Masahiro Kiuchi

Prediction of drug-drug interactions of zonisamide metabolism in humans from in vitro data

Objective: The purposes of this study were to identify the P450 enzyme (CYP) responsible for zonisamide metabolism in humans by using expressed human CYPs and to predict drug interaction of zonisamide in vivo from in vitro data. Methods: Ten expressed human CYPs and human liver microsomes were used in the experiments for the identification of enzymes responsible for zonisamide metabolism and for the prediction of drug-drug interactions of zonisamide metabolism in humans from in vitro data, respectively. Two-sulfamoylacetyl phenol, a reductive metabolite of zonisamide, was measured by the HPLC method. Results: From the experiments using ten expressed human CYPs, CYP2C19, CYP3A4 and CYP3A5 were shown to be capable of catalyzing zonisamide reduction. However, an intrinsic clearance, Vmax/kM, of CYP3A4 was much higher than those of CYP2C19 and CYP3A5. From the point of view of enzyme amount in human liver CYPs isoform and their intrinsic clearance, it was suggested that CYP3A4 is mainly responsible for zonisamide metabolism in human CYPs. Zonisamide metabolism in human liver microsomes was markedly inhibited by cyclosporin A, dihydroergotamine, ketoconazole, itraconazole, miconazole and triazolam. We estimated the possibility and degree of change of zonisamide clearance in vivo in clinical dose range from in vitro inhibition constant of other drugs against zonisamide metabolism (Ki) and unbound inhibitor concentration in blood (Iu) in clinical usage. Clearance of zonisamide was maximally estimated to decrease by 31%, 23% and 17% of the clearance without inhibitors i.e. ketoconazole, cyclospolin A and miconazole, respectively. Fluconazole and carbamazepine are estimated to decrease by 5–6% of the clearance of zonisamide. On the other hand, there may be lack of interaction of zonisamide metabolism by dihydroergotamine, itraconazole and triazolam in clinical dose range. Conclusion: We demonstrated that: (1) zonisamide is metabolized by recombinant CYP3A4, CYP2C19 and CYP3A5, (2) the metabolism is inhibited to a variable extent by known CYP3A4/5 substrates and/or inhibitors in human liver microsomes, and (3) in vitro-in vivo predictive calculations suggest that several compounds demonstrating CYP3A4-affinity might cause in vivo drug-drug interactions with zonisamide.

Protective immunity induced by vaccination with SAG1 gene-transfected cells against Toxoplasma gondii-infection in mice

To develop a vaccine by augmenting the protective cellular immunity against Toxoplasma gondii (T. gondii)‐infection, T. gondii SAG1 gene‐transfectants were established by using RMA.S (H‐2b), a murine transporter associated with the antigen processing (TAP) molecule‐deficient lymphoma line, as a host antigen‐presenting cell (APC). Immunization of C57BL/6 mice with the SAG1‐transfected RMA.S induced CD8+ cytotoxic T lymphocytes (CTL) specific for not only SAG1‐transfected RMA.S but also T. gondii‐infected RMA.S, and elicited protective responses to infection with a virulent T. gondii strain, RH.

Regional differences in homicide patterns in five areas of Japan

This article describes regional differences in the homicide patterns which occurred in Sapporo City and the surrounding area, and in Akita, Ibaraki, Chiba and Toyama prefectures in Japan. Information collected from each case of homicide included factors such as age, sex of the victim and assailant, causes of death, disposition of the offender, relationship between assailant and victim, reasons for criminal action, et al. The statistical features of homicidal episodes among the five different regions showed considerable variation, as follows. The mean death rates for homicide (number of victims per 100,000 of population) during the period 1986-1995 were 0.44 (Sapporo), 0.8 (Akita), 0.58 (Toyama), 0.7 (Ibaraki) and 0.75 (Chiba), respectively. Close family relationship between the victim and assailant was observed in the homicidal acts which occurred in Sapporo, Akita and Toyama. Assailants relationship to victim was commonly extra-familial in Ibaraki and Chiba-neighboring megalopolis Tokyo, where some events of murder by a foreigner occurred. Homicide by female assailant, murder by mentally abnormal killers and homicide-suicide events were closely associated with family members. And these factors contributed to the considerable number of victims in Sapporo, Akita and Toyama. But, this close family relationship of the victim to the assailant did not correspond with the elevation in the number of deaths, and it was rather inversely related to the higher death rates recognized in Ibaraki and Chiba. This comparative study suggested that rapid urbanization considerably affects regional differences in homicide patterns.

The different mobility of complementary strands depends on the proportion AC/GT

The electrophoretic mobility of DNA fragments on denaturing polyacrylamide gel depends on various factors. One of these is the base composition of a single-stranded DNA (ssDNA). We confirmed that one strand and its complementary strand of polymerase chain reaction (PCR) products migrated with different mobilities in all alleles detected at 12 out of the 13 short tandem repeat (STR) loci studied. The mobility differences between complementary strands (MD) were also observed regardless of end-polishing with Pfu DNA polymerase. MD was therefore not influenced by additional nucleotides to each strand of the PCR products. We then analyzed the relation between MD and the base composition using one representative allele at each of the 13 loci. The results indicated that MD was affected by the adenine plus cytosine (AC) content in the ssDNA and was proportional to the values of the AC content divided by the guanine plus thymine (GT) content in the AC-rich strand (the proportion AC/GT). When the proportion AC/GT was well-balanced, MD decreased. The same tendency was observed even in the end-polished strands. In this study, the electrophoretic mobility of an ssDNA on denaturing polyacrylamide gels was shown to depend on the proportion AC/GT. Unless the same side of the PCR products is labelled in the context of a PCR-based STR typing, distinct alleles may be mistaken for identical ones because of the different mobility of complementary strands. Accordingly, the labelled strand should be described if only one strand of the PCR products is detected. When using an allelic ladder marker as a size standard, the labelled side should be unified between STR alleles and the allelic ladder alleles.

Trithorax complex component Menin controls differentiation and maintenance of T helper 17 cells

Significance Epigenetic modifications, including various histone modifications, play important roles in regulating gene expression. The Trithorax group (TrxG) complex induces permissive histone modifications to activate transcription. We herein investigate the role for Menin, a component of the TrxG complex, in T helper (Th) cell differentiation, and find a critical role for Menin in differentiation and maintenance of Th17 cells. Menin is required for Th17 cell differentiation in vitro through the direct regulation of Il17a expression. Menin controls IL-17–mediated pathology in vivo. Menin is also required to maintain expression of Rorc, the gene encoding RORγt, a key transcription factor for Th17 cell function. Thus, Menin orchestrates Th17 cell differentiation and function by regulating both induction and maintenance of target gene expression. Epigenetic modifications, such as posttranslational modifications of histones, play an important role in gene expression and regulation. These modifications are in part mediated by the Trithorax group (TrxG) complex and the Polycomb group (PcG) complex, which activate and repress transcription, respectively. We herein investigate the role of Menin, a component of the TrxG complex in T helper (Th) cell differentiation and show a critical role for Menin in differentiation and maintenance of Th17 cells. Menin−/− T cells do not efficiently differentiate into Th17 cells, leaving Th1 and Th2 cell differentiation intact in in vitro cultures. Menin deficiency resulted in the attenuation of Th17-induced airway inflammation. In differentiating Th17 cells, Menin directly bound to the Il17a gene locus and was required for the deposition of permissive histone modifications and recruitment of the RNA polymerase II transcriptional complex. Interestingly, although Menin bound to the Rorc locus, Menin was dispensable for the induction of Rorc expression and permissive histone modifications in differentiating Th17 cells. In contrast, Menin was required to maintain expression of Rorc in differentiated Th17 cells, indicating that Menin is essential to stabilize expression of the Rorc gene. Thus, Menin orchestrates Th17 cell differentiation and function by regulating both the induction and maintenance of target gene expression.

Immunohistochemical study of the kidneys after severe muscular injury

Abstract Severe muscular injury sometimes causes renal failure, and myoglobin in skeletal muscle is known to induce toxic free oxygen radicals in the kidneys. The relationship between the immunohistochemical expression of myoglobin and the scavenger copper/zinc superoxide dismutase (Cu/Zn-SOD) was investigated in kidneys taken from two autopsy groups, a group with tourniquet shock (n = 4), and a group with severely injured skeletal muscle (n = 18). Paraffin-embedded kidney sections were used for immunohistochemical staining by the avidin-biotin-complex (ABC) method using antibodies against myoglobin and Cu/Zn-SOD. Detection of the two antigens was analyzed qualitatively. In most cases of tourniquet shock in which the survival time was considered to be relatively long, myoglobin staining was positive and Cu/Zn-SOD was negative. Among the seven cases of severely injured skeletal muscle in which the survival period was considered to be relatively short, positive staining was detected immunohistochemically for both myoglobin and Cu/Zn-SOD. Moreover, in most of the cases in this group that showed acute tubular necrosis, immunohistochemical staining was negative for both markers, whereas positive staining was found for most of the cases in which the kidneys were revealed to be normal by HE staining. These findings suggest that when myoglobin enters the kidneys via the circulation, Cu/Zn-SOD reacts to eliminate free radicals, but is depleted by consumption in the long run, and that there might be a relationship between these histological findings and immunohistochemical expression.

Amphiregulin-Producing Pathogenic Memory T Helper 2 Cells Instruct Eosinophils to Secrete Osteopontin and Facilitate Airway Fibrosis

Summary Memory T cells provide long‐lasting protective immunity, and distinct subpopulations of memory T cells drive chronic inflammatory diseases such as asthma. Asthma is a chronic allergic inflammatory disease with airway remodeling including fibrotic changes. The immunological mechanisms that induce airway fibrotic changes remain unknown. We found that interleukin‐33 (IL‐33) enhanced amphiregulin production by the IL‐33 receptor, ST2hi memory T helper 2 (Th2) cells. Amphiregulin‐epidermal growth factor receptor (EGFR)‐mediated signaling directly reprogramed eosinophils to an inflammatory state with enhanced production of osteopontin, a key profibrotic immunomodulatory protein. IL‐5‐producing memory Th2 cells and amphiregulin‐producing memory Th2 cells appeared to cooperate to establish lung fibrosis. The analysis of polyps from patients with eosinophilic chronic rhinosinusitis revealed fibrosis with accumulation of amphiregulin‐producing CRTH2hiCD161hiCD45RO+CD4+ Th2 cells and osteopontin‐producing eosinophils. Thus, the IL‐33‐amphiregulin‐osteopontin axis directs fibrotic responses in eosinophilic airway inflammation and is a potential target for the treatment of fibrosis induced by chronic allergic disorders. Graphical Abstract Figure. No caption available. HighlightsPathogenic memory Th2 cells induce amphiregulin (Areg) via IL‐33Areg reprograms the transcriptome of eosinophils toward an inflammatory stateAreg‐reprogramed eosinophils produce osteopontin to trigger airway fibrosisAreg+ Th2 cells and osteopontin+ eosinophils co‐localize in fibrotic ECRS polyps &NA; Asthma is a chronic allergic inflammatory disease with airway remodeling including fibrotic changes. Morimoto and colleagues find that the IL‐33‐ST2‐amphiregulin‐EGRF‐osteopontin axis directs fibrotic responses in chronic allergic inflammation with the involvement of airway epithelial cells, pathogenic memory Th2 cells, and inflammatory eosinophils in both mouse and human.

Menin Controls the Memory Th2 Cell Function by Maintaining the Epigenetic Integrity of Th2 Cells

Posttranslational modifications of histones are well-established epigenetic modifications that play an important role in gene expression and regulation. These modifications are partly mediated by the Trithorax group (TrxG) complex, which regulates the induction or maintenance of gene transcription. We investigated the role of Menin, a component of the TrxG complex, in the acquisition and maintenance of Th2 cell identity using T cell–specific Menin-deficient mice. Our gene expression analysis revealed that Menin was involved in the maintenance of the high expression of the previously identified Th2-specific genes rather than the induction of these genes. This result suggests that Menin plays a role in the maintenance of Th2 cell identity. Menin directly bound to the Gata3 gene locus, and this Menin-Gata3 axis appeared to form a core unit of the Th2-specific gene regulatory network. Consistent with the phenotype of Menin-deficient Th2 cells observed in vitro, Menin deficiency resulted in the attenuation of effector Th2 cell–induced airway inflammation. In addition, in memory Th2 (mTh2) cells, Menin was found to play an important role in the maintenance of the expression of Th2-specific genes, including Gata3, Il4, and Il13. Consequently, Menin-deficient mTh2 cells showed an impaired ability to recruit eosinophils to the lung, resulting in the attenuation of mTh2 cell–induced airway inflammation. This study confirmed the critical role of Menin in Th2 cell–mediated immune responses.

A fatal case of shotgun injury caused by one pellet.

A 73-year-old man was shot to death due to the misjudgment of his comrade while deer hunting. There were two wounds on the victims body. One was a gunshot wound which had perforated his left brachium and constituted the entry wound on the outer brachium and the exit wound on the axilla. The other was the re-entry wound on the axilla just on the inner side of the former exit wound. These wounds were made by one pellet from nine-pellet-type buckshot ammunition. No other gunshot injury was found.