Summary Both interferon-gamma-producing type 1 T helper (Th1)- and interleukin-17 (IL-17)-producing Th17 cells have been proposed to be involved in anti-fungal host defence. Although invasive aspergillosis is one of the most severe human fungal infections, little is known regarding the relative importance of the Th1 versus Th17 cellular immune pathways for the human anti-Aspergillus host defence. Using human peripheral blood mononuclear cells and a system consisting of monocyte-derived macrophages with lymphocytes, we found that Aspergillus fumigatus is a weak inducer of human IL-17 but induces a strong Th1 response. These data were validated by the very low IL-17 levels in bronchoalveolar lavage fluid and serum of patients with invasive aspergillosis. Surprisingly, live A. fumigatus reduced IL-17 production induced by mitogenic stimuli. This effect was mediated through the propensity of A. fumigatus to metabolize tryptophan and release kynurenine, which modulates the inflammatory response through inhibition of IL-17 production. In conclusion, A. fumigatus does not stimulate production of IL-17 and human host defence against aspergillosis may not rely on potent Th17 responses.

BACKGROUND: Angiotensin-converting enzyme (ACE) insertion/deletion (I/D) polymorphism-related differences in ACE concentration do not result in differences in angiotensin levels. METHODS AND RESULTS: To investigate whether this relates to differences in the contribution of the ACE C-domain and N-domain, we quantified, using the C-domain-selective inhibitors quinaprilat and RXPA380, and the N-domain-selective inhibitor RXP407, the contribution of both domains to the metabolism of angiotensin I, bradykinin, the C-domain-selective substrate Mca-BK(1-8), and the N-domain-selective substrate Mca-Ala in serum of IIs, DDs, and 'hyperACE' subjects (i.e., subjects with increased ACE due to enhanced shedding). During incubation with angiotensin I, the highest angiotensin II levels were observed in sera with the highest ACE activity. This confirms that ACE is rate-limiting with regard to angiotensin II generation. C-domain-selective concentrations of quinaprilat fully blocked angiotensin I-II conversion in DDs, whereas additional N-domain blockade was required to fully block conversion in IIs. Both domains contributed to bradykinin hydrolysis in all subjects, and the inhibition profile of RXP407 when using Mca-Ala was identical in IIs and DDs. In contrast, the RXPA380 concentrations required to block C-domain activity when using Mca-BK (1-8) were three-fold higher in IIs than DDs. CONCLUSION: The contributions of the C-domain and N-domain differ between DDs and IIs, and RXPA380 is the first inhibitor capable of distinguishing D-allele ACE from I-allele ACE. The lack of angiotensin II accumulation in DDs in vivo is not because of the often quoted concept that ACE is a nonrate-limiting enzyme. It may relate to the fact that in IIs both the N-domain and C- domain generate angiotensin II, whereas in DDs only the C-domain converts angiotensin I.

Serum paraoxonase-1 (PON1) is a high-density lipoprotein-associated enzyme that can inhibit low-density lipoprotein (LDL) oxidation in vitro. The role of PON1 in vivo still remains to be clarified. We investigated the effect of PON1 genotype (-107C > T and 192Q > R), concentration, paraoxonase activity, and arylesterase activity on the early phase of lipid peroxidation in plasma samples of 110 patients with heterozygous familial hypercholesterolemia. The degree of lipid oxidation was assessed by quantitation of oxidized-linoleic acid (the most abundant fatty acid present in LDL) using high performance liquid chromatography. We found a significant inverse correlation between paraoxonase activity and the oxidized-linoleic acid concentration (r =-0.22, P = 0.03), independent of baseline linoleic acid levels. These findings support an anti-oxidative role for PON1 in patients with FH, and thus may give insight into the functioning of PON1 in vivo.

BACKGROUND: Ubiquinol is a sensitive redox marker in the first line of the antioxidative defence mechanism and is increasingly being measured in oxidation studies. Because of its apparent instability during storage and processing, we compared various storage conditions. METHOD: Blood was collected from three volunteers into tubes containing EDTA; it was then separated at 4 degrees C and cryopreserved with saccharose (final concentration 6 g/L). Aliquots were stored with or without glutathione or butylated hydroxytoluene at -20 degrees C and -80 degrees C. RESULTS AND CONCLUSION: Ubiquinol in samples stored at -20 degrees C was not stable; however, it was stable when stored at -80 degrees C, even without addition of antioxidant. By contrast, alpha-tocopherol was stable under all conditions studied.

OBJECTIVE: Margarine with added plant sterols lowers plasma cholesterol levels. It is of importance to know whether these margarines can be used safely in carriers of a hereditary disorder with increased absorption of plant sterols. DESIGN: In an open feeding study of 8 weeks with a 2-week run-in period and 2 final weeks as a washout period on control margarine (0.3% plant sterols), two obligate heterozygous parents of a patient with classical sitosterolaemia were subjected for 4 weeks to a diet containing margarine enriched with plant sterols (8%). Fasting blood samples were taken weekly. Primary outcomes were plasma lipid and lipoprotein levels and plant sterol levels. RESULTS: Both parents were hyperlipidaemic. Total plasma cholesterol levels were decreased by 11 and 12%, respectively, after 4 weeks of the consumption of 40 g day(-1) of plant sterol-enriched margarine. This was mainly due to changes in LDL-cholesterol, whereas the other lipoproteins, including lipoprotein(a), were unaffected. Total plant sterol levels increased maximally 139% from 0.31 to 0.82% of total sterols in the father, and maximally 83% from 0.32 to 0.66% of total sterols in the mother. CONCLUSION: An intake of around 3 g day(-1) of plant sterols by subjects heterozygous for phytosterolaemia increased campesterol or sitosterol levels in blood to similar levels as found in normal subjects. In addition, plasma cholesterol levels were reduced to the same extent as in normal or hypercholesterolaemic individuals.

In previous work we identified a transfer/diffusion process occurring in the postprandial state that more or less contributes to the accumulation of beta-VLDL in familial dysbetalipoproteinemia (FD). Here we present a new theoretical concept underlying chylomicron processing developed on the basis of extended quantitative analyses of fat loading experiments, with both vitamins A and E, performed in patients with familial combined hyperlipidemia (FCH) in comparison to patients with FD and control subjects. Recovery of triglycerides from the fat load in the plasma triglyceride pool was <4%, indicating a very effective lipolysis process with an active remnant generation. Vitamin A from the fat load was, over 48 h, quantitatively recovered in the plasma lipoprotein pool; vitamin E was recovered to 2241%. Nevertheless, transfer/diffusion of both vitamins showed similar patterns. At equilibrium, their contents correlated strongly with the lipoprotein concentrations, the slopes being similar for control subjects and both groups of patients. Only in those FD patients with the highest lipid values, did the vitamin A/lipoprotein mass ratio in the Sf>100 fraction deviate from the total group mean. In the Sf 15-100 fraction, most specific for 'remnants', vitamin A/cholesterol ratios for all subjects were uniform proving that beta-VLDL formation is a thermodynamic process regulated by concentration gradients and the lipophilicity of lipoprotein constituents, not a typical feature for patients with FD. In patients with FD, vitamin A in the plasma pool was recovered excessively (276%) in line with recognition in various pools as a result of the transfer/diffusion process in plasma.

OBJECTIVE: To measure plasma thiol levels in women with normal pregnancies, women with preeclampsia, and nonpregnant controls to define plasma thiol's effect on glutathione homeostasis and pathophysiology of preeclampsia. METHODS: Total plasma cysteine, gamma-glutamylcysteine, homocysteine, cysteinylglycine, and glutathione levels were measured in ten nonpregnant women, ten women with normotensive pregnancies, and 20 women with preeclampsia at delivery. RESULTS: Median total plasma levels of all thiols in normotensive pregnant women were significantly lower than in nonpregnant women. Median total plasma cysteine and homocysteine levels in women with preeclampsia were significantly higher compared with pregnant controls (254 versus 190 micromol/L, P <.001; and 13.3 versus 8.4 micromol/L, P <.02, respectively), whereas glutathione levels were significantly lower in women with preeclampsia compared with those in pregnant controls (5.1 versus 6.3 micromol/L, P <.05). CONCLUSION: In women with preeclampsia, homocysteine and cysteine levels, which are lowered in normotensive pregnancy, were comparable to levels in nonpregnant women, whereas glutathione levels were lower. Those results suggest that in women with preeclampsia, glutathione use is higher or its synthesis is disturbed. Therefore, glutathione might affect pathophysiology of preeclampsia.

Vitamins A and E differ in hydrophobicity. When added to a fat load more or less specific labeling of chylomicrons and their remnants can be expected and this allows to approach the mechanism of postprandial lipemia from a new sight of view. Applied in a study in which 20 patients participated (eight patients with familial dysbetalipoproteinemia (FD), six patients with familial combined hyperlipidemia (FCH) and six controls) we found that vitamin A, no longer paralleled the apo B-48 concentrations from 9 h after a fat load, especially in the remnant fraction with Sf 15-100. Qualitatively, the distribution of vitamin A to the more dense fractions mirrored that of vitamin E, but the latter was more rapid. Both vitamins at the maximum of remnant-accumulation, at 14 h after the fat load, correlated with the cholesterol content of the remnant fraction. For vitamin E there was a similar concentration dependent distribution to all other lipoprotein fractions. The results confirm our view that the lipoprotein mechanism can be regarded as a dynamic system. During regular episodes following the meals, exogenous fat is, like the vitamins, distributed over all endogenously formed lipoproteins.This transfer process results in the formation of beta-VLDL and contributes to the pathogenesis of FCH and FD.

To gain more insight into the accumulation of beta-very low density lipoprotein (beta-VLDL) in familial dysbetalipoproteinemia (FD), we followed the courses of the levels of retinyl palmitate (rp), alpha-tocopherol (alpha-T) and apolipoprotein (apo) B-48 in various lipoprotein fractions for up to 48 h in eight patients with FD and six normolipidemic control subjects after an oral fat load (50 g fat/m2 containing 150000 IU of rp and 5000 IU of alpha-T). Alpha-T was added because of its rapid transfer to other lipoproteins. Fasting apo B-48 concentration in FD was normal to strongly elevated, dependent on the fasting lipid concentrations. 3 h after fat loading, total apo B-48 content did not abnormally increase; while the apo B-100 content in the triglyceride-rich lipoprotein fraction remained stable. The levels of both vitamins increased considerably, especially in the remnant fraction (Sf 15-100), which in due course exclusively contained apo B-100 in most hyperlipidemic patients. This, together with the observation that peaks for rp and alpha-T were observed 3-6 h later than for apo B-48 strongly suggests that both vitamins transfer or diffuse rapidly towards the apo B-100 containing VLDL. RP is thus more a marker for this process, which also comprises chylomicron lipids, than a specific marker for chylomicrons. This process, first described here, appears decisive in the pathogenesis of FD.

Dietary supplementation with n-3 fatty acids from fish oil alleviates inflammation in various chronic inflammatory disease states. Reductions in the production of pro-inflammatory cytokines interleukin 1 beta (IL-1 beta), tumour necrosis factor alpha (TNF-alpha), and interleukin 6 (IL-6) have been seen in humans after short-term n-3 fatty acid supplementation. We investigated long-term effects of dietary n-3 fatty acids on circulating cytokine concentrations and on ex vivo stimulated whole-blood production of IL-1 beta, TNF-alpha and interleukin 1 receptor antagonist (IL-1Ra), the naturally occurring antagonist of IL-1. A total of 58 monks with a mean age of 56 years were randomized into four groups and their diets were supplemented with 0, 3, 6, or 9 g of fish oil, providing 0, 1.06, 2.13 or 3.19 g of n-3 fatty acids per day. Subjects received equal amounts of saturated fatty acids, vitamin E and cholesterol. Compliance was excellent and erythrocyte fatty acid profiles closely reflected the amounts of n-3 fatty acids ingested. In the group receiving 9 g of fish oil per day, no influence of n-3 fatty acids on circulating cytokine concentrations was observed relative to placebo. Endotoxin-stimulated whole-blood cytokine production was measured at 26 and 52 weeks after the start and at 4, 8 and 26 weeks after cessation of supplementation. In all groups, the production of IL-1 beta and IL-1Ra was higher during supplementation than afterwards. However, no differences in cytokine production were noted between the placebo group and the various treatment groups at any point in time. Our results suggest that long-term supplementation of fish oil does not affect ex vivo cytokine production in man.