A Genome-Wide Comparative Evolutionary Analysis of Herpes Simplex Virus Type 1 and Varicella Zoster Virus

Herpes simplex virus type 1 (HSV-1) and varicella zoster virus (VZV) are closely related viruses causing lifelong infections. They are typically associated with mucocutaneous or skin lesions, but may also cause severe neurological or ophthalmic diseases, possibly due to viral- and/or host-genetic factors. Although these viruses are well characterized, genome-wide evolutionary studies have hitherto only been presented for VZV. Here, we present a genome-wide study on HSV-1. We also compared the evolutionary characteristics of HSV-1 with those for VZV. We demonstrate that, in contrast to VZV for which only a few ancient recombination events have been suggested, all HSV-1 genomes contain mosaic patterns of segments with different evolutionary origins. Thus, recombination seems to occur extremely frequent for HSV-1. We conclude by proposing a timescale for HSV-1 evolution, and by discussing putative underlying mechanisms for why these otherwise biologically similar viruses have such striking evolutionary differences

Glutamine and glutathione at ICU admission in relation to outcome

Glutamine depletion is demonstrated to be an independent predictor of hospital mortality in ICU (intensive care unit) patients. Today glutamine supplementation is recommended to ICU patients on parenteral nutrition. In addition to glutamine, glutathione may be a limiting factor in ICU patients with MOF (multiple organ failure). To study the prevalence of glutamine and glutathione depletion an observational study was performed. The results were analysed in relation to mortality and the conventional predictors of mortality outcome, APACHE II (Acute Physiology and Chronic Health Evaluation II) and SOFA (Sequential Organ Failure Assessment). Consecutive patients admitted to the ICU at Karolinska University Hospital Huddinge were studied. Patient admission scoring of APACHE II and SOFA were registered as well as mortality up to 6 months. Plasma glutamine concentration and whole blood glutathione status at admittance were analysed. The admission plasma glutamine concentrations were totally independent of the conventional risk scoring at admittance, and a subnormal concentration was an independent predictor of mortality. In addition, glutathione redox status was also an independent mortality predictor, but here a normal ratio was the risk factor. In both cases the mortality risk was mainly confined to the post-ICU period. A low plasma concentration of glutamine at ICU admission is an independent risk factor for post-ICU mortality. The possible benefit of extending glutamine supplementation post-ICU should be evaluated prospectively

DLA Class II Alleles Are Associated with Risk for Canine Symmetrical Lupoid Onychodystropy (SLO)

Symmetrical lupoid onychodystrophy (SLO) is an immune-mediated disease in dogs affecting the claws with a suggested autoimmune aethiology. Sequence-based genotyping of the polymorphic exon 2 from DLA-DRB1, -DQA1, and -DQB1 class II loci were performed in a total of 98 SLO Gordon setter cases and 98 healthy controls. A risk haplotype (DRB1*01801/DQA1*00101/DQB1*00802) was present in 53% of cases and 34% of controls and conferred an elevated risk of developing SLO with an odds ratio (OR) of 2.1. When dogs homozygous for the risk haplotype were compared to all dogs not carrying the haplotype the OR was 5.4. However, a stronger protective haplotype (DRB1*02001/DQA1*00401/DQB1*01303, OR = 0.03, 1/OR = 33) was present in 16.8% of controls, but only in a single case (0.5%). The effect of the protective haplotype was clearly stronger than the risk haplotype, since 11.2% of the controls were heterozygous for the risk and protective haplotypes, whereas this combination was absent from cases. When the dogs with the protective haplotype were excluded, an OR of 2.5 was obtained when dogs homozygous for the risk haplotype were compared to those heterozygous for the risk haplotype, suggesting a co-dominant effect of the risk haplotype. In smaller sample sizes of the bearded collie and giant schnauzer breeds we found the same or similar haplotypes, sharing the same DQA1 allele, over-represented among the cases suggesting that the risk is associated primarily with DLA-DQ. We obtained conclusive results that DLA class II is significantly associated with risk of developing SLO in Gordon setters, thus supporting that SLO is an immune-mediated disease. Further studies of SLO in dogs may provide important insight into immune privilege of the nail apparatus and also knowledge about a number of inflammatory disorders of the nail apparatus like lichen planus, psoriasis, alopecia areata and onycholysis

Clinical pharmacokinetics of intravenous ethanol : Relationship between the ethanol space and total body water

Introduction: Total body water (TBW) is an important parameter in pathological states where the normal regulation of fluid balance is impaired (e.g., during critical illness, congestive heart failure, bum injury and renal insufficiency). Dilution of water isotopes, such as deuterium oxide (D2O), is considered die gold standard method for measuring TBW in humans. However this procedure requires skilled staff, expensive equipment and the results are seldom available in a timely fashion, which makes it less suitable for clinical applications. Ethanol is completely miscible with water and is thought to distribute into the TBW. The ethanol volume of distribution at steady state (Vss) can be estimated by pharmacokinetic analysis of the concentration-time profile. Ethanol can be measured in expired breath with high precision and this non- invasive method of analysis could provide an attractive alternative with the prospect of bedside monitoring of Vss within 4-6 hours. The aim. of this thesis was to develop an appropriate pharmacokinetic model for intravenous ethanol administration and to identify experimental factors that impact on the results. With this background, we compared Vss. determined by ethanol dilution with TBW determined by D20 dilution. The complicated absorption kinetics of ethanol (e.g. variable gastric emptying and first-pass metabolism) was avoided by use of the intravenous route of administration. Material and Methods: Forty-six healthy volunteers (20 women and 26 men) received intravenous infusions of ethanol (0.4- 0.6 g/kg body weight) in 15-60 minutes. The concentration of ethanol was measured in end-expired breath by infrared spectrometry and in blood and urine by headspace gas chromatography. Specimens of blood and breath were obtained at 5-15 min intervals for 3-6 hours post-dosing. The concentration of D20 in plasmawater was measured with isotope ratio mass spectrometry. Subjective feelings of inebriation were measured with a visual analogue scale. A number of pharmacokinetic models were developed and evaluated in the course of this thesis. The effect of eating a meal on ethanol kinetics was investigated in crossover studies by giving the same dose of ethanol in fed and fasted states. The magnitude and time-course of arterio-venous differences in ethanol concentration were determined to assess the importance of local peripheral vasodilatation and vasoconstriction on results. This was achieved by warming the sampling hand in a heating box or cooling the hand in cold water during ethanol infusion experiments. The impact of ~ling site on pharmacokinetic parameters of ethanol was investigated by simultaneous sampling in expired breath, arterial blood and venous blood. Inter- and intra-individual variations in precision of the estimates of Vss. and TBW were studied by making repeat infusions of ethanol a few days apart, and use of analysis of variance. Results and discussion: The two-compartment model with parallel Michaelis-Menten kinetics and first-order rend elimination provided an excellent prediction of the entire concentration-time profiles of ethanol and proved to be theoretically superior to the other kinetic models tested. A good reproducibility was obtained for all pharmacokinetic parameters, especially for Vss and the maximal metabolic rate (Vmax). Eating a meal increased the rate of ethanol metabolism by 30-60% and this confounding factor needs to be considered in clinical studies when Vss. for ethanol is estimated. Peripheral vasoconstriction caused a lowering of the concentrations of ethanol in venous blood, which also impacts on the pharmacokinetic parameters of ethanol. Measuring ~1 in breath agreed more closely with arterial blood concentrations than with venous blood. The pharmacokinetic parameters of ethanol could be measured with equally high precision in expired breath and venous blood. An ethanol dose of 0.4 g/kg body weight infused in 15 minutes led to unacceptable inebriation in some volunteer subjects. The precision of estimating Vss by ethanol dilution was about the same as for measuring TBW by D20 dilution (SD 0.8-.1.1 litres). However, me observed a systematic bias of -13% between Vss and TBW (D20). A likely explanation for this finding might be that water in the body, at least in part, is structured in such a way that ethanol is prevented from complete equilibration. Conclusions: A two-compartment model with parallel Michaelis-Menten and first-order renal elimination gave an excellent fit to the concentration-time profile of ethanol after intravenous ~on. Feeding state and peripheral circulation affected the estimated model parameters. Sampling and analysis of breath provided results with the same high precision as venous blood for estimating ethanol Vss. The non-invasive nature of breath sampling makes it more suitable for clinical purposes. The systematic bias of-13% between the Vss and TBW by D20 dilution suggests that ethanol does not distribute uniformly into the TBW. The reason for this discrepancy remains to be established

Isolation and characterization of regulatory peptides and bioactive compounds

Isolation of peptides and other bioactive compounds is an important and often necessary step to get the total information about their structures. This is demonstrated by a number of different characterizations in this thesis. Bioactive peptides and small organic molecules can act as signaling substances and messengers in multicellular organisms and are fundamental to higher forms of life. The following bioactive peptides and compounds were studied. 1) Different assays can be used for detection of novel peptides. Chemical assays are simple, but still robust, and have proven successful in several cases. In this thesis, an assay was developed to detect peptides that contain tryptophan. An N-terminal dipeptide Gly-Trp is characteristic for galanin, which was cleaved off, separated and visualized. In this manner a novel form of galanin from chicken was detected and isolated. 2) Antibacterial peptides were first found in insects and later in mammals. Many of these peptides are basic, some are Pro/Arg-rich (e.g. peptide PR-39). Investigation of basic peptides from pig spleen, resulted in the isolation of a new variant form of NK-lysin, previously only detected in a cDNA library from porcine bone marrow. PR-39 was identified by a method suitable for Pro/Arg-rich peptides using ladder sequence analysis with mass spectrometry. 3) Another biological assay, involving cAMP production in cell culture of SK-N-MC cells, was used to screen peptide fractions, and resulted in the isolation of a novel form of the hormone PYY, with a phosphate group attached to the Ser-13 residue. This finding demonstrates the benefit of peptide isolation to find posttranslational modifications not directly obvious from the corresponding DNA sequence. Using nano-electrospray tandem mass spectrometry, the phosphorylated form of porcine PYY was identified and the modification localized to Ser- 13. 4) Specific antibodies raised against peptides are a valuable tool in peptide chemistry. We have isolated PEC-60 from pig and rat brains with a method that combines column purification procedures with the specificity of a radioimmunoassay (RIA) and the sensitivity of mass spectrometry to directly identify the peptide. The results show that PEC-60, like many other peptides, is localized in the gastrointestinal tract and also in the central nervous system. The specific regional brain distribution may imply a specific function. 5) Using RIA and HPLC purification, 4 novel forms of modified galanin were isolated and characterized by mass spectrometry. The modified forms contained beta-aspartic shifts and oxidized tyrosine. 6) Hypoglycemic agents have been described in plants. Gynostemma pentaphyllum (Cucurbitaceae), an East-Asian herb, has been reported to have different activities, such as antitumor, cholesterol-lowering, immunopotentiating, hypoglycemic and antioxidant effects. We have isolated a novel insulin-releasing substance from this extract, determined the structure by NMR and mass spectrometry and characterized the effects on insulin release. We found that it is a novel saponin and we named it phanoside. The results provide us with new knowledge about localization, structure, and processing of hormones and bioactive molecules