Koichi Ariyoshi

Molecular mimicry by Helicobacter pylori CagA protein may be involved in the pathogenesis of H. pylori‐associated chronic idiopathic thrombocytopenic purpura

The eradication of Helicobacter pylori often leads to platelet recovery in patients with chronic idiopathic thrombocytopenic purpura (cITP). Although this clinical observation suggests the involvement of H. pylori, little is known about the pathogenesis of cITP. We initially examined the effect of H. pylori eradication on platelet counts in 20 adult Japanese cITP patients. Then, using platelet eluates as the probe in immunoblot analyses, we examined the role of molecular mimicry in the pathogenesis of cITP. Helicobacter pylori infection was detected in 75% (15 of 20) of cITP patients. Eradication was achieved in 13 (87%) of the H. pylori‐positive patients, seven (54%) of which showed increased platelet counts within the 4 months following treatment. Completely responsive patients also showed significant declines in platelet‐associated immunoglobulin G (PAIgG) levels. Platelet eluates from 12 (nine H. pylori‐positive and three H. pylori‐negative) patients recognized H. pylori cytotoxin‐associated gene A (CagA) protein, and in three completely responsive patients, levels of anti‐CagA antibody in platelet eluates declined after eradication therapy. Cross‐reactivity between PAIgG and H. pylori CagA protein suggests that molecular mimicry by CagA plays a key role in the pathogenesis of a subset of cITP patients.

Four New Pyruvate Kinase (PK) Variants and a Classical PK Deficiency

Summary. Four new red‐cell pyruvate kinase (PK) variants are presented along with one case of so‐called classical type PK deficiency. PK ‘Tokyo II’ had a low activity, Km (PEP) and Vmax, but a normal urea stability and only slight deviation from normal in neutralization tests by antiserum. It had a normal nucleotide specificity, abnormal electrophoretic mobility (fast moving) and the variant was associated with a mild haemolytic anaemia. PK ‘Maebashi’ had a low activity, high Km (PEP), low Vmax urea instability, decreased reactivity to antiserum, normal electrophoretic mobility, normal nucleotide specificity and was associated with a moderate haemolytic anaemia. PK ‘Tsukiji’ had low activity, high Km (PEP), markedly high Vmax, urea instability, decreased reactivity to antiserum, abnormal electrophoretic mobility (fast moving) and grossly abnormal nucleotide specificity especially abnormal behaviour to ADP. The haemolytic process in this case was moderate to severe. PK ‘Ube’ was electrophoretically abnormal (fast moving) but otherwise had normal characteristics and the propositus was healthy and not anaemic. PK ‘Ube’ was found by electrophoretic screening for genetic PK polymorphism. In the classical type PK deficiency, the usual red‐cell PK (PK‐R1 and PK‐R2) was not demonstrable by electrophoresis but instead M2‐type PK was present, presumably by compensatory process. Kinetic studies confirmed that the patients red‐cell PK consisted of M2‐type PK. This patient had a severe haemolytic anaemia.

MEK kinase 1 mediates the antiapoptotic effect of the Bcr-Abl oncogene through NF-κB activation

Bcr-Abl tyrosine kinase, a chimeric oncoprotein responsible for chronic myelogenous leukemia, constitutively activates several signal transduction pathways that stimulate cell proliferation and prevent apoptosis in hematopoietic cells. The antiapoptotic function of Bcr-Abl is necessary for hematopoietic transformation, and also contributes to leukemogenesis. Herein, we show for the first time that cell transformation induced by Bcr-Abl leads to increased expression and kinase activity of MEK kinase 1 (MEKK1), which acts upstream of the c-Jun N-terminal kinase (JNK), extracellular signal regulated kinase (ERK) and NF-κB signaling pathways. Inhibition of MEKK1 activity using a dominant-negative MEKK1 mutant (MEKK1km) diminished the ability of Bcr-Abl to protect cells from genotoxin-induced apoptosis, but had no effect on the proliferation of Bcr-Abl-transformed cells. Expression of MEKK1km also reduced NF-κB activation, and inhibited antiapoptotic c-IAP1 and c-IAP2 mRNA expression in response to the genotoxin. By contrast, neither JNK nor ERK activation was affected. These results indicate that MEKK1 is a downstream target of Bcr-Abl, and that the antiapoptotic effect of Bcr-Abl in chronic myelogenous leukemia cells is mediated via the MEKK1-NF-κB pathway.

Elevation of serum high‐mobility group box 1 protein during granulocyte colony‐stimulating factor‐induced peripheral blood stem cell mobilisation

High mobility group box 1 (HMGB1) is a non‐histone protein involved in maintaining the architecture of chromatin. HMGB1 also acts extracellularly as a cytokine, in processes such as inflammation, cell migration and stem cell recruitment. The involvement of HMGB1 in granulocyte colony‐stimulating factor (G‐CSF)‐induced mobilisation of haematopoietic stem cells was investigated in 21 healthy donors. G‐CSF treatment significantly elevated serum HMGB1 levels, which increased from 1·16 ± 0·86 ng/ml, before treatment, to 31·1 ± 5·99 ng/ml, after treatment. These findings suggest HMGB1 may play a role during the mobilisation of stem cells from the bone marrow into the systemic circulation.

Increased serum levels of high‐mobility group box 1 protein in patients who developed acute graft‐versus‐host disease after allogeneic hematopoietic stem cell transplantation

To the Editor: High-mobility group box protein 1 (HMGB1) is a nonhistone nuclear protein that performs a dual function. Inside the cell, HMGB1 binds DNA, regulates transcription, and determines chromosomal architecture. Outside the cell, HMGB1 activates the innate immune system and mediates a wide range of physiological and pathological responses (1). HMGB1 is released when cells are damaged. It is also actively released from monocytes or macrophages exposed to lipopolysaccharide (LPS), proinflammatory cytokines, or nitric oxide. HMGB1 reportedly transmits signals via Toll-like receptor (TLR)2, TLR4, TLR9, and the receptor for advanced glycation end-products (2). Acute graft-versus-host disease (aGVHD) is the major cause of morbidity and mortality in patients who have undergone allogeneic hematopoietic stem cell transplantation (HSCT). The pathophysiology of aGVHD can be considered to be a three-step process involving the interaction of the innate and adaptive immune systems: (I) tissue damage to the recipient as a result of the radiation ⁄ chemotherapy pretransplant conditioning regimen, (II) donor T-cell activation and clonal expansion, and (III) release of cellular and inflammatory factors (3). Our aim was to determine the association of HMGB1 with the pathogenesis of aGVHD. We enrolled 29 consecutive patients who had undergone allogeneic HSCT for a variety of disorders at our hospital. Patient characteristics are summarized in Table 1. In the time period starting from before HSCT up to discharge from our hospital, venous blood was collected once a week without documented infections. The serum samples were frozen and stored at )80 C until the assays were performed. The serum level of HMGB1 was measured in duplicate by using enzyme-linked immunosorbent assay following the manufacturer’s protocol (Shino-Test Corporation, Kanagawa, Japan). The serum HMGB1 levels in the inactive state (before HSCT and in the absence of aGVHD after HSCT; GVHD)) were compared to the active state aGVHD (grades I–III; GVHD+) (Fig. 1). The serum HMGB1 levels in the active state of aGVHD (median 1.41 ng ⁄mL, IQR 0.06– 3.27 ng ⁄mL) were significantly higher than those in the inactive state (median 0.15 ng ⁄mL, IQR 0–1.06 ng ⁄mL) (P = 0.0002, Mann–Whitney U test). The serum levels of HMGB1 in patients with a higher aGVHD grade tended to be greater than those in patients with a lower aGVHD grade; however, these differences were not statistically significant (grade I: median 0.93 ng ⁄mL, IQR 0.06–2.95 ng ⁄mL; grade II: median 1.17 ng ⁄mL, IQR 0.47–2.35 ng ⁄mL; grade III: median 1.41 ng ⁄mL, IQR 1.25–2.36 ng ⁄mL; Kruskal–Wallis test). We have shown, for the first time, that serum HMGB1 levels are elevated in patients who develop aGVHD after allogeneic HSCT. Our data suggest that HMGB1 may be involved in the pathogenesis of aGVHD. High-dose preparative regimens damage tissues, particularly in the gut, allowing LPS from bacteria in the bowel to leak into adjacent tissues and the bloodstream. Distinct classes of conserved microbial molecules and damaged cell elements, including HMGB1, probably activate TLRs on Table 1 Characteristics of patients

Effects of Surface Concentration on Drying Behavior of Carbohydrate Solutions

ABSTRACT As the surface properties of the drying materials are very important not only for the drying rate but also for the quality change during drying, the effects of surface concentration on the drying behavior of liquid foods (sugar solutions) were investigated by isothermal drying experiments and by numerical calculation experiments. The isothermal drying experiments with gelled sugar solution systems (sucrose and maltodextrin) were carried out at various relative humidity (RH) values (RH = 0 to 84%). Separate experiments were carried out for determination of the desorption isotherms. The isothermal drying curves of sugar solutions at RH = 0 to 51% were very similar. Numerical simulations also showed that the drying curves of these sugars at the surface concentration = 0 and 0.1 are almost the same, although the concentration distributions are different. When a small amount of gelatin was added to sugar solutions, the drying rate decreased remarkably as the gelatin might form a thin film (skin) near the surface, and consequently the retention of ethanol increased.

Limited detectability of linezolid-resistant Staphylococcus aureus by the Etest method and its improvement using enriched media.

The aim of this study was to evaluate Etest for detectability of linezolid-resistant meticillin-resistant Staphylococcus aureus (MRSA). The MIC of linezolid obtained by the Etest method in 18 linezolid-resistant strains of MRSA was compared with that obtained using standard agar and broth dilution methods according to Clinical and Laboratory Standards Institute guidelines. The mean linezolid MIC obtained by Etest in 18 linezolid-resistant strains of MRSA using Mueller-Hinton (MH) agar was 12.6-fold lower than that obtained by the agar dilution method, with the result that 78 % of the linezolid-resistant strains were incorrectly classified as linezolid-susceptible. The MIC of linezolid by Etest on brain-heart infusion (BHI) agar had a mean value 2.5-fold lower than that obtained by the agar dilution method, suggesting that replacing MH agar with BHI agar considerably improved the detectability of linezolid-resistant MRSA. Use of blood agar (MH agar supplemented with 5 % sheep blood) and 48 h of incubation resulted in 100 % agreement with the agar and broth dilution methods. Thus, this study revealed that the Etest on MH agar and BHI agar yielded false-negative results in a significant fraction of the linezolid-resistant MRSA. Hence, the use of blood agar and prolonged incubation is highly recommended for the accurate detection of linezolid-resistant MRSA using Etest.

Activation of the unfolded protein response in primary acute myeloid leukemia cells.

The unfolded protein response (UPR), which plays an important role in maintaining homeostasis of the endoplasmic reticulum (ER), is known to be activated in various solid tumors [1]. The role of the UPR in different forms of cancer or metastasis remains poorly characterized, and it is unclear whether UPR activation in cancer is due solely to microenvironmental stress, or to other mechanisms. The influence of the UPR on leukemogenesis remains largely uninvestigated. We previously reported that the UPR is activated in Philadelphia (Ph)-positive leukemia cells using cell lines and primary cells from Ph-positive acute lymphoid leukemia (ALL) patients [2]. In the present study, we investigated whether the UPR is activated in another type of acute leukemia, primary acute myeloid leukemia (AML).

Increased serum levels of matrix metalloproteinase-9 in acute graft-versus-host disease after allogeneic haematopoietic stem cell transplantation

Matrix metalloproteinases (MMPs) have been implicated in a variety of normal and pathological conditions that involve matrix degradation and remodelling. We investigated the role of MMPs in acute graft-versus-host disease (aGVHD) in 29 patients who had undergone allogeneic haematopoietic stem cell transplantation. The present study showed that the serum levels of MMP-9, but not those of MMP-2, significantly correlated with the occurrence and severity of aGVHD. Moreover, immunohistochemical analysis of the cutaneous lesions of patients with aGVHD revealed an increased number of inflammatory cells positive for MMP-9. These results suggest that MMP-9 might play an important role in the pathogenesis of aGVHD.