Intervention of Online Percent Coefficient of Variation Reporting System Reduces the Variability of Tacrolimus Trough Concentration in Kidney Transplant Recipients.

BACKGROUND: The online percent coefficient of variation reporting system could monitor the variation of tacrolimus trough level (T0) and identify kidney transplant recipients (KTRs) with a higher percent coefficient of variation (%CV) instantly. Consequently, transplant doctors and pharmacists could take actions to improve drug variability. The purpose of this study was to determine the efficacy of the system for higher intrapatient variability of T0 in KTRs. METHODS: The T0 data were collected with KTRs routinely followed up at an outpatient clinic between June 2016 and November 2016. The %CV was calculated with T0 data within 6 months before and after the index date. The last outpatient clinic visit date was before December 1, 2016. The KTRs with %CV of T0 greater than 22% were enrolled. RESULTS: The study consisted of 183 KTRs (96 male, 87 female), the median age was 50 years (interquartile range [IQR], 41.0-57.0), and the median years post-kidney transplantation was 7 years (IQR, 3.0-12.4). The median T0 and creatinine level at baseline were 6.09 ng/mL (IQR, 4.80-7.52) and 1.33 mg/dL (IQR, 1.03-1.72), respectively. After the intervention, the median %CV of T0 was significantly lower than before, 32% (IQR, 26%-42%) vs 22% (IQR, 15%-33%), P < .001. The average improvement of %CV was also significantly better in KTRs with %CV ≥ 30% (median, from 41% to 25%) than KTRs with %CV between 22% and 30% (median, from 26% to 20%), P < .001. CONCLUSIONS: The results of this study indicate that continuously aggressive intervention with an online %CV reporting system effectively improves intrapatient variability of T0 in KTRs.

Secondary prophylaxis treatment versus on-demand treatment for patients with severe haemophilia A: comparisons of cost and outcomes in Taiwan.

This study compared secondary prophylaxis treatment with on-demand treatment for severe haemophilia A in Taiwan. Fifty patients from one medical centre were evaluated over a 5-year period. Differences in annual bleed rates and factor VIII (FVIII) utilization were assessed between patients receiving secondary prophylaxis and patients receiving FVIII concentrates on-demand. Results were then used as inputs in a pharmacoeconomic model to predict outcomes of future haemophilia therapy strategies in Taiwan. The median annual number of total bleeding episodes was significantly lower in the 13 (26%) patients who received secondary prophylaxis than in the 37 patients who received FVIII on-demand (7.76 vs. 31.91, P < 0.0001). The between-group difference in median annual factor VIII utilization was statistically significant (1824.41 IU kg(-1) for the prophylaxis group and 1324.81 IU kg(-1) for the on-demand group, P < 0.01). It was estimated that approximately $2 million (USD) per year would be added to the cost of treatment by having all severe haemophilia A patients in Taiwan receive secondary prophylaxis instead of on-demand therapy while 12,566 bleeding will be prevented. It is recommended that National Health Insurance officials utilize these data to evaluate the benefits of enhanced treatment strategies and before making substantial policy changes to haemophilia care in Taiwan.

Correlation between Klebsiella pneumoniae carrying pLVPK-derived loci and abscess formation.

Klebsiella pneumoniae-caused liver abscess (KLA) is an emerging infectious disease. However, factors other than K1-specific loci that contribute to the pathogenesis of this disease have not been identified. pLVPK is a 219,385-bp plasmid of K. pneumoniae CG43, an invasive K2 strain associated with KLA. We aimed in this study to evaluate the involvement of pLVPK in K. pneumoniae virulence and its clinical significance in abscess formation. A pLVPK-cured CG43 was isolated and its virulence was examined in a mouse model. The prevalence of pLVPK-derived loci terW, iutA, rmpA, silS, and repA was investigated in 207 clinical isolates by screening with specific primers. Loss of pLVPK abolished the ability of K. pneumoniae to disseminate into extraintestinal sites and, consequently, attenuated abscess formation in mice. Primary K. pneumoniae abscess isolates (n = 94) were more likely to be terW (+)-iutA (+)-rmpA (+)-silS (+) than those related to non-abscess infections (n = 113) (62% vs. 27%; p < 0.0001). Logistic regression analysis indicated that the presence of the terW-rmpA-iutA-silS loci was a significant risk factor (odds ratio, 4.12; 95% confidence interval, 2.02-8.4; p < 0.0001) for abscess formation. pLVPK is a determinant for K. pneumoniae virulence and infection with strains carrying the pLVPK-derived terW-rmpA-iutA-silS loci may predispose patients to abscess formation.

Delayed airway obstruction secondary to inadvertent arterial puncture during percutaneous central venous cannulation.

We report a case of fatal respiratory complication secondary to central venous cannulation in a 63-year-old male patient, which came up gradually and insidiously following an accidental puncture of carotid artery in an attempt to cannulate the right internal jugular vein. He died 14 h after the mishap due to severe upper airway obstruction. The nature of the vascular laceration was still obscure.

Experimental System for X-ray Cone-Beam Microtomography

: A laboratory test of X-ray tomography employing a diverging beam of X-rays rather than the usual parallel X-ray beam is described. We chose to test and demonstrate the advantages of divergent beam tomography by imaging an extracted juvenile human premolar using an ordinary dental X-ray source and a cooled CCD camera. Experiments with a three-piece cover-glass sample and with the human tooth demonstrated that three-dimensional reconstruction can be achieved at 34 µm per pixel resolution employing an X-ray tube spot 800 µm in its smallest direction without requiring close contact with the fluorescent screen. Reconstruction of a 256 x 256 pixel single-plane image from 100 projection images took only 45 sec on a personal computer with a Pentium 166 MHz processor. We have also demonstrated a volume reconstruction of 256 x 256 x 256 voxels from the data. Successful extension of this work to submicrometer projection X-ray microscopy is predicted. Improved resolution of medical tomography is another possible application.

The superimposed effects of chronic phrenicotomy and cervical spinal cord hemisection on synaptic cytoarchitecture in the rat phrenic nucleus.

The present study was carried out to determine the effects of a combined peripheral phrenicotomy and rostral spinal cord hemisection on the synaptic architecture in the ipsilateral rat phrenic nucleus. Young adult female Sprague-Dawley rats were divided into a hemisection-alone and two hemisection-plus-phrenicotomy (HPP) groups. In all animals, DiI, a fluorescent carbocyanine dye was injected into the left hemidiaphragm to retrogradely label the ipsilateral phrenic motoneurons. In the HPP groups, left intrathoracic phrenicotomies were carried out at 2 and 4 weeks prior to sacrificing. Hemisection-alone animals were not subjected to phrenicotomy. In all animals, a left C2 spinal cord hemisection was performed 24 h prior to death. Quantitative morphometric analysis of the phrenic nucleus showed that the number of synapses contacting phrenic profiles is significantly less in the HPP (2 week) group as compared to the hemisection-alone group, but this number returns to a level not significantly different from the hemisection-alone value in the HPP (4 week) group. The results suggest that the transient change in the number of synapses might contribute to the differential expression of the crossed phrenic phenomenon documented in another group of animals subjected to the same surgical procedures. Furthermore, the different stages of glial reaction induced by phrenicotomy/spinal cord hemisection might underlie the change in synaptic number.

The autophagic and endocytic pathways converge at the nascent autophagic vacuoles.

We used an improved cryosectioning technique in combination with immunogold cytochemistry and morphometric analysis to study the convergence of the autophagic and endocytic pathways in isolated rat hepatocytes. The endocytic pathway was traced by continuous uptake of gold tracer for various time periods, up to 45 min, while the cells were incubated in serum-free medium to induce autophagy. Endocytic structures involved in fusion with autophagic vacuoles (AV) were categorized into multivesicular endosomes (MVE) and vesicular endosomes (VE). Three types of AV--initial (AVi), intermediate (AVi/d), and degradative (AVd)--were defined by morphological criteria and immunogold labeling characteristics of marker enzymes. The entry of tracer into AV, manifested as either tracer-containing AV profiles (AV+) or fusion profiles (FP+) between AV and tracer-positive endosomal vesicles/vacuoles, was detected as early as 10 min after endocytosis. The number of AV+ exhibited an exponential increase with time. FP+ between MVE or VE and all three types of AV were observed. Among the 112 FP+ scored, 36% involved VE. Of the AV types, AVi and AVi/d were found five to six times more likely to be involved in fusions than AVd. These fusion patterns did not significantly change during the period of endocytosis (15-45 min). We conclude that the autophagic and endocytic pathways converge in a multistage fashion starting within 10 min of endocytosis. The nascent AV is the most upstream and preferred fusion partner for endosomes.

Three-dimensional laser scanning two-photon fluorescence confocal microscopy of polymer materials using a new, efficient upconverting fluorophore.

Three-dimensional confocal imaging of polymer samples was achieved by the use of two-photon excited fluorescence in both positive and negative contrast modes. The fluorophore was a new and highly efficient two-photon induced upconverter, resulting in improved signal strength at low pumping power. Because of the relatively long wavelength of the excitation source (798 nm from a mode-locked Ti:Sapphire laser), this technique shows a larger penetration depth into the samples than provided by conventional single-photon fluorescence confocal microscopy. Single-photon and two-photon images of the same area of each sample show significant differences. The results suggest the possibility of using two-photon confocal microscopy, in conjunction with highly efficient fluorophores, as a tool to study the surface, interface, and fracture in material science applications.

Improving structural integrity of cryosections for immunogold labeling.

Cryosections of aldehyde-fixed material prepared according to Tokuyasu are a good substrate for immunocytochemistry. However, structural defects occur that limit the resolution of this approach. We found that the step during which sections are thawed and transferred from the cryochamber to the supporting film on an EM grid is most critical for structural preservation. Surface tension of the transfer medium, on which sections are spread during thawing, can easily damage their structure by overstretching. By substituting a mixture of methylcellulose and sucrose for the conventional sucrose transfer medium, we were able to alleviate the problem of overstretching, thus improving greatly the structural integrity of thin cryosections. Also, material extraction from the sections after thawing causes structural damage, particularly when cross-linking is deficient. Incorporation of uranyl acetate in the transfer medium can then further help to maintain the structural integrity of the sections during the immunolabeling procedure. Excellent ultrastructure was featured in sections picked up and dried directly in methylcellulose/uranyl acetate mixtures. Such preparations can provide new insight into subcellular details and is an efficient back-up for immunolabeled sections in respect of their morphology. Cryosections from fresh frozen tissue can be preserved for immunolabeling by using transfer media that contain fixatives. This approach may have advantages if chemical fixation of tissue is thought to induce morphological artifacts or antigen redistribution.

The superimposed effects of chronic phrenicotomy and cervical spinal cord hemisection on glial cytoarchitecture in the rat phrenic nucleus.

This study was conducted to determine the effects of chronic phrenicotomy on spinal hemisection-induced morphological plasticity occurring in the phrenic nucleus. Young adult rats were divided into a hemisection-alone and two hemisection-plus-phrenicotomy (HPP) groups. HPP animals received a left phrenicotomy two or four weeks prior to sacrificing; whereas hemisection-alone animals did not. All animals received a left C2 spinal hemisection 24 hours prior to death. Quantitative morphometric analysis of the phrenic nucleus showed significant reductions in phrenic dendritic size and the number of dendrodendritic appositions in HPP (two week) animals and in the length of dendrodendritic appositions in HPP (four week) animals. Significant increases in microglial area fraction in HPP (two week) animals and in astroglia area fraction in HPP (four week) animals were also detected. The results suggest that the alterations in the spinal hemisection-induced dendrodendritic apposition formation is most likely influenced by the different stages of the glial reactions induced by the chronic phrenicotomy/spinal hemisection.

Ultrastructural and immunocytochemical characterization of autophagic vacuoles in isolated hepatocytes: effects of vinblastine and asparagine on vacuole distributions.

The interactions between the autophagic and the endocytic degradation pathways were investigated by means of immunogold labeling of autophagic vacuoles (AVs) in ultrathin frozen sections from isolated rat hepatocytes. AVs were identified by their autophagocytosed contents of the degradation-resistant cytosolic enzyme CuZn-superoxide dismutase (SOD). Another cytosolic enzyme, carbonic anhydrase (CAIII), was rapidly degraded in the lysosomes, making the vacuolar CAIII/SOD ratio useful as a rough indicator of the progress of autophagic-lysosomal degradation. Lysosomes could be recognized by the presence of the lysosomal membrane glycoprotein lgp120, which was absent from hepatocytic endosomes. Endocytic inputs into the AVs were detected by the presence of gold-conjugated bovine serum albumin (BSA-gold), taken up by fluid-phase endocytosis. All vacuoles recognized morphologically as AVs were SOD-positive, as were essentially all of the lysosomes (96%). The majority (72%) of the lysosomes also labeled positively for BSA within 2 h of endocytosis. The data are thus compatible with the notion that all lysosomes can engage in both autophagic and endocytic degradation. Lgp120 appeared to distinguish well between lysosomes and nonlysosomal AVs: the lgp120-negative AVs (nonlysosomes) had a CAIII/SOD ratio identical to that of the cytosol, indicating that no degradation had occurred. In the lgp120-positive AVs (lysosomes), the ratio was only 43% of the cytosolic value, consistent with substantial CAIII degradation. Among the nonlysosomal AVs (about one-third of all AVs), one-half were BSA-positive, suggesting that early AVs (autophagosomes) and suggesting that early AVs (autophagosomes) and intermediary AVs (amphisomes) that had fused with endosomes were equally abundant. These morphological data thus support previous biochemical evidence for a prelysosomal meeting of the autophagic and endocytic pathways. The microtubule inhibitor vinblastine inhibited the autophagic influx to the lysosomes, causing an accumulation of autophagosomes and a reduction in average lysosomal size. Vinblastine also inhibited the endocytic flux, thereby precluding the formation of amphisomes and of BSA-positive lysosomes. High concentrations (20 mM) of asparagine induced swelling of amphisomes and of BSA-positive lysosomes, probably reflecting an acidotropic effect of ammonia generated by asparagine deamination. Asparagine also caused an accumulation of autophagosomes, amphisomes, and BSA-negative lysosomes, presumably as a result of impaired fusion with the swollen BSA-positive lysosomes. The two agents thus appear to perturb the autophagic-endocytic-lysosomal vacuole dynamics by different mechanisms, making them useful in the further study of these complex organelle interactions.

[The effects of anticonvulsants on the epileptiform discharges--study by 24-hour ambulatory cassette EEG system].

Thirty-three epileptic children who had epileptiform discharges in conventional 16 channel EEG study were enrolled in this study. The patients have never been treated with medication at first visit. Then patient were put on ambulatory monitoring EEG with the Oxford Medilog 9-channel cassette EEG system, which included six symmetrical scalp leads, one EOG, one Chin EMG and one channel for time signal. The EEG record was processed under the rule of the international standard method in terms of 10-20 system. The 24 hours EEG cassettes which recorded before and after antiepileptic drugs therapy were reviewed by an experienced electroencephalographer on a video play back unit. The number and duration of spikes or spike wave discharges were calculated. Among those 22 cases who had studied completed, there are 11 cases with generalized spike waves (5 with irregular spike waves, 3 with regular 3 hz spike waves, 1 with multiple spike waves, 1 with slow sharp waves and 1 with abortive spike waves), and 11 cases with focal spike activities (5 with bilateral central or centrotemporal spikes, 4 with temporal spikes and 2 with occipital spike discharges). After anticonvulsants therapy, 9 cases (82%) out of those 11 cases with generalized spike waves, the spike waves were disappeared; 2 cases (18%) revealed no significant change in spike rate and duration.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative assessment of the effect of chronic phrenicotomy on the induction of the crossed phrenic phenomenon.

The present study was carried out to determine if chronic peripheral phrenicotomy has a functional influence on the plasticity that is normally demonstrated by phrenic motoneurons in the spinal cord following spinal cord injury. Young adult female Sprague-Dawley rats were divided into an experimental and a control group. Left intrathoracic phrenicotomies were carried out at 1, 2, 3, and 4 weeks prior to induction of the crossed phrenic phenomenon and crossed phrenic nerve activity recording in the experimental group. Control animals were not subjected to chronic phrenicotomy. In each animal the crossed phrenic phenomenon was induced by left C2 spinal cord hemisection and turning off the ventilator. The reflex-induced activity in the phrenic nerve ipsilateral to hemisection is defined as "crossed phrenic nerve activity." All animals were subjected to spinal cord hemisection 24 h before crossed phrenic nerve activity recording. The results showed that there is a transient but statistically significant depression of crossed phrenic nerve activity at 2 weeks postphrenicotomy and a recovery to the normal activity level at 4 weeks postphrenicotomy. One control experiment was carried out to assess the effects of phrenicotomy on respiratory activity that is normally present in the phrenic nerve (i.e., not reflex-induced). This "primary respiratory nerve activity" is different from crossed phrenic nerve activity in that the phrenic motoneurons are driven by different bulbospinal respiratory pathways. The results indicated a marked decrease in primary respiratory nerve activity at 1 week after phrenicotomy with no significant recovery by the 4th week after phrenicotomy.(ABSTRACT TRUNCATED AT 250 WORDS)

Guillain-Barré syndrome in children: a cooperative study in Taiwan.

Seventy-two children with Guillain-Barré syndrome (GBS), diagnosed at 11 major teaching hospitals in Taiwan during the period 1986-1990, were studied retrospectively. There were 44 males and 28 females ranging in age from 7 months to 15 years. Preceding events could be traced in 61 patients (85%), including antecedent infection in 59 patients and previous vaccination in 2. As well as the consistent pictures of progressive weakness and generalized hyporeflexia, there were sensory complaints (26%), cranial nerve lesions (46%), respiratory failure (14%) and autonomic dysfunction (25%). Motor symptoms reached a maximum within 20 days in 88% of the patients, with the plateau lasting less than 2 weeks in 75%, and became stable within 3 months in 76%. Overall outcome showed complete recovery in 73% of the patients within 6 months after onset. Four (5.6%) had recurrence, and there was no mortality. The present study revealed that the annual incidence of GBS in Taiwan can be estimated roughly as 0.66 per 100,000 and that the course of childhood GBS is relatively benign.

Expression of functional growth hormone receptor in a mouse L cell line infected with recombinant vaccinia virus.

The growth hormone receptor is a member of a large family of receptors including the receptors for prolactin and interleukins. Upon binding to one molecule of growth hormone two growth hormone receptor polypeptides dimerize. We have expressed the rabbit growth hormone receptor DNA in transfected mouse L cells infected with polymerase T7-producing vaccinia virus. The growth hormone receptor was synthesized as a 85-kDa protein and transported to the cell surface. Western blotting and metabolic labeling combined with immunoprecipitation using a rabbit antibody probably directed against the cytosolic domain of the receptor showed that its expression was dependent on both transfection of the growth hormone receptor DNA and vaccinia infection. Binding studies with 125I-labeled growth hormone demonstrated specific binding sites at the cell surface 20 h after transfection. Permeabilization with saponin showed that the growth hormone receptor binding sites were almost exclusively present at the cell surface with little intracellularly. Chemical crosslinking of the 125I-growth hormone complex resulted in a 180-kDa complex which could specifically be immunoprecipitated with the antiserum. Electron microscopic immunocytochemistry confirmed the presence of growth hormone receptor at the cell surface. Furthermore, specific growth hormone receptor antigen was also associated with intracellular membranes. These results thus show that this transient transfection system will be useful for cell biological studies of growth hormone receptor regulation.

Ultrastructure and glycoconjugate histochemistry of the lens capsule during lens regeneration from the iris in the Newt.

The lens capsule of the regenerating lens develops from the basal lamina of the iris epithelium. As the lens differentiates and grows in size, the lens capsule increases in thickness by the formation of successive layers of basal laminar material resulting in a structure composed of increasing numbers of parallel lamellae. This initial arrangement may be lost and a more homogeneous composition of fine granules and filaments may characterize some parts of the capsule in older lenses. The developing lens capsule stains with the PAS and PAPD methods but not with the PAPS, AB and HID methods. Therefore, it appears to contain only neutral, non-acidic glycoconjugates (probably glycoproteins) which may contain fucose, galactose or mannose. This conclusion is supported by the incorporation of 3H-fucose into the developing lens capsule of regenerating lenses beginning at a stage with initial lens fiber formation. Except for the apparent absence of acidic glycoconjugates in the regenerating lens capsule, these events are similar to those described for developing lenses in chick and mammalian embryos. The differentiation of lens fibers by elongation, loss of organelles and accumulation of fine, electron-dense particles in the cytoplasm also parallels embryonic, lens fiber differentiation.

Early-stage development of auditory center: an experimental study of auditory evoked electrophysiologic recordings from fetal and newborn guinea pigs.

Five guinea pig fetuses at 8 weeks' gestation (term is 9 weeks) were tested for auditory brain stem response (ABR) generated by earphones placed outside the maternal abdomen. The results obtained from these fetuses were compared with the ABRs in 10 term newborn guinea pigs on the 1st and 20th day after delivery. The ABRs indicated a decreasing peak latency over wave III with increasing age. The interpeak latency to wave III-V decreased markedly, with very little change in wave I-III. The results suggested rapid maturation of the upper brain stem and midbrain during late gestation and in newborns. They also exhibited an increasing peak latency of ABRs from both fetuses and newborns with decreasing stimulus intensity.

Distribution of CuZn superoxide dismutase in rat liver.

Morphological and cell fractionation approaches were used to establish unambiguously the distribution of CuZn superoxide dismutase (CuZn SOD) in rat hepatocytes. Immunocytochemical observations revealed a primarily cytoplasmic localization of the enzyme. While only trace amounts were found in cell organelles like mitochondria and peroxisomes, lysosomes were labelled stronger than the cytoplasm. The presence of CuZn SOD in lysosomes was also identified in cell fractions of normal and Triton WR-1339-treated rats. Microscopic studies showed that the distribution of CuZn SOD was not affected by Triton, but the Triton treatment induced an apparent increase in the number and size of lysosomes with electron lucent contents which corresponded with a shift of lysosomes to low buoyant density fractions. The majority of CuZn SOD originally present in the mixed mitochondrial-peroxisomal-lysosomal fractions of the normal liver comigrated with the main peak of the lysosomal marker, beta-hexosaminidase, after Triton treatment. The peroxisomal marker, catalase, did not migrate with the CuZn SOD/beta-hexosaminidase-rich fractions in livers from Triton-treated animals. These results confirm earlier observations in rat liver cells, showing that CuZn SOD, a primarily cytosolic enzyme, accumulates in lysosomes. It is not present in significant amounts in rat hepatocyte peroxisomes.

Effects of phenobarbital on leukocyte activation: membrane potential, actin polymerization, chemotaxis, respiratory burst, cytokine production, and lymphocyte proliferation.

Leukocyte activation is known to involve cell membrane potential changes. Phenobarbital, an anesthetic and anticonvulsant that can inhibit neuronal membrane depolarization, may also affect leukocyte activation. Measuring membrane potential, actin polymerization, chemotaxis, superoxide production, lymphocyte proliferation, intracellular calcium concentration, and cytokine production, we found that phenobarbital at a concentration of 15-30 micrograms/ml, which is considered a therapeutic serum level for controlling seizures, did not affect polymorphonuclear neutrophil (PMN) activation. At levels higher than 100 micrograms/ml, phenobarbital significantly suppressed formylmethionyl-leucyl-phenylalanine (fMLP)-induced chemotaxis. Concentrations greater than 300 micrograms/ml also inhibited phorbol myristate acetate-stimulated membrane potential change. In contrast, 30 micrograms/ml phenobarbital significantly inhibited lymphocyte proliferation stimulated by phytohemagglutinin (PHA) and pokeweed mitogen. This concentration of phenobarbital also suppressed the increase of intracellular free calcium induced by PHA. However, only a higher concentration of phenobarbital (300 micrograms/ml) was able to inhibit PHA-induced interleukin-2 (IL-2) production and suppress the proliferation of PHA-induced IL-2 receptor-bearing lymphocytes. These results suggest that concentrations of phenobarbital associated with anticonvulsive levels do not affect PMN activation but suppress lymphocyte activation, possibly by affecting intracellular signal transduction.