Immunohistochemical, morphological and ultrastructural resemblance between dendritic cells and folliculo-stellate cells in normal human and rat anterior pituitaries

Immunolabeling of cryo-sections of human anterior pituitaries obtained at autopsy, and of cryo-sections of freshly prepared rat anterior pituitaries, with a panel of monoclonal antibodies against markers of the monocyte/dendritic cell/macrophage lineage, reveals in both species a characteristic pattern of immunopositive cells, among which many cells with dendritic phenotype are found. Cells characterized by marker expression of MHC-class II determinants and a dendritic morphology are present in both human and rat anterior pituitary. Markers characteristic of dendritic cells such as the L25 antigen and the OX62 antigen were present in anterior pituitaries from human and rat respectively. The population of MHC-class II expressing dendritic cells of the rat anterior pituitary is compared at the ultrastructural level with the folliculo-stellate cell population, which cell type has been previously characterized by its distinctive ultrastructure and immunopositivity for the S100 protein. Using immune-electron microscopy of rat anterior pituitaries fixed with periodate-lysine-paraformaldehyde, we were able to distinguish non-granulated cells expressing MHC-class II determinants, whereas no MHC-class II expression was found in the granulated endocrine cells. Using double immunolabeling of cryo-sections of these rat AP with 25 nm and 15 nm gold labels, we demonstrated an overlap between the populations of MHC-class II-expressing and S100 protein-expressing cells. Furthermore, MHC-class II-expressing and S100-positive cells showed ultrastructural characteristics that have been previously ascribed to folliculo-stellate cells. At the light microscopical level in the rat AP, a proportion of 10 to 20% of the S100-positive cells was found immunopositive for the MHC-class II marker OX6. In the hu

Plasma alpha(2) macroglobulin is increased in nephrotic patients as a result of increased synthesis alone

Background. alpha(2) Macroglobulin (alpha(2)M), a protease inhibitor, is often increased in plasma of patients with the nephrotic syndrome. Although it has been speculated that synthesis is increased, no direct measurements have been performed. Methods. alpha(2)M synthesis in both normal subjects (N = 4) and nephrotic patients (N = 7) were measured using endogenous labeling with C-13 valine in order to establish the mechanism of increased plasma level in the nephrotic syndrome and the relationship between alpha(2)M synthesis rate and plasma concentration over a wide range of plasma concentration values. A primed (15 mu mol/kg)/continuous (15 mu mol/kg/hr) infusion was administered for six hours. Blood samples were collected at different intervals and at each time point alpha(2)M was isolated from EDTA plasma using immunoprecipitation and SDS-polyacrylamide gel electrophoresis (PAGE). Care was taken to ensure that the alpha(2)M used for combustion had not been subjected to proteolysis. The rate of appearance of C-13 valine derived from the isolated alpha(2)M was measured by gas chromatography combustion isotope ratio mass spectrometry. Results. Plasma alpha(2)M was significantly elevated in nephrotic subjects (3.13 +/- 0.33 g/liter) versus controls (1.64 +/- 0.15 g/liter; P = 0.012). The alpha(2)M fractional synthesis rate [(FSR), which is equal to fractional catabolic rate (FCR) in steady state] was the same in the two groups: 2.70 +/- 0.18%/day for the nephrotic patients versus controls 2.74 +/- 0.21%/day. However, the alpha(2)M absolute synthesis rate (ASR) was significantly (P = 0.012) increased in the patients (3.69 +/- 0.33 mg/kg/day) versus controls (2.06 +/- 0.35 mg/kg/day). Plasma alpha(2)M concentration correlated directly to its ASR (r(2) = 0.821; P 0.0001; N = 11). Conclusions. Increased plasma alpha(2)M concentration in nephrotic patients is therefore a result of increased synthesis alone

New method for faecal fat determination by mid-infrared spectroscopy, using a transmission cell:an improvement in standardization

Current techniques used in clinical laboratories for faecal fat determination, such as the Van de Kamer method, are not very accurate or precise. This became apparent when results obtained by different laboratories were compared, and could explain the disappointing performance of near-infrared and mid-infrared spectroscopy since the accuracy of these techniques depends upon the accuracy of the calibration used (i.e. inaccurate wet chemical analysis). In order to improve standardization, we developed and tested a new quantitative method in three laboratories, based on Fourier transform infrared (FT-IR) spectroscopy. Fatty acids were extracted from faecal samples with acidified petroleum ether-ethanol and the extracts were dried and dissolved in chloroform. An infrared spectrum of the extracts was recorded in the range 4000-650 cm(-1), using an infrared transmission cell. Standard mixtures of stearic and palmitic acids (65:35) were used for calibration. Quantification was based on the absorbance band of the CH2 group (2855 cm(-1)) of free fatty acids and fatty acid glycerol esters. The calibration curve showed excellent linearity. The correlation coefficient between the titrimetric Van de Kamer and FT-IR methods was 0.96 (y = 1.12x-0.02, standard error of prediction = 0.89 g% fat). No significant difference was found when the FT-IR results of 28 faecal samples from patients were compared between two different university hospital laboratories. The new FT-IR method, using primary standards, is simple and rapid, and provides satisfactory intra- and inter-laboratory precision for the diagnosis and monitoring of steatorrhoea

Proportionate increase of fibrinogen and albumin synthesis in nephrotic patients:Measurements with stable isotopes

Hyperfibrinogenemia is a common feature of the nephrotic syndrome, and contributes to increased tendency for thrombosis and atherosclerosis. Its genesis is not certain, but the increase in liver fibrinogen mRNA in nephrotic rats indicates increased synthesis. Data in humans are scarce. We presently compared synthesis rates of fibrinogen and albumin in nephrotic adults (N = 7; plasma albumin 22.3 +/- 0.7 g/liter, proteinuria 12 g/day) and healthy control subjects (N = 8) using a primed/continuous infusion of the stable isotope L-[1-C-13]valine for six hours. Absolute synthesis rate (ASR) of fibrinogen was 31 +/- 3 mg/kg/day in nephrotic subjects and 21 +/- 1 mg/kg/day in control subjects (P <0.05), and positively correlated with plasma fibrinogen (P = 0.0317). The plasma fibrinogen pool was disproportionately increased in the nephrotic patients (271 +/- 30 mg/kg) compared to the controls (126 +/- 8 mg/kg), suggesting decreased fractional catabolic rate as well. The ASR of albumin was increased from 71 +/- 4 mg/kg/day in the controls to 160 +/- 19 mg/kg/day in the patients (P <0.0001), and strongly correlated with the ASR of fibrinogen (P = 0.0046). Plasma alpha(2)-macroglobulin was also elevated and correlated with the albumin synthesis rate, whereas plasma serum amyloid A and C-reactive protein were not elevated. These data suggest that in nephrotic patients the increased albumin synthesis is associated with an increase in synthesis of a specific and coordinated group of proteins, among which is fibrinogen

A single food bolus stimulates albumin synthesis in growing piglets

Background: A stable isotope tracer method to quantify the synthesis of proteins of hepatic origin in response to feeding is described. The response of albumin synthesis on one mixed meal in a piglet model was investigated and the intragastric and intravenous administration modes of C-13-valine were compared. Methods: The fasting and postprandial fractional synthesis rates (FSRs) of albumin in 15 piglets were measured while infusion rates of C-13-valine were changed in anticipation of the increased appearance of the tracee after a single liquid food bolus (30 mL/kg infant formula). C-13-valine enrichments in albumin hydrolysates at regular time intervals were determined with gas chromatography-combustion isotope ratio mass spectrometry. Results: The intravenous mode (n = 8) showed constant plasma alpha-ketoisovalerate tracer-to-tracee ratios (coefficient of variation range: 1-8%), and a 27% increase in albumin FSR after the food bolus (mean FSR +/- standard error [SE]: fasting 14.4% +/- 1.6% vs. postprandial 18.3% +/- 2.2% per day; P <0.005). In the intragastric mode (n = 7), albumin FSR calculated from the mean precursor values increased 32% after feeding (fasting 14.6% +/- 1.5% vs. postprandial 19.3% +/- 1.6% per day; P = 0.005), despite absence of constant alpha-ketoisovalerate enrichment (coefficient of variation range: 15-31%). The FSRs were not significantly different between both infusion modes. Conclusions: A mixed food bolus increases albumin FSR in growing piglets by approximately 30%, irrespective of the tracer administration route. The concept of anticipated precursor steady state is applicable to study changes of hepatic protein synthesis after a single meal. The intragastric mode of tracer administration can be applied as a less invasive method to measure tissue specific protein synthesis in children