Thyrotropin-releasing hormone cannot be detected in plasma from normal subjects

Different laboratories report widely varying average levels of TRH in plasma from normal man. However, arguments can be raised against the methods used to characterize the substances measured in all previous studies. Using a sensitive RIA and methanol extraction, immunoreactive TRH (iTRH) in serum from normal subjects averaged 49 +/- 26 pg/ml (mean +/- SD; n = 6). When SP-Sephadex purification was introduced, iTRH decreased to 1.8 +/- 0.7 pg/ml. These low quantities did not further decrease when incubated with TRH-degrading enzymes, demonstrating that the remaining iTRH was not identical to synthetic TRH. Investigations of iTRH in pools of normal plasma indicate that the level of true TRH in normal human plasma is below 0.4 pg/ml.     

FAS promoter polymorphisms correlate with activity grade in hepatitis C patients

OBJECTIVE: Hepatocytes are susceptible to FAS-mediated apoptosis. The impact of polymorphisms in the FAS gene on histopathological features of HCV infection was therefore investigated. DESIGN/METHODS: Three single-nucleotide polymorphisms in the FAS promoter were assessed in 190 patients with chronic hepatitis C. Associations between FAS haplotypes and fibrosis stage and activity grade were tested by univariate and multivariate analyses. RESULTS: While there was no correlation between FAS promoter genotype and fibrosis stage, patients carrying the GCA haplotype (P=0.03, Fisher's exact test) and those homozygous for the GTG haplotype (P = 0.06) tended to have lower activity scores. Logistic regression showed that these associations were independent of patient age, sex and alcohol consumption. In a logistic regression model incorporating only male gender (odds ratio 2.1, 95% confidence interval 1.1-4.1 P = 0.04), the presence of the GCA haplotype (OR 0.31 95% CI 0.13-0.78 P = 0.01), and GTG homozygosity (OR 0.26 95% CI 0.08-0.83 P = 0.02), all three factors were independently correlated with activity grade. Furthermore, the GTG haplotype appeared to have lower promoter activity than the wild type GTA haplotype in a hepatocellular carcinoma cell line. CONCLUSIONS: Genetic polymorphism in the FAS gene may account for some of the histopathological variability in chronic hepatitis C.     

Liver cell-membrane specific antibodies in autoimmune hepatitis patients detected by flow cytometry

In this study, we identified autoantibody in patients with autoimmune hepatitis against liver cell membrane using flow cytometry. After incubation of one of the hepatoblastoma cell lines, Hep G2, with serum from a patient, and the addition of FITC-labeled anti-human Ig antibody, anti-membrane antibodies were analyzed by flow cytometry. By our method, the antibody in serum can react only with autoantigens on the cell surface. Furthermore, propidium iodide staining enabled us to exclude the possibility of crossreaction of antibodies against dead cells. The relative fluorescence intensity in 12 patients with autoimmune chronic active hepatitis was significantly higher than that in 20 normal subjects, six primary biliary cirrhosis (PBC), 18 chronic viral hepatitis, and 11 systemic lupus erythematosus (SLE). The normal level of fluorescence intensity provided by sera from the SLE patients indicated that antibody binding to the liver cell membrane was not derived from Fc-mediated immune complex capture. These findings demonstrated that this flow cytometric technique provides a simple and accurate method for the detection of autoantibodies against liver cell membrane.     

Diet-induced alteration in the activity of plasma lipid transfer protein in normolipidemic human subjects.

Studies were performed to investigate the effect of diets rich in oleic or linoleic acids on the activity of plasma cholesteryl ester transfer protein (CETP) in normolipidemic subjects. Previous to the test diets, all subjects consumed a baseline diet rich in saturated fatty acids ("sat-diet") for 17 days. The test diets, rich in either monounsaturated fatty acids ("mono-diet") or rich in polyunsaturated fatty acids ("poly-diet"), were given for 5 weeks to 52 normolipidemic healthy volunteers. The activity of CETP was measured, using a method independent of endogenous plasma lipoproteins, as the rate of exchange of radioactive cholesteryl oleate between labelled LDL and unlabelled HDL. The "mono-diet" induced a statistically significant decrease in CETP activity (from 115 +/- 20 to 102 +/- 19 units/ml plasma, P less than 0.01), while the small decrease on the "poly-diet" (from 111 +/- 23 to 107 +/- 22 units/ml plasma) did not reach significancy. The percentual decrease in CETP activity induced by the "mono-diet" was higher than that induced by the "poly-diet" as was also found for the decrease in LDL cholesterol. In both diet groups a positive correlation was found between changes in CETP activity and changes in plasma total or (VLDL + LDL) cholesterol. The results suggest that high levels of dietary monounsaturated fatty acids may result in decreased plasma CETP activity, as well as LDL cholesterol levels. The mechanisms of these effects, and their possible interrelations, remain to be established.     

Molecular hybridization study of plasma hepatitis B virus DNA from different carriers

We have used molecular hybridization techniques to show that hepatitis B virus (HBV) DNA from different individuals may have substantial differences in sequence homology. Seven specimens of HBV DNA were isolated from plasma of different blood donors. Samples were applied as dots to membranes and nick-translated to form probes. Densitometry of the radioautograms showed that hybridization was most extensive with probe prepared from the same specimen. The hybridization bias was statistically significant (P less than .02) and visible to the naked eye. Hybridization to probes that were digested with nuclease S1 before nick translation did not eliminate the bias. Nor was the bias related to the d/y subdeterminants; on the average, 15 other specimens hybridized equally well to probes prepared from HBV with the same or different subdeterminant. Many specimens among 37 other serum samples showed greater or lesser degrees of homology to different probes, as demonstrated by reprobing of samples fixed to nylon membranes.     

Effect of LDL and serum from diabetic subjects on DNA synthesis in HIT-cells in culture

The effect of lipoprotein-deficient serum (LPDS) with and without added low-density lipoprotein (LDL), isolated from diabetic subjects, on the replication of SV40-transformed islet cells (HIT cells) was investigated. Whole serum as well as LPDS preparations stimulated DNA synthesis maximally when added to the culture medium at a final concentration of 0.1%. The addition of LDL at 25 and 175 g protein/ml medium did not cause further stimulation. On the contrary, the higher concentrations resulted in a significant inhibition. These results suggest that previously observed stimulation of DNA synthesis in smooth muscle cells by LDL from diabetic subjects is most likely due to the presence of growth factors in the serum of these patients and not to LDL per se.     

Prolactin stimulates creatine kinase activity and DNA synthesis in explants of human amnion

To characterize the action of hPRL and human placental lactogen on the amnion, decidua and placenta, we examined the effects of these hormones on the brain type isozyme of creatine kinase in cultured explants of these tissues from normal deliveries. In the amnion, hPRL (1 mg/l) caused a 1.8-fold increase in creatine kinase specific activity in 24 h, whereas hGH (1 mg/l) or human placental lactogen (1 mg/l) had no effect; oPRL (1 mg/l) also caused a 2.5-fold increase in creatine kinase activity. Neither hPRL, human placental lactogen nor hGH had a significant effect on creatine kinase activity in the placenta or decidua. [3H]thymidine incorporation into DNA increased in parallel to the stimulation of creatine kinase activity. The predominant isozyme of creatine kinase in both the unstimulated and stimulated explants was the brain type isozyme. Creatine kinase activity in the amniotic tissue increased significantly 2 h after hPRL treatment and reached its highest value at 4 h. The enzyme activity in the amnion rose with increasing hPRL dose and showed a significant increase at physiologic concentrations as low as 0.01 mg/l. This study, therefore, provides evidence for biological action of prolactin in amniotic tissue, suggesting that the amnion is physiologically responsive to prolactin.     

Liver injury is associated with enhanced regulatory T-cell activity in patients with chronic hepatitis B

Chronic hepatitis B virus (HBV) infection is associated with impairment of HBV-specific immune responses. Recently, it has been shown that regulatory T (Treg) cells downregulate HBV-specific immune responses but their role in chronic hepatitis B is still controversial. We hypothesized that liver injury enhances the influence of Treg cells on HBV-specific immune responses. The frequency of Treg cell and the in vitro expansion of HBV-specific CD8+ T cell detected by the tetramer method were investigated in 79 patients with chronic hepatitis B. Thirty-three healthy volunteers were enrolled to measure the frequency of Treg cell as controls. The results showed that in chronic hepatitis B cases, the frequency of Treg cells in peripheral blood was significantly higher than that in normal volunteers. The higher level of serum transaminase was associated with higher frequency of Treg cells, which both had a linear correlation relationship. HBV-DNA level, HBe status, age and sex had no statistical association with Treg cell frequency. Furthermore, in patients with higher serum transaminase levels, the expansion of HBV-specific CD8+ T cells was higher after removal of Treg cells when compared with patients with lower serum transaminase levels. In conclusion, our data indicate a significant association between serum transaminase level and frequency/activity of Treg cells. Based on this observation, we propose that liver-injury enhances Treg cell frequency/activity in chronic hepatitis B patients.     

Regulation of plasma lactate concentration in resting human subjects

We evaluated the relative contributions of glucose, insulin, and the rate of glucose disposal to the regulation of the plasma lactate concentration. Rates of glucose disposal were measured in 88 separate studies in whole body and across the forearm at varying plasma insulin (9, 50, 160, and 1,800 microU/mL) concentrations, and at each insulin concentration at four different glucose concentrations (90, 160, 250, and 400 mg/dL) in healthy male subjects. The rate of glucose disposal was positively correlated with the plasma lactate concentration (r = .83, n = 88, P less than .0001). When the plasma lactate concentration was adjusted for the rate of glucose disposal, plasma glucose or insulin concentrations did not contribute significantly to the residual variation in plasma lactate. When plasma lactate concentrations were compared at matched rates of glucose disposal, the lactate levels were similar regardless of whether glucose disposal was induced by hyperglycemia or hyperinsulinemia. At the lowest glucose and insulin concentrations, forearm tissues released lactate, but at all other glucose and insulin concentrations, no significant net lactate flux was observed. After subtraction of the rate of forearm glucose disposal from whole-body glucose disposal, the plasma lactate concentration correlated with the remaining, extramuscular, rate of glucose disposal (r = .60, P less than .0001). These data suggest that in resting normal subjects the plasma lactate concentration may be determined by the rate of glucose disposal in extramuscular tissues, rather than the ambient glucose or insulin concentration.     

ACAT activity in freshly isolated human mononuclear cell homogenates from hyperlipidemic subjects.

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) catalyzes the esterification of cholesterol in human mononuclear cells (MNC). In order to assess the relationship between lipid levels and ACAT activity in circulating MNC, we measured the rate of [14C]oleoyl-CoA incorporation into cholesterol ester in freshly isolated MNC homogenates from hyperlipidemic subjects. Baseline, off-treatment results obtained in 14 hypertriglyceridemic subjects (eight type IV and six type III) and seven subjects with familial hypercholesterolemia (FH) due to the same deletion of greater than 10 kb on the low-density lipoprotein (LDL)-receptor gene were compared with values determined in 12 healthy normolipidemic subjects. The rate of cholesterol esterification was 45 +/- 28 pmol/5 min/mg cell protein in healthy normolipidemic controls. This rate was significantly higher in type IV subjects (84 +/- 52 pmol/5 min/mg cell protein, P less than .05) and FH subjects (67 +/- 25 pmol/5 min/mg cell protein, P less than .05). The values were more dispersed in type III subjects; the mean value for the group (72 +/- 46 pmol/5 min/mg cell protein) was not statistically different from the control. Hypertriglyceridemic patients were then treated with 6 g/d of omega-3 fatty acids. This resulted in a significant reduction in plasma total triglycerides and very-low-density lipoprotein (VLDL)-cholesterol in both type III subjects (-57% and -51%, P less than .05) and type IV subjects (-62% and -62%, P less than .01). The reduction in VLDL concentration was associated with a significantly lower ACAT activity in MNC homogenates from type IV subjects (from 84 +/- 52 to 60 +/- 36 pmol/5 min/mg cell protein, P less than .05), but not from type III hypertriglyceridemic subjects (from 72 +/- 46 to 73 +/- 36 pmol/5 min/mg cell protein). In conclusion, we found that cholesterol esterification in human MNC is elevated in hyperlipidemic subjects and can be decreased with normalization of lipid levels. However, ACAT activity changes occurring with treatment are heterogeneous among hyperlipidemic subjects, suggesting that factors other than plasma lipid level reduction affect ACAT activity in vivo.     

Smoking and interleukin-1 activity released from human alveolar macrophages in healthy subjects

To evaluate the activation of alveolar macrophages from smoking, we studied interleukin-1 (IL-1) activity released from alveolar macrophages in eight healthy smokers, compared to 12 healthy nonsmokers. We used 24-hour culture supernatants containing IL-1 of bronchoalveolar lavage fluid (BALF) macrophages/blood monocytes with or without LPS stimulation. Using C3H/HeJ thymocyte PHA costimulation assay, we found that IL-1 activity released from LPS stimulated BALF macrophages was significantly higher in smokers (2.39 +/- 0.33 U/ml) than in nonsmokers (1.47 +/- 0.19 U/ml, p less than 0.05). We also detected IL-1 inhibitory activity in supernatants by using IL-1 inhibitory assay. The inhibitory activity was higher in nonsmokers than in smokers especially under LPS stimulation. The presence of inhibitory factors other than prostaglandin-E2 was suggested from the differential response to the addition of indomethacin into cultures from nonstimulated and LPS-stimulated supernatants of BALF macrophages.     

Effect of human beta-endorphin on plasma aldosterone concentrations in normal human subjects

beta-Endorphin recently was proposed as a possible physiological stimulus of aldosterone secretion based on studies in animals. Since human beta-endorphin (beta h-endorphin) does not contain the ACTH-(4-10) homology common to other ACTH-related neuropeptides that stimulate aldosterone, its mechanism of stimulation might differ from that of the other peptides. In the present study, we infused beta h-endorphin into six normal subjects under carefully controlled conditions at dosage levels several orders of magnitude higher than endogenous levels. No increase in plasma aldosterone was found in these subjects ingesting a normal sodium intake despite the fact that other biological actions of beta h-endorphin were manifest. By contrast, an equimolar infusion of ACTH-(1-24) caused a significant increase in plasma aldosterone. These studies do not support a significant role for beta h-endorphin in control of aldosterone secretion in man and are consistent with the concept that the ACTH-(4-10) amino acid sequence, common to ACTH, beta-lipotropin, gamma-lipotropin, beta MSH, and alpha MSH, is a major determinant of their aldosterone-stimulating capacity.     

Circadian rhythm of plasma beta-thromboglobulin in healthy human subjects.

The circadian variation in platelet function may play a role in the morning increase of coronary thrombotic complications. The circadian variation of plasma beta-thromboglobulin levels--as an indicator of in vivo platelet activation--was investigated in seven healthy volunteers using a commercial radioimmunoassay kit. There was a statistically significant rhythm culminating with an amplitude of 30 micrograms/l (95% tolerance interval/15-45 micrograms/l) at 10.00 hours (95% tolerance interval/07.00-13.00 hours). The morning increase of plasma beta-thromboglobulin indicates an increased in vivo platelet activation at this time of day. This finding could correlate with the morning peak in the onset of myocardial infarction. It might also help in timing therapy of diseases which are associated with platelet hyperactivation.