Conformational changes in a Vernier zone region: Implications for antibody dual specificity

Understanding the determinants of antibody specificity is one of the challenging tasks in antibody development. Monospecific antibodies are still dominant in approved antibody therapeutics but there is a significant body of work to show that multispecific antibodies can increase the overall therapeutic effect. Dual-specific or “Two-in-One” antibodies can bind to two different antigens separately with the same antigen-binding site as opposed to bispecifics, which simultaneously bind to two different antigens through separate antigen-binding units. These nonstandard dual-specific antibodies were recently shown to be promising for new antibody-based therapeutics. Here, we physicochemically and structurally analyzed six different antibodies of which two are monospecific and four are dual-specific antibodies derived from monospecific templates to gain insight about dual-specificity determinants. These dual-specific antibodies can target both human epidermal growth factor receptor 2 and vascular endothelial growth factor at different binding affinities. We showed that a particular region of clustered Vernier zone residues might play key roles in gaining dual specificity. While there are minimal intramolecular interactions between a certain Vernier zone region, namely LV4 and LCDR1 of monospecific template, there is a significant structural change and consequently close contact formation between LV4-LCDR1 loops of derived dual-specific antibodies. Although Vernier zone residues were previously shown to be important for humanization applications, they are mostly underestimated in the literature. Here, we also aim to resurrect Vernier zone residues for antibody engineering efforts.     

Do alignment thresholds define a critical boundary in long-range detection facilitation with co-linear lines?

Thresholds for line contrast detection (experiment 2) were measured with a two-alternative temporal forced-choice procedure as a function of the spatial position of a vertical target line with regard to two co-linear context lines. The different spatial positions of the target line corresponded to values near the position discrimination threshold (experiment 1) reflecting the just detectable lateral offset, or non-co-linearity, between the context lines which were vertically separated by about 100 minutes of visual arc. Target and context lines were vertically separated by about 30 minutes of arc, had equal contrast polarity in one case, and opposite contrast polarity in the other. Strong line contrast detection facilitation is found at perceptually co-linear target locations. This facilitation decreases noticeably at a horizontal target offset that corresponds to the alignment threshold measured with the context lines. The effects are independent of the relative contrast polarity of target and context and, as shown in a third experiment, also independent of both the relative length or number of lines, and the magnitude of their absolute co-axial separation. This independence seems to hold, provided individual line length and co-axial distance between lines are larger than what appears to be the lower limit of the long-range spatial domain for orientation or contour integration (i.e. 20 minutes of arc), as determined by previous studies. The findings reported here suggest that alignment thresholds are likely to define a critical lateral boundary in long-range detection facilitation with co-linear lines. They support models of contour integration based on interactions between neural mechanisms that integrate local signals of contrast, orientation, and relative position or end-to-end alignment. Such mechanisms may help to explain the formation of representations of virtual contours and object contours in human perception.     

Cortical magnification within human primary visual cortex correlates with acuity thresholds

We measured linear cortical magnification factors in V1 with fMRI, and we measured visual acuity (Vernier and grating) in the same observers. The cortical representation of both Vernier and grating acuity thresholds in V1 was found to be roughly constant across all eccentricities. We also found a within-observer correlation between cortical magnification and Vernier acuity, further supporting claims that Vernier acuity is limited by cortical magnification in V1.     

Asymmetries in oriented-line detection indicate two orthogonal filters in early vision.

Visual detection of a line target differing in orientation from a background of lines may be achieved speedily and effortlessly. Such performance is assumed to occur early in vision and to involve filter mechanisms acting in parallel over the visual field. This study establishes orientational limits on this performance and analytically derives some generic properties of the underlying filters. It was found that, in brief displays, target orientation detection thresholds increased approximately linearly with background orientation, from minima at 0 degree (vertical) and 90 degrees, whereas background orientation detection thresholds decreased approximately linearly with target orientation, from maxima at 0 degree and 90 degrees. Target and background threshold functions were exactly antisymmetric. These data are shown to indicate a model of early line processing dominated by two classes of orientation-sensitive filter with axes close to the vertical and horizontal and orientation-tuning half-widths each of approximately 30 degrees at half-height.     

Vernier acuity predicted from changes in the light distribution of the retinal image

If two thin bars of different luminance are placed side by side, their joint spatial position in a Vernier alignment task is determined simply by their relative luminances. The threshold luminance contrast difference required to produce a just detectable change in spatial position corresponds to a spatial shift of 5-20 arcsec in the centroid of the retinal light distribution, depending upon contrast relative to the background. This technique may be used to measure acuity with a display that has a spatial resolution considerably worse than the Vernier offset threshold. We have also extended the centroid technique to components that differ both in wavelength and luminance. Colour was found to make no essential difference to the task. Taking into account the spread of light in the retinal image, the manifest contrast thresholds are equivalent to threshold intensity increments between adjacent foveal receptors of less than 1% comparable to the values reported by Hecht and Mintz for dark line detection.     

Relative localization in the human fovea: radial-tangential anisotropy

A distinction is drawn between two intrinsic directions within a simple spatial configuration. The line joining two elements is the radial direction and orthogonal to it is the tangential direction. Separation and bisection discrimination and just-detectable differences in line length are examples of radial thresholds. Vernier and alignment detection are tangential thresholds. Neural processing along these two intrinsic directions differs. There is a strong 'oblique effect' for tangential thresholds and virtually none for radial thresholds. Flank interaction impairs tangential but not radial thresholds.     

Horizontal-vertical filters in early vision predict anomalous line-orientation identification frequencies.

A characteristic of early visual processing is a reduction in the effective number of filter mechanisms acting in parallel over the visual field. In the detection of a line target differing in orientation from a background of lines, performance with brief displays appears to be determined by just two classes of orientation-sensitive filter, with preferred orientations close to the vertical and horizontal. An orientation signal represented as a linear combination of responses from such filters is shown to provide a quantitative prediction of the probability density function for identifying the perceived orientation of a target line. This prediction was confirmed in an orientation-matching experiment, which showed that the precision of orientation estimates was worst near the vertical and horizontal and best at about 30 degrees each side of the vertical, a result that contrasts with the classical oblique effect in vision, when scrutiny of the image is allowed. A comparison of predicted and observed frequency distributions showed that the hypothesized orientation signal was formed as an opponent combination and horizontal and vertical filter responses.     

The central performance drop in texture segmentation: a simulation based on a spatial filter model

 This article presents a computational model of early visual information processing that attempts to account for the central performance drop (CPD) in texture segmentation. CPD is the finding that detection performance on short stimulus displays of line textures using orientation differences to set off the target is not maximal at the foveal center but in parafoveal areas. A comparison between a simulation and psychophysical experimental data supported the assumption that the CPD may be explained by properties of spatial frequency channels whose band-pass filter characteristics are not constant over the retina but differ with eccentricity in a defined manner. The model provided satisfactory predictions of experimental data based on densely or widely spaced line elements in texture fields. It is concluded that preattentive texture analysis might be performed by a relatively small number of simple spatial filters. Received: 14 November 1996 / Accepted in revised form: 3 June 1997     

Thermodynamic consequences of mutations in vernier zone residues of a humanized anti-human epidermal growth factor receptor murine antibody, 528

To investigate the role of Vernier zone residues, which are comprised in the framework regions and underlie the complementarity-determining regions (CDRs) of antibodies, in the specific, high affinity interactions of antibodies with their targets, we focused on the variable domain fragment of murine anti-human epidermal growth factor receptor antibody 528 (m528Fv). Grafting of the CDRs of m528Fv onto a selected framework region of human antibodies, referred to as humanization, reduced the antibody's affinity for its target by a factor of 1/40. The reduction in affinity was due to a substantial reduction in the negative enthalpy change associated with binding. Crystal structures of the ligand-free antibody fragments showed no noteworthy conformational changes due to humanization, and the loop structures of the CDRs of the humanized antibodies were identical to those of the parent antibodies. Several mutants of the CDR-grafted (humanized) variable domain fragment (h528Fv), in which some of the Vernier zone residues in the heavy chain were replaced with the parental murine residues, were constructed and prepared using a bacterial expression system. Thermodynamic analyses of the interactions between the mutants and the soluble extracellular domain of epidermal growth factor receptor showed that several single mutations and a double mutation increased the negative enthalpy and heat capacity changes. Combination of these mutations, however, led to somewhat reduced negative enthalpy and heat capacity changes. The affinity of each mutant for the target was within the range for the wild-type h528Fv, and this similarity was due to enthalpy-entropy compensation. These results suggest that Vernier zone residues make enthalpic contributions to antigen binding and that the regulation of conformational entropy changes upon humanization of murine antibodies must be carefully considered and optimized.     

Vernier acuity with noisy lines: Estimation of relative position uncertainty

Vernier acuity experiments were carried out to obtain estimates of the minimal number of spatial samples extrated, and of the equivalent noise figure characterizing inherent limitations of the visual system in relative position detection tasks. Results are interpreted by modelling the visual system as an ideal detector limited by external noise and internal position uncertainty resulting from structural limitations and neural noise. Accordingly, we estimate that less than ten samples per line are extracted by the observer and that the equivalent noise figure or position uncertainty is of the order of 20 arc s.     

Perceptual segregation and retinal position

This study is concerned with the dependence of perceptual segregation performance on the retinal position at which the performance is evaluated. Segregation performance consisted in detecting a target texture composed of line elements of constant length and orientation embedded in a background texture. The background texture was made up of the same line elements as the target texture but the background line elements were set at 90 deg to the target elements. Results showed that, at least for 40- and 50-ms presentation times, this task could be much more effectively completed outside the fovea centralis than within this area. These findings indicate that extrafoveal areas of the retina may make a significant and previously underestimated contribution to perceptual segregation.     

Temporal consistency is currency in shifts of transient visual attention

Background

Observers respond more accurately to targets in visual search tasks that share properties with previously presented items, and transient attention can learn featural consistencies on a precue, irrespective of its absolute location.

Methodology/Principal Findings

We investigated whether such attentional benefits also apply to temporal consistencies. Would performance on a precued Vernier acuity discrimination task, followed by a mask, improve if the cue-lead times (CLTs; 50, 100, 150 or 200 ms) remained constant between trials compared to when they changed? The results showed that if CLTs remained constant for a few trials in a row, Vernier acuity performance gradually improved while changes in CLT from one trial to the next led to worse than average discrimination performance. The results show that transient attention can quickly adjust to temporal regularities, similarly to spatial and featural regularities. Further experiments show that this form of learning is not under voluntary control.

Conclusions/Significance

The results add to a growing literature showing how consistency in visual presentation improves visual performance, in this case temporal consistency.     

A space-variant filter model of texture segregation: Parameter adjustment guided by psychophysical data

 This article presents a space-variant version of a standard spatial filter model of texture segregation of the “back-pocket” type (i.e., two filter layers with an intermediate pointwise nonlinearity). The model was tested with psychophysical data from experiments with line textures in which target lines differed in orientation from background lines. The textures were presented briefly and then masked. Segregation performance was evaluated along the horizontal meridian up to retinal eccentricities of about 10 deg. Data are reported from two experiments with different line densities (Kehrer 1989) and two experiments with different orientation contrasts between target lines and background lines (Kehrer 1990). Segregation performance proved to depend strongly on these texture variations, and it peaked several degrees from fixation in all cases. The filter model provided satisfactory predictions of experimental data when model parameters were adjusted appropriately. It is concluded (1) that filter models defined in strictly spatial terms (i.e., without temporal properties) offer a sufficient framework to account for the psychophysical data and (2) that the particular course of the performance curve (i.e., the performance peak outside the central region) must be attributed to the characteristics of second-layer filters. Received: 28 June 2001 / Accepted in revised form: 10 October 2002 / Published online: 13 February 2003 Correspondence to: L. Kehrer (e-mail: lothar.kehrer@uni-bielefeld.de) Acknowledgements. This work was supported by Grant Ke 388/3-2 from the Deutsche Forschungsgemeinschaft (DFG). We wish to thank Jonathan Harrow for improving the English text and an anonymous reviewer for many constructive comments.     

On the Structure of the Scaffolding Core of Bacteriophage T4 and Its Role in Head Length Determination

The scaffolding core in bacteriophages is a temporary structure that plays a major role in determining the shape of the protein shell that encapsulates the viral DNA. In the currently accepted structure for the scaffolding core in bacteriophage T4, there is a symmetry mismatch between the protein shell, which has fivefold symmetry, and the scaffolding core, which is believed to consist of six helical chains. Alternate structures for the scaffolding core in T4 are investigated. Starting with the hypothesis that the shell and a 10-helix core would have matching symmetry, a Vernier mechanism is proposed that explains the previously unexplained behavior of the length determination process in giant head mutants of T4. Other possible Vernier mechanisms for core structures containing six and eight helices are also explored.     

Matching the orientation of dot patterns with real, interpolated, and extrapolated lines

The perception of the orientation of random-dot patterns was studied using four different matching tasks. Homogeneous, elongated patterns and patterns containing Moiré effects were used. One of the tasks implied linear extrapolation and two others implied linear interpolation of the matching line. The fourth task was identical with those used in previous studies by the authors on this topic. Systematic deviations from the axes of orientation of the patterns were observed for the latter task when compared with the former ones. When a short matching line, implying linear extrapolation, was used performance by subjects tended to be more inaccurate than in the other matching tasks. The linear interpolation tasks, in which the matching line was determined by either two collinear distant short lines or by two distant dots, provided more accurate and stable performance than the other two tasks. The results are discussed from the point of view of global orientation perception derived from an image function of the stimuli.     

Selective and divided attention in animals

This article reviews some of the research on attentional processes in animals. In the traditional approach to selective attention, it is proposed that in addition to specific response attachments, animals also learn something about the dimension along which the stimuli fall (e.g., hue, brightness, or line orientation). More recently, there has been an attempt to find animal analogs to methodologies originally applied to research with humans. One line of research has been directed to the question of whether animals can locate a target among distracters faster if they are prepared for the presentation of the target (search image and priming). In the study of search image, the target is typically a food item and the cue consists of previous trials on which the same target is presented. In research on priming effects, the cue is typically different from the target but is a good predictor of its occurrence. The study of preattentive processes shows that perceptually, certain stimuli stand out from distracters better than others, depending not only on characteristics of the target relative to the distracters, but also on relations among the distracters. Research on divided attention is examined with the goal of determining whether an animal can process two elements of a compound sample with the same efficiency as one. Taken together, the reviewed research indicates that animals are capable of centrally (not just peripherally) attending to selective aspects of a stimulus display.     

Multiple groups of orientation-selective visual mechanisms underlying rapid orientated-line detection.

Visual search for an edge or line element differing in orientation from a background of other edge or line elements can be performed rapidly and effortlessly. In this study, based on psychophysical measurements with ten human observers, threshold values of the angle between a target and background line elements were obtained as functions of background-element orientation, in brief masked displays. A repeated-loess analysis of the threshold functions suggested the existence of several groups of orientation-selective mechanisms contributing to rapid orientated-line detection; specifically, coarse, intermediate and fine mechanisms with preferred orientations spaced at angles of approximately 90 degrees, 35 degrees, and 10 degrees-25 degrees, respectively. The preferred orientations of coarse and some intermediate mechanisms coincided with the vertical or horizontal of the frontoparallel plane, but the preferred orientations of fine mechanisms varied randomly from observer to observer, possibly reflecting individual variations in neuronal sampling characteristics.     

Postdictive modulation of visual orientation

The present study investigated how visual orientation is modulated by subsequent orientation inputs. Observers were presented a near-vertical Gabor patch as a target, followed by a left- or right-tilted second Gabor patch as a distracter in the spatial vicinity of the target. The task of the observers was to judge whether the target was right- or left-tilted (Experiment 1) or whether the target was vertical or not (Supplementary experiment). The judgment was biased toward the orientation of the distracter (the postdictive modulation of visual orientation). The judgment bias peaked when the target and distracter were temporally separated by 100 ms, indicating a specific temporal mechanism for this phenomenon. However, when the visibility of the distracter was reduced via backward masking, the judgment bias disappeared. On the other hand, the low-visibility distracter could still cause a simultaneous orientation contrast, indicating that the distracter orientation is still processed in the visual system (Experiment 2). Our results suggest that the postdictive modulation of visual orientation stems from spatiotemporal integration of visual orientation on the basis of a slow feature matching process.     

Effects of an exercise programme for chronically ill and mobility-restricted elderly with structured support by the general practitioner's practice (HOMEfit) - study protocol of a randomised controlled trial

Background

After more than half a century of modern psychopharmacology, with billions of dollars spent on antidepressants annually world-wide, we lack good evidence to guide our everyday decisions in conducting antidepressant treatment of patients with major depression. First we did not know which antidepressant to use as first line treatment. Second we do not know which dosage we should be aiming at with that antidepressant. Because more than half of the patients with major depression starting treatment do not remit after adequate trial with the first agent, they will need a second line treatment. Dose escalation, augmentation and switching are the three often recommended second line strategies but we do not know which is better than the others. Moreover, we do not know when to start considering this second line treatment. The recently published multiple-treatments meta-analysis of 12 new generation antidepressants has provided some partial answers to the first question. Starting with these findings, this proposed trial aims to establish the optimum 1st line and 2nd line antidepressant treatment strategy among adult patients with a non-psychotic unipolar major depressive episode.

Methods

SUN(^_^)D, the Strategic Use of New generation antidepressants for Depression, is an assessor-blinded, parallel-group, multi-centre randomised controlled trial. Step I is a cluster-randomised trial comparing titration up to the minimum vs maximum of the recommended dose range among patients starting with sertraline. The primary outcome is the change in the Patient Health Questionnaire (PHQ)-9 scores administered by a blinded rater via telephone at week 1 through 3. Step II is an individually randomised trial comparing staying on sertraline, augmentation of sertraline with mirtazapine, and switching to mirtazapine among patients who have not remitted on the first line treatment by week 3. The primary outcome is the change in the PHQ-9 scores at week 4 through 9. Step III represents a continuation phase to Steps I and II and aims to establish longer-term effectiveness and acceptability of the above-examined treatment strategies up to week 25. The trial is supported by the Grant-in-Aid by the Ministry of Health, Labour and Welfare, Japan.

Discussion

SUN(^_^)D promises to be a pragmatic large trial to answer important clinical questions that every clinician treating patients with major depression faces in his/her daily practices concerning its first- and second-line treatments.

Trial registration

ClinicalTrials.gov: NCT01109693