An F. C. Ruiter

Maintenance of Peripheral Naive T Cells Is Sustained by Thymus Output in Mice but Not Humans

Parallels between T cell kinetics in mice and men have fueled the idea that a young mouse is a good model system for a young human, and an old mouse, for an elderly human. By combining in vivo kinetic labeling using deuterated water, thymectomy experiments, analysis of T cell receptor excision circles and CD31 expression, and mathematical modeling, we have quantified the contribution of thymus output and peripheral naive T cell division to the maintenance of T cells in mice and men. Aging affected naive T cell maintenance fundamentally differently in mice and men. Whereas the naive T cell pool in mice was almost exclusively sustained by thymus output throughout their lifetime, the maintenance of the adult human naive T cell pool occurred almost exclusively through peripheral T cell division. These findings put constraints on the extrapolation of insights into T cell dynamics from mouse to man and vice versa.

Sparse production but preferential incorporation of recently produced naïve T cells in the human peripheral pool

In mice, recent thymic emigrants (RTEs) make up a large part of the naïve T cell pool and have been suggested to be a distinct short-lived pool. In humans, however, the life span and number of RTEs are unknown. Although 2H2O labeling in young mice showed high thymic-dependent daily naïve T cell production, long term up- and down-labeling with 2H2O in human adults revealed a low daily production of naïve T cells. Using mathematical modeling, we estimated human naïve CD4 and CD8 T cell half-lives of 4.2 and 6.5 years, respectively, whereas memory CD4 and CD8 T cells had half-lives of 0.4 and 0.7 year. The estimated half-life of recently produced naïve T cells was much longer than these average half-lives. Thus, our data are incompatible with a substantial short-lived RTE population in human adults and suggest that the few naïve T cells that are newly produced are preferentially incorporated in the peripheral pool.

In vivo Dynamics of Stable Chronic Lymphocytic Leukemia Inversely Correlate with Somatic Hypermutation Levels and Suggest No Major Leukemic Turnover in Bone Marrow

Although accumulating evidence indicates that chronic lymphocytic leukemia (CLL) is a disease with appreciable cell dynamics, it remains uncertain whether this also applies to patients with stable disease. In this study, (2)H(2)O was administered to a clinically homogeneous cohort of nine stable, untreated CLL patients. CLL dynamics in blood and bone marrow were determined and compared with normal B-cell dynamics in blood from five healthy individuals who underwent a similar (2)H(2)O labeling protocol. Average CLL turnover rates (0.08-0.35% of the clone per day) were approximately 2-fold lower than average B-cell turnover rates from healthy individuals (0.34-0.89%), whereas the rate at which labeled CLL cells in blood disappeared (0.00-0.39% of B cells per day) was approximately 10-fold lower compared with labeled B cells from healthy individuals (1.57-4.24% per day). Leukemic cell turnover variables inversely correlated with the level of somatic hypermutation of the CLL clone (IgVH mutations). Although CLL cells in bone marrow had a higher level of label enrichment than CLL cells in blood, no difference between proliferation rates and proapoptotic and antiapoptotic profiles of CLL cells from these compartments was observed. These data suggest that, in stable disease, there is a biological relationship between the degree of somatic hypermutation of the CLL clone and its dynamics in vivo. Furthermore, in contrast to lymph nodes, the bone marrow does not seem to be a major CLL proliferation site.

Liver progenitor cell line HepaRG differentiated in a bioartificial liver effectively supplies liver support to rats with acute liver failure

A major roadblock to the application of bioartificial livers is the need for a human liver cell line that displays a high and broad level of hepatic functionality. The human bipotent liver progenitor cell line HepaRG is a promising candidate in this respect, for its potential to differentiate into hepatocytes and bile duct cells. Metabolism and synthesis of HepaRG monolayer cultures is relatively high and their drug metabolism can be enhanced upon treatment with 2% dimethyl sulfoxide (DMSO). However, their potential for bioartificial liver application has not been assessed so far. Therefore, HepaRG cells were cultured in the Academic Medical Center bioartificial liver (AMC-BAL) with and without DMSO and assessed for their hepatic functionality in vitro and in a rat model of acute liver failure. HepaRG-AMC-BALs cultured without DMSO eliminated ammonia and lactate, and produced apolipoprotein A-1 at rates comparable to freshly isolated hepatocytes. Cytochrome P450 3A4 transcript levels and activity were high with 88% and 37%, respectively, of the level of hepatocytes. DMSO treatment of HepaRG-AMC-BALs reduced the cell population and the abovementioned functions drastically. Therefore, solely HepaRG-AMC-BALs cultured without DMSO were tested for efficacy in rats with acute liver failure (n = 6). HepaRG-AMC-BAL treatment increased survival time of acute liver failure rats ∼50% compared to acellular-BAL treatment. Moreover, HepaRG-AMC-BAL treatment decreased the progression of hepatic encephalopathy, kidney failure, and ammonia accumulation. These results demonstrate that the HepaRG-AMC-BAL is a promising bioartificial liver for clinical application.

Effects of insulin on ketogenesis following fasting in lean and obese men.

The ketone bodies (KBs) D‐3‐hydroxybutyrate (D‐3HB) and acetoacetate (AcAc) play a role in starvation and have been associated with insulin resistance. The dose–response relationship between insulin and KBs was demonstrated to be shifted to the right in type 2 diabetes patients. However, KB levels have also been reported to be decreased in obesity. We investigated the metabolic adaptation to fasting with respect to glucose and KB metabolism in lean and obese men without type 2 diabetes using stable glucose and D‐3HB isotopes in a two‐step pancreatic clamp after 38 h of fasting. We found that D‐3HB fluxes in the basal state were higher in lean compared to obese men: 15.2 (10.7–27.1) vs. 7.0 (3.5–15.1) µmol/kg lean body mass (LBM)·min, respectively, P < 0.01. No differences were found in KB fluxes between lean and obese volunteers during the pancreatic clamp (step 1: 6.9 (1.8–12.0) vs. 7.4 (4.2–17.8) µmol/kg LBM·min, respectively; and step 2: 2.9 (0–7.2) vs. 3.4 (0.85–18.7) µmol/kg LBM·min, respectively), despite similar plasma insulin levels. Meanwhile, peripheral glucose uptake was higher in lean compared to obese men (step 1: 15.2 (12.3–25.6) vs. 14.7 (11.9–22.7) µmol/kg LBM·min, respectively, P ≤ 0.05; and step 2: 12.5 (7.0–17.3) vs. 10.8 (5.2–15.0) µmol/kg LBM·min, respectively, P ≤ 0.01). These data show that obese subjects who display insulin resistance on insulin‐mediated peripheral glucose uptake have the same sensitivity for the insulin‐mediated suppression of ketogenesis. This implies differential insulin sensitivity of intermediary metabolism in obesity.

Alanine administration does not stimulate gluconeogenesis in preterm infants

Gluconeogenesis partially depends on sufficient precursor supply, and plasma alanine concentrations are generally low in preterm infants. Stimulation of gluconeogenesis may contribute to the prevention of hypoglycemia, an important clinical problem in these infants. In this study we evaluated the effect of extra precursor supply on gluconeogenesis in preterm infants. In 11 infants, gestational age < or = 32 weeks, glucose production rate (GPR) and gluconeogenesis were measured using the [6,6-(2)H(2)]glucose dilution technique and mass isotopomer distribution analysis with [2-(13)C]glycerol, respectively. Unlabeled glucose was administered throughout the study period at a rate of 22 micromol. kg(-1). min(-1). Five infants received alanine (1.5 mg. kg(-1). min(-1)) during the last 3 hours of the study protocol, and 6 infants served as controls. In the control group the rate of gluconeogenesis and GPR remained constant at 4.0 +/- 0.3 micromol. kg(-1). min(-1) and 8.3 +/- 0.6 micromol. kg(-1). min(-1), respectively. In the alanine group plasma alanine concentrations increased from 45 +/- 23 to 829 +/- 115 micromol/L (P =.001); gluconeogenesis and GPR did not differ from control: 3.8 +/- 0.2 micromol. kg(-1). min(-1) and 6.4 +/- 2.0 micromol. kg(-1). min(-1), respectively. We conclude that administration of the gluconeogenic precursor alanine does not stimulate gluconeogenesis in preterm infants, despite a sharp increase in plasma alanine concentrations. We speculate either a restricted capacity of the enzymes involved in the gluconeogenic pathway or a low secretion rate of glucoregulatory hormones as causative mechanisms involved in the gluconeogenic pathway in the preterm neonate.

Determination of unbound prednisolone, prednisone and cortisol in human serum and saliva by on-line solid-phase extraction liquid chromatography tandem mass spectrometry and potential implications for drug monitoring of prednisolone and prednisone in saliva

Prednisolone (PLN) and prednisone (PN) are widely used glucocorticoids. Drug monitoring of PLN and PN is not routinely done owing to the need for multiple blood sampling and challenging measurement of unbound PLN and PN in blood. Here we present a robust method for quantification of cortisol, PLN and PN in serum, ultrafiltrate and saliva by on-line solid-phase extraction LC-MS/MS. The method is linear for the three analytes over the range of 6-1400 nmol/L for serum and 2-450 nmol/L for ultrafiltrate and saliva. Within-run precision of all three analytes was <10% and total precision was <15%. This method was applied to create time-concentration profiles of cortisol, PLN and PN after an oral dose of prednisolone in a healthy volunteer. Salivary levels of PLN correlated well with ultrafiltrate levels (p < 0.01), while this correlation was only marginal for PN (p = 0.052). The PN/PLN ratio was significantly higher in saliva than in ultrafiltrate and serum (p < 0.01). Addition sums of both metabolites in saliva showed excellent correlation with those of ultrafiltrate (p < 0.01). These findings have not been presented before and may have important implications for future studies concerning drug monitoring of PLN and PN in saliva.

The effect of carbohydrate and fat variation in euenergetic diets on postabsorptive free fatty acid release

Diet composition and energy content modulate free fatty acid (FFA) release. The aim of this study was to evaluate the dose-response effects of euenergetic variations in dietary carbohydrate and fat content on postabsorptive FFA release. The rate of appearance (Ra) of palmitate was measured by infusion of [2,2-2H2]palmitate after an overnight fast in six healthy men on three separate occasions, i.e. after 7 d on euenergetic control, high-carbohydrate and high-fat diets. The protein content and composition was identical for each diet. Postabsorptive plasma fatty acid concentrations were not different between the high-carbohydrate and control diets (0.36 (se 0.07) v. 0.43 (se 0.04) mmol/l), but were increased after the high-fat diet (0.75 (se 0.09) mmol/l, (P<0.01 compared with the other diets). Ra palmitate was not different between the high-carbohydrate and control diets (1.36 (se 0.20) v. 1.47 (se 0.15) micromol/kg per min). However, Ra palmitate was increased to 2.36 (se 0.26) micromol/kg per min after the high-fat diet (P<0.01 compared with the other diets). The fatty acid flux and whole-body fat oxidation were not affected by the high-carbohydrate diet compared with the control diet, but were increased by 67 and 47 % respectively, on the high-fat diet (P<0.01 compared with the other diets). A euenergetic high-fat diet results in increased postabsorptive FFA release and fat oxidation, whereas a euenergetic high-carbohydrate diet does not affect these variables of fat metabolism.

Simultaneous gas chromatographic determination of concentration and isotopic enrichment of fatty acids in human plasma using flame ionization and mass spectrometric detection

Free fatty acids (FFAs) are important not only because they provide substrate for oxidation but also because they have the potential to regulate several metabolic and hormonal processes. Using stable isotope tracers, these processes can be studied. Here we present a gas chromatographic method to measure FFA concentrations and enrichments after extraction from plasma and subsequent derivatization in one analytical run, using both flame ionization and mass-selective detection. For concentration determinations intra-assay variation ranged from 1.5 to 4.9%, inter-assay variation ranged from 3 to 11%. Intra- and inter-assay variations of the enrichment determination of palmitic acid were 1.4 and 0.9%, respectively.

Determination of a steroid profile in heel prick blood using LC–MS/MS

BACKGROUND The aim of this study was to improve the sensitivity of the congenital adrenal hyperplasia (CAH) neonatal screening by including second-tier steroid profiling on a DBS using LC-MS. RESULTS We developed a method to measure the steroid profile in DBS and established gestational age-specific reference ranges of cortisol, cortisone, 11-deoxycortisol, 21-deoxycortisol, 17-hydroxyprogesterone, testosterone, Δ4-androstenedione, corticosterone and 11-deoxycorticosterone using 450 heel prick samples of neonates, participating in the Dutch Screening Program. Analyzing 92 cards with a positive CAH screening showed that only 21-deoxycortisol was 100% specific for diagnosed CAH patients. CONCLUSION Steroid precursors can be measured in DBS and we suggest to implement the method as a second tier testing for CAH in The Netherlands.