Isolation, characterization, and experimental infection of Streptococcus gallolyticus subspecies pasteurianus from commercial turkeys with acute septicemia: a pilot study.

Streptococcus gallolyticus (SG) is a Gram-positive cocci found as commensal gut flora in animals and humans. SG has emerged as a cause of disease in young poults between 1 and 3 wk of age. SG is associated with septicemia resulting in acute mortality with no premonitory signs in turkeys. Three SG isolates were obtained from clinical field cases of acute septicemia of commercial turkeys and used in three independent experiments. In Experiment 1, embryos were inoculated 25 d of embryogenesis with varying concentrations of SG1, SG2, or SG3. In Experiment 2, day of hatch, poults were inoculated with varying concentrations using different routes of administration of SG1, SG2, or SG3. In Experiment 3, day of hatch, poults were inoculated with only isolate SG1 using different paths. Poults were randomly selected for necropsy on d 8 and d 15 and sampled to collect spleen, heart, and liver for SG on d 21, the remaining poults were necropsied and cultured. Samples were plated on Columbia nalidixic acid and colistin agar (CNA) (40°C, 18-24 h). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) confirmed suspect colonies. Data were analyzed using the chi-square test of independence, testing all possible combinations to determine significance (P < 0.05). Weight data were subjected to ANOVA using JMP with significance (P < 0.05). No differences were found in BW or BWG on d 0, 8, 15, or 22. Splenomegaly, focal heart necrosis, and pericarditis were observed in all groups in experiments 1 through 3. In Experiment 3, only airsacculitis was observed in a negative control in separate isolation (P > 0.05). On d 21 of Experiment 3, increased (P < 0.05) recovery of SG from spleens were observed in co-housed negative controls, as well as poults challenged by oral gavage (P > 0.05 for d 7 and d 14). These results confirm numerous previous studies indicating that SG subsp. pasteurianus is a primary infectious microorganism that causes septicemia in young poults.

Increased intracellular cyclic adenosine monophosphate inhibits T lymphocyte-mediated cytolysis by two distinct mechanisms.

Cholera toxin (CT), but not pertussis toxin (PT), treatment of cloned murine CTL inhibited target cell lysis in a dose-dependent fashion. The effects of CT were mimicked by forskolin and cyclic adenosine monophosphate (cAMP) analogues. Inhibition of cytotoxicity by CT and cAMP analogs was mediated in part by attenuation of conjugate formation. Additionally, both CT and cAMP analogs blocked the increase in intracellular Ca2+ induced by stimulation of the TCR complex by mAbs. These findings indicate that cAMP inhibits the activity of CTL by two distinct mechanisms and suggests a role for this second messenger in CTL-mediated cytolysis.

The effect of proximity on open-loop accommodation responses measured with pinholes.

Open-loop accommodation levels were measured in 41 healthy, young subjects using a Shin-Nippon SRW-5000 autorefractor in the three viewing conditions: a small physical pinhole pupil (SP), an optically projected pinhole in Maxwellian view (MV) and in the dark (DF). The target viewed through the pinholes was a high-contrast letter presented at 0 D vergence in a +5 D Badal lens system. Overall, results showed that SP open-loop accommodation levels were significantly higher than MV and DF levels. Subjects could be divided into two distinct subgroups according to their response behaviour: responders to the proximal effect of the small physical pinhole (SP accommodation > MV accommodation) and non-responders to the proximal effect of the small physical pinhole (SP accommodation approximately MV accommodation). Correlation analysis demonstrated that open-loop accommodation for both pinhole conditions was correlated with DF for the responders, while for the non-responders SP and MV accommodation were correlated, but were not related to DF accommodation. This suggests that under open-loop conditions some individuals' accommodation levels are mainly affected by proximal and cognitive factors (responders) while others are guided primarily by the presence of the more distal target (non-responders). In conclusion, MV reduces the proximal effect of the physical pinhole and produces open-loop accommodation responses which are more consistent than SP and DF responses.

Increased intracellular Ca2+ induces Ca2+ influx in human T lymphocytes.

One current hypothesis for the initiation of Ca2+ entry into nonelectrically excitable cells proposes that Ca2+ entry is linked to the state of filling of intracellular Ca2+ stores. In the human T lymphocyte cell line Jurkat, stimulation of the antigen receptor leads to release of Ca2+ from internal stores and influx of extracellular Ca2+. Similarly, treatment of Jurkat cells with the tumor promoter thapsigargin induced release of Ca2+ from internal stores and also resulted in influx of extracellular Ca2+. Initiation of Ca2+ entry by thapsigargin was blocked by chelation of Ca2+ released from the internal storage pool. The Ca2+ entry pathway also could be initiated by an increase in the intracellular concentration of Ca2+ after photolysis of the Ca(2+)-cage, nitr-5. Thus, three separate treatments that caused an increase in the intracellular concentration of Ca2+ initiated Ca2+ influx in Jurkat cells. In all cases, Ca(2+)-initiated Ca2+ influx was blocked by treatment with any of three phenothiazines or W-7, suggesting that it is mediated by calmodulin. These data suggest that release of Ca2+ from internal stores is not linked capacitatively to Ca2+ entry but that initiation is linked instead by Ca2+ itself, perhaps via calmodulin.

Inhibition of human prostate cancer proliferation in vitro and in a mouse model by a compound synthesized to block Ca2+ entry.

Accelerated Ca2+ entry may be one component of the pathway regulating the proliferative phenotype of some types of cancer. Thus, a pharmacological agent with the ability to retard Ca2+ influx in susceptible cancers might inhibit proliferation of them by a cytostatic mechanism rather than by inducing cytotoxicity. We have developed a chemical synthetic scheme that has produced a small library of novel compounds that block Ca2+ entry induced by occupancy of the P2 receptor in two prostate cancer cell lines and inhibit proliferation of these cells in vitro. One of the agents, named TH-1177, was used to treat severe combined immunodeficient mice inoculated with the human prostate cancer line PC-3. Although the doses used and treatment schedule were chosen arbitrarily, treatment extended the mean life span of mice bearing tumors by up to 38%. Treatment of mice without cancer at doses 18 times that used in mice with tumors was not associated with any obvious toxicity, either grossly or on histological examination. These results suggest that novel cytostatic agents with efficacy against human prostate cancer cells can be developed by chemical synthesis of agents directed at the Ca2+ entry pathway.

Calmodulin regulation of Ca2+ entry in Jurkat T cells.

We have previously proposed a role for calmodulin (CaM) in the regulation of initiation of Ca2+ entry in Jurkat T cells, as well as in the regulation of the current that mediates Ca2+ entry, IT. In this report, we provide evidence for the mechanism of CaM action. We have previously shown that activation-induced Ca2+ entry into Jurkat T cells is mediated by a current we have called IT. In the whole cell variation, but not the perforated patch variation, of the patch clamp technique, this current is short-lived (under 6 min) suggesting that the current is under the control of a diffusible component of the cytosol. Addition of CaM to the whole cell recording pipette solution maintained IT for up to 20 min, suggesting that CaM may be this diffusible component. Pharmacological inhibitors of CaM blocked the augmentation of IT normally induced by an activating stimulus. Cells electroporated in the presence of anti-CaM antibodies had reduced influx of extracellular Ca2+, with no change in release of Ca2+ from the internal stores. These observations suggest that T cell receptor engagement initiates Ca2+ influx by a pathway that likely includes CaM, which may in turn regulate IT. Influx of extracellular Ca2+ is required for cellular proliferation, and inhibition of CaM by pharmacological inhibitors reduced cellular proliferation. This same inhibition of proliferation was seen in cells electroporated with anti-CaM antibodies. This suggests that inhibition of CaM and/or IT may be a target for therapeutic inhibition of inappropriate T cell proliferation.

A voltage-gated calcium channel is linked to the antigen receptor in Jurkat T lymphocytes.

Activation of T lymphocytes results in an increase in intracellular Ca2+ due in large part to influx of extracellular Ca2+. Using the patch clamp technique, an inward current in Jurkat T lymphocytes was observed upon depolarization from a holding potential of -90 mV but not from -60 mV. This whole-cell current was insensitive to tetrodotoxin, carried by Ba2+, and blocked by Ni2+. Occupancy of the T lymphocyte antigen receptor increased the current's magnitude. These data suggest that antigen receptor-induced Ca2+ entry in T lymphocytes may be mediated by a voltage-regulated Ca channel.

Hemolytic activity of adenylate cyclase toxin from Bordetella pertussis.

Adenylate cyclase (AC) toxin from B. pertussis enters eukaryotic cells where it produces supraphysiologic levels of cAMP. Purification of AC toxin activity [(1989) J. Biol. Chem. 264, 19279] results in increasing potency of hemolytic activity and electroelution of the 216-kDa holotoxin yields a single protein with AC enzymatic, toxin and hemolytic activities. AC toxin and E. coli hemolysin, which have DNA sequence homology [(1988) EMBO J. 7, 3997] are immunologically cross-reactive. The time courses of hemolysis elicited by the two molecules are strikingly different, however, with AC toxin eliciting cAMP accumulation with rapid onset, but hemolysis with a lag of greater than or equal to 45 min. Finally, osmotic protection experiments indicate that the size of the putative pore produced by AC toxin is 3-5-fold smaller than that of E. coli hemolysin.

Accommodation responses and ageing.

PURPOSE: To study the impact of age on accommodation dynamics. METHODS: Monocular accommodation responses were measured continuously using a modified Canon Auto Ref R1 infrared optometer. The stimulus was a single letter oscillating sinusoidally between 2.38 and 1.33 D providing a stimulus amplitude of 0.52 D, about a mean level of 1.86 D. Response characteristics were used to quantify gain and phase. Step responses were also recorded between these stimulus vergence levels for calibration purposes and to measure reaction and response times. Nineteen visually normal subjects 18 to 49 years of age participated, and 11 frequencies were used in the range 0.05 to 1.0 Hz. A key feature of the experimental design was to use a stimulus vergence range that lay within the amplitude of accommodation of all the observers. RESULTS: Accommodation gain reduced and phase lag increased with age, particularly at the higher frequencies used. No strongly significant change with age was found for reaction and response times or accommodation velocity, and results were similar for both far-to-near and near-to-far responses. Response amplitude for the step change in target vergence declined with age, and substantial differences were found between the measured and predicted (from reaction time) phase lags at 1.0 Hz as a function of age. Young observers showed a phase lag that was shorter than predicted, whereas older observers' measured phase lags were considerably larger than predicted. CONCLUSIONS: Results show that for a target oscillating sinusoidally in a predictable manner at a modest amplitude, the main ageing effects occur in phase lag, which is appreciably longer than predicted from reaction times in the older observers. The effects of ageing on gain were not as marked. Although responses to small step changes do reduce with age, there is no evidence of increased response times with ageing. In general, accommodation function in the middle-aged eye is quite robust despite a dwindling amplitude of accommodation. These results provide evidence of accommodative vigor in youth and a slowing of accommodation with ageing.

Objective concurrent measures of open-loop accommodation and vergence under photopic conditions.

PURPOSE: To investigate the relationship between photopic open-loop accommodation and vergence and the effect of mental effort on these positions. METHODS: Twenty subjects (11 men and 9 women) viewed monocularly a photopic (25 cdm-2), high-contrast (90%) Maltese cross-target for 3 minutes, through a 0.5-mm pinhole drilled into an infrared filter. Accommodation was measured objectively at 1-second intervals using a Canon Autoref R-1 infrared optometer, and vergence was recorded continuously and objectively using a differential infrared eye tracking system. RESULTS: Under passive viewing conditions there was a significant correlation between photopic open-loop accommodation and vergence (R = 0.671, P = 0.0012); for the majority of subjects the imposition of mental effort shifted the passive levels of both open-loop accommodation and vergence, but these shifts were unsystematic and uncorrelated (R = 0.259, P = 0.270). The active open-loop positions of accommodation and vergence were also found to be correlated (R = 0.692, P = 0.0007). CONCLUSIONS: The influence of proximal stimuli can explain the correlations identified between photopic open-loop positions of accommodation and vergence. The uncorrelated responses of the accommodation and vergence systems to mental effort are likely to reflect interactions between various spatiotopic stimuli including mental effort and perceived proximity.

Functional visual loss in amblyopia and the effect of occlusion therapy.

PURPOSE: The aim of this study was to define the nature of functional visual loss in amblyopia and to identify those subjects whose amblyopia is chiefly due to one or more of the following deficits: abnormal contour interaction, abnormal eye movements, abnormal contrast perception, or positional uncertainty. METHODS: Fifty amblyopic children with a mean age of 5.6+/-1.3 years were referred from diverse sources. In addition to routine orthoptic and optometric evaluation the principal visual deficits in the amblyopic eye of each subject were identified using the following measures of visual acuity: high contrast linear, single optotype, repeat letter and low contrast linear, plus Vernier and displacement thresholds. These measures were repeated as the children underwent a prescribed occlusion therapy regime, after parental consent. RESULTS: All amblyopic subjects demonstrated a functional loss in each of the tests used, and occlusion therapy appeared to improve all aspects of the amblyopia. High contrast visual acuity was not always the primary deficit in visual function, and when amblyopic subjects were divided according to their primary visual loss, this visual function was found to show the greatest improvement with treatment. CONCLUSIONS: These results suggest that to successfully identify the primary visual deficit and monitor the success of occlusion therapy it is necessary to assess other aspects of visual function in amblyopia.

Red cell survival studies in a patient with anti-Tca.

A radiolabeled, allogenic red cell survival study was performed on a patient who lacked the Cromer-related antigen Tca and who had developed the corresponding antibody. Red cell survival was 92% at 1 hour and 88% at 24 hours. Monocyte monolayer assays (MMA) and IgG subclass determinations were performed on samples from: (1) 1965, the period of initial antibody formation; (2) approximately two years before the red cell survival study; and (3) four months after the study. All samples reacted w+ to 1+ by the antiglobulin test. The earliest sample contained IgG1, IgG2, and IgG4 anti-Tca. Because there were 20.5% reactive monocytes in the MMA (normal range 0-3%), this antibody may have produced extravascular red cell destruction. In contrast to the initial example, the samples before and after the red cell survival study both contained IgG2 and IgG4 subclasses with 1.3% and 2.2% MMA reactivity, respectively. The current pattern of antibody subclass, the lack of reactivity in the MMA, and a red cell survival of 88% at 24 hours indicate that short-term transfusion support would have been well tolerated. This contrasts to the in vitro results obtained with the earliest sample, which suggest a clinically significant antibody. This appears to be the first report of a red cell alloantibody that remained serologically reactive but underwent a loss of its IgG1 fraction, which appeared capable of red cell destruction based on the MMA results.

Multicompartment analysis of the effects of plasmapheresis. Application to lipid kinetics in humans.

The quantitative effect of plasmapheresis on concentrations of intravascular solutes is described by a minimal two-compartment model, which allows exit to and entry from a nonplasma body pool of the solute. This novel method is simple and capable of determining endogenous metabolic turnover of physiologically important plasma constituents in both therapeutic and experimental settings. The model was suggested by the therapeutic use of plasmapheresis in hyperlipidemia, and the turnover rates of triglyceride and cholesterol were calculated for one patient treated with chronic plasmapheresis. It is concluded that at least a two-compartment model is necessary to quantitatively describe the effect of plasmapheresis on any substance that undergoes appreciable endogenous turnover (metabolic clearance rate greater than 1.1 times plasmapheresis rate when both rates are in units of volume per time). Calculation of endogenous turnover rate with the use of measurements of the concentration of a solute in plasma before and after plasmapheresis and in the total plasmapheresate and measurement of the volume of plasma removed, volume of plasma substitute infused, and total plasmapheresis time is detailed. This method avoids the use of isotopic labeling experiments. In turn, the endogenous turnover rate may predict the efficacy of therapeutic plasmapheresis.

Early events in target-cell lysis by cytotoxic T cells.

Using ratio-imaging fluorescence microscopy, we have investigated the changes in intracellular Ca2+ [( Ca2+]i) that occurred in cytotoxic T lymphocytes (CTL) upon target-cell binding. This process resulted in a rapid increase in [Ca2+]i, which was localized in the region of the CTL in contact with the target cell. This increase was mediated both by influx from the external medium as well as by release from intracellular stores. Although the magnitude of the initial increase in [Ca2+]i was not dependent upon the presence of extracellular Ca2+, influx was necessary for sustained elevation of [Ca2+]i. Inasmuch as target-cell lysis by the CTL clone used is dependent on extracellular Ca2+, this suggests that a prolonged elevation of [Ca2+]i is necessary for lytic function. It was also shown that the increase in [Ca2+]i and its subsequent decay show several pulsations. The mechanism by which these variations are generated and their possible function is not known. Finally, a role for K+ efflux in the control of the antigen-induced increase in [Ca2+]i was demonstrated. Thus it is becoming clear that signal transduction in CTL is remarkably complex, involving several ionic species and second messengers.

Accommodative microfluctuations and pupil diameter.

The nominally steady-state accommodation response exhibits temporal variations which can be characterized by two dominant regions of activity; a low frequency component (LFC < 0.6 Hz) and a high frequency component (1.0 < or = HFC < or = 2.1 Hz). There is no consensus as to the relative contribution made by each of the frequency components of the microfluctuations to the control of steady-state accommodation. We investigate the effect of variations in artificial pupil diameter (0.5, 1, 2, 3, 4 and 5 mm pupils) on the microfluctuations of accommodation, while three young emmetropic subjects view, monocularly, a photopic high contrast Maltese cross target placed at a dioptric distance equal to their open-loop accommodation level. Average power spectra were calculated for five accommodation signals, each of 10 sec duration, collected for each viewing condition at a sampling rate of 102.4 Hz using a continuously recording infrared objective optometer. For artificial pupil diameters < or = 2 mm the power of the LFC was found to increase as a function of reducing pupil diameter, while for artificial pupil diameters > 2 mm the LFC was found to be relatively constant. No systematic change in the HFC with varying artificial pupil diameter was observed. Changes in the root-mean-square (r.m.s.) value of the fluctuations with varying pupil diameter were significant (one-way ANOVA, F = 8.507, P = 0.0001, d.f. = 89) and showed a similar form to the changes in the LFC.(ABSTRACT TRUNCATED AT 250 WORDS)

Screening for amblyopia: a comparison of paediatric letter tests.

AIMS/BACKGROUND: The measurement of visual acuity is the most widely accepted indicator of amblyopia and is thought by some to be the only effective screening test. The aim of this study was to investigate the effectiveness of the traditional single optotype Sheridan-Gardiner test (SGT) in the measurement of visual acuity and the detection of amblyopia, compared with the log based linear format Glasgow acuity cards (GAC). METHODS: In the present study visual acuity was measured monocularly in 702 primary 1 schoolchildren using both acuity tests. RESULTS: A significant difference was found in the mean (SD) visual acuity measured with GAC (0.9 (0.08) modified logMAR) and SGT (1.13 (0.09) modified logMAR), df = 632, t = -59.08, p = 0.0001. The majority of children (89.3%) achieved visual acuities better than 6/6 in either eye when using the single optotype test. If the 95% confidence limits for a significant interocular difference in acuity are used as-criteria for the detection of unilateral amblyopia, GAC were found to be the most sensitive, correctly identifying 100%, while SGT identified 55% of the children with unilateral amblyopia. CONCLUSION: The results of this study highlight several problems with both the test format and testing procedure in the present school screening system.

The influence of tinted lenses upon ocular accommodation.

We determined the effect upon accommodative responses of tinted lenses prescribed for the relief of visual discomfort in a group of five long term lens wearers. Static and dynamic responses were measured under four viewing conditions (1) prescribed tinted lens (2) neutral density filter (3) tinted lens of complementary colour and (4) no absorptive lens. While similarity and normality of the mean stimulus-response functions between the four viewing conditions were evident, the low frequency component of the accommodation microfluctuations was significantly greater while viewing the target in the 'no lens' viewing condition. These increases in the low frequency components (LFC) of the accommodation may be a subtle indicator of visual stress in these patients. Colour specificity is not supported by this finding.

Visual function thresholds in children.

PURPOSE: The aim of the present study was to establish a range of values for individual visual function measurements in Primary 1 schoolchildren. METHODS: 93 visually normal children with a mean age of 5.4 +/- 0.3 years were recruited with parental consent. Measures of visual acuity obtained included 1) High Contrast (CAT) 2) Low Contrast (LC) 3) Single Letter (SLA) and 4) Repeat Letter Acuity (RRL). These measurements were randomised and obtained during the same visit. Hyperacuity thresholds were obtained at a separate visit, using both Vernier and oscillatory motion displacement stimuli (OMDT). Re-test data for all tests was collected from a random sample of 30 children after a period of at least two weeks. RESULTS: For all measures the distribution of scores was found to be normal. All visual acuity scores are in log units. Mean ( +/- SD) values for each test were as follows; CAT = 0.11 +/- 0.07; LC = 0.38 +/- 0.08; RRL = 0.04 +/- 0.07; SLA = -0.02 +/- 0.09; Vernier threshold = 69 +/- 21 arcsec; OMDT = 60 +/- 9 arc sec. The confidence limits of the test-retest discrepancies were as follows; CAT = +/- 0.05; LC = +/- 0.07; RRL = +/- 0.04; SLA = +/- 0.04; Vernier = +/- 17 arcsec; OMDT = +/- 6 arcsec. The confidence limits of the interocular discrepancies were as follows; CAT = +/- 0.08; LC = +/- 0.08; RRL = +/- 0.08; SLA = +/- 0.11; Vernier = +/- 14 arcsec; OMDT = +/- 11 arcsec. CONCLUSIONS: To fully describe an individual's visual capacity it is important that new tests of visual function are developed based on emerging knowledge of visual physiology. In this study confidence limits can be constructed for each visual function measure, test-retest and interocular discrepancies using the normal distributions found above. This will provide the clinician with further age-matched values and contribute to our understanding of functional visual development.

The effect of abnormal fixational eye movements upon visual acuity in congenital nystagmus.

PURPOSE: The purpose of this study was to investigate the role that abnormal eye movements play in the degradation of visual acuity. METHODS: Visual acuity was measured monocularly in 10 normal subjects (26.7+/-4.3 years) and 5 subjects with congenital nystagmus (34.9+/-8.8 years), using Regan Repeat Letter charts (RRL) and a logMAR based test (LogMAR Crowded Acuity Test (CAT)) while eye movements were continuously recorded using a commercially available infrared limbal eye tracker (Type 54, Optoelectronic Developments, UK). The eye tracker was controlled via a virtual oscilloscope (Viewdac, Keighly Instruments, UK) on an IBM PC clone (Opus Technology 486). RESULTS: The mean visual acuity obtained with RRL was significantly higher than that obtained by CAT in the subjects with congenital nystagmus. A significant correlation was found between the root mean square value of the nystagmus waveform and the angular extent of CAT. Linear regression analysis revealed a correlation between the duration of the foveation periods and the linear acuity of the subjects with congential nystagmus. The nystagmus waveforms also demonstrated increased amounts of high frequency components (HFC: >3.0 Hz) when compared to the normal eyes. CONCLUSIONS: The results of this study reveal 1) RRL charts provide a measure of a subjects resolution limit which is less dependent on eye movements; 2) the duration of foveation periods has a significant effect on visual acuity measurements obtained using a linear test chart format; 3) the predominance of high frequency components in the congenital nystagmus waveforms lead to short foveation periods adding to the degree of fixation instability.

Grating detection and orientation discrimination in amblyopia.

PURPOSE: We examined whether the misperceptions associated with amblyopic visual perception can be revealed under natural viewing conditions by comparing the ability to detect the presence of a grating with the ability to identify the grating orientation. METHODS: Grating detection and orientation discrimination performance (horizontal versus vertical) were determined, using stimuli that consisted of sinusoidal gratings of fixed contrast (75%) but with variable spatial frequency. A total of four amblyopic subjects (two strabismic and two non-strabismic) and four age-matched normals participated in the experiment. RESULTS: Psychometric functions for grating detection and orientation identification were found to be closely matched in the normal subjects and in all four amblyopic subjects, indicating that orientation could be correctly identified at detection threshold. CONCLUSIONS: The absence of orientation uncertainty in the psychophysical data for the amblyopic observers is not consistent with the several previous reports of spatial aliasing in the central field of amblyopes. Our results suggest that non veridical visual perception in central amblyopic vision can not be revealed under natural viewing conditions by comparing the ability to detect the presence of a grating with the ability to identify its orientation. Possible reasons for the failure of this technique to reveal spatial aliasing in amblyopes are discussed.