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The key to developing a therapeutic vaccine for chronic hepadnavirus infection lies in the characteristics of the host-immune response which leads to clearance of acute infection. Groups of 28-day-old ducks which had been surgically bursectomized (n = 10) or thymectomized (n = 13) on the day of hatch or were untreated (n = 21) were inoculated with 10(9) viral genome equivalents (vge) DHBV, then bled twice a week, and euthanased 40 days later. Serum and liver were tested for DHBV DNA and total leukocytes and peripheral blood mononuclear cells (PBMCs) counted. Liver and spleen sections were either stained with hematoxylin and eosin, and graded for inflammation or stained with peroxidase-labeled anti-human CD3 antibody and examined for T lymphocyte distribution. PBMC counts were similar in all groups. DHBV infection combined with bursectomy increased significantly, while thymectomy decreased significantly the total leukocyte count. The spleen and liver bursectomy increased T lymphocyte number while B cells were decreased. Converse changes were observed in thymectomized ducks. Histological evidence of hepatitis was present in infected control and bursectomized ducks but not in the uninfected control or infected thymectomized ducks. In control animals, DHBV challenge caused viremia in 17 and persistent infection in 11 (56%). Fewer thymectomized ducks (3/13, 23%) and significantly more (100%) bursectomized ducks remained persistently infected (P < 0.001). Unexpectedly, bursectomy led to persistence of infection while clearance of infection occurred normally in thymectomized ducks despite decreased T lymphocyte numbers. This suggests that clearance requires T and B lymphocyte collaboration., ((c) 2006 Wiley-Liss, Inc.)

Background: The purpose of this study was to investigate the regulatory effect of a small molecule selectin inhibitor in the liver by examining the functional, structural, and survival response of animals subjected to hemorrhagic shock and to determine the liver infiltration of neutrophils and the regulation of chemokine expression. Selectins play an important role in the development of the lesions associated with ischemia/reperfusion and hemorrhagic shock. Blocking individually the selectin family of adhesion molecules with monoclonal antibodies has resulted in better organ function and survival. To our knowledge, there are no studies demonstrating the beneficial effect of multiple selectin blockade with a small molecule inhibitor under conditions of hemorrhagic shock., Methods: Forty-eight Sprague-Dawley rats were subjected to hemorrhagic shock. Three groups of animals were included (n = 16/group), i.e., the sham, control, and treated groups, which received a small molecule selectin inhibitor (TBC-1269) at 25 mg/kg body weight after the bleeding began. The following parameters were evaluated: fluid requirements during resuscitation, liver injury tests (aspartate aminotransferase, alanine aminotransferase), liver histology and myeloperoxidase, and macrophage inflammatory protein-2 mRNA and cytokine-induced neutrophil chemoattractant mRNA in liver tissue, and animal survival at 3 days. Statistical analysis included Student's t test and analysis of variance when indicated., Results: Significant improvement in liver function and histology was noted in the treated group. Survival was also improved, although it is not known whether liver failure was the most proximate cause of lethality. Infiltration of neutrophils, measured by tissue myeloperoxidase, was significantly decreased in livers of treated animals. No significant changes were noted in fluid requirements. The small molecule selectin inhibitor group showed a down-regulating effect on liver macrophage inflammatory protein-2 and cytokine-induced neutrophil chemoattractant mRNA expression associated with less accumulation of neutrophils in the liver., Conclusion: This study supports the role that selectins play in the pathogenesis of hemorrhagic shock. The mechanism of protection seen after multiple selectin blockade (TBC-1269) centered, in part, around the infiltration of liver neutrophils, probably dependent on the induction of macrophage inflammatory protein-2 and cytokine-induced neutrophil chemoattractant mRNA expression in liver tissue.

NMR detection of multiply labeled compounds in biological samples is often used to follow metabolic pathways. Detection of protons bound to 13C atoms offers a more sensitive approach than direct 13C detection, but generally results in the loss of carbon-carbon coupling information. We have modified an HSQC sequence to refocus the carbon chemical shifts in order to obtain a proton-correlated 13C homonuclear J spectrum, which allows us to measure singly and doubly labeled compounds in the same spectrum., (Copyright 1999 Academic Press.)

Background: The selectin family of adhesion molecules plays a key role in the neutrophil-mediated injury observed after ischemia and reperfusion. In our study, we investigated the effects of TBC-1269, a novel small-molecule, nonoligosaccharide inhibitor of P-, E-, and L-selectin binding, in the liver inflammatory response after 90 minutes of warm ischemia., Study Design: Total liver ischemia was produced in Sprague-Dawley rats for 90 minutes using an extracorporeal portosystemic shunt. The animals were divided into five groups including: the sham (group 1), ischemic control (group 2) receiving only the vehicle, and the treated groups receiving TBC-1269 at a dose of 25 mg/kg at different times of administration: 15 minutes before reperfusion (group 3), at reperfusion (group 4), and 15 minutes after reperfusion (group 5). The following indices were analyzed: 7-day survival, liver injury tests, liver tissue myeloperoxidase as an index of neutrophil infiltration, and liver histology., Results: TBC-1269 treated groups experienced a significant increase in survival compared with controls. Best overall survival, 70%, was observed when TBC-1269 (Texas Biotechnology Corporation, Houston, TX) was administered 15 minutes before reperfusion (p < 0.05). This group also showed a marked decrease (p < 0.05) in liver enzyme levels at 6 hours after reperfusion. Neutrophil migration was also significantly ameliorated (81%), as reflected by decreased myeloperoxidase levels. We observed improved histologic damage scores in the treated group compared with controls (p < 0.05)., Conclusions: A small-molecule selectin inhibitor (TBC-1269) had a protective effect in livers subjected to 90 minutes of warm hepatic ischemia and 6 hours of reperfusion by decreasing neutrophil infiltration, migration and subsequent tissue damage. The best protective effect was achieved when the compound was administered 15 minutes before reperfusion. These findings offer a new therapeutic alternative for protection against ischemia and reperfusion injury.

Liver regeneration is significantly impaired in rats with both alpha-adrenergic hepatic denervation and hereditary vasopressin deficiency. This may implicate a direct role for these agonists in the process of compensatory hyperplasia. The mitogenic capacities of norepinephrine, vasopressin and hepatocyte growth factor (HGF), either alone or in combination were investigated by [3H]thymidine incorporation into hepatocyte cultures prepared from normal and regenerating rat livers. The results show that normal hepatocytes incorporate less [3H]thymidine in response to HGF than do regenerating hepatocytes. In addition, physiological concentrations of vasopressin cause a synergistic stimulation of [3H]thymidine uptake in rat liver cells in the presence of HGF.

The lymphomatous liver contains high levels of phosphoethanolamine. This compound is a precursor of the phospholipid, phosphatidylethanolamine. The kinetics of phosphatidylethanolamine synthesis has been studied by 13C NMR spectroscopy of extracts of the lymphomatous mouse liver following the administration of (13C2)ethanolamine. The concentrations of (13C2)ethanolamine, (13C2)phosphoethanolamine, and (13C2)phosphatidylethanolamine were fitted to kinetic models and the maximal activities of the enzymes in the synthetic pathway were estimated. Phosphatidylethanolamine synthesis in the normal liver appears to be limited by the supply of ethanolamine. In the lymphomatous liver, both ethanolamine kinase and PE:CTP cytidylyltransferase had lower activities than in the normal liver, and there was evidence for a higher activity of ethanolamine base-exchange enzyme.

The transverse relaxation time of water protons is shortened by the presence of iron. This shortening depends on the amount and the environment of iron in the sample. We have developed a method for measuring short transverse relaxation time noninvasively by magnetic resonance spectroscopy. To evaluate magnetic resonance spectroscopy as a means of assessing hepatic iron content in patients with transfusional iron overload, we compared the results obtained with this method with those obtained by other means of assessing total body iron content. The correlation between the liver biopsy iron concentration and 1/transverse relaxation time was highly significant (r = 0.95, p < 0.004, n = 6) for iron loads up to 3% dry weight. The correlation between serum ferritin and 1/transverse relaxation time was also significant, but the correlation coefficient was much lower (r = 0.67, p < 0.002, n = 20). The correlation between 24-hr urinary iron excretion and 1/transverse relaxation time was not significant, nor was that between AST and 1/transverse relaxation time. We conclude that magnetic resonance spectroscopic determination of the transverse relaxation time of hepatic water is an accurate method of measuring liver iron content, especially when the iron content is below 3%. Because it is a noninvasive method that is associated with negligible side effects, it could provide clinicians with an excellent means of assessing the effectiveness of the various therapeutic strategies used in the management of patients with iron overload.

Eighteen patients with acetaminophen poisoning were studied with 31P magnetic resonance spectroscopy to measure phosphorus-containing metabolites in their livers. The concentrations of all magnetic resonance-detectable metabolites fell in parallel with a decrease in the synthetic ability of the liver, indicated by the prothrombin time ratio (international normalized ratio). In particular, ATP fell to about 20% of its normal concentration in severely affected patients, as did the phosphodiester signal, which is thought to arise mainly from the endoplasmic reticulum in the liver. The correlation between ATP levels and international normalized ratio suggests that the international normalized ratio is a more accurate measure of the number of viable hepatocytes than are other biochemical tests.

Aspartyl (asparaginyl) beta-hydroxylase which specifically hydroxylates 1 Asp or Asn residue in certain epidermal growth factor-like domains of a number of proteins, has been previously purified to apparent homogeneity from detergent-solubilized bovine liver microsomes (Wang, Q., VanDusen, W. J., Petroski, C. J., Garsky, V. M., Stern, A. M., and Friedman, P. A. (1991) J. Biol. Chem. 266, 14004-14010). Three oligonucleotides, corresponding to three amino acid sequences of the purified hydroxylase, were used to screen bovine cDNA libraries. Several overlapping positive cDNA clones containing a full length open reading frame of 754 amino acids encoding a 85-kDa protein were isolated, and a cDNA, containing the full length open reading frame, was constructed from two of these clones. The resulting clone was then transcribed and translated in vitro to produce recombinant protein which possessed Asp beta-hydroxylase activity. These results constitute proof that the protein purified from bovine liver is an Asp beta-hydroxylase. Comparisons of deduced amino acid sequences of two other alpha-ketoglutarate-dependent dioxygenases, prolyl-4-hydroxylase and lysyl hydroxylase, with that of Asp beta-hydroxylase showed no significant homologies. Indeed, Asp beta-hydroxylase appears to be unique as no striking homology was found with known protein sequences. Furthermore, structural predictions derived from the deduced amino acid sequence are in accord with earlier Stokes' radius and sedimentation coefficient determinations of the enzyme, suggesting that the enzyme contains a relatively compact carboxyl-terminal catalytic domain and an extended amino terminus. This amino-terminal region has a potential transmembrane type II signal-anchor domain that could direct the catalytic domain into the lumen of the endoplasmic reticulum.

An oral load of 20 mg/kg galactose produces significant changes in the 31P magnetic resonance spectrum of the liver of a galactosemic patient. The peak at 5.2 ppm (which includes inorganic phosphate and galactose-1-phosphate) increased on two occasions to about twice its original size 60 min after galactose administration. An oral load of 10 mg/kg galactose given to a second patient produced no discernible changes at 30 min. We have also used an animal model of galactose intolerance, in which galactose metabolism in rats was blocked by the acute administration of ethanol. Studies in vivo and in vitro showed that the increase in the peak at 5.2 ppm was largely due to galactose-1-phosphate. We have shown in this preliminary study that small amounts of galactose can produce significant elevation of hepatic galactose-1-phosphate, which can be detected by 31P magnetic resonance spectroscopy.

31P-NMR spectra of regenerating rat liver in vivo show increases in resonance intensities in the phosphomonoester (PME) region and decreases in the phosphodiester (PDE) region as early as 12 h post partial hepatectomy, which return to normal by 8 days. The compounds primarily responsible for these changes have been identified in perchloric acid extracts as the phosphomonoester phosphoethanolamine and the phosphodiester glycerophosphoethanolamine (GPE), indicating altered phosphatidylethanolamine metabolism. A corresponding increase in diacylglycerol (DAG) levels during regeneration indicates a possible role for a phosphatidylethanolamine-specific phospholipase C in cellular proliferation. These results suggest that changes in phospholipid metabolites previously associated with neoplastic tissue can also be induced by normal tissue undergoing rapid cellular proliferation. The spectral changes observed in the regenerating rat liver are similar to changes seen in spectra from the livers of human patients in several disease states, indicating that 31P-NMR may allow non-invasive study of cell turnover in liver disease.

Hepatic infiltration by lymphoma can be difficult to detect by conventional methods. We have studied 22 patients in vivo 31P magnetic resonance spectroscopy of the liver and compared the results with the clinical staging and assessment of liver involvement by computed tomography (CT), ultrasound (US), and liver function tests (LFTs). We find that the phosphomonoester (PME) to ATP, and the PME to Pi ratios are the best indication of liver involvement as in all the patients with liver involvement apparent on CT or US, these ratios were elevated (greater than 2 s.d. above the control mean). Of the patients with deranged LFTs but normal CT or US, five out of nine showed increased PME/ATP and PME/Pi ratios, and in the patients with normal LFTs and normal CT or US, three out of eight patients had raised PME ratios. Extracts of lymphomatous lymph nodes contain high concentrations of phosphoethanolamine which suggests that this compound is responsible for the increase in the PME peak. Eleven patients were studied again after chemotherapy, and those with initially raised PME/ATP and PME/Pi ratios all showed a decrease in these ratios towards normal. The patients with initially normal ratios showed no changes.

The hyperuricemia responsible for the development of gouty arthritis results from a wide range of environmental factors and underlying genetically determined aberrations of metabolism. 31P magnetic resonance spectroscopy studies of children with hereditary fructose intolerance revealed a readily detectable rise in phosphomonoesters with a marked fall in inorganic phosphate in their liver in vivo and a rise in serum urate in response to very low doses of oral fructose. Parents and some family members heterozygous for this enzyme deficiency showed a similar pattern when given a substantially larger dose of fructose. Three of the nine heterozygotes thus identified also had clinical gout, suggesting the possibility of this defect being a fairly common cause of gout. In the present study this same noninvasive technology was used to identify the same spectral pattern in 2 of the 11 families studied with hereditary gout. In one family, the index patient's three brothers and his mother all showed the fructose-induced abnormality of metabolism, in agreement with the maternal inheritance of the gout in this family group. The test dose of fructose used produced a significantly larger increment in the concentration of serum urate in the patients showing the changes in 31P magnetic resonance spectra than in the other patients with familial gout or in nonaffected members, thus suggesting a simpler method for initial screening for the defect.

Measurement of arterial and liver tissue oxygen tension (PO2) in animals subjected to hemorrhagic shock demonstrates a significant (p less than 0.001) decrease in tissue PO2 while PaO2 remains essentially unchanged. In fact, marked increase in PaO2 fail to increase tissue PO2 to control levels, demonstrating that the act of increasing FIO2 and/or PaO2 is inadequate treatment of decreased tissue oxygenation in marginal or low flow states. Measurement of tissue PO2 in a variety of clinical situations seems warranted to allow alterations of therapy to improve flow when indicated by inadequate tissue oxygenation. The application of this simple but extremely useful technic should result in improved survival rates in critically ill patients.