Pharmacokinetics of dexmedetomidine combined with therapeutic hypothermia in a piglet asphyxia model.

BACKGROUND: The highly selective α2 -adrenoreceptor agonist, dexmedetomidine, exerts neuroprotective, analgesic, anti-inflammatory and sympatholytic properties that may be beneficial for perinatal asphyxia. The optimal safe dose for pre-clinical newborn neuroprotection studies is unknown. METHODS: Following cerebral hypoxia-ischaemia, dexmedetomidine was administered to nine newborn piglets in a de-escalation dose study in combination with hypothermia (whole body cooling to 33.5°C). Dexmedetomidine was administered with a loading dose of 1 µg/kg and maintenance infusion at doses from 10 to 0.6 µg/kg/h. One additional piglet was not subjected to hypoxia-ischaemia. Blood for pharmacokinetic analysis was sampled pre-insult and frequently post-insult. A one-compartment linear disposition model was used to fit data. Population parameter estimates were obtained using non-linear mixed effects modelling. RESULTS: All dexmedetomidine infusion regimens led to plasma concentrations above those associated with sedation in neonates and children (0.4-0.8 µg/l). Seven out of the nine piglets with hypoxia-ischaemia experienced periods of bradycardia, hypotension, hypertension and cardiac arrest; all haemodynamic adverse events occurred in piglets with plasma concentrations greater than 1 µg/l. Dexmedetomidine clearance was 0.126 l/kg/h [coefficient of variation (CV) 46.6.%] and volume of distribution was 3.37 l/kg (CV 191%). Dexmedetomidine clearance was reduced by 32.7% at a temperature of 33.5°C. Dexmedetomidine clearance was reduced by 55.8% following hypoxia-ischaemia. CONCLUSIONS: Dexmedetomidine clearance was reduced almost tenfold compared with adult values in the newborn piglet following hypoxic-ischaemic brain injury and subsequent therapeutic hypothermia. Reduced clearance was related to cumulative effects of both hypothermia and exposure to hypoxia. High plasma levels of dexmedetomidine were associated with major cardiovascular complications.

Determinants of outcomes following acute child encephalopathy and encephalitis: pivotal effect of early and delayed cooling.

BACKGROUND: Acute encephalopathy/encephalitis is one of the most important causatives of mortality and neurological deficit during childhood. The aim of this retrospective observational study was to investigate clinical variables and therapeutic options associated with the outcome of children with acute encephalopathy/encephalitis. METHODS: Relationships between the clinical information at admission and the neurological outcome evaluated using Pediatric Cerebral Performance Category Scale (PCPC) at 12 months after admission were assessed in 43 patients who were treated at 10 Japanese paediatric intensive care units. RESULTS: Sixteen patients were cared for at normothermia, whereas mild hypothermia was applied to 27 children. In univariate analysis, ages ≤ 18 months, marked elevation in serum lactate dehydrogenase (LD) and aspartate transaminase, diagnosis of either acute necrotising encephalopathy or haemorrhagic shock and encephalopathy syndrome and longer hypothermic periods were associated with increased risks of death or severe neurological deficit, whereas hypothermia showed pivotal effects: the outcome of children cooled after 12 h of diagnosis was statistically invariant with normothermic children, but was significantly worse compared with children cooled ≤ 12 h. In multivariate analysis, younger ages and elevated serum LD were associated with adverse outcomes, whereas early initiation of cooling was related to favourable outcomes. For normothermic children, PCPC scores were dependent on the computed tomographic findings suggestive of cerebral oedema, serum LD levels and Glasgow Coma Scale at admission. For hypothermic children, PCPC scores depended on longer delays in cooling initiation. CONCLUSION: Without therapeutic hypothermia, the outcome of children was determined by variables suggestive of the severity of encephalopathy/encephalitis at admission. Hypothermia may have pivotal impacts on the outcome of children according to the timing of cooling initiation following acute encephalopathy/encephalitis.

Formulation and evaluation of lactoferrin bioadhesive tablets.

For the treatment of chronic inflammation in the oral cavity, we attempted to develop bioadhesive tablets of bovine lactoferrin (B-LF) which has antibacterial properties and immune regulatory functions. B-LF tablets containing pectin, tamarind gum or carboxymethylcellulose (CMC) were prepared by direct compression. Tablets consisting of B-LF, pectin and xylitol passed through 60- or 100-mesh sieves were also prepared. The tablets containing CMC had insufficient bioadhesive force. Although the tablets containing tamarind gum showed the longest residence time in the oral cavity, an unpleasant taste gradually developed. The tablets containing pectin showed the highest value of bioadhesive force and the taste was acceptable. The characteristics of the B-LF tablets were improved by adding an appropriate amount of xylitol and using the ingredients sieved by a 100-mesh sieve. The therapeutic effect was evaluated by using rats with an ulcer on the oral mucosa. In the present study, swelling on the periphery of the ulcer was observed after administration of the B-LF tablets, and then the ulcer has reduced overall.

Single cell reporter assay using cell surface displayed Vargula luciferase.

Reporter genes such as firefly luciferase are common tools to monitor gene expression in various systems. As reporter gene represents the expression level of the gene of interest with its enzyme activity, firefly luciferase is most frequently used because its luminescent activity is highly sensitive and less time consumable for assay. However, since firefly luciferase is expressed internally in the cell, lysis of the cell is a critical step, and thus it is difficult to monitor the gene expression level continuously. In this report, we utilized secretive Vargula hilgendorfii luciferase modified to cell surface displayed one by fusing with human EGFR transmembrane sequence. This modified Vargula luciferase was expressed on cell surface without losing its bioluminescent activity. Co-transfection with secretive alkaline phosphatase showed that the behaviors of cell surface displayed Vargula luciferase and secretive alkaline phosphatase are comparable to each other. Furthermore, the luminescence of a single cell expressing cell surface displayed Vargula luciferase can be monitored by using photon counting CCD camera, which indicates that this reporter gene can monitor gene expression in a single cell without cell lysis.

Engineering of functional chimeric protein G-Vargula luciferase.

Luciferase of Vargula hilgendorfli is infinitely stable at room temperature in dried state, and its light-emitting reaction is very simple. These unique characteristics of Vargula luciferase have prompted us to engineer chimeric protein, the other moiety chosen for conjugation being streptococcal protein G. A single domain of protein G which binds to IgG of a wide range of species was fused at the N-terminal region of Vargula luciferase. Unexpectedly, we found that the chimeric protein expressed in mammalian COS-1 cells had no IgG-binding ability, probably due to some sort of interaction between the two moieties or some conformational preferences of the IgG-binding domain of protein G when fused to Vargula luciferase. Here we report how we regained the IgG binding of protein G, by the intervention of three alpha-helices of protein A between protein G and luciferase. To our knowledge, the new chimeric protein provides the first reported model of this kind.

Truncation of Vargula luciferase still results in retention of luminescence.

Significant amino acid sequence homology in two regions of Vargula hilgendorfii to one in apoaequorin was reported. The intra-amino acid homology in Vargula luciferase between residues 81-312 and 321-540 was 19.3%, and each of this intra-homologous region contained the region homologous to apoaequorin. In order to prove the possibility that only one of the homologous regions is sufficient for luminescence, we have produced a chimeric protein comprising of only the N-terminal homologous region of Vargula luciferase fused to protein A. Comparison of the luminescence of this truncate luciferase indicated that there was 38.5% retention in the bioluminescence of luciferase when compared to that of the mature form of luciferase. This fact may have interesting implications for further study of engineering luciferase.

Expression of a bifunctional chimeric protein A-Vargula hilgendorfii luciferase in mammalian cells.

We have designed and constructed a novel chimeric protein that consisted of a single domain of protein A and luciferase derived from sea-firefly Vargula hilgendorfii with the goal of obtaining a heterofunctional immunological tool. The structural gene of luciferase was fused to the 3' terminus of the D domain gene of protein A with/without a short linker of five amino acids. The resulting constructs under the transcriptional regulation of the Rous sarcoma virus (RSV) promoter, were expressed transiently in simian COS-1 and stably in Chinese hamster ovary (CHO) cells. The properties of the resultant chimeric protein were characterized. The results indicated that the dual properties of the chimeric protein could be retained only after the introduction of a linker of (Gly)4 Ser between the two conjugated moieties. Moreover, the chimeric protein was found to retain at least 50% of the specific activity as compared with the non-fused luciferase. The future prospect of the usage of this chimeric protein in the field of diagnostics was further evaluated by performing bioluminescent immunoassays.

Detection of monocyte chemotactic and activating factor (MCAF) in normal blood and urine using a sensitive ELISA.

We developed a highly sensitive enzyme-linked immunosorbent assay (ELISA) for human monocyte chemotactic and activating factor (MCAF), an inflammatory cytokine that plays an important role in the recruitment of blood monocytes to areas of inflammation. The ELISA, which is based on a sandwich method using two newly-developed monoclonal antibodies, could quantitatively detect MCAF in the range between 2.5 pg/ml (50 fg/sample) to 300 pg/ml after incubation for a total of 2 h, and showed no cross-reactivity with various structurally-related IL-8 superfamily proteins. It was not affected by blood or urine components non-specifically, and thus was directly applicable to clinical specimens. When serum and urine samples from healthy subjects were measured, they all turned out to contain detectable levels of MCAF (more than 30 pg/ml). By gel-filtration column chromatography analysis, MCAF in the body fluids was eluted as a single peak at the position corresponding to the molecular weight of 10 kD, suggesting that it exists as a monomer form, free from carrier proteins. The established ELISA here is expected to be effectively used for the further investigations on the relationship of MCAF with various inflammatory diseases.

Three-dimensional crystal structure of recombinant murine interferon-beta.

The crystal structure of recombinant murine interferon-beta (IFN-beta) has been solved by the multiple isomorphous replacement method and refined to an R-factor of 20.5% against 2.6 A X-ray diffraction data. The structure shows a variant of the alpha-helix bundle with a new chain-folding topology, which seems to represent a basic structural framework of all the IFN-alpha and IFN-beta molecules belonging to the type I family. Functionally important segments of the polypeptide chain, as implied through numerous gene manipulation studies carried out so far, are spatially clustered indicating the binding site(s) to the receptor(s). Comparison of the present structure with those of other alpha-helical cytokine proteins, including porcine growth hormone, interleukin 2 and interferon gamma, indicated either a topological similarity in chain folding or a similar spatial arrangement of the alpha-helices.

New crystal form of recombinant murine interferon-beta.

Although we have reported (Matsuda, S., Kawano, G., Itoh, S., Mitsui, Y., and Iitaka, Y. (1986) J. Biol. Chem. 261, 16207-16209) that recombinant murine interferon-beta produced in Escherichia coli was crystallized in an orthorhombic space group C222(1) using polyethyleneglycol 8000 as precipitant, the crystals had an insufficient resolution and a marked tendency for orientational disorder around the c axis. We now report that another form of murine interferon-beta crystals with little disorder was obtained in the presence of dioxane using ammonium sulfate as precipitant. The new crystals belong to a hexagonal space group P6(1) or P6(5) with a = b = 71.4 A and c = 79.6 A having only one murine interferon-beta molecule in an asymmetric unit. The crystals are reasonably stable to x-rays and significantly diffract up to 2.2 A resolution when a synchrotron beam is used.

Crystallization and preliminary X-ray studies of recombinant murine interferon-beta.

Recombinant murine interferon-beta produced in Escherichia coli was purified and crystallized in an orthorhombic space group C222(1) with a = 61.67 A, b = 55.62 A, and c = 92.16 A. The crystals with a slight tendency for orientational disorder around the c axis diffract at least up to 3.3-A resolution. The crystallizability and the fact that the crystallographic asymmetric unit contains only one molecule of murine interferon-beta strongly indicate that the present preparation (Matsuda, S., Utsumi, J., and Kawano, G. (1986) J. Interferon Res., in press) of recombinant murine interferon-beta is predominantly homogeneous with respect to chemical, tertiary, and quaternary structures.

Purification and characterization of recombinant mouse interferon-beta.

Recombinant mouse interferon-beta (rMuIFN-beta) produced in Escherichia coli was purified to homogeneity and characterized. The purified protein exhibited a single band of Mr 19,900 on SDS-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions, and also exhibited a single band on native polyacrylamide gel electrophoresis at pH 4.3. The observed molecular weight corresponded to that of the polypeptide moiety of natural MuIFN-beta of Mr 19,700. The amino acid composition and the amino-terminal sequence of the purified rMuIFN-beta were identical to those predicted from cDNA sequence. These results indicate that the purified protein is a nonglycosylated MuIFN-beta, which forms no disulfide-linked dimer and probably exists as a monomeric form.

Production of recombinant mouse beta-interferon.

A plasmid was constructed to express a mouse beta-interferon (IFN-beta) in Escherichia coli under the control of the modified tryptophan (trp) promoter. E. coli carrying the plasmid were cultivated in a minijar fermentor and synthesized up to 2.7 X 10(6) IU/ml of antiviral activity. At the end of the cultivation the cells became elongated and curved.