Routine noninvasive prenatal screening for fetal RHD in plasma of RhD-negative pregnant women-2 years of screening experience from Denmark.

OBJECTIVE: Prenatal and postnatal RhD prophylaxis reduces the risk of RhD immunization in pregnancies of RhD-negative women. Based on the result from prenatal screening for the fetal RHD gene, prenatal RhD prophylaxis in Denmark is targeted to RhD-negative women who carry an RhD-positive fetus. Here, we present a 2-year evaluation of a nationwide prenatal RHD screening. METHODS: Blood samples were drawn from RhD-negative women in gestational week 25. DNA was extracted from maternal plasma and analyzed for the RHD gene. The prenatal RHD results were compared with the serological typing of newborns in 12,668 pregnancies. Early compliance was assessed for 690 pregnancies. RESULTS: The sensitivity for the detection of fetal RHD was 99.9% (95% CI: 99.7-99.9%). Unnecessary recommendation of prenatal RhD prophylaxis was avoided in 97.3% of the women carrying an RhD-negative fetus. Fetuses that were seropositive for RhD were not detected in 11 pregnancies (0.087%). The sample uptake percentage was 84.2%, and the compliance for prenatal anti-D administration was 93.2%. CONCLUSION: The high sensitivity, maintained over 2 years, underlines the reliability of routine prenatal fetal RHD screening in RhD-negative pregnant women, specifically at 25 weeks of gestation. The remaining challenges are logistical and are related to program compliance.

Aspects of present and future data presentation in Scandiatransplant.

Scandiatransplant is the Nordic organ exchange organization having existed for almost 40 years. With close collaboration between transplant centers in the Nordic countries, it has been valuable to ensure the optimal usage of available organs. The heart is the most often exchanged organ within the collaboration. It has been decided to create a priority for hyperimmunized kidney patients for compulsory exchange of organs from deceased donors. The age of the deceased organ donors has changed from younger to older donors. The evaluation of deceased kidney transplantations and deceased liver transplantations from 1995 to 2007 is shown for 4 countries. Iceland by itself is performing living donor kidney transplantations with great intensity. Scandiatransplant will make efforts to present more data than just transplantation to yield a more complete picture of organ transplantation.

Compartmentation of cytosolic dehydrogenases studied by transfer of tritium from labelled substrates into lactate in rat hepatocytes.

This paper describes the transfer of tritium from [2-(3)H]xylitol or (1R)-[1-(3)H]ethanol into lactate in cells from fed rats either untreated or triiodothyronine-treated. The labelling pattern of lactate during the metabolism of [2-(3)H]xylitol or (1R)-[1-(3)H]ethanol follows the equation L = K(1 - e-t/tau) (mumol tritium/mumol lactate). The yield in lactate together with the minimum value of the total flux of reducing equivalents are used to estimate the specific radioactivity of NADH. We have calculated the lactate dehydrogenase-catalysed oxidation rate of NADH from the experimental values of lactate labelling and the specific radioactivity of NADH. We found the calculated flux of reducing equivalents from NADH to pyruvate to be of the same order of magnitude whether labelled ethanol or labelled xylitol was metabolized. We found the flux to be only a few percent of the maximal activity of lactate dehydrogenase. The results obtained suggest that the cytoplasm can be regarded as one compartment, containing a single pool of NAD(H).

Ethanol metabolism and lipid synthesis by isolated liver cells from fed rats.

1. The fatty acid synthesis in isolated liver cells from fed rats was studied with tritiated water as the radioactive precursor. The cells incorporated 3H20 at a rate of 1.26 mumol per min per g packed cells. 2. Addition of ethanol caused a 20% decrease in the incorporation of tritium into fatty acids. The decrease was correlated to the increase in the NAD-redox level. Probably, the decreased tritium incorporation into fatty acids during ethanol metabolism is due to a decrease in the specific activity of the NADPH used for the synthesis of fatty acids, rather than to a real inhibition of the fatty acid synthesis. 3. Ethanol oxidation via NADPH-consuming pathways and ethanol per se at a concentration of 80 mM had no effect upon the incorporation of tritium into fatty acids. 4. Fructose in a concentration of 15 mM inhibited the fatty acid synthesis by 75%, and this inhibition was further augmented by ethanol. 5. The ioslated rat liver cells oxidized ethanol at a rate of 2.72, 2.93 and 3.48 mumol per min per g packed cells at 5, 20 and 80 mM ethanol, respectively. Fructose had no effect upon ethanol oxidation neither at low nor at high concentrations of ethanol. 6. Ethanol oxidation via the non alcohol dehydrogenase pathway(s) may involve a transfer of reducing equivalents from mitochondrial NADH to cyctosolic NADP+ as judged from measurements of metabolite levels. This conclusion is supported by determinations of 14C yield in glucose from [1-14C] ethanol, and the results are taken as evidence for the presence of hydrogen shuttle activity during metabolism of ethanol, catalyzed by the NAD-dependent alcohol dehydrogenase. A metabolic scheme is proposed to account for the observed changes at low and high concentrations of ethanol.

Quantification of protein turnover in primary cultures of rat hepatocytes.

1. A radioactive tracer method, based on [3H]valine, for determination in parallel experiments of degradation of cellular proteins and synthesis of both cellular and secreted proteins in cultured rat hepatocytes was developed with special emphasis on methods of calculation, the number of protein pools and maintenance of nitrogen balance. 2. An extracellular concentration of 2 mM valine ensured a specific activity of the precursor pool for protein synthesis, which was constant during the experimental period and practically identical to that of extracellular valine. 3. Amino acid concentrations in the culture medium used were not rate-limiting for the synthesis of proteins. 4. The rate of labelling of the cellular protein pool during 8 days was in accordance with first-order saturation kinetics, which together with a constant ratio between labelling of soluble and membrane bound proteins, is compatible with a single pool of cellular proteins. 5. Protein degradation can be accurately measured by the release from prelabelled proteins of [3H]valine in the presence of 2 mM extracellular valine, if a 1 h chasing period is included in the experimental design. 6. The constancy of the degradation constant (kd) during protein labelling for up to 8 days, is in accordance with the existence of only one pool of cellular protein. 7. The cultured hepatocytes were in nitrogen balance during the experimental period of 8 days as reflected in a constant protein content per cell. The absolute rates of degradation and synthesis of cellular protein were identical, which confirm the validity of the method described.

Inhibition of fatty acid synthesis in rat hepatocytes by exogenous polyunsaturated fatty acids is caused by lipid peroxidation.

Rat hepatocyte long-term cultures were utilized to investigate the impact of different polyunsaturated fatty acids (PUFA) on the insulin-induced de novo fatty acid synthesis in vitro. The addition of 0.5 mM albumin-complexed oleic, linoleic, columbinic, arachidonic, eicosapentaenoic or docosahexaenoic acid resulted in a marked suppression of fatty acid synthesis. By evaluation of cell viability (determined as the leakage of lactate dehydrogenase (LDH) it turned out, that the antioxidant used (50 microM alpha-tocopherol phosphate) had a low antioxidant activity, resulting in cytotoxic effects by the peroxidized PUFA. Arachidonic acid and eicosapentaenoic acid showed a dose- and time-dependent cytotoxicity. Two other antioxidants: 50 microM alpha-tocopherol acid succinate and 1 microM N,N'-diphenyl-1,4-phenylenediamine, both proved more efficient than alpha-tocopherol phosphate. There was a significant correlation between LDH-leakage and inhibition of fatty acid synthesis. Lipid peroxidation, measured as thiobarbituric acid-reactive substances, also showed a significant correlation with the degree of inhibition of fatty acid synthesis. Furthermore, PUFA had no inhibitory effect on fatty acid synthesis when peroxidation was minimized by the use of proper antioxidants. These data indicate that PUFA in vitro inhibit the insulin-induced de novo fatty acid synthesis in hepatocytes from starved rats, due to cytotoxic effects caused by lipid peroxidation.

Involvement of PI 3-kinase and activated ERK in facilitating insulin-stimulated triacylglycerol synthesis in hepatocytes.

Triacylglycerol synthesis was studied in hepatocytes isolated from fasted/refed rats by EDTA perfusion. Insulin induced a 1.5-fold increase in glucose incorporation into triacylglycerol. Insulin-stimulated triacylglycerol synthesis and insulin-stimulated protein kinase B/Akt activity were inhibited by the phosphatidylinositol 3-kinase inhibitors wortmannin and LY 294002, and the mitogen-activated protein kinase kinase inhibitor PD 98059. Inhibition of p70 ribosomal protein-S6 kinase with rapamycin was without effect. Insulin-stimulated pyruvate dehydrogenase activity was abolished by phosphatidylinositol 3-kinase inhibitors. No effect of insulin on acetyl CoA carboxylase activity was observed.

Selected activities in Scandiatransplant.

Scandiatransplant is the Nordic organ exchange organization. It has existed for 35 years and it is owned by all organ transplantation hospital departments in the five Nordic countries--Denmark, Finland, Iceland, Norway, and Sweden. The use of living organ donors for kidney transplantation has become a more common procedure not only in Norway but also in Sweden and Denmark. For the first time, in 2003, one transplant center performed relatively more living donor kidney transplantations than with deceased donors. The overall organ transplant activity reveals a remarkably stable situation in the area covered by Scandiatransplant. Scandiatransplant as an organ exchange organization has changed from a solely kidney exchange organization to an organization in which the more immediate vital organs as liver and heart are exchanged more commonly than kidneys.

Cod liver oil inhibits neutrophil and monocyte chemotaxis in healthy males.

Epidemiological evidence suggests a reduced rate of chronic inflammatory diseases and ischaemic heart disease in populations with a high consumption of fish. This has been ascribed to the high content in sea food of polyunsaturated fatty acids (PUFAs), belonging to the n - 3 family. We have studied neutrophil and monocyte chemotaxis in 12 healthy males before and after 6 weeks supplementation with cod liver oil, corresponding to 5.3 g n - 3 PUFAs daily. Neutrophil and monocyte chemotaxis were investigated using the under agarose technique with N-formyl-methionyl-leucyl-phenylalanine (N-FMLP) and autologous serum as chemoattractants. Neutrophil chemotaxis towards both chemoattractants and monocyte chemotaxis towards N-FMLP were significantly reduced after supplementation with cod liver oil.

Real-time scanning and image analysis. A fast method for the determination of neutrophil orientation under agarose.

We describe a newly developed method for fast determination of neutrophil chemotaxis and orientation in concentration gradients of chemotactic factors. The system implements video-based real-time scanning and image analysis of neutrophil migration under agarose, using an interactive easy-to-use computer program. Two methods for determining cell orientation are presented. No statistically significant difference between the methods was found. The analysis program distinguishes between chemokinetic and chemotactic behaviour of the cells (P less than 0.01).

Contribution of non-ADH pathways to ethanol oxidation in hepatocytes from fed and hyperthyroid rats. Effect of fructose and xylitol.

The metabolism of (1R)[1-3H]ethanol, [2-3H]lactate or [2-3H]xylitol was studied in hepatocytes from fed or T3-treated rats in the presence or absence of fructose or xylitol. The yields of tritium in ethanol, lactate, water, glycerol and glucose were determined. A simple model, describing the metabolic fate of tritium from these substrates is presented. The model allows estimation of the ethanol oxidation rate by the non-alcohol dehydrogenase pathways from the relative yield of tritium in water and glucose. The calculations are based on a comparison of the fate of the 1-proR-hydrogen of ethanol and the hydrogen bound to carbon 2 of lactate (or xylitol) under identical condition. In our calculations we have taken into account that the reactions catalyzed by lactate dehydrogenase and alcohol dehydrogenase are reversible and that lactate or ethanol labelled during the metabolism of the other tritiated substrates will contribute to the tritium found in water. The contribution of non-ADH pathways to ethanol oxidation varied from 10 to 50% and was correlated to changes in the lactate/pyruvate ratio from 80 to 500. In T3-treated rats the activity of non-ADH pathways were greater than in fed rats for the same lactate/pyruvate ratio.

The content and activity of cytochrome P-450 in long-term culture of hepatocytes from male and female rats.

The content of cytochrome P-450 and the capacity for O-demethylation have been measured in cultures of hepatocytes from male and female rats for a period of 21 days. The effect of dexamethasone, insulin, glucagon, phenobarbital and hemin was investigated. In hepatocytes from female rats the content of cytochrome P-450 was unchanged after one day of culture. From day 1 to day 3 the content of cytochrome P-450 decreased by 65% and only the combined addition of dexamethasone, phenobarbital and hemin diminished the fall. After the initial fall, addition of 0.1 microM dexamethasone resulted in a stable value. Addition of 1 microM dexamethasone or 1 mM phenobarbital gave rise to an induction of cytochrome P-450 (285%). The high level of cytochrome P-450 was maintained for 3 weeks. In hepatocytes from male rats the content of cytochrome P-450 decreased by 40% after one day of culture. From day 1 to day 3 the content decreased by 45% and the decrease continued irrespective of the presence of hormones and/or phenobarbital. The O-demethylase activity in cultures of hepatocytes from female rats correlated to the cytochrome P-450 content independent of medium composition and age of the cultures, whereas no correlation was found in cultures from male rats. The present study demonstrates that hepatocytes from female rats in cultures retain O-demethylase activity for at least 3 weeks and that, with the experimental conditions used, the response to the hormones and inducers is different for hepatocytes from male and female rats.

Regulation by growth hormone and glucocorticoid of testosterone metabolism in long-term cultures of hepatocytes from male and female rats.

The activities of 2-, 6 beta-, 7 alpha- and 16 alpha-testosterone hydroxylase and 5 alpha-testosterone reductase were measured in intact hepatocytes from male and female rats cultured for 8 days in a modified Waymouth medium supplemented with 0.1 or 1.0 microM dexamethasone with or without addition of 1 microgram/mL growth hormone. During culture of hepatocytes from female rats the activity of the male-specific 16 alpha-testosterone hydroxylase increased. This increase was significantly inhibited at day 8 by 1 microM dexamethasone as well as by growth hormone. Furthermore, in cultures of hepatocytes from male rats, the activity of the constitutive 16 alpha-testosterone hydroxylase was decreased by 1 microM dexamethasone as well as by growth hormone. The induction of 6 beta-testosterone hydroxylase by dexamethasone was suppressed by growth hormone in hepatocytes from both male and female rats, while the 7 alpha-testosterone hydroxylase activity was unaffected by culture time, hormone additions and gender. The decrease in female-specific 5 alpha-reductase activity with culture time in hepatocytes from female rats was significantly attenuated by growth hormone at 0.1 microM dexamethasone. The effects of growth hormone on testosterone hydroxylase activities in hepatocyte cultures from male and female rats are in accordance with the concept of growth hormone as a "feminization signal". The results suggest that the glucocorticoid-dependent expression of the male constitutive 16 alpha-hydroxylase requires periods of low levels of growth hormone.

Turpentine- and fibrin-induced pleuritis in nude mice. Histopathological, cellular and biochemical changes.

Turpentine was injected into the right pleural cavity of nude immune-incompetent mice, causing a temporary irritative exudative pleuritis. A transient occurrence of so-called rheumatoid arthritis cells was observed in the pleural fluid together with parallel characteristic biochemical changes. In similar experiments in nude mice, however, immunization followed by intrapleural application of bovine fibrin showed irritative "dry" pleuritis without the presence of rheumatoid arthritis cells. This is in contrast with previous results from similar experiments done using normal mice. The conclusion from the present experiments is that in nude immune-incompetent mice only the non-immunological, turpentine-induced pleuritis will generate cellular and biochemical changes typical of the rheumatoid disease in patients, while the fibrin-induced pleuritis fails to show similar changes. This suggests that the rheumatoid-like pleural changes described in the present experiments in nude mice have a non-immunological basis.

Cytokine plasma levels and lymphocyte subsets in patients with newly diagnosed insulin-dependent (type 1) diabetes mellitus before and following initial insulin treatment.

Changes in the plasma concentrations of interleukin-1 beta (IL-1 beta), tumour necrosis factor alpha (TNF alpha), interleukin 2 (IL-2), and lymphocyte subsets were investigated in 19 persons with newly diagnosed (type 1) insulin-dependent diabetes mellitus (IDDM) from admission to hospital prior to insulin treatment and following 1 week and 1 month of treatment. Furthermore, the cytokines were measured 16-28 months after the presentation of IDDM. The mean TNF alpha values were all within the normal range, but demonstrated a slight increase in all the samples taken (Friedman 0.06). The mean plasma IL-1 beta value was initially at the upper normal limit, but gradually increased significantly from admission to hospital to 1 week, 1 month, and 16-28 months afterwards (Friedman 0.031). No IL-2 activity was detectable in the majority of the samples. No significant changes in total leukocyte and lymphocyte counts were found. The lymphocyte subsets (CD5+, CD8+, CD4+, CD16+, CD20+, HLA-DR+) did not show any significant changes from admission to after the start of insulin treatment. It is concluded that the gradual increase in IL-1 beta and TNF alpha plasma levels may reflect an ongoing autoimmune inflammatory reaction at the onset of IDDM.

Rheumatoid arthritis cells and biochemical changes in turpentine-induced pleuritis in rabbits.

When turpentine was instilled into the right pleural cavity in rabbits a pleural effusion developed in half of the animals, with a low pH, low glucose concentration, high lactic dehydrogenase activity and the constant presence of rheumatoid arthritis cells in the affected pleural cavity. The biochemical values in the pleural fluid were significantly different from the values for normal pleural fluid obtained by a special microtechnique. These changes resulting from the experimentally induced, simple, irritative turpentine pleuritis are similar to the findings in the pleural effusion in human rheumatoid pleuritis; this implies that such changes are probably non-specific and without evidence of an immunological background.

Effect of physical exercise on cytokines and lymphocyte subpopulations in human peripheral blood.

To examine the effect of intensive physical exercise on interleukin 2 (IL-2), tumor necrosis factor alpha (TNF alpha) and lymphocyte subsets, eleven elite and well-conditioned runners were tested in relation to a five-kilometer race. IL-2 was significantly decreased (p less than 0.01) immediately after the exercise and significantly increased after 24 hours (p less than 0.05), compared to the pre-exercise values taken at steady state. TNF alpha was significantly increased after 2 hours (p less than 0.05), and returned to habitual values after 24 hours. In the steady state at rest, elevation of HLA-DR+ cells was observed in all runners compared with control subjects (p less than 0.05), indicating a persistent activation of lymphoid cells. In connection with exercise a significant increase in NK cells (CD16+) was observed (p less than 0.01). The T-helper/T-suppressor (CD4+/CD8+) ratio was significantly reduced in connection with physical activity (p less than 0.01). In seven runners the ratio was reduced to a value of less than one. This decrease was observed immediately after the exercise, followed by increased ratios 2 hours later (p less than 0.01), due to oppositely directed quantitative changes of the CD4+ and CD8+ cell populations. After 24 hours the ratios returned to habitual levels. Furthermore, we confirmed an increase in the total number of granulocytes in connection with exercise (p less than 0.01), and observed a decrease in absolute numbers of lymphocytes two hours after exercise (p less than 0.01). We emphasize the importance of obtaining information about physical activity within the previous 24 hours before measuring white blood cell parameters.

Prospective study of anticardiolipin antibodies in immunized and untreated women with recurrent spontaneous abortions.

OBJECTIVE: To investigate whether active leukocyte immunization increases levels of anticardiolipin antibodies in women with recurrent spontaneous abortions. To assess the impact of anticardiolipin antibodies on pregnancy outcome in these women. DESIGN: Patients who had received various treatments in an ongoing randomized trial were studied prospectively. SETTING: A department of clinical immunology investigating women with recurrent spontaneous abortions from all over Denmark. PATIENTS: Eighty-nine patients with unexplained recurrent spontaneous abortions whose pregnancies had been completed during the course of the trial. INTERVENTIONS: After randomization, 44 patients were actively immunized with husband's or third party leukocytes, and 27 patients received placebo. Eighteen patients received anticoagulation therapy in pregnancy. MAIN OUTCOME MEASURES: Changes in levels of immunoglobulin (Ig)M class and IgG class anticardiolipin antibodies after active immunization. Frequency of new miscarriages in patients who were positive or negative for anticardiolipin antibodies. RESULTS: Neither IgM nor IgG anticardiolipin antibodies changed significantly after active immunization (P greater than 0.2). The interim results of the immunization trial showed a success rate of 68% in the treated group versus 56% in the placebo group (not significantly different). Relative risk of miscarriage in anticardiolipin antibody-positive patients compared with anticardiolipin antibody-negative patients was 1.3 (95% confidence interval 0.7 to 2.2; P = 0.4) in the combined study groups. CONCLUSIONS: Patients eligible for active immunization did not exhibit significant changes in anticardiolipin antibody levels subsequent to the treatment. The treatment did not seem to provide any overall benefit with respect to pregnancy outcome. Prospectively, the risk of miscarriage in patients positive for anticardiolipin antibodies was not significantly increased.

Circulating levels of pregnancy zone protein: normal range and the influence of age and gender.

Serum levels of pregnancy zone protein (PZP) were measured in 506 apparently normal males and 329 normal non-pregnant females, the age range being 18 to 70 years. The estimations of PZP were performed by sensitive radio-rocket-line immunoelectrophoresis. The distribution of the data had a marked positive skew in both sexes which was reduced following logarithmic transformation. Serum concentrations in both men and women were found to increase significantly with advancing age. This increase and the mean concentrations were significantly higher in females.

Primary cultures of rat hepatocytes.

With the advent in 1969 of the collagenase-perfusion technique for the high-yield preparation of isolated, differentiated hepatocytes (1), easy establishment of primary cultures of hepatocytes was made possible. Since then, this experimental system has been increasingly used in many research fields.