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BackgroundRecent increases in the number of patients with non-alcoholic steatohepatitis (NASH) warrant the identification of biomarkers for early detection of hepatocellular carcinoma (HCC) associated with NASH (NASH-HCC). IgM-free apoptosis inhibitor of macrophage (AIM), which generally associates with IgM in blood and exerts its biological function by dissociation from IgM, may serve as an effective biomarker for NASH-HCC. Here, we established a fully automatic and high-throughput electrochemiluminescence immunoassay (ECLIA) to measure IgM-free AIM and investigated its efficacy in diagnosing NASH-HCC and viral HCC.MethodsIgM-free AIM levels were measured in 212 serum samples from patients with, or without, HCC related to NASH, hepatitis B virus, and hepatitis C virus, using ECLIA. We also developed an ECLIA for measuring both IgM-free and IgM-bound AIM and investigated the existing form of AIM in blood by size-exclusion chromatography.ResultsIgM-free AIM levels were significantly higher in the HCC group than in the non-HCC group, regardless of the associated pathogenesis. Moreover, the area under the receiver operating curve for IgM-free AIM was greater than that for conventional HCC biomarkers, alpha-fetoprotein or des-γ-carboxy prothrombin, regardless of the cancer stage. ECLIA counts of IgM-free AIM derived from samples fractionated by size-exclusion chromatography were significantly higher in patients with NASH-HCC than in healthy volunteers and in patients with non-alcoholic fatty liver and NASH.ConclusionsSerum IgM-free AIM may represent a universal HCC diagnostic marker superior to alpha-fetoprotein or des-γ-carboxy prothrombin. Our newly established ECLIA could contribute to further clinical studies on AIM and in vitro HCC diagnosis.

Circulating immunoglobulin M (IgM) exists in a pentameric form, possessing a polyreactive nature that responds not only to foreign antigens but also to autoantigens; thus, it is involved in both beneficial and detrimental immune responses, including protection from infection and the progression of autoimmunity. On the other hand, IgM also behaves as a carrier of the apoptosis inhibitor of macrophage (AIM) protein, storing a large amount of the inactivated form of AIM in the blood through this association. Under different disease conditions, AIM can dissociate from IgM locally or systemically to exert its function, inducing the removal of various biological debris such as excess fat, bacteria, cancer cells or dead cell debris. Most typically, upon induction of acute kidney injury (AKI), IgM-free AIM is filtered by the glomerulus in the kidney, which stimulates the clearance of intraluminal dead cells debris at the obstructed proximal tubules, thereby facilitating the repair of kidney injury. Interestingly, cats exhibit a deficiency in AIM release from IgM, which may increase their susceptibility to renal failure. Conversely, association with AIM inhibits IgM binding to the Fcα/μ receptor on follicular dendritic cells at the splenic germinal center, thereby protecting the IgM immune complex from Fcα/μ receptor-mediated internalization, which supports IgM-dependent antigen presentation to B cells and stimulates high-affinity IgG antibody production. The regulation of AIM-IgM binding, resulting from the discovery of reciprocal actions between AIM and IgM, could lead to the development of novel therapies against different diseases.

Crossover stenting of femoral bifurcation raises the concern of jeopardizing the deep femoral artery (DFA) entry, thereby increasing future risk of limb-threatening ischemia and amputation. This retrospective multicenter study compared clinical outcomes of crossover stenting and non-crossover stenting for ostial superficial femoral artery (SFA) lesions. We reviewed 125 limbs in 103 patients with successful stent implantation for ostial SFA lesions and allocated them to two groups, based on whether the stent crossed over the DFA orifice (CO, n = 54) or not (NC, n = 71). The decision of applying whether CO or NC was at the operators' discretion. Primary endpoints were incidences of major amputation and acute limb ischemia (ALI) at 24 months, and secondary endpoints were incidences of death, target lesion revascularization (TLR), composite of amputation or death, and major adverse limb events which was a composite of major amputation, ALI, TLR, or death at 24 months. Baseline characteristics were similar between the groups. Major amputation occurred only in the NC group, while ALI occurred only in the CO group. Kaplan-Meier estimation showed no significant differences in incidences of major amputation (NC: 3.0% vs. CO: 0.0%, p = 0.21), ALI, or any of the secondary endpoints. However, there was a trend towards higher incidence of ALI in the CO group (NC 0.0% vs. CO 3.9%, p = 0.11). Crossover stenting did not result in a significant difference in major amputation compared to non-crossover stenting within 24 months. However, it showed a trend towards higher incidence of ALI.

Apoptosis inhibitor of macrophage (AIM) expressed on macrophages prolongs inflammation by protecting macrophages from apoptosis. Most circulating AIM co-exists with immunoglobulin M (IgM). AIM’s pathophysiological role in relation to IgM remains unclear. Here we evaluated the glomerular expression/deposition of AIM and IgM in the kidney using immunohistochemistry and its associations with clinical manifestations in 43 patients with biopsy-confirmed kidney diseases. Kidney biopsy tissue from all patients was immunostained for AIM and IgM. Staining patterns and percent stained areas within the glomeruli were determined. Cells expressing AIM were identified by co-staining with macrophage and endothelial cell surface markers. Correlations between staining results and clinical parameters were evaluated using univariate and multivariate analyses. AIM was deposited in various areas, such as mesangial and capillary area. A part of AIM expression was localized to CD68-positive macrophages in the glomerulus. Amount of glomerular expression was positively correlated with urinary protein in patients with severe proteinuria (urinary protein ≥0.5 g/day) and kidney dysfunction [estimated glomerular filtration ratio (eGFR)

Acute kidney injury (AKI) is associated with prolonged hospitalization and high mortality, and it predisposes individuals to chronic kidney disease. To date, no effective AKI treatments have been established. Here we show that the apoptosis inhibitor of macrophage (AIM) protein on intraluminal debris interacts with kidney injury molecule (KIM)-1 and promotes recovery from AKI. During AKI, the concentration of AIM increases in the urine, and AIM accumulates on necrotic cell debris within the kidney proximal tubules. The AIM present in this cellular debris binds to KIM-1, which is expressed on injured tubular epithelial cells, and enhances the phagocytic removal of the debris by the epithelial cells, thus contributing to kidney tissue repair. When subjected to ischemia-reperfusion (IR)-induced AKI, AIM-deficient mice exhibited abrogated debris clearance and persistent renal inflammation, resulting in higher mortality than wild-type (WT) mice due to progressive renal dysfunction. Treatment of mice with IR-induced AKI using recombinant AIM resulted in the removal of the debris, thereby ameliorating renal pathology. We observed this effect in both AIM-deficient and WT mice, but not in KIM-1-deficient mice. Our findings provide a basis for the development of potentially novel therapies for AKI.

Apoptosis inhibitor of macrophage (AIM, encoded by cd5l) is a multi-functional circulating protein that has a beneficial role in the regulation of a broad range of diseases, some of which are ameliorated by AIM administration in mice. In blood, AIM is stabilized by association with IgM pentamers and maintains its high circulating levels. The mechanism regulating the excessive accumulation of blood AIM remains unknown, although it is important, since a constitutive increase in AIM levels promotes chronic inflammation. Here we found a physiological AIM-cleavage process that induces destabilization of AIM and its excretion in urine. In blood, IgM-free AIM appeared to be cleaved and reduced in size approximately 10 kDa. Cleaved AIM was unable to bind to IgM and was selectively filtered by the glomerulus, thereby excreted in urine. Amino acid substitution at the cleavage site resulted in no renal excretion of AIM. Interestingly, cleaved AIM retained a comparable potency with full-length AIM in facilitating the clearance of dead cell debris in injured kidney, which is a key response in the recovery of acute kidney injury. Identification of AIM-cleavage and resulting functional modification could be the basis for designing safe and efficient AIM therapy for various diseases.

Renal failure is one of the most important social problems for its incurability and high costs for patients’ health care. Through clarification of the underlying mechanism for the high susceptibility of cats to renal disease, we here demonstrates that the effective dissociation of serum AIM protein from IgM is necessary for the recovery from acute kidney injury (AKI). In cats, the AIM-IgM binding affinity is 1000-fold higher than that in mice, which is caused by the unique positively-charged amino-acid cluster present in feline AIM. Hence, feline AIM does not dissociate from IgM during AKI, abolishing its translocation into urine. This results in inefficient clearance of lumen-obstructing necrotic cell debris at proximal tubules, thereby impairing AKI recovery. Accordingly, mice whose AIM is replaced by feline AIM exhibit higher mortality by AKI than in wild-type mice. Recombinant AIM administration into the mice improves their renal function and survival. As insufficient recovery from AKI predisposes patients to chronic, end-stage renal disease, feline AIM may be involved crucially in the high mortality of cats due to renal disease. Our findings could be the basis of the development of novel AKI therapies targeting AIM-IgM dissociation, and may support renal function in cats and prolong their lives.

In the thymi of WT1-transgenic (Tg) mice with the 17AA+/KTS- spliced form of the Wilms tumor gene WT1 driven by the lck promoter, the frequencies of CD4-CD8- double-negative (DN) thymocytes were significantly increased relative to those in normal littermates. Of the 4 subsets of CD4-CD8- DN thymocytes, the DN1 (CD44+CD25-) subset increased in both frequency and absolute cell number, whereas the DN2 (CD44+CD25+) and DN3 (CD44-CD25+) subsets decreased, indicating the blocking of thymocyte differentiation from the DN1 to the DN2 subsets. Furthermore, CD4-CD8+ T-cell receptor (TCR) γδ T-cells increased in both frequency and absolute cell number in the spleen and peripheral blood of the WT1-Tg mice relative to those of normal littermates. The CD8 molecules of these CD4-CD8+ TCR78 T-cells were CD8as, suggesting that they originated from the thymus. These results are the first direct evidence demonstrating that the WT1 gene is involved in the development and differentiation of T-lineage cells.IntJHematol. 2003;77:463-470.

MafB is a transcription factor that induces myelomonocytic differentiation. However, the precise role of MafB in the pathogenic function of macrophages has never been clarified. Here we demonstrate that MafB promotes hyperlipidemic atherosclerosis by suppressing foam-cell apoptosis. Our data show that MafB is predominantly expressed in foam cells found within atherosclerotic lesions, where MafB mediates the oxidized LDL-activated LXR/RXR-induced expression of apoptosis inhibitor of macrophages (AIM). In the absence of MafB, activated LXR/RXR fails to induce the expression of AIM, a protein that is normally responsible for protecting macrophages from apoptosis; thus, Mafb-deficient macrophages are prone to apoptosis. Haematopoietic reconstitution with Mafb-deficient fetal liver cells in recipient LDL receptor-deficient hyperlipidemic mice revealed accelerated foam-cell apoptosis, which subsequently led to the attenuation of the early atherogenic lesion. These findings represent the first evidence that the macrophage-affiliated MafB transcription factor participates in the acceleration of atherogenesis.

The effects of aging temperature on the microstructure and mechanical properties of a newly designed martensitic precipitation hardening stainless steel, which is 1.8Cu-15.9Cr-7.3Ni-1.2Mo-low C, N steel, for improving the toughness, ductility and corrosion resistance of stainless steel of 1000 MPa grade tensile strength were experimentally investigated. The specimen aged at 753 K for 14.4 ks has a typical lath martensitic structure with about 12% interlath austenite, while the specimens aged at 813 K and 853 K for 14.4 ks have the lamellar duplex microstructure of the reverted austenite and the aging hardened martensite. The formation process of reverted austenite is controlled by diffusion of Ni in martensite. The mean size of precipitates which are enriched with Cu increases with rising aging temperature, however, it is about 30 nm even after aging at 853 K for 14.4 ks. The specimens aged at 813 K and 853 K for 14.4 ks, in which the reversion of martensite to austenite is observed, have the excellent combinations of strength, ductility and toughness.

The critical minimum size of stable precipitate in the vicinity of the edge of miscibility gap is experimentally determined for Ni3Al precipitate particles in the Ni-Al and Cu4Ti particles in the Cu-Ti binary alloy systems using the macroscopic composition gradient method recently proposed. The results obtained are as follows: the critical nucleus size shows a rapid increase to several tens of nanometers in a very narrow composition region less than 0.3 at. pct from the phase boundary. Such a big critical size of the nucleus is statistically rationalized by the conventional nucleation theories, but remains kinetically unreasonable.

The effect of retained austenite (γ) on the microstructure and mechanical properties of a martensitic precipitation hardening stainless steel was experimentally investigated, whose chemical composition was Fe-1.8Cu-15.9Cr-7.3Ni-1.2Mo-0.08Nb-low C, N (mass %). The microstructures of all specimens consist of a typical lath martensite with interlath films of the retained γ, which is not reverted with aging. Cu-rich precipitates which may contribute to precipitation hardening can not clearly be observed. The tensile properties and Charpy absorbed energy are linearly approximated to the amount of retained γ as follows: 0.2% Y.S. (MPa) = 1192.3 − 13.6 × γ%, T.S. (MPa) = 1250.1 − 9.3 × γ%, El. (%) = 12.16 + 0.43 × γ%, R.A. (%) = 64.25 + 0.14 × γ%, and A.E. (J) = 72.5 + 0.8 × γ%. The introduction of retained γ is not beneficial to the fatigue limit. An excellent combinations of strength, ductility and toughness obtained in the present work is attributed to the introduction of retained γ and also to the chemical composition of the specimen used.

The spinodal decomposition of the β Ti-Cr binary alloys system is still questionable, because there are only rare experimental and thermodynamics data; moreover, simulation results for a real alloy system have not been found. Transmission electron microscopy (TEM) and quantitative computer simulations based on Khachaturyan’s diffusion equation have been employed to study the microstructural evolution occurring in the metastable β Ti-Cr alloys. Our study results reveal that the metastable β Ti-Cr undergoes a phase separation reaction β → titanium rich (β 1)+chromium rich (β 2) through a spinodal decomposition. The coherent two-phase fields show extremely fine platelike precipitates distributed homogenously through the bcc matrix, which are parallel to the {100} plane. Those precipitates are highly elastic induced from the first step of the phase separation. There is good agreement between the observed microstructure and the simulation results. The mode of formation of α phase is also discussed.

A computer simulation, based on the Khachaturyan diffusion equation, is presented to develop the kinetics of the morphological evolution of the A2/B2 second-order phase transition in a binary solid solution. The evolution of the occupation probability, as a function of composition, shows a good similarity to the actual micrographs experimentally obtained based on the macroscopic composition gradient method. These results are used in parallel with the results of the evolution of the long-range order parameter as a function of the ageing time, to verify a new concept of the ordering behaviour inside the ordered phase.

The phase separation in iron-rich Fe-Si-Ge ternary alloys was investigated by means of transmission electron microscopy. The phase separations into A2 and D03 were recognized in several alloys in addition to the phase separation into B2 and D03 which has already been found in Fe-Si and Fe-Ge binary systems. On the basis of TEM observations, isothermal section diagrams are proposed for the iron-rich corner at 823, 873, 923 and 973 K. The A2 + D03 two-phase field exists in a band-shaped region at 823 K. With increase in temperature,the A2 + D03 field gradually shrinks and is eventually replaced at 973 Kwith the B2 + D03 field, which extends from both the Fe-Si and Fe-Ge binary sides. The phase diagrams were well reproduced by the calculations based on the so-called Bragg-Williams-Gorsky approximation, taking account of the chemical and magnetic pair interactions up to the second nearest neighbour. The effects of the magnetism on the equilibria are discussed.

Recently, Khachaturyan’s group proposed a new calculation method for phase decomposition on the basis of the Onsager equation. In the present study, we modified the Khachaturyan diffusion equation to allow simulation of the phase decomposition in actual alloy systems. Two-dimensional (2-D) computer calculations are performed for the phase decompositions of Al-Zn, Cu-Co, and Fe-Mo binary systems by using the thermodynamic data related to the equilibrium phase diagrams. The calculated microstructures are very similar to the actual micrographs experimentally obtained for these alloys.

Phase separations in iron-rich Fe-Al-Co ternary alloys were investigated by means of transmission electron microscopy, differential thermal analysis and magnetization measurement. Two kinds of phase separations have been found at 923 K; A2+B2 and B2+B2*. The former occurs in a tongue-shaped composition region ranging from Fe-12 at % Al-15 at % Co to Fe-35 at % Al-45 at % Co and the latter appears in the small region bordering on the Fe-Al binary side of the A2+B2 field. These two-phase fields have theoretically been evaluated on the basis of the so-called Bragg-Williams-Gorsky model taking account of not only chemical but also magnetic interactions. The magnetic ordering accounts for the expansion of the A2+B2 coexistent region. In the measurement of magnetic properties, large increases in coercive force, Hc, and residual magnetic flux density, Br, were observed in the two-phase microstructures.

The so-called bifurcation diagrams for γ′ precipitate particles in nickel-based alloys were obtained by calculating the energy state for a pair of particles on the basis of the bifurcation theory. Based on the bifurcation diagrams, we have proposed a new parameter for describing the two-phase structure containing coherent particles. The parameter is defined as the mean particle radius at the intersection of the energy ridge and the line ofR=−0.5 orR=0.5 in the bifurcation diagram (R≡(r α−r β)/(r α+r β), wherer α andr β are the radii of α and β), and is symbolized by¯r * ±0.5. Because the energy state of the paired α and β is maximum at¯r * ±0.5 whenR=±0.5,¯r * ±0.5 is just like the watershed and hence we have termed it “structureshed”. This parameter successfully describes the effects of elastic energy as well as surface energy on the microstructural changes during coarsening of precipitate particles.

Computer simulations of phase decomposition were performed for the Cu-Co alloy system on the basis of the non-linear diffusion equation. In the calculations, the modified regular solution approximation was adopted, i.e. the composition and temperature dependences of the interaction parameter, Ω, between the nearest neighbour atoms were taken into account and the mobility of atoms was defined as a function of solute composition. The phase decompositions were successfully computed for the Cu-Co alloys. The calculation method proposed here is applicable to many actual alloy systems.

Theoretical analysis based on the calculation of phase diagrams was employed for Fe-Si-Co and Fe-Si-Al ordering systems to clarify the necessity for the occurrence of phase separation in Fe-base ternary ordering systems. The free energy of Fe-base ternary ordering alloys where B2 and D03 ordered structures are formed is evaluated statistically using a pairwise interaction approximation up to second nearest neighbours, taking into account not only the atomic interaction but also the magnetic interaction, based on the Bragg-Williams-Gorsky model. The calculated phase diagrams are consistent with the experimentally obtained ones. The phase diagram calculation in this work is useful to predict the equilibrium states of the ternary ordering systems. The phase separation in ordering alloys is caused by the contribution of excess free energies due to ordering. The influences of ferromagnetism on the two-phase regions are also demonstrated.

Phase decomposition behaviour of liquid-quenched β (bcc) type Ti-Cr alloys was investigated by means of transmission electron microscopy and hardness measurements. It was found that decomposition of β to β1 (Ti-rich, bcc) + β2 (Ti-lean, bcc) takes place in the intermediate composition range of the Ti-Cr system. This experimental result proves the theoretical prediction made by Menon and Aaronson, but the observed β1 + β2 two-phase field expands towards higher temperatures than the predicted binodal line. The coherent β1 + β2 two-phase state exhibits the so-called 〈100〉 modulated structure and it was concluded that the formation of such a structure is a result of spinodal decomposition of the β-phase. We obtained time-temperature-transformation (TTT) diagrams of β-type Ti-30, 40 and 50 at % Cr alloys. A typical sequence of structural change is β → coherent β1 + β2 → incoherent β1 + β2 → incoherent β1 + β2 + grain boundary precipitates → stable state of β + TiCr2 or α + TiCr2. Not all the states in the above sequence appear, depending on alloy composition, liquid-quenching rate and ageing temperature.

Insulin-dependent diabetes mellitus is characterized by the infiltration of lymphocytes into the islets of Langerhans of the pancreas (insulitis) followed by destruction of insulin-secreting beta-cells leading to overt diabetes. The best model for the disease is the non-obese diabetic (NOD) mouse. Two unusual features of the class II major histocompatibility complex (MHC) of the NOD mouse are the absence of I-E and the presence of unique I-A molecules (I-ANOD), in which aspartic acid at position 57 of the beta-chain is replaced by serine. This feature is also found in the HLA-DQ chain of many Caucasians with insulin-dependent diabetes mellitus. We have previously reported that the expression of I-E prevents the development of insulitis in NOD mouse. Here we report that the expression of I-Ak (A alpha kA beta k) in transgenic NOD mice can also prevent insulitis, and that this protection is seen not only when the I-A beta-chain has aspartic acid as residue 57, but also when this residue is serine. These results show that the single amino-acid substitution at position 57 of the I-A beta-chain from aspartic acid to serine is not sufficient for the development of the disease.

Several important conclusions can be drawn from these experiments and are as follows. First, not only the lack of I-E but also the presence of unique I-A are involved in the development of autoimmune insulitis in a NOD mouse. Second, the mechanism of prevention in I-Ak transgenic mice might be different from that in I-E transgenic mice, although the negative selection of autoreactive T cells in thymus has been suggested in I-E transgenic mice. Third, the single amino acid substitution from Asp to Ser at position 57 of the Aβ chain is not sufficient for development of insulitis. Other amino acids including residue 56 of the Aβ chain as well as Aα chain are important in the development of insulitis and diabetes. However, the amino acid at position 57 may be involved in the conformational change of I-A molecule in such a way as to alter the interaction with the T cell receptor, leading to the positive or negative selection of autoreactive T cells. Fourth, the ectopic expression of class II molecules on β cells may not be the initial event for the generation of insulitis.

Apoptosis inhibitor expressed by macrophages (AIM) is a murine macrophage-specific protein and belongs to the macrophage scavenger receptor cysteine-rich domain superfamily. AIM has been introduced as the inducer of resistance to thymocyte apoptosis [1]. Because apoptosis of inflammatory cells plays a pivotal role in inflammation [2], we have applied a mouse model to address potential involvement of AIM in the process of granulomatous inflammation in vivo.

Elastic interaction energies between two identical inhomogeneous plates in an anisotropic matrix were calculated with respect to particle separation distance and alignment orientation on the basis of the microscopic theory of elasticity developed by Yamauchi and de Fontaine. Four types of plate-arrangement along the cube directions; two plates whose faces were mutually parallel, two plates which were arranged on a cube plane as a raft, two plates which were perpendicularly arranged with face—edge configuration and two plates which were perpendicularly aligned with edge—edge configuration were energetically stable, while other arrangements were not stable. Compared with experimental observations on the local arrangement of γ′plates in a Ni-Al single crystal, the calculated results were confirmed to be qualitatively correct.

New “interfaces” are produced on the slip plane when a crystal with continuous composition fluctuation arising from spinodal decomposition is deformed by slip. In this work, the energy of such “interfaces” is evaluated for both modulated and mottled structures, and their effects on slip behaviour are discussed. It is concluded that the contribution of this “interfacial energy” is large enough to account for the age-hardening concomitant with spinodal decomposition.

Influences of the external tension and compression on the morphological changes of γ′ precipitates during coarsening in an Ni-15 at. % Al single crystal were experimentally investigated, and theoretical evalutions on the energetically favourable shape and its orientation were also derived based upon the anisotropic elasticity theory. The shape transformation with growth in size is experimentally found to follow the sequence: cuboid→rod→ plate, under all annealing conditions, i.e. compression-annealing, tension-annealing and external stress-free annealing. However, the external stresses give preferential orientation to such microstructures. The tension of the [001] direction aligns the rods and plates along the tension axis, while the compression aligns them on a plane perpendicular to the compression axis. Theoretical evaluations based upon the anisotropic elasticity for the energetically favourable shapes and orientations of γ′ are consistent with experimental results except in the appearance of rods after compression-annealing. This discrepancy is understood by a conception that the shape transformation is influenced not only by energetics in the elastic strain and interfacial energies, but also by elastic interaction energy.

Stable structures of Fe-Si-Al ternary alloys and Fe-Si and Fe-Al binary alloys containing up to about 40 at% solute atoms were investigated by means of transmission electron microscopy. The following results were obtained. Two types of phase separation, B2+DO3 andα + DO3 were observed in the alloys whose compositions lie in a narrow band connecting Fe-10 to 14 at% Si with Fe-20 to 25 at % Al and also in the neighbourhood of a Fe-30 at%Al-3 at% Si alloy. Such compositions of the alloys are located in the phase boundary of B2 and DO3 single phases orα and DO3 single phases. The phase separation in the Fe-Si-Al and Fe-Si alloys produce the 〈100〉 modulated structure which differs from the morphology formed by the phase separation of the Fe-Al system.