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PROBLEM: We assessed the expression of natural killer (NK) receptors in recurrent aborters before and after immunotherapy using their husbands’ peripheral blood mononuclear cells (PBMCs). METHOD OF STUDY: Using stored PBMCs from recurrent aborters before and after the immunotherapy, the expression of NK receptors, CD158a, CD158b, CD159 and CD94, were analyzed using monoclonal antibodies for respective receptors. The diversity of killer activatory receptors (KARs) and killer inhibitory receptors (KIRs) was also examined using reverse transcriptase‐polymerase chain reaction (RT‐PCR)‐single strand conformation polymorphism (SSCP) method. RESULTS: In recurrent aborters, no apparent changes in NK receptor expression and the balance between KARs and KIRs were found before and after the immunotherapy. CONCLUSION: The allo‐human leukocyte antigen (HLA)–stimulation caused by the immunotherapy for recurrent aborters did not affect the expression of NK receptors and the ratio of KARs to KIRs regardless of the outcome of subsequent pregnancies, suggesting that recurrent aborters may benefit from the immunotherapy through mechanisms unrelated to alteration in NK receptor status.

PROBLEM AND METHOD OF STUDY: We have shown that Tokishakuyaku‐san (Toki) and Sairei‐to (Sai) enhance T helper‐1 (Th1) cytokine release from peripheral blood mononuclear cells (PBMCs); thereby, they could be a therapeutic means in the treatment of autoimmunity related recurrent abortion in which T helper‐2 (Th2) polarization is exaggerated, the condition purported to benefit from these herbal medicines. However, an open question is whether these medicines might enhance Th1 cytokine release in decidual tissues and thereby stimulate the killer activity, thus, working counterproductively by accelerating maternal alloimmune reactions toward fetal tissues. To address this, we examined the effects of these medicines on the release of cytokines from decidual mononuclear cells (DMCs) in comparison with PBMCs on the assumption that they might act differently on these cell types. The effects of these medicines were investigated as related to human leukocyte antigen (HLA)‐G, a nonclassical HLA class I antigen expressed on trophoblasts and a putative crucial player involved in fetomaternal immune interplay.
 RESULTS: Regarding Th1 cytokines, Toki marginally increased the release of tumor necrosis factor (TNF)‐α, but not interferon (IFN)‐γ from DMCs while Sai did not affect the release of both. Both Toki and Sai were without effect in modulating the release of interleukin (IL)‐4, a member of Th2 cytokines. Interestingly, the presence of HLA‐G reduced the release of Th1 cytokines from DMCs regardless of the addition of Toki, Sai or none. These findings are in sharp contrast with PBMCs on which these medicines seem to act so as to enhance Th1 polarization and attenuate Th2 polarization.
 CONCLUSION: Differential effects of Toki and Sai on the release of Th1/Th2 cytokines between DMCs and PBMCs may afford the rationale of these medicines in the treatment of autoimmunity‐related recurrent abortion.

PROBLEM: The aim of this study was to investigate the gene frequencies and shared alleles of human leukocyte antigen (HLA)‐E gene in Japanese couples with or without recurrent abortion.
 METHOD OF STUDY: Polymerase chain reaction (PCR)‐single strand conformation polymorphism (SSCP) analysis was carried out to detect polymorphism in exon 3 of the HLA‐E gene in 30 Japanese couples with recurrent abortion and 38 normal Japanese couples with proven fertility.
 RESULTS: No point mutation was detected in exon 3 of HLA‐E in both recurrent aborters and normal controls. HLA‐EG and HLA‐ER alleles were detected with frequencies of 66.7% and 33.3% in couples with recurrent abortion and 69.2% and 30.8% in normal couples, respectively. The gene frequency of HLA‐EG was higher than that of HLA‐ER, which is contrary to that found in Caucasian, African–American and Hispanic people but similar to Chinese people. The frequency of each allele was not significantly different between recurrent aborters and normal controls. The number of shared alleles between each couple with recurrent abortion is not significantly different from that with normal controls.
 CONCLUSION: Allele frequencies of HLA‐E were suggested to be different in Asian people from those in other ethnic people. In light of no specific distribution pattern in recurrent aborters, HLA‐E polymorphism does not seem to play a role in the pathogenesis of recurrent abortion.

PROBLEM: To better understand the role of human leukocyte antigen (HLA)‐G in regulating the T helper (Th)1/Th2 cytokine balance, one of key conditions in determining the fate of pregnancy, we asked whether the presence of HLA‐G protein altered the release of cytokines from both decidual mononuclear cells and peripheral blood mononuclear cells (PBMCs).
 METHOD OF STUDY: The amounts of cytokines released from decidual mononuclear cells and PBMCs were compared in the presence or absence of HLA‐G‐expressing cells.
 RESULTS: When cocultured with HLA‐G‐expressing cells, the amounts of tumor necrosis factor‐α and interferon‐γ released from decidual mononuclear cells and PBMCs were decreased, while the amounts of interleukin (IL)‐4 from PBMCs was increased, with IL‐4 release from decidual mononuclear cells being unchanged.
 CONCLUSIONS: Upon contact with HLA‐G, decidual mononuclear cells, and PBMCs as well, modulate their ability to release cytokines in a way that may shift the Th1/Th2 balance towards relative Th2 dominance, suggesting a role for HLA‐G in maintaining pregnancy.

PROBLEM AND METHOD OF STUDY: To get insight into the basis for the empirical usage of herbal medicines in the treatment of recurrent abortion, we examined whether Tokishakuyaku‐san (Toki) and Sairei‐to (Sai) modulate T helper‐1 (Th1) and T helper‐2 (Th2) cytokine release from peripheral blood mononuclear cells (PBMCs). The effects of these medicines were investigated as related to human leukocyte antigen (HLA)‐G, a non‐classical HLA class I antigen expressed on trophoblasts and a putative crucial player involved in fetomaternal immune interplay.
 RESULTS: Toki and Sai increased the release of Th1 group cytokines, tumor necrosis factor (TNF)‐α and interferon (IFN)‐γ while preserving the inhibitory effect of HLA‐G on the release of these cytokines. As for Th2 group cytokine release, Toki was without effect in modulating interleukin (IL)‐4 release, regardless of the presence of HLA‐G, whereas Sai nullified the effect of the presence of HLA‐G to stimulate the release of IL‐4 without affecting its release in the absence of HLA‐G.
 CONCLUSION: Toki and Sai may have therapeutic potential, particularly in autoimmunity‐related recurrent abortion where Th2 response is pathologically enhanced, but not in recurrent abortion involving alloimmune fetomaternal derangement, a condition of, rather, an enhanced Th1 response.

PROBLEM: The appropriate modality of immunotherapy with the husband's mononuclear cells in women with a history of recurrent abortion who aborted despite the immunotherapy performed before pregnancy was explored.

PROBLEM: The objective was to clarify whether lymphocytes in decidual tissue express only killer inhibitory receptors (KIRs) or both KIRs and killer activatory receptors (KARs).

PROBLEM: We have shown the attenuated human leukocyte antigen (HLA)‐G expression on trophoblasts and an aberrant expression of interleukin (IL)‐2, a cytotoxic cytokine, in decidual tissue in preeclampsia, where deteriorated trophoblastic invasion into decidual layers may constitute a crucial pathogenesis. We hypothesized that the absence of HLA‐G might make trophoblasts susceptible to compromise by IL‐2.

PROBLEM: The purpose of this study was to clarify whether there is a difference between the allele frequency of human leukocyte antigen (HLA)‐G in healthy Japanese people and that of Japanese couples with habitual abortion.

Summary: The myotome of early chick embryos was investigated histochemically by means of the acetylcholinesterase (AChE) reaction.Light-microscopically, at the cervical level, the myotome was first recognized and AChE activity demonstrated at stage 13 (2 day-old embryo). Subsequently, the myotome elongated ventro-laterally along the inner surface of the dermomyotome and reached the ventro-lateral end of the dermomyotome at stage 17 to 18 (3 day-old embryo). AChE activity in the myotome showed subsequent increase in intensity during the course of development. The myotome consisted mainly of AChE-positive cells displaying enzymatic activity along the nuclear membrane and within the cytoplasm. In contrast, almost all cells of the dermomyotome and the interstitial cells were AChE-negative.Electron-microscopically, the myotome cells of the 2 day-old embryo and the cells in the dorso-medial portion of the myotome of the 3 day-old embryo were morphologically undifferentiated; AChE activity was detected in the nuclear envelope and in single short profiles of the endoplasmic reticulum (ER). On the other hand, in the 3 day-old embryo the cells in the ventro-lateral portion of the myotome showed AChE activity in the nuclear envelope, numerous profiles of the ER and some Golgi complexes. These AChE-positive cells were regarded as developing myogenic cells based on their morphological characteristics.The present findings indicate (i) that the appearance of AChE activity in the cytoplasm is the first sign of the differentiation of myogenic cells, and (ii) that in these myogenic cells the increase in AChE activity is based on the development of the ER.

PROBLEM: In search for pathogenesis of recurrent abortion, we examined whether lymphocytes/macrophages from women with recurrent abortion exhibited an aberrant ability to release cytokines upon the direct contact of human leukocyte antigen (HLA)‐G.

Abstract:  The developmental expression of occludin was studied biochemically in whole chick embryos to determine when intercellular tight junctions develop. Occludin mRNA was first detected after 3 days of incubation by the reverse transcriptase-polymerase chain reaction. On northern blot analysis, although occludin mRNA was not discernible in 3-day-old embryos, weak but clear expression was noted on day 4 of incubation and increased dramatically in 5-day-old embryos. Occludin was not detectable on days 3 or 4 of incubation by western blot analysis, and was first detected in 5-day-old embryos. In addition, the expression of occludin was examined immunohistochemically in the gastrointestinal tract of 3- to 21-day-old embryos. Immunoreactivity for occludin was not expressed on day 3 of incubation. On day 4 of incubation, weak immunoreactivity was demonstrated in the gastrointestinal tract, and gradually became stronger with development. By day 11 of incubation, a positive immunoreaction was obtained only on the apical surfaces of the epithelial cells, i.e., at the junctional complexes, while weak immunoreactivity was diffusely distributed throughout the epithelial cells. The possible roles of occludin in the developing gastrointestinal tract are discussed.

Intralimb incoordination needs to be distinguished from interlimb incoordination, which is observed in Parkinson's disease (PD). With this aim, intra and inter-limb coordination impairment in the lower limbs were measured in 13 patients with cerebellar ataxia during pedaling of an ergometer with left and right pedals that can be rotated independently. Intralimb incoordination in patients with cerebellar ataxia was compared with interlimb incoordiation in patients with PD. We concluded that impairment of intralimb coordination in ataxia patients leads to inability to maintain pedaling amplitude and speed, but unlike in PD, right and left coordination was roughly preserved. Our method may be useful for assessing the severity of ataxia and detecting hidden parkinsonism in ataxic patients.

PROBLEM: The objective of this study was to establish an enzyme‐linked immunosorbent assay (ELISA) system, in an attempt to quantify the amount of human leukocyte antigen (HLA)‐G protein in amniotic fluid.

In this study, the ionic mass transfer phenomenon of Al3+in a sulfuric acid (pH 1) accompanying the anodization of an Al electrode was investigated using a digital holographic interferometric microscope. The anodization of an Al electrode is very popular technique to functionalize Al plates however, the anodization mechanism in the initial stage has not yet been understood, especially for the ionic mass transfer phenomenon. The investigation revealed the refractive index profile corresponding to the Al3+concentration profile in the electrolyte. The Al3+concentration on the electrode surface increased because of the Al dissolution in the initial stage of the anodization, and the thickness of the diffusion layer was proportional to the square root of time. The Al3+surface concentration started to decrease after approximately 20 s probably because the Al dissolution reaction was hindered, and the Al oxide layer formation progressed. Additionally, ex situ scanning electron microscopy observation showed pores on the electrode surface at 80 s, which indicated that the pore formation was initiated from approximately 40 s with the progress of the oxide film formation. This research must be a steppingstone in order to understand the mechanism of anodization of Al.