Viable bacterial colonization is highly limited in the human intestine in utero.

Mucosal immunity develops in the human fetal intestine by 11-14 weeks of gestation, yet whether viable microbes exist in utero and interact with the intestinal immune system is unknown. Bacteria-like morphology was identified in pockets of human fetal meconium at mid-gestation by scanning electron microscopy (n = 4), and a sparse bacterial signal was detected by 16S rRNA sequencing (n = 40 of 50) compared to environmental controls (n = 87). Eighteen taxa were enriched in fetal meconium, with Micrococcaceae (n = 9) and Lactobacillus (n = 6) the most abundant. Fetal intestines dominated by Micrococcaceae exhibited distinct patterns of T cell composition and epithelial transcription. Fetal Micrococcus luteus, isolated only in the presence of monocytes, grew on placental hormones, remained viable within antigen presenting cells, limited inflammation ex vivo and possessed genomic features linked with survival in the fetus. Thus, viable bacteria are highly limited in the fetal intestine at mid-gestation, although strains with immunomodulatory capacity are detected in subsets of specimens.

Short-term effects of rapid pacing on mRNA level of voltage-dependent K(+) channels in rat atrium: electrical remodeling in paroxysmal atrial tachycardia.

BACKGROUND: Atrial fibrillation causes electrophysiological changes of the atrium, thereby facilitating its maintenance. Although the expression of ion channels is modulated in chronic atrial fibrillation, it is yet unknown whether paroxysmal atrial fibrillation can also lead to electrical remodeling by affecting gene expression. METHODS AND RESULTS: To examine the short-term effects of rapid pacing on the mRNA level of voltage-dependent K(+) channels, high-rate atrial pacing was performed in Sprague-Dawley rat hearts. Total RNA was prepared from the atrial appendages from 0 to 8 hours after the onset of pacing, and mRNA levels of Kv1.2, Kv1. 4, Kv1.5, Kv2.1, Kv4.2, Kv4.3, erg, KvLQT1, and minK were determined by RNase protection assay. Among these 9 genes, the mRNA level of the Kv1.5 channel immediately and transiently increased, with bimodal peaks at 0.5 and 2 hours after the onset of pacing. Conversely, the pacing gradually and progressively decreased the mRNA levels of the Kv4.2 and Kv4.3 channels. The increase of Kv1.5 and the decrease of Kv4.2 and Kv4.3 mRNA levels were both rate dependent. In correspondence with the changes in the mRNA level, Kv1. 5 channel protein transiently increased in the membrane fraction of the atrium during a 2- to 8-hour pacing period. Electrophysiological findings that the shortening of the action potential produced by 4-hour pacing was almost abolished by a low concentration of 4-aminopyridine implied that the increased Kv1.5 protein was functioning. CONCLUSIONS: Even short-term high-rate atrial excitation could differentially alter the mRNA levels of Kv1.5, Kv4.2, and Kv4.3 in a rate-dependent manner. In particular, increased Kv1.5 gene expression, having a transient nature, implied the possible biochemical electrical remodeling unique to paroxysmal tachycardia.

Relation between aging and circadian variation of paroxysmal atrial fibrillation.

The purpose of this study was to determine whether aging influences the circadian variation of nonvalvular paroxysmal atrial fibrillation (AF). Among 31,200 consecutive Holter monitorings recorded between January 1988 and March 1997, we detected 212 patients who had paroxysmal AF in a drug-free state. These patients were divided into 2 groups according to their age: < or = 60 years old (94 patients) and >60 years old (118 patients). In each group, the sum of the duration of each AF episode and the probability of onset, maintenance, and termination of AF were determined as hourly data and compared between the 2 groups. The time distribution of AF showed remarkable age dependence, with a well-modulated and monophasic circadian rhythm in the younger group in contrast to a toneless triphasic rhythm in the older group. Among the onset, maintenance, and termination of the arrhythmia, the most obvious age-dependence was observed in the circadian variation of onset. In the younger group, there were triple peaks with the highest one in the night, whereas the older group exhibited a single peak in the daytime. In contrast, the probabilities of maintenance and termination showed similar circadian patterns between the groups, although their amplitudes were significantly reduced in the older group. Thus, aging significantly influenced the circadian variation of paroxysmal AF, with the most prominent effect on its onset, leading to more random time-distribution of AF with increasing age. These results extend to paroxysmal AF the concept that aging disrupts rhythmicity, suggesting age-dependent differences in its pathophysiology.

An in vitro investigation of the suitability of press-coated tablets with hydroxypropylmethylcellulose acetate succinate (HPMCAS) and hydrophobic additives in the outer shell for colon targeting.

To develop a new colon targeting formulation, which can suppress drug release completely during 12 h in the stomach and release the drug rapidly after a lag time of 3+/-1 h in the small intestine, the use of press-coated tablets with hydroxypropylmethylcellulose acetate succinate (HPMCAS) in the outer shell was investigated. The release of diltiazem hydrochloride (DIL) as a model drug contained in the core tablets in the 1st fluid (pH 1.2) was suppressed by preparing with higher compression force, but the lag time in the 2nd fluid (pH 6.8) could not exceed 1.5 h. Therefore, to improve the dissolution characteristics, the effects of addition of various hydrophobic additives to HPMCAS were examined. All of the additives examined suppressed the release rate in the 1st fluid, and prolonged the lag time in the 2nd fluid compared to HPMCAS alone. However, although none of the additives examined fulfilled all of the desired criteria, magnesium stearate (MgSt) and calcium stearate (CaSt) showed interesting effects; the former suppressed drug release completely in 1st fluid, while the latter markedly prolonged the lag time in 2nd fluid. To integrate the merits of each additive, press-coated tablets with a powder mixture of HPMCAS, MgSt and CaSt in the outer shell (HMC tablets) were prepared and in vitro tests were performed. The results indicated that HMC tablets with a mixing ratio of 80% HPMCAS, 5-15% MgSt and 15-5% CaSt in the outer shell met the desired criteria and the lag time in 2nd fluid could also be controlled from 2 to 9 h. At a mixing ratio of 80% HPMCAS, 10% MgSt and 10% CaSt, the dissolution profiles of DIL in 1st fluid and 2nd fluid were not remarkably affected by agitation intensity, and addition of bile salts, pretreatment time or anticipated higher pH except for pH 6.0, respectively. These results indicated the usefulness of HMC tablets with the desirable functions for colon-targeting formulations.

Studies on applicability of press-coated tablets using hydroxypropylcellulose (HPC) in the outer shell for timed-release preparations.

Press-coated tablets, containing diltiazem hydrochloride (DIL) in the core tablet and coated with hydroxypropylcellulose (HPC) as the outer shell, were examined for applicability as timed-release tablets with a predetermined lag time and subsequent rapid drug release phase. Various types of press-coated tablets were prepared using a rotary tabletting machine and their DIL dissolution behavior was evaluated by the JP paddle method. The results indicated that tablets with the timed-release function could be prepared, and that the lag times were prolonged as the viscosity of HPC and the amount of the outer shell were increased. The lag times could be controlled widely by the above method, however, the compression load had little effect. Two different kinds of timed-release press-coated tablets that showed lag times of 3 and 6 h in the in vitro test (denoted PCT(L3) and PCT(L6), respectively) were administered to beagle dogs. DIL was first detected in the plasma more than 3 h after administration, and both tablets showed timed-release. The lag times showed a good agreement between the in vivo and in vitro tests in PCT(L3). However, the in vivo lag times were about 4 h in PCT(L6) and were much shorter than the in vitro lag time. The dissolution test was performed at different paddle rotation speeds, and good agreement was obtained between the in vivo and in vitro lag times at 150 rpm. This suggested that the effects of gastrointestinal peristalsis and contraction should also be taken into consideration for the further development of drug delivery systems.

Effect of magnesium stearate or calcium stearate as additives on dissolution profiles of diltiazem hydrochloride from press-coated tablets with hydroxypropylmethylcellulose acetate succinate in the outer shell.

Effect of magnesium stearate (MgSt) or calcium stearate (CaSt) on the dissolution profiles of diltiazem hydrochloride in the core of press-coated (PC) tablets with an outer shell composed of hydroxypropylmethylcellulose acetate succinate (HPMCAS) was evaluated by porosity and changes in IR spectra of tablets. In JP first fluid (pH 1.2), the lag time increased with decreasing porosity and was greatest by the addition of MgSt to HPMCAS. While, in JP second fluid (pH 6.8), it increased with decreasing porosity by the addition of CaSt, but hardly changed by the addition of MgSt. Thus, using tablets prepared with the same composition as the outer shell, the changes in IR spectra and uptake amount of the dissolution media after immersion in first fluid and second fluid were determined. The results suggested that some physicochemical interaction occur between MgSt and HPMCAS in tablets with HPMCAS and MgSt and the uptake increased markedly in each dissolution medium. These phenomena seem to cause a prolongation of lag time in first fluid but a shortening of it in second fluid in PC tablets with HPMCAS and MgSt. In contrast, CaSt and HPMCAS did not show such interactions and increased the hydrophobic properties of the outer shell. Consequently, the lag time was only slightly prolonged in first fluid, however, markedly prolonged in second fluid due to suppression of second fluid penetration into micro pores in the outer shell and HPMCAS gel formation on the surface in PC tablets with HPMCAS and CaSt.

Drug release from and mechanical properties of press-coated tablets with hydroxypropylmethylcellulose acetate succinate and plasticizers in the outer shell.

Dissolution profiles of diltiazem hydrochloride (DIL) contained in core tablets from press-coated (PC) tablets with hydroxypropylmethylcellulose acetate succinate (HPMCAS) and plasticizers-adsorbent in the outer shell were investigated. Although, on the addition of triethyl citrate (TEC), triacetin (TA), and acetyltriethy citrate (ATEC) as plasticizers, DIL release was suppressed completely in first fluid (pH 1.2) for 10 h, it was not suppressed in HPMCAS on the addition of dibutyl sebacate (DBS) and acetylated monoglyceride. On the other hand, DIL in second fluid (pH 6.8) was released rapidly after a lag time in all the PC tablets. Water-soluble plasticizers such as TEC, TA, and ATEC showed greater compatibility to HPMCAS, and the results were consistent with suppression of DIL release in first fluid. Furthermore, as to PC tablets with HPMCAS and TEC-adsorbent, the DIL release in second fluid did not change after pretreatment in first fluid by the paddle-beads methods. To evaluate the resistance of the outer shell against such a mechanical impact, tablets with HPMCAS, HPMCAS and TEC- or DBS-adsorbent (H, HT, or HD tablets, respectively) were prepared. In compressive load-strain curves after immersion in first fluid, wet crushing strength was lower in the order of HT > H > HD tablets. Also, the curves of HT tablets at 3 and 21 h after immersion were quite different from those of other tablets, and it was hard to find crushing points. These results suggested that the resistance of the outer shell was due to plastic deformation properties involving some interaction between HPMCAS and TEC.

Preparation of enteric coated timed-release press-coated tablets and evaluation of their function by in vitro and in vivo tests for colon targeting.

As a new oral drug delivery system for colon targeting, enteric coated timed-release press-coated tablets (ETP tablets) were developed by coating enteric polymer on timed-release press-coated tablets composed of an outer shell of hydroxypropylcellulose and core tablet containing diltiazem hydrochloride (DIL) as a model drug. The results of the in vitro dissolution tests in JP 1st fluid (pH 1.2) and JP 2nd fluid (pH 6.8) indicated that these tablets showed both acid resistance and timed-release. To clarify whether ETP tablets could have been of use in the gastrointestinal tract, ETP tablets with a layer of phenylpropanolamine hydrochloride (PPA) (a marker of gastric emptying) between the enteric coating layer and outer shell were prepared, and were administered to beagle dogs. The gastric emptying time and lag time after gastric emptying were evaluated by determining the times at which PPA and DIL first appeared in the plasma (TFA(PPA) and TFA(DIL), respectively). TFA(PPA) and TFA(DIL) were about 4 and 7 h, respectively. This value of TFA(PPA) indicated that ETP tablets displayed acid resistance in the stomach as well as in JP Ist fluid. Subtraction of TFA(PPA) from TFA(DIL) gave a value of about 3 h which agreed well with the lag time determined by in vitro dissolution test in JP 2nd fluid. Also, the results seemed to be in accordance with the time at which the tablets reached the colon after gastric emptying. Therefore, ETP tablets seemed to be an effective tool for oral site-specific delivery including targeting of the colon.

Chromosomal diagnosis in each individual blastomere of 5- to 10-cell bovine embryos derived from in vitro fertilization.

Chromosomal normality and sex were diagnosed in each blastomere of bovine embryos derived from in vitro fertilization (IVF). Bovine embryos developing to the 5- to 10-cell stage were separated into individual blastomeres with 0.5% protease. After treatment with 100 ng/mL vinblastine sulfate for 8 to 10 h, they were prepared for chromosome samples. In total, 33 bovine embryos and 185 blastomeres were examined. Chromosomal normality was analyzed in 43.8% (81/185) of the blastomeres and 60.6% (20/33) of the embryos; while chromosomal anomalies were found in 16 (80%, 16/20) of the embryos, 5 haploid embryos and 11 mosaic (n/2n) embryos. Mosaicism characteristic of the opposite sex in X-and Y-chromosomes was found in 2 haploid embryos, and that of a Y-chromosome and of XX chromosomes in 1 n/2n embryo. Various sex-chromosome compositions were also observed in the other 10 chromosomal mosaic n/2n embryos.

Molecular analysis of esterase D polymorphism.

We have analyzed the esterase D (EsD) polymorphism at the nucleic acid level. Two common alleles, EsD1 and EsD2, are characterized by the substitution of one amino acid (Gly-to-Glu), which is caused by the point mutation of one nucleotide (G-to-A). Individuals exhibiting the EsD1 and EsD 2 phenotypes are homozygotes for EsD 1 and EsD 2 cDNAs, respectively. Individuals showing the EsD 2-1 phenotype have two kinds of cDNAs, viz., EsD 1 and EsD 2. The point mutation difference between the cDNAs of the EsD1 and EsD2 alleles results in a different SspI digestion site. A restriction fragment length polymorphism caused by this difference with respect to the SspI digestion site makes it possible to determine the EsD phenotype using DNA samples extracted from forensic materials with no EsD enzymatic activity.

Enhancing effect of medium-chain triglycerides on intestinal absorption of d-alpha-tocopherol acetate from lecithin-dispersed preparations in the rat.

The effect of formulations of lecithin-dispersed preparation on the absorption of d-alpha-tocopherol acetate (VEA) from the small intestine was investigated in rats. When lecithin-dispersed preparations containing VEA or polysorbate 80 (PS-80)-solubilized solution of VEA were intraduodenally administered, VEA was hydrolyzed to d-alpha-tocopherol (VE) and was not detected in the plasma nor in the thoracic lymph. The maximum plasma concentration (Cmax) of VE after the intraduodenal administration of a preparation consisting of VEA, soybean phosphatidylcholine (PC) and medium-chain triglycerides (MCTG) (VEA/PC/MCTG, 5/16/1 by weight) was highest among the VEA preparations, and PS-80-solubilized solution gave the lowest Cmax. AUC of VE up to 24 h was also increased by the addition of MCTG to VEA/PC preparation. In the thoracic duct-fistula rat, the transport of VE into the thoracic lymph was increased by the administration of the VEA/PC/MCTG preparation significantly more than the VEA/PC preparation; the cumulative amounts of VE recovered in the thoracic lymph up to 24 h were 23.2 +/- 0.5% and 10.9 +/- 1.5% of dose, respectively. The plasma concentration of VE was not increased in the thoracic duct-fistula rat even after the intraduodenal administration of VEA preparations, suggesting that VE is not transported directly to the systemic circulation, but by way of the lymphatic route. The lymphatic transport of VE following the intraduodenal administration of VEA/PC/MCTG preparation was markedly diminished by the simultaneous administration of Pluronic L-81 emulsion, an inhibitor of chylomicron formation. It is suggested that the chylomicron is essential to the lymphatic transport of VE from VEA preparations.

Further investigations of enhancing effect of medium-chain triglycerides on d-alpha-tocopherol acetate absorption from lecithin-dispersed preparations in rat small intestine.

The effect of medium-chain triglycerides (MCTG) in lecithin-dispersed d-alpha-tocopherol acetate (VEA) preparation on intestinal absorption of VEA was investigated in rats using the in situ loop experiment. When VEA preparations containing soybean phosphatidylcholine (PC) and various amounts of MCTG were administered, the amount of VEA absorbed in 2 h was not significantly different among them. However, the amounts of total VE, sum of VEA and d-alpha-tocopherol (VE), remaining in the luminal fluid and the intestinal tissue were dependent on the MCTG content. Total VE remaining in the intestinal tissue after the administration of a VEA/PC/MCTG (5/16/1 by weight) preparation was nearly twice of VEA/PC (5/16) preparation, although the effect of MCTG varied with the increase or decrease in the MCTG content. Moreover, the increased tissue accumulation of total VE by the VEA/PC/MCTG (5/16/1) preparation resulted in an increase in the plasma VE concentration after the removal of the luminal fluid. No effect of pretreatment with PC/MCTG (1/1) dispersion on the tissue accumulation of total VE from VEA/PC (5/16) preparation was observed. Furthermore, the plasma concentration of VE from VEA and/or VE taken up in the tissue from the VEA/PC preparation was not increased by the treatment with the PC/MCTG dispersion. These results suggest that MCTG should coexist in the luminal VEA preparation to enhance the mucosal uptake. A similar enhancing effect was also observed by the addition of the metabolite of MCTG, a medium-chain fatty acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrophoretic analysis of bile proteins from patients with and without gallstones.

Bile proteins from 82 cases with various types of gallstones (pure cholesterol stones, cholesterol stones with a small amount of other substances, mixed stones and pigment stones) and 9 controls without gallstones were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A unique bile protein with a molecular mass of 32 kDa was identified. The 32 kDa protein could be stained on immunoblotting membranes using immunoperoxidase with concanavalin A. The 32 kDa protein was found in all controls but only in a portion (22.2-36.4%) of the patients with gallstones of various types. Following electrophoretic elution from the SDS-PAGE gels, the 32 kDa protein was analyzed by reversed phase--high-performance liquid chromatography, yielding three peaks for controls and only two peaks for patients with gallstones.

Comparison of methods of immobilization to enzyme-linked immunosorbent assay plates for the detection of sugar chains.

The immobilization of carbohydrates for solid-phase assays, including enzyme-linked immunosorbent assay (ELISA), is difficult because they are hydrophilic. We developed four new methods for the immobilization of oligosaccharides. ELISA plates were first coated with methyl vinyl ether-maleic anhydride copolymer (MMAC) and an excess of active anhydride groups was introduced. They were subsequently reacted, in four different ways, to bind oligosaccharides. In method 1, the anhydride groups were reacted with hydrazide groups, in the presence of adipic acid dihydrazide, and then coupled to the reducing ends of sugar chains by reductive amination. In method 2, the anhydride groups were reacted with p-aminophenyl glycoside obtained by reduction with p-nitrophenyl glycoside. In method 3, the anhydride groups were reacted with 1, 6-hexamethylenediamine. Aminooxy groups were coupled to the amino groups introduced and then aminooxyacetic acid with carbodiimide and ligated to oligosaccharides by oxime formation. In method 4, stereospecifically aminated oligosaccharides reacted with the anhydride groups. We compared, in solid-phase assays systems, the ability of lectins to detect oligosaccharides immobilized with either one of these four new methods or one of the two methods previously described. Detection of sugars with lectins is useful because, in most cases, they recognize sugars stereospecifically. The immobilization method should therefore be carefully selected to avoid changing the configuration and substitution in C-1.

Uniqueness of pilsicainide in class Ic antiarrhythmics.

Pilsicainide, a class Ic agent, is known to be an effective drug particularly for treating atrial tachyarrhythmias. However, its electrophysiological effects on the atrium have not been well studied. To characterize the electrophysiologic effects of pilsicainide on atrial myocytes in class Ic drugs, we examined the effects of this drug on membrane currents in single rabbit atrial myocytes using the tight-seal whole cell voltage-clamp technique. Under the current-clamp condition, pilsicainide did not affect the action potential duration at therapeutic ranges (< or = 3 microM) and slightly shortened it at higher concentrations (> or = 10 microM). These observations were quite different from those with other class Ic agents including flecainide and propafenone which prolong the atrial action potential duration. The drug did not affect the resting membrane potential. Under the voltage-clamp condition, pilsicainide inhibited the transient outward current (Ito) that is more prominent in the atrium than in the ventricle in a concentration-dependent manner. However, in contrast to other class Ic agents, the inhibition to Ito by pilsicainide was observed only at much higher concentrations (IC50-300 microM) and did not affect the inactivation time-course of Ito. Moreover, the drug (10 microM) did not significantly affect the Ca2+, delayed rectifier K+, inward rectifying K+, acetylcholine-induced K+ or ATP-sensitive K+ currents. From these results pilsicainide could be differentiated as a pure Na+ channel blocker from other class Ic agents with diverse effects on membrane currents and should be recognized accordingly in clinical situations.

Infradian rhythm of paroxysmal atrial fibrillation. A case report.

To examine the circadian and infradian rhythms of paroxysmal atrial fibrillation, the time and date of 85 arrhythmic attacks occurring over a period of 4 years were analyzed in a patient with reliable symptoms. In the hourly analysis, a remarkable circadian rhythm similar to the reported population circadian rhythm was observed. On a day basis, the distribution of the intervals between 2 successive episodes showed a significant departure from the exponential distribution, indicating the arrhythmia was not a simple probabilistic phenomenon. Spectrum analysis revealed a prominent peak occurring at about 0.3 cycles/day, suggesting a possible circasemiseptan rhythm. Thus, in this patient, paroxysmal atrial fibrillation was not a random event when observed not only from an hour incremental perspective but also from a day incremental perspective, suggesting the circadian and infradian rhythms of this arrhythmia.

Is the QT interval an indicator of autonomic state?

Prolonged QT interval is suggested to indicate an increased risk of sudden cardiac death in certain clinical conditions such as diabetes mellitus. We investigated whether the individual QT interval is an indicator of an autonomic state. An ambulatory 24-hour ECG was recorded in 53 subjects from different clinical backgrounds. Power spectral components of heart rate variability (HRV) and the QT interval were regressively obtained at a heart rate of 60, 70, 80, 90, or 100 beats per minutes (bpm). Log values of the high-frequency component of HRV (HF: 0.15-0.50 Hz, a scale of cardiac parasympathetic tone) failed to show a relationship with the QT interval. In contrast, the QT interval at a heart rate of 90 bpm and 100 bpm showed a significant correlation with the log values of the low-frequency component (LF: 0.04-0.15 Hz) and the log[LF/HF], i.e., a putative scale of sympathetic tone (100 bpm: QT vs logLF: r = 0.414, p < 0.005, QT vs log[LF/HF]: 0.416, p < 0.002). Also, attenuated rate-dependent QT shortening was associated with greater logLF and log[LF / HF] values at a heart rate of 80, 90, or 100 bpm. These results suggest that the QT interval at a moderate heart rate (approximately 90-100 / min) and the degree of rate-dependent QT shortening are related to individual sympathetic tone.

A wide "gap" in retrograde conduction through a concealed accessory atrioventricular pathway depending on ventricular pacing sites.

We present a 57-year-old man with Wolff-Parkinson-White syndrome who exhibited a wide "gap" in retrograde conduction through a concealed atrioventricular accessory pathway. The appearance of the wide "gap" depended on the ventricular pacing sites. While ventricular extrastimuli at a basic cycle length of 600 msec from the right ventricular outflow tract consistently conducted to the atria, retrogradely through the accessory pathway, those from the right ventricular apex repeatedly revealed disappearance of the retrograde conduction at the wide coupling intervals from 550 to 380 msec. The mechanisms of this rare "gap"-like phenomenon are discussed in this paper.