Long-term systemic administration of human recombinant interleukin-1beta induces a dose-dependent fall in circulating parathyroid hormone in rats

The synergism/antagonism between interleukin (IL)-1beta and parathyroid hormone (PTH) has been the subject of in vitro and in vivo work, but a possible direct action of the cytokine on PTH release has not been reported. We have investigated the effect of a continuous infusion of human recombinant IL-1beta (rIL-1beta) on circulating PTH during a 14-day period in 7-week-old female rats. This time interval was chosen in order to exclude initial hypercalcemia and to enable data collection under steady-state conditions. Five groups of 20 animals each had miniosmotic pumps (Alzet 2002, 200 microl) implanted subcutaneously and primed to release either distilled water (controls) or 100, 500, 1,000 and 2, 000 ng/24 h of rIL-1beta. Blood was drawn on days 1 and 14 for PTH, corticosterone and Ca2+ determinations. Adequate biological activity of the infused rIL-1beta was supported by elevated rectal temperature records and significant elevations of plasma corticosterone on day 14. The 100-ng dose had no effect but 500-2, 000 ng rIL-1beta/24 h significantly reduced plasma PTH in a dose-dependent manner down to 54% of basal value (20.4 +/- 1.1 vs. 15.3 +/- 1.4 pg/ml for 500 ng, p < 0.005; 20.5 +/- 1.3 vs 12.3 +/- 1.1 for 1,000 ng, p < 0.001, and 19.5 +/- 2.0 vs. 10.6 +/- 1.1 pg/ml for 2,000 ng, p < 0.0008). Despite these findings, no differences in blood Ca2+ could be detected between treated animals and controls. The following conclusions can be inferred from the foregoing: Systemic administration of rIL-1beta to rats induced a dose-dependent fall in circulating PTH without altering calcemia, calling into question the biological relevance of the former finding. Although the recorded PTH depression may indeed not have been severe enough to cause hypocalcemia, it can be hypothesized that osteoclast activation by rIL-1beta would enhance bone mineral release into the pool compensating for depressed PTH activity.

A quantitative phenytoin GC-MS method and it's validation for samples from human ex situ brain microdialysis, blood and saliva using solid-phase extraction

This study describes the development and validation of a gas chromatography-mass spectrometry (GC-MS) method to identify and quantitate phenytoin in brain microdialysate, saliva and blood from human samples. A solid-phase extraction (SPE) was performed with a nonpolar C8-SCX column. The eluate was evaporated with nitrogen (50°C) and derivatized with trimethylsulfonium hydroxide before GC-MS analysis. As the internal standard, 5-(p-methylphenyl)-5-phenylhydantoin was used. The MS was run in scan mode and the identification was made with three ion fragment masses. All peaks were identified with MassLib. Spiked phenytoin samples showed recovery after SPE of ≥94%. The calibration curve (phenytoin 50 to 1,200 ng/mL, n = 6, at six concentration levels) showed good linearity and correlation (r² > 0.998). The limit of detection was 15 ng/mL; the limit of quantification was 50 ng/mL. Dried extracted samples were stable within a 15% deviation range for ≥4 weeks at room temperature. The method met International Organization for Standardization standards and was able to detect and quantify phenytoin in different biological matrices and patient samples. The GC-MS method with SPE is specific, sensitive, robust and well reproducible, and is therefore an appropriate candidate for the pharmacokinetic assessment of phenytoin concentrations in different human biological samples.

Acute Stress Reduces Wound-Induced Activation of Microbicidal Potential of Ex Vivo Isolated Human Monocyte-Derived Macrophages

Background Psychological stress delays wound healing but the precise underlying mechanisms are unclear. Macrophages play an important role in wound healing, in particular by killing microbes. We hypothesized that (a) acute psychological stress reduces wound-induced activation of microbicidal potential of human monocyte-derived macrophages (HMDM), and (b) that these reductions are modulated by stress hormone release. Methods Fourty-one healthy men (mean age 35±13 years) were randomly assigned to either a stress or stress-control group. While the stress group underwent a standardized short-term psychological stress task after catheter-induced wound infliction, stress-controls did not. Catheter insertion was controlled. Assessing the microbicidal potential, we investigated PMA-activated superoxide anion production by HMDM immediately before and 1, 10 and 60 min after stress/rest. Moreover, plasma norepinephrine and epinephrine and salivary cortisol were repeatedly measured. In subsequent in vitro studies, whole blood was incubated with norepinephrine in the presence or absence of phentolamine (norepinephrine blocker) before assessing HMDM microbicidal potential. Results Compared with stress-controls, HMDM of the stressed subjects displayed decreased superoxide anion-responses after stress (p’s <.05). Higher plasma norepinephrine levels statistically mediated lower amounts of superoxide anion-responses (indirect effect 95% CI: 4.14–44.72). Norepinephrine-treated HMDM showed reduced superoxide anion-production (p<.001). This effect was blocked by prior incubation with phentolamine. Conclusions Our results suggest that acute psychological stress reduces wound-induced activation of microbicidal potential of HMDM and that this reduction is mediated by norepinephrine. This might have implications for stress-induced impairment in wound healing.

Potent innate immune response to pathogenic leptospira in human whole blood.

BACKGROUND Leptospirosis is caused by pathogenic spirochetes of the genus Leptospira. The bacteria enter the human body via abraded skin or mucous membranes and may disseminate throughout. In general the clinical picture is mild but some patients develop rapidly progressive, severe disease with a high case fatality rate. Not much is known about the innate immune response to leptospires during haematogenous dissemination. Previous work showed that a human THP-1 cell line recognized heat-killed leptospires and leptospiral LPS through TLR2 instead of TLR4. The LPS of virulent leptospires displayed a lower potency to trigger TNF production by THP-1 cells compared to LPS of non-virulent leptospires. METHODOLOGY/PRINCIPAL FINDINGS We investigated the host response and killing of virulent and non-virulent Leptospira of different serovars by human THP-1 cells, human PBMC's and human whole blood. Virulence of each leptospiral strain was tested in a well accepted standard guinea pig model. Virulent leptospires displayed complement resistance in human serum and whole blood while in-vitro attenuated non-virulent leptospires were rapidly killed in a complement dependent manner. In vitro stimulation of THP-1 and PBMC's with heat-killed and living leptospires showed differential serovar and cell type dependence of cytokine induction. However, at low, physiological, leptospiral dose, living virulent complement resistant strains were consistently more potent in whole blood stimulations than the corresponding non-virulent complement sensitive strains. At higher dose living virulent and non-virulent leptospires were equipotent in whole blood. Inhibition of different TLRs indicated that both TLR2 and TLR4 as well as TLR5 play a role in the whole blood cytokine response to living leptospires. CONCLUSIONS/SIGNIFICANCE Thus, in a minimally altered system as human whole blood, highly virulent Leptospira are potent inducers of the cytokine response.

A quantitiative LC-MS/MS method for the measurement of arachidonic acid, prostanoids, endocannabinoids, N-acylethanolamines and steroids in human plasma.

Free arachidonic acid is functionally interlinked with different lipid signaling networks including those involving prostanoid pathways, the endocannabinoid system, N-acylethanolamines, as well as steroids. A sensitive and specific LC-MS/MS method for the quantification of arachidonic acid, prostaglandin E2, thromboxane B2, anandamide, 2-arachidonoylglycerol, noladin ether, lineoyl ethanolamide, oleoyl ethanolamide, palmitoyl ethanolamide, steroyl ethanolamide, aldosterone, cortisol, dehydroepiandrosterone, progesterone, and testosterone in human plasma was developed and validated. Analytes were extracted using acetonitrile precipitation followed by solid phase extraction. Separations were performed by UFLC using a C18 column and analyzed on a triple quadrupole MS with electron spray ionization. Analytes were run first in negative mode and, subsequently, in positive mode in two independent LC-MS/MS runs. For each analyte, two MRM transitions were collected in order to confirm identity. All analytes showed good linearity over the investigated concentration range (r>0.98). Validated LLOQs ranged from 0.1 to 190ng/mL and LODs ranged from 0.04 to 12.3ng/mL. Our data show that this LC-MS/MS method is suitable for the quantification of a diverse set of bioactive lipids in plasma from human donors (n=32). The determined plasma levels are in agreement with the literature, thus providing a versatile method to explore pathophysiological processes in which changes of these lipids are implicated.

DARC shuttles inflammatory chemokines across the blood-brain barrier during autoimmune central nervous system inflammation

The Duffy antigen/receptor for chemokines, DARC, belongs to the family of atypical heptahelical chemokine receptors that do not couple to G proteins and therefore fail to transmit conventional intracellular signals. Here we show that during experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, the expression of DARC is upregulated at the blood-brain barrier. These findings are corroborated by the presence of a significantly increased number of subcortical white matter microvessels staining positive for DARC in human multiple sclerosis brains as compared to control tissue. Using an in vitro blood-brain barrier model we demonstrated that endothelial DARC mediates the abluminal to luminal transport of inflammatory chemokines across the blood-brain barrier. An involvement of DARC in experimental autoimmune encephalomyelitis pathogenesis was confirmed by the observed ameliorated experimental autoimmune encephalomyelitis in Darc(-/-) C57BL/6 and SJL mice, as compared to wild-type control littermates. Experimental autoimmune encephalomyelitis studies in bone marrow chimeric Darc(-/-) and wild-type mice revealed that increased plasma levels of inflammatory chemokines in experimental autoimmune encephalomyelitis depended on the presence of erythrocyte DARC. However, fully developed experimental autoimmune encephalomyelitis required the expression of endothelial DARC. Taken together, our data show a role for erythrocyte DARC as a chemokine reservoir and that endothelial DARC contributes to the pathogenesis of experimental autoimmune encephalomyelitis by shuttling chemokines across the blood-brain barrier.

Effects of opioids on human serotonin transporters.

The serotonin (5-hydroxtryptamine, 5-HT) system plays a role in analgesia and emesis. The aim of this study was to test whether opioids or ketamine inhibit the human 5-HT transporter and whether this increases free plasma 5-HT concentrations. HEK293 cells, stably transfected with the human 5-HT transporter cDNA, were incubated with morphine, hydromorphone, fentanyl, alfentanil, pethidine (meperidine), tramadol, ketamine, and the reference substance citalopram (specific 5-HT transporter inhibitor). The uptake of [(3)H]5-HT was measured by liquid scintillation counting. In a second series of experiments, study drugs were incubated with plasma of ten healthy blood donors and change of 5-HT plasma-concentrations were measured (ELISA). The end point was the inhibition of the 5-HT transporter by different analgesics either in HEK293 cells or in human platelets ex vivo. Tramadol, pethidine, and ketamine suppressed [(3)H]5-HT uptake dose-dependently with an IC50 of 1, 20.9, and 230 μM, respectively. These drugs also prevented 5-HT uptake in platelets with an increase in free plasma 5-HT. Free 5-HT concentrations in human plasma were increased by citalopram 1 μM, tramadol 20 μM, pethidine 30 μM, and ketamine 100 μM to 280 [248/312]%, 269 [188/349]%, and 149 [122/174]%, respectively, compared to controls without any co-incubation (means [95 % CI]; all p < 0.005). No change in both experimental settings was observed for the other opioids. Tramadol and pethidine inhibited the 5-HT transporter in HEK293 cells and platelets. This inhibition may contribute to serotonergic effects when these opioids are given in combination, e.g., with monoamine oxidase inhibitors or selective serotonin reuptake inhibitors.

A high-affinity natural autoantibody from human cord blood defines a physiologically relevant epitope on the FcepsilonRIalpha.

Natural Abs represent the indigenous immune repertoire and are thus present at birth and persist throughout life. Previously, human autoantibodies to the alpha domain of the high-affinity IgE receptor (FcepsilonRIalpha) have been isolated from Ab libraries derived from normal donors and patients with chronic urticaria. To investigate whether these anti-FcepsilonRIalpha Abs are present in the germline repertoire, we constructed a phage Fab display library from human cord blood, which represents the naive immune repertoire before exposure to exogenous Ags. All isolated clones specific to the FcepsilonRIalpha had the same sequence. This single IgM Ab, named CBMalpha8, was strictly in germline configuration and had high affinity and functional in vitro anaphylactogenic activity. Inhibition experiments indicated an overlapping epitope on the FcepsilonRIalpha recognized by both CBMalpha8 and the previously isolated anti-FcepsilonRIalpha Abs from autoimmune and healthy donors. This common epitope on FcepsilonRIalpha coincides with the binding site for IgE. Affinity measurements demonstrated the presence of Abs showing CBMalpha8-like specificity, but with a significantly lower affinity in i.v. Ig, a therapeutic multidonor IgG preparation. We propose a hypothesis of escape mutants, whereby the resulting lower affinity IgG anti-FcepsilonRIalpha Abs are rendered less likely to compete with IgE for binding to FcepsilonRIalpha.

Microstructure and Cerebral Blood Flow within White Matter of the Human Brain: A TBSS Analysis.

BACKGROUND White matter (WM) fibers connect different brain regions and are critical for proper brain function. However, little is known about the cerebral blood flow in WM and its relation to WM microstructure. Recent improvements in measuring cerebral blood flow (CBF) by means of arterial spin labeling (ASL) suggest that the signal in white matter may be detected. Its implications for physiology needs to be extensively explored. For this purpose, CBF and its relation to anisotropic diffusion was analyzed across subjects on a voxel-wise basis with tract-based spatial statistics (TBSS) and also across white matter tracts within subjects. METHODS Diffusion tensor imaging and ASL were acquired in 43 healthy subjects (mean age = 26.3 years). RESULTS CBF in WM was observed to correlate positively with fractional anisotropy across subjects in parts of the splenium of corpus callosum, the right posterior thalamic radiation (including the optic radiation), the forceps major, the right inferior fronto-occipital fasciculus, the right inferior longitudinal fasciculus and the right superior longitudinal fasciculus. Furthermore, radial diffusivity correlated negatively with CBF across subjects in similar regions. Moreover, CBF and FA correlated positively across white matter tracts within subjects. CONCLUSION The currently observed findings on a macroscopic level might reflect the metabolic demand of white matter on a microscopic level involving myelination processes or axonal function. However, the exact underlying physiological mechanism of this relationship needs further evaluation.

Tracking of plasma lipids and lipoproteins, within-person and year-to-year variability in plasma cholesterol levels among participants from age 8 to 18 in Project HeartBeat!

Coronary heart disease remains the leading cause of death in the United States and increased blood cholesterol level has been found to be a major risk factor with roots in childhood. Tracking of cholesterol, i.e., the tendency to maintain a particular cholesterol level relative to the rest of the population, and variability in blood lipid levels with increase in age have implications for cholesterol screening and assessment of lipid levels in children for possible prevention of further rise to prevent adulthood heart disease. In this study the pattern of change in plasma lipids, over time, and their tracking were investigated. Also, within-person variance and retest reliability defined as the square root of within-person variance for plasma total cholesterol, HDL-cholesterol, LDL-cholesterol, and triglycerides and their relation to age, sex and body mass index among participants from age 8 to 18 years were investigated. ^ In Project HeartBeat!, 678 healthy children aged 8, 11 and 14 years at baseline were enrolled and examined at 4-monthly intervals for up to 4 years. We examined the relationship between repeated observations by Pearson's correlations. Age- and sex-specific quintiles were calculated and the probability of participants to remain in the uppermost quintile of their respective distribution was evaluated with life table methods. Plasma total cholesterol, HDL-C and LDL-C at baseline were strongly and significantly correlated with measurements at subsequent visits across the sex and age groups. Plasma triglyceride at baseline was also significantly correlated with subsequent measurements but less strongly than was the case for other plasma lipids. The probability to remain in the upper quintile was also high (60 to 70%) for plasma total cholesterol, HDL-C and LDL-C. ^ We used a mixed longitudinal, or synthetic cohort design with continuous observations from age 8 to 18 years to estimate within person variance of plasma total cholesterol, HDL-C, LDL-C and triglycerides. A total of 5809 measurements were available for both cholesterol and triglycerides. A multilevel linear model was used. Within-person variance among repeated measures over up to four years of follow-up was estimated for total cholesterol, HDL-C, LDL-C and triglycerides separately. The relationship of within-person and inter-individual variance with age, sex, and body mass index was evaluated. Likelihood ratio tests were conducted by calculating the deviation of −2log (likelihood) within the basic model and alternative models. The square root of within-person variance provided the retest reliability (within person standard deviation) for plasma total cholesterol, HDL-C, LDL-C and triglycerides. We found 13.6 percent retest reliability for plasma cholesterol, 6.1 percent for HDL-cholesterol, 11.9 percent for LDL-cholesterol and 32.4 percent for triglycerides. Retest reliability of plasma lipids was significantly related with age and body mass index. It increased with increase in body mass index and age. These findings have implications for screening guidelines, as participants in the uppermost quintile tended to maintain their status in each of the age groups during a four-year follow-up. The magnitude of within-person variability of plasma lipids influences the ability to classify children into risk categories recommended by the National Cholesterol Education Program. ^