Latency characteristics of an EPO and LLT mutant of pseudorabies virus.
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Animals↗
Biomedical subjects
A K Cheung
Publications and source records attributed to A K Cheung.
Pseudorabies virus EPO is functionally homologous to varicella-zoster virus ORF61 protein and herpes simplex virus type 1 ICPO.
Pseudorabies virus (PRV) early protein O (EPO) is the homolog of varicella-zoster virus (VZV) open reading frame 61 (ORF61) protein and herpes simplex virus type 1 (HSV-1) ICPO. A PRV EPO deletion mutant grows poorly in cell culture, suggesting that EPO plays a critical role in the viral replicative cycle. In this study, we have shown that the growth defect of an EPO deletion mutant was complemented in Vero cells expressing VZV ORF61 protein or HSV-1 ICPO. In transient expression assays PRV EPO, like VZV ORF61 protein and HSV-1 ICPO, transactivates a variety of promoters from PRV, VZV, HSV, and unrelated viruses. These data indicate that PRV EPO is functionally homologous to VZV ORF61 protein and HSV-1 ICPO.
Animals↗
Ultrafiltration and solute kinetics using low sodium peritoneal dialysate.
Low sodium peritoneal dialysate has been reported to enhance sodium loss and alleviate signs of fluid overload in continuous ambulatory peritoneal dialysis patients. To elucidate the mechanisms involved, we compared ultrafiltration and solute kinetics using low sodium dialysate (LNaD; 105 mEq/liter sodium, 2.5% glucose, 348 mOsm/liter), conventional dialysate with equal osmolality (CD1.5; 132 mEq/liter sodium, 1.5% glucose, 348 mOsm/liter) and conventional dialysate with equal glucose concentration (CD2.5; 132 mEq/liter sodium, 2.5% glucose, 403 mOsm/liter). A 2 liter, six hour exchange of each dialysate was performed on separate days in 10 chronic peritoneal dialysis patients. Transperitoneal solute diffusion was assessed by calculating the permeability-area product (PA) of the peritoneal membrane from the dependence of plasma and dialysate solute concentrations on tie. Net fluid removed using LNaD of 190 +/- 90 (SEM) ml was similar to that using CD2.5 (250 +/- 90 ml) but higher (P < 0.01) than that using CD1.5 (-200 +/- 60 ml). Sodium loss was higher using LNaD (72 +/- 11 mEq, P < 0.01) and CD2.5 (41 +/- 12 mEq, P < 0.05) than using CD1.5 (-18 +/- 8 mEq). Changes in plasma sodium concentration were small during each dwell and were not different among the study dialysates. PA values for urea (23.4 +/- 1.6 ml/min), creatinine (10.0 +/- 1.0 ml/min), and glucose (10.3 +/- 1.3 ml/min) were similar when determined in each dialysate. The PA value for sodium (7.6 +/- 1.5 ml/min) could only be accurately determined in LNaD. We conclude that: (1) net fluid removed is greater using LNaD than CD1.5 despite similar osmolalities because LNaD has a higher glucose concentration and glucose is a more effective osmotic solute than sodium; (2) sodium loss when using LNaD is enhanced by both diffusion and convection; and (3) sodium diffuses across the peritoneum slower than urea, creatinine and glucose. These data suggest that LNaD alleviates signs of fluid overload by increasing net fluid removal and enhancing sodium loss.
Adult↗
A deletion at the UL/IR junction reduces pseudorabies virus neurovirulence.
A recombinant pseudorabies virus (PRV), designated LLT beta delta 2, which contains a 3-kbp deletion spanning the junction of the unique long and internal repeat sequences was constructed. Compared with the parental strain and a virus rescued for the deleted sequences, LLT beta delta 2 exhibited similar replication characteristics in tissue culture. When inoculated intranasally in swine, LLT beta delta 2 was significantly reduced in virulence and did not produce neurological signs characteristic of PRV infection. LLT beta delta 2 replicated efficiently at the site of inoculation and in peripheral nervous tissues, but replication was restricted in the central nervous system. These results indicate the presence of a PRV neurovirulence determinant in the vicinity of the junction.
Animals↗
Determination of circulating blood volume by continuously monitoring hematocrit during hemodialysis.
Dialysis-induced hypovolemia occurs because the rate of extracorporeal ultrafiltration exceeds the rate of refilling of the blood compartment. The purpose of this study was to evaluate a method for calculating circulating blood volume (BV) during hemodialysis (HD) from changes in hematocrit (Hct) shortly (2 to 10 min) before and after ultrafiltration (UF) was abruptly stopped. Hct was monitored continuously during 93 HD treatment sessions in 16 patients by an optical technique and at selected times by centrifugation of blood samples. Total plasma protein and albumin concentrations were also measured at selected times. Continuously monitored Hct correlated with Hct determined by centrifugation (R = 0.89, N = 579). Relative changes in BV determined by continuously monitored Hct were not different from those determined by total plasma protein concentration (P = 0.05; N = 273). Calculated BV at the start of dialysis (4.1 +/- 1.3 L) was not different (P = 0.18, N = 12) from that derived anthropometrically from the patient's dry weight (4.6 +/- 0.8 L), and calculated BV when UF was stopped was 3.2 +/- 0.5 L (46 +/- 7 ml/kg body wt). These latter estimates of BV are consistent with those determined previously by dilution techniques in HD patients. It was concluded that (1) relative changes in BV assessed by continuously monitored Hct were unbiased and (2) BV can be determined noninvasively during HD by continuously monitoring Hct and temporarily stopping UF.
Blood Volume↗
Investigation of pseudorabies virus DNA and RNA in trigeminal ganglia and tonsil tissues of latently infected swine.
Pseudorabies virus (PRV) nucleic acids in the trigeminal ganglia and tonsils of swine latently infected with the virus were analyzed. By use of DNA-polymerase chain reaction (PCR), 14 of 14 trigeminal ganglia and 12 of 14 tonsils were positive for PRV genomes. By use of RNA-PCR, RNA containing the large latency transcript splice junction were detected in 4 of 4 trigeminal ganglia and 4 of 5 tonsils. In general, results of both PCR procedures indicated that the amounts of PRV DNA and RNA per microgram of cellular nucleic acids were higher in trigeminal ganglia than in tonsils. Identification of peripheral tissues that harbor latent PRV is an important asset for PRV research. The presence of large latency transcript in tonsil tissues, in the absence of virus replication, is a critical characteristic, which indicates that the tonsil is a site of PRV latency. For diagnostic purposes, animals need not be euthanatized to obtain their nervous tissue to determine latency; instead, tonsil biopsy specimens could be obtained from live animals for analysis. For pathogenesis, studies, multiple specimens obtained sequentially from the same animal would be available for examination for the duration of the experiment.
Animals↗
Identification of the pseudorabies virus UL4 and UL5 (helicase) genes.
The DNA sequence of the left end of the pseudorabies virus (PRV) BamHI-G fragment and a short adjacent sequence from the BamHI-C fragment (0.641-0.664 map units) were determined. Two open reading frames were identified and designated PRV UL4 and UL5 because they exhibit amino acid sequence homology to the herpes simplex virus type 1 UL4 and UL5 open reading frames. The PRV UL4 open reading frame codes for a 145-amino acid polypeptide of unknown function. The deduced PRV UL5 gene product is 833 amino acid residues and exhibits sequence characteristics of a helicase, including six sequence motifs which are conserved for a superfamily of helicases. PRV UL5 also contains a putative leucine zipper motif which is not present in other herpesvirus helicase sequences. PRV UL4 and UL5 are transcribed in the opposite orientation with respect to the UL1, UL2, UL3, and UL3.5 gene cluster located at the right end of BamHI-G.
Amino Acid Sequence↗
Soluble complement receptor type 1 inhibits complement activation induced by hemodialysis membranes in vitro.
A variety of bioincompatible events that occur during hemodialysis have been attributed to complement activation. However, cause-effect relationships have been based primarily on indirect evidence and the results of in vitro studies, because an acceptable method for inhibiting complement activation during clinical hemodialysis has been unavailable. Methods for inactivating complement in vitro are available, but the most commonly used of these techniques (heat inactivation and chelation of divalent cations) lack specificity. A recombinant, soluble form of human complement receptor type 1 (sCR1) has been developed recently and shown to inhibit complement activation in vivo. Here, we report studies aimed at determining the effects of sCR1 on dialysis-induced complement activation and neutrophil degranulation. In a concentration dependent fashion, sCR1 inhibited plasma complement activation by cuprophan membrane in vitro. Using a maximally inhibitory concentration (30 micrograms/ml), sCR1 blocked generation of C3a(desArg) by cuprophan, cellulose acetate, and polymethylmethacrylate membranes by 90%, 84%, and 84%, respectively. In contrast, elastase release (a measure of neutrophil degranulation) was inhibited by 70%, 70%, and 44%, respectively, suggesting that dialysis-induced neutrophil activation is mediated in part by noncomplement dependent mechanisms. Both heat- and EDTA-treatment of plasma abolished dialysis membrane-induced complement activation, but these treatments also affected noncomplement dependent components of the degranulation process. These observations show that, compared with other commonly used methods for inhibiting dialysis induced complement activation, sCR1 is more specific. An additional advantage of sCR1 is its potential for use in the clinical setting.
Biocompatible Materials↗
Quantitation of dialysis. The importance of membrane and middle molecules.
Despite the availability of calcitriol and recombinant erythropoietin to replace two major endocrine functions of the kidneys, mortality in chronic hemodialysis patients remains substantially higher than that in the general population. This suggests that most, if not all, patients are underdialyzed. While increasing small solute clearance by dialysis using urea kinetics as a guide improves clinical outcome, 'adequate' treatment using current techniques is only an arbitrary definition, since solute removal by dialysis remains far inferior to that of the normal native kidneys. In recent years, significant advances have been made in the structural and functional characterization of toxic uremic solutes that are in the higher molecular size range ('middle molecules'). Several lines of evidence suggest that, in addition to small solutes, removal of middle molecules is also advantageous. The potential beneficial effects of synthetic high flux hemodialysis membranes are probably attributed to both their solute clearance profiles as well as their biocompatibility characteristics.
Equipment Design↗
Detection of the large latency transcript of pseudorabies virus by RNA-PCR and its potential in diagnosis.
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Animals↗
Effect of thrombosis on complement activation and neutrophil degranulation during in vitro hemodialysis.
Coagulation proteins are known to affect the complement system and neutrophils. To assess the influence of thrombosis on complement and neutrophils during hemodialysis, whole human blood obtained from normal donors and anticoagulated with either 0.8 or 2.0 U/mL of heparin was recirculated for 4 h through cellulose acetate hollow fibers in vitro. Plasma fibrinopeptide A (FPA) levels were measured to assess thrombosis, whereas the activation of the complement system was evaluated at various stages by determining plasma C3a(desArg), C5a(desArg), and SC5b-9 concentrations. The activation of circulating neutrophils was assessed by quantitation of the extracellular release of the intragranular proteins elastase and lactoferrin. Thrombosis was observed when 0.8 U/mL of heparin was used, as indicated by a 36-fold increase in (FPA) levels at 240 min of recirculation and occasional grossly visible blood clots. In contrast, no increase in FPA or clot formation was observed with 2.0 U/mL of heparin. The higher dose of heparin was also associated with a lesser increase in plasma C3a(desArg) and C5a(desArg) concentrations, an observation that is compatible with either an inhibitory effect of heparin or a stimulatory effect of coagulation on the complement system. Plasma elastase or lactoferrin concentrations increased during hemodialysis but were not dependent on heparin dosage at any time. It was concluded that anticoagulation with higher heparin concentration inhibits complement activation in the hemodialysis circuit, but it does not affect neutrophil degranulation.
Anticoagulants↗
Characterization of a pseudorabies virus that is defective in the early protein 0 and latency genes.
A recombinant pseudorabies virus that is defective in the early protein 0 (EP0) and large latency transcript (LLT) genes was constructed. A portion of the EP0 and LLT genes was replaced by the lacZ gene of Escherichia coli that had been placed under the control of the pseudorabies virus gX gene promoter. This recombinant virus produces smaller size plaques and yields less virus than does the parent virus on Madin-Darby bovine kidney cells. Although the time course of virus replication and release into the medium of the recombinant and parent viruses are similar, the recombinant virus did not reach as high a titer. Similar to the parent virus, the recombinant virus replicates in the upper segment of the respiratory tract of swine, but the amount of progeny viruses produced is significantly reduced. The data indicated that the EP0 and LLT genes of pseudorabies virus are nonessential for replication. Virus that lacks these 2 genes has impaired growth in tissue culture and is attenuated for swine, compared with the parent virus.
Animals↗
Criteria and standardization for biocompatibility.
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Equipment and Supplies↗
Complement activation as index of haemodialysis membrane biocompatibility: the choice of methods and assays.
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Complement Activation↗
Atherogenic lipids and lipoproteins in hemodialysis patients.
Dyslipidemia may contribute to atherosclerosis in hemodialysis patients. While hypertriglyceridemia is relatively common in this population, hypercholesterolemia is not. Since abnormalities in various plasma cholesterol fractions and lipoproteins have been associated with an increased incidence of cardiovascular disease in the nonuremic population, we examined these abnormalities to determine whether they occur in patients with chronic renal failure. Twenty-four patients on maintenance hemodialysis were studied. We found that, despite relatively low plasma total cholesterol levels, a substantial number of patients had low high-density lipoprotein cholesterol, low apolipoprotein AI, and high apolipoprotein B levels. Furthermore, approximately 40% and 30%, respectively, of the patients had elevated plasma levels of lipoprotein(a) and remnants of chylomicron and very low-density lipoprotein. Lipoprotein(a) levels could not be predicted from any of the variables that were studied. The abnormal plasma levels of these potentially atherogenic lipids and lipoproteins suggest that they may contribute to the high incidence of cardiovascular diseases in the hemodialysis population.
Adult↗
Beta2 integrins are required for neutrophil degranulation induced by hemodialysis membranes.
An untoward consequence of hemodialysis is degranulation of peripheral blood neutrophils. The mechanisms that mediate this process, however, have not been conclusively identified. In the present study, the participation of complement activation and beta 2 integrins (CD11/CD18) in hemodialysis-induced neutrophil degranulation was investigated. Incubation of neutrophils with cuprophan membrane stimulated the release of very small amounts of the cytoplasmic granular protein, elastase. This release was markedly enhanced by the addition of plasma. Inactivation of complement reduced degranulation by approximately 60%, but the contribution of anaphylatoxins C3a and C5a to the degranulation process was modest. Treatment of plasma with EDTA completely abolished neutrophil degranulation in the presence of cuprophan membrane. Further, when incubated with plasma and cuprophan membrane, neutrophils that are deficient in beta 2 integrins released only 10% as much elastase as normal cells. Together, these observations strongly suggest that one or more members of the beta 2 integrin family of receptors is essential for cuprophan membrane-induced neutrophil degranulation and that both complement-related and noncomplement-related factors serve as receptor ligands.
Cations, Divalent↗
Atherosclerosis in chronic renal failure.
Atherosclerotic cardiovascular disease is a significant cause of morbidity and mortality in patients with chronic renal failure. It is unclear, however, if atherosclerosis in fact occurs at a higher incidence compared with the nonuremic population matched for age, hypertension, and diabetes mellitus or if it occurs at an accelerated rate following the onset of end-stage renal disease. The extent of true atherosclerotic lesions, versus clinically diagnosed "atherosclerosis," in patients with chronic renal failure is equally unclear. Potentially, the uremic state per se, the dialysis treatment, and factors unrelated to renal failure may participate in atherogenesis. The relative contribution of each of these factors is unknown. In this review, we discuss the pathology of "atherosclerotic" lesions in patients with chronic renal failure and the putative factors involved in atherogenesis in this population and describe the results of available studies examining the issue of accelerated atherosclerosis in uremia.
Arteriosclerosis↗
A 3' coterminal gene cluster in pseudorabies virus contains herpes simplex virus UL1, UL2, and UL3 gene homologs and a unique UL3.5 open reading frame.
We have determined the nucleotide sequence and transcription pattern of a group of open reading frames of pseudorabies virus (PRV), which are located at the right end of the BamHI-G fragment from 0.664 to 0.695 map units in the unique long region of the genome. Nucleotide sequence analysis revealed four open reading frames. The first three correspond in genome location to the herpes simplex virus type 1 (HSV-1) open reading frames UL1, which codes for glycoprotein L (gL); UL2, which codes for a uracil-DNA glycosylase; and UL3, which codes for a polypeptide of unknown function. The fourth open reading frame, UL3.5, is not present in the HSV-1 genome. Northern (RNA) blot analysis with oligonucleotide and cDNA probes revealed four abundant mRNA species of 3.3, 2.7, 1.8, and 0.9 kb, which are likely to yield polypeptides encoded by the UL1, -2, -3, and -3.5 open reading frames, respectively. All four transcripts were of the early-late kinetic class, transcribed in the same direction, and 3' coterminal. The UL2 and UL3 genes of PRV and HSV-1 have significant amino acid sequence homology, while the UL1 genes are positional homologs and the UL3.5 gene is unique to PRV.
Amino Acid Sequence↗