Eternacept for the treatment of patients with rheumatoid arthritis and concurrent interstitial lung disease.

WHAT IS KNOWN AND OBJECTIVE: Tumour necrosis factor-α (TNF-α)-blocking agents are increasingly used in the management of refractory rheumatoid arthritis (RA). Although effective, they are associated with rare but potentially fatal adverse effects, including interstitial lung disease (ILD). In patients with pre-existing ILD, eternacept (ETN) monotherapy is often regarded as a suitable choice. Other anti-TNF-α blockers such as infliximab and adalimumab, are used in combination therapy with methotrexate (MTX) in most of the cases. We report on a case of fatal exacerbation of ILD in a patient given ETN monotherapy and review the literature on ETN-associated ILD. METHODS: We report on a case of a 75-year-old male with RA who developed severe ILD after the introduction of ETN, and we undertook a literature search to identify other reports of similar cases. We then critically assessed those reports. RESULTS AND DISCUSSION: In addition to our case, 11 other patients have been reported to have developed ILD in association with the use of ETN. Six patients had pre-existing ILD. Although four patients received MTX, eight patients developed severe ILD without MTX. Ten patients recovered after termination of ETN, although two patients died. WHAT IS NEW AND CONCLUSION: Although ETN is often regarded as safe for patients with ILD, our case and the literature reports suggest that caution is still required.

Improvement in multipass Thomson scattering system comprising laser amplification system developed in GAMMA 10/PDX.

The multipass Thomson scattering (MPTS) technique is one of the most useful methods for measuring low-electron-density plasmas. The MPTS system increases Thomson scattering (TS) signal intensities by integrating all multipass (MP) signals and improving the TS time resolution by analyzing each pass signal. The fully coaxial MPTS system developed in GAMMA 10/potential-control and diverter-simulator experiments has a polarization-based configuration with image-relaying optics. The MPTS system can enhance Thomson scattered signals for improving the measurement accuracy and megahertz-order time resolution. In this study, we develop a new MPTS system comprising a laser amplification system to obtain continuous MP signals. The laser amplification system can improve degraded laser power and return an amplified laser to the MP system. We obtain continuous MP signals from the laser amplification system by improving the laser beam profile adjuster in gas scattering experiments. Moreover, we demonstrate that more MP signals and stronger amplified MP signals can be achieved via multiple laser injections to the laser amplification system in the developed MP system comprising a laser amplification system.

Development of a laser amplification system for the multi-pass Thomson scattering system for GAMMA 10/PDX.

The multi-pass Thomson scattering (MPTS) system is a useful technique for increasing the Thomson scattering (TS) signal intensities and improving the TS diagnostic time resolution. The MPTS system developed in GAMMA 10/PDX has a polarization-based configuration with an image relaying system. The MPTS system has been constructed for enhancing the Thomson scattered signals for the improvement of measurement accuracy and the megahertz sampling time resolution. However, in the normal MPTS system, the MPTS signal intensities decrease with the pass number because of the damping due to the optical components. Subsequently, we have developed a new MPTS system with the laser amplification system. The laser amplification system can improve the degraded laser power after six passes in the multi-pass system to the initial laser power. For the first time worldwide, we successfully obtained the continued multi-pass signals after the laser amplification system in the gas scattering experiments.

Insertion of a 5' truncated L1 element into the 3' end of exon 44 of the dystrophin gene resulted in skipping of the exon during splicing in a case of Duchenne muscular dystrophy.

We report here the second evidence of retrotransposition of L1, which was found inserted into the dystrophin gene of a patient, causing Duchenne muscular dystrophy (DMD). When the PCR was used to amplify a region of the dystrophin gene encompassing exon 44 from genomic DNA of two Japanese brothers with DMD, it was found to be approximately 600 bp larger than expected. Both the normal and the abnormally large products were amplified from the DNA of their mother. However, the maternal grandparents did not have the abnormal allele, and the mutation must therefore have occurred in the mother. Analysis of nucleotide sequence of the amplified product from a patient disclosed that the insertion was present zero to two bases upstream from the 3' end of exon 44 and that two to four bases of the exon sequence were deleted from the insertion site. The insertion sequence was found to be composed of 606-608 bp and to be almost identical to the inverse complement of 3' portion of the L1 retrotransposon consensus sequence. The dystrophin gene transcript from peripheral lymphocytes of one of the patients was analyzed by using reverse transcription/semi-nested PCR. The size of the amplified product encompassing exon 42 to 46 was smaller than expected. Sequencing of the amplified product disclosed that the sequence of exon 43 was directly joined to that of exon 45. Exon 44 of the transcript was thus shown to be skipped during splicing. This novel mutation of the dystrophin gene has important implications regarding retrotransposition of an active L1 element and provides a new insight into the origins of mutations in the dystrophin gene.

Identification of a novel first exon in the human dystrophin gene and of a new promoter located more than 500 kb upstream of the nearest known promoter.

The dystrophin gene, which is mutated in patients with Duchenne and Becker muscular dystrophies, is the largest known human gene. Five alternative promoters have been characterized until now. Here we show that a novel dystrophin isoform with a different first exon can be produced through transcription initiation at a previously unidentified alternative promoter. The case study presented is that of a patient with Duchenne muscular dystrophy who had a deletion extending from the 5' end of the dystrophin gene to exon 2, including all promoters previously mapped in the 5' part of the gene. Transcripts from lymphoblastoid cells were found to contain sequences corresponding to exon 3, indicating the presence of new promoter upstream of this exon. The nucleotide sequence of amplified cDNA corresponding to the 5' end of the new transcript indicated that the 5' end of exon 3 was extended by 9 codons, only the last (most 3') of which codes for methionine. The genomic nucleotide sequence upstream from the new exon, as determined using inverse polymerase chain reaction, revealed the presence of sequences similar to a TATA box, an octamer motif and an MEF-2 element. The identified promoter/exon did not map to intron 2, as might have been expected, but to a position more than 500 kb upstream of the most 5' of the previously identified promoters, thereby adding 500 kb to the dystrophin gene. The sequence of part of the new promoter region is very similar to that of certain medium reiteration frequency repetitive sequences. These findings may help us understand the molecular evolution of the dystrophin gene.

Measurement of effect of electron cyclotron heating in a tandem mirror plasma using a semiconductor detector array and an electrostatic energy analyzer.

Temporally and spatially resolved soft x-ray and end-loss-electron analyses of the electron cyclotron heated plasmas are carried out by using a semiconductor detector array and an electrostatic energy analyzer in the GAMMA 10 tandem mirror. The flux and the energy spectrum of the end loss electrons are measured by a multi-grid energy analyzer. Recently, the electron cyclotron heating power modulation experiments have been started in order to generate and control the high heat flux and to make the edge localized mode-like intermittent heat load pattern for the divertor simulation studies by the use of these detectors for electron properties.

Analysis of 5'-flanking region of human Smad4 (DPC4) gene.

Among the transducers of the transforming growth factor (TGF)-beta/bone morphogenetic protein (BMP) receptor signaling proteins, Smad4 and Smad1 act together in BMP 2/4 signaling pathways, and Smad4 and Smad2 act in TGF-beta/activin signaling. To investigate how the Smad4 gene is regulated at the transcriptional level, we cloned and characterized its 5'-flanking region. The major transcription start site mapped by primer extension analysis was 132 bp upstream of the translation start site. The promoter region lacked canonical TATA and GC boxes; it did, however, contain a TATA-like structure (TAAAAT) 32 bp upstream of the transcription start site. A consensus sequence for homeoprotein HoxA-5 (TTTAAAAATTA) was identified at 171 bp upstream of the transcription start site. Within 600 bp upstream of the transcription start site, two Pit-1 and four F2F binding sites were found. One putative AP-1 site was located at -1129. These findings suggest that these homeoproteins could conduct their signals specifically by controlling both inter- and intracellular signal transduction pathways.

Interleukin-6 sensitizes multiple myeloma cell lines for apoptosis induced by interferon-alpha.

OBJECTIVE: Interleukin-6 (IL-6) is a multifunctional cytokine affecting growth and survival of normal B cell lineage and multiple myeloma cells. To test the hypothesis that IL-6, as well as other hematopoietic growth factors, may enhance apoptosis of target cells, we investigated the effect of IL-6 on myeloma cells in the presence of IFN-alpha, which is prescribed for patients with multiple myeloma. MATERIALS AND METHODS: Four myeloma cell lines, PCM6, NOP-2, U266, RPMI8226 were tested. We determined the induction of apoptosis by flow cytometry, using an FITC-Annexin V. RESULTS: IFN-alpha induced apoptosis on myeloma cell lines, and this apoptosis was further enhanced in the presence of IL-6, via activation of caspase 3. During induction of this apoptosis, the expression of c-Myc and Fas increased. The addition of IL-6 further increased the expression of Fas, but not that of c-Myc. Bcl-2, Bcl-x, and p53 were not affected by the addition of IL-6 and/or IFN-alpha. Addition of a PI-3-K inhibitor interfered with the enhancing effect of IL-6 on the apoptosis induced by IFN-alpha. CONCLUSION: We propose that IL-6 has the death signal, as well as growth promoting effects, and that PI-3-K may play a key role in the induction of apoptosis by IL-6.

A novel deletion of the dystrophin S-promoter region cosegregating with mental retardation.

A contiguous deletion of the S-promoter/first S exon and its downstream exon 56 of the dystrophin gene was identified in a Japanese dystrophinopathy family in which two brothers and their half brother were affected. Characteristically, severe mental retardation cosegregated with the deletion even though they grew up in different environments. Furthermore, mild cerebral atrophy was observed by CT scan and MRI in the eldest brother.

Amino-terminal deletion of 53% of dystrophin results in an intermediate Duchenne-Becker muscular dystrophy phenotype.

We report a Japanese boy with muscular dystrophy whose clinical symptoms were intermediate between those usually considered typical of Duchenne and Becker muscular dystrophies. The patient had a large inframe deletion extending from exons 3 to 41 of the dystrophin gene, which would be expected to cause the production of a dystrophin protein composing only 53% of the normal polypeptide chain. Such an inframe deletion would be expected to cause Becker muscular dystrophy. We did not obtain evidence for alternative splicing or for RNA editing. Immunocytochemical analysis of skeletal muscle showed that a dystrophin-related polypeptide was detectable with antibody directed against the carboxyl-terminal part of the polypeptide but not with antibodies directed against the amino-terminal part, although labeling by antibody against the carboxyl-terminal was faint and patchy. The severity of the disease in this case may be due to the lack of the amino-terminal, actin-binding domain of dystrophin.

Glut1 expression in T1 and T2 stage colorectal carcinomas: its relationship to clinicopathological features.

Glucose uptake is mediated by glucose transporter (Glut) proteins, which exhibit altered expression in a variety of malignant neoplasms. Glut1 expression is thought to be a potential marker for malignant transformation. The aim of the present study was to investigate the expression of Glut1 protein in colorectal adenomas, T1 and T2 stage carcinomas. Immunohistochemical detection of Glut1 protein was examined in 141 formalin-fixed and paraffin-embedded colorectal tumour specimens (57 adenomas, 84 carcinomas). The degree of Glut1 immunostaining of a specimen was graded according to the proportion of Glut1-positive cells in it; absent (positive cells are 0%), weakly positive (less than 10%), moderately positive (10-50%), and strongly positive (more than 50%). Glut1 expression was present in 18% of the adenomas with low-grade dysplasia, and in 63% of the adenomas with high-grade dysplasia. The positivity in such lesions was usually weak, but was moderate in 8% of the adenomas with high grade dysplasia. For the carcinomas, there were significant correlations between Glut1-positivity and depth of invasion (T1 45% versus T2 74%, P<0.01), histological differentiation (well 49% versus moderately to poorly 74%, P< 0.05) and morphological type (polypoid 42% versus depressed 73%, P< 0.05), if the cut-off value was set at 10% of cells. In conclusion, we clarified the relationship between Glut1 expression and clinicopathological features in T1 and T2 stage colorectal carcinomas, and our results suggested a high malignant potential of the depressed-type carcinoma.

Variation in MT expression in early-stage depressed-type and polypoid-type colorectal tumours.

Metallothionein (MT) expression is observed in various carcinomas, but its role is not fully understood. To clarify the clinicopathological significance of MT, 87 colorectal adenomas and 128 early-stage carcinomas were immunohistochemically analysed for MT expression. The degree of MT immunostaining of a specimen was graded according to the proportion of MT-positive cells; negative (<5%) and positive (focally 5-50%, diffusely >50%). MT expression significantly decreased with tumour development. For carcinomas, MT-positivity was significantly associated with depth of invasion (T1 60% versus T2 33%; P<0.01), vascular involvement (positive 35% versus negative 61%; P<0.01) and morphology (polypoid 62% versus depressed 26%; P<0.01). Regarding MT-positive distribution, the diffuse-positive rate in MT-positive polypoid lesions was 28%, while MT-positive depressed lesions were all diffusely stained (P<0.01). In conclusion, our results suggested that decreasing MT expression is an early event in colorectal carcinogenesis and may reflect local invasion. Furthermore, MT-positive distribution may reflect genetic differences between the polypoid and depressed-type.

An enzyme-linked immunosorbent assay for antibodies to native and denatured DNA.

A modification of the enzyme-linked immunosorbent assay (ELISA) is described, that permits determination of antibodies to native DNA (nDNA). The same approach can be used to measure antibodies to denatured DNA (dDNA). Poor binding of nDNA to the polystyrene solid phase has presented difficulties in using the ELISA method for assaying anti-nDNA activity (Engvall, 1976), but we find that precoating of the solid phase with protamine sulfate circumvents this problem. Assays for anti-dDNA are also enhanced by the use of protamine sulfate coated tubes. We have used the ELISA method to assay 15 SLE and 27 non-SLE sera for anti-nDNA and anti-dDNA activity. The results are compared with those obtained using the GF/A glass fiber filter assay, previously described by Lewis et al. (1973).

Characterization of neutral glycosphingolipids from fetal human brain: evidence for stage-specific expression of the globo, ganglio, and neolacto series in the central nervous system.

Neutral glycosphingolipids were isolated from normal human fetal brains, at 22 to 23 weeks gestation. They were identified as monohexosylceramides, lactosylceramide, and glycolipids belonging to the globo (globotriaosylceramide) and ganglio (gangliotriaosylceramide) series. In addition, considerable amounts of neolactotetraosylceramide and III3-alpha-fucosyl-neolactotetraosylceramide were detected. Although neutral glycolipids of the globo, ganglio, and neolacto series have been demonstrated in the brains of cases with some sphingolipidoses, they are not present in appreciable amounts in differentiated normal brain. Therefore, the present and previous observations would imply that the metabolism of these glycolipid series actively occurs in the normal brain at an early stage of differentiation and continues thereafter in the brain in the case of some sphingolipidoses. The diseased brain is most probably accompanied by a disturbance of differentiation.

Properties, sequence, and synthesis in Escherichia coli of 1-aminocyclopropane-1-carboxylate deaminase from Hansenula saturnus.

The plant hormone ethylene is generated from a unique precursor, 1-aminocyclopropane-1-carboxylate (ACC). In previous studies, ACC deaminase, which degrades ACC to alpha-ketobutyrate and ammonia, was found in four strains of Pseudomonas, characterized, and sequenced. To verify the wider distribution of ACC deaminase in microorganisms, we purified and sequenced ACC deaminase from the yeast Hansenula saturnus. The purified enzyme was active toward ACC, D-serine and dl-coronamic acid, indicating the same stereospecificity as the Pseudomonas enzyme, but unlike the bacterial enzyme it was not active toward beta-chloro-D-alanine and O-acetyl-D-serine. Analyses of peptides from proteolytic digests of the purified and modified ACC deaminase covered more than 90% of its amino acid sequence and showed a blocked N-terminal residue as N-acetylserine. A cDNA encoding the ACC deaminase was isolated from H. saturnus cells incubated in alpha-aminoisobutyrate medium, and sequenced. The yeast enzyme has 441 amino acid residues, of which 60 to 63% are identical to those of reported Pseudomonas enzymes. The open reading frame encoding ACC deaminase was subcloned into pET-11d and expressed in Escherichia coli BL21 (DE3) as an active enzyme.

Angiosarcoma arising from skeletal haemangiomatosis in an atomic bomb survivor.

The authors report a unique case in which an angiosarcoma arose from skeletal haemangiomatosis in a 72 year old man. This patient had a history of atomic bomb irradiation more than 50 years ago. Radiographically, the patient had multiple sclerotic foci of benign haemangiomas in the pelvis, the sacrum, and the left femur. The patient developed a high grade angiosarcoma in the left pubic bone. It is thought that atomic bomb irradiation played an important role in the development of the malignant lesion.

Effect of milk on absorption of norfloxacin in healthy volunteers.

The authors studied the effect of milk on the bioavailability of norfloxacin in six healthy male volunteers in a randomized crossover trial. After an overnight fast, 200 mg of norfloxacin was given with 200 mL of water or milk. Area under the curve (AUC) of norfloxacin with milk was significantly (P < 0.01) smaller than that with water. The mean peak serum concentration was decreased to 60% after oral administration of norfloxacin with milk (P < 0.01). The apparent volume of distribution at central compartment (Vc/f) value of norfloxacin was significantly (P < 0.05) increased with milk. Milk exhibits a clinically significant effect on norfloxacin absorption.

Aberrant BCR-ABL transcript with intronic insertion in a patient with philadelphia chromosome-positive chronic myeloid leukemia: implications for disease progression.

The BCR-ABL fusion gene is important for the leukemogenesis of chronic myeloid leukemia (CML). A relationship between types of BCR-ABL transcripts in CML and clinical features has been proposed. We present here a patient with CML who carried an aberrant BCR-ABL transcript with an intronic sequence insert. A 26-year-old woman was diagnosed as having Philadelphia chromosome (Ph) positive CML. Reverse transcription polymerase chain reaction detected an atypically large BCR-ABL mRNA transcript. Sequencing revealed a 589bp insertion consisting of a 5' portion of BCR intron b2 and a 3' portion of ABL intron 1b between BCR exon b2 and ABL exon a2. Although the typical b2a2 transcript was undetectable initially, it appeared after intensive chemotherapy. The aberrant transcript presumably arose as a result of a lack of splicing, and chemotherapy might modify the disease course by selecting the subpopulation of the CML clone expressing typical BCR-ABL mRNA dominantly.

Central nervous system involvement and generalized muscular atrophy in occipital horn syndrome: Ehlers-Danlos type IX. A first Japanese case.

Occipital horn syndrome (OHS, Ehlers-Danlos syndrome type IX) belongs to the category of the copper metabolism disorders and is at present being investigated biochemically as is Menkes' disease. Unlike Menkes' disease, most patients with OHS have mild submentality. We report a case of OHS with severe central nervous system involvement and muscular atrophy in a 34-year-old male. He had psychomotor retardation and seizures since early childhood and now presented severe mental retardation and generalized muscular atrophy in addition to characteristic facial appearance, hyperelasticity of the skin and joint subluxation. Laboratory investigations revealed a low serum copper and ceruloplasmin level as well as intestinal non-absorption of copper. Radiographic imaging showed occipital exostoses, bladder diverticula, tortuosity of the peripheral vein and osteoporosis of the skeletal bones. The activity of lysyl oxidase, a copper-enzyme involved in cross-link formation in collagen, was found to be decreased in a skin-biopsy specimen. Electron-microscopic investigation of a muscle biopsy showed irregularity of the myofibrillar network and accumulation of concentric laminated bodies in the subsarcolemmal regions.

Delayed expression of dystrophin on regenerating muscle from two siblings with Becker muscular dystrophy.

We present here a unique expression of dystrophin on biopsied muscle from 2 siblings with Becker muscular dystrophy (BMD). They had neither muscle weakness nor atrophy. Clustered dystrophin-deficient fibers were constituted to regenerating basophilic fibers (mainly type 2C fiber) based on histochemical stainings. We speculate that the developmental delay in the expression of dystrophin is a characteristic finding in regenerating fibers from asymptomatic and young BMD patients, such as the siblings in this report.