Safety evaluation of the use of calcineurin inhibitor to prenatal and postpartum women in Japan from a health administrative database.

BACKGROUND: To investigate the use of calcineurin inhibitors (CNIs) in pregnant Japanese women and to evaluate their safety in infants. METHODS: Data were extracted from the claims database of the Japan Medical Data Center. The prevalence of CNIs was evaluated 180 days before pregnancy onset, during pregnancy, and within180-days post partum. We investigated the characteristics of the infants, including the presence of major malformations and their diagnoses, for 1 year after birth. RESULTS: A total of 91,865 pregnancies in 80,049 women were included. Fifty-three women were prescribed CNIs between 180-day before pregnancy onset and 180-day postpartum; 35 of the 53 women were prescribed the drugs during pregnancy, and 10 of their infants were born preterm. Three were diagnosed with major congenital malformations, such as patent ductus arteriosus. Six preterm infants presented with infant respiratory distress syndrome. CONCLUSIONS: No congenital anomalies were clearly attributable to the use of CNIs during pregnancy.

Paternal height has an impact on birth weight of their offspring in a Japanese population: the Japan Environment and Children's Study.

This study examines the relationship between paternal height or body mass index (BMI) and birth weight of their offspring in a Japanese general population. The sample included 33,448 pregnant Japanese women and used fixed data, including maternal, paternal and infant characteristics, from the Japan Environment and Children's Study (JECS), an ongoing nationwide birth cohort study. Relationships between paternal height or BMI and infant birth weight [i.e., small for gestational age (SGA) and large for gestational age (LGA)] were examined using a multinomial logistic regression model. Since fetal programming may be a sex-specific process, male and female infants were analyzed separately. Multivariate analysis showed that the higher the paternal height, the higher the odds of LGA and the lower the odds of SGA in both male and female infants. The effects of paternal BMI on the odds of both SGA and LGA in male infants were similar to those of paternal height; however, paternal height had a stronger impact than BMI on the odds of male LGA. In addition, paternal BMI showed no association with the odds of SGA and only a weak association with the odds of LGA in female infants. This cohort study showed that paternal height was associated with birth weight of their offspring and had stronger effects than paternal BMI, suggesting that the impact of paternal height on infant birth weight could be explained by genetic factors. The sex-dependent effect of paternal BMI on infant birth weight may be due to epigenetic effects.

Rapid tumor imaging by active background reduction using biotin-bearing liposomes and avidin.

Tumor imaging with labeled liposomes is slow; although they reach the tumor quickly, their blood clearance is slow, and the high blood background hinders early imaging. We have developed a rapid tumor imaging technique based on the active removal of liposomes from the circulation by using the avidin-biotin system. 67Ga- or 111In-labeled liposomes with biotin molecules bound on the surface were administered to mice bearing sarcoma 180, and avidin was administered 2 h later. The strong affinity between biotin and avidin initiated the aggregation of liposomes, resulting in their rapid removal from the circulation by the reticuloendothelial system, and the blood level of radioactivity was dramatically reduced without any change of the tumor level. Consequently, the tumor:blood ratio reached 14-18 only 2.5 h after liposome injection. Increased accumulation in the liver was also observed. By this method, an acceptable tumor image could be obtained no more than 2 h after administration of labeled liposomes.

Stimulation of melanogenesis in murine melanoma cells by 2-mercapto-1-(beta-4-pyridethyl) benzimidazole (MPB).

The effects of 2-mercapto-1-(beta-4-pyridethyl) benzimidazole (MPB), one of the benzimidazole derivatives designed for a nucleic acid analogue, on melanogenesis of murine B16-F10 melanoma cell lines were investigated. MPB (40 microM) induced a striking dendricity in B16 melanoma cells within 12 h and maximal dendricity between 48 and 72 h. The stimulation of melanin synthesis was observed after only 2 days of treatment together with a dose-dependent growth inhibition. Moreover, MPB increased the activity of tyrosinase through the expression of tyrosinase mRNA without increasing the intracellular cyclic AMP content. MPB-induced melanogenesis was inhibited by novel protein kinase A inhibitors, KT-5720 and H-85. These findings indicate that MPB stimulated B16 cells to terminally differentiate and may be a useful drug in studying the regulation of melanogenesis.

Identification of mutations in the hepatocyte nuclear factor (HNF)-1 alpha gene in Japanese subjects with IDDM.

One form of maturity-onset diabetes of the young, MODY3, is characterized by a severe insulin secretory defect, compared with MODY2, a glucokinase-deficient diabetes. It has recently been shown that mutations of the gene encoding the transcription factor hepatocyte nuclear factor (HNF)-1 alpha cause MODY3. Because of the rapid progress to overt diabetes and the high prevalence of required insulin treatment in patients with MODY3, we screened the HNF-1 alpha gene for mutations in Japanese subjects with IDDM. Ten exons and flanking introns of the HNF-1 alpha gene in these subjects were amplified by polymerase chain reaction and direct sequencing of the products. Mutations were identified in three (5.5%) of the 55 unrelated subjects with IDDM. A missense mutation of R272H (replacement of Arg by His in codon 272) in the DNA binding domain of HNF-1 alpha was found in a subject who developed IDDM 1 year after diagnosis of NIDDM at 8 years of age. A frameshift mutation of P291 fsinsC (insertion of a C in a polyC tract around codon 291 for Pro), which would generate a mutant truncated protein of 340 amino acids, was found in a subject who started insulin treatment when hyperglycemia and ketonuria were noticed at 13 years of age. A missense mutation of R583G (replacement of Arg by Gly in codon 583) in the transactivation domain of HNF-1 alpha was found in a subject with sudden-onset IDDM at 20 years of age. None of these mutations were present in 100 nondiabetic subjects (200 normal chromosomes). These results indicate that the HNF-1 alpha gene defects could lead to the development of not only early-onset NIDDM but also IDDM, implicating the importance of subclassification of HNF-1 alpha-deficient IDDM from a classical type of autoimmune-based IDDM in Japanese.

Hepatocyte growth factor in vitreous fluid of patients with proliferative diabetic retinopathy and other retinal disorders.

OBJECTIVE: To determine whether hepatocyte growth factor (HGF) is elevated in the vitreous fluid of patients with proliferative diabetic retinopathy (PDR). RESEARCH DESIGN AND METHODS: Vitreous fluid samples were obtained at the time of vitreoretinal surgery from 73 eyes of PDR patients and from 17 eyes of nondiabetic patients (control subjects) who had macular hole, rhegmatogenous retinal detachment, or epiretinal membrane (9, 4, and 4 eyes, respectively) but no associated proliferative vitreoretinopathy Stages of PDR were classified as active or quiescent. Concentrations of HGF and vascular endothelial growth factor (VEGF) in vitreous fluid were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Intravitreous concentrations of HGF (median [range]) were significantly higher in diabetic patients with PDR (6.00 ng/ml [0.75-22.21) than in control patients (2.86 ng/ml [0.75-5.801). Intravitreous concentrations of VEGF were also higher in diabetic patients with PDR (1.62 ng/ml [0.15-7.91) than in control patients (0.16 ng/ml [0.160.29]). Both VEGF and HGF concentrations were significantly higher in patients with active retinopathy than in those with quiescent retinopathy However, vitreous concentrations of HGF were unrelated to those of VEGE CONCLUSIONS: We found that levels of HGF in vitreous fluid of PDR patients are significantly higher than in nondiabetic patients and that the levels of HGF are elevated in the active PDR stage. This suggests that HGF stimulates or perpetuates neovascularization in PDR.

Optimal radiolabeled liposomes for tumor imaging.

UNLABELLED: We conducted a systematic study of the effects of liposome formulation and encapsulated radionuclides on imaging ability. METHODS: Various types of liposomes were prepared and labeled with 67Ga, 111In or 99mTc. Their tumor-imaging potential was evaluated in terms of tumor accumulation and tumor-to-blood ratios of radioactivity delivered by the liposomes. Mouse sarcoma 180 and Ehrlich solid tumor were the tumor models. RESULTS: Liposomes could be labeled rapidly and with high efficiency, which was sufficient for clinical application. Tumor accumulation of liposome-encapsulated radionuclides that have intrinsic tumor affinity, such as 67Ga-NTA or 111In-NTA, was larger than that of the other nuclides. Liposomes that were fairly small, cholesterol-rich and composed of so-called rigid phospholipids, could deliver large amounts of encapsulated radionuclides to the tumor. We also found that tumor uptake of such liposomes was large and their blood retention was prolonged. Liposomal lipid dose also influenced tumor delivery and blood retention. The results suggest that these factors extended liposomal blood retention and, consequently, increased tumor uptake of the liposomes and tumor delivery of encapsulated radionuclides. Not all liposomes with long blood retention, however, are suitable for tumor imaging. Incorporation of monosialo-ganglioside in the liposomal membrane greatly extended blood retention but increased tumor uptake only slightly and, consequently, made the tumor-to-blood value worse. One of the 67Ga-labeled liposome formulations resulted in high tumor uptake and tumor-to-blood ratios in various tumor models as well as clearly visualized tumors clearly in sarcoma 180-bearing mice. CONCLUSION: For tumor imaging with radiolabeled liposomes, we should choose liposomal formulations and dose to give prolonged blood retention for large tumor delivery. We must then select liposomes that give good tumor-to-blood values. For the best results, the radionuclide should have intrinsic tumor affinity. Labeled liposomes that meet these criteria result in excellent tumor images.

Assessment of antioxidative ability in brain: technetium-99m-meso-HMPAO as an imaging agent for glutathione localization.

UNLABELLED: To visualize the regional localization of glutathione (GSH) in the brain, the relationship between the concentrations of tissue GSH and uptake of [99mTc]meso-hexamethyl propyleneamine oxime ([99mTc] meso-HMPAO) or [99mTc]d,l-hexamethyl propyleneamine oxime ([99mTc]d,l-HMPAO) was studied in mice. METHODS: The uptake of [99mTc]meso-HMPAO in the mouse brain was decreased to 35% of control paralleling the decrease in GSH content by pre-loading of diethyl maleate (DEM), an agent to reduce GSH. In contrast, pre-treatment with DEM scarcely affected the 99mTc-d,l-HMPAO uptake in the brain. RESULTS: The DEM treatment decreased the GSH content in liver, kidney, spleen, fat and lung but did not affect the uptake of [99mTc]meso-HMPAO in those tissues except lung. The images of rat brain acquired with a gamma camera showed a significant reduction of [99mTc]meso-HMPAO uptake by DEM treatment. CONCLUSION: Technetium-99m-meso-HMPAO may be a potential tool to assess GSH content and to estimate antioxidative ability in the brain.

Steroid-induced short term diabetes in chick embryo: reversible effects of insulin on metabolic changes and cataract formation.

PURPOSE: To determine the reversible effect of insulin on glucocorticoid (GC)-induced cataract formation in relation to systemic metabolic changes in the developing chick embryo. METHODS: Hydrocortisone sodium succinate (HC; 0.25 micromoles) was administered to 15-day-old embryos followed by administration of long-acting recombinant human insulin, 4 and 28 hours later. At the indicated time after HC administration, the incidence of cataractous lenses and any changes in the components of the lenses, liver, and blood were determined. RESULTS: At 48 hours after HC administration, the following observations were made: opacification of lenses; an elevation of glucose and lipids in the blood and lenses; an increase in lipid peroxide (LPO) in the blood, liver, and lenses; a decrease in glutathione (GSH) in the lens and liver (at 24 hours after HC administration); and a depletion of adenosine triphosphate (ATP) in the liver. These changes in response to HC administration were reversed by a double application of insulin. CONCLUSIONS: Insulin antagonizes GC-induced gluconeogenesis, stimulates glycolysis, and ultimately leads to recovery of decreased activity in the citric acid cycle. The restoration of ATP by the recovered citric acid cycle may facilitate de novo synthesis of GSH, which in turn may diminish GC-induced elevation of LPO in the liver. Thus, the metabolic changes in response to HC-accelerated gluconeogenesis in the liver, which can be reversed by insulin, are likely to produce oxidative stress that leads to cataract formation. GC-induced metabolic changes in the liver, which are antagonized by insulin, may relate to production of one of the risk factors for cataract formation.

Glucocorticoid-induced cataract in chick embryo monitored by Raman spectroscopy.

Glucocorticoid-induced cataract lens in chick embryo was monitored by laser Raman spectroscopy. The lens opacity that appeared in chick embryo is a reversible one. Raman spectra show no significant change in the relative content of water or secondary structure of the proteins upon lens opacification. The intensity ratios of tyrosine doublet bands in Raman spectra between clear and opaque lens portions are changes. This change is reversible, and is interpreted as a protein-water phase separation that occurred during lens opacification.

Analysis of glucose levels during glucocorticoid-induced cataract formation in chick embryos.

When 15-day-old developing chick embryos were administered hydrocortisone hemisuccinate sodium (HC; 0.25 mumol/egg), the content of glucose in the lens markedly increased from around 6 hr, and reached about 25-30-fold above the matched control at 24-48 hr. Thereafter, the glucose level declined and returned to the control level by 100 hr. The profile of lenticular glucose levels was similar to that of the appearance and disappearance of lens opacification. Prednisolone, as well as HC, produced cataract and the elevation of glucose in the lenses. Cortexolone and cortisone, which have weak or negligible glucocorticoid activity in developing chick embryo, could neither produce cataract nor the elevation of glucose in the lenses. An attempt was made to find similarity between this glucocorticoid-induced cataract and sugar cataract known in mammals. In both control and HC-induced cataract (stage IV-V) obtained 48 hr after HC administration, sorbitol, fructose, and glycosylation of protein could not be detected. Dehydration was observed in HC-induced cataractous lens. These data demonstrate that the glycosylation of lenticular protein and the accumulation of polyol were not involved in glucocorticoid-induced cataract formation in developing chick embryos. These results suggest a relationship between the elevation of glucose and cataract formation. However, when cataract formation was blocked by ascorbic acid treatment, the glucose level remained high. Therefore, any relationship between glucose level and cataract may be complex or indirect.

Effect of MPG on glucocorticoid-induced cataract formation in developing chick embryo.

Cataract in the developing chick embryo can be easily produced by administration of high doses of glucocorticoid and the cataract is preceded by a decreased level of glutathione in the lens (Nishigori et al, Exp Eye Res 36:617, 1983). In an attempt to prevent cataract formation, various natural and synthetic sulfhydryl compounds, glutathione, cysteine, ergothioneine, penicillamine, cysteamine and N-(2-mercaptopropionyl)glycine, were applied to hydrocortisone-treated developing chick embryos. N-(2-mercaptopropionyl)glycine showed the most potent delaying activity against cataract formation and also lessened the decrease of glutathione level in the lens. However, except for cysteamine, the other compounds tested had little or no effect.

Effects of sex hormones and glucocorticoids on the induction of a drug metabolizing enzyme by phenobarbital in chick embryo.

Elevation of aniline hydroxylase activity by phenobarbital administration was examined in liver of chick embryo pretreated with sex hormones and glucocorticoids. It was found that androgens and progestins did not alter elevation of aniline hydroxylase activity by phenobarbital. However, the phenobarbital effect was prevented by pretreatment with estrogens and enhanced by that of glucocorticoids. These results may indicate that the hormonal environment should be considered when administering agents that induce drug-metabolizing enzymes.

Preventive effect of pyrroloquinoline quinone (PQQ) on biliverdin accumulation of the liver of chick embryo after glucocorticoid administration.

Administration of a high dose of hydrocortisone hemisuccinate sodium (0.25 mumol/egg) to 15-day-old fertilized hen's eggs caused accumulation of biliverdin in the embryonic liver. The responses were 10 to 20-fold higher than controls within 48 hr as a result of stimulation of biliverdin synthesis in the liver and the decrease of biliverdin excretion from the liver. This accumulation was effectively prevented by PQQ, possibly through the enhancement of biliverdin excretion from the liver to the gallbladder. This PQQ action was due to its preventive effect against the decrease of glutathione in the liver caused by glucocorticoid since glutathione is suggested to play a role in elimination of bile components from the liver.

Pyrroloquinoline quinone (PQQ) inhibits the expression of tyrosinase mRNA by alpha-melanocyte stimulating hormone in murine B16 melanoma cells.

The biological functions of pyrroloquinoline quinone (PQQ), a bacterial redox coenzyme and potent radical scavenger, have not been elucidated in mammals. In this paper, we studied the effects of PQQ on tyrosinase activity and subsequent melanogenesis in murine B16-F10 melanoma and found that alpha-Melanocyte stimulating hormone (MSH)-induced melanogenesis was inhibited by 6.3 to 25 microM PQQ in a dose-dependent manner. Moreover, PQQ inhibited MSH-induced tyrosinase activity by suppressing tyrosinase mRNA expressed by MSH. However, PQQ had no effect on MSH-stimulated cyclic adenosine 3', 5'-monophosphate (cAMP) production. These observations suggest that PQQ inhibits the expression of tyrosinase mRNA at a post receptor level and that PQQ may be useful in investigating hormone actions mediated by cAMP.

Preventive effects of pyrroloquinoline quinone on formation of cataract and decline of lenticular and hepatic glutathione of developing chick embryo after glucocorticoid treatment.

When 0.25 mumol of hydrocortisone succinate sodium (HC) was administered to 15-day-old fertile eggs, almost all lenses of chick embryos treated with HC for 48 hr were classified as cataract stage IV-V (95%). A triple application of potassium pyrroloquinoline quinone (PQQ) (1.25 mumol/egg) at 3, 10 and 20 hr after HC treatment showed a preventive effect against the HC-induced cataract formation (I:45%, II:25%, III: 30%). PQQ also prevented the decline of GSH in the lens caused by HC. The decline of GSH in liver 24 hr after HC administration was prevented by PQQ. These data indicate that PQQ can modify HC-induced effects and that the preventive effect of PQQ against HC-induced decline of hepatic GSH seemed to influence HC-induced events in lens.

ProMMP-9 (92 kDa gelatinase) in vitreous fluid of patients with proliferative diabetic retinopathy.

Matrix metalloproteinases (MMPs) are implicated in tissue destruction during various pathophysiologic conditions. The vitreous body is a gel-like extracellular matrix that undergoes liquefaction during aging and pathological processes. To investigate the pathogenic role of MMPs in proliferative diabetic retinopathy (PDR), we studied 73 eyes from PDR patients and 25 eyes from patients with non-diabetic ocular diseases. Vitreous MMPs were measured by zymography. Retinopathy was assessed by ophthalmoscopy and PDR was classified into 3 stages, 'naked', 'active', and 'quiescent'. Although proMMP-9 was expressed in only 8% (2/25) of non-diabetic patients, it was expressed in more than 80% (38/47) of 'active' PDR patients and still expressed in 60% (9/15) of those with 'quiescent' PDR. Vascular endothelial growth factor (VEGF) in vitreous fluids was undetectable (<0.16 ng/ml) in most of the non-diabetic patients, and was maximally elevated in the 'active' PDR patients (mean=2.20 ng/ml, range; 0.16-7.61), declining in patients with 'quiescent' PDR (1.04 ng/ml, 0.16-3.77). These results suggest that MMP-9 is one of the noteworthy factors in relation to the progress of PDR, as well as angiogenic cytokines such as VEGF.

Alteration of hepatic lipidperoxide levels during cataract formation caused by glucocorticoids in developing chick embryos.

The level of hepatic lipidperoxides in chick embryos was determined during cataract formation resulting from glucocorticoid treatment. When 15 day old chick embryos were administered 0.25 mumol of hydrocortisone acetate their hepatic lipidperoxide level, determined by thio-barbituric acid, increased after a lag time of 20 hr and reached approximately 8-fold of control at 48 hr after the treatment. These studies indicate that the peroxidation of lipid in tissues should be considered in elucidating mechanisms of action or adverse effects of glucocorticoids.

Establishment of a chick embryo shell-less culture system and its use to observe change in behavior caused by nicotine and substances from cigarette smoke.

Early detection and removal of harmful factors are essential to the proper physical and psychological development of the fetus, presumably showing the effects during the prenatal period and after birth. As one procedure to aid in understanding such factors, we have established a shell-less culture system for video monitoring to observe change in behavior of 7-day-old chick embryos. Nicotine and aqueous cigarette smoke extract (ACSE) were selected for the present experiments, and the results showed a complete stoppage of swing-like movements by administrations of 10 microg nicotine and 1xACSE, possibly displaying paralytic symptoms. Quantitative analysis of nicotine in 1xACSE indicated that more than 10 microg of nicotine were contained in 100 microl of the extract. The present system, although in initial stage of development, may be a useful preliminary screening procedure for perhaps supervision and warning about the environment surrounding pregnant women.

Pigmentation of glucocorticoid-induced cataractous lenses of developing chick embryos on heating.

Cataractous lenses produced by hydrocortisone or prednisolone treatment in developing chick embryos turned golden brown on heating at 100 degrees C for 60 min. Analysis of an 80% ethanol fraction from the lenses, which contained the pigment-forming substance(s), demonstrated that the heat-induced pigmentation was related to the appearance of opacity of the lens after glucocorticoid administration and suggested that the pigmentation occurred by the Maillard reaction between glucose and amino acids.