Isoniazid-induced Immune Thrombocytopenia.

Drug-induced thrombocytopenia occurs through immune-mediated platelet destruction, and its management is challenging during tuberculosis treatment. Although rifampicin is the most common drug causing thrombocytopenia, isoniazid can also cause thrombocytopenia. We herein report a 75-year-old man who developed thrombocytopenia during tuberculosis treatment. Platelet-associated immunoglobulin G and a drug-induced lymphocyte stimulation test for isoniazid were positive; no other causes of thrombocytopenia were identified. The patient was diagnosed with isoniazid-induced immune thrombocytopenia, and the platelet count normalized after isoniazid discontinuation. We describe the immunological mechanism of thrombocytosis due to isoniazid, an uncommon cause of thrombocytopenia that physicians should be aware exists.

Miction-induced Hypertension Disclosed by Home Blood Pressure Measurement in a Patient with Small Paraganglioma.

A 46-year-old woman complained of a 10-year history of headache, nausea, a precordial oppressive feeling and shortness of breath on miction. She had noted a marked elevation in her blood pressure after miction using home blood pressure measurement. Her catecholamine levels were less than twice the value of the normal upper limit. Several imaging modalities detected a urinary bladder tumor, and 123I-metaiodobenzylguanidine scintigraphy showed positive accumulation. The diagnosis of urinary bladder paraganglioma was confirmed by partial cystectomy. We must keep in mind that paroxysms and hypertension associated with miction are important diagnostic clues of pheochromocytoma/paraganglioma. Home blood pressure measurement was very useful for detecting hypertension in this case.

The enhancement of antibody concentration and achievement of high cell density CHO cell cultivation by adding nucleoside.

Recently, with the dramatic increase in demand for therapeutic antibodies, Chinese hamster ovary (CHO) cell culture systems have made significant progress in recombinant antibody production. Over the past two decades, recombinant antibody productivity has been improved by more than 100-fold. Medium optimization has been identified as an important key approach for increasing product concentrations. In this study, we evaluated the effects of deoxyuridine addition to fed-batch cultures of antibody-expressing CHO cell lines. Furthermore, we investigated the effects of combined addition of deoxyuridine, thymidine, and deoxycytidine. Our results suggest that addition of these pyrimidine nucleosides can increase CHO cell growth, with no significant change in the specific production rate. As a result of the increased cell growth, the antibody concentration was elevated and we were able to achieve more than 9 g/L during 16 days of culture. Similar effects of nucleoside addition were observed in fed-batch cultures of a Fab fragment-expressing CHO cell line, and the final Fab fragment concentration was more than 4 g/L. This nucleoside addition strategy could be a powerful platform for efficient antibody production.

Identification of regulatory motifs in the CHO genome for stable monoclonal antibody production.

Chinese hamster ovary (CHO) cell lines are widely used for therapeutic protein production. When a transgene is integrated into the genome of a CHO cell, the expression level is highly dependent on the site of integration because of positional effects such as gene silencing. To overcome negative positional effects and establish stable CHO cell lines with high productivity, several regulatory DNA elements are used in vector construction. Previously, we established the CHO DR1000L-4N cell line, a stable and high copy number Dhfr gene-amplified cell line. It was hypothesized that the chromosomal location of the exogenous gene-amplified region in the CHO DR1000L-4N genome contains regulatory motifs for stable protein production. Therefore, we isolated DNA regulatory motifs from the CHO DR1000L-4N cell line and determined whether these motifs act as an insulator. Our results suggest that stable expression of a transgene can be promoted by the CHO genome sequence, and it would be a powerful tool for therapeutic protein manufacturing.

Development of an electrochemical cholesterol sensor system for food analysis.

In this article, we report on a food-cholesterol monitoring sensor based on a non-enzymatic approach. Amorphous and single-crystal gold electrodes were modified with an alkanethiol self-assembled monolayer to quantify it by voltammetry. We first discuss the basic characteristics of these sensors and provide more information about the instrument developed by JSK Co. This instrument is a battery-operated handheld voltammetric analyzer, which mounts a sensor chip to monitor cholesterol contents in food samples. The sensor showed excellent linearity with the cholesterol concentration; egg-yolk samples were analyzed to give an excellent agreement between the values obtained by the sensor (1.4 mM) and chromatography (1.1 mM).

Effect of three types of mixed anesthetic agents alternate to ketamine in mice.

Ketamine is usually used for murine anesthesia in animal experiments with other anesthetics for its sedation and analgesic effects. However, ketamine was categorized as a narcotic drug in Japan on January 1, 2007. After this act came into effect, a narcotic handling license became necessary for using and possessing ketamine. Pentobarbital sodium, which is also used for laboratory animal experiments as Nembutal, is no longer being manufactured. For these reasons, other anesthetic agents that can be used without a license are needed. In this paper, we examined the use of anesthetics other than ketamine and pentobarbital sodium. A combination anesthetic (M/M/B: 0.3/4/5) was prepared with 0.3 mg/kg of medetomidine, 4.0 mg/kg of midazolam, and 5.0 mg/kg of butorphanol. The anesthetics were administered to male ICR mice by intraperitoneal injection. In order to assess anesthetic depth and duration, we stimulated the mice directly after loss of righting reflexes to recovery of these same reflexes and then recorded four parameters--a tail pinch reflex, a pedal withdrawal reflex in the forelimbs, a pedal withdrawal reflex in the hindlimbs, and corneal reflex. Each parameter was scored, and the anesthetic depth, expressed by the total score, was summed. The surgical anesthesia duration of M/M/B: 0.3/4/5 mg/kg was almost identical to the surgical anesthetic duration with a ketamine and xylazine mixture (80-8 mg/kg). These data suggested that mice can be anesthetized by M/M/B: 0.3/4/5 as an alternate to ketamine. We thus can recommend M/M/B: 0.3/4/5 for murine surgical anesthesia.

Orally administered rutin inhibits the gene expression of Th2 cytokines in the gut and lung in aged mice.

Rutin is one of the flavonoids derived from plants such as buckwheat and is well known as a powerful antioxidant. To determine whether dietary rutin could modulate mucosal immunity, we examined the gene expression of Th1/Th2 cytokines and the receptors in the gut and lung. Aged (18 months old, 18 M) C3H/HeN female mice were orally administered rutin for 10 days. The small intestine and lung were taken and analyzed by real-time PCR for gene expression. Interleukin (IL)-13 and IL-13Rα2 gene expression was significantly low (P<0.05 respectively) in the small intestine of aged rutin-fed mice. Meanwhile, there was no change in interferon γ gene expression between control and rutin-fed mice. IL-13 gene expression was also downregulated in the lung. To examine the mechanism of the inhibitory effect of rutin on Th2 cytokines in aged mice, intestinal nitric oxide synthase (NOS) expression was evaluated. Rutin inhibited inducible NOS (NOS2) gene expression, but not neuronal NOS and endothelial NOS. Gene analysis of cells collected from the small intestine by laser capture dissection revealed that NOS2 expression was significantly inhibited in crypt regions. Thus, rutin might be effective against a Th2-dominant profile through NOS2 inhibition in aged mice.

Detection of prolonged regional myocardial systolic dysfunction after exercise-induced myocardial ischemia by strain echocardiography with high frame rate tissue Doppler echocardiography.

BACKGROUND: Strain echocardiography has enabled quantification of regional myocardial systolic function objectively and is less influenced by tethering effects and cardiac translational artifact than Doppler tissue imaging. Although strain echocardiography has been applied for the detection of inducible ischemia during dobutamine stress, it has not been fully applied to exercise stress echocardiography (ESE) because of technical difficulties. Prolonged myocardial systolic dysfunction after exercise-induced ischemia has been shown previously. Thus, we designed this study to evaluate whether the myocardial strain analysis can detect myocardial ischemia by the assessment of prolonged regional left ventricular (LV) dysfunction in ESE. METHODS: We performed ESE with myocardial strain imaging system in 20 consecutive patients who had exercise Tl-201 single photon emission computed tomography (SPECT). Myocardial strain curves were obtained at six segments in mid LV walls from the apical approach before and 5 min after ESE. We measured the duration from the R wave in the electrocardiogram to the timing of peak systolic strain corrected by the square root of the RR interval (TPSc). We finally calculated the differences of TPSc (ΔTPSc) before ESE and 5 min after ESE. The results were compared with SPECT as a reference standard. RESULTS: A receiver operating characteristic curve demonstrated that a ΔTPSc cutoff value of 70 ms had a sensitivity of 80% and a specificity of 84% for the detection of myocardial ischemia. CONCLUSIONS: Prolonged regional LV systolic dysfunction assessed by ESE with strain analysis was useful for the detection of myocardial ischemia.

A diagnostic method for pneumocystis carinii a causative agent of pneumonia in immunodeficient rats.

Immunodeficient animals are in demand in current biomedical research, and they contribute to medical progress. In Pneumocystis infections, a specific histological diagnostic tool may be immunochemistry (IC). However, it was recently reported that the antibody (3F6) was not suitable for detecting Pneumocystis in rats. We purchased another antibody [PNC007] from a commercial source for IC. We could detect positive signals at identical locations with IC and Toluidine blue O in lungs of infected rats. These results corresponded to the results obtained with PCR. We should study the relationship between unexpected positive signals seen in IC and trophic forms in lungs of infected rats. We could clinically diagnose pneumonia caused by Pneumocystis carinii with the diagnostic method we developed.

Identification and analysis of specific chromosomal region adjacent to exogenous Dhfr-amplified region in Chinese hamster ovary cell genome.

Chinese hamster ovary (CHO) cells are widely used for the stable production of recombinant proteins. Gene amplification techniques are frequently used to improve of protein production, and the dihydrofolate reductase (DHFR) gene amplification system is most widely used in the CHO cell line. We previously constructed a CHO genomic bacterial artificial chromosome (BAC) library from a mouse Dhfr-amplified CHO DR1000L-4N cell line and one BAC clone (Cg0031N14) containing the CHO genomic DNA sequence adjacent to Dhfr was selected. To identify the specific chromosomal region adjacent to the exogenous Dhfr-amplified region in the CHO cell genome, we performed further screening of BAC clones to obtain other Dhfr-amplified regions in the CHO genome. From the screening by high-density replica filter hybridization using a digoxigenin-labeled pSV2-dhfr/hGM-CSF probe, we obtained 8 new BAC clones containing a Dhfr-amplified region. To define the structures of the 8 BAC clones, Southern blot analysis, BAC end sequencing and fluorescence in situ hybridization (FISH) were performed. These results revealed that all the selected BAC clones contained a large palindrome structure with a small inverted repeat in the junction region. This suggests that the obtained amplicon structure in the Dhfr-amplified region in the CHO genome plays an important role in exogenous gene amplification.

Right ventricular histological substrate and conduction delay in patients with Brugada syndrome.

The reported pathogenesis of Brugada syndrome is phase 2 reentry resulting from shortening of the epicardial action potential duration at the right ventricular outflow tract (RVOT). However, several studies have revealed a high incidence of ventricular late potentials and high rate of ventricular fibrillation (VF) induced by programmed ventricular stimulation (PVS). The aim of the present study was to evaluate the role of slow conduction at the RVOT for the initiation of VF by PVS and any underlying pathological conditions in Brugada syndrome. Endocardial mapping of the RVOT and endomyocardial biopsy of the right ventricle were performed in 25 patients with Brugada syndrome with inducible VF. Late potentials were positive in 11 of the 25 (44%) patients. Low-amplitude fragmented and delayed electrograms were recorded at the RVOT in 13 of 18 (72.2%) patients. Histologic examination of the biopsy samples revealed fatty tissue infiltration, interstitial fibrosis, lymphocyte infiltration, and/or myocyte disorganization in 13 patients. Slow conduction at the RVOT may contribute to the induction of VF by PVS in Brugada syndrome. Various pathomorphologic changes may contribute to slow conduction at the RVOT.

Combined administration of (L)-cystine and (L)-theanine enhances immune functions and protects against influenza virus infection in aged mice.

Cell-mediated and humoral immune responses are attenuated with aging. Intracellular glutathione (GSH) levels also decrease with aging. Previously, we have reported that combined administration of (L)-cystine and (L)-theanine enhances antigen-specific IgG production, partly through augmentation of GSH levels and T helper 2-mediated responses in 12-week-old mice. These findings suggest that combined administration of (L)-cystine and (L)-theanine to aged mice improves immune responses via increase of GSH synthesis. Here, we examined the effects of combined administration of (L)-cystine and (L)-theanine on antigen-specific antibody production and influenza virus infection in aged mice. Combined administration of these amino acids for 14 days before primary immunization significantly enhanced the serum antigen-specific IgM and IgG levels in 24-month-old mice. Furthermore, 13-month-old mice co-treated with these amino acids orally for 10 days had significantly lower lung viral titers than controls at 6 days after influenza virus infection. In addition, this co-treatment also significantly prevented the weight loss associated with infection. Enhancement of anti-influenza-virus IgG antibodies by combined administration of (L)-cystine and (L)-theanine was seen 10 days after infection. The significantly elevated serum interleukin-10/interferon-gamma ratio and gamma-glutamylcysteine synthetase mRNA expression, which is the rate-limiting enzyme of GSH synthesis, in the spleen 3 days after infection may have contributed to the observed beneficial effects. These results suggest that combined administration of (L)-cystine and (L)-theanine enhances immune function and GSH synthesis which are compromised with advanced age, and may become a useful strategy in healthy aging.

Bacterial artificial chromosome library for genome-wide analysis of Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cell lines are widely used for scientific research and biotechnology. A CHO genomic bacterial artificial chromosome (BAC) library was constructed from a mouse dihydrofolate reductase (DHFR) gene-amplified CHO DR1000L-4N cell line for genome-wide analysis of CHO cell lines. The CHO BAC library consisted of 122,281 clones and was expected to cover the entire CHO genome five times. A CHO chromosomal map was constructed by fluorescence in situ hybridization (FISH) imaging using BAC clones as hybridization probes (BAC-FISH). Thirteen BAC-FISH marker clones were necessary to identify all the 20 individual chromosomes in a DHFR-deficient CHO DG44 cell line because of the aneuploidy of the cell line. To determine the genomic structure of the exogenous Dhfr amplicon, a 165-kb DNA region containing exogenous Dhfr was cloned from the BAC library using high-density replica (HDR) filters and Southern blot analysis. The nucleotide sequence analysis revealed a novel genomic structure in which the vector sequence containing Dhfr was sandwiched by long inverted sequences of the CHO genome.

Comparison of determinations of left atrial volume by the biplane area-length and Simpson's methods using 64-slice computed tomography.

OBJECTIVES: There is increasing evidence that left atrial (LA) size is an important predictor of adverse cardiovascular outcomes such as atrial fibrillation, stroke, and congestive heart failure. The aim of this study was to determine whether there is a difference in results of quantification of LA volume by the area-length and Simpson's methods using multislice computed tomography (MSCT). METHODS AND RESULTS: The study population consisted of 51 patients with sinus rhythm (sinus group) and 20 patients with atrial fibrillation (af group) clinically indicated for MSCT angiography for evaluation of coronary arteries. Maximum LA volume, obtained at end-systole from the phase immediately preceding mitral valve opening, was measured using the area-length and Simpson's methods. In the sinus group, the mean LA volumes, indexed to body surface area, were 48.4+/-17.9 ml/m(2) with the area-length method and 48.3+/-17.0 ml/m(2) with the Simpson's method. In the af group, the mean indexed LA volumes with the area-length method and the Simposon's method were 91.5+/-47.5 ml/m(2) and 90.3+/-45.9 ml/m(2), respectively. LA volumes calculated by the area-length method exhibited a strong linear relationship and agreement with those calculated using Simpson's method in both the groups (sinus group: r=0.99, P<0.0001, af group: r=0.99, P<0.0001). CONCLUSIONS: The area-length method is a simple and reproducible means of assessment of LA volume. Standardization of LA volume assessment using MSCT is important for serial follow-up and meaningful communication of results of testing among institutions and physicians.

Automated assessment of left atrial function from time-left atrial volume curves using a novel speckle tracking imaging method.

BACKGROUND: Using a novel speckle-tracking imaging method, time-left atrial (LA) volume curves (TLAVCs) can be automatically obtained. The aim of this study was to evaluate whether this method can be used for the measurement of LA function with TLAVCs. METHODS: In 10 normal subjects and 20 patients, apical 4-chamber images were obtained. Maximum volume, reservoir volume, conduit volume, booster pump volume, and minimum volume were measured from TLAVCs. The results were compared with those obtained by the manual tracing method on every frame during 1 cardiac cycle. RESULTS: There was good agreement between the speckle-tracking imaging and manual methods for maximum LA volume (r = 0.98, P < .001), reservoir volume (r = 0.82, P < .001), conduit volume (r = 0.87, P < .001), booster pump volume (r = 0.80, P < .001), and minimum volume (r = 0.98, P < .001). The time to obtain TLAVCs was significantly shorter with the speckle-tracking imaging method (64 +/- 22 seconds) than with the manual method (22 +/- 4 minutes). CONCLUSION: TLAVCs obtained using the speckle-tracking imaging method can be used for the rapid and noninvasive automated quantitation of LA function.

Combined effect of pulmonary vein isolation and ablation of cardiac autonomic nerves for atrial fibrillation.

This study was designed to determine whether endocardial high-frequency stimulation at the pulmonary vein (PV) antrums can localize cardiac autonomic ganglionated plexi (GP) and whether ablation at these sites can evoke a vagal response and provide a long-term benefit after PV isolation (PVI) for atrial fibrillation (AF). Radiofrequency ablation of each PV antrum was performed in 21 patients with paroxysmal AF (n = 17) or persistent (n = 4) AF. In 8 patients with paroxysmal AF, a ring electrode catheter was placed at each PV antrum. High-frequency stimulation prolonged the R-R interval in 6 of 8 patients at the left superior (LS) PV, in 3 of 8 patients at the left inferior (LI) PV, in 3 of 8 patients at the right superior (RS) PV, and in 3 of 8 patients at the right inferior (RI) PV. A decrease in sinus rate > 20% was observed in 4 of 21 patients during LS PVI, in 2 of 21 patients during RS PVI, and in 1 of 2 patients during RI PVI. Atrioventricular block or a > 5 second pause was observed in 5 of 21 patients during LS PVI. AF recurred during the follow-up period in 5 of the 16 patients (31%) who had no atrioventricular block or > 5 second pause during PVI but did not recur in 5 patients in whom atrioventricular block or a > 5 second pause developed during PVI. GP can be identified by endocardial stimulation. The AF recurrence rate is decreased when a vagal response is achieved by radiofrequency ablation.

Full-motion two- and three-dimensional pulmonary vein imaging by intracardiac echocardiography after pulmonary vein isolation.

BACKGROUND: The pulmonary veins (PVs) are topographically complex and motile, so angiographic visualization of the PVs anatomy is limited. An imaging technique that accurately portrays the pulmonary vein (PV) anatomy would be valuable during and after catheter ablation procedures. PURPOSE: We investigated whether three-dimensional (3D) intracardiac echocardiography (ICE) can visualize radiofrequency (RF)-induced tissue changes after PV isolation. METHODS: We performed 3D ICE studies with a 9F, 9-MHz ICE catheter after segmental or extended PV isolation. The ICE catheter was placed 3-4 cm inside the PV ostium and mounted onto a pullback device. Sequential two-dimensional (2D) images of the full length of the vein were obtained in 0.3 mm steps with cardiac and respiratory cycle gating. Each image was fed into a computer, and the aggregate data set was reconstructed into a 3D, full-motion image. RF lesion location and lesion size were studied on 67 pullback images from 29 patients. RESULTS: The 2D and 3D reconstruction was possible for 27 left superior PVs, 13 left inferior PVs, 26 right superior PVs, and one right inferior PV. The ablation site was identified 3-7 mm inside the PV ostium, and a 1/2 - 4/5 circumferential area was ablated with no clinically relevant stenosis. No significant differences were found on the ablated area or ablation site between segmental and extensive PV isolation. CONCLUSION: The 2D and 3D ICE of the PVs provides detailed anatomical information of the proximal PVs, and RF-induced tissue changes in the PV wall can be visualized by ICE.

Electrophysiological properties of the atrium after cardioversion of chronic atrial fibrillation: relation to the plasma brain natriuretic peptide level.

BACKGROUND: Brain natriuretic peptide (BNP) level has been shown to increase in patients with chronic atrial fibrillation (CAF) without overt heart failure (HF). Although atrial electrical remodeling associated with CAF has been described, little is known about the effects of the BNP level on the electrophysiological properties in CAF patients. METHODS AND RESULTS: In 42 CAF patients without overt HF, the atrial monophasic action potential duration (MAPD) at pacing cycle lengths (CLs) of 300-800 msec and P-wave signal-averaged electrograms were recorded after cardioversion. The MAPDs for all CLs were significantly longer in patients with a BNP concentration greater than the 50th percentile (group 1, BNP = 215 +/- 118.2 pg/mL) than in patients with a concentration less than the 50th percentile (group 2, BNP = 68.3 +/- 20.9 pg/mL), resulting in a similar value in the MAPDs at CLs of 350 and 600 msec for group 1 and the control patients (n = 8). The slope value of the MAPDs between CLs of 350 and 600 msec was normal in group 1, but slightly lower in group 2 than in group 1 and control patients. The filtered P-wave duration did not differ between the two groups. CONCLUSIONS: These electrophysiological characteristics related to the BNP level suggest that the atrial repolarization may be affected by a latent ventricular dysfunction.

Electrocardiographic and electrophysiologic characteristics in patients with Brugada type electrocardiogram and inducible ventricular fibrillation: single center experience.

BACKGROUND: The study examined the electrocardiographic and electrophysiologic characteristics in relation to programmed ventricular stimulation (PVS)-induced ventricular fibrillation (VF) in patients with Brugada syndrome. METHODS AND RESULTS: Thirty-four patients with a Brugada-type electrocardiogram (ECG) were enrolled. Twelve patients had a type 1 ECG, 12 had a type 2 ECG, and 10 had a type 3 ECG. PVS was performed with up to 2 ventricular premature beats from the right ventricular apex and outflow tract at 2 basic cycle lengths (600 and 400 ms). VF was induced in 17 of 23 (74%) asymptomatic patients and 10 of 11 (91%) symptomatic patients (p<0.05). The 27 patients in whom VF was induced by PVS and 7 patients without inducible VF were followed up for 47.1+/-33.7 months. One sudden death occurred during the follow-up period among asymptomatic patients with inducible VF, and no sudden death occurred among patients without inducible VF. CONCLUSIONS: In conclusion, inducibility of ventricular arrhythmia is high in patients with Brugada syndrome, but it does not correlate with clinical presentation. Few arrhythmic events occur during follow up. However, the present study data suggest that electrophysiologic study-induced VF does not predict arrhythmic events during follow up.

Endocardial electrograms from the right ventricular outflow tract after induced ventricular fibrillation in patients with Brugada syndrome.

BACKGROUND: The pathogenesis of Brugada syndrome (BS) is reported to be phase 2 reentry resulting from shortening of the action potential duration at the epicardial site of the right ventricular outflow tract (RVOT). However, several reports have shown a high incidence of ventricular late potentials (LPs) and a high rate of induction of ventricular fibrillation (VF) by programmed ventricular stimulation (PVS) among patients with BS. The aim of this study was to investigate the role of slow conduction for the initiation of VF by PVS in these patients. METHODS AND RESULTS: Endocardial mapping of the RVOT was conducted in 17 patients in whom VF was induced by PVS from the RV apex or RVOT; 11 patients had a positive LP. In 10 patients, RV mapping showed that low-amplitude fragmented and delayed potentials (DPs) were recorded at the RVOT below the pulmonary valve (PV) or between the PV and His bundle electrogram recording site. Electrograms recorded after PVS showed a high incidence of fractionated and disorganized DPs that lead to VF. CONCLUSIONS: Slow conduction at the RVOT may contribute to the induction of VF by PVS. However, the role of slow conduction in spontaneous VF remains controversial.