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.

Characterization of a caspase-3-substrate kinome using an N- and C-terminally tagged protein kinase library produced by a cell-free system.

Caspase-3 (CASP3) cleaves many proteins including protein kinases (PKs). Understanding the relationship(s) between CASP3 and its PK substrates is necessary to delineate the apoptosis signaling cascades that are controlled by CASP3 activity. We report herein the characterization of a CASP3-substrate kinome using a simple cell-free system to synthesize a library that contained 304 PKs tagged at their N- and C-termini (NCtagged PKs) and a luminescence assay to report CASP3 cleavage events. Forty-three PKs, including 30 newly identified PKs, were found to be CASP3 substrates, and 28 cleavage sites in 23 PKs were determined. Interestingly, 16 out of the 23 PKs have cleavage sites within 60 residues of their N- or C-termini. Furthermore, 29 of the PKs were cleaved in apoptotic cells, including five that were cleaved near their termini in vitro. In total, approximately 14% of the PKs tested were CASP3 substrates, suggesting that CASP3 cleavage of PKs may be a signature event in apoptotic-signaling cascades. This proteolytic assay method would identify other protease substrates.

In vitro activation of the corticosteroid ciclesonide in animal nasal mucosal homogenates.

Ciclesonide, a new corticosteroid for allergic rhinitis, is administered as an inactive parent compound that is converted by esterases to the pharmacologically active metabolite, desisobutyryl-ciclesonide (des-CIC). This study investigated the in vitro activation of ciclesonide in nasal mucosa of multiple animal species. Nasal mucosal homogenates from rats, guinea-pigs, rabbits and dogs were incubated with ciclesonide 0.5 micromol/l (0.271 microg/ml) or 5 micromol/l (2.71 microg/ml) for up to 120 min. Concentrations of ciclesonide and des-CIC were measured by high-performance liquid chromatography with tandem mass spectrometry. Ciclesonide was metabolized to des-CIC in nasal mucosal homogenates of each species. The initial velocities of des-CIC formation ranged from 0.0038 to 0.0150 nmol/min/mg protein and 0.0319 to 0.0983 nmol/min/mg protein in nasal mucosal homogenates incubated with ciclesonide 0.5 micromol/l and 5 micromol/l, respectively. Furthermore, the initial velocities of ciclesonide metabolism ranged from 0.0032 to 0.0142 nmol/min/mg protein and 0.0445 to 0.1316 nmol/min/mg protein in nasal mucosal homogenates incubated with ciclesonide 0.5 micromol/l and 5 micromol/l, respectively. This study confirms that ciclesonide is converted to des-CIC in nasal mucosal homogenates without any marked differences among animal species.

Semicontinuous measurements of organic carbon and acidity during the Pittsburgh Air Quality Study: implications for acid-catalyzed organic aerosol formation.

Laboratory evidence suggests that inorganic acid seed particles may increase secondary organic aerosol yields secondary organic aerosol (SOA) through heterogeneous chemistry. Additional laboratory studies, however, report that organic acidity generated in the same photochemical process by which SOA is formed may be sufficient to catalyze these heterogeneous reactions. Understanding the interaction between inorganic acidity and SOA mass is important when evaluating emission controls to meet PM2.5 regulations. We examine semicontinuous measurements of organic carbon (OC), elemental carbon (EC), and inorganic species from the Pittsburgh Air Quality Study to determine if we can detect coupling in the variations of inorganic acidity and OC. We were not able to detect significant enhancements of SOA production due to inorganic acidity in Western Pennsylvania most of the time, but its signal might have been lost in the noise. If we assume a causal relationship between inorganic acidity and OC, reductions in OC for Western Pennsylvania that might result from drastic reductions in inorganic acidity were estimated to be 2 +/- 4% by a regression technique, and an upper bound for this geographic area was estimated to be 5 +/- 8% based on calculations from laboratory measurements.

Two interactive genes responsible for a new inherited cataract (RCT) in the mouse.

We discovered a mutant mouse, RCT (Rinshoken cataract), with a new congenital cataract in strain SJL/J. The opacity of the lens associated with microphthalmia could be observed visually at 3 to 3.5 months of age. Marked degeneration of the lens, including loss of the fine structure of the lens fibers and swelling of epithelial cells with vacuoles of various sizes in the cortex, but no other defects except photoreceptor degeneration in the retina, was detected. Histological change in the lens was first observed at 2 days after birth. No sex-related differences were detected, and normal phenotypes in the F1 progeny of RCT and normal mice indicated that the cataract was recessive. The chromosomal location of the causative gene was determined by interval mapping by using intersubspecific backcross progeny of RCT and MSM/Ms, an inbred strain from the Japanese wild mouse Mus musculus molossinus. Backcross progeny were divided into three groups according to phenotype: mice (1) with an early-onset cataract, which can be detected visually as in RCT mice, (2) with a late-onset cataract, which can be detected histologically but not visually, and (3) with a normal lens. Three phenotypes were found to be expressed by allele combinations of two recessive genes, rct and mrct (a modifier of rct). The rct locus essential for the onset of the cataract was tightly linked to D4Mit278 on Chromosome (Chr) 4 with no recombination. The mrct locus was closely linked to D5Mit239 (chi2 = 66.3, P << 0.00001) on Chr 5.

[Deficits of Morris water maze learning in rats with striatal kainic acid lesions].

This study investigated effects of intrastriatal kainic acid administration, which induces selective neuronal cell death in the striatum sparing passing nerve fibers and terminals, on the acquisition of spatial learning in Morris water maze. Rats treated with bilateral kainic acid (0.5-1.0 microgram) into the striatum were trained to escape to a hidden platform under the water. Compared with control rats, kainic acid (1.0 microgram)-lesioned rats showed significantly longer escape latency in the place navigation, and in the following probe test lower rate of swimming within the quadrant where the platform had been placed. In addition, kainic acid (1.0 microgram)-lesioned rats showed longer latency in the cue navigation in which rats were trained to escape to a visible platform above the water. These deficits were caused by their tendency to swim along the wall of pool before approaching the platform and not by their swim difficulty. Results suggest that striatal neurons or neural circuits containing these neurons play an important role in the acquisition of spatial learning task, and the nature of this performance impairment was discussed in terms of both learning and attention deficits.

Maternal inheritance of mouse mtDNA in interspecific hybrids: segregation of the leaked paternal mtDNA followed by the prevention of subsequent paternal leakage.

The transmission profiles of sperm mtDNA introduced into fertilized eggs were examined in detail in F1 hybrids of mouse interspecific crosses by addressing three aspects. The first is whether the leaked paternal mtDNA in fertilized eggs produced by interspecific crosses was distributed stably to all tissues after the eggs' development to adults. The second is whether the leaked paternal mtDNA was transmitted to the subsequent generations. The third is whether paternal mtDNA continuously leaks in subsequent backcrosses. For identification of the leaked paternal mtDNA, we prepared total DNA samples directly from tissues or embryos and used PCR techniques that can detect a few molecules of paternal mtDNA even in the presence of 10(8)-fold excess of maternal mtDNA. The results showed that the leaked paternal mtDNA was not distributed to all tissues in the F1 hybrids or transmitted to the following generations through the female germ line. Moreover, the paternal mtDNA leakage was limited to the first generation of an interspecific cross and did not occur in progeny from subsequent backcrosses. These observations suggest that species-specific exclusion of sperm mtDNA in mammalian fertilized eggs is extremely stringent, ensuring strictly maternal inheritance of mtDNA.

Four dominant loci for the vascular responses by the antitumor polysaccharide, lentinan.

Lentinan, beta-1,6;1,3-glucan, showing an antitumor effect against mouse solid type tumors, can induce marked vascular dilation and hemorrhage (VDH) in very localized areas such as the ears, feet, and tails of mice in the early stages after its administration (Maeda et al. 1984). VDH has been found to be one of the T-cell-mediated responses triggered by lentinan. We reported previously that the responsiveness of mice to lentinan with respect to VDH induction is controlled by a dominant gene(s), Ltn2 (formerly), and that no sex difference was observed (Maeda et al. 1991). To determine the chromosomal location of the Ltn2 gene(s), we typed genomic DNAs of 193 N2 segregants of crosses between a high responder MA/MyJ and a low responder AKR/J by the polymerase chain reaction-simple sequence length polymorphism technique using 83 chromosome-specific microsatellite markers. We identified one major gene (Ltnr3) and three minor genes (Ltnr4, Ltnr5, and Ltnr6) responsible for the VDH induction. Ltnr3 was closely linked to D6Mit135 on chromosome 6 (P <0.00000) and Ltnr4, Ltnr5, and Ltnr6 to D9Mit161 on chromosome 9 (P <0.00032), D15Mit147 on chromosome 15 (P <0.00014) and D16Mit4 on chromosome 16 (P <0.00014), respectively.

Polygenic control of the expression of biological activities of an antitumor polysaccharide, lentinan.

Genetic studies were carried out on two in vivo responses of lentinan, delayed type-acute phase responses (DT-APR) and vascular dilation and hemorrhage (VDH). Linkage analyses showed that DT-APR was controlled by two recessive genes, ltnr1 and ltnr2, which were mapped on chromosome 3 and 11, respectively. VDH was also found to be controlled by polygenes. One dominant major gene, Ltnr3, and three dominant minor genes, Ltnr4, Ltnr5, and Ltnr6, were mapped on chromosomes 6, 9, 15 and 16, respectively.

The effect of pH, hydrogen peroxide and temperature on the stability of human monoclonal antibody.

The stability of human monoclonal antibody (C23), which is being developed as a passive immunotherapeutic agent against human cytomegalovirus, was investigated. C23 (about 2 mg ml-1) was incubated under sterile conditions for 14 days in buffers with different pH values (ranging from 4-10), in hydrogen peroxide solutions with different concentrations (0.01% or 0.1%), and in saline at 8 degrees C or 37 degrees C. Samples were collected on days 0, 3, 7 and 14, and various physicochemical or biological methods were used to determine the changes in in C23. These methods included turbidity (absorbance at 408 nm and transmittance at 580 nm), pH, size-exclusion high performance liquid chromatography (HPLC), hydroxyapatite HPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, matrix-assisted laser desorption ionization time-of-flight mass spectrometry and virus neutralization assay. Using these methods, the possible degradation processes of C23 were initially characterized. Deamidation, oxidation, fragmentation, covalent cross-links and aggregation were observed as major degradation routes. These results gave useful information for the manufacturing process and quality control of C23.

Two genes controlling acute phase responses by the antitumor polysacch aride, lentinan.

Lentinan, a beta-1,6;1,3-glucan, is tumor-specific for transplantable mouse solid-type tumors and it also stimulates the production of acute phase proteins (APPs). The APP response to lentinan is of the delayed type (DT-APR) and differs from that to lipopolysaccharide, which is acute. We found that the responses were genetically controlled in mice and that low responsiveness is dominant (Maeda et al. 1991). Using 123 segregants of crosses between SWR/J (a high responder) and Mus spretus (a low responder), we analyzed the linkage between DT-APR responsiveness and the DNA polymerase chain reaction-simple sequence length polymorphism (PCR-SSLP) phenotype using 80 chromosome-specific microsatellite markers. We identified two loci (ltn1.1 and ltn1.2) responsible for DT-APR. ltn1.1 is closely linked to D3Mit11 on chromosome 3 and ltn1.2 to D11Nds9 on chromosome 11 (P <0.001). The linkage analysis also suggested that ltn1.2 is the major determinant for DT-APR. Correlation between lentinan-specific IL-6 mRNA expression (the late expression) controlled recessively and DT-APR induction suggests that the ltn1 loci control some process(es) of IL-6 expression in the regulation step before NF-IL6.

Elimination of paternal mitochondrial DNA in intraspecific crosses during early mouse embryogenesis.

To examine whether mtDNA is uni- or biparentally transmitted in mice, we developed an assay that can detect sperm mtDNA in a single mouse embryo. In intraspecific hybrids of Mus musculus, paternal mtDNA was detected only through the early pronucleus stage, and its disappearance co-incided with loss of membrane potential in sperm-derived mitochondria. By contrast, in interspecific hybrids between M. musculus and Mus spretus, paternal mtDNA was detected throughout development from pronucleus stage to neonates. We propose that oocyte cytoplasm has a species-specific mechanism that recognizes and eliminates sperm mitochondria and mtDNA. This mechanism must recognize nuclearly encoded proteins in the sperm midpiece, and not the mtDNA or the proteins it encodes, because sperm mitochondria from the congenic strain B6.mtspr, which carries M. spretus mtDNA on background of M. musculus (B6) nuclear genes, were eliminated early by B6 oocytes as in intraspecific crosses. We conclude that cytoplasmic genomes are transmitted uniparentally in intraspecific crosses in mammals as in Chlamydomonas and that leakage of parental mtDNA is limited to interspecific crosses, which rarely occur in nature.

[Efficacy of smoking cessation instruction for general smokers at an annual physical examination].

A randomized controlled trial was conducted to evaluate the efficacy of smoking cessation instruction given to general smokers at an annual physical examination. Four hundred and twenty-six male and 42 female clients were randomly assigned to an intervention group (I), and 413 males and 76 females were assigned to a control (C) group. The I group was given an approximately 2-minute smoking cessation instruction by physicians, answered a quiz concerning tobacco, chose their own behavioral goals and were handed a leaflet on how to quit smoking. Subjects in both groups responded to a self-administered questionnaire and a 6-month and 12-month follow-up was performed by post card or telephone. The I group received an encouragement card one month after instruction and abstainers of the I group were awarded a telephone card at the 6-month follow-up. The results were as follows: 1) The male I group exhibited 7.3% (6 months), 10.1% (12 months) abstinence rates and the male C group 4.4% (6 months), 5.3% (12 months), respectively. The difference in 12-month abstinence rates was statistically significant. 2) The female I group exhibited abstinence rates of 16.7% (6 months), 23.8% (12 months) and the female C group 14.5% (6 months), 17.1% (12 months), respectively. 3) A multivariate analysis of smoking cessation showed that lower nicotine dependency, strong determination for smoking cessation, and being female were significant factors for abstinence at the 6-month follow-up. At 12 months, the smoking cessation instruction also became a significant factor. These data suggest that a simple smoking cessation instruction at an annual physical examination was effective for general smokers.

Inhibitory effects of prostacyclin analogue, TFC-132, on aortic neointimal thickening in vivo and smooth muscle cell proliferation in vitro.

Effects of a new prostacyclin analogue, TFC-132, on neointimal thickening following intimal mechanical injury and on the proliferation of cultured aortic smooth muscle cells (SMCs) were studied. The intimal injury was induced by indwelling of polyethylene tubing for 24 h in the rabbit aorta. Rabbits were killed 10 days after drawing out the tubing. TFC-132 (0.6 mg/kg or 1.2 mg/kg) was given orally at 8-h intervals through the experiment. The serum concentrations of the analogue rose significantly 1 and 2 h after administration. The mean intimal thickening in the TFC-132 treated groups was significantly thinner than in the control one. Human aortic SMCs were cultured and 3H-thymidine incorporation into DNA (DNA synthesis) was measured at the varying concentrations of TFC-132. The analogue inhibited DNA synthesis of cultured SMCs at 10(-6) and 10(-5) M. These data indicate that a new prostacyclin analogue, TFC-132, has an inhibitory effect on the neointimal thickening after intimal injury and on the aortic SMC proliferation.

Platinum/carbon multilayer reflectors for soft-x-ray optics.

We have fabricated platinum/carbon (Pt/C) multilayer reflectors with 2d spacaings between 50 and 200 Å, using an electron-beam evaporator. We investigated the effects of 2d values, the number of layer pairs, substrate temperature, coatings, and the long-term stability on the reflectivity performance by using characteristic x rays and monochromatized synchrotron radiation in the 0.8-8-keV region. In this study we show that Pt/C multilayers with 10-20 layer pairs exhibit high and stable soft-x-ray reflectivity. The interfacial roughness was measured in the range of 5 Å and becomes lower for structures deposited at liquid-nitrogen temperatures. Coating these reflectors with a 100-Å-thick platinum layer increased the grazing angle reflectivity without significantly lowering the Bragg peak reflectivity.

Quantification of 5-fluoro-2'-deoxyuridine in plasma by gas chromatography and negative-ion chemical ionization mass spectrometry.

A sensitive and specific method using gas chromatography and negative-ion chemical ionization mass spectrometry is described for the determination of 5-fluoro-2'-deoxyuridine (FdUrd) in plasma. The method is based on the formation of the pentafluoropropionyl derivative of FdUrd and of its stable isotope as internal standard after sample clean-up by solid-phase extraction and purification by high-performance liquid chromatography. Quantification in plasma was possible down to 300 pg/ml. The method was applied to the analysis of plasma levels of FdUrd in mice and dogs.

Development of radioimmunoassay for TG-51, a new anti-ulcer drug, and its application.

A sensitive and specific radioimmunoassay for a new anti-ulcer drug TG-51 has been developed and applied to the evaluation of its pharmacokinetics in humans. The antiserum was raised in rabbits against an immunogen of N-Acetyl-TG-51 coupled to human serum albumin. The radioactive compound was prepared by acetylating TG-51 with 3H-Acetic anhydride. The separation of free and antibody-bound N-Acetyl-TG-51 was performed by the dextran coated charcoal technique. TG-51 in biological fluids could be quantitatively converted to N-Acetyl-TG-51 prior to radioimmunoassay. This assay system made it possible to ascertain values of 3 ng/ml of TG-51 in plasma using 100 microliters of samples. Results were in good agreement with a high performance liquid chromatography method (HPLC), and the detection limit was raised 25 fold. The accuracy and repoducibility were also satisfactory. By use of this assay method, plasma levels of TG-51 could be determined after a single oral administration of clinical doses of human volunteers.

Detection of myocardial infarction in the presence of Wolff-Parkinson-White syndrome by QRST isoarea map in dogs.

The possibility of detecting myocardial infarction (MI) in the presence of Wolff-Parkinson-White (WPW) syndrome by means of body surface QRST isoarea maps was studied in eight dogs. Eighty-seven body surface ECGs were recorded simultaneously. Recordings were taken during right atrial (RA) and right atrial and right ventricular (RA + RV) sequential pacing, which simulated WPW syndrome, during control periods and at 1-hour intervals for up to 5 hours after occlusion of the left anterior descending coronary artery. In ECGs during the RA drive, diagnostic findings of MI such as abnormal Q waves were observed but became obscure during the RA + RV drive. On the contrary, the QRST values over the anterior chest during both drives were positive soon after coronary occlusion, decreased gradually as time passed, and became abnormally negative after 5 hours. The QRST isoarea maps during RA and RA + RV pacing showed quite similar patterns and were highly correlated with each other throughout this study (r greater than 0.95). These findings demonstrate that localized abnormalities resulting from MI are evident in QRST isoarea maps even in the presence of preexcitation and fusion.

Effect of a new anti-ulcer drug, (3-[p-(trans-4-aminomethylcyclohexylcarbonyl)-phenyl] propionic acid hydrochloride, TEI-5103) on prostaglandin (PG) levels in rat gastric mucosa.

1. Effect of TEI-5103 on the levels of various prostaglandins (PG's) in rat gastric mucosa was studied by HPLC and RIA methods. 2. When administrated in vivo, TEI-5103 significantly increased the level of prostaglandin E2 (PGE2) but only slightly increased that of prostaglandin I2 (PGI2) or thromboxane A2 (TXA2). 3. In the water-immersion stress model, the PGE2 level in control animals was increased immediately after stress and then decreased gradually to the initial level. Indomethacin (5 mg/kg, s.c.) sustained a lower PGE2 level during the experiment, as well as aggravated the lesions. 4. TEI-5103 (400 mg/kg, p.o.) increased the PGE2 level just before stress, and it resulted in the prevention of lesion formation. 5. TEI-5103 inhibited the activity of 15-OH-PG-dehydrogenase from guinea pig lungs, but did not affect the activity of phospholipase A2 from porcine pancreas.