Suitability of FTIR to distinguish pure cultures of problematic mould species from closely related species in the meat industry.

AIMS: The aim of the study was to apply Fourier Transform Infrared spectroscopy (FTIR) as a rapid screening method for moulds in a specific food production environment (cured meat) and to evaluate whether the method was sufficiently accurate to distinguish Penicillium species that constitute a hazard for the food quality and safety (Penicillium solitum and Penicillium nordicum) from closely related species. METHODS AND RESULTS: FTIR was applied to classify the indigenous mycobiota of two production sites for dried and cured meat products in Norway. Results showed that FTIR was suitable to analyse large amounts of data. While correct classification rates varied depending on the species, overall results indicated that FTIR was able to distinguish the undesired mould species P. solitum and P. nordicum from other species and may hence present an option for rapid screening of large numbers of samples to identify changes in mould composition on site. CONCLUSIONS: FTIR presents a potential method for detecting changes in levels of undesired fungi in meat-processing environments. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study that applies FTIR to a specific food production environment and it increases the knowledge on both possibilities and limitations of the method in classification of fungi.

Etiology and attitudes: beliefs about the origins of homosexuality and their implications for public policy.

Using survey data from the 2008 election cycle, this article updates and extends analysis of public attitudes regarding various aspects of homosexuality. Continued expansion of public belief in a biological root to homosexuality is found, and variations in such opinions are explored. Public attitudes toward the emerging issue of gay adoption is also examined, finding both similarities with and important differences from attitudes toward same-sex civil unions, although both are profoundly influenced by underlying attitudes regarding the causes of homosexuality.

Anthropometric correlates of basketball free-throw shootings by young girls.

The purpose of this analysis of data from a larger investigation was to assess effects of anthropometric factors on free throw shooting performance of 15 girls from Michigan and 18 from Puerto Rico. Subjects performed 60 free throws (10 trials x 3 ball sizes x 2 basket heights). Correlations were low, with two exceptions, .53 between shooting performance at the low basket and grip strength (as measured by hand grip dynamometer) for girls from Michigan, and .49 for hand width and performance at the low basket for girls from Michigan.

Identification of amino acid residues associated with modulation of flavin-containing monooxygenase (FMO) activity by imipramine: structure/function studies with FMO1 from pig and rabbit.

The activity of the flavin-containing monooxygenase (FMO) can be modulated by a number of nitrogen-containing compounds in a manner that is both isoform and modulator-dependent. We now show that the direction (activation or inhibition) and extent of modulation can also be dependent on substrate concentration. Imipramine activates methimazole metabolism catalyzed by rabbit FMO1 or FMO2 at methimazole concentrations greater than 50 or 100 microM, respectively, and inhibits at lower methimazole concentrations. The extent of the activation increases as the substrate concentration increases, and the extent of inhibition increases as the substrate concentration decreases. With either inhibition or activation, the magnitude of the effect shows a similar, direct dependency on imipramine concentration. In contrast, imipramine inhibits the metabolism of methimazole catalyzed by pig FMO1 at all substrate concentrations. The structural basis for this unique ortholog difference between the responses of rabbit and pig FMO1 to imipramine was studied by random chimeragenesis and site-directed mutagenesis. Results with chimeras indicated that modulation of FMO1 activity by imipramine is controlled to a great extent by two areas of the FMO primary structure (residues 381-432 and 433-465). Four amino acids in these regions (positions 381, 400, 420 and 433) and one additional residue (position 186) were identified by site-directed mutagenesis as primary determinants of the imipramine response. When the residues at these positions in rabbit FMO1 are exchanged for the corresponding residues of pig FMO1, a mutant with the functional properties of pig FMO1 is produced. Our results suggest that the response of FMO1 to imipramine involves a distribution between two sites that is regulated by structural features that do not alter the overall binding. The inhibition observed, although it appears to be competitive, likely does not involve competition for a binding site since alteration of imipramine metabolism has no effect on the parameters of methimazole metabolism.

Quantitation and kinetic properties of hepatic microsomal and recombinant flavin-containing monooxygenases 3 and 5 from humans.

Variable amounts of flavin-containing monooxygenase isoforms 3 and 5 (FMO3 and FMO5) are present in microsomal preparations from adult, male, human liver. Quantitation with monospecific antibodies and recombinant isoforms as standards showed levels of FMO3 and of FMO5 that ranged from 12.5 to 117 and 3.5 to 34 pmol/mg microsomal protein, respectively. The concentration of FMO3 was greater than that of FMO5 in all samples, but the ratio of FMO3 to FMO5 varied from 2:1 to 10:1. Human hepatic microsomal samples also showed variable activities for the S-oxidation of methimazole. This activity was associated totally with FMO3; no participation of FMO5 was apparent. This conclusion was supported by several lines of evidence: first, the catalytic efficiency of FMO3 with methimazole was found to be approximately 5000 times greater than that of FMO5; second, the rate of metabolism showed a direct, quantitative relationship with FMO3 content; third, the plot of the relationship between metabolism and FMO3 content extrapolated close to the origin. A second reaction, the N-oxidation of ranitidine, exhibited a much higher Km with recombinant FMO3 than did methimazole (2 mM vs. 35 microM). However, a direct relationship between this reaction and FMO3 content in human hepatic microsomal preparations was also apparent. This result shows that even with a high Km substrate, FMO3-catalyzed metabolism can account for the majority of the product formation with some drugs. Our findings demonstrate that the contribution of FMO isoforms to human hepatic drug metabolism can be assessed quantitatively on the basis of the characteristics of the enzymes expressed in Escherichia coli.

Oxidation of cysteine S-conjugates by rabbit liver microsomes and cDNA-expressed flavin-containing mono-oxygenases: studies with S-(1,2-dichlorovinyl)-L-cysteine, S-(1,2,2-trichlorovinyl)-L-cysteine, S-allyl-L-cysteine, and S-benzyl-L-cysteine.

Rabbit liver microsomes catalyzed the highly stereoselective, NADPH- and time-dependent S-oxidation of S-benzyl-L-cysteine (SBC), S-allyl-L-cysteine (SAC), S-(1,2-dichlorovinyl)-L-cysteine (DCVC), and S-(1,2,2-trichlorovinyl)-L-cysteine (TCVC) to their respective sulfoxides. Methimazole, a flavin-containing mono-oxygenase (FMO) substrate, inhibited S-oxidation of all four conjugates. The cytochrome P450 inhibitor 1-benzylimidazole did not affect SAC, SBC, or DCVC S-oxidation but inhibited the S-oxidation of TCVC. Solubilization of microsomes also inhibited TCVC activity, whereas SBC, SAC, and DCVC activities were not affected. Because these results suggested that FMOs were the major catalysts of SBC, SAC, and DCVC sulfoxidations, the four conjugates were evaluated as substrates for cDNA-expressed rabbit FMO isoforms FMO1, FMO2, FMO3, and FMO5. At equimolar concentrations (10 mM), FMO1 S-oxidized SBC and SAC, but no sulfoxides were detected with DCVC or TCVC. FMO3 S-oxidized all four conjugates. Km values determined with FMO3 were comparable to Km values from rabbit liver microsomes. S-Oxidation by FMO2 was detected only with SAC, and no sulfoxides were detected in incubations with FMO5. These results show that FMO isoforms can catalyze cysteine conjugate S-oxidation and that the specific isoform involved depends on the structure of the cysteine conjugate. The cysteine conjugates with more nucleophilic sulfur atoms, SAC and SBC, were much better FMO substrates than those having the less nucleophilic sulfur atoms DCVC and TCVC. The sulfoxides of TCVC and DCVC were reactive toward GSH, whereas the sulfoxides of SBC and SAC were not reactive. These results provide evidence for different chemical reactivities of these sulfoxides.

Characterization of the methionine S-oxidase activity of rat liver and kidney microsomes: immunochemical and kinetic evidence for FMO3 being the major catalyst.

Methionine is oxidized to methionine sulfoxide by rat liver and kidney microsomes in an O2- and NADPH-dependent manner. In all microsomal assays, no methionine sulfone was detected. Use of a monoclonal antibody to rat liver cytochrome P-450 reductase, various cytochrome P-450 and peroxidase inhibitors, antioxidants, and competitive flavin-containing monooxygenase (FMO) substrates suggested that methionine sulfoxidation was exclusively mediated by FMOs. At 5 mM methionine, the d-isomer of methionine sulfoxide was preferentially detected over the l-isomer in both liver (ratio, 5:1) and kidney microsomes (ratio, 12:1); however, at 30 to 40 mM methionine concentrations, the diastereomeric ratio was reduced to approximately 3:1 in both tissues. The Vmax/K(m) ratios determined for the liver and kidney microsomes were similar. Because cDNA-expressed rabbit FMO3 and FMO1 were previously shown to preferentially catalyze methionine and S-benzyl-L-cysteine (SBC) sulfoxidations, respectively, these substrates were used to isolate two distinct S-oxidase activities from the same rat liver microsomal preparation. The purified activities have apparent molecular weights of approximately 55 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The findings that the methionine S-oxidase reacted intensely with antibodies raised against rabbit FMO3 and the SBC S-oxidase reacted intensely with antibodies raised against rabbit FMO1 provide evidence for these activities being associated with FMO3 and FMO1, respectively. The apparent methionine K(m) determined with the purified methionine S-oxidase was 3.4 mM, whereas the apparent methionine K(m) determined with the purified SBC S-oxidase was 48 mM. The methionine sulfoxide d:l diastereomeric ratio obtained with methionine S-oxidase was 15:1, whereas the diastereomeric ratio obtained with SBC S-oxidase was only 2:1. These results provide strong evidence for the expression of both FMO1 and FMO3 in rat liver microsomes and suggest that FMO3 is the major catalyst of methionine sulfoxidation in rat liver and kidney microsomes.

Expression of xenobiotic-metabolizing enzymes in cultured rat tracheal epithelial cells.

Rat tracheal epithelial (RTE) cells were cultured on membrane support with and without retinoic acid (RA). In early (6-day-old) cultures, the epithelium is a monolayer or bilayer of undifferentiated cells and secretes little mucuslike product either in the absence or presence of RA. In late (12- to 15-day-old) cultures, the epithelium differentiates as a mucociliary epithelium in the presence of RA and as a squamous epithelium in the absence of RA. The purpose of our study was to determine whether a number of xenobiotic enzymes are expressed in these cultures and whether their expression depends on the state of differentiation. Enzyme expression was characterized by electrophoresis and immunoblotting as a function of time in culture and phenotypic differentiation. Cytochrome P450 1A1 was not expressed in freshly harvested RTE cells. This isoenzyme was induced in rats by gavage with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or by exposure of early RTE cell cultures to TCDD, provided RA was also added to the cultures. Cytochrome P450 2B1 was observed in freshly isolated RTE cells, but not in early or late RTE cultures. In contrast, expression of NADPH-cytochrome P450 reductase was decreased in early cultures, but was increased in well-differentiated cultures. Flavin-containing monooxygenase was detected in lung tissue, but not in freshly harvested or cultured RTE cells. Glutathione S-transferase (GST) mu and pi were expressed in freshly harvested RTE cells. GST pi was expressed in early and late cultures, whereas GST mu was expressed in late cultures, but could not be found in early cultured RTE cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of flavin-containing monooxygenase 5 (FMO5) cloned from human and guinea pig: evidence that the unique catalytic properties of FMO5 are not confined to the rabbit ortholog.

Several full-length clones encoding the human and guinea pig orthologs of flavin-containing monooxygenase 5 (FMO5) have been isolated from libraries constructed with hepatic mRNA. The clones were detected by hybridization with the cDNA encoding FMO5 expressed in rabbit. The human and guinea pig cDNAs encode for proteins of 533 amino acids that contain putative pyrophosphate binding domains characteristic of mammalian FMOs. The sequences derived for the human and guinea pig FMO5 proteins are 87% identical and are 85 and 82% identical, respectively, to the sequence of rabbit FMO5. As is the case with other FMOs, FMO5 in human and guinea pig is encoded by multiple transcripts. Rabbit FMO5 expressed in Escherichia coli was purified and used to elicit antibodies in goat. These antibodies detected FMO5 in samples from livers of adult humans, rabbits, and guinea pigs and fetal livers of humans. The human and guinea pig forms of FMO5 were expressed in E. coli and characterized. Neither enzyme effectively catalyzed the metabolism of methimazole, a general FMO substrate; however, both were active with n-octylamine. The responses of the human FMO5 and guinea pig FMO5 to detergent, ions and elevated temperature are all similar to the responses described for rabbit FMO5. These results indicate that the unique properties of FMO5 from rabbit are species-independent and that this form of the flavin-containing monooxygenase is not readily classified as a drug-metabolizing enzyme.

Detection of antibodies to avian leukosis/sarcoma viruses and reticuloendotheliosis viruses in humans by western blot assay.

Serologic evidence of antibodies in humans to avian leukosis/sarcoma viruses (ALSV) and reticuloendotheliosis viruses (REV) has in general been negative. Because of the difficulty in infecting mammalian cells in vitro with these viruses, it is generally held that they do not infect humans. We first provided presumptive evidence of serologic response to these viruses in human sera of workers in poultry slaughtering plants, using an ELISA. We now provide confirmatory evidence using Western blot assay. Our results show that exposed poultry workers and subjects with no occupational exposure to these viruses have antibodies in their sera specifically directed against ALSV p27, p19, p15, and p12 antigens. In addition, we demonstrate evidence of serologic response to REV. This is the first time definitive evidence of exposure to ALSV or REV has been demonstrated in human sera. The significance of this is not known. Further investigation into whether these findings mean that virus has been integrated into the human genome is needed, to assess the public health implications of these results.

Unique distribution profiles of glutathione S-transferases in regions of kidney, ureter, and bladder of rabbit.

BACKGROUND: Glutathione S-transferases detoxify a broad range of exogenous compounds, but are important also in the metabolism of endogenous compounds. Physiologically relevant substrates are the endoperoxide and hydroperoxide metabolites of arachidonic acid that play important roles in many tissues including the kidney. EXPERIMENTAL DESIGN: We used immunohistochemical and immunoblotting techniques in a systematic study of renal localization of four rabbit enzymes that represent three major mammalian cytosolic glutathione S-transferase classes, alpha, pi, and mu. RESULTS: The two alpha-class enzymes (rbGST alpha I, rbGST alpha II) were distributed discretely in kidney, ureter, and bladder, while pi and mu were widely distributed in the renal system. Immunohistochemical localization in paraffin sections with antibodies specific for rbGST alpha I or rbGST alpha II demonstrated that no compartment of the renal system contained both enzymes. Collecting ducts of the inner medulla and all epithelial cells of the kidney pelvis, ureter, and bladder contained rbGST alpha I. All cells lining proximal tubules contained rbGST alpha II. No other compartment of the renal system exhibited immunoreactivity with anti-rbGST alpha II. Antibody specific for pi reacted with cells lining nephrons, ureter, and bladder and with endothelial cells throughout the renal system. Localization of pi was most prominent in the collecting ducts of medulla and in the epithelial cells lining the kidney pelvis, ureter, and bladder. As anti-mu did not react in tissue sections, distribution of mu was determined by immunoblotting. Immunoblots of cytosolic preparations from whole kidney, cortex, medulla, and epithelia of ureter, bladder, and kidney pelvis were prepared and tested with each of the 4 antibodies. This second localization method confirmed the distribution data from tissue sections for rbGST alpha I, rb GST alpha II, and pi; also, it demonstrated that the staining observed in tissue was specifically for each enzyme. mu was detected in all the renal cytosolic preparations except those from the epithelium of the kidney pelvis. CONCLUSIONS: The discrete renal distribution of rbGST alpha I and rbGST alpha II and their distinct catalytic activities with prostaglandin substrates suggest important roles for these enzymes in prostaglandin-dependent renal functions.

Hepatitis C: looking at a virus that hasn't been seen.

Hepatitis C (HCV) is the first virus to be discovered by molecular cloning without direct use of biological or biophysical methods. HCV was first recognised in 1974 as non-A, non-B (NANB) hepatitis resulting from blood transfusions. It took almost 15 years to identify it successfully--by detecting a clone in a library of cDNA prepared from the nucleic acids extracted from plasma known to be infectious for chimpanzees. The clone was derived from a positive-sense RNA of about 10,000 nucleotides, with a long open reading frame encoding for a polyprotein of about 3000 amino acids. The structure of the RNA and the encoded polyprotein had properties similar to known flaviviruses and pestiviruses. Functions of viral proteins produced by proteolytic cleavage of the polyprotein are estimated by analogy with known viruses of similar genomic organisation. Each of the HCV proteins has been produced in recombinant organisms and used as an antigen in immunoassays to investigate serological responses during the course of infection. Seroconversions to both structural and non-structural antigens are observed during the course of disease but typical diagnostic serological patterns have not yet evolved. Immunoassays for HCV antibodies reacting with recombinant antigens are used widely for screening blood donations and for studying the epidemiology of HCV infection. Comparisons of the nucleic acid sequences from different isolates of HCV have shown considerable variability throughout the genome. The importance of this genomic heterogeneity will be a challenging problem for the future.

Interstitial pulmonary macrophages produce platelet-derived growth factor that stimulates rat lung fibroblast proliferation in vitro.

Alveolar macrophages from humans and several animal species produce factors in vitro that modulate fibroblast growth and have been proposed as mediators of interstitial pulmonary fibrosis. Pulmonary interstitial macrophages (IMs) have not been studied previously in this regard. Pulmonary IMs were isolated from prelavaged rat lungs by enzymatic digestion of tissue and subsequent differential adherence of cells to culture dishes. The ability of IMs to release modulators of fibroblast growth into the culture medium was assessed by measuring [3H]thymidine incorporation into DNA and/or nuclear labeling of early-passage rat lung fibroblasts exposed to medium conditioned by IMs. The percentages of nuclei labeled in fibroblast cultures exposed to interstitial macrophage-conditioned medium (IMCM) alone did not significantly differ from that observed in controls, but fibroblasts exposed to IMCM supplemented with 2% platelet-poor plasma showed a 2.6-fold increase in labeling, indicating that IMCM contains predominantly "competence" growth factor activity. Similar results were obtained using purified human platelet-derived growth factor (PDGF). The level of growth factor activity released by IMs increased in cells that had phagocytized iron spheres during the culture period. In addition, fractionation of IMCM by high-performance liquid chromatography demonstrated most of the growth factor activity at a relative molecular mass of about 35 kd. Subsequent quantitative analysis of the fractions by an enzyme immunoassay for PDGF demonstrated that IMCM contains a homologue of human PDGF. These results show that IMs are capable of producing a PDGF-like growth factor for autologous fibroblasts and that release of this factor is enhanced by exposure to an insoluble inorganic particle. Because PDGF is a potent growth factor for fibroblasts and is released by IMs, it is essential to ask in future studies whether this or similar macrophage products play a significant role in mediating fibroblast proliferation in vivo.

Distribution of cytochrome P450 1A1 and NADPH-cytochrome P450 reductase in lungs of rabbits treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin: ultrastructural immunolocalization and in situ hybridization.

Induction of cytochrome P450 1A1 (P450 1A1) in a variety of tissues is a well established consequence of exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Although localization of the induced protein within the lung has been described, the precise intracellular distribution of the enzyme is not clear. Analysis of tissue sections, microsomal proteins, and mRNA from lungs of treated and untreated rabbits established that P450 1A1 had been induced by treatment with TCDD. Rabbit lungs from animals treated with TCDD were examined with immunocytochemistry and in situ hybridization, to identify the cell types that contain P450 1A1 and those that contain mRNA encoding P450 1A1. Endothelial cells of the entire vascular bed of rabbit lung reacted markedly with anti-P450 1A1. Likewise, cells lining both arteries and veins, as well as capillary endothelial cells, reacted strongly with the cDNA probe for mRNA encoding P450 1A1. Clara cells at all levels of airway labeled prominently for both P-450 1A1 and P450 1A1 mRNA. In addition, type 2 cells, alveolar macrophages, and to a lesser degree, ciliated cells reacted with the cDNA probe. P450 reductase, which is required for P450 activity, has previously been identified in Clara cells, type 2 cells, and alveolar macrophages, but not in endothelium of rabbit lung. We have now obtained similar results for the localization of mRNA encoding P-450 reductase. This finding brings into question the function of P450 1A1 in endothelium.

Perceptual asymmetry in psychosis-prone college students: evidence for left-hemisphere overactivation.

The hypothesis that psychosis-prone students demonstrate a pattern of exaggerated perceptual asymmetry across both left- and right-hemisphere dichotic-listening tasks (consonant-vowel [CV] and tonal contour discrimination) was investigated. Subjects who scored high on the Perceptual Aberration or Magical Ideation scale or both (n = 20) demonstrated a significantly exaggerated right-ear advantage on a CV task in relation to normal control subjects (n = 27) but showed a reduced left-ear advantage on a tone task. The hypothesis of exaggerated functional lateralization across hemispheres in the psychosis-prone subjects was not supported, but the results are consistent with a hypothesis of left hemisphere overactivation in this sample.

Cellular localization of flavin-containing monooxygenase in rabbit lung.

A specific form of flavin monooxygenase has been identified in the lungs of a number of species. Distribution of the pulmonary flavin-containing monooxygenase (FMOp) is of interest because it oxidatively metabolizes a wide variety of nitrogen-, sulfur-, and phosphorous-containing xenobiotics, some of which form highly toxic reactive intermediates. We have identified the nonciliated bronchiolar epithelial (Clara) cell as the predominant location for this enzyme in rabbit lung. In addition, protein in ciliated, endothelial, type I, and type II cells and in tracheal lining layer reacted with antibodies to FMOp. In all these cell types antigen was found associated with cytoplasmic organelles, and in the Clara cell antigen was most concentrated in areas rich in smooth endoplasmic reticulum. Staining of ciliated surfaces was also observed at both the light and electron microscopy levels. Extracellular antigen was also apparent in tracheal lining layer smeared onto glass slides. We compared the location of the FMOp with that of two enzymes of the cytochrome P-450 monooxygenase system (studied here and elsewhere), cytochrome P450 IIB (P450 IIB), and NADPH cytochrome P450 reductase (reductase), and concluded that (1) FMOp is detected in all cells where P450 IIB and reductase are both present (Clara, type II, and ciliated); (2) FMOp and P450 IIB, but not reductase, are detected in endothelial cells; (3) P450 IIB alone is detected in the plasma membrane, cilia, and microvillae of ciliated cells and plasma membrane of endothelial cells; and (4) FMOp alone is detected in type I cells.

Lung cell toxicity experimentally induced by a mixed dust from Mexicali, Baja California, Mexico.

Lung disease caused by nonoccupational exposures to inorganic particles from the soil has been reported in several areas of the world. We tested the toxic potential of dust samples from a Mexican city (Mexicali) that is frequently affected by dust storms and is geographically related to the area of San Diego, CA, where constituents of the soil have been reported to be fibrogenic. We found that samples of Mexicali dust are a mixture of approximately 75% potassium aluminum silicates (illite) and approximately 20% silica. Respirable size particles were highly hemolytic and induced lactic dehydrogenase release from alveolar macrophages exposed in vitro. Animals instilled intratracheally with the dust developed a multifocal interstitial lung disease associated with deposits of the aluminum silicates, which were identified by X-ray microanalysis. Inhalation studies in rats demonstrated that the majority of particles were deposited preferentially at the first alveolar duct bifurcations. Twenty-four hours later, numerous particles had been ingested by alveolar macrophages that had migrated to those sites of deposition. It is proposed that alveolar macrophages are attracted to the deposited particles by complement fragments since Mexicali dust is capable of activating complement proteins from both serum and bronchoalveolar lavage. Activation resulted in alveolar macrophage chemotaxis. Mexicali dust induced biological activities and lung changes similar to those of asbestos and silica, suggesting that this material could be an etiologic agent of pulmonary fibrosis in exposed individuals.

Hepatitis C virus: the major causative agent of viral non-A, non-B hepatitis.

A 'blind' recombinant immunoscreening approach, of general application to studies of infectious diseases, was used to clone and identify the genome of the previously uncharacterized non-A, non-B hepatitis (NANB) virus. This agent is a positive-stranded RNA virus that appears to be distantly related to the flaviviridae family. A recombinant viral antigen (C100-3) was used to develop a capture assay for circulating antibody. Data obtained using this assay indicate that this agent, termed the hepatitis C virus (HCV), is the major cause of post-transfusion, community-acquired and cryptogenic, NANB. Anti-C100-3 antibody appears to be directed towards dominant, non-structural viral epitopes. It is a non-neutralising antibody that develops generally late in infection and is a particularly good marker of chronic, persistent viraemia. Many asymptomatic but infectious blood donors can now be detected using this antibody assay. HCV is associated with the development of hepatocellular carcinoma and possibly, other liver diseases.

Comparison of arachidonic acid metabolism by pulmonary intravascular and alveolar macrophages exposed to particulate and soluble stimuli.

Pulmonary intravascular macrophages, as prominent components of the pulmonary mononuclear phagocyte system, could be significant mediators of lung inflammation. We have shown that intravascular and alveolar macrophages metabolize exogenous arachidonic acid to its inflammatory metabolites via the lipoxygenase and cyclooxygenase pathways after exposure to the calcium ionophore A23187. In this study, we compare the metabolism of endogenous arachidonic acid by porcine intravascular and alveolar macrophages after exposure to soluble and particulate stimuli. Since intravascular and alveolar macrophages are exposed to various stimuli in vivo, it is essential to know the range of inflammatory mediators that these cells can generate. Alveolar macrophages attached to plastic and exposed to the various stimuli produced prostaglandin F2 alpha, 12-hydroxyheptade-catrienoic acid (HHT), hydroxyeicosatetraenoic acids (HETE), and leukotriene B4. In contrast, adherent and stimulated intravascular macrophages produced several cyclooxygenase products and lipoxygenase products including 5-HETE, 12-HETE, and leukotriene B4. Both macrophages released large amounts of arachidonic acid upon exposure to each stimulant. Intravascular macrophages that were adherent to plastic or were stimulated with glass, asbestos, or A23187 released significantly (p less than 0.05) more metabolized arachidonic acid than similarly treated alveolar macrophages. The major cyclooxygenase metabolite released by alveolar macrophages was prostaglandin 2 alpha, whereas HHT was the primary metabolite of intravascular macrophages. The major lipoxygenase metabolite released by both macrophage types was 5-HETE, but intravascular macrophages also released substantial amounts of 12-HETE and leukotriene B4. In both macrophage preparations, lipoxygenase products composed most released metabolites. After exposure to iron, asbestos, and A23187 intravascular macrophages released significantly more (p less than 0.05) lipoxygenase metabolites than alveolar macrophages. However, in alveolar macrophages, chrysotile asbestos induced greater activity by the cyclooxygenase pathway than by the lipoxygenase pathway. Both asbestos and iron spheres induced release of arachidonic acid and its metabolites, but the most potent stimulants in both macrophage preparations were A23187, zymosan, and lipopolysaccharide. We conclude that stimulated intravascular macrophages use both cyclooxygenase and lipoxygenase pathways to metabolize endogenous arachidonic acid, that these macrophages are metabolically more active than alveolar macrophages, and that both macrophage types are induced to metabolize arachidonic acid by various particulate and soluble stimuli. Furthermore, we have shown that intravascular macrophages predominantly utilize the lipoxygenase rather than cyclooxygenase pathways to metabolize endogenous arachidonic acid.