Paracetamol versus paracetamol-codeine in the treatment of post-operative dental pain: A randomized, double-blind, prospective trial

Background: Codeine is frequently added to paracetamol to treat post-operative dento-alveolar pain; studies have shown effectiveness in relief of post-operative pain at high doses but at the expense of central nervous and gastrointestinal side effects. There has been no trial to compare the efficacy and safety of paracetamol 1000mg with paracetamol 1000mg combined with codeine 30mg. Method. A randomized, single centre, double-blind prospective parallel group trial was performed to compare paracetamol 1000mg with paracetamol 1000mg with codeine 30mg for the relief of pain following surgical removal of impacted third molars, and analysed on an intention-to-treat (ITT) basis. Eighty-two patients were assigned randomly to receive either drug for a maximum of three doses. Patients recorded their pain intensity one hour after surgery and hourly thereafter for 12 hours. Results: The average increase in pain intensity over 12 hours was significantly less in patients receiving paracetamol plus codeine than in those receiving paracetamol alone (p=0.03) -1.81cm/h compared with 0.45cm/h - a difference of 1.13cm/h (95 per cent Cl: 0.18 to 2.08). Of the patients who received the paracetamol codeine combination, 62 per cent used escape medication compared with 75 per cent of those on paracetamol alone (p=0.20). There was no significant difference between the two groups in the proportion of patients experiencing adverse events (P=0.5). Conclusion: A combination of 1000mg paracetamol and 30mg codeine was significantly more effective in controlling pain for 12 hours following third molar removal, with no significant difference of side effects during the 12 hour period studied.

Improved detection of lacZ reporter gene expression in transgenic epithelia by immunofluorescence microscopy

The bacterial lacZ gene is commonly used as a reporter for the in vivo analysis of gene regulation in transgenic mice. However, several laboratories have reported poor detection of beta-galactosidase (the lacZ gene product) using histochemical techniques, particularly in skin. Here we report the difficulties we encountered in assessing lacZ expression in transgenic keratinocytes using classic X-gal histochemical protocols in tissues shown to express the transgene by mRNA in situ hybridization. We found that lacZ reporter gene expression could be reliably detected in frozen tissue sections by immunofluorescence analysis using a beta-galactosidase-specific antibody. Moreover, we were able to localize both transgene and endogenous gene products simultaneously using double-label immunofluorescence. Our results suggest that antibody detection of beta-galactosidase should be used to verify other assays of lacZ expression, particularly where low expression levels are suspected or patchy expression is observed.

The Mallory body as an aggresome: In vitro studies

Prior in vivo studies supported the concept that Mallory bodies (MBs) are aggresomes of cytokeratins 8 and 18. However, to test this hypothesis an in vitro model is needed to study the dynamics of MB formation. Such a study is difficult because MBs have never been induced in tissue culture. Therefore, MBs were first induced in vivo in drug-primed mice and then primary cultures of hepatocytes from these mice were studied. Two approaches were utilized: 1. Primary cultures were transfected with plasmids containing the sequence for cytokeratin 18 (CK 18) tagged with green fluorescent protein (GFP). 2. Immunofluorescent staining was used to localize the ubiquitin-proteasome pathway components involved in MB-aggresome complex formation in primary hepatocyte cultures. The cells were double stained with a ubiquitin antibody and one of the following antibodies: CK 8, CK 18, tubulin, mutant ubiquitin (UBB+ 1), transglutaminase, phosphothreonine, and the 20S and 26S proteasome subunits P25 and Tbp7, respectively. In the first approach, fluorescence was observed in keratin filaments and MBs 48 h after the cells were transfected with the CK 18 GFP plasmid. Nascent cytokeratin 18 was preferentially concentrated in MBs. Less fluorescence was observed in the normal keratin filaments. This indicated that MBs continued to form in vitro. The immunofluorescent staining of the hepatocytes showed that CK 8 and 18, ubiquitin, mutant ubiquitin (UBB+ 1), P25, Tbp7, phosphothreonine, tubulin, and transglutaminase were all located at the border or the interior of the MB. These results support the concept that MBs are aggresomes of CK 8 and CK 18 and are a result of inhibition of the ubiquitin-proteasome pathway of protein degradation possibly caused by UBB+ 1. (C) 2002 Elsevier Science.

GH treatment in adults with chronic liver disease: A randomized, double-blind, placebo-controlled, cross-over study

Patients with chronic liver disease (CLD) are catabolic and GH-resistant. The effects of supraphysiological recombinant human GH (rhGH; 0.2 IU.kg(-1).d(-1)) treatment in adults with CLD were assessed in a randomized, double-blind, placebo-controlled cross-over trial (4-wk dietary run-in, 4-wk treatment, and 2-wk wash-out phases). Nine adults with mild- to moderate-severity CLD participated (median age, 49 yr; three males and six females; Child's classification A in six and B in three). Biopsy-proven etiologies were: alcohol (four patients), primary biliary cirrhosis (three patients), non-A, non-B, non-C hepatitis (one patient), and cryptogenic (one patient). Treatment with rhGH increased serum IGF-I (median increase over placebo, +93 mug.liter(-1); P = 0.004), IGF-binding protein-3 (+0.9 mg.liter(-1): P = 0.004), and acid labile subunit (+10.7 nM; P = 0.004). Total body potassium (+8.0 g; P = 0.023), body weight (+1.6 kg; P = 0.008), and total body water (by bioelectrical impedance; +4.9 kg; P = 0.004) increased. Resting metabolic rate (+313 ml.kg(-1).min(-1); P = 0.004) and lipid oxidation (+1072.0 kcal.d(-1); P = 0.032) increased. Metabolic changes included increased fasting plasma glucose (+1.2 mm; P = 0.008), insulin (+33.8 mU.liter(-1); P = 0.004), C-peptide (+0.7 nM; P = 0.004), and free-fatty acids (+0.1 mEq.liter(-1); P = 0.04). Clinical side effects included worsening edema and ascites. Hepatocellular function did not change. Therefore, rbGH treatment in CLD: 1) overcame hepatic GH resistance; 2) may have improved whole-body protein catabolism; 3) increased lipolysis and lipid oxidation; 4) increased insulin resistance; and 5) had potent antinatriuretic effects. Long-term safety and efficacy require further assessment.

Detection of West Nile virus antigen in mosquitoes and avian tissues by a monoclonal antibody-based capture enzyme immunoassay

An antigen capture immunoassay to detect West Nile (WN) virus antigen in infected mosquitoes and avian tissues has been developed. With this assay purified WN virus was detected at a concentration of 32 pg/0.1 ml, and antigen in infected suckling mouse brain and laboratory-infected mosquito pools could be detected when the WN virus titer was 10(2.1) to 10(3.7) PFU/0.1 ml. In a blindly coded set of field-collected mosquito pools (n = 100), this assay detected WN virus antigen in 12 of 18 (66.7%) TaqMan-positive pools, whereas traditional reverse transcriptase PCR detected 10 of 18 (55.5%) positive pools. A sample set of 73 organ homogenates from naturally infected American crows was also examined by WN virus antigen capture immunoassay and TaqMan for the presence of WN virus. The antigen capture assay detected antigen in 30 of 34 (88.2%) TaqMan-positive tissues. Based upon a TaqMan-generated standard curve of infectious WN virus, the limit of detection in the antigen capture assay for avian tissue homogenates was approximately 10(3) PFU/0.1 ml. The recommended WN virus antigen capture protocol, which includes a capture assay followed by a confirmatory inhibition assay used to retest presumptive positive samples, could distinguish between the closely related WN and St. Louis encephalitis viruses in virus-infected mosquito pools and avian tissues. Therefore, this immunoassay demonstrates adequate sensitivity and specificity for surveillance of WN virus activity in mosquito vectors and avian hosts, and, in addition, it is easy to perform and relatively inexpensive compared with the TaqMan assay.

Equine respiratory viruses in foals in New Zealand

AIMS: To identify the respiratory viruses that are present among foals in New Zealand and to establish the age at which foals first become infected with these viruses. METHODS: Foals were recruited to the study in October/ November 1995 at the age of 1 month (Group A) or in March/ April 1996 at the age of 4-6 months (Groups B and C). Nasal swabs and blood samples were collected at monthly intervals. Nasal swabs and peripheral blood leucocytes (PBL) harvested from heparinised blood samples were used for virus isolation; serum harvested from whole-blood samples was used for serological testing for the presence of antibodies against equine herpesvirus (EHV)-1 or -4, equine rhinitis-A virus (ERAV), equine rhinitis-B virus (ERBV), equine adenovirus 1 (EAdV-1), equine arteritis virus (EAV), reovirus 3 and parainfluenza virus type 3 (PIV3). Twelve foals were sampled until December 1996; the remaining 19 foals were lost from the study at various times prior to this date. RESULTS: The only viruses isolated were EHV 2 and EHV 5. EHV 2 was isolated from 155/157 PBL samples collected during the period of study and from 40/172 nasal swabs collected from 18 foals. All isolations from nasal swabs, except one, were made over a period of 2-4 months from January to April (Group A), March to April (Group B) or May, to July (Group C). EHV 5 was isolated from either PBL, nasal swabs, or both, from 15 foals on 32 occasions. All foals were positive for antibodies to EHV 1 or EHV 4, as tested by serum neutralisation (SN), on at least one sampling occasion and all but one were positive for EHV 1 antibodies measured by enzyme-linked immunosorbent assay (ELISA) on at least one sampling occasion. Recent EHV 1 infection was evident at least once during the period of study in 18/23 (78%) foals for which at least two samples were collected. SN antibodies to ERBV were evident in 19/23 (83%) foals on at least one sampling occasion and 15/23 foals showed evidence of seroconversion to ERBV Antibodies to ERAV were only detected in serum samples collected from foals in Group A and probably represented maternally-derived antibodies. Haemagglutination inhibition (HI) antibody titres greater than or equal to 1:10 to EAdV-1 were evident in 21/23 (91%) foals on at least one sampling occasion and 16/23 foals showed serological evidence of recent EAdV-1 infection. None of the 67 serum samples tested were positive for antibodies to EAV, reovirus 3 or PIV3. There was no clear association between infection with any of the viruses isolated or tested for and the presence of overt clinical signs of respiratory disease. CONCLUSIONS: There was serological and/or virological evidence that EHV-1, EHV-2, EHV-5, EAdV-1 and ERBV infections were present among foals in New Zealand. EHV-2 infection was first detected in foals as young as 3 months of age. The isolation of EHV-2 from nasal swabs preceded serological evidence of infection with other respiratory viruses, suggesting that EHV-2 may predispose foals to other viral infections.

Viruses associated with outbreaks of equine respiratory disease in New Zealand

AIM: To identify viruses associated with respiratory disease in young horses in New Zealand. METHODS: Nasal swabs and blood samples were collected from 45 foals or horses from five separate outbreaks of respiratory disease that occurred in New Zealand in 1996, and from 37 yearlings at the time of the annual yearling sales in January that same year. Virus isolation from nasal swabs and peripheral blood leukocytes (PBL) was undertaken and serum samples were tested for antibodies against equine herpesviruses (EHV-1, EHV-2, EHV-4 and EHV-5), equine rhinitis-A virus (ERAV), equine rhinitis-B virus (ERBV), equine adenovirus 1 (EAdV-1), equine arteritis virus (EAV), reovirus 3 and parainfluenza virus type 3 (PIV3). RESULTS: Viruses were isolated from 24/94 (26%) nasal swab samples and from 77/80 (96%) PBL samples collected from both healthy horses and horses showing clinical signs of respiratory disease. All isolates were identified as EHV-2, EHV-4, EHV-5 or untyped EHV Of the horses and foals tested, 59/82 (72%) were positive for EHV-1 and/or EHV-4 serum neutralising (SN) antibody on at least one sampling occasion, 52/82 (63%) for EHV-1-specific antibody tested by enzyme-linked immunosorbent assay (ELISA), 10/80 (13%) for ERAV SN antibody, 60/80 (75%) for ERBV SN antibody, and 42/80 (53%) for haemagglutination inhibition (HI) antibody to EAdV-1. None of the 64 serum samples tested were positive for antibodies to EAV, reovirus 3 or PIV3. Evidence of infection with all viruses tested was detected in both healthy horses and in horses showing clinical signs of respiratory disease. Recent EHV 2 infection was associated with the development of signs of respiratory disease among yearlings [relative risk (RR) = 2.67, 95% CI = 1.59-4.47, p = 0.0171]. CONCLUSIONS: Of the equine respiratory viruses detected in horses in New Zealand during this study, EHV 2 was most likely to be associated with respiratory disease. However, factors other than viral infection are probably important in the development of clinical signs of disease.

Avian paramyxoviruses and influenza viruses isolated from mallard ducks (Anas platyrhynchos) in New Zealand

A comprehensive study using virological and serological approaches was carried out to determine the status of live healthy mallard ducks (Anas platyrhynchos) in New Zealand for infections with avian paramyxoviruses (APMV) and influenza viruses (AIV). Thirty-three viruses isolated from 321 tracheal and cloacal swabs were characterized as: 6 AIV (two H5N2 and four H4N6), 10 APMV-1 and 17 APMV-4. Of 335 sera samples tested for AIV antibodies, 109 (32.5%) sera were positive by nucleoprotein-blocking ELISA (NP-B-ELISA). Serum samples (315) were examined for antibody to APMV-1, -2, -3, -4, -6, -7, -8, -9 by the haemagglutination inhibition test. The largest number of reactions, with titres up to greater than or equal to 1/64, was to APMV-1 (93.1%), followed by APMV-6 (85.1%), APMV-8 (56%), APMV-4 (51.7%), APMV-7 (47%), APMV-9 (15.9%), APMV-2 (13.3%) and APMV-3 (6.0%). All of the H5N2 isolates of AIV and the APMV-1 isolates from this and earlier New Zealand studies had low pathogenicity indices assessed by the Intravenous Pathogenicity Index (IVPI) with the result 0.00 and Intracerebral Pathogenicity Index (ICPI) with results 0.00-0.16. Partial genomic and antigenic analyses were also consistent with the isolates being non-pathogenic. Phylogenetic analysis of the 10 APMV-1 isolates showed 9 to be most similar to the reference APMV-1 strain D26/76 originally isolated in Japan and also to the Que/66 strain, which was isolated in Australia. The other isolate was very similar to a virus (MC 110/77) obtained from a shelduck in France.

Comparison of adsorption capacity of p-Cresol & p-Nitrophenol by activated carbon in single and double solute

Adsorption of p-Cresol and p-Nitrophenol by untreated activated carbon in single and multisolute solutions was carried out at 301 K and at controlled pH conditions. In acidic conditions, well below the pK(a) of both solutes, it was observed that the adsorbate solubility and the electron density of aromatic rings influenced the extent of adsorption by affecting the extent of London dispersion forces. The fitted parameters obtained from single-solute Langmuir equation show that Q(max) and the adsorption affinity of carbon for the compound with low pK(a) decrease more significantly. In higher solution pH conditions, on the other hand, it was found that electrostatic forces played a significant role on the extent of adsorption. The presence of another compound decreases Q(max) and the adsorption affinity of carbon for the principal compound. The effect of pH, on the carbon surface and on the solute molecules, must be considered. Adsorption of the solute at higher pH values was found to be dependent on the concentration of anionic form of the solute. The isotherm data were fitted to the Langmuir isotherm equation for both single and double solute solutions.

Comparison of an ELISA and an LC/MS/MS method for measuring tacrolimus concentrations and making dosage decisions in transplant recipients

This study compared an enzyme-linked immunosorbent assay (ELISA) to a liquid chromatography-tandem mass spectrometry (LC/MS/MS) technique for measurement of tacrolimus concentrations in adult kidney and liver transplant recipients, and investigated how assay choice influenced pharmacokinetic parameter estimates and drug dosage decisions. Tacrolimus concentrations measured by both ELISA and LC/MS/MS from 29 kidney (n = 98 samples) and 27 liver (n = 97 samples) transplant recipients were used to evaluate the performance of these methods in the clinical setting. Tacrolimus concentrations measured by the two techniques were compared via regression analysis. Population pharmacokinetic models were developed independently using ELISA and LC/MS/MS data from 76 kidney recipients. Derived kinetic parameters were used to formulate typical dosing regimens for concentration targeting. Dosage recommendations for the two assays were compared. The relation between LC/MS/MS and ELISA measurements was best described by the regression equation ELISA = 1.02 . (LC/MS/MS) + 0.14 in kidney recipients, and ELISA = 1.12 . (LC/MS/MS) - 0.87 in liver recipients. ELISA displayed less accuracy than LC/MS/MS at lower tacrolimus concentrations. Population pharmacokinetic models based on ELISA and LC/MS/MS data were similar with residual random errors of 4.1 ng/mL and 3.7 ng/mL, respectively. Assay choice gave rise to dosage prediction differences ranging from 0% to 30%. ELISA measurements of tacrolimus are not automatically interchangeable with LC/MS/MS values. Assay differences were greatest in adult liver recipients, probably reflecting periods of liver dysfunction and impaired biliary secretion of metabolites. While the majority of data collected in this study suggested assay differences in adult kidney recipients were minimal, findings of ELISA dosage underpredictions of up to 25% in the long term must be investigated further.

A novel bioassay for human somatogenic activity in serum samples supports the clinical reliability of immunoassays

OBJECTIVE Because there is discordance between different immunoassay values for serum hGH, and because clinical state may not correlate with immunoreactive hGH, we have developed an assay to accurately measure serum hGH somatogenic bioactivity. The results of this assay were compared with the Elegance two-site ELISA assay across 135 patient samples in a variety of clinical states. DESIGN The somatogenic assay was based on stable expression of hGH receptor in the murine BaF line, allowing these cells to proliferate in response to hGH. To eliminate interference by other growth factors in serum, we created a specific antagonist of the hGH receptor (similar to Trovert or Pegvisomant) which allowed us to obtain a true measure of hGH somatogenic activity by subtraction of the activity in the presence of the antagonist. The assay was carried out in microtiter plates over 24 h, with oxidation of a chromogenic tetrazolium salt (MTT) as the endpoint. PATIENTS These encompassed a number of different clinical conditions related to short stature, including idiopathic short stature, neurosecretory dysfunction and renal failure, as well as obese patients on dietary restriction and normal volunteers. MEASUREMENTS In addition to the colourimetric (MTT) response to hGH, we measured free hGH by stripping out GHBP-bound hGH using beads coupled to a monoclonal antibody to the GHBP (GH binding protein). All samples were measured in both bioassay and ELISA assay. RESULTS This bioassay was sensitive (5 mU/l or 2 mug/l) and precise, and not subject to interference by the GHBP. There was a good correlation (r = 0.95) between bioactivity and immunoactivity across clinical states. There was, however, an increased bioactivity during secretory peaks (over 25 mU/l), which has been reported previously for the Nb2 bioassay. Free hGH did not correlate with clinical state. CONCLUSIONS Because the results of the Elegance ELISA and the bioassay correlate well, even though there is greater bioactivity at higher hormone concentrations, it is evident that an appropriate immunoassay is able to act as a reliable indicator for clinical assessment. In those rare cases where bio-inactive GH exists, our bioassay should provide an appropriate means to demonstrate this.