Serum C-reactive protein (CRP) concentrations in healthy beagle dogs of various ages and in pregnant beagles were measured by enzyme-linked immunosorbent assay (ELISA). Serum CRP concentrations were 1.5-16.0 µg/ml (mean 7.9 ± 3.4 µg/ml) in male, and 1.8-18.9 µg/ml (mean 8.3 ± 4.0 µg/ml) in female dogs. No significant sex-related differences were observed in the values. Further, there were no significant age-related differences either. Serum CRP concentrations increased during pregnancy. The concentration of serum CRP in pregnant dogs peaked at 70.2-90.4 µg/ml (mean 77.5 ± 7.1 µg/ml) 30 or 45 days after ovulation, demonstrating two characteristic features of CRP concentration change in pregnant dogs.

Lipoxygenase was purified homogeneously from cups of Pleurotus ostreatus by Sephacryl S-400 HR gel filtration, Dyematrex Green A affinity, and DEAE-Toyopearl 650M ion-exchange chromatographies. The molecular weight of the enzyme was estimated to be 67,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 66,000 by gel filtration; the isoelectric point was pH 5.1. The optimum pH and temperature of the enzymatic activity were 8.0 and 25 degrees C, respectively. The enzyme contained non-heme iron, and a thiol group seemed to be involved in its activity. The K(m), V(max), and k(cat) values of the enzyme for linoleic acid were 0.13 mM, 23.4 micromol.min(-1).mg(-1), and 25.7 s(-1), respectively. The enzyme showed high specificity toward linoleic acid. When linoleic acid was incubated with the enzyme, 13-hydroperoxy-9Z,11E-octadecadienoic acid was found to be the main oxidative product.

Mycobacterium avium subsp. paratuberculosis (MAP) is the established causative agent of Johne's disease in cattle and other ruminants, and it has also been speculated to be a putative etiological agent of several human autoimmune diseases. It is acknowledged that dairy products deriving from infected animals play a role (could be vehicles) in exposing humans to MAP. MAP could stimulate the human immune system by means of their complex antigen (in the case of lipids, multivalent antigens) and may modulate it, acting as adjuvant molecules such as Freund's complete adjuvant. The immune system might be abnormally stimulated by the constant presence of MAP antigens (for example, in the dairy products), and this might be particularly relevant in genetically predisposed individuals. However, there is limited understanding about the current human exposure to MAP. The present study analyzed the antibody recognition profile of MAP lipophilic antigens in a cohort of 126 healthy Japanese. We measured the serum levels of total immunoglobulin G (IgG) and subclasses targeting MAP surface antigens through ethanol vortex indirect enzyme-linked immunosorbent assay (EVELISA) by using serum absorbed with Mycobacterium phlei. Elevated IgG (especially IgG1 and IgG4) responses were observed in 14% of the sera. To assess the specificity of EVELISA, the same samples were analyzed by means of a commercially available Johnelisa II kit. It was noteworthy that a high degree of correlation was observed when comparing the two methodologies (rs=0.7, p<0.0001). Moreover, in order to investigate the specificity of the binding, inhibition assay experiments were carried out also searching for antibodies against Bacillus Calmette-Guérin antigens, but no cross-reaction was observed. The result obtained represents the first evidence implying that the Japanese population is exposed to MAP, and additionally the existence of a foodborne chain of exposure that transmits MAP antigens to humans.

Biological monitoring of workers exposed to N,N-dimethylformamide (DMF) was carried out by determination of the urinary metabolites, N-methylformamide (MF, mainly from N-hydroxymethylformamide) and N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC), which were derived from two different routes of metabolism of the solvent. The urinary levels of MF increased rapidly at the start of the work shift, and decreased almost to zero within 24 h after the beginning of the last exposure. The highest level was found between the end of the afternoon shift and bedtime. AMCC levels remained constant over the consecutive work days and increased after the cessation of exposure, with the peak concentration being observed at 16-40 h after the cessation of exposure. AMCC levels at the beginning of the next morning shift were closely correlated with personal exposure levels of DMF in air, although the correlation of MF and DMF in air was highest in the urine at the end of the shift. Hence urinary AMCC represents an index of the average exposure during several preceding work days and may indicate the internal dose. By contrast, MF represents an index of daily exposure.

The elimination half-lives of in Interleukin-6 (IL-6) and cytokine-induced neutrophil chemoattractant-1 (CINC-1) in rats after inflammatory stimulation were investigated. Five male Sprague-Dawley rats were used (age, 9 weeks; body weight, 235–375 g). Turpentine oil was intramuscularly injected at a dose of 2 mL/kg body weight to induce acute inflammation. Blood was collected pre-injection and 6, 12, 24, 36, 48, 60, 72, 84, and 96 h after the turpentine oil injection. Serum concentrations of IL-6, CINC-1, and α2-macroglobulin (α2M) were measured by enzyme-linked immunosorbent assay. Half-lives were calculated as 0.693/elimination rate constant. The serum concentration of α2M peaked at 48 h after turpentine oil injection. Serum concentrations of IL-6 and CINC-1 increased and peaked at 12 and 24 h, respectively. The terminal elimination half-lives of IL-6 and CINC-1 were 15.5 and 29.9 h, respectively. The half-life of CINC-1 was significantly longer than that of IL-6 (P=0.006). These results suggested that these cytokines synthesized in response to inflammatory stimulation were rapidly eliminated in rats. The serum concentrations of these cytokines should be measured at an early stage if these cytokines will be used as surrogate inflammatory markers instead of acute-phase proteins.

The kinetics of a 2 -macroglobulin (a2M) and a 1 -acid glycoprotein (AAG) in rats repeatedly stimulated with intramuscular injections of turpentine oil at doses 0.05 and 0.4 mL/rat were investigated. Mean serum levels of a2M peaked at 48 h after the first turpentine oil injection, reaching 1.74 and 2.36 mg/mL at 0.05 and 0.4 mL/rat, respectively. AAG peaks were also observed at 48 h after injection, and the mean values were 2.02 and 2.53 mg/mL, respectively. These peak values of a2M and AAG differed significantly between the 0.05 and 0.4 mL/rat injection groups. Mean serum levels of interleukin-6 (IL-6) at 0.05 mL/rat were 52.61 pg/mL at 12 h, 48.86 pg/mL at 36 h and 81.93 pg/mL at 84 h after the first injection. Mean IL-6 serum levels at 0.4 mL/rat were 215.24 pg/mL at 12 h, 56.33 pg/mL at 36 h and 39.25 pg/mL at 84 h after the first injection. Mean serum levels of cytokineinduced chemoattractant-1 (CINC-1) at a dose of 0.05 mL/rat were 5.70 ng/mL at 12 h, 5.58 ng/mL at 36 h and 4.58 ng/mL at 84 h after the first injection. Mean serum levels of CINC-1 after injection at 0.4 mL/rat were 11.57 ng/mL at 12 h, 4.68 ng/mL at 36 h and 4.42 ng/mL at 84 h. Serum levels of IL-6 differed significantly at 12, 24, 72 and 84 h, while those of CINC-1 differed significantly at 12, 24, 48 and 96 h between the 0.05 and 0.4 mL/rat injection groups. Differences in peak serum levels in the 0.05 and 0.4 mL/rat groups were attributed to differences in the production of IL-6 and CINC-1, which are thought to contribute to a2M and AAG production.

Abstract. Serum alpha 1-acid glycoprotein (AAG) levels were measured in healthy beagles of various ages (66 male and 74 female) by turbidimetric immunoassay (TIA), and then separately -in pregnant beagles -by single radial immunodiffusion (SRID). The first experiment revealed that serum AAG levels ranged from 40 to 960 µg/ml (mean of 322 ± 202 µg/ml) in male dogs, and from 47 to 833 µg/ml -in female dogs (mean of 316 ± 199 µg/ ml), without any significant sex-or age-related variation. The second experiment, however, revealed that serum AAG levels increased in all pregnant beagles and peaked in the middle of gestation at 250-1,000 µg/ml (mean of 634 ± 246 µg/ml). In 7 of 8 dogs the AAG levels peaked about 45 days after ovulation. Despite a high value of 1,210-1,360 µg/ml being observed for serum AAG levels in 3 pregnant beagles inoculated with Staphylococcus aureus, its levels in umbilical cord blood were below the detection limit of SRID (40 µg/ml).

The relationship between intensity of inflammatory stimulation and production of α2-macroglobulin (α2M) and α1-acid glycoprotein (AAG) in rats was investigated. Sprague-Dawley rats were injected with turpentine oil at doses of 0.05, 0.2 or 0.4 mL/rat. Serum levels of α2M, interleukin (IL)-6 and cytokine-induced neutrophil chemoattractant-1 (CINC-1) were measured by enzyme-linked immunosorbent assay, and AAG was measured by single radial immunodiffusion. Peak serum levels of α2M and AAG in rats injected at 0.05 mL/rat were significantly lower than those at 0.2 or 0.4 mL/rat. However, no significant differences were observed for peak serum levels of these acute-phase proteins between 0.2 and 0.4 mL/rat. Furthermore, peak serum levels of IL-6 and CINC-1 in rats injected at 0.05 mL/rat were significantly lower than those at 0.2 or 0.4 mL/rat. Thus, the production of these acute-phase proteins has upper limits, even under increased strength of inflammatory stimulation in rats injected with turpentine oil.