Identification and typing of Francisella tularensis with a highly automated genotyping assay.

A PCR assay was developed to genotypically characterize Francisella tularensis and F. novicida. An integrated and partially redundant set of markers was selected to provide positive identification of these species, identify subspecies of F. tularensis and genotype 14 variable number tandem repeat (VNTR) markers. Assay performance was evaluated with 117 Francisella samples. Sample DNA was amplified, and the masses of the PCR products were determined with electrospray ionization/time of flight mass spectrometry (ESI-MS). The base compositions of the PCR amplicons were derived from these high-accuracy mass measurements and contrasted with databased information associated with each of the 25 assay markers. Species and subspecies determinations for all samples were fully concordant with results from established typing methods, and VNTR markers provided additional discrimination among samples. Sequence variants were observed with a number of assay markers, but these did not interfere with sample characterization, and served to increase the genetic diversity detected by the assay.

Identification and characterization of Francisella species from natural warm springs in Utah, USA.

AIMS: To characterize Francisella isolated from two natural warm springs in Utah and compare them to a strain isolated from a patient with probable exposure to one of the springs in 2001. METHODS AND RESULTS: A total of 39 presumptive Francisella isolates were obtained from two springs, Wasatch Hot Spring and Hobo Warm Spring, just north of Salt Lake City, Utah. All isolates were characterized by a combination of biochemical and molecular analyses, including novel PCR/electrospray ionization-mass spectrometry (ESI-MS) typing assays. Thirty-one were identified as F. philomiragia, while the remaining eight were identified as F. tularensis ssp. novicida. Phylogenetic analysis of the 16S rRNA sequences revealed 27 isolates, which clustered with F. philomiragia, albeit into two distinct clades. The remaining isolates clustered along with other F. tularensis strains including the Utah clinical isolate. Testing with the PCR/ESI-MS assays confirmed the identities of the isolates, but both yielded DNA signatures distinct from that of the clinical isolate. CONCLUSION: We were successful in isolating several Francisella strains from natural warm springs; however, none appeared to genetically match the original 2001 clinical isolate. SIGNIFICANCE AND IMPACT OF THE STUDY: This work highlights the presence of viable, potentially pathogenic Franscisella species living in the unique environmental niche of natural warm springs.

Performance of a gradient-based shift estimator in a spatially sparse data environment: tracking the sub-pixel motion of fluorescent particles.

Through a series of numerical simulations, we investigate the suitability of a relatively new gradient-based particle-tracking algorithm for efficiently quantifying sub-pixel shifts of fluorescently labelled cells or particles from a sequence of video microscopy images. The algorithm excels at estimating sub-0.5 pixel per frame shifts in both data-dense (e.g. laser speckle imaging) and data-sparse (e.g. fluorescence imaging) applications. No upsampling (i.e. interpolation) is required to achieve the sub-pixel shift resolution, and thus the approach avoids the complexity and potential errors associated with the interpolation process. An efficient MATLAB sub-routine is provided for implementing the algorithm.

Lidar system model for use with path obscurants and experimental validation.

When lidar pulses travel through a short path that includes a relatively high concentration of aerosols, scattering phenomena can alter the power and temporal properties of the pulses significantly, causing undesirable effects in the received pulse. In many applications the design of the lidar transmitter and receiver must consider adverse environmental aerosol conditions to ensure the desired performance. We present an analytical model of lidar system operation when the optical path includes aerosols for use in support of instrument design, simulations, and system evaluation. The model considers an optical path terminated with a solid object, although it can also be applied, with minor modifications, to cases where the expected backscatter occurs from nonsolid objects. The optical path aerosols are characterized by their attenuation and backscatter coefficients derived by the Mie theory from the concentration and particle size distribution of the aerosol. Other inputs include the lidar system parameters and instrument response function, and the model output is the time-resolved received pulse. The model is demonstrated and experimentally validated with military fog oil smoke for short ranges (several meters). The results are obtained with a lidar system operating at a wavelength of 0.905 microm within and outside the aerosol. The model goodness of fit is evaluated using the statistical coefficient of determination whose value ranged from 0.88 to 0.99 in this study.

Processing algorithms for tracking speckle shifts in optical elastography of biological tissues.

Parametric and nonparametric data processing schemes for analyzing translating laser speckle data used to investigate the mechanical behavior of biological tissues are examined. Cross-correlation, minimum mean square estimator, maximum likelihood, and maximum entropy approaches are discussed and compared on speckle data derived from cortical bone samples undergoing dynamic loading. While it was not the purpose of this paper to demonstrate that one processing technique is superior to another, maximum likelihood and maximum entropy approaches are shown to be particularly useful when the observed speckle motion is small.

The heritability of age-related cortical cataract: the twin eye study.

PURPOSE: A classical twin study was performed to establish the relative importance of genes and environment in cortical cataract. METHODS: Five hundred six pairs of unselected female twin volunteers (226 monozygotic and 280 dizygotic) with a mean age of 62 years (range, 49-79 years) were examined. Cortical cataract was assessed using the slit-lamp-based Oxford Clinical Cataract Classification and Grading System (clinical grading) and the Wilmer Automated Grading System, which analyzed digital retroillumination images of subjects' lenses (digital grading). The worse eye categorized score for each individual was used in maximum likelihood path modeling of the correlations within twin pairs. These correlations were used to determine the underlying liability to cortical cataract. RESULTS: Prevalence of significant cortical cataract (>/=5% of lens area) was similar in monozygotic and dizygotic twins, occurring in 19.4% and 20.6% with the clinical grading system and 24% and 23% using the digital grading system, respectively. Modeling suggested liability to cortical cataract is explained by additive and dominant genes, individual environment, and age. Estimates of the broad sense heritability of cortical cataract were 58% (95% confidence interval [CI], 51%-64%) for the clinical grading system and 53% (95% CI, 45%-60%) for the digital system. Dominant genes were estimated to contribute to 38% (95% CI, 1%-64%) of the genetic effect with the clinical grading and 53% (95% CI, 28%-60%) with the digital grading. Individual environment explained 26% and 37% and age 16% and 11% of cortical cataract variance in clinical and digital gradings, respectively. CONCLUSIONS: Genetic effects are important in the development of cortical cataract and involve the action of dominant genes.

Digital image capture and automated analysis of posterior capsular opacification.

PURPOSE: To develop and validate a digital imaging and analysis technique for assessing the extent of posterior capsular opacification after cataract surgery. METHODS: Retroillumination images of the posterior capsule were obtained by using a digital camera mounted on a slit lamp. The images were analyzed using an available image analysis software program. The image acquisition and analysis techniques were tested for face validity, reproducibility, and the ability to detect progression of capsular opacity over time. RESULTS: Digital retroillumination images were obtained without patient discomfort. Automated analysis of images correlated well with clinical grading both at slit lamp examination and when looking at the images themselves (Spearman's correlation coefficient >0.7). Analysis of images taken at different times showed high reproducibility (intraclass correlation >0.9), and the system was able to identify progression of capsular opacity over a 2-year period with a mean increase of 15.8% in progressors versus an increase of 0.6% in nonprogressors (P < 0.05). CONCLUSIONS: Digital retroillumination images of the posterior capsule can be obtained reliably, and automated analyses correlate well with clinical assessment. The system presented here uses commercially available instruments and software, and it is practical for use in longitudinal studies of posterior capsule opacification. It is reliable, easy to use, and can detect small changes in the percentage area covered by posterior capsule opacification over time.

Racial differences in lens opacities: the Salisbury Eye Evaluation (SEE) project.

The purpose of this study was to determine racial differences in the prevalence of different types of lens opacities and cataract surgery. Between 1993 and 1995, the Salisbury Eye Evaluation (SEE) Project enrolled a representative sample of 2,520 community-dwelling persons aged 65-84 years in Salisbury, Maryland, 26.4% of whom were African-American. Participants received a full eye examination, and photographs were taken for documentation of lens status. Photographs were graded using a standardized grading system for the presence of cortical, nuclear, or posterior subcapsular cataract (PSC) opacification in at least one eye. The odds of having cortical opacities were 4.0 times greater among African Americans than among Caucasians (95% confidence interval (CI) 3.3-4.8). Caucasians were significantly more likely to have nuclear opacities (odds ratio = 2.1, 95% CI 1.7-2.6) and PSC opacities (odds ratio = 2.5, 95% CI 1.7-3.6). The odds of cataract surgery were 2.8 times higher among Caucasians, but these differences did not explain the differences in the prevalence of different types of lens opacities by racial group. With lower rates of nuclear and PSC opacities than Caucasians, African Americans may have a lower demand for cataract surgery. However, even with these differences, there is still significant unnecessary loss of vision due to cataract among older African Americans, for whom programs to ensure access to surgical care are indicated.

Sunlight exposure and risk of lens opacities in a population-based study: the Salisbury Eye Evaluation project.

CONTEXT: Exposure to UV-B radiation in sunlight has been shown to increase the risk of cataract formation in high-risk occupational groups, but risk to the population has not been quantified. OBJECTIVES: To determine the ocular exposure to UV-B radiation in sunlight for a population of older persons and to determine the association between UV-B and lens opacities. DESIGN: The Salisbury Eye Evaluation project, a population-based cohort of older adults. SETTING: Salisbury, Md. PARTICIPANTS: A total of 2520 community-dwelling 65-year-old to 84-year-old adults in Salisbury, Md, from 1993 to 1995, of whom 26.4% were African Americans. MAIN OUTCOME MEASURE: Association of photographically documented cortical opacity 3/16 or greater in at least 1 eye with ocular UV-B exposure, reported in Maryland sun-years of exposure. RESULTS: The odds of cortical opacity increased with increasing ocular exposure to UV-B (odds ratio [OR], 1.10; 95% confidence interval [CI], 1.02-1.20). The relationship was similar for women (OR, 1.14; 95% CI, 1.00-1.30) and for African Americans (OR, 1.18; 95% CI, 1.04-1.33). Analyses of the ocular dose by each age group after the age of 30 years showed no vulnerable age group, suggesting damage is based on cumulative exposure. CONCLUSIONS: Although this population of older Americans has relatively low ocular exposure to UV-B in sunlight, there is still an association between ocular exposure and increasing odds of cortical opacity. Our study found an association among African Americans, which, to our knowledge, has not been reported previously. All sex and racial groups would benefit from simple methods to avoid ocular sun exposure.

New objective classification system for nuclear opacification.

We have developed an autonomous objective classification scheme for degree of nuclear opacification. The algorithm was developed by using a series of color 35-mm slides acquired with a Topcon photo slit-lamp microscope and use of standard camera settings. The photographs were digitized, and first, and second-order gray-level statistics were extracted from within circular regions of the nucleus. Classifications of severity were performed by using these features as input to a neural network. Training versus classification performance was tested by using photographs of different eyes, and test/retest classification reproducibility was evaluated by using paired photographs of the same eyes. We demonstrate good performance of the classifier against subjective assessments rendered by the Wilmer grading system [Invest. Ophthalmol. Visual Sci. 29, 73 (1988)] and markedly better test/retest reproducibility.

Positive selection induces CD4 promoter and enhancer function.

Developmental expression of the CD4 gene in mature T cells is controlled by at least four transcriptional control elements: a promoter, two enhancers and a silencer. In this report we use a transgenic approach to study the mechanisms in which these elements interact to convey appropriate tissue- and cell-specific expression at all stages of T cell development. Our data indicate that the control of CD4 gene expression requires the interaction of multiple elements functioning in different combinations at different stages of T cell development. Expression of the CD4 gene in immature CD4+CD8+ thymocytes requires a third enhancer element located in the 3' flanking region of the CD4 gene. Interestingly, the CD4 promoter and proximal/distal enhancers first begin to function at the HSAlo CD69lo H-2Khi CD4 single-positive stage; cells of this phenotype are believed to have survived positive selection. These data indicate that the CD4 promoter and late enhancer elements are induced by positive selection; thus, the final maturation process is an active event that requires the initiation of a novel program of gene expression.

Visible and ultraviolet-B ocular-ambient exposure ratios for a general population. Salisbury Eye Evaluation Project Team.

PURPOSE: To estimate the numerical value of the ocular-ambient exposure ratio (OAER) (ratio of the facial exposure to that on a horizontal plane) as a function of wavelength band, season, and job category and to establish the effect of various modifiers, such as geography and the use of hats, for use in general population studies. METHODS: Two hundred sixty-four persons within several job categories representing the jobs in our Salisbury, Maryland, population were instrumented with ultraviolet-B (UVB) and visible band sensors for 1 complete day. Studies were done over all four seasons, both with and without hats. RESULTS: OAERs in the UVB wavelength band generally are higher than in the visible (13% versus 6%), display no significant variation with job category, show a seasonal effect (highest in the winter-spring [18%], lowest in the summer [10%], and intermediate in the fall [14%]), and are reduced 34% by the use of hats. In the visible wavelength band, OAERs are affected weakly by job function, although this variation is not significant, display a seasonal effect with three seasons as in the UVB, and are not affected significantly by the use of hats. In neither the UVB nor the visible portions of the spectrum did the authors find an effect on the OAER due to photophobia or eye color. CONCLUSIONS: With the authors' exposure model, the authors have at their disposal a valuable tool for exploring the relation between UVB, UVA, and visible radiation and a number of age-related eye diseases.

Assessment of ocular exposure to ultraviolet-B for population studies. Salisbury Eye Evaluation Project Team.

We have developed an empirical model with which to estimate the ocular exposure in the UVB wavelength band. This model incorporates aspects of personal behavior, geographic location, season and wavelength that have been developed from population-based data. This model, in conjunction with job history interviews, allows the estimate of cumulative exposures from age 30 in our population-based study. We present data on average annual exposure by age, gender, race, education and reported photophobia. The exposures in the general population are considerably lower than exposures reported in previous works based on occupational groups. There is a statistically significant difference between males and females, with females having lower exposures. African-Americans have median exposures similar to whites. Exposures decrease with education and with reported photophobia. These data provide the basis for characterizing lifetime exposure for the general population. The variability of exposure is much greater than found in occupational groups and includes numbers in the lowest exposure. The model should permit determination of a dose-response relationship with eye disease, even to the lower dose ranges.

Asymmetric redundancy in CD4 silencer function.

We and others have defined a transcriptional silencer critical for the proper expression of the CD4 gene at all stages of T cell development. In this report, we use biochemical techniques to identify three different factor-binding sites within the CD4 silencer, denoted sites I, II, and III. Using transgenic analyses, we determine that although all three factor-binding sites are important for silencer activity, there is significant redundancy in that the presence of either site II alone, or the combination of sites I and III permits silencer function. Thus, our data indicate that the mechanism of function of the CD4 silencer is extremely complex. Further biochemical analyses indicate that the factor binding to site II has the same sequence specificity as a factor binding to an E box site in the CD4 enhancer; thus, a member of the bHLH factor family may be important in mediating silencer function.

Noncontact microstrain measurements in orthodontic wires.

A technique is presented that allows for highly sensitive and highly accurate measurements of microstrain in biomaterials. It is particularly useful for measuring strains in materials where the use of conventional extensometers or strain gauges is impracticable, such as reinforcement fibers and orthodontic wires and in hostile environments. The technique is based on the well known technique of observing translating laser speckle with a linear-array charge-coupled device (CCD) camera. However, it employs a relatively new data processing algorithm involving a two-dimensional frequency transform of the data. Advantages of the technique include: insensitivity to slow surface microstructure changes, insensitivity to zero-mean noise processes, compact design, modest resolution requirements, and the fact that it is truly noncontact. Strain rate measurements were made on an 0.028-gauge round stainless steel orthodontic wire as an example of this technique. The Young's modulus of the wire based on the speckle technique was 2.04 x 10(11) Nm(-2), which is very close to the textbook value.

The development of personal dosimeters for use in the visible and ultraviolet wavelengths regions. The Salisbury Eye Evaluation Team.

We have designed, developed, and field evaluated a two-part personal dosimeter for estimating ocular exposure to visible and ultraviolet radiation. The detector head id compact enough to be mounted on the bow of eyeglass frames, and the recorder is small enough to be carried in a shirt pocket. The detector head consists of a silicon photodiode with filter (Schott glass GG400) and diffuser for visible light measurements and a polysulphone film badge for ultraviolet-B (UVB, 290-320 nm) measurements. The measurements correspond well to those obtained using ambient monitors and should be useful for studies of ocular UVB exposure.

A Myc-associated zinc finger protein binding site is one of four important functional regions in the CD4 promoter.

The CD4 promoter plays an important role in the developmental control of CD4 transcription. In this report, we show that the minimal CD4 promoter has four factor binding sites, each of which is required for full function. Using biochemical and mutagenesis analyses, we determined that multiple nuclear factors bind to these independent sites. We determined that an initiator-like sequence present at the cap site and an Ets consensus sequence are required for full promoter function. We also demonstrate that the Myc-associated zinc finger protein (MAZ) appears to be the predominant factor binding to one of these sites. This last site closely resembles the ME1a1 G3AG4AG3 motif previously shown to be a critical element in the P2 promoter of the c-myc gene. We therefore believe that the MAZ transcription factor is also likely to play an important role in the control of developmental expression of the CD4 gene.

Ocular and dermatologic health effects of ultraviolet radiation exposure from the ozone hole in southern Chile.

OBJECTIVES: This study sought to investigate numerous reports emanating from Punta Arenas, Chile (population 110,000, latitude 53 degrees S), that associated acute ocular and dermatologic disease in humans and animals with excess ultraviolet-B (UV-B) exposure in the setting of the thinning of the ozone column. METHODS: Ophthalmologic and dermatologic records in Punta Arenas were systematically reviewed to enumerate sentinel diagnoses potentially associated with UV-B exposure, ocular examinations on representative animal populations were performed, and the ambient UV-B exposure in the region during the time of maximal thinning was estimated. RESULTS: No increase in patient visits or conditions attributable to UV-B exposure was seen for periods of known ozone depletion compared with control periods. Although ambient UV-B exposure was 1.6 to 2.3 times the habitual exposure on individual days, this excess exposure conferred only a 1% increase in annual exposure on the region. CONCLUSION: This study does not support existing lay reports of ocular and dermatologic disease in humans and animals that had been associated with the ozone hole over southern Chile.

Results of ophthalmic examinations of 29 alpacas.

Analysis of the results of ophthalmic examinations of 29 alpacas (Lama pacos) revealed a direct relationship among coat color, iris color, and ocular fundus pigmentation. Alpacas with light-pigmented coats had iris color that included combinations of gray, blue, and brown, and had reduced pigmentation of the ocular fundi. Alpacas with dark coats had brown irides and pigmented ocular fundi. Eleven alpacas had evidence of ophthalmic conditions, including bilateral conjunctivitis (1); formation of a superficial corneal scar (1); formation of posterior synechiae and anterior cataracts (4); development of a deep corneal scar with anterior synechiae, buphthalmia, a subluxated lens, and cataracts (1); development of focal incipient primary cataracts (2); formation of vitreous opacities (1); and development of a unilateral optic nerve coloboma (1).

Role of antigen-presenting cells in the polarized development of helper T cell subsets: evidence for differential cytokine production by Th0 cells in response to antigen presentation by B cells and macrophages.

Immune challenges can elicit polarized responses skewed towards the development of T helper type 1 (Th1) or Th2 T cell subsets. To determine if distinct antigen-presenting cells (APC) populations might selectively influence Th subset development, we studied the role of two key APC populations, B cells and macrophages, in the differentiation of effector Th populations from naive precursor Th in vitro. Antigen (Ag)-specific, naive CD4+ T cells were enriched from a mouse strain, AND, bearing a transgenic alpha/beta T cell receptor (TCR) encoding reactivity with pigeon cytochrome c peptide 88-104. Peptide Ag was used throughout these studies so that differences in the uptake and processing by the two APC populations would not influence the results. Both APC populations, activated B cells and bone marrow-derived macrophages, supported the development of effector Th having the capacity to secrete high levels of cytokines when restimulated. Regardless of APC population present during effector development, exogenous interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) had dominant effects on Th subset development. Thus, with both APC populations, effector Th generated in the presence of IFN-gamma acquired a Th1-type cytokine profile, Th generated with IL-4 acquired a Th2-type cytokine profile, and Th generated without IFN-gamma or IL-4 acquired a Th0-type cytokine profile. B cells and macrophages also had equivalent APC function in the restimulation of Th1 and Th2-like effectors, since only minor differences in cytokine production were noted for these effector populations when restimulated with the two APC populations. However, in 8 of 19 experiments, the Th0-like effector population generated in the presence of IL-2 differentially responded to restimulation with B cells and macrophages, secreting significantly more IFN-gamma when restimulated with B cells, and significantly more IL-4 when restimulated with macrophages. We also found that Th effector populations recultured in IFN-gamma or IL-4 assumed a more Th1 or Th2-like phenotype, respectively, regardless of their initial cytokine profile. We conclude that through a subtle capacity to skew cytokine production by a Th0 subset, different APC may selectively influence Th subset development under conditions of prolonged or chronic stimulation in an autocrine fashion.