bluuebunny/crossref_metadata_2025 huggingface dataset:
Spontaneous eyelid movements during human sleep: a possible ponto‐geniculo‐occipital analogue?
Authors: Conduit Russell , Crewther Sheila Gillard , Bruck Dorothy , Coleman Grahame
Published: March 2003

The aim of the present study was to investigate whether eye lid movements (ELMs) were temporally related to the activity of other skeletal musculature and to proposed analogues of ponto‐geniculo‐occipital (PGO) waves during human sleep. Electroencephalogram (EEG), laryngeal‐masseter electromyogram (EMG), electrooculgram (EOG), peri‐orbital integrated potentials (PIPs), middle ear muscle activity (MEMA), ankle flexion (AF) and respiration (RESP) were monitored with ELMs during one night's sleep. Results showed that ELMs always occurred during full arousal and movement time. The ELMs that occurred during sleep were most prominent during rapid eye movement (REM) sleep, occurred at higher frequency just before REM, and were observed synchronously with other PGO analogues, supporting the notion that ELMs may be an indicator of PGO activity in humans. Of the ELMs observed during sleep, 16% showed changes in EOG, PIP, MEMA, AF and RESP simultaneously, suggesting generalized muscle activation. This coactivation of muscle activity suggested that the relationship between the muscular measures and PGO activity might be an indirect one, possibly mediated by alerting mechanisms, previously shown to be related to PGO waves in animals. Such an interpretation is consistent with the use of ELM as a widely accepted measure of the eye‐blink startle response in awake human subjects.
Comparison of plasma pharmacokinetics and bioavailability of ceftiofur sodium and ceftiofur hydrochloride in pigs after a single intramuscular injection
Authors: Brown  , Hanson  , Mignot  , Millérioux  , Hamlow  , Hubbard  , Callahan  , Kausche
Published: March 2003

Ceftiofur sodium, a broad‐spectrum cephalosporin, is active against gram‐positive and gram‐negative pathogens of veterinary importance. Two studies were designed to compare the intramuscular bioavailability of the current sodium salt and the new hydrochloride salt in pigs at doses of either 3 mg or 5 mg ceftiofur equivalents (CE)/kg body weight.

 Twenty‐six healthy young pigs were selected for these two‐period, two‐treatment crossover studies, 12 for the 3 mg/kg study and 14 for the 5 mg/kg study. Each animal received one intramuscular (i.m.) injection of ceftiofur sodium and one i.m. injection of ceftiofur hydrochloride with a 14‐day washout period between the two treatments. Blood samples were collected serially for up to 96 h postinjection. Plasma samples were then analysed using a validated assay that measures ceftiofur and all desfuroylceftiofur‐related metabolites by high‐performance liquid chromatography.

 In the 3 mg/kg dosage study, average maximum plasma concentration (Cmax) after administration of ceftiofur sodium was 15.8 ± 3.40 μg/mL at 0.4–4 h after injection. After administration of ceftiofur hydrochloride, the Cmax was 11.8 ± 1.67 μg/mL at 1–4 h after injection. Concentrations of ceftiofur and metabolites 72 h after the injection were 0.392 ± 0.162 μg/mL for ceftiofur hydrochloride and 0.270 ± 0.118 μg/mL for ceftiofur sodium. The mean area under the curve (AUC), from time 0 to the limit of quantitation (AUC0‐LOQ) after ceftiofur hydrochloride administration, was 216 ± 28.0 μg·h/mL, compared to 169 ± 45.4 μg·/mL after ceftiofur sodium administration. The calculated time during which plasma concentrations remained above 0.02 μg/mL (t>0.2) was 85.3 ± 10.6 h for ceftiofur sodium and 77.2 ± 10.7 h for ceftiofur hydrochloride.

 In the 5 mg/kg dosage study, Cmax after administration of ceftiofur sodium was 28.3 ± 4.45 μg/mL at 0.33–2 h after injection. After administration of ceftiofur hydrochloride, the Cmax was 29.7 ± 6.72 μg/mL at 0.66–2 h after injection. Concentrations of ceftiofur and metabolites 96 h after the injection were 0.274 ± 0.0550 μg/mL for ceftiofur hydrochloride and 0.224 ± 0.0350 μg/mL for ceftiofur sodium. The mean AUC0‐LOQ after ceftiofur hydrochloride administration was 382 ± 89.8 μg.h/mL compared to 302 ± 54.4 μg.h/mL after ceftiofur sodium administration. The t>0.2 was 78.9 ± 9.65 h for ceftiofur sodium and 94.2 ± 8.64 h for ceftiofur hydrochloride.

 Based on the similarity of the pharmacokinetic parameters of the sodium and hydrochloride formulations of ceftiofur, similar therapeutic efficacy can be inferred for the two products.
Absorption kinetics and bioavailability of cephalexin in the dog after oral and intramuscular administration
Authors: Carli  , Anfossi  , Villa  , Castellani  , Mengozzi  , Montesissa
Published: March 2003

The pharmacokinetics of cephalexin, a first generation cephalosporin, were investigated in dogs using two formulations marketed for humans, but also often employed by practitioners for pet therapy. Cephalexin was administered to five dogs intravenously and intramuscularly as a sodium salt and by the oral route as a monohydrate. The dosage was always 20 mg/kg of active ingredient. A microbiological assay withSarcina luteaas the test organism was adopted to measure cephalexin concentrations in serum.

The mean residence time (MRT) median values after intravenous (i.v.), intramuscular (i.m.) and oral administration (p.o.) were 86 min, 200 min, and 279 min, respectively.

After i.m. and oral dosing the peak serum concentrations (24.2 ± 1.8 μg/mL and 20.3 ± 1.7 μg/mL, respectively) were attained at 90 min in all dogs and bioavailabilities were 63 ± 10% and 57 ± 5%, respectively.

The time course of the cephalexin serum concentrations after oral administration was best described by a model incorporating saturable absorption kinetics of the Michaelis‐Menten type; thus in the gastrointestinal tract of dogs a carrier mediated transport for cephalexin similar to that reported in humans, may exist.

The predicted average serum concentrations of cephalexin after repeated i.m and oral administration indicated that, in order to maintain the therapeutic concentrations, the 20 mg/kg b.w. dosage should be administered every 6–8 h.
Correlation between serum concentrations following continuous intravenous infusion of dexmedetomidine or medetomidine in cats and their sedative and analgesic effects
Authors: Ansah  , Raekallio  , Vainio
Published: March 2003

Dexmedetomidine (DEX) may have some therapeutic advantages over the racemate medetomidine (MED). Here we have examined how serum concentrations of DEX correlate with some of its anaesthetic effects. Cats (n = 6) were administered with a continuous stepwise intravenous (i.v.) infusion of DEX or MED on different occasions in a cross‐over design. Maintenance infusion rates (mg/kg/min) used were: DEX = 0.25 (MED = 0.50); DEX = 1 (MED = 2) and DEX = 4 (MED = 8) for infusion steps 1, 2 and 3, respectively. Each maintenance infusion lasted at least 50 min and was preceded with a loading dose. There was no significant difference between serum DEX and 0.5 serum MED concentrations at any dose level nor was there a significant difference between serum DEX and the (entire) serum MED concentrations. There was no significant difference between DEX and MED for sedation, analgesia, muscular relaxation and heart and respiratory rates. For both DEX and MED, serum drug concentration and analgesia were dose‐dependent and sedation increased until the end of infusion step 2 (dose level 2) and decreased at the end of step 3 (dose level 3). Muscular relaxation was not dose‐dependent. We conclude that increasing the blood concentration of DEX or MED beyond a certain level decreases the level of sedation instead of increasing it even though analgesia increases. The rate at which DEX and MED are metabolized in cats may not be the same.
The binding and distribution of albendazole and its principal metabolites in Giardia duodenalis
Authors: Oxberry M. E. , Reynoldson J. A. , Thompson R. C. A.
Published: March 2003

Trophozoites of the protozoan parasite Giardia duodenalis were exposed to various albendazole concentrations for 4 h, washed, fixed and incubated with antibodies raised against albendazole and its two major metabolites albendazole sulphoxide and albendazole sulphone. Tubulin antibodies were also used. A peroxidase‐ or FITC‐conjugated secondary antibody was used to detect the primary antibody with transmission electron microscopy or confocal laser scanning microscopy, respectively. Albendazole, a benzimidazole compound, was detected in the mid‐dorsal region of trophozoites, albendazole sulphoxide in the posterior‐dorsal region and albendazole sulphone in clusters above the median bodies. Tubulin was recognised in the ventral disk. This is the first indication that G. duodenalis may be capable of metabolising albendazole and the potential path of the metabolised drug traced within the trophozoite. Fluorescence measurements revealed that albendazole sulphoxide binding decreased and albendazole sulphone binding increased with exposure of the trophozoites to increasing albendazole concentration. This indicates that if albendazole was being metabolised by trophozoites, it occurred to a greater extent following exposure to higher albendazole concentrations.
Pharmacokinetics of gentamicin in mares in late pregnancy and early lactation
Authors: SANTSCHI E. M. , PAPICH M. G.
Published: March 2003

The disposition of drugs may differ between pregnant and nonpregnant animals, necessitating a change in dosage. We hypothesized that volume of distribution or clearance may be different for aminoglycoside antibiotics in pregnant mares vs. nonpregnant lactating mares. To examine this hypothesis, we administered gentamicin sulfate to seven Thoroughbred and Quarterhorse mares on two occasions, followed by plasma drug gentamicin assay and pharmacokinetic analysis. The first dose was administered 1–4 weeks before parturition (mean weight 578 kg) and the second dose was administered in the period 1–4 weeks after parturition (mean weight 518 kg). The dose administered at each time was approximately 6.6 mg/kg, intravenously (i.v.). Plasma gentamicin concentrations were determined using fluorescence polarization immunoassay and pharmacokinetic analysis was performed using a two‐compartment open model. The plasma concentration vs. time profiles and total area‐under‐the‐curve were almost identical for mares at late gestation vs. early lactation. Mean volume of distribution at steady‐state was 0.15 (±0.02) and 0.16 (±0.03) L/kg, systemic clearance was 1.06 (±0.17) and 1.11 (±0.17) mL/kg/min, and mean (harmonic) elimination half‐life was 2.2 and 2.1 h, for pregnant and nonpregnant mares, respectively. We concluded that there were no differences in drug distribution and clearance between pregnant and nonpregnant lactating mares. Gentamicin was also assayed in plasma of newborn foals after an injection of 6.6 mg/kg to three of the mares within 60 min of parturition. Gentamicin was undetectable in plasma samples from these foals and, therefore, apparently does not cross the placenta of mares at term.
Influence of verapamil on the efflux and metabolism of 14C moxidectin in cultured rat hepatocytes
Authors: Dupuy J. , Larrieu G. , Sutra J. F. , Eeckhoutte C. , Alvinerie M.
Published: March 2003

Moxidectin (MOX) is an antiparasitic drug widely used in cattle, sheep and companion animals. As a result of the implication of cytochrome P450 3 A in the metabolism of MOX and the role of competitor substrates of P‐glycoprotein (Pgp) in modification of the bioavailability of endectocides, we studied the influence of verapamil (a multidrug‐resistance reversing agent) on the metabolism of 14C moxidectin in cultured rat hepatocytes over 72 h. The metabolism of MOX remained low: 10.79 ± 1.99% of the total 14C moxidectin for the main detected metabolite in verapamil‐treated cells and 7.17 ± 0.74% for the control cells after 24 h. The main detected metabolite in rat hepatocytes was the same as that detected in rat hepatic microsomes (the C29 monohydroxymethyl metabolite). Verapamil increased the quantity of MOX in the cells after 24, 48 and 72 h. Examination of the Area Under the concentration time Curve (AUC) of the main detected metabolite revealed a significant increase in the exposure of cells to MOX after verapamil treatment throughout the experiment. It is hypothesized that verapamil interfered with MOX as a substrate for Pgp during the initial incubation period. After this initial interaction, verapamil metabolites were able to interfere with Pgp. This experiment demonstrated the implication of Pgp in the transport of MOX and allowed prediction of the drug–drug interactions which might modify the bioavailability of endectocides.
Disposition kinetics of lactoferrin in milk after intramammary administration
Authors: KUTILA T. , PYÖRÄLÄ S. , KAARTINEN L. , VAHTOLA K. , MYLLYKOSKI L. , SALONIEMI H.
Published: March 2003

Disposition kinetics of lactoferrin (Lf) purified from cheese whey was studied in the milk of Finnish Ayrshire cows after intramammary administration of 1 g of Lf into one udder quarter. Intramammary administration of 1 g of Lf increased Lf concentration in milk for several hours. Mean elimination half‐life of Lf was 2.2 h and a mean maximum concentration of 6.3 g/L was reached between 1 and 4 h. After 8 h of administration, Lf concentrations in milk decreased to almost the same level as before the infusion. Forty‐eight hours postinfusion, the mean Lf concentration was again higher than in the milk samples taken before the infusion of Lf, being on average 1.5 g/L. Lactoferrin caused some local tissue irritation in the udder quarter. Severity of the irritation reactions varied between cows. The udder quarters of primiparous cows reacted faster than those of multiparous cows, but irritation reactions decreased more rapidly in the older cows than in primiparous cows. The cows had no general signs such as fever or anorexia. The somatic cell count returned to baseline level 4 days after the administration.
The effects of body composition on the pharmacokinetics of subcutaneously injected ivermectin and moxidectin in pigs
Authors: CRAVEN J. , BJØRN H. , HENNESSY D. R. , FRIIS C.
Published: March 2003

Craven, J., Bjørn, H., Hennessy, D. R., Friis, C. The effects of body composition on the pharmacokinetics of subcutaneously injected ivermectin and moxidectin in pigs. J. vet Pharmacol. Therap.25, 227–232.

Macrocyclic lactones are characterized by their long persistence in animals because of their extensive distribution into fat. This study examined the influence of body condition on the disposition of ivermectin (IVM) and moxidectin (MXD) in blood and fat following subcutaneous (s.c.) drug administration. `Fat' and `thin' lines of pigs were established using two different diets. All animals were then injected with either MXD or IVM at 300 μg/kg and blood samples were taken at regular intervals until slaughter. Two IVM‐treated animals from each diet group were slaughtered at either 3 days or 3 weeks posttreatment. Two MXD‐treated animals from each diet group were slaughtered at 3 days, 3, 6 or 9 weeks after treatment. Samples of backfat were taken from all animals at slaughter. Fluorescence HPLC was used to determine the concentrations of MXD or IVM in the plasma and fat samples. The plasma IVM concentration peaked more rapidly in the thin IVM treated pigs compared with the fat pigs. The concentration of IVM in backfat was significantly lower in the thin animals slaughtered 3 weeks after treatment. The MXD plasma concentration peaked within the first hour in both the thin and fat groups, but from 12 h posttreatment there was a higher MXD concentration in the plasma of the fat pigs resulting in MXD being detectable in these pigs for 28 days compared with only 17 days in the thin pigs. Despite this difference in plasma persistence no differences were seen in the MXD concentration of backfat between fat and thin animals. Body condition influenced the kinetic disposition of IVM and MXD following s.c. drug administration with both drugs being less persistent in thin compared with fat animals.
Efficacy of trimethoprim‐sulfadoxine against Escherichia coli in a tissue cage model in calves
Authors: Greko C. , Bengtsson B. , Franklin A. , Jacobsson S.‐O. , Wiese B. , Luthman J.
Published: March 2003

Tissue cages implanted subcutaneously in calves were infected with Escherichia coli. Twenty‐four hours later, the calves were treated either with single doses of 2.5 + 12.5 or 5 + 25 mg/kg trimethoprim (TMP) + sulfadoxine (SDX) or with five doses of 7.5 + 37.5 mg/kg TMP + SDX at 12‐h intervals. In addition, one cage in each of three calves in the highest dose group was infected 3 h after initiation of treatment. Untreated calves were kept as controls. Concentrations of TMP and SDX in plasma and tissue cage fluid (TCF) and counts of viable bacteria in TCF were determined. In the highest dose group, concentrations of TMP in TCF remained above the minimum inhibitory concentration of the test strain for 94–101 h and peak to minimum inhibitory concentration (MIC) ratio was close to 10. In spite of this, an effect of treatment was noted only in cages infected after initiation of treatment. In vitro studies and analysis of thymidine content in serum and TCF from calves suggest that levels of thymidine in TCF are high enough to antagonize the antibacterial effect of TMP. The results indicate that soft tissue infections in secluded infection sites of calves are refractory to treatment with TMP + SDX.
Structure and function of the encapsidation signal of hepadnaviridae
Authors: Kramvis A. , Kew M. C.
Published: March 2003

The hepatitis B virus (HBV) and other members of the hepadnaviridae replicate by reverse transcription of an RNA intermediate, pregenomic RNA (pgRNA). pgRNA is also translated into core protein and polymerase (reverse transcriptase) protein. Before being reverse transcribed, pgRNA is sequestrated from the cytoplasm by being packaged, together with polymerase, into subviral particles composed of core protein. For pgRNA to be encapsidated, its 5′ end is folded into a stem–loop structure, known as the encapsidation signal or epsilon (ɛ). This stable bipartite stem–loop structure contains a bulge and an apical loop. Besides encapsidation, ɛ is involved in the activation of polymerase, in template restriction and in the initiation of DNA synthesis by reverse transcription. HBV DNA encoding ɛ forms part of the template that is translated into the precore/core fusion protein that is in turn post‐translationally modified to produce hepatitis B e antigen (HBeAg). The DNA encoding ɛ may be recombinogenic. Mutations within ɛ can affect its function and sequence conservation within ɛ in natural isolates is therefore high. ɛ could provide a practical target for antiviral therapy.
Persistence of hepatitis C virus in a human megakaryoblastic leukaemia cell line
Authors: Li X. , Jeffers L. J. , Garon C. , Fischer E. R. , Scheffel J. , Moore B. , Reddy K. R. , DeMedina M. , Schiff E. R.
Published: March 2003

Thrombocytopenia is a frequent clinical finding in patients with hepatitis C virus (HCV) infection. Platelets from patients with HCV infection have been identified as carriers of HCV RNA in our previous studies. The present study was designed to further investigate the possibility of HCV replication in megakaryoblasts from which platelets are eventually released. A megakaryoblastic cell line (MEG‐01), established from a chronic myelogenous leukaemia patient 13years ago, was used for this study. The MEG‐01 cells were inoculated with fresh serum from a patient with HCV infection and renamed MEG‐01‐I cells. Surprisingly, both MEG‐01 and MEG‐01‐I were positive by HCV reverse transcription–polymerase chain reaction (RT–PCR) for the existence of HCV RNA and minus‐strand HCV RNA, regardless of inoculation. This was further confirmed by in situ RT–PCR. The HCV antigens, such as core, envelope, and non‐structural (NS)3 and NS4, were also present in both cell lines, as identified by Western blotting and indirect immunofluorescence staining. In addition, virus‐like particles were observed by electron microscopy in the MEG‐01 cell line as well as in the MEG‐01‐I cell line. These findings indicate that the megakaryoblasts are vulnerable to HCV infection and that replication of HCV can occur in these cells. This may help us to better understand the pathogenesis of thrombocytopenia in patients with HCV infection. The MEG‐01 cell line, which may have been continuously shedding HCV for years, should be a useful model for experimental research into HCV.
Detection of antibodies against hepatitis C virus in saliva: a marker of viral replication
Authors: Cameron S. O. , Wilson K. S. , Good T. , McMenamin J. , McCarron B. , Pithie A. , Fox R.
Published: March 2003

Hepatitis C surveillance has been restricted owing to the lack of a sensitive antibody assay for saliva. The aim of our study was to develop and evaluate a screening assay for hepatitis C antibody in saliva specimens. Serum/saliva pairs were collected from 115 hepatitis C‐positive patients. A modified hepatitis C antibody assay for saliva was developed and linked to testing carried out in the diagnostic laboratory. Correlation between the presence of antibody in serum and in saliva was poor (100%vs 85%). However, of 98 patients who were saliva antibody positive, 96 (98%) were also serum hepatitis C RNA positive and two (2%) were serum hepatitis C RNA negative. Hence, the correlation between a positive salivary antibody test and the serum hepatitis C RNA status of intravenous drug users suggests that this test could be used as a surrogate marker for hepatitis C viraemia in epidemiological studies.
The NS3/4A proteinase of the hepatitis C virus: unravelling structure and function of an unusual enzyme and a prime target for antiviral therapy
Authors: Bartenschlager R.
Published: March 2003

The hepatitis C virus (HCV) is a major causative agent of transfusion‐acquired and sporadic non‐A, non‐B hepatitis worldwide. Infections most often persist and lead, in ≈ 50% of all patients, to chronic liver disease. As is characteristic for a member of the familyFlaviviridae, HCV has a plus‐strand RNA genome encoding a polyprotein, which is cleaved co‐ and post‐translationally into at least 10 different products. These cleavages are mediated, among others, by a virally encoded chymotrypsin‐like serine proteinase located in the N‐terminal domain of non‐structural protein 3 (NS3). Activity of this enzyme requires NS4A, a 54‐residue polyprotein cleavage product, to form a stable complex with the NS3 domain. This review will describe the biochemical properties of the NS3/4A proteinase, its X‐ray crystal structure and current attempts towards development of efficient inhibitors.
Hepatitis C virus mixed genotype infection in patients on haemodialysis
Authors: Qian  , Natov  , Pereira  , Lau
Published: March 2003

Hepatitis C virus (HCV) has been classified into different genotypes/subtypes with demonstrated clinical implications. Whether there is biological difference between genotypes is unknown. We determined HCV genotype in 120 anti‐HCV‐positive patients with end‐stage renal disease and on haemodialysis, by both serological assay (which showed evidence of previous exposure) and by two molecular assays: restriction fragment length polymorphism (RFLP) and line‐probe reverse hybridization (LiPA). In mixing experiments, RFLP and LiPA was able to detect the minor HCV genotypes (if present) in 5–30% and 1–2% of the viral population, respectively. Of the 120 patients studied, genotype‐specific antibodies were detected in 50 (42%), and eight patients had reactivities to peptides derived from multiple genotypes (genotypes 1 and 2 and/or 3). Only genotype 1 infection was found by RFLP/LiPA in these eight patients with reactivities to multiple HCV genotypes. One‐hundred and five of the 120 (88%) patients were positive for HCV RNA by reverse transcription–polymerase chain reaction (RT–PCR) analysis and 14 were found to have mixed genotype infection. Follow‐up serum samples (4–21 months later) were available in five patients (genotype 1a with another genotype/subtype). All five patients had a reduced number of HCV genotypes detected during follow‐up; four of the five patients still had detectable genotype 1a, and one patient lost genotype 1a and was positive for genotype 2b only. These data showed that HCV mixed infection can be reliably detected by molecular methods and, in patients with end‐stage renal disease and mixed genotype infection, there is a trend that during follow‐up, HCV genotype 1 may prevail, or ‘take over’ the genotype 2 and 3 infection.
RNA‐dependent RNA polymerase activity encoded by GB virus‐B non‐structural protein 5B
Authors: Zhong  , Ingravallo  , Wright‐Minogue  , Uss  , Skelton  , Ferrari  , Lau  , Hong
Published: March 2003

Phylogenetic analysis and polyprotein organization comparison have shown that GB virus‐B (GBV‐B) is closely related to hepatitis C virus (HCV). In this study, the coding region for GBV‐B non‐structural protein 5B (NS5B) was isolated by reverse transcription–polymerase chain reaction (RT–PCR) from pooled serum of GBV‐B‐infected tamarins. Expression of soluble GBV‐B NS5B protein in Escherichia coli was achieved by removal of a 19‐amino acid hydrophobic domain at the C‐terminus of the protein. The truncated GBV‐B NS5B (NS5BΔCT19) was purified to homogeneity and shown to possess an RNA‐dependent RNA polymerase (RdRp) activity in both gel‐based and scintillation proximity assays. NS5BΔCT19 required the divalent cation Mn2+ for enzymatic activity, at an optimal concentration of 15 m
M. Interestingly, Mg2+, at concentrations up to 20 m
M, did not support the GBV‐B NS5B activity. This differs from HCV NS5B where both Mn2+ and Mg2+ can support RdRp activity. Zn2+ was found to inhibit the activity of GBV‐B NS5B, with a 50% inhibitory concentration (IC50) of 5–10 μM. Higher concentrations of monovalent salts (NaCl or KCl > 100 m
M) and glycerol (> 3%) were also inhibitory. NS5BΔCT19 was able to bind to RNA homopolymers, but utilized most efficiently poly(C), the one with the lowest binding affinity for RNA synthesis. Mutational analysis of GBV‐B NS5B demonstrated the importance of several conserved sequence motifs for enzymatic activity. Based on sequence homology (≈ 37% identity and 52% similarity) between GBV‐B and HCV NS5B proteins, the active GBV‐B RdRp provides a good surrogate assay system for HCV polymerase studies.
Expression of interferon‐α subtypes in peripheral mononuclear cells from patients with chronic hepatitis C: a role for interferon‐α5
Authors: Larrea E. , Alberdi A. , Castelruiz Y. , Boya P. , Civeira M.‐P. , Prieto J.
Published: March 2003

Interferon (IFN)‐α is a family of antiviral proteins encoded by different genes. The biological significance of the existence of various IFN‐α subtypes is not clear. We have investigated the interferon system in chronic hepatitis C virus (HCV) infection, a disease that responds to interferon‐α2 therapy in only a limited proportion of cases. We analysed the expression of interferon regulatory factor (IRF)‐1, IRF‐2, and IFN‐α subtypes in nonstimulated and Sendai virus‐stimulated peripheral blood mononuclear cells (PBMC) from HCV infected patients and healthy controls. We observed that the IRF‐1 mRNA and IRF‐1/IRF‐2 ratios were increased in PBMC from hepatitis C patients with respect to normal subjects. Sendai virus stimulation of PBMC led to a significant increase in the levels of IRF‐1, IRF‐2 and IFN‐α mRNAs and in the production of IFN‐α protein with respect to basal values in healthy controls as well as in patients with HCV infection. In addition, we found that while natural HCV infection induced increased IFN‐α5 expression in PBMC, in vitro infection of these cells with Sendai virus caused a raise in the expression of IFN‐α8 in both patients and normal controls. In summary, our results indicate that virus‐induced activation of the IFN system in human PBMC is associated with selective expression of individual IFN‐α subtypes, IFN‐α5 being the specific subtype induced in PBMC from patients with chronic HCV infection.
Zinc supplementation enhances the response to interferon therapy in patients with chronic hepatitis C
Authors: Takagi H. , Nagamine T. , Abe T. , Takayama H. , Sato K. , Otsuka T. , Kakizaki S. , Hashimoto Y. , Matsumoto T. , Kojima A. , Takezawa J. , Suzuki K. , Sato S. , Mori M.
Published: March 2003

We evaluated the synergistic effect of zinc supplementation on the response to interferon (IFN) therapy in patients with intractable chronic hepatitis C in a pilot study using natural IFN‐α with or without zinc. No clinical differences were observed between patients treated with IFN alone (n=40) and IFN with polaprezinc (IFN + Zn, n=35). All patients were positive for HCV genotype Ib and had more than 105 copies of the virus/mL serum. Ten million units of natural IFN‐α was administered daily for 4 weeks followed by the same dose every other day for 20 weeks. In the IFN + Zn group, patients received an additional dose of 150 mg/day polaprezinc orally throughout the 24‐week IFN course. No additional side‐effects of polaprezinc were noted but four out of 40 IFN alone treatment and three out of 35 IFN + Zn group withdrew because of side‐effects. Complete response (CR) was defined as negative HCV RNA in the serum on PCR and normal aminotransferase level 6 months after therapy. Incomplete response (IR) was normal liver enzyme and positive serum HCV RNA. Both of them were evaluated at the 6 months after the completion of the treatment. Patients with higher levels of serum HCV (more than 5 × 105 copies/mL) had little response in both treatment groups. Patients with moderate amount of HCV (105 to 4.99 × 105/mL) showed high response rates in combination group (CR: 11/27, 40.7%; CR + IR 15/27, 64.3%), better than IFN alone (CR: 2/15, 18.2%; CR + IR: 2/15, 18.2%). Serum zinc levels were higher in patients with IFN + Zn group than in the IFN group. Our results indicate that zinc supplementation enhances the response to interferon therapy in patients with intractable chronic hepatitis C.
Prediction of progressive liver fibrosis in hepatitis C infection by serum and tissue levels of transforming growth factor‐β
Authors: Kanzler S. , Baumann M. , Schirmacher P. , Dries V. , Bayer E. , Gerken G. , Dienes H.‐P. , Lohse A. W.
Published: March 2003

Although many patients with chronic viral hepatitis C infection suffer from progressive liver disease, the rate of fibrosis progression is highly variable and some patients do not show any measurable progression. However, our ability to predict which patients progress is very limited. Since transforming growth factor‐β (TGF‐β) is a key mediator of liver fibrogenesis, we assessed the predictive role of TGF‐β for fibrogenesis in chronic hepatitis C. We studied 39 patients with chronic hepatitis C in whom two liver biopsies were taken at least 12 months apart, and who did not receive therapy during this period. TGF‐β was measured by bioassay and by ELISA in serum samples taken at the time of the first biopsies, and TGF‐β was determined semiquantitatively by immunostaining of liver biopsy sections. Fibrosis was scored blinded in the biopsy samples by two pathologists independently. There was a close correlation between TGF‐β serum levels and the rate of fibrosis progression. Patients with no progression of fibrosis had significantly lower (59 ng/mL ± 22) TGF‐β serum levels than patients with progressive disease (115 ng/mL ± 20), and a TGF‐β level below 75 ng/mL was predictive for stable disease. Immunohistology for TGF‐β in biopsy samples was also predictive for progressive liver disease with fibrosis progression found in those patients displaying staining of hepatocytes and sinusoidal cells. No such correlation was found with other markers such as procollagen III peptide, viral load or transaminase levels. These results further support the role of TGF‐β in liver fibrogenesis, and offer an opportunity to predict clinical disease progression, which may help in selecting patients who are in need of therapeutic interventions.
Fas‐mediated hepatocyte apoptosis is increased by hepatitis C virus infection and alcohol consumption, and may be associated with hepatic fibrosis: mechanisms of liver cell injury in chronic hepatitis C virus infection
Authors: Pianko S. , Patella S. , Ostapowicz G. , Desmond P. , Sievert W.
Published: March 2003

Epidemiological studies have established that heavy alcohol consumption in persons with chronic hepatitis C virus (HCV) infection is associated with advanced liver disease, including cirrhosis. The aims of this study were to evaluate the relationship between alcohol consumption and hepatocyte apoptosis in HCV‐infected patients and to determine the role of Fas in HCV‐mediated apoptosis. Liver tissue from 44 HCV‐infected patients with variable alcohol consumption, and 10 normal control subjects who did not consume alcohol was examined for hepatocyte apoptosis, proliferation and Fas expression. Alcohol consumption was assessed using the ‘Lifetime Drinking History’ alcohol questionnaire. HCV RNA, alanine aminotransferase (ALT) and ferritin were also assessed in addition to demographic data. Hepatocyte apoptosis was significantly greater in HCV‐infected patients compared to controls. Expression of Fas (CD95) was found in HCV patients but not in controls. The degree of Fas expression correlated with hepatocyte apoptosis as detected by terminal UTP nick end labelling (TUNEL). Active ethanol consumption led to a significant increase in hepatocyte apoptosis. Fas expression correlated with fibrosis in HCV‐infected patients who were not actively drinking ethanol. In summary, HCV leads to increased apoptotic cell death in hepatocytes. Programmed cell death can be further up‐regulated by active ethanol consumption. The correlation between Fas expression and TUNEL supports the hypothesis that the Fas–Fas ligand interaction is the major mechanism for HCV‐induced hepatocyte apoptosis.
Long‐term evaluation of recombinant interferon α2b in the treatment of patients with hepatitis B e antigen‐negative chronic hepatitis B in Taiwan
Authors: Lin C.‐C. , Wu J.‐C. , Chang T.‐T. , Huang Y.‐H. , Wang Y.‐J. , Tsay S.‐H. , Chow N.‐H. , Chang F.‐Y. , Lee S.‐D.
Published: March 2003

The effect of interferon (IFN) on hepatitis B e antigen (HBeAg)‐negative chronic hepatitis B (CHB) has not been fully investigated in Chinese patients. We enrolled 58 HBeAg‐negative CHB Chinese patients with hepatitis B viremia in Taiwan to evaluate the response to IFN. 30 patients received recombinant IFN 5 million units 3 times weekly for 6–10 months, and 28 patients who refused IFN treatment served as controls. Rates of virological response and biochemical response were higher in the treated group at the end of treatment (57% vs 18%, P=0.006, and 73% vs 29%, P=0.002, respectively). Both effects were superior in the treated group at 6 months after IFN withdrawal (virological: 30% vs 7%, P=0.06; biochemical: 47% vs 7%, P=0.002). Improvement of liver histological activities with persistently biochemical response was found in 65% of the treated patients. After a mean of 32 months’ follow‐up, virological response was rarely maintained (17% vs 4%, P=0.228) but biochemical response was better in the treated group (27% vs 4%, P=0.039). None of the treated patients but five controls developed severe complications of CHB during the follow‐up period. A larger total IFN dosage or a younger age (≤ 40 years) were associated with ‘sustained’ virological response. Younger age and higher baseline alanine transaminase values (≥ 120 Ul–1) were related to ‘sustained’ biochemical response.
Hepatitis C and human immune deficiency coinfection at the era of highly active antiretroviral therapy
Authors: Pol S. , Vallet‐Pichard A. , Fontaine H.
Published: March 2003

Interactions between human immunodeficiency virus (HIV) and hepatitis C virus (HCV) have been widely studied before the introduction of highly active antiretroviral therapies (HAART). We reviewed the potential impact of HAART on hepatitis C as well as the interactions between HIV and HCV therapies. Physicians should be aware of the potential risk of: (i) symptomatic liver disease in HCV‐HIV‐coinfected patients at the era of triple antiretroviral therapy; (ii) potential liver deterioration paralleling immune restoration; (iii) lack of impact of triple antiretroviral therapy on HCV load; and (iv) potential drug‐related hepatitis which may modify the natural history of HCV‐related liver disease. Liver biopsies should be performed regularly in these patients in order to identify patients with severe liver disease who require early initiation of anti‐HCV therapy under close monitoring of their immune status. Treatment is, to date, based on the combination of ribavirin and interferon with an expected sustained response rate around 25%. An important unresolved issue is to better delineate the temporal place of anti‐HCV and anti‐HIV antiviral therapies. At least in coinfected patients with significant liver disease, namely necro‐inflammatory activity and/or fibrosis ≥ 2, we believe that anti‐HCV therapy is the priority since it lessens the risk of drug‐induced hepatitis and of hepatitis due to immune restoration.
Occult hepatitis B virus infection and its clinical implications
Authors: Hu Ke‐Qin
Published: March 2003

Occult hepatitis B virus (HBV) infection is characterized by presence of HBV infection with undetectable hepatitis B surface antigen (HBsAg). Serum HBV level is usually less than 104 copies/mL in these patients. Diagnosis of occult HBV infection requires sensitive HBV‐DNA PCR assay. Several possibilities have been hypothesized as the mechanisms of occult HBV infection. These include: (i) mutations of HBV‐DNA sequence; (ii) integration of HBV‐DNA into host's chromosomes; (iii) infection of peripheral blood mononuclear cells by HBV; (iv) formation of HBV‐containing immune complex; (v) altered host immune response; and (vi) interference of HBV by other viruses. The precise prevalence of occult HBV infection remains to be defined. The clinical implications of occult HBV infection involve different clinical aspects. First of all, occult HBV infection harbours potential risk of HBV transmission through blood transfusion, haemodialysis, and organ transplantation. Second, it may serve as the cause of cryptogenic liver disease, contribute to acute exacerbation of chronic hepatitis B, or even fulminant hepatitis. Third, it is associated with development of hepatocellular carcinoma. Fourth, it may affect disease progression and treatment response of chronic hepatitis C. Most of the previous studies utilized retrospective observation without control groups, and lacked direct association of occult HBV infection with specific pathological changes and disease progression. Highly sensitive, quantitative, and functional molecular analyses of HBV, combined with a well‐designed prospective clinical assessment will provide the best approach for the future study of occult HBV infection.
Matrix metalloproteinases and their inhibitors in acute viral hepatitis
Authors: Koulentaki M. , Valatas V. , Xidakis K. , Kouroumalis A. , Petinaki E. , Castanas E. , Kouroumalis E.
Published: March 2003

Matrix metalloproteases (MMPs) and their inhibitors are effector molecules involved in extracellular matrix remodelling. The serum profile for these proteolytic enzymes and their inhibitors during acute self‐limiting viral hepatitis has not been studied. We therefore determined serum concentrations of MMP‐1, MMP‐3, MMP‐2, MMP‐9 and their inhibitors (tissue inhibitors of metalloproteinase) TIMP‐1, TIMP‐2 and α2 macroglobulin (AMG) in the serum of patients during the icteric stage of self‐limiting acute viral hepatitis. Transforming growth factor‐beta (TGF‐β) and interleukin (IL)‐10, two cytokines involved in the regulation of MMPs and TIMPs were also assessed. Nineteen patients (12 men, seven women) with a mean age of 29.9 years (range 16–65 years) participated in the study. Fifteen had hepatitis B virus (HBV, two HCV and two HAV infection. The values of patients were compared with those obtained from 15 blood donor controls (eight men, seven women), mean age 36.2 years (range 18–55 years). Serum levels of TGF‐β, IL‐10, MMP‐1, MMP‐3, MMP‐2, MMP‐9, TIMP‐1 and TIMP‐2 were assessed by ELISA. MMP‐2 and MMP‐9 were also measured by a zymogram protease assay. α2 macroglobulin (AMG) was measured by nephelometry. Compared with the healthy controls the mean serum concentrations of all MMPs were significantly decreased in the acute hepatitis patients. There was no difference in the serum concentration of TIMP‐1 between patients and the controls. Serum levels of TIMP‐2 (P < 0001), TGF‐β (P < 0.05), IL‐10 (P < 0.001) and AMG (P < 0001) were increased in patients compared to healthy controls. A statistically significant negative correlation by linear regression analysis was found between AMG and MMP‐1 (P=0003). The decreased levels of MMPs observed, together with normal and increased levels of TIMP‐1 and TIMP‐2, may indicate an attempt to limit matrix degradation at this stage of disease resolution. The increased levels of the anti‐inflammatory cytokines IL‐10 and TGF‐β might be the underlying mechanism responsible for the above effect. AMG inhibition especially for MMP‐1 may play an additional important role.
Mapping and characterization of B cell linear epitopes in the conservative regions of hepatitis C virus envelope glycoproteins
Authors: Olenina L. V. , Nikolaeva L. I. , Sobolev B. N. , Blokhina N. P. , Archakov A. I. , Kolesanova E. F.
Published: March 2003

Forty‐eight overlapping octapeptides covering highly conservative regions of E1 and E2 hepatitis C virus (HCV) envelope proteins were synthesized and tested by ELISA against different groups of sera obtained from HCV‐infected patients. All sera from patients with acute infection, except a single case of serum reactivity with the region HINRTALN, were nonreactive with any peptide. Sera obtained from chronic patients reacted with 12 peptides from five selected regions. Two immunodominant B epitopes were found, one being the precisely mapped antigenic site RMAWDM positioned inside the earlier shown immunodominant epitope from E1, and the second site, PALSTGLIH from E2, detected for the first time. New minor antigenic site was determined as PTDCFRKH from E2. We found only minor seroreactivity for one of the putative sites involved in CD81 binding, PYCWHYAP.
A phylogenetic‐tree analysis elucidating nosocomial transmission of hepatitis C virus in a haemodialysis unit
Authors: Kokubo S. , Horii T. , Yonekawa O. , Ozawa N. , Mukaide M.
Published: March 2003

summary. Nosocomial transmission of hepatitis C virus (HCV) subtype 1b involving 11 haemodialysis patients occurred in a haemodialysis unit in Japan in March 2000. Sequencing of the HCV‐E1 region (411‐bp) and phylogenetic‐tree analysis showed near identity between HCV isolates derived from these patients and a haemodialysis patient who was known to be HCV‐positive. The mode of transmission could not be conclusively established, but retrospective analysis suggested that the sharing of contaminated multidose vials of heparin‐saline solutions, which were prepared in the Haemodialysis Center using accidentally contaminated instruments such as needles, may have been responsible for the outbreak. To prevent transmission of HCV in a haemodialysis unit, it may be important to observe strictly standard precautions and to prepare all medications in the Pharmacy. After these measures were taken, no new seroconversions and no new nosocomial transmissions of HCV have been observed in our haemodialysis unit.
A multicentre, randomized study to evaluate the safety and efficacy of histamine dihydrochloride and interferon‐α‐2b for the treatment of chronic hepatitis C
Authors: Lurie Y. , Nevens F. , Aprosina Z. G. , Fedorova T. A. , Kalinin A. V. , Klimova E. A. , Ilan Y. , Maevskaya M. V. , Warnes T. W. , Yuschuk N. D. , Hellstrand K. , Gehlsen K. R.
Published: March 2003

summary. Interferon‐ (IFN‐)α is currently the standard of care treatment for patients with chronic hepatitis C virus (HCV) infection. A significant part of the benefit of IFN‐α in chronic hepatitis C is believed to be related to the activation of lymphocytes such as T cells and natural killer (NK) cells, which participate in the elimination of infected cells. Histamine dihydrochloride (HDC) has been shown to potentiate the IFN‐α‐induced activation of T cells and NK cells by a mechanism that involves the protection of these lymphocytes against oxygen radical‐induced functional inhibition and apoptosis. This study was designed to examine the efficacy and safety of HDC in combination with IFN‐α‐2b in treatment‐naïve patients with chronic HCV infection. All patients received IFN‐α‐2b, 3 MIU, three times weekly via subcutaneous injection, and were randomized to one of four HDC regimens (1 mg of either: once a day, three times a week; once a day, five times a week; twice a day, three times a week or; twice a day, five times a week). The doses of HDC in combination with IFN‐α‐2b resulted in sustained viral response rates ranging from 31% to 38%. Sustained biochemical response rates ranged from 28% to 41% across the four treatment groups. Patients infected with HCV genotype 1, and those with high baseline viral levels, which are characteristics associated with poor prognosis, had sustained virologic response rates ranging from 18% to 42% and 15% to 39%, respectively. Combination treatment was generally well tolerated. We propose that the potential benefit of HDC + IFN therapy for chronic HCV infection should be the focus of further investigation.
Interspecific hybridization and genetic variability of Phlebotomus sandflies
Authors: Ghosh K. N. , Mukhopadhyay J. M. , Guzman H. , Tesh R. B. , Munstermann L. E.
Published: March 2003

The first successful hybridization is reported between Phlebotomus papatasi and P. duboscqi, two important Old World sandfly vectors of leishmaniasis and other diseases. Laboratory strains of P. papatasi and P. duboscqi were separable by six diagnostic enzyme loci: Est‐3, Idh‐1, Mdh‐2, Mpi, Tre‐1 and Tre‐3. Hybrids between the two species were verified by the recovery of heterozygous isozyme patterns for the diagnostic loci. No F2 or backcross progeny were obtained. P. papatasi was separated from P. bergeroti by three diagnostic enzyme loci: Est‐3, Mpi and Pgd. The isozyme patterns of P. bergeroti contain elements of both P. duboscqi and P. papatasi, although seven diagnostic loci (Est‐3, Idh‐1, Me, Mpi, Pgd, Tre‐1 and Tre‐3) separated P. bergeroti from P. duboscqi. Genetic variability profiles of the three species were established for 20 enzyme loci. Three geographically distant strains of P. papatasi from Calcutta, Maharashtra and Israel had isozyme genetic distances of < 0.05. The recently established Calcutta strain showed an unexpectedly low genetic variability with only one (Idh‐2) of 20 loci being polymorphic (average heterozygosity of 1.9%) in contrast to 5–8 polymorphic loci (10–12% heterozygosity) in the Maharashtra and Israel strains. Mass and single pair crosses between the three P. papatasi strains were fertile with normal progeny numbers. Thus we found no signs of speciation in P. papatasi.