Comparative genomic identification and validation of ß-defensin genes in the Ovis aries genome.

BACKGROUND: ß-defensins are small, cationic, antimicrobial peptides found in species across the plant and animal kingdoms. In addition to microbiocidal activity, roles in immunity as well as reproduction have more recently been documented. ß-defensin genes in Ovis aries (domestic sheep) have been poorly annotated, having been identified only by automatic gene prediction algorithms. The objective of this study was to use a comparative genomics approach to identify and characterise the ß-defensin gene repertoire in sheep using the bovine genome as the primary reference. RESULTS: All 57 currently predicted bovine ß-defensin genes were used to find orthologous sequences in the most recent version of the sheep genome (OAR v4.0). Forty three genes were found to have close genomic matches (>70% similarity) between sheep and cattle. The orthologous genes were located in four clusters across the genome, with 4 genes on chromosome 2, 19 genes on chromosome 13, 5 genes on chromosome 20 and 15 genes on chromosome 26. Conserved gene order for the ß-defensin genes was apparent in the two smaller clusters, although gene order was reversed on chromosome 2, suggesting an inversion between sheep and cattle. Complete conservation of gene order was also observed for chromosome 13 ß-defensin orthologs. More structural differences were apparent between chromosome 26 genes and the orthologous region in the bovine reference genome, which is known to be copy-number variable. In this cluster, the Defensin-beta 1 (DEFB1) gene matched to eleven Bovine Neutrophil beta-Defensin (BNBD) genes on chromosome 27 with almost uniform similarity, as well as to tracheal, enteric and lingual anti-microbial peptides (TAP, EAP and LAP), suggesting that annotation of the bovine reference sequence is still incomplete. qPCR was used to profile the expression of 34 ß-defensin genes, representing each of the four clusters, in the ram reproductive tract. Distinct site-specific and differential expression profiles were detected across the reproductive tract of mature rams with preferential ß-defensin gene expression in the epididymis, recapitulating observations for orthologous genes in other species. CONCLUSIONS: This is the first comprehensive analysis of ß-defensin genes encoded by the ovine reference sequence, and the first report of an expanded repertoire of ß-defensin genes in this species. The preferential expression of these genes in the epididymis suggests a role in fertility, possibly providing immunoprotection for sperm within the female reproductive tract.

Changes in shear wave speed pre- and post-induction of labor: a feasibility study.

OBJECTIVE: To explore the feasibility of using shear wave speed (SWS) estimates to detect differences in cervical softening pre- and post-ripening in women undergoing induction of labor. METHODS: Subjects at 37-41 weeks' gestation undergoing cervical ripening before induction of labor were recruited (n = 20). Examinations, performed prior to administration of misoprostol and 4 h later included Bishop score, transvaginal ultrasound measurement of cervical length, and 10 replicate SWS measurements using an ultrasound system equipped with a prototype transducer (128 element, 3 mm diameter, 14 mm aperture) attached to the clinician's hand. Subjects were divided into two groups, 'not-in-labor' and 'marked-progression', based on cervical evaluation at the second examination. Measurements were compared via individual paired hypotheses tests and using a linear mixed model, with the latter also used to compare groups. Spearman's rank correlation coefficient was used to compare SWS with Bishop score. The linear mixed model can take into account clustered data and accommodate multiple predictors simultaneously. RESULTS: The Wilcoxon signed-rank paired test established a significant difference in pre- and post-ripening SWS, with mean SWS estimates of 2.53 ± 0.75 and 1.54 ± 0.31 m/s, respectively (P < 0.001) in the not-in-labor group (decrease in stiffness) and 1.58 ± 0.33 and 2.35 ± 0.65 m/s for the marked-progression group (increase in stiffness). The linear mixed model corroborated significant differences in pre- and post-ripening measurements in individual subjects (P < 0.001) as well as between groups (P < 0.0001). SWS estimates were significantly correlated with digitally-assessed cervical softness and marginally correlated with Bishop score as assessed by Spearman's rank correlation coefficient. CONCLUSIONS: In-vivo SWS estimates detected stiffness differences before and after misoprostol-induced softening in term pregnancies. This ultrasonic shear elasticity imaging technique shows promise for assessing cervical softness.

Estimation of shear wave speed in the human uterine cervix.

OBJECTIVES: To explore spatial variability within the cervix and the sensitivity of shear wave speed (SWS) to assess softness/stiffness differences in ripened (softened) vs unripened tissue. METHODS: We obtained SWS estimates from hysterectomy specimens (n = 22), a subset of which were ripened (n = 13). Multiple measurements were made longitudinally along the cervical canal on both the anterior and posterior sides of the cervix. Statistical tests of differences in the proximal vs distal, anterior vs posterior and ripened vs unripened cervix were performed with individual two-sample t-tests and a linear mixed model. RESULTS: Estimates of SWS increase monotonically from distal to proximal longitudinally along the cervix, they vary in the anterior compared to the posterior cervix and they are significantly different in ripened vs unripened cervical tissue. Specifically, the mid position SWS estimates for the unripened group were 3.45 ± 0.95 m/s (anterior; mean ± SD) and 3.56 ± 0.92 m/s (posterior), and 2.11 ± 0.45 m/s (anterior) and 2.68 ± 0.57 m/s (posterior) for the ripened group (P < 0.001). CONCLUSIONS: We propose that SWS estimation may be a valuable research and, ultimately, diagnostic tool for objective quantification of cervical stiffness/softness.

Correlation length ratio as a parameter for determination of fiber-like structures in soft tissues.

Quantitative ultrasound methods can provide valuable information about the microstructure of a material or tissue. This works well when the common assumptions of homogeneity, isotropy, and diffuse scattering conditions are valid. In biological tissues, however, these assumptions are often violated because the microstructure of biological tissues is often heterogeneous and anisotropic. The microstructure of biological tissues can change with disease, and therefore accurate identification and description of a tissue's microstructure can offer important clinical insight. To address the challenge of evaluating the microstructure of biological tissues, here we introduce a novel parameter called the correlation length ratio (CLR), a ratio of lateral to axial correlation lengths for backscattered echo signals. We developed it to determine the presence of fiber-like structures in soft tissues by comparing this value in tissue to a threshold determined from a reference material that is homogeneous, isotropic, and provides diffuse scattering. We tested this novel parameter in phantoms with spherical scattering sources, in an anisotropic phantom (containing elongated fibers), and in human biceps muscle. We found that the CLR accurately detected the presence of elongated structures in both the anisotropic phantom and muscle. These results encourage further exploration of this novel parameter in microstructurally complex tissues.

The impact of laterally coupled grating microstructure on effective coupling coefficients.

Lithographic fabrication may be used to define laterally coupled gratings of high refractive index contrast on waveguide ridges, eliminating the need for regrowth steps in such distributed feedback lasers. These may be made more amenable to fabrication by employing higher-order gratings. Reliable exploration of the laser design space requires that the radiating partial waves be accurately incorporated in numerical simulations. We modify the coupled-mode approach to fully consider the two-dimensional cross section, analyzing rectangular, sinusoidal, triangular and trapezoidal grating shapes. Effective coupling coefficients are determined for grating orders from first to third. We show that, by tailoring the grating microstructure, effective coupling coefficients up to double that of a 0.5 duty cycle rectangular grating can be achieved. The actual grating microstructure of an as-fabricated grating was analyzed and its effective coupling coefficient predicted as [Formula: see text]. This was found to be in excellent agreement with the value extracted from the amplified spontaneous emission spectrum, [Formula: see text].

Removing bacteria from hospital surfaces: a laboratory comparison of ultramicrofibre and standard cloths.

We compared the ability of ultramicrofibre-woven cloths with conventional cloths moistened with water only, for their ability to remove several types of organisms relevant to hospital-acquired infections from a variety of surfaces in hospitals. We showed that ultramicrofibre cloths consistently outperformed conventional cloths in their decontamination ability, across all surfaces, and irrespective of whether the bacteria were coated on to the surfaces with phosphate-buffered saline (PBS) or PBS containing horse serum to simulate real-life soiling. The ability of the cloths to remove bacteria from surfaces was assessed by contact plating and colony formation, and by swabbing and measurement of ATP bioluminescence. The results suggest potential for use of ultramicrofibre in healthcare environments. Further studies are required, however, to define accurately how these cloths, which are designed to be used without detergent or biocides, might be capable of safe and effective deployment and recycling in the healthcare environment.

Instrument for determining the complex shear modulus of soft-tissue-like materials from 10 to 300 Hz.

Accurate determination of the complex shear modulus of soft tissues and soft-tissue-like materials in the 10-300 Hz frequency range is very important to researchers in MR elastography and acoustic radiation force impulse (ARFI) imaging. A variety of instruments for making such measurements has been reported, but none of them is easily reproduced, and none have been tested to conform to causality via the Kramers-Kronig (K-K) relations. A promising linear oscillation instrument described in a previous brief report operates between 20 and 160 Hz, but results were not tested for conformity to the K-K relations. We have produced a similar instrument with our own version of the electronic components and have also accounted for instrumental effects on the data reduction, which is not addressed in the previous report. The improved instrument has been shown to conform to an accurate approximation of the K-K relations over the 10-300 Hz range. The K-K approximation is based on the Weichert mechanical circuit model. We also found that the sample thickness must be small enough to obtain agreement with a calibrated commercial rheometer. A complete description of the improved instrument is given, facilitating replication in other labs.

Acoustic backscatter and effective scatterer size estimates using a 2D CMUT transducer.

Compared to conventional piezoelectric transducers, new capacitive microfabricated ultrasonic transducer (CMUT) technology is expected to offer a broader bandwidth, higher resolution and advanced 3D/4D imaging inherent in a 2D array. For ultrasound scatterer size imaging, a broader frequency range provides more information on frequency-dependent backscatter, and therefore, generally more accurate size estimates. Elevational compounding, which can significantly reduce the large statistical fluctuations associated with parametric imaging, becomes readily available with a 2D array. In this work, we show phantom and in vivo breast tumor scatterer size image results using a prototype 2D CMUT transducer (9 MHz center frequency) attached to a clinical scanner. A uniform phantom with two 1 cm diameter spherical inclusions of slightly smaller scatterer size was submerged in oil and scanned by both the 2D CMUT and a conventional piezoelectric linear array transducer. The attenuation and scatterer sizes of the sample were estimated using a reference phantom method. RF correlation analysis was performed using the data acquired by both transducers. The 2D CMUT results indicate that at a 2 cm depth (near the transmit focus for both transducers) the correlation coefficient reduced to less than 1/e for 0.2 mm lateral or 0.25 mm elevational separation between acoustic scanlines. For the conventional array this level of decorrelation requires a 0.3 mm lateral or 0.75 mm elevational translation. Angular and/or elevational compounding is used to reduce the variance of scatterer size estimates. The 2D array transducer acquired RF signals from 140 planes over a 2.8 cm elevational direction. If no elevational compounding is used, the fractional standard deviation of the size estimates is about 12% of the mean size estimate for both the spherical inclusion and the background. Elevational compounding of 11 adjacent planes reduces it to 7% for both media. Using an experimentally estimated attenuation of 0.6 dB cm(-1) MHz(-1), scatterer size estimates for an in vivo breast tumor also demonstrate improvements using elevational compounding with data from the 2D CMUT transducer.

The efficacy of the inorganic copper-based biocide CuWB50 is compromised by hard water.

AIMS: We sought to explain the unexpected failure of the inorganic copper-based biocide CuWB50 to effectively decontaminate microfibre cleaning cloths that became contaminated with Acinetobacter lwoffii. METHODS AND RESULTS: CuWB50 was diluted using distilled water or tap water obtained from two different ICUs. Microtitre plate assays were used to determine the minimum inhibitory concentration (MIC) for the implicated A. lwoffii. pH and oxidation-reduction potential (ORP) tests were performed and representative water samples were chemically analysed. When diluted in distilled water, the CuWB50 MIC for A. lwoffii was 9 mg l(-1) but in tap water from each ICU it was 37 and 75 mg l(-1) at hardness levels of 246 and 296 mg CaCO(3) l(-1) respectively. CuWB50-distilled water solutions consistently had a lower pH and higher ORP than CuWB50-tap water solutions. CONCLUSIONS: Hard water adversely affects the biocidal efficacy of CuWB50. SIGNIFICANCE AND IMPACT OF THE STUDY: Unintentional environmental contamination is a risk when using wet microfibre cloths. This occurred when cloths were stored in CuWB50 overnight combined with the unintentional but erroneous use of tap water. This study emphasizes the need for clearly documented cleaning protocols embedded within a culture of adequate training and constant supervision of cleaning staff.

A parallelizable real-time motion tracking algorithm with applications to ultrasonic strain imaging.

Ultrasound-based mechanical strain imaging systems utilize signals from conventional diagnostic ultrasound systems to image tissue elasticity contrast that provides new diagnostically valuable information. Previous works (Hall et al 2003 Ultrasound Med. Biol. 29 427, Zhu and Hall 2002 Ultrason. Imaging 24 161) demonstrated that uniaxial deformation with minimal elevation motion is preferred for breast strain imaging and real-time strain image feedback to operators is important to accomplish this goal. The work reported here enhances the real-time speckle tracking algorithm with two significant modifications. One fundamental change is that the proposed algorithm is a column-based algorithm (a column is defined by a line of data parallel to the ultrasound beam direction, i.e. an A-line), as opposed to a row-based algorithm (a row is defined by a line of data perpendicular to the ultrasound beam direction). Then, displacement estimates from its adjacent columns provide good guidance for motion tracking in a significantly reduced search region to reduce computational cost. Consequently, the process of displacement estimation can be naturally split into at least two separated tasks, computed in parallel, propagating outward from the center of the region of interest (ROI). The proposed algorithm has been implemented and optimized in a Windows system as a stand-alone ANSI C++ program. Results of preliminary tests, using numerical and tissue-mimicking phantoms, and in vivo tissue data, suggest that high contrast strain images can be consistently obtained with frame rates (10 frames s(-1)) that exceed our previous methods.

Hapten-induced enhancement of monoclonal antibody binding in ELISA.

We have recently reported [Hall T. J., Stenzel-Poore M. and Rittenburg M. B., Sixth International Congress of Immunology, Toronto. Abstract 2.15.27, p. 108 (1986)] on the fine specificity analysis of six monoclonal antibodies (MAbs) which utilize lambda 1 (lambda 1) light chains, produced from BALB/c mice immunized with PC-KLH. Binding of these MAbs to PC-histone in ELISA was inhibited by the PC analogue nitrophenylphosphocholine (NPPC) but not by PC itself, thus distinguishing these group II antibodies from the group I PC binding myeloma proteins such as TEPC 15 and MOPC 603 [Leon and Young, (1971) Biochemistry 10, 1424-1429]. Three of these lambda MAbs showed a reproducible 2-4 fold increase in binding in ELISA at certain concns (10-100 microM) of NPPC and one of them (PCG2b-3) was chosen to examine this observation further.

A newly characterized group of anti-phosphocholine antibodies: group III antibodies.

Four anti-phosphocholine (PC) monoclonal antibodies (MAbs) are described that can be clearly distinguished from group I and II anti-PC antibodies [Chang et al. (1982) J. Immun. 128, 702-706]. They bind optimally and suboptimally PC-histone coated ELISA plates equally well, in contrast to group I and II MAbs which bind much less well to suboptimally coated plates. On optimally coated plates their fine specificity for PC analogues is group II-like, whereas on suboptimally coated plates it is group I-like; group I and II MAbs each maintain their own characteristic fine specificity patterns on either type of plate. At the molecular level, all four MAbs utilize VK24 light chains and share a 5.2 kb rearranged band in Southern blot analysis (using a JH probe and EcoRI restricted DNA). The term "group III" is proposed to define and distinguish these anti-PC antibodies.

A model for the hapten-induced enhancement of monoclonal antibody binding in ELISA.

The unusual binding properties of three anti-phosphocholine (PC) monoclonal antibodies (MAbs) in ELISA has been reported [Hall T. J. (1987) Molec. Immun. 24, 773-777]. These MAbs gave reproducible 2-4 fold increases in binding to PC-protein coated plates in the presence of PC (approximately 2 mM), nitrophenyl PC (approximately 10 micron) and other PC analogues. A model is proposed here in which the complementary and opposite binding of hapten and PC-protein in the combining sites of these MAbs explains the hapten enhanceable binding in ELISA.

A novel in vitro assay for mouse IgE.

A simple and sensitive assay for screening mouse IgE-producing hybridomas is described. Rat basophil leukemia (RBL) cells (either live or glutaraldehyde fixed) were sensitized by mouse monoclonal IgE and then rosetted with antigen-coated sheep red blood cells. The assay was established using a mouse monoclonal IgE anti-DNP and was able to detect as little as 5 ng/ml IgE. The rosette assay is also a useful technique for studying the binding of mouse IgG monoclonal (MC) antibodies to RBL cells.

Inhibition of human natural killer cell activity by prostaglandin D2.

We have studied the effect of prostaglandin (PG) D2 on human natural killer (NK) cell activity using 51Cr-labelled K562 target cells. PGD2 caused a dose dependant inhibition of NK cell activity, producing maximal, non-toxic inhibition at a concentration of 1 X 10(-6) M. This inhibition was not due to interference with effector:target cell binding (E:TCB) and was seen using Percoll-enriched NK cells. A time dependent inhibition of NK cell activity was seen when dibutyryl cyclic AMP (DiBcAMP) and PGD2 were added in parallel to the NK cell assay suggesting that PGD2 directly inhibits NK cell activity via the adenylate cyclase system which modulates the killing process.

Lack of effect of LTB4 on human natural killer cell activity and T lymphocyte transformation.

Human natural killer (NK) cell activity was not significantly affected by leukotriene (LT) B4 over a wide concentration range (10(-6)-10(-14) M), whether added directly to the NK cell assay or after preincubation of effector cells for 2 h with the drug before addition of Cr labelled K562 target cells. In addition, LTB4 did not affect the kinetics of NK cell mediated cytotoxicity. Addition of LTB4 (10(-6)-10(-10) M) to concanavalin A (Con A) or phytohaemagglutinin (PHA)-stimulated human peripheral blood mononuclear cells (PBMC) had no significant effect on proliferation measured by tritiated thymidine incorporation, using both optimal and sub-optimal mitogen concentrations. Whilst it is clear that LTB4 is an important mediator of inflammation involving polymorphonuclear (PMN) leukocytes in vivo, it does not affect NK cell or T cell function in vitro.

Lithium counteracts histamine suppression of human T cell mitogenesis.

Human T cell proliferative responses to concanavalin A (conA) were suppressed by approximately 50% by histamine (100 microM). In contrast, LiCl (1 or 3 mM) potentiated T cell responses by about 50%, but 10 mM LiCl had no significant effect on T cell proliferation. Histamine suppression was not significantly affected by the presence of potentiating concentrations of LiCl, whereas 10 mM LiCl completely abrogated histamine suppression.

Multiple synchronous primaries of the gastrointestinal tract: a molecular case report.

Six synchronous gastrointestinal primaries were identified in a 70 year old male with no known cancer predisposition syndrome or recognized risk factors except alcohol abuse. These specimens appeared to be independent and unrelated by gross and histopathological examination. In order to further evaluate the six tumors, we analyzed selected DNA sequences for alterations in the K-ras oncogene and p53 tumor suppressor gene. In addition, three loci were analyzed to determine microsatellite instability. Using the polymerase chain reaction, single stranded conformational polymorphism, and DNA sequencing, we demonstrated that each primary manifests genetic characteristics typical of the tissue of origin. In addition, one primary, a moderately differentiated colon adenocarcinoma, exhibited mutations not detected in the other specimens. This study suggests that these synchronous primaries arose independently and progressed along different carcinogenic pathways.