Prediction of postnatal clinical course in primary congenital dilated cardiomyopathy.

BACKGROUND: The aim of this study was to investigate the prediction of postnatal prognosis using fetal and perinatal data in patients with primary congenital dilated cardiomyopathy (PCDCM), and to estimate the incidence of this disease. METHODS: We examined correlations between fetal or perinatal data and postnatal clinical course in a multicenter retrospective study of eight patients with PCDCM. Incidence was calculated in a population-based study. RESULTS: All patients developed heart failure at a median of 8 days (range, 0-43 days), and six patients died or required extracorporeal artificial heart therapy at a median of 67 days (range, 0-92 days). The cardiothoracic area ratio from fetal echocardiography, the Apgar score, and the standard deviation of birth weight correlated significantly with the date at onset of heart failure. However, no data correlated with survival. Cumulative incidence of PCDCM was calculated as 1.21 per 100 000 total births (95% confidence interval, 0.37 to 2.06). CONCLUSIONS: Primary congenital dilated cardiomyopathy has a poor prognosis, but cardiothoracic area ratio from fetal echocardiography, body weight at birth, and Apgar score correlate with the timing of the onset of heart failure, and these indicators might therefore be useful for peri- and postnatal management.

Aortic valve replacement using Konno procedure for prior Yasui procedure.

A 2-year-old girl underwent conversion to the Konno procedure by removing the Damus-Kaye-Stansel anastomosis after the neonatal Yasui procedure for an interrupted aortic arch with left ventricular outflow tract stenosis. Her postoperative course was uneventful. However, left ventricular outflow tract restenosis occurred due to narrowed ventricular septal defect and moderate neoaortic regurgitation from the old pulmonary valve. The Konno procedure was performed by removing the Damus-Kaye-Stansel anastomosis for left ventricular outflow tract restenosis and neoaortic regurgitation and performing right ventricular outflow tract reconstruction and ventricular septal defect closure. Left ventricular outflow tract restenosis was not observed.

A case of biatrial drainage of the right superior vena cava.

Biatrial drainage of the right superior vena cava resulting from a sinus venosus defect is a rare congenital malformation. It can result in severe desaturation although a sinus venosus defect usually presents as a left-to-right shunt. A male baby was noted to have cyanosis while nursing and was referred to us for medical treatment on his second day of life. Echocardiography showed that most of the blood flowing through the superior vena cava drained into the left atrium. He underwent successful surgery to correct this defect at the age of 2 years.

Neuroradiologic findings in Sotos syndrome.

Sotos syndrome is a well-known anomaly syndrome characterized by overgrowth, characteristic facial gestalt, and developmental delay, and haploinsufficiency of the NSD1 gene has been revealed as one of the major genetic causes. However, there have been only a few reports on neuroradiologic findings by computed tomography (CT) or magnetic resonance imaging (MRI), and functional examination of the brain has not been reported. We examined three cases with typical Sotos syndrome, which also were confirmed by genetic analysis with a specific probe for the NSD1 gene. The results of MRI showed the characteristic features that have been reported previously. The findings obtained by using single-photon emission computed tomography and magnetic resonance spectroscopy suggested an association between mental delay and behavioral tendency in Sotos syndrome and immaturity in frontal brain function.

MR imaging and 1H-MR spectroscopy of a case of van der Knaap disease.

Van der Knaap disease, characterized by megalencephalic leukoencephalopathy and subcortical cysts, is a rare and recently defined condition. We discuss here the MR image (MRI) and MR spectroscopy (MRS) features in a 30-year-old man with S93L homozygous mutation in the MLC1 gene. MRI demonstrated high intensity diffuse white matter with T2-weighted image and subcortical cysts in the parietal and temporal lobes and MRS showed mildly reduced N-acetylaspartate (NAA) in areas of severe T2 elongation with a long TE sequence. A peak of lactate/lipid was indicated at a chemical shift of 1.3 ppm with a short TE sequence. The peak for myo-inositol was normal in areas of severe and mild T2 elongation with short TE MRS. These findings suggest that demyelination progresses slowly in van der Knaap disease and that MRS with long and short TE is useful for the evaluation of neural metabolization associated with van der Knaap disease.

Sweet's syndrome in a neonate with non-B54 types of human leukocyte antigen.

BACKGROUND: Sweet's syndrome (acute febrile neutrophilic dermatosis) is characterized by fever, polymorphonuclear leukocytosis of blood, painful plaques on the limbs, face and neck, and histologically a dense dermal infiltration with mature neutrophils. Sweet's syndrome is often a complication of hematologic malignant disease or drug-induced sensitivity reactions and has a significant susceptibility correlated with certain human leukocyte antigen (HLA). METHODS: A 5-week-old Japanese girl with Sweet's syndrome confirmed by skin biopsy was successfully treated and HLA analysis was performed. RESULTS: The patient was one of the youngest patients reported with Sweet's syndrome, suggesting the importance of the genetic background. Although the HLA types of the patient did not have B54, which was reported as a significant susceptibility correlation, structural analysis of the patient's HLAs suggested a similar possible motif for the bound peptides. CONCLUSION: Studies on the HLA bound peptides and HLA structural analysis for patients with Sweet's syndrome would be valuable for understanding the molecular mechanism of the pathogenesis.

Development of fluorescence-linked immunosorbent assay for high throughput screening of interferon-gamma.

BACKGROUND: Human interferon-gamma (hIFN-gamma) is produced by lymphocytes and has a variety of biological properties. Measurement of hIFN-gamma is widely used for various immunological responses for allergic or autoimmune diseases. Enzyme-linked immunosorbent assay (ELISA) is an established immunoassay used to quantify cellular metabolites or cytokines. ELISA requires many incubation and wash steps and is not practically suitable for screening large numbers of samples. METHODS: We have developed a fluorescence-linked immunosorbent assay (FLISA) method for the detection of hIFN-gamma. We measured the 50% inhibitory concentration (IC50) value of the hIFN-gamma production by interleukin (IL)-18 binding protein and anti-IL-18 monoclonal antibody. The IC50 described by FLISA was compared with that by ELISA. RESULTS: We developed a new system for measuring hIFN-gamma using Allophycocyanine (APC) fluorescent protein and compared it with the previous method using Cy5.5. The proposed FLISA had a smaller coefficient of variation than ELISA, and the means of coefficient of variation using the same samples measured by ELISA and FLISA were, respectively, 11.1% and 3.8%, suggesting that the edge effect often giving non-specific results may be smaller in FLISA than in ELISA. CONCLUSIONS: The improved FLISA system proposed is ideally suited for efficient measurements of hIFN-gamma. This homogeneous and multiplex method will be a powerful tool for high throughput screening for drug discovery research.

Molecular mechanism of a temperature-sensitive phenotype in peroxisomal biogenesis disorder.

Peroxisomal biogenesis disorders include Zellweger syndrome and milder phenotypes, such as neonatal adrenoleukodystrophy (NALD). Our previous study of a NALD patient with a marked deterioration by a fever revealed a mutation (Ile326Thr) within a SH3 domain of PEX13 protein (Pex13p), showing a temperature-sensitive (TS) phenotype in peroxisomal biogenesis. Clinical TS phenotypes also have been reported in several genetic diseases, but the molecular mechanisms still remain to be clarified. The immunofluorescent staining with anti-Pex13p antibody also revealed TS phenotype of the I326T mutant protein itself in the patient cells. Protease digestion of the recombinant Pex13p-SH3 domain showed an increase of protease susceptibility, suggesting a problem of mutant protein fold. Conformational analyses against urea denaturation using urea gradient gel electrophoresis or fluorescence emission from tryptophan residue revealed that the mutant protein should be easily unfolded. Far-UV circular dichroism (CD) spectra demonstrated that both wild-type and the mutant protein have antiparallel beta-sheets as their secondary structure with slightly different extent. The thermal unfolding profiles measured by CD showed a marked lower melting temperature for I326T protein compared with that of wild-type protein. Analysis of the protein 3D-structure indicated that the Ile326 should be a core residue for folding kinetics and the substitution of Ile326 by threonine should directly alter the kinetic equilibrium, suggesting a marked increase of the unfolded molecules when the patient had a high fever. Structural analyses of the protein in the other genetic diseases could provide an avenue for better understanding of genotype-phenotype correlations.

Optimized gene synthesis and high expression of human interleukin-18.

Human interleukin-18 (hIL-18), originally known as an IFN-gamma-inducing factor, is a recently cloned cytokine that is secreted by Kupffer cells of the liver and by stimulated macrophages. We have previously established a method of expression and purification of IL-18. The yield however remains low and the insufficient expression of a heterologous protein could be due to skewed codon usage between the expression host and the cDNA donor. The sequence of mature hIL-18 has 37 a.a. rare codons for Escherichia coli in a total of 157 a.a. To overcome this problem, gene synthesis was performed with optimized codons for the expression host E. coli. The final yield of the hIL-18 protein with optimized codons was about five times higher than the yield with the native sequence. Using a minimal medium, this system produces large quantities of labeled proteins that can be used in NMR analysis. Our simple and efficient production system can be applied to the production of other cytokines for new structural and therapeutic use.

Generation of highly stable IL-18 based on a ligand-receptor complex structure.

Human interleukin-18 (hIL-18), initially cloned as an IFN-gamma-inducing factor, has a key role in many inflammatory diseases. We have previously developed a high production system for correctly folded active hIL-18 protein, leading to the revelation of the 3D-structure and the receptor binding mode. These findings can strongly indicate the experimental and medical applications of IL-18; however, the recombinant protein is prone to be inactivated forming multimers. Recently, therapeutic approaches using recombinant IL-18 have shown the effectiveness for treatment of cancer; indicating the necessity of a more stable protein for therapy with intertrial reliability. Here we have generated a highly stable hIL-18 with replacement of cysteine by serine based on the tertiary structure and the binding mechanism, retaining the biological activity. Similar rational designs can be applied to develop new therapeutic molecules of other cytokines.

Systematic optimization of active protein expression using GFP as a folding reporter.

Many recombinant proteins have been used as drugs; however, human proteins expressed using heterologous hosts are often insoluble. To obtain correctly folded active proteins, many optimizations of expression have been attempted but usually are found to be applicable only for specific targets. Interleukin-18 (IL-18) has a key role in many severe disorders including autoimmune diseases, and therapeutic approaches using IL-18 have been reported. However, production of IL-18 in Escherichia coli resulted in extensive inclusion body formation and previous conventional screenings of expression conditions could obtain only a condition with a low yield. To address the problem, we applied a folding reporter system using green fluorescent protein (GFP) for screening of the expression conditions for hIL-18. The established system efficiently screened many conditions, and optimized conditions for the expression of hIL-18 significantly enhanced the final yield of the active protein. Systematic screening using a GFP reporter system could be applied for the production of other proteins and in other organisms.

The structure and binding mode of interleukin-18.

Interleukin-18 (IL-18), a cytokine formerly known as interferon-gamma- (IFN-gamma-) inducing factor, has pleiotropic immunoregulatory functions, including augmentation of IFN-gamma production, Fas-mediated cytotoxicity and developmental regulation of T-lymphocyte helper type I. We determined the solution structure of IL-18 as a first step toward understanding its receptor activation mechanism. It folds into a beta-trefoil structure that resembles that of IL-1. Extensive mutagenesis revealed the presence of three sites that are important for receptor activation: two serve as binding sites for IL-18 receptor alpha (IL-18Ralpha), located at positions similar to those of IL-1 for IL-1 receptor type I (IL-1RI), whereas the third site may be involved in IL-18 receptor beta (IL-18Rbeta) binding. The structure and mutagenesis data provide a basis for understanding the IL-18-induced heterodimerization of receptor subunits, which is necessary for receptor activation.