4 weeks after transplantation or as early as the day of transplantation were associated with significantly increased risk of TA-TMA. In contrast, thrombomodulin, a potential marker for TA-TMA, was not helpful to predict the occurrence of TA-TMA in our study. In addition, we directly detected glomerular deposition of NETs in 2 TA-TMA patients. Increased NET levels are a significant risk factor for TA-TMA, suggesting that NET level is a useful biomarker for TA-TMA.

Carbonato-bridged Ni(II)2Ln(III)2 (Ln(III) = Gd(III), Tb(III), Dy(III)) complexes generated by atmospheric CO2 fixation and their single-molecule-magnet behavior: [(µ4-CO3)2{Ni(II)(3-MeOsaltn)(MeOH or H2O)Ln(III)(NO3)}2]·solvent [3-MeOsaltn = N,N'-bis(3-methoxy-2-oxybenzylidene)-1,3-propanediaminato].

Atmospheric CO2 fixation of [Ni(II)(3-MeOsaltn)(H2O)2]·2.5H2O [3-MeOsaltn = N,N'-bis(3-methoxy-2-oxybenzylidene)-1,3-propanediaminato], Ln(III)(NO3)3·6H2O, and triethylamine occurred in methanol/acetone, giving a first series of carbonato-bridged Ni(II)2Ln(III)2 complexes [(µ4-CO3)2{Ni(II)(3-MeOsaltn)(MeOH)Ln(III)(NO3)}2] (1Gd, 1Tb, and 1Dy). When the reaction was carried out in acetonitrile/water, it gave a second series of complexes [(µ4-CO3)2{Ni(II)(3-MeOsaltn)(H2O)Ln(III)(NO3)}2]·2CH3CN·2H2O (2Gd, 2Tb, and 2Dy). For both series, each Ni(II)2Ln(III)2 structure can be described as two di-µ-phenoxo-bridged Ni(II)Ln(III) binuclear units bridged by two carbonato CO3(2-) units to form a carbonato-bridged (µ4-CO3)2{Ni(II)2Ln(III)2} structure. The high-spin Ni(II) ion has octahedral coordination geometry, and the Ln(III) ion is coordinated by O9 donor atoms from Ni(II)(3-MeOsaltn), bidentate NO3(-), and one and two oxygen atoms of two CO3(2-) ions. The NO3(-) ion for the first series roughly lie on Ln-O(methoxy) bonds and are tilted toward the outside, while for the second series, the two oxygen atoms roughly lie on one of the Ln-O(phenoxy) bonds due to the intramolecular hydrogen bond. The temperature-dependent magnetic susceptibilities indicated a ferromagnetic interaction between the Ni(II) and Ln(III) ions (Ln(III) = Gd(III), Tb(III), Dy(III)) for all of the complexes, with a distinctly different magnetic behavior between the two series in the lowest-temperature region due to the Ln(III)-Ln(III) magnetic interaction and/or different magnetic anisotropies of the Tb(III) or Dy(III) ion. Alternating-current susceptibility measurements under the 0 and 1000 Oe direct-current (dc) bias fields showed no magnetic relaxation for the Ni(II)2Gd(III)2 complexes but exhibited an out-of-phase signal for Ni(II)2Tb(III)2 and Ni(II)2Dy(III)2, indicative of slow relaxation of magnetization. The energy barriers, Δ/kB, for the spin flipping were estimated from the Arrhenius plot to be 12.2(7) and 6.1(3) K for 1Tb and 2Tb, respectively, and 18.1(6) and 14.5(4) K for 1Dy and 2Dy, respectively, under a dc bias field of 1000 Oe. Compound 1Dy showed relatively slow relaxation of magnetization reorientation even at zero dc applied field with Δ/kB = 6.6(4) K.

Differing impacts of pretransplant serum ferritin and C-reactive protein levels on the incidence of chronic graft-versus-host disease after allogeneic hematopoietic stem cell transplantation.

Studies have suggested an association between pretransplant serum levels of ferritin and C-reactive protein (CRP) and complications of allogeneic hematopoietic stem cell transplantation (HSCT). To evaluate the prognostic impact of these biomarkers on the development of acute and chronic graft-versus-host disease (GVHD), we retrospectively studied 211 patients who underwent allogeneic HSCT for hematologic diseases at our institution. The cumulative incidence rate of chronic GVHD at 3 years was 40.7 %. In the multivariate analysis, elevated CRP levels (≥2 mg/L) were significantly associated with a high incidence of chronic GVHD, whereas high ferritin levels (≥880 ng/mL) showed a tendency, though not statistically significant, to association with a low incidence of chronic GVHD. No significant association was observed between the pretransplant serum ferritin or CRP levels and the incidence of acute GVHD. Multivariate analysis indicated that high pretransplant serum ferritin levels were significantly associated with increases in treatment-related mortality and relapse rates. Overall, an elevated pretransplant serum ferritin level, but not an elevated serum CRP level, is a strong risk factor for overall mortality (hazard ratio, 2.16; P = 0.002). Our results also indicate that pretransplant serum CRP levels may be a useful biomarker for predicting the risk of chronic GVHD.

Acidic milieu augments the expression of hepcidin, the central regulator of iron homeostasis.

Hepcidin is the central regulator of body iron homeostasis, and dysregulation of hepcidin expression causes various clinical disorders, such as anemia and hemochromatosis. Various stimuli, including iron load and interleukin-6, are involved in the regulation of hepcidin expression. We previously reported that serum hepcidin levels were high in patients with end-stage renal disease, compared with healthy subjects. Since metabolic acidosis is commonly observed in these patients, we hypothesized that acidic milieu might augment hepcidin expression. In this study, we investigated the effect of changes in the pH of the microenvironment on hepcidin expression in human hepatoma and leukemia cell lines. We found that hepcidin expression in these cells was augmented by the acidic milieu created with lactic acid, hydrochloric acid and excess carbon dioxide. Acidic milieu did not clearly enhance hepcidin promoter activity, but rather stabilized hepcidin transcript in the hepatoma cells. We speculate that metabolic acidosis may contribute in part to the elevation of serum hepcidin levels in patients with end-stage chronic kidney disease. Further studies are needed to elucidate the association between acidosis and hepcidin expression in various clinical settings.

A case of congenital dyserythropoietic anemia type 1 in a Japanese adult with a CDAN1 gene mutation and an inappropriately low serum hepcidin-25 level.

We describe the first case of genetically diagnosed congenital dyserythropoietic anemia (CDA) type 1 in a Japanese man. The patient had hemolytic anemia since he was a child, and he developed diabetes, hypogonadism, and liver dysfunction in his thirties, presumably from systemic iron overload. When he was 48 years old a diagnosis was finally made by genetic analysis that revealed a homozygous mutation of CDAN1 gene (Pro1129Leu). His serum hepcidin-25 level was inappropriately low. We conclude that physicians should be aware of the possibility of CDA in a patient with anemia and systemic iron overload at any age.

Durable hematological response and improvement of nephrotic syndrome on thalidomide therapy in a patient with refractory light chain deposition disease.

Light chain deposition disease (LCDD) is a rare disease for which an optimal treatment is not yet available. Here, we report the clinical course of a 32-year-old woman with LCDD who was successfully treated with thalidomide. She presented with nephrotic syndrome. Based on the renal biopsy findings and the presence of monoclonal immunoglobulin light chains in her serum and urine, LCDD was diagnosed. Prednisolone and cytotoxic chemotherapy used for multiple myeloma proved ineffective. We initiated administration of thalidomide (100 mg daily) and dexamethasone (20 mg for 4 days per month). After 8 months of treatment, she achieved complete hematological remission, defined as the disappearance of monoclonal protein and a normalized free light chain ratio, which led to improvement of her renal insufficiency. She has shown sustained hematological and organ response for 31 months with thalidomide therapy. Thus, thalidomide therapy seems to be a promising approach to the treatment of LCDD.