Reduced interferon-gamma secretion by natural killer cells from rats susceptible to postviral chronic airway dysfunction.

After parainfluenza type 1 (Sendai) virus infection as weanlings, Brown Norway (BN), unlike Fischer 344 (F344), rats develop an asthma-like phenotype. Reduced postinfection interferon (IFN)-gamma levels in bronchoalveolar lavage fluid from BN weanlings and the prevention of chronic airway sequelae in BN rats by IFN-gamma treatment led to the hypothesis that cells from BN weanlings have a reduced ability to secrete IFN-gamma. After stimulation with Sendai virus or interleukin (IL)-12, splenocytes from uninfected BN weanlings secreted significantly less IFN-gamma than did splenocytes from F344 weanlings (P < 0.005), as determined by enzyme-linked immunosorbent assay. Because levels of potential IFN-gamma-secreting cells in the spleen differed between the strains, natural killer (NK) cells, an important IFN-gamma source during early antiviral responses, were purified from spleens of uninfected weanlings. When stimulated with IL-12, BN NK cells secreted significantly less IFN-gamma than did F344 NK cells (P < 0.001). Incubation of NK cells from either strain with IL-12 and IL-18 resulted in synergistic increases in IFN-gamma production, but BN cells still secreted significantly less IFN-gamma than did F344 cells (P < 0.05). Similarly, after incubation with either IFN-alpha or IFN-alpha plus IL-18, BN NK cells secreted significantly less IFN-gamma than did F344 NK cells (P < 0.05). Therefore, reduced IFN-gamma secretion by NK cells in BN weanlings may play a role in the development of postviral chronic airway dysfunction.

Direct determination of free triiodothyronine (T3) in undiluted serum by equilibrium dialysis/radioimmunoassay (RIA).

UNLABELLED: We have devised a practical, sensitive, and reliable assay for measurement of free T3 concentration in serum. The assay employs a convenient and disposable plastic equilibrium dialysis cell and a buffer that resembles the in vivo biochemical environment (Nelson JC, Tomei RT 1988 Clin Chem 34:1737). A 200-microliters aliquot of serum was dialyzed against 2.4 mL buffer at 37 degrees C for 18 +/- 2 h and T3 was quantified by RIA of about 1.0-mL aliquot of the dialysate buffer. The detection threshold of the RIA approximated 2 pg/ml permitting accurate measurement of > 200 pg/dL of free T3 directly. Serum specimens that contained less free T3 were spiked with 200 ng/dL of non-radioactive T3 prior to dialysis. Free T3 in the dialysate of these samples was divided by total T3 in serum (after spiking) to determine percent free T3. Free T3 was calculated by multiplying percent free T3 and serum total T3 (before spiking). Free T3 concentration (pg/dL) did not differ appreciably in a serum pool when tested both with and without spiking with exogenous T3. The between assay coefficient of variation of three specimens tested over an 8-month period averaged 20%. Serum free T3 concentration (pg/dL) was [mean +/- SD (n), range, p] [293 +/- 12 (39), 154-440] in normal subjects. It was significantly increased [742 +/- 87 (13), 525-1700, p < 0.001] in hyperthyroidism and significantly decreased in nonthyroidal illness [NTI, 138 +/- 26 (9), 53-320, p < 0.001], cord blood serum [124 +/- 7.5 (11), 93-353, p < 0.001], and third trimester of pregnancy [214 +/- 26 (8), 93-253, p < 0.02]. Serum free T3 concentration varied widely in hypothyroidism 274 +/- 92 (10), 10-923, NS]. CONCLUSIONS: We have described a practical method and initial results of direct measurements of free T3 concentration in health and disease.

Differential requirements for cellular cytoskeleton in human macrophage complement receptor- and Fc receptor-mediated phagocytosis.

We investigated the requirement for cellular cytoskeleton in CR- and FcR-mediated phagocytosis by human monocyte-derived macrophages (M phi). Inhibition of actin microfilament (MF) assembly and stability by cytochalasins B and D completely inhibited M phi phagocytosis of sheep E coated with C3b (EC3b), iC3b (EC3bi), and IgG (EIgG) via CR1, CR3, and FcR, respectively. Ligand-binding to either CR or FcR was not effected by cytochalasins. Nocodazole (NOC), which prevents microtubule (MT) polymerization, and taxol, which causes random polymerization of MT inhibited M phi phagocytosis of EC3b(i) but not EIgG. However, the combination of taxol (5 x 10(-4) M) and NOC (2 x 10(-6) M) augmented M phi CR-mediated phagocytosis. In addition, agents known to increase intracellular cGMP augmented phagocytosis of EC3b(i). Conversely, agents that increase intracellular cAMP inhibited CR-mediated phagocytosis. These agents had no effect on FcR-mediated phagocytosis, and did not effect ligand-binding to CR or FcR. PMA markedly enhanced CR- but not FcR-mediated phagocytosis, and augmentation of CR-mediated phagocytosis by PMA was inhibited by both CD and NOC. In contrast, the synthetic diacylglycerol, 1-oleoyl-2-acetoyl-sn-3-glycerol augmented, and inhibitors of protein kinase C inhibited M phi phagocytosis via CR and FcR. These data indicate that for adherently cultured human M phi: 1) binding of ligand-coated E to CR or FcR does not require an intact cytoskeleton; 2) intact actin microfilament are required for phagocytosis via CR and FcR; 3) phagocytosis via CR1 and CR3 but not FcR is dependent on MT assembly; 4) PMA most likely augments CR-mediated phagocytosis through promotion of MT assembly; and 5) PKC activity is involved in the phagocytic signal generated by both CR and FcR.

Deposition of C3b and iC3b onto particulate activators of the human complement system. Quantitation with monoclonal antibodies to human C3.

Monoclonal antibodies were used to determine the number and molecular form of C3 bound to particulate activators of the complement (C) system by human serum. Sheep erythrocytes (E) coated with IgM (EIgM) and IgG (EIgG) were used to study activation of the classical pathway (CP). Yeast (Y), rabbit erythrocytes (ER), and five species of bacteria (Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae type 3, Streptococcus pyogenes, and Hemophilus influenzae type b) were used to study activation of the alternative pathway (AP). The deposition of C3b onto EIgM and EIgG incubated in C7-deficient human serum was dependent on the serum concentration. At all serum concentrations tested, there was complete conversion of C3b to iC3b. Kinetic analysis of C3b deposition and conversion to iC3b indicated that these events occurred almost simultaneously; the reaction was completed by 15 min. The deposition of C3 onto the AP activators ER and Y was also dependent on serum concentration, and ER, but not Y, required the presence of Mg-EGTA and thus the activation of only the AP. C3b deposition and conversion to iC3b on Y was complete in 15 min, with 82% of bound C3 converted to iC3b. For ER, maximum C3 deposition required 30 min in both the presence and absence of Mg-EGTA. However, after 1 h of incubation, 74% of bound C2 was iC3b in the absence of Mg-EGTA, compared with only 52% in the presence of Mg-EGTA. Thus, even on AP activators, a large portion of C3b may be converted to iC3b, and this conversion is probably controlled by elements on the particle's surface. Studies with the five species of bacteria yielded similar results. Approximately 3-5 X 10(4) molecules of C3 were bound per microorganism, with opsonization being completed in 30 min. Remarkably, only 16-28% of bound C3 was in the form of iC3b, even after 2 h of incubation. The presence or absence of Mg-EGTA, or the addition of purified CR1 to the reaction mixture, did not significantly effect the ratio of C3b to iC3b. Finally, SDS-PAGE and autoradiography of particle-bound 125I-C3 fragments confirmed that there was no conversion of iC3b to C3d,g or C3d. The data obtained about the opsonization of bacteria suggest that the predominant form of C3 that is encountered by inflammatory phagocytes may be C3b.

The effect of chronic marginal vitamin C deficiency on the rate of secretion and the removal of plasma triglycerides in guinea-pigs.

In guinea-pigs, chronic borderline vitamin C deficiency leads to hypertriglyceridaemia and to the accumulation of triglycerides in the liver. We investigated the triglyceride secretion rate by determining the rate of accumulation of triglycerides in the plasma following a Triton WR 1339 block of the their removal from the plasma (experiment 1) and the rate of the removal of triglycerides from the plasma by mathematical analysis of the specific plasma triglyceride activity - time curve after the administration of 3H-glycerol (experiment 2). In the 14th and 16th week of the experiment it was found that borderline vitamin C deficiency slowed down the triglyceride secretion rate, prolonged the half-time and reduced fractional plasma triglyceride turnover. The balance shift between the rate of the uptake and removal of very low density lipoprotein triglycerides (VLDL-TG) in the plasma compartment (greater retardation of the catabolic phase of kinetics) is probably the reason why triglycerides accumulate in the plasma and liver compartment of guinea-pigs with borderline vitamin C deficiency.

Effect of infrequent feeding and increased physical activity on cholesterol kinetics in the rat.

The authors studied the effect of chronic physical exercise (running in a rotating drum at 850 m/hour, 5 times a week for 16 weeks) on the size of the cholesterol body pools and on cholesterol kinetics in adult male Wistar rats fed on a standard diet either ad libitum or 2 hours daily [33 weeks]. These data were obtained by mathematical analysis of the curve expressing the correlation of specific plasma cholesterol activity to time after a single dose of cholesterol-4-14C. Chronic physical stress and infrequent feeding, as separate experimental stimuli, both caused cholesterol to shift from the blood plasma at a higher rate and reduced the size of one or both cholesterol body pools (with quick or slow turnover, pools A and B). Physical exercise also reduced fractional cholesterol turnover in pool A. When the two stimuli were combined, i.e. in infrequently fed and chronically stressed rats, the rate of the cholesterol shift from the blood plasma slowed down, the total and irreversible shift of cholesterol from pool A diminished and the production rate in this pool also fell.

Reduction of gallstone formation by ascorbic acid in hamsters.

The addition of 0.5% of ascorbic acid to the lithogenic diet of golden hamsters whose body pool was labelled with 26-14C-cholesterol, lowered the formation of gallstones, the cholesterol concentration and half-life in blood plasma and in the liver, and accelerated cholesterol transformation to bile acids.