Survey and genetic characterization of Vibrio cholerae in Apalachicola Bay, Florida (2012-2014).

AIMS: A small outbreak of gastroenteritis in 2011 in Apalachicola Bay, FL was attributed to consumption of raw oysters carrying Vibrio cholerae serotype O75. To better understand possible health risks, V. cholerae was surveyed in oysters, fish and seawater, and results were compared to data for Vibrio vulnificus and Vibrio parahaemolyticus. METHODS AND RESULTS: Enrichment protocols were used to compare prevalence of V. cholerae (0, 48, 50%), V. vulnificus (89, 97, 100%) and V. parahaemolyticus (83, 83, 100%) in fish, seawater and oysters respectively. Compared to other species, Most probable number results indicated significantly (P < 0·001) lower abundance of V. cholerae, which was also detected more frequently at lower salinity, near-shore sites; other species were more widely distributed throughout the bay. Genes for expression (ctxA, ctxB) and acquisition (tcpA) of cholera toxin were absent in all strains by PCR, which was confirmed by whole genome sequencing; however, other putative virulence genes (toxR, rtxA, hlyA, opmU) were common. Multi-locus sequence typing revealed 78% of isolates were genetically closer to V. cholerae O75 lineage or other non-O1 serogroups than to O1 or O139 serogroups. Resistance to amoxicillin, kanamycin, streptomycin, amikacin, tetracycline and cephalothin, as well as multidrug resistance, was noted. CONCLUSIONS: Results indicated minimal human health risk posed by V. cholerae, as all isolates recovered from Apalachicola Bay did not have the genetic capacity to produce cholera toxin. Vibrio cholerae was less prevalent and abundant relative to other pathogenic Vibrio species. SIGNIFICANCE AND IMPACT OF THE STUDY: These studies provide important baseline observations for V. cholerae virulence potential regarding: (i) genetic relatedness to V. cholerae O75, (ii) antibiotic resistance and (iii) prevalence of multiple virulence genes. These data will serve as a biomonitoring tool to better understand ecosystem status and management if bacterial densities and virulence potential are altered by environmental and climatic changes over time.

A Novel Nano-approach for Targeted Inner Ear Imaging.

During the last decade, there have been major improvements in imaging modalities and the development of molecular imaging in general. However detailed inner ear imaging still provides very limited information to physicians. This is unsatisfactory as sensorineural hearing loss is the main cause of permanent hearing loss in adults and at least 134 genetic mutations that result in congenital hearing loss have been identified. We are still unable, in most cases where gross anatomical changes are not observed, to determine the exact cause of hearing loss at a cellular or molecular level in patients using non-invasive techniques. This limitation in inner ear diagnostic modalities is a major obstacle behind the delay in discovering treatments for many of the causes of sensorineural hearing loss. This paper initially investigated the use of targeted gold nanoparticles as contrast agents for inner ear imaging. These nanoparticles have many useful characteristics such as being easy to target and possessing minimal cytotoxicity. We were able to detect the nanoparticles diffusing in the hair cells using confocal microscopy. Regrettably, despite their many admirable characteristics, the gold nanoparticles were unable to significantly enhance CT imaging of the inner ear. Consequently, we investigated liposomal iodine as a potential solution for the unsatisfactory CT contrast obtained with the gold nanoparticles. Fortunately, significant enhancement of the micro-CT image was observed with either Lugol's solution or liposomal iodine, with Lugol's solution enabling fine inner ear structures to be detected.

Application of chitosan microparticles for mitigation of Salmonella in agricultural water.

AIM: The activity of chitosan microparticles (CM) was examined using a matrix of conditions in order to assess the efficacy of CM as a mitigation against various strains of Salmonella enterica in agricultural water. METHODS AND RESULTS: Different concentrations of CM (0, 0·01, 0·1, 0·2, 0·3% w/v) were examined for antimicrobial activity against log vs stationary phase cells of Salmonella and at different conditions of temperature, salinity and pH. Results showed greatest activity with 0·3% CM at pH 7, 25-37°C without additional of salt. Significant reductions in Salmonella levels were also achieved in natural pond water, although decreases were reduced compared to sterile water. All serotypes were sensitive to CM, with minimal inhibitory concentrations ranging from 0·0031 to 0·0250% w/v. Phylogenic analysis of Javiana strains showed increased resistance appeared in multiple genetic lineages. CONCLUSION: Conditions demonstrating greatest CM activity were compatible with agricultural practices. Although sensitivity to CM varied among Salmonella strains, all strains were sensitive under conditions examined in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: This research indicated that CM, a natural compound with minimal environmental impact, could be an effective alternative for mitigating Salmonella in agricultural water applications.

beta-Catenin initiates tooth neogenesis in adult rodent incisors.

beta-Catenin signaling is required for embryonic tooth morphogenesis and promotes continuous tooth development when activated in embryos. To determine whether activation of this pathway in the adult oral cavity could promote tooth development, we induced mutation of epithelial beta-catenin to a stabilized form in adult mice. This caused increased proliferation of the incisor tooth cervical loop, outpouching of incisor epithelium, abnormal morphology of the epithelial-mesenchymal junction, and enhanced expression of genes associated with embryonic tooth development. Ectopic dental-like structures were formed from the incisor region following implantation into immunodeficient mice. Thus, forced activation of beta-catenin signaling can initiate an embryonic-like program of tooth development in adult rodent incisor teeth.

Ultra-short echo-time MRI detects changes in bone mineralization and water content in OVX rat bone in response to alendronate treatment.

In this work we hypothesize that bisphosphonate treatment following ovariectomy manifests in increased phosphorus and decreased water concentration, both quantifiable nondestructively with ultra-short echo-time (UTE) (31)P and (1)H-MRI techniques. We evaluated this hypothesis in ovariectomized (OVX) rats undergoing treatment with two regimens of alendronate. Sixty female four-month-old rats were divided into four groups of 15 animals each: ovariectomized (OVX), OVX treatment groups ALN1 and ALN2, receiving 5 microg/kg/day and 25 microg/kg/day of alendronate, and a sham-operated group (NO) serving as control. Treatment, starting 1 week post-surgery, lasted for 50 days at which time animals were sacrificed. Whole bones from the left and right femora were extracted from all the animals. (31)P and (1)H water concentration were measured by UTE MRI at 162 and 400 MHz in the femoral shaft and the results compared with other measures of mineral and matrix properties obtained by (31)P solution NMR, CT density, ash weight, and water measured by dehydration. Mechanical parameters (elastic modulus, EM, and ultimate strength, US) were obtained by three-point bending. The following quantities were lower in OVX relative to NO: phosphorus concentration measured by (31)P-MRI (-8%; 11.4+/-0.9 vs. 12.4+/-0.8%, p<0.005), (31)P-NMR (-4%; 12.8+/-0.4 vs. 13.3+/-0.8 %, p<0.05) and micro-CT density (-2.5%; 1316+/-34 vs. 1349+/-32 mg/cm(3), p=0.005). In contrast, water concentration by (1)H-MRI was elevated in OVX relative to NO (+6%; 15.5+/-1.7 vs. 14.6+/-1.4 %, p<0.05). Alendronate treatment increased phosphorus concentration and decreased water concentration in a dose-dependent manner, the higher dose yielding significant changes relative to values found in OVX animals: (31)P-MRI (+14%; p<0.0001), (31)P-NMR (+9%; p<0.0001), ash content (+1.5%; p<0.005), micro-CT mineralization density (+2.8%; p<0.05), and (1)H-MRI, (-19%, p<0.0001). The higher dose raised phosphorus concentration above and water concentration below NO levels: (31)P-MRI (+6%; p<0.05), (31)P-NMR (+5%; p=0.01), ash content (+1.5%; p=0.005), (1)H-MRI (-14%; p<0.0001), and drying water (-10%; p<0.0005). Finally, the group means of phosphorus concentration were positively correlated with EM and US (R(2)> or =0.98, p<0.001 to p<0.05) even though the pooled data from individual animals were not. The results highlight the implications of estrogen depletion and bisphosphonate treatment on mineral composition and mechanical properties and the potential of solid-state MR imaging to detect these changes in situ in an animal model of rat ovariectomy.

Construction and calibration of a 50 T/m z-gradient coil for quantitative diffusion microimaging.

q-Space imaging is capable of providing quantitative geometrical information of structures at cellular resolution. However, the size of restrictions that can be probed hinges on available gradient amplitude and places very high demands on gradient performance. In this work we describe the design and construction of a small, high-amplitude (50 T/m) z-gradient coil, interfaced with a commercial 9.4 T microimaging system. We also describe a method to calibrate the coil for quantitative measurements of molecular diffusion at very high-gradient amplitudes. Calibration showed linear current response up to 50 T/m, with a gain=1.255 T/m/A. The z-gradient coil was combined with the commercial x- and y-gradients for tri-axial imaging, and its performance was demonstrated by ADC maps of free water and by q-space experiments on water sequestered around polystyrene microspheres (4.5 microm diameter), which showed the expected diffraction peak. In addition, diffusion-weighted images of a fixed mouse spinal cord illustrated the capability of this coil for quantitative imaging of tissue microstructure.

Structural MRI of carotid artery atherosclerotic lesion burden and characterization of hemispheric cerebral blood flow before and after carotid endarterectomy.

Collateral circulation plays a major role in maintaining cerebral blood flow (CBF) in patients with internal carotid artery (ICA) stenosis. CBF can remain normal despite severe ICA stenosis, making the benefit of carotid endarterectomy (CEA) or stenting difficult to assess. Before and after surgery, we assessed CBF supplied through the ipsilateral (stenotic) or contralateral ICA individually with a novel hemisphere-selective arterial spin-labeling (ASL) perfusion MR technique. We further explored the relationship between CBF and ICA obstruction ratio (OR) acquired with a multislice black-blood imaging sequence. For patients with unilateral ICA stenosis (n = 19), conventional bilateral labeling did not reveal interhemispheric differences. With unilateral labeling, CBF in the middle cerebral artery (MCA) territory on the surgical side from the ipsilateral supply (53.7 +/- 3.3 ml/100 g/min) was lower than CBF in the contralateral MCA territory from the contralateral supply (58.5 +/- 2.7 ml/100 g/min), although not statistically significant (p = 0.09). The ipsilateral MCA territory received significant (p = 0.02) contralateral supply (7.0 +/- 2.7 ml/100 g/min), while ipsilateral supply to the contralateral side was not reciprocated. After surgery (n = 11), ipsilateral supply to the MCA territory increased from 57.3 +/- 5.7 to 67.3 +/- 5.4 ml/100 g/min (p = 0.03), and contralateral supply to the ipsilateral MCA territory decreased. The best predictor of increased CBF on the side of surgery was normalized presurgical ipsilateral supply (r(2) = 0.62, p = 0.004). OR was less predictive of change, although the change in normalized contralateral supply was negatively correlated with OR(excess) (=OR(ipsilateral) - OR(contralateral)) (r(2) = 0.58, p = 0.006). The results demonstrate the effect of carotid artery stenosis on blood supply to the cerebral hemispheres, as well as the relative role of collateral pathways before surgery and redistribution of blood flow through these pathways after surgery. Unilateral ASL may better predict hemodynamic surgical outcome (measured by improved perfusion) than ICA OR.

Reproducibility and error sources of micro-MRI-based trabecular bone structural parameters of the distal radius and tibia.

The mechanical competence of trabecular bone is significantly determined, next to material density, by its three-dimensional (3D) structure. Recent advances in micromagnetic resonance imaging (micro-MRI) acquisition and processing techniques allow the 3D trabecular structure to be analyzed in vivo at peripheral sites such as the distal radius and tibia. The practicality of micro-MRI-based noninvasive virtual bone biopsy (VBB) for longitudinal studies of patients hinges on the reproducibility of the derived structural parameters, which largely determine the size of the effect that can be detected at a given power and significance level. In this paper, the reproducibility of micro-MRI-derived trabecular bone structure measures was examined by performing repeat studies in six healthy subjects in whom the distal aspects of the radius and tibia were scanned with a 3D spin-echo sequence at 137 x 137 x 410 microm3 voxel size. Bone volume fraction (BV/TV) and digital topological analysis (DTA) structural parameters including the topological bone surface-to-curve ratio (SCR) and topological erosion index (TEI) were evaluated after subjecting the raw images to a cascade of processing steps. The average coefficient of variation was 4-7% and was comparable for the two anatomic sites and for all parameters measured. The reliability expressed in terms of the intraclass correlation coefficient ranged from 0.95 to 0.97 in the radius and 0.68 to 0.92 in the tibia. Error analysis based on simulations suggests involuntary patient motion, primarily rotation, to be the chief source of imprecision, followed by failure to accurately match the analysis volumes in repeat studies.

Identification of a group 1-like capsular polysaccharide operon for Vibrio vulnificus.

Virulence of Vibrio vulnificus correlates with changes in colony morphology that are indicative of a reversible phase variation for expression of capsular polysaccharide (CPS). Encapsulated variants are virulent with opaque colonies, whereas phase variants with reduced CPS expression are attenuated and are translucent. Using TnphoA mutagenesis, we identified a V. vulnificus CPS locus, which included an upstream ops element, a wza gene (wza(Vv)), and several open reading frames with homology to CPS biosynthetic genes. This genetic organization is characteristic of group 1 CPS operons. The wza gene product is required for transport of CPS to the cell surface in Escherichia coli. Polar transposon mutations in wza(Vv) eliminated expression of downstream biosynthetic genes, confirming operon structure. On the other hand, nonpolar inactivation of wza(Vv) was specific for CPS transport, did not alter CPS biosynthesis, and could be complemented in trans. Southern analysis of CPS phase variants revealed deletions or rearrangements at this locus. A survey of environmental isolates indicated a correlation between deletions in wza(Vv) and loss of virulent phenotype, suggesting a genetic mechanism for CPS phase variation. Full virulence in mice required surface expression of CPS and supported the essential role of capsule in the pathogenesis of V. vulnificus.

Effects of chemopreventive and antitelomerase agents on the spontaneous immortalization of breast epithelial cells.

BACKGROUND: Activation of telomerase is an early event in the development of breast and other cancers that may lead to cell immortalization, a critical and rate-limiting step in cancer progression. Breast epithelial cells from women with Li-Fraumeni syndrome (LFS) immortalize spontaneously and reproducibly in culture. We, therefore, tested whether immortalization of these cells could be prevented by treating them with chemopreventive agents and by inhibiting telomerase activity. METHODS: Noncancerous, preimmortal breast epithelial cells derived from a patient with LFS were treated for 3 months with nontoxic concentrations of the chemopreventive agents oltipraz, difluoromethylornithine, tamoxifen, and retinoic acid or with two different telomerase inhibitors. The frequency of spontaneous immortalization of LFS-derived cells was estimated by an approach based on fluctuation analyses. Statistical analyses were two-sided. RESULTS: The frequency of spontaneous immortalization events of LFS-derived breast epithelial cells was reduced by long-term treatment with retinoic acid (P<0.001) or tamoxifen (P<0.05) compared with solvent-treated cells. The frequency of immortalization was also reduced by treating LFS-derived cells with an antitelomerase antisense oligonucleotide (P<0.001) or by inducing the cells to express a dominant negative mutant of telomerase (P<0.025) compared with cells treated with a control oligonucleotide or with empty vector, respectively. CONCLUSIONS: Treatment of preimmortal LFS breast epithelial cells with chemopreventive and antitelomerase agents decreased the frequency of spontaneous immortalization in vitro. These studies validate the application of a new cell culture model system to screen the effects of novel chemopreventive agents by use of cell immortalization as an end point. The results also suggest that the telomerase ribonucleoprotein complex may be an important molecular target for breast cancer prevention.

The bile acid synthetic gene 3beta-hydroxy-Delta(5)-C(27)-steroid oxidoreductase is mutated in progressive intrahepatic cholestasis.

We used expression cloning to isolate cDNAs encoding a microsomal 3beta-hydroxy-Delta(5)-C(27)-steroid oxidoreductase (C(27) 3beta-HSD) that is expressed predominantly in the liver. The predicted product shares 34% sequence identity with the C(19) and C(21) 3beta-HSD enzymes, which participate in steroid hormone metabolism. When transfected into cultured cells, the cloned C(27) 3beta-HSD cDNA encodes an enzyme that is active against four 7alpha-hydroxylated sterols, indicating that a single C(27) 3beta-HSD enzyme can participate in all known pathways of bile acid synthesis. The expressed enzyme did not metabolize several different C(19/21) steroids as substrates. The levels of hepatic C(27) 3beta-HSD mRNA in the mouse are not sexually dimorphic and do not change in response to dietary cholesterol or to changes in bile acid pool size. The corresponding human gene on chromosome 16p11.2-12 contains six exons and spans 3 kb of DNA, and we identified a 2-bp deletion in the C27 3beta-HSD gene of a patient with neonatal progressive intrahepatic cholestasis. This mutation eliminates the activity of the enzyme in transfected cells. These findings establish the central role of C(27) 3beta-HSD in the biosynthesis of bile acids and provide molecular tools for the diagnosis of a third type of neonatal progressive intrahepatic cholestasis associated with impaired bile acid synthesis.

Evidence of a role for the Q151L mutation and the viral background in development of multiple dideoxynucleoside-resistant human immunodeficiency virus type 1.

The majority of human immunodeficiency virus type 1 (HIV-1)-infected patients treated with zidovudine (AZT) plus zalcitabine (ddC) and didanosine (ddI) develop AZT resistance mediated by mutations such as T215Y and M41L. Only a small proportion of patients develop multiple dideoxynucleoside resistance (MDNR) mediated by the Q151M mutation. To gain insight into the factors responsible for the low frequency of selection of Q151M, we evaluated the replication capabilities of recombinant viruses carrying two possible intermediates (151L or 151K) of the Q151M mutation generated in different reverse transcriptase (RT) genetic backgrounds. The 151L and 151K mutations were introduced by site-directed mutagenesis in RTs from two patient-derived HIV-1 isolates that had either wild type (WT) Q or the Q151M (posttreatment isolate) mutation. For comparison, both mutations were also introduced in a laboratory-adapted HIV-1 strain (HIV-1(HXB2)). Analysis of replication capabilities showed that both 151L and 151K were lethal in RT genetic backgrounds of the WT isolate and in HIV-1(HXB2). In contrast, 151L but not 151K allowed virus replication in RT backgrounds of the posttreatment isolate. Three mutations (V35I, S68G, and I178M) were present in the RT background of the posttreatment isolate but not in the WT isolate. Introduction of S68G in the RT of both the WT isolate and HIV-1(HXB2) partially restored replication capacity of recombinants carrying the 151L mutation. The S68G mutation alone did not confer a significant replicative disadvantage in WT viruses. Like HIV-1(151M), HIV-1(151L) RT was found to have six- to eightfold resistance to AZT-triphosphate (TP), ddA-TP, and ddC-TP, indicating an MDNR phenotype. However, HIV-1(151L) was found to be less fit than HIV-1(151M), which may explain the preferential selection of HIV-1(151M) observed in vivo. The demonstrated ability of HIV-1(151L/68G) to replicate and the associated MDNR suggest that 151L is a potential intermediate of Q151M. The dependence of HIV-1(151L) on other mutations, such as S68G, for replication may explain the low frequency of the Q151M-mediated pathway of resistance.

In vivo MR micro imaging with conventional radiofrequency coils cooled to 77 degrees K.

Cryogenically cooled conventional surface coils are shown to provide significant signal-to-noise ratio (SNR) gains for MR micro imaging of tissue structure in vivo. Measurements are described which employ a simple, all-polyvinyl chloride (PVC) vacuum dewar capable of maintaining a bath of liquid nitrogen around the coil, within 5 mm of the tissue to be imaged. Images acquired in vivo at 64 MHz with a 2-cm diameter copper coil cooled to 77 K demonstrated a gain in SNR of approximately 2.7 +/- 0.3 relative to those obtained with the same coil at room temperature under otherwise identical conditions. This increase is consistent with the reduction in coil resistance and the minor contribution to overall resistance from the imaging object. The performance of the coil is illustrated with images from the human finger and rabbit eye and potential applications are discussed.

Nucleotide sequence variability in the nontranscribed spacer of the rRNA locus in the oyster parasite Perkinsus marinus.

We examined the sequence variability of the nontranscribed spacer (NTS) and internal-transcribed spacer (ITS1 and ITS2) domains of the rRNA locus of Perkinsus marinus from Maryland, Florida, and Louisiana. The sequence of P. marinus DNA including the 5S rRNA, NTS, small subunit (SSU) rRNA, ITSI, and ITS2 regions confirmed their contiguity in the rRNA locus and revealed differences at 28 positions with the SSU rRNA sequences published earlier. The 307-bp polymerase chain reaction (PCR)-amplified fragments from the NTS domain of the various P. marinus isolates revealed the presence of 2 distinct sequences, designated as types I and II, that differed at 6 defined nucleotide positions. Based on these differences, nested PCR and restriction enzyme digests were used to distinguish between the 2 types. Sequences of the ITS1 and ITS2 domains of samples from either NTS type I (n = 3) or type II (n = 3) showed no variation and were identical to published sequences. Frequencies of the P. marinus NTS sequence types I and II in infected oysters varied with the geographic origin of the samples. All Maryland samples examined (n = 19) corresponded to the NTS type I sequence, the type II was the most frequent in the Florida samples (n = 17), and both types were about equally represented in the Louisiana samples (n = 19), with both sequence types found in individual oyster specimens. Although it has been suggested that P. marinus is diploid, it remains to be determined if both NTS sequence types can be present in a single P. marinus trophozoite.

Differential expression of Vibrio vulnificus capsular polysaccharide.

Vibrio vulnificus is a human pathogen whose virulence has been associated with the expression of capsular polysaccharide (CPS). Multiple CPS types have been described; however, virulence does not appear to correlate with a particular CPS composition. Reversible-phase variation for opaque and translucent colony morphologies is characterized by changes in CPS expression, as suggested by electron microscopy of cells stained nonspecifically with ruthenium red. Isolates with opaque colony morphologies are virulent and appear to be more thickly encapsulated than naturally occurring translucent-phase variants, which have reduced, patchy, or absent CPS. Previously, we have shown that the virulence of translucent-phase variants was intermediate between opaque-phase variants and acapsular transposon mutants, suggesting a correlation between virulence and the amount of CPS expressed. In the present study, CPS expression of phase variants and genetically defined mutants of V. vulnificus M06-24/O was examined by using a CPS-specific monoclonal antibody with an enzyme-linked immunosorbent assay, flow cytometry, and immunoelectron microscopy. Semiquantitative analyses of CPS expression correlated well among these assays, confirming that the translucent-phase variant was intermediate in CPS expression and retained type I CPS-specific epitopes. Cell surface expression of CPS varied with the growth phase, increasing during logarithmic growth and declining in stationary culture. Significantly greater CPS expression (P = 0.026) was observed for cells grown at 30 degrees C than for those at 37 degrees C. These studies confirm that phase variation and virulence in V. vulnificus correlate with the amount of CPS expressed and demonstrate the fluidity of bacterial polysaccharide expression in response to environmental conditions.

Species-specificity and sensitivity of a PCR-based assay for Perkinsus marinus in the eastern oyster, Crassostrea virginica: a comparison with the fluid thioglycollate assay.

We examined the species-specificity and sensitivity of a polymerase chain reaction (PCR)-based assay for Perkinsus marinus and compared its overall performance with the fluid thioglycollate medium (FTM) assay on oyster (Crassostrea virginica) hemolymph, mantle, and rectum samples. Our results indicated that the PCR-based methodology is species-specific because Perkinsus olseni, Perkinsus atlanticus, and Perkinsus spp. DNAs were not amplified with the PCR primers developed for P. marinus diagnosis. The sensitivity of the PCR method, as assessed through spike/recovery experiments, was established by the detection of as few as 1 cell of P. marinus in 30 mg of oyster tissue. Tissue samples from naturally infected oysters analyzed both by the FTM and PCR assay suggested that the latter was more sensitive for the diagnosis of P. marinus. Positive results for P. marinus infection ranged from 70% to 83% by FTM and from 92% to 100% by PCR, depending on the tissue examined. Therefore, species-specificity and sensitivity of the NTS-based PCR assay validate its use as a tool for assessment of P. marinus in mollusks.

Magnetic resonance chemical shift microimaging of aging human skin in vivo: initial findings.

BACKGROUND/AIMS: In recent decades, interest has increased in the chronological and environmental factors governing the aging of skin. Various methods have been used for determining water and lipid content of human skin as a function of subject age. Magnetic resonance chemical shift imaging (CSI) offers a noninvasive technique for observing detailed distributions of water, lipids and other chemicals in the skin, and thus may be useful in dermatogerontology. METHODS: Human skin was examined in vivo on nine healthy volunteers, both male and female. Localized (1) H spectra of the skin were obtained from voxels 78 µm thick and parallel to the skin surface. Unique water and lipid profiles were observed for different individuals, showing the composition and microstructure of epidermis, dermis and hypodermis in each subject. These allowed the quantification of skin thickness in vivo by the first appearance of triacylglyceride olefinic protons at the dermal-hy-podermal junction, or alternatively by the degree of lipid infiltration into dermis. RESULTS: The relative concentration of free water in the skin, normalized to skin thickness, was observed to be slightly greater in older subjects and also in tanned subjects. More significantly, a microstructural feature common to every subject, i.e., the position of a possible capillary plexus in the dermis, showed migration toward the skin surface with advancing age. CONCLUSIONS: Such observations are consistent with previous studies of skin aging by other techniques and show promise for CSI in dermatogerontology as a non-invasive means for determination of skin water in vivo.

Magnetic resonance coil for (31) P spectroscopy of skin over curved body surfaces.

BACKGROUND/AIMS: Increasing signal-to-noise ratio for skin spectroscopy means increasing area, but increased area usually means resorting to Fourier window techniques to prevent overwhelming muscle contamination. The aim of this work was to develop a simple coil that would allow large areas of skin to be sampled while limiting muscle contamination simply by the coil's geometry. METHODS: The coil design was arrived at by using simulations that were calibrated by phantom tests with prototype units. It was then fabricated by using an etched circuitboard technique and tested on three volunteers for comparison with predicted performance. RESULTS: The new coil acquires magnetic resonance signals from a 140 cm(2) contoured layer of surface tissue with sufficient selectivity that 81% of the signal comes from tissue less than 1.9 mm deep. For skin and fat thicknesses of 1.1 and 3.1 mm, respectively, tending toward a worst case in-vivo situation, muscle contamination is only 3.3%. With the new coil, in 1 h, it was possible to directly acquire (31) P skin spectra comparable to those formerly obtained in 4 h in two separate acquistions that then had to be subtracted to obtain the desired skin spectra. CONCLUSION: Increasing the area of skin sampled while maintaining selectivity to limit muscle contamination, relying simply on coil geometry, is practical using a parallel configuration of long narrow strip coils.

Release of tumor necrosis factor alpha in response to Vibrio vulnificus capsular polysaccharide in in vivo and in vitro models.

Vibrio vulnificus produces a severe septic shock syndrome in susceptible individuals. Virulence of the bacterium has been closely linked to the presence of a surface-exposed acidic capsular polysaccharide (CPS). To investigate whether CPS plays an additional role in pathogenesis by modulating inflammatory-associated cytokine production, studies were initiated in a mouse model and followed by investigations of cytokine release from human peripheral blood mononuclear cells (PBMCs). Mouse tumor necrosis factor alpha (TNF-alpha) could be detected in serum up to 12 h postinoculation in animals challenged with the encapsulated parent strain MO6-24/O. The unencapsulated strain CVD752 was quickly eliminated by the animals, thus preventing a direct association between serum TNF-alpha levels and the presence or absence of the CPS. Purified CPS from MO6-24/O when injected into D-galactosamine-sensitized mice was a more immediate inducer of TNF-alpha than an equivalent quantity of MO6-24/O lipopolysaccharide (LPS). Both V. vulnificus CPS and V. vulnificus LPS induced inflammation-associated cytokine responses from primary human PBMCs in vitro. CPS elicited TNF-alpha from PBMCs in a dose-dependent manner, with maximal induction at 6 to 10 h, and was not inhibited by polymyxin B. Expression of interleukin-6 (IL-6) mRNAs was also induced in the presence of CPS. Interestingly, while adherent PBMCs secreted high levels of TNF-alpha after stimulation with LPS, they secreted little TNF-alpha in response to CPS. These studies provide evidence that V. vulnificus CPS directly stimulates the expression and secretion of proinflammatory cytokines by murine and human cells and suggest that CPS activation of PBMCs operates through a cellular mechanism distinct from that of LPS.

Phosphorus metabolites in human skin and muscle obtained by phosphorus-31 magnetic resonance spectroscopy.

BACKGROUND/AIMS: Characterization of skin phosphometabol-ism in vivo by non-invasive magnetic resonance spectroscopy (MRS) might allow assessment of ischemic or irradiative damage. METHODS: Based on a study of 17 healthy volunteers, we present statistical analyses for intra- and inter-tissue metabolite concentration ratios, as well as for pH and [Mg(2+) ]. RESULTS: Results contrasted steady-state energy metabolism in skin and muscle, showing lower phosphocreatine/ATP, higher percentage of inorganic phosphate, phosphodiesters and phos-phomonoesters, and higher pH and [Mg(2+) ] in skin than in muscle. CONCLUSION: Results were consistent with known skin physiology and structure and suggested an inverse relationship between skin phosphodiester levels and melanin.