Dengue virus-infected human dendritic cells reveal hierarchies of naturally expressed novel NS3 CD8 T cell epitopes.

Detailed knowledge of dengue virus (DENV) cell-mediated immunity is limited. In this study we characterize CD8(+) T lymphocytes recognizing three novel and two known non-structural protein 3 peptide epitopes in DENV-infected dendritic cells. Three epitopes displayed high conservation (75-100%), compared to the others (0-50%). A hierarchy ranking based on magnitude and polyfunctionality of the antigen-specific response showed that dominant epitopes were both highly conserved and cross-reactive against multiple DENV serotypes. These results are relevant to DENV pathogenesis and vaccine design.

Creatine feeding does not enhance intramyocellular glycogen concentration during carbohydrate loading: an in vivo study by 31P- and 13C-MRS.

The main aim of this study was to examine the hypothesis that creatine (Cr) feeding enhances myocellular glycogen storage in humans undergoing carbohydrate loading. Twenty trained male subjects were randomly assigned to have their diets supplemented daily with 252 g of glucose polymer (GP) and either 21 g of Cr (CR-GP, n = 10) or placebo (PL-GP, n = 10) for 5 days. Changes in resting myocellular glycogen and phosphocreatine (PCr) were determined with Magnetic Resonance Spectroscopy (13C- and 31P-MRS, respectively). After CR-GP, the levels of intramyocellular glycogen increased from 147 +/- 13 (standard error) mmol x (kg wet weight(-1)) to 172 +/- 13 m mol x (kg wet weight)(-1), while it increased from 134 +/- 17 mmol x (kg wet weight)(-) to 182 +/- 17 mmol x (kg wet weight)(-1) after PL-GP; the increments in intramyocellular glycogen concentrations were not statistically different. The increment in the PCr/ATP ratio after CR-GP (+ 0.20 +/- 0.12) was significantly different compared to PL-GP (- 0.34 +/- 0.16) (p < 0.05). The present results do not support the hypothesis that Cr loading increases muscle glycogen storage.

Identification and mapping of keratinocyte muscarinic acetylcholine receptor subtypes in human epidermis.

Acetylcholine mediates cell-to-cell communications in the skin. Human epidermal keratinocytes respond to acetylcholine via two classes of cell-surface receptors, the nicotinic and the muscarinic cholinergic receptors. High affinity muscarinic acetylcholine receptors (mAChR) have been found on keratinocyte cell surfaces at high density. These receptors mediate effects of muscarinic drugs on keratinocyte viability, proliferation, adhesion, lateral migration, and differentiation. In this study, we investigated the molecular structure of keratinocyte mAChR and their location in human epidermis. Polymerase chain reaction amplification of cDNA sequences uniquely present within the third cytoplasmic loop of each subtype demonstrated the expression of the m1, m3, m4, and m5 mAChR subtypes. To visualize these mAChR, we raised rabbit anti-sera to synthetic peptide analogs of the carboxyl terminal regions of each subtype. The antibodies selectively bound to keratinocyte mAChR subtypes in immunoblotting membranes and epidermis, both of which could be abolished by preincubating the anti-serum with the peptide used for immunization. The immunofluorescent staining patterns produced by each antibody in the epidermis suggested that the profile of keratinocyte mAChR changes during epidermal turnover. The semiquantitative analysis of fluorescence revealed that basal cells predominantly expressed m3, prickle cells had equally high levels of m4 and m5, and granular cells mostly possessed m1. Thus, the results of this study demonstrate for the first time the presence of m1, m3, m4, and m5 mAChR in epidermal keratinocytes. Because keratinocytes express a unique combination of mAChR subtypes at each stage of their development in the epidermis, each receptor may regulate a specific cell function. Hence, a single cytotransmitter, acetylcholine, and muscarinic drugs may exert different biologic effects on keratinocytes at different stages of their maturation.

Choline acetyltransferase, acetylcholinesterase, and nicotinic acetylcholine receptors of human gingival and esophageal epithelia.

A non-neuronal cholinergic system that includes neuronal-like nicotinic acetylcholine receptors (nAChRs) has recently been described in epithelial cells that line the skin and the upper respiratory tract. Since the use of nicotine-containing products is associated with morbidity in the upper digestive tract, and since nicotine may alter cellular functions directly via nAChRs, we sought to identify and characterize a non-neuronal cholinergic system in the gingival and esophageal epithelia. mRNA transcripts for alpha3, alpha5, alpha7, and beta2 nAChR subunits, choline acetyltransferase, and the asymmetric and globular forms of acetylcholinesterase were amplified from gingival keratinocytes (KC) by means of polymerase chain-reactions. These proteins were visualized in the gingival and esophageal epithelia by means of specific antibodies. Variations in distribution and intensity of immunostaining were found, indicating that the repertoire of cholinergic enzymes and receptors expressed by the cells changes during epithelial maturation, and that an upward concentration gradient of free acetylcholine exists. Blocking of the nAChRs with mecamylamine resulted in reversible loss of cell-to-cell adhesion, and shrinking and rounding of cultured gingival KC. Activation of the receptors with acetylcholine or carbachol caused stretching and peripheral ruffling of the cytoplasmic aprons, and formation of new intercellular contacts. These results demonstrate that both the keratinizing epithelium of attached gingiva and the non-keratinizing epithelium lining the upper two-thirds of the esophageal mucosa possess a non-neuronal cholinergic system. The nAChRs expressed by these epithelia are coupled to regulation of cell adhesion and motility, and may provide a target for the deleterious effects of nicotine.

Noninvasive measurement of muscle high-energy phosphates and glycogen concentrations in elite soccer players by 31P- and 13C-MRS.

PURPOSE: The purpose of this study was to measure noninvasively the absolute concentrations of muscle adenosine triphosphate [ATP], phosphocreatine [PCr], inorganic phosphate (Pi), and glycogen [Gly] of elite soccer players. METHODS: Magnetic resonance spectroscopy (31P- and 13C-MRS) was used to measure the concentrations of metabolites in the calf muscles of 18 young male players [age = 17.5 +/- 1.0 (SD) yr]. RESULTS: Average muscle [PCr] and [ATP] were 17.8 +/- 3.3 and 6.0 +/- 1.2 mmol x (kg wet weight)(-1), respectively. The ratios of Pi/PCr and PCr/ATP were 0.15 +/- 0.05 and 3.00 +/- 0.26, respectively. The muscle [Gly] was 144 +/- 54 mmol x (kg wet weight)(-1). There was a high correlation (r = 0.93, P < 0.0001) between muscle ATP and PCr concentrations, but there was no correlation between [Gly] and [PCr] or [ATP]. The concentrations of the different metabolites determined in the present study with noninvasive MRS methods were within the ranges of values reported in human muscle from biochemical analysis of muscle biopsies. CONCLUSION: MRS methods can be utilized to assess noninvasively the muscle energetic status of elite soccer players during a soccer season. The high correlation between ATP and PCr might be indicative of fiber type differences in the content of these two metabolites.

Muscle glycogen degradation during simulation of a fatiguing soccer match in elite soccer players examined noninvasively by 13C-MRS.

PURPOSE: The purpose of this research project was to noninvasively determine individual muscle glycogen [Gly] degradation during a test intended to predict individual fatigue in intense soccer matches. METHODS: The [Gly] of the calf muscles of 17 elite soccer players [age = 17.4 +/- 0.8 (SD)] were measured with 13C-MRS before and after an alternating velocity test to exhaustion. Blood samples were taken before and 3 min after the test for determination of blood metabolites. RESULTS: Average muscle [Gly] was 135 +/- 53 mmol x (kg wet weight)(-1) before and 87 +/- 27 mmol x (kg wet weight)(-1) (P < 0.001) after exhaustion (42 +/- 25 min). There was a high correlation (r = 0.87, P < 0.0001) between muscle [Gly] at rest and net muscle [Gly] utilized. There was also a more moderate correlation (r = 0.62, P < 0.01) between net muscle [Gly] used and time to exhaustion during the soccer-specific test. There was some evidence of correlation (r = 0.42, P = 0.09) between resting [Gly] and time to exhaustion. Plasma lactate increased (P < 0.001) from 0.8 +/- 0.4 before the test to 2.5 +/- 1.0 mmol x L(-1) at exhaustion, whereas ammonia was raised (P < 0.0001) from 44.1 +/- 10.3 to 89.7 +/- 14.9 micromol x L(-1). Similarly, plasma free fatty acids were elevated (P < 0.0001) from 148 +/- 106 to 797 +/- 401 micromol x L(-1), and glycerol was increased (P < 0.0001) from 48.3 +/- 17.7 to 182.2 +/- 61.8 micromol x L(-1). Insulin levels (11.9 +/- 3.7 vs 11.7 +/- 4.8 microU x mL(-1)) remained the same. Creatine kinase levels increased (P < 0.0001) from 486 +/- 501 to 640 +/- 548 micromol x L(-1) after the test. CONCLUSIONS: We conclude that exhaustion during soccer-specific performance is related to the capacity to utilize muscle [Gly]. The results underline the importance of dietary counseling (glycogen loading and resynthesis strategies) and proper training to enhance the glycogen levels and glycogenolytic capacity of the players.

Main sources of indoor radon in the Swiss Central Alps.

To estimate the amount of radon gas transferred from the water to the indoor atmosphere of dwellings in the Swiss Central Alps, the radon concentrations of tap water of about 50 public water supplies were analysed. The radon levels of water in alpine communities fit a lognormal distribution with a geometric mean of 26.5 Bq/1 and a geometric standard deviation of 3.15. Radon monitoring in typical homes in the Swiss Midland revealed a significant influence of wind velocity and temperature difference indoor/outdoor on the indoor radon-daughter levels during the winter period.

Correlation among the terrestrial gamma radiation, the indoor air 222Rn, and the tap water 222Rn in Switzerland.

The external gamma radiation and the indoor air Rn (222Rn) concentration were measured in 55 houses of the South East Grisons, the Urseren valley, and the Upper Rhine valley (crystalline subsoils) and in 39 houses of the Molasse basin and the Helvetic nappes (sedimentary subsoils). In homes located on a crystalline subsoil, a mean cellar gamma level of 1.40 mGy y-1 was measured, which is twice the mean gamma level of 0.70 mGy y-1 found in homes built on a sedimentary subsoil. The cellar 222Rn gas concentration is about six times higher in houses with a crystalline subsoil (1232 Bq m-3) than in houses with a sedimentary subsoil (201 Bq m-3). Although a weak correlation is observed between the mean gamma radiation levels and mean cellar 222Rn gas concentrations for the five subregions investigated, the gamma levels and the 222Rn gas concentrations do not correlate for single homes. For the population living on the ground floor of a house with a crystalline subsoil, the gamma radiation and the indoor air 222Rn lead to estimated mean exposures of 1.16 mSv and 9.44 mSv effective dose equivalent per year, respectively. In houses with a sedimentary subsoil, these mean exposures lead to 0.68 mSv y-1 and 3.22 mSv y-1, respectively. A mean tap water 222Rn content of 38.3 Bq L-1 and 10.4 Bq L-1 was measured in 31 villages with a crystalline subsoil and 73 villages with a sedimentary subsoil, respectively. Radon-222 degasing from the tap water into the indoor air leads to an additional exposure of about 0.11 mSv y-1 and 0.03 mSv y-1 in homes with a crystalline subsoil and homes with a sedimentary subsoil, respectively.

Influence of subsoil geology and construction technique on indoor air 222Rn levels in 80 houses of the central Swiss Alps.

The indoor 222Rn level depends mainly on the subsoil geology, the cellar floor permeability, the cellar aeration, the air-tightness of the homes, and the aeration habits of the occupants. These five parameters and the 222Rn levels in the cellar and in the living room on the ground floor were compiled in 80 one- or two-family houses of the central Swiss Alps. The 222Rn levels were measured with passive alpha track detectors. Houses located on a granite, ortho-gneiss or verrucano subsoil have a cellar 222Rn level that is on the average 4.4 times higher than houses which are built on grey-schist or sediments. The cellar level is on the average 5.4 times higher if the cellar has partially a gravel or earth floor than if the whole cellar surface is covered with a concrete floor. Energy-efficient, highly air-tightened homes have a living room level that is on the average 1.8 times higher than normally insulated conventional homes. In the cellars and the living rooms of the 80 houses considered, arithmetic mean 222Rn levels of 724 Bq m-3 (20 pCi L-1) and 178 Bq m-3 (4.8 pCi L-1), respectively, were found. In the central Swiss Alps 222Rn and 222Rn decay products lead to an estimated mean exposure of 5.3 mSv effective dose equivalent per year.

Increased indoor Rn levels due to actinide containing mineral collections displayed in living areas.

Collection of crystals ranging from quartz to U or Th containing minerals is a widespread spare time activity in Switzerland. Radon emanation from stones displayed in showcases in the living area may contribute considerably to elevated Rn levels in the indoor air. Time-averaged Rn gas measurements in 35 homes of subscribers to a journal for mineral collectors showed an unexpected statistically significant increase of 98 Bq m-3 in the Rn level of the room containing the collections compared to the levels measured in a control room on the same floor. Using ICRP Publication 50 conversion factors for indoor exposure to Rn decay products, the additional effective dose equivalent contracted was estimated to amount to an average of 2.7 mSv y-1. Although the 220Rn emanation rate, as measured in the display cases, was considerable in several cases, the dose from 220Rn decay products in the living area remained always a small fraction of the Rn dose. Remedial actions for crystal collections containing considerable amounts of U- or Th-based minerals are suggested.

Indoor Rn levels in different geological areas in Switzerland.

The distribution of indoor Rn concentrations in different geological areas in Switzerland was studied using passive alpha-track detectors. Measurements involving a sample of 400 single-family homes were made in the cellar, on the ground floor and the first floor, respectively. On the basis of a pilot survey, the country was divided into four zones in which the Rn distribution in houses was analyzed separately. The indoor exposure to Rn and Rn decay products is quite variable from region to region. The geology of the different areas was found to be an important factor in determining the mean value Rn levels. In the basin north of the Alps, where the population centers are located, a median Rn gas level of 47 Bq m-3 for the living area was found. The arithmetic mean value of 60 Bq m-3 in this region leads to an annual effective dose equivalent of about 1.8 mSv. For the population living in alpine areas, an arithmetic mean value exceeding 200 Bq m-3 will lead to an annual effective dose equivalent in the range of 6 mSv. The estimated exposure to Rn and Rn decay products for the upper one-percentile of the homes in the most affected alpine region even exceeds the annual limit of 50 mSv effective dose equivalent for occupational exposure.

Comparison of methods for the determination of absolute metabolite concentrations in human muscles by 31P MRS.

In order to determine metabolite concentrations in human skeletal muscles by in vivo 31P MRS, different quantification methods were analyzed with regard to the accuracy and reproducibility of results and the simplicity of handling. Each quantification method comprised a calibration strategy and a localization technique. Extensive in vivo and in vitro tests showed that homonuclear phantom-based calibration strategies yielded significantly more accurate (lower systematic errors) and more reproducible (lower statistical errors) concentration estimates than heteronuclear strategies using internal water as a concentration standard. Additionally, the former strategies are easier to handle than the latter. Localization with the volume-selective sequence ISIS yielded slightly more reproducible results than localization by surface coil. We conclude that phosphorus metabolite concentrations are determined most accurately with phantom-based calibration strategies in combination with ISIS localization (measurement errors approximately 5-7%).

Heating effects of metallic implants by MRI examinations.

Magnetic radiofrequency fields applied in magnetic resonance imaging examinations induce electrical currents in metallic implants. These eddy currents may heat up the implants and thus may be capable of causing localized tissue heating. The rf power deposition and the joule heating of the implant can be calculated by solving Maxwell's equations for the specific problem. First, extreme in vitro worst-case experiments were performed with a large and very thin aluminum sheet, which was placed in a 1.5-T MRI device in a position parallel to the magnetic rf field. In agreement with the theoretical results the temperature rise of a thermally insulated sheet amounted to only 0.08 degrees C after a 15-min MRI examination at 64 MHz. No temperature rise in the aluminum sheet could be measured for a sheet immersed in a saline solution. Second, in vitro experiments with a hip joint prosthesis and an osteosynthetic plate were performed to confirm the theoretical results, which predict nearly no temperature rise in the metallic implants. No temperature rise in the implants could be measured.

Comparison of calibration strategies for the in vivo determination of absolute metabolite concentrations in the human brain by 31P MRS.

Cerebral concentrations of phosphorus metabolites can be assessed non-invasively by 31P MRS provided the metabolite signals are calibrated with the signal of a standard of known concentration. The reliability of the concentration estimates depends mainly on the strategy of calibration. Three strategies were compared by assessing the concentrations both in a test dummy and in the brain of volunteers. The first strategy utilized tissue water as an internal heteronuclear concentration standard. The second and third strategies used as phosphorus solution as an external homonuclear standard; this solution was either put into a reference bottle placed on top of the head or into a simulation phantom measured instead of the head. Localization was always achieved with the ISIS pulse sequence. The two external homonuclear strategies achieved a higher accuracy (mean error approximately 5%) and reproducibility (mean SD approximately 8%) of the concentration estimates than the internal heteronuclear strategy (mean error approximately 11%; mean SD approximately 15%).

Assessment of absolute metabolite concentrations in human tissue by 31P MRS in vivo. Part I: Cerebrum, cerebellum, cerebral gray and white matter.

Absolute metabolite concentrations were determined in four different brain regions using phosphorus magnetic resonance spectroscopy (31P MRS) on 10 healthy adult volunteers. Localized spectra were collected simultaneously from the cerebellum and the cerebrum and, later, from deep white matter and cortical gray matter by means of a two-volume ISIS pulse sequence and a Helmholtz-type RF-coli. Each brain spectrum was quantified with a calibration spectrum from a head-shaped simulation phantom. A time-domain fitting routine was used to process the fully relaxed data. Several metabolite concentrations (mmol/liter) differed significantly between the cerebrum and the cerebellum (PME = 3.2 +/- 0.3 and 4.0 +/- 0.6, PCr = 2.9 +/- 0.3 and 3.9 +/- 0.4, NTP = 2.9 +/- 0.2 and 2.6 +/- 0.2, respectively) and between cortical gray matter and deep white matter (PME = 3.1 +/- 0.4 and 4.3 +/- 0.8, PDE = 10.1 +/- 2.5 and 14.2 +/- 2.6, respectively). The concentration of free magnesium ion was found to be similar in all four brain regions (0.53 +/- 0.21 mmol/liter) but the intracellular pH was significantly higher in the cerebellum (7.04 +/- 0.03) than in the cerebrum (6.99 +/- 0.02).

Assessment of absolute metabolite concentrations in human tissue by 31P MRS in vivo. Part II: Muscle, liver, kidney.

Absolute metabolite concentrations were assessed in the muscle, the liver, and the kidney of healthy human volunteers by 31P MRS. Fully relaxed in vivo spectra were acquired with a surface coil and were localized with an adiabatic ISIS pulse sequence. The spectra were quantified with a subsequent measurement of a calibration phantom and were processed iteratively in the time domain. The following mean metabolite concentrations (mmol/liter) were measured in the resting male calf muscle (n = 9), in the fasting liver (n = 12), and in the orthotopic kidney (n = 5): [PME] = 2.0 +/- 0.6, 3.8 +/- 0.7, and 2.6 +/- 0.9, [Pi] = 2.9 +/- 0.3, 1.8 +/- 0.3, and 1.6 +/- 0.4, [PDE] = 3.8 +/- 0.8, 9.7 +/- 1.5, and 4.9 +/- 1.1, [PCr] = 22.0 +/- 1.2, 0, and 0, [NTP] = 5.7 +/- 0.4, 2.9 +/- 0.4, and 2.0 +/- 0.3, respectively. Several interesting findings are to be emphasized: The concentrations of Pi, PCr, and NTP were 20% lower in the muscle of women than of men. In addition, the pHi was significantly lower in female muscle (6.99 +/- 0.03) than in male muscle (7.05 +/- 0.03). The pHi in the liver (7.12 +/- 0.09) and in the kidney (7.09 +/- 0.08) were higher than in the muscle of both genders. The free magnesium concentration (mmol/liter) was higher in the liver (1.40 +/- 0.64) than in the kidney (0.79 +/- 0.39) and in the muscle (0.52 +/- 0.10).

Developmental changes of phosphorus metabolite concentrations in the human brain: a 31P magnetic resonance spectroscopy study in vivo.

Phosphorus magnetic resonance spectroscopy is a noninvasive method to investigate brain metabolism in vivo. ATP generally serves as an internal concentration standard for the quantification of the various phosphorus metabolites, because the ATP concentration in mammalian brains is assumed to be age independent. This presumption is based on observations made in the biochemical analysis of the developing rat brain. In the present study, metabolite concentrations were assessed with an external concentration standard. Each brain spectrum was quantified using a calibration spectrum that was acquired from a phantom after the in vivo brain measurement. Fully relaxed localized brain spectra were obtained from 16 neonates (2-28 d), 17 infants (6-20 mo), and 28 adults (22-58 y). The metabolite concentrations (in mmol/L) changed from neonates to adults: phosphomonoester from 4.5 to 3.5, inorganic phosphate from 0.6 to 1.0, phosphodiester from 3.2 to 11.7, phosphocreatine from 1.4 to 3.4, and ATP from 1.6 to 2.9. We conclude that 1) the ATP concentration in the human brain almost doubles between neonates and adults, and 2) ATP may not be used as an age-independent internal concentration standard.

Toward fully automatic estimation of in vivo 31P spectra.

Fitting a model to an experimental spectrum is a difficult nonlinear estimation problem. The solution presented here is to start an iterative search procedure sufficiently close to the optimal model parameter set. This is achieved by providing tissue-dependent a priori peak information and by a novel correlation method to get good primary estimates of the resonance and phase offset parameters. The resulting estimation procedure is fully automatic and has proven to be robust for 31P data.