Hepatic TLR2 & TLR4 expression correlates with hepatic inflammation and TNF-α in HCV & HCV/HIV infection.

Signalling activated by Toll-like receptors (TLRs) can result in the production of tumour necrosis factor alpha (TNF-α) which is implicated in hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infection. No study has examined or compared hepatic expression of TLRs in both HCV and HCV/HIV. Liver and peripheral blood mononuclear cells (PBMCs) were obtained from HCV & HCV/HIV-infected patients and PBMCs from HIV-infected patients. Liver RNA was analysed by microarray and reverse transcription quantitative PCR (RT-qPCR). PBMCs were analysed by flow cytometry. Associations with hepatic histology and infection type were sought. Forty-six HCV, 20 HIV and 27 HCV/HIV-infected patients were recruited. Increasing Metavir inflammatory activity score was associated with increased hepatic TLR mRNA by RT-qPCR: TLR2 (P ≤ 0.001), TLR4 (P = 0.008) and TNF-α (P ≤ 0.001). A high degree of correlation was seen between hepatic mRNA expression of TNF-αvs TLR2 (r(2) = 0.66, P < 0.0001) and TLR4 (r(2) = 0.60, P < 0.0001). No differences in TLR gene or protein expression was observed between HCV, HCV/HIV- or HIV-infected groups. Hepatic TLR2, TLR4 and TNF-α mRNA are associated with hepatic inflammation in both HCV and HCV/HIV infection. High correlation between TNF-α and TLR2/TLR4 suggests a role for the innate immune response in TNF-α production. Activation of the innate immune response appears to be independent of infection type.

Determination of immunoglobulin G in bovine colostrum and milk powders, and in dietary supplements of bovine origin by protein G affinity liquid chromatography: collaborative study.

An AOAC collaborative study was conducted to evaluate an affinity LC procedure for measuring immunoglobulin G (IgG) in selected dairy powders. The powders were extracted with 0.15 M sodium chloride solution and the pH was adjusted to 4.6 to precipitate caseins, which would otherwise lead to an overestimation of IgG. The analyte was then bound to a commercially available Protein G affinity cartridge and selectively eluted with a glycine buffer at pH 2.5. Detection was at 280 nm and quantification was made against a calibration curve prepared from bovine serum IgG. The samples analyzed included the likely matrixes for which this assay will find commercial use, namely, high- and low-protein-content colostrum powders, tablets containing colostrum powder, and some IgG-containing dairy powders; milk protein isolate, whey protein concentrate, and skim milk powder. Eleven laboratories provided data for the study and assayed blind duplicates of six materials. The repeatability RSD values ranged from 2.1 to 4.2% and the reproducibility RSD values ranged from 6.4 to 18.5%. The Protein G method with casein removal has adequate reproducibility for measuring IgG in colostrum-derived powders that are traded on the basis of IgG content as a colostral marker.

Changes in plasma vitamin levels in the first 48 hours after onset of acute myocardial infarction.

To establish whether plasma vitamin measurements made after acute myocardial infarction (AMI) can be used in case-control studies of coronary artery disease, the short-term effect of AMI on plasma concentrations of 25-hydroxyvitamin D3, beta-carotene, vitamin E and retinol was investigated. Sequential measures of these vitamins were made during the first 48 hours after AMI in 13 patients admitted to the hospital within 4 hours after the onset of symptoms. Plasma levels of 25-hydroxyvitamin D did not change significantly during the first 12 hours after onset of symptoms. Beta-carotene levels increased significantly (p less than 0.05) during the first 12 hours and then decreased, whereas levels of vitamin E and retinol progressively decreased during the first 48 hours by 26 and 25%, respectively. These results suggest that, of these vitamins, only plasma measurements of 25-hydroxyvitamin D3 collected within 12 hours of onset of symptoms may provide reliable information for case-control studies of AMI.

Characterization of 5-hydroxytryptamine receptors mediating mucosal secretion in guinea-pig ileum.

1. The receptor subtypes through which 5-hydroxytryptamine (5-HT) increases electrolyte secretion across the mucosa of guinea-pig ileum were studied. 2. Flat sheep preparations of guinea-pig mucosa plus submucosa were placed in Ussing chambers and the short circuit current (ISC), an index of net electrogenic electrolyte transport across the mucosa, was measured under voltage clamp conditions. 3. Low concentrations of 5-HT (10-300 nM) evoked monophasic increases in ISC which were significantly reduced by hyoscine (100 nM), tetrodotoxin (TTX, 300 nM) and the 5-HT2 receptor antagonist, ketanserin (3-300 nM). 4. Higher concentrations of 5-HT (1-10 microM) produced biphasic responses which were reduced by hyoscine (100 nM), TTX (300 nM), ketanserin (3-300 nM) and also by the 5-HT3 receptor antagonists, granisetron (1 microM) and ICS 205-930 (100 nM). 5. 2-Methyl-5-HT (1-100 microM) and alpha-methyl-5-HT (30 nM-30 microM), agonists at 5-HT3 and 5-HT2 receptors respectively, also evoked ISC increases. These responses were reduced by hyoscine (100 nM) and abolished by TTX (300 nM) and the respective receptor antagonists, granisetron (1 microM) and ketanserin (30 nM). 6. The 5-HT4 receptor antagonist, SDZ 205-557 (300 nM) had no effect on the response to 5-HT. 7. The TTX-resistant response to 5-HT was not affected by 5-HT2,3 or 4 receptor antagonists. 8. These results indicate that 5-HT mediates secretion partly by an action on 5-HT3 receptors located on cholinergic and noncholinergic secretomotor neurones, partly by an action on higher affinity'5-HT2-like' receptors predominantly on noncholinergic neurones, and partly by a direct action on the epithelium.

Vaccination trials in Australia and Argentina confirm the effectiveness of the EG95 hydatid vaccine in sheep.

Experimental vaccine trials against hydatid disease have been undertaken in sheep using the EG95 recombinant vaccine. Challenge infection was with viable Echinococcus granulosus eggs obtained from a New Zealand isolate (dog/sheep cycle), an Australian isolate (dingo/wallaby cycle) and an Argentine isolate (dog/sheep cycle). Vaccination with EG95 conferred a high degree of protection against challenge with all three parasite isolates (protection range 96-100%). Taken together, the trials demonstrated that 86% of vaccinated sheep were completely free of viable hydatid cysts when examined approximately 1 year after challenge infection. Vaccination reduced the number of viable cysts by 99.3% compared with unvaccinated controls. These results suggest that the EG95 vaccine could have wide applicability as a new tool for use in hydatid control campaigns.

Characterization of 5-HT receptors mediating contraction and relaxation of the longitudinal muscle of guinea-pig distal colon in vitro.

A range of agonists and antagonists were used to characterize the receptors through which 5-hydroxytryptamine (5-HT) contracts and relaxes the longitudinal muscle of segments of guinea-pig distal colon, in vitro. 5-HT contracted the longitudinal muscle over the concentration range 10(-9) to 10(-4) mol/l. The 5-HT3 receptor agonist, 2-methyl-5-HT, produced concentration dependent contractions over the range 10(-6) to 10(-4) mol/l. 5-methoxytryptamine, an agonist at 5-HT4 receptors, caused contractions over a concentration range of 10(-8) to 10(-4) mol/l. The 5-HT4 antagonist, SDZ 205-557 (5 x 10(-7) mol/l) substantially suppressed the responses to low concentrations of 5-HT and to 5-methoxytryptamine, but had no effect on the responses to higher concentrations of 5-HT. In contrast, the 5-HT3 antagonist, granisetron (10(-6) mol/l), blocked the effect of 2-methyl-5-HT and substantially depressed responses to high concentrations of 5-HT, but had no effect on lower concentrations of 5-HT. Granisetron produced a small reduction in the response to 5-methoxytryptamine. Tetrodotoxin (TTX) (3 x 10(-7) mol/l) almost abolished the response to 5-methoxytryptamine and markedly suppressed the response to 2-methyl-5-HT, but the responses to 5-HT were only partially reduced. The 5-HT1 antagonist, methiothepin (10(-6) mol/l) depressed the response to 5-HT (10(-7) to 10(-4) mol/l) and blocked its TTX insensitive component. The 5-HT2 antagonist, ketanserin, in concentrations up to 10(-5) mol/l, had no effect on the contractions evoked by 5-HT.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzymatic determination of free carnitine in milk and infant formulas.

A simple spectrophotometric assay for free carnitine in milk and supplemented infant formulas has been developed. After acid extraction, carnitine is measured enzymatically through its reaction with carnitine acetyltransferase coupled with acetyl coenzyme A and dithiobenzoate (DTNB). The manually performed method is rapid, accurate, and convenient for routine quality-control analysis of infant formulas. The chemistry is also suitable for automation, for greatly enhanced throughput.

Taurine analysis in milk and infant formulae by liquid chromatography: collaborative study.

A collaborative study was conducted on a liquid chromatographic (LC) method for determination of taurine in infant formula and milk powders. Twenty laboratories participated in the analysis of 8 blind duplicates over the range of approximately 3-60 mg/100 g sample. The method involved protein removal, conversion to the dansyl-derivative, and isocratic LC separation with UV and/or fluorescence detection. Following outlier treatment, overall mean RSDR has been estimated at 7.00% for supplemented products with a HORRAT value of 1.1. The poorer precision at endogenous levels establishes a lower limit of determination of about 5 mg/100 g. An overall mean RSDr:RSDR value of 0.7 for all products demonstrated acceptable performance.

Determination of vitamin K in milk and infant formulas by liquid chromatography: collaborative study.

A simple procedure for determination of vitamin K1 was developed for routine compliance monitoring of supplemented infant formula and measurement of endogenous levels in milk and milk powders. Samples are digested with lipase and extracted into hexane; and aliquot is evaporated, reconstituted in methanol, and analyzed by reversed-phase LC. Post-column zinc reduction of phylloquinone facilitates detection by fluorescence. The procedure was subjected to an AOAC collaborative study involving 8 materials, each in blind duplicate, across the range of 5-120 micrograms/100 g solids and including NIST 1846 reference material. Thirty-three laboratories returned valid data which were then statistically analyzed for outliers and precision parameters. Mean RSDR (%) was 6.53 (4.33-10.94), with a mean HORRAT value of 0.33 (0.23-0.43) and RSDr:RSDR ratio of 0.74. K1 isomers (cis and trans) were aggregated with conventional C18 columns, but may be selectively estimated with use of the C30 column.

Determination of choline in milk and infant formulas by enzymatic analysis: collaborative study.

A collaborative study was conducted on a coupled enzymatic-spectrophotometric method for the determination of choline in infant formula and milk powders. Twenty-nine laboratories participated in the analysis of 8 blind duplicates over the range of 45-175 micrograms/100 g sample. After the combined acid hydrolysis-phospholipase release of choline, incubation with choline oxidase in the presence of peroxidase and phenol generates a quinoneimine chromophore with 4-aminoantipyrine. Following raw data screening, overall mean RSDR was estimated at 5.14% (range, 4.26-6.34%) with a HORRAT value of 0.91 (range, 0.76-1.00) and an overall mean RSDr:RSDR value of 0.53 (range, 0.42-0.74). The method was also compared with alternative independent analytical techniques for several of the collaborative study samples.

Liquid chromatographic determination of vitamin K1 in infant formulas and milk.

Vitamin K1 in infant formulas and milk products is determined by reversed-phase liquid chromatography (LC) with UV detection. The sample is hydrolyzed enzymatically, and the vitamin is extracted with hexane. Fractionation by normal-phase semi-preparative LC is followed by analytical LC, with quantitation by the internal standard technique. Recovery of the analyte was 97.4 +/- 2.8%. Linearity was established between 0.05 and 4.0 micrograms/mL. The limit of quantitation is 0.5 microgram/100 g for milk powder, which allows the method to quantitate endogenous levels of vitamin K1.

Determination of niacin in infant formula by solid-phase extraction and anion-exchange liquid chromatography.

A peer-verified, solid-phase extraction (SPE)/anion exchange liquid chromatographic method is presented for the determination of niacin in milk-based and soy-based infant formula. Analysis is in 3 steps: test sample digestion, extraction/cleanup, and liquid chromatography (LC). Digestion uses a standard AOAC digestion procedure that involves autoclaving at 121 degrees C for 45 min in (1 + 1) H2SO4 to free endogenous niacin from protein and to convert added niacinamide to niacin. The digest solution is adjusted to pH 6.5 with 7.5M NaOH. Acidification to pH <1.0 with (1 + 1) H2SO4 precipitates the protein. The clarified solution is then filtered, and the filtrate is brought to volume. SPE of niacin is accomplished by passing an aliquot of the digest solution through an aromatic sulfonic acid-SPE (ArSCX-SPE) column. After the column is washed with methanol and water to remove extraneous material, the niacin is eluted with 0.25M sodium acetate/acetic acid buffer at pH 5.6. An anion-exchange polystyrene-divinylbenzene column with 0.1 M sodium acetate/acetic acid buffer at pH 4.0 is used for LC. Niacin is determined by UV detection at 260 nm. A standard curve is prepared by passing known amounts of niacin through the ArSCX-SPE columns used for niacin extraction. The following values for x and relative standard deviation (RSD) were obtained for National Institute of Standards and Technology Standard Reference Material (NIST SRM) 1846 Infant Formula with a certified value for niacin of 63.3 +/- 7.6 microg/g: Submitting laboratory.-- x = 59.7 +/- 4.0 microg/g; RSD = >6.7%; confidence interval (CI) = +/- 1.4 microg/g; n = 27. Peer laboratory.--x = 56.6 +/- 6.6 microg/g; RSD = >11.7%; CI =+/- 4.1 microg/g; n = 8.

Determination of biotin and folate in infant formula and milk by optical biosensor-based immunoassay.

Biomolecular interaction analysis was evaluated for the automated analysis of biotin- and folate-supplemented infant formulas and milk powders. The technique was configured as a biosensor-based, nonlabeled inhibition immunoassay using monoclonal antibodies raised against analyte-conjugate. Sample extraction conditions were optimized and antibodies were evaluated for cross-reactivity. Performance parameters included a quantitation range of 2-70 ng/mL, recoveries of 86-102%, agreement against assigned reference values for National Institute of Standards and Technology Standard Reference Material 1846, between-laboratory reproducibility relative standard deviation of 9.1% for biotin and 8.1% for folate, respectively, and equivalence against reference microbiological assay methods for both analytes.

Determination of free myo-inositol in milk and infant formula by high-performance liquid chromatography.

A high-performance liquid chromatographic method is described for the determination of supplemental myo-inositol in infant formulas based on cows' milk. The technique incorporates pre-column derivatization with phenylisocyanate followed by reversed-phase gradient chromatography and ultraviolet detection. The protocol was also applied to a survey of free inositol at endogenous levels in the milk of cows, goats and humans. Nutritional preparations containing inositol at pharmacological levels are also amenable to a simplified isocratic analysis.

Antioxidant analysis in edible oils and fats by normal-phase high-performance liquid chromatography.

Following a simple dilution in the appropriate phase, the sample is injected directly onto either of two normal-phase high-performance liquid chromatography systems (3,5-di-tert.-butyl-4-hydroxytoluene or 3-tert.-butyl-4-hydroxyanisole-tert.-butylhydroquinone) with UV detection at 280 nm. An isocratic ternary mobile phase, incorporating acetonitrile as the polar modifier, has been found to facilitate such an approach, thereby avoiding the discriminatory and recovery problems inherent in other techniques requiring prior sample manipulations. The three most commonly used antioxidants may be estimated at levels down to 3 ppm (3,5-di-tert.-butyl-4-hydroxytoluene or 3-tert.-butyl-4-hydroxyanisole) and 10 ppm (tert.-butylhydroquinone) within 30 min.

Vitamin K in milk and infant formulas: determination and distribution of phylloquinone and menaquinone-4.

A method is described for the determination of phylloquinone and menaquinones following enzymatic digestion, extraction and a single-stage HPLC technique utilizing post-column reduction with zinc and fluorescence detection. The technique is applicable to both routine compliance control of phylloquinone supplemented infant formula powders (30-150 micrograms per 100 g) and fundamental studies of the K vitamins at endogenous levels in fluid milks (0-5.0 micrograms per 100 g). Analytical figures of merit include a detection limit of 30 micrograms on-column, recoveries greater than 98% for both K1 and MK4, an RSDR of 2.35% (K1) and 2.32% (MK4) and a regression correlation of 0.9932 for a wide range of infant formulas when compared against an alternative HPLC-UV technique. MK4 and 2',3'-dihydrophylloquinone, both with undefined bioactivity, were detected at measurable levels in a range of infant formulas. Although the higher menaquinones were found to be essentially absent in the milk of several species, the significant presence of MK4 relative to K1 has been confirmed in all milks examined, with both dominant forms correlated during early lactation in the cow. These observations suggest an as yet unrecognized physiological function for MK4 in infant nutrition.

Synthetic peptides induce antibody against a host-protective antigen of Echinococcus granulosus.

The immunogenicity of four synthetic peptides was investigated in sheep. The sequences of the peptides (6, 12/13, 21/22 and 24) were derived from linear, antibody-binding epitopes of the EG95 recombinant protein, a host-protective antigen of the parasite Echinococcus granulosus. Sheep were immunised with either free peptide or peptide conjugated to diphtheria toxoid. All sheep responded to both conjugated and unconjugated forms of the peptides. For two of the four peptides (6 and 21/22), the amount of antibody elicited was significantly greater for the conjugated form of the peptides than for the corresponding unconjugated forms. For the other two peptides (12/13 and 24), peak antibody levels to both forms of the peptide were equivalent. Maximal antibody titres against peptides 6, 12/13 and 21/22 were established after only one immunisation and were not boosted by a second dose. Antisera to all four peptides reacted with the recombinant antigen, and three of the four peptides generated antibodies, which bound to the native parasite oncosphere antigen. Antisera raised against the peptides were unable to kill the parasite in in vitro culture, although each of the peptides could be used to affinity purify lethal antibody from antisera raised against the recombinant protein. These results indicate that peptides 6, 12/13, 21/22 and 24 of the EG95 recombinant vaccine are immunogenic and suggest that they are associated with host-protective epitopes.

Assessment of protective immune responses against hydatid disease in sheep by immunization with synthetic peptide antigens.

Four synthetic peptides which comprise the immunodominant linear epitopes of the EG95 recombinant protein, were investigated for their ability to induce host-protective immunity against Echinococcus granulosus in sheep. Sheep were immunized with either free peptide or peptide conjugated to diphtheria toxoid and challenge infected with E. granulosus eggs. All of the peptides elicited specific antibody, but these did not kill the parasite in in vitro culture assays, nor did the peptides induce protection against challenge infection. In contrast, anti-EG95 antibodies affinity purified against each of the 4 peptides were lethal to the parasite in in vitro culture. These affinity-purified antibodies were shown to contain specific antibody to both peptide and EG95. In in vitro inhibition assays, the peptides did not diminish anti-EG95 antibody binding to EG95 or parasite lysis in oncosphere killing assays. These results suggest that the fine specificities of antibodies raised against the recombinant protein are different to those raised against the peptide immunogens and that the majority of the antibody induced by vaccination with EG95 is raised against conformational determinants.

Protection against hydatid disease induced with the EG95 vaccine is associated with conformational epitopes.

This paper describes attempts to map the location of host-protective epitopes of a recombinant vaccine antigen by assessing the ability of truncated regions of the antigen to elicit protective immune responses in sheep. Sheep were immunised with three truncated regions (EG95-1, EG95-2 and EG95-3) of the hydatid vaccine antigen, EG95. These regions overlapped each other and corresponded to amino acids 1-70 (EG95-1), 51-106 (EG95-2) and 89-153 (EG95-3) of the full length recombinant protein. Each region elicited antibody which reacted with the parent antigen, although these reactivities were a small proportion of the level of reactivity generated by immunisation with the full length antigen. Antisera raised against each of the truncated proteins reacted with the native parasite antigen. In vaccination and parasite challenge trials in sheep, none of the truncated regions elicited significant protection against challenge infection or antibody which was lethal to the parasite in vitro. Antibodies from sheep immunised with the combination of all three overlapping truncations elicited a comparatively low but significant level of lysis of the parasite in vitro. These antigens did not inhibit anti-EG95 antibody reactivity with EG95 nor did they inhibit in vitro oncosphere killing induced by anti-EG95 antibodies. These results indicate that the major part of the immune response induced by EG95 vaccination is directed against conformational epitopes and that the host-protective epitope(s) is/are conformational.

Inhibition of neutral endopeptidase, the degradative enzyme for natriuretic peptides, in rat kidney after oral SCH 42495.

1. Inhibition of neutral endopeptidase (NEP), the degradative enzyme for atrial natriuretic peptide, was studied in vitro and in vivo using a previously characterized NEP inhibitor radioligand, 125I-labelled RB104. 2. SCH 42354, the active di-acid of the ethylester prodrug, SCH 42495, caused a concentration-dependent displacement of 125I-labelled RB104 from rat renal NEP. The concentration of SCH 42354 that displaced 50% of radioligand bound to the enzyme NEP (IC50) was 3.3 +/- 0.1 nmol/l (mean +/- SEM). Enalaprilat, an angiotensin converting enzyme inhibitor, did not displace 125I-labelled RB104 in concentrations up to 10 mumol/l. 3. In adult normotensive Sprague-Dawley rats, oral SCH 42495 (3-300 mg/kg) caused significant inhibition of renal NEP (P < 0.001). SCH 42495 had no effect on renal or plasma angiotensin converting enzyme activity, but high-dose SCH 42495 (300 mg/kg) caused a significant increase in plasma renin activity (P < 0.01). 4. In a time course study, oral SCH 42495 (30 mg/kg) caused rapid (within 30 min) and significant inhibition of renal NEP for up to 48 h (P < 0.001). No changes in plasma atrial natriuretic peptide or plasma angiotensin converting enzyme activity were seen. 5. These data provide evidence that short-term administration of the NEP inhibitor SCH 42495 results in inhibition of renal NEP and does not inhibit the circulating or the tissue renin-angiotensin system. The NEP inhibitor radioligand 125I-labelled RB104, is a useful tool to study tissue NEP inhibition after administration of NEP inhibitors.