Human S-cone electroretinograms obtained by silent substitution stimulation.

We used triple silent substitution stimuli to characterize human S-cone electroretinograms (ERGs) in normal trichromats. Short-wavelength-cone (S-cone) ERGs were found to have different morphological features and temporal frequency response characteristics compared to ERGs derived from L-cones, M-cones, and rod photoreceptors in normal participants. Furthermore, in two cases of retinal pathology, blue cone monochromatism (BCM) and enhanced S-cone syndrome (ESCS), S-cone ERGs elicited by our stimuli were preserved and enhanced, respectively. The results from both normal and pathological retinae demonstrate that triple silent substitution stimuli can be used to generate ERGs that provide an assay of human S-cone function.

The morphology of human rod ERGs obtained by silent substitution stimulation.

PURPOSE: To record transient ERGs from the light-adapted human retina using silent substitution stimuli which selectively reflect the activity of rod photoreceptors. We aim to describe the morphology of these waveforms and examine how they are affected by the use of less selective stimuli and by retinal pathology. METHODS: Rod-isolating stimuli with square-wave temporal profiles (250/250 ms onset/offset) were presented using a 4 primary LED ganzfeld stimulator. Experiment 1: ERGs were recorded using a rod-isolating stimulus (63 ph Td, rod contrast, C rod = 0.25) from a group (n = 20) of normal trichromatic observers. Experiment 2: Rod ERGs were recorded from a group (n = 5) using a rod-isolating stimulus (C rod = 0.25) which varied in retinal illuminance from 40 to 10,000 ph Td. Experiment 3: ERGs were elicited using 2 kinds of non-isolating stimuli; (1) broadband and (2) rod-isolating stimuli which contained varying degrees of L- and M-cone excitation. Experiment 4: Rod ERGs were recorded from two patient groups with rod monochromacy (n = 3) and CSNB (type 1; n = 2). RESULTS: The rod-isolated ERGs elicited from normal subjects had a waveform with a positive onset component followed by a negative offset. Response amplitude was maximal at retinal illuminances <100 ph Td and was virtually abolished at 400 ph Td. The use of non-selective stimuli altered the ERG waveform eliciting more photopic-like ERG responses. Rod ERGs recorded from rod monochromats had similar features to those recorded from normal trichromats, in contrast to those recorded from participants with CSNB which had an electronegative appearance. CONCLUSIONS: Our results demonstrate that ERGs elicited by silent substitution stimuli can selectively reflect the operation of rod photoreceptors in the normal, light-adapted human retina.

L- and M-cone input to 12Hz and 30Hz flicker ERGs across the human retina.

We recorded L- and M-cone isolating ERGs from human subjects using a silent substitution technique at temporal rates of 12 and 30 Hz. These frequencies isolate the activity of cone-opponent and non-opponent post-receptoral mechanisms, respectively. ERGs were obtained using a sequence of stimuli with different spatial configurations comprising; (1) circular stimuli of different sizes which increased in 10° steps up to 70°diameter, or (2) annular stimuli with a 70° outer diameter but with different sized central ablations from 10° up to 60°. L- and M-cone isolating ERGs were obtained from five colour normal subjects using a DTL fibre electrode. Fourier analysis of the ERGs was performed and we measured the amplitude of the first harmonic of the response. For 12 Hz ERGs the L:M cone response amplitude ratio (L:M(ERG)) was close to unity and remained stable irrespective of the spatial configuration of the stimulus. The maintenance of this balanced ratio points to the existence of cone selective input across the human retina for the L-M cone opponent mechanism. For 30 Hz the L:M(ERG) ratio was greater than unity but varied depending upon which region of the retina was being stimulated. This variation we consider to be a consequence of the global response properties of M-cone ERGs rather than representing a real variation in L:M cone ratios across the retina.

Naming versus matching and the stability of unique hues.

It is known that there is a distortion of hue and saturation in the peripheral visual field. In a previous study, when an asymmetric matching paradigm was used, four hues in the blue, red, yellow and green regions of colour space were unchanged and these were referred to as peripherally invariant (Parry et al., J Opt Soc Am A, 23, 2006, 1586). Three of these invariant hues were similar to unique blue, red and yellow. However, for most observers there was a marked difference between unique and invariant green. To investigate this apparent paradox, we have measured unique hues using a range of eccentricities and colourimetric purities. An asymmetric matching and a 4-AFC paradigm were used to establish peripherally invariant and unique hues, respectively. In the asymmetric matching task the observer matched a peripheral spot with a para-foveal spot, for 24 different hues at 18° eccentricity. In the 4-AFC paradigm, 41 hues were presented 20 times at three purities (0.5, 0.75 and 1.0) and three eccentricities (18°, 10° and 1°). The observer had to name the hues as red, blue, green or yellow. Unique hues were found to be constant with eccentricity and purity. The unique green, established with 4-AFC, was found to differ from the invariant green, determined using the matching task. However, red, blue and yellow invariant hues correspond well with unique hues. The data suggest that different mechanisms mediate the matching of green compared with the identification of unique hues. This is similar to the difference between detection and discrimination of spectral stimuli: the detection process is dominated by the cone opponent mechanisms and is most sensitive, whereas more central processes, serving unique hues, influence discrimination.

A behavioural investigation of human visual short term memory for colour.

We examined visual short term memory (VSTM) for colour using a delayed-match-to-sample paradigm. In these experiments we measured the effects of increasing inter-stimulus interval (ISI), varying between 0 and 10 s, on the ability of five colour normal human observers to make colour matches between a reference and subsequently presented test stimuli. The coloured stimuli used were defined by different chromatic axes on the isoluminant plane of DKL colour space. In preliminary experiments we used a hue scaling procedure to identify a total of 12 colour stimuli which served as reference hues in the colour memory experiments: four stimuli were exemplars of red, green, blue and yellow colour appearance categories, four were located between these categories and a further four were located on the cardinal axes that isolated the activity of the cone-opponent mechanisms. Our results demonstrate that there is a reduction in the ability of observers to make accurate colour matches with increasing ISIs and that this reduced performance was similar for all colour stimuli. However, the shifts in hue that were measured between the reference and matched test stimuli were significantly greater for the cardinal stimuli compared to those measured for the stimuli defined by the hue scaling procedure. This deterioration in the retention of hue in VSTM for stimuli that isolate cone-opponent mechanisms may be a reflection of the reorganisation of colour processing that occurs in the cortex where colour appearance mechanisms become more prominent.

Nasal-temporal differences in cone-opponency in the near peripheral retina.

The purpose of this study is to establish whether nasal-temporal differences in cone photoreceptor distributions are linked to differences in colour matching performance in the two hemi-fields. Perceived shifts in chromaticity were measured using an asymmetric matching paradigm. They were expressed in terms of hue rotations and relative saturation changes and also in terms of activation levels of L-M or S-(L+M) cone-opponent channels. Up to 19 degrees eccentricity there was little difference in chromaticity shifts between nasal and temporal retina for either channel. For matches beyond 19 degrees L-M activation is significantly lower in the nasal field and the S-(L+M) channel was equally activated in both fields. The data are consistent with the asymmetric distribution of L- and M-cones in the nasal and temporal retinae.

Summation characteristics of the detection of compound gratings.

Many classical experiments have shown that two superimposed gratings are more easily detected than a single grating, in keeping with probability theory. Here we test the rules for the detection of 2-component compound gratings by extending the range of parameters used in previous experiments. Two complementary methods of deriving summation indices are described. Data are presented so that the conditions for the transition from probability to neural summation are easily identified. True probability summation occurs only when grating contrasts are carefully perceptually equalised and spatial frequency differs by more than a factor of 2. A wide range of contrast ratios of the component gratings were explored such that gratings were at different contrasts, relative to respective thresholds. We find clear evidence of suppressive interactions when the compound gratings are composed of a close to threshold low frequency component and a below-threshold higher spatial frequency component.

L- and M-cone isolating ERGs: LED versus CRT stimulation.

Using double silent substitution, it is possible to generate L-cone and M-cone isolating electroretinograms (ERGs) on a CRT. A major limitation of the technique is that the depth of modulation of cone classes is limited by the restricted luminance of the phosphors and their spectral overlap. To address this problem we have ported the technique to a four-color LED Ganzfeld stimulus (Diagnosis ColorDome). This allows higher retinal illuminances, higher contrasts, and triple silent substitution. With careful control over the retinal area stimulated, we show that the same data can be recorded from both CRT and LED stimuli when luminance, size and cone contrast are kept constant. Importantly, the different temporal profiles of the two devices do not influence the ERG amplitude and phase plots. We present data over a much wider range of luminances (up to about 10,000 trolands) and contrasts with the LED stimulator than previously reported with CRT screens. We conclude that the close resemblance between data obtained with an LED stimulator and with a CRT screen indicate that the differences have a purely physiological origin.

Reaction time measures of adaptation to chromatic contrast.

Simple reaction times (RTs) were measured to brief temporally blurred (total onset 570 ms) Gaussian isoluminant chromatic patches (s.d. 0.5 degrees) whose chromaticities lay along the cardinal chromatic axes (0 degrees, 90 degrees, 180 degrees, and 270 degrees in MBDKL color space). Bipolar adapting stimuli were employed (0 degrees versus 180 degrees or 90 degrees versus 270 degrees). These were larger Gaussian blobs (s.d. 1 degree), modulating sinusoidally between the two hues at 1 Hz. Throughout, the background was illuminant "C" (x = 0.31, y = 0.316, L = 12.5). In a single run, a series of 64 or 32 stimuli were presented without adaptation, followed by 64 or 32 stimuli each of which was preceded by 3 s of adaptation, either along the same or the orthogonal chromatic axis. Finally, 192 or 128 RTs were recorded to measure the time course of recovery from adaptation. Both adapting and test stimuli were presented at fixed supra-threshold contrasts. The effect of adaptation was seen as a lengthening of the RT, which occurred in the first few seconds of the adaptation period. After cessation of adaptation, there was a similarly rapid shortening of RT, although full recovery took 60-90 s. Adaptation gain functions suggested that the S-(L + M) system was less prone to adaptation than L-M.

Cone opponency in the near peripheral retina.

Changes of color perception in the peripheral field are measured using an asymmetric simultaneous matching paradigm. The data confirm previous observations in that saturation changes can be neutralized if the test target is increased in size. However, this compensation does not apply to hue shifts. We show that some hues remain unchanged with eccentricity whereas others exhibit substantial changes. Here the color shifts are plotted in terms of a second-stage cone opponent model. The data suggest that the S-L+M channel is more robust to increasing eccentricity than the L-M channel. Observations are interpreted in terms of the known underlying morphological and physiological differences in these channels.

Photoreceptor topography and cone-specific electroretinograms.

It is implicit in many cone-specific ERG studies that the amplitude is proportional to the numbers of cones stimulated. The objective of these experiments was to test this idea by comparing ERGs obtained from different areas of the retina with histological data on cone-density distributions. The histology (Curcio et al., 1990) shows that the cumulative number of cones in the human retina increases exponentially with stimulus diameter between 0- and 40-deg eccentricity. L-, M-, and (L+M) cone-driven 30-Hz ERGs were obtained from a series of stimuli with one of the following configurations: (1) Circular stimuli of different angular subtense up to 70-deg diameter. (2) Annuli with 70-deg outer diameter but variable inner diameter. (3) Annuli of constant area but increasing eccentricity. Cone contrasts were equalized for each stimulus condition. The modulated and nonmodulated regions of the screen had the same mean hue and luminance. The data suggest that the L+M cone ERG amplitude increases with stimulus diameter in direct proportion to the estimated number of cones stimulated. Furthermore, the total L+M responses appear to be predicted from individual L and M responses by simple linear summation for both the disc and annular stimuli.

Effect of foveal tritanopia on reaction times to chromatic stimuli.

To investigate the effect of foveal inhomogeneities on sensitivity to chromatic stimuli, we measured simple reaction times (RTs) and detection thresholds to temporally and spatially blurred isoluminant stimuli at retinal eccentricities from 0 deg to 8 deg. Three color-normal subjects participated. Contrast gain was derived from the slope of the RT versus contrast function. With a Gaussian spatial distribution (S.D. = 0.5 deg) and modulation between white (CIE x, y, L = 0.31, 0.316, 12.5 cd x m(-2)) and blue (MBDKL 90 deg), gain was maximal at about 2-deg eccentricity and declined by approximately 1 log unit towards the center and the periphery. The red (0 deg) and green (180 deg) cardinal axes showed maximum gain in the center, whilst the yellow (270 deg) data were intermediate. Although the spatial extent of the Gaussian spot was much larger than the S-cone free zone, we wished to determine whether foveal tritanopia was responsible for the marked drop in sensitivity to the 90-deg stimulus. To align the color vector along a tritan line, we used a smaller disk (0.3 deg) with a blurred edge and measured detection threshold, rotating the vector until minimum central sensitivity was obtained. Other workers have used transient tritanopia or minimally distinct border to similar effect. By repeating this at different locations in color space, a group of vectors were obtained. These converged near to the S-cone co-punctal point, evidence that they lay along tritan confusion lines. These threshold findings were then confirmed using the RT-derived contrast gain function. The tritan vectors were less pronounced as stimulus size increased. With the vector optimized to produce foveal tritanopia, the RT gain versus eccentricity functions for the 90-deg and 270-deg stimuli both fell markedly in the center and periphery, and sensitivity peaked at about 3-deg eccentricity. There are some similarities between these findings and the underlying photoreceptor distributions. As a result, there is a greater difference in gain between red-green and blue-yellow systems in the center than in the near periphery. We conclude that the RT versus contrast function is a sensitive index of foveal opponency.

Regression of canine oral papillomas is associated with infiltration of CD4+ and CD8+ lymphocytes.

Canine oral papillomavirus (COPV) infection is used in vaccine development against mucosal papillomaviruses. The predictable, spontaneous regression of the papillomas makes this an attractive system for analysis of cellular immunity. Immunohistochemical analysis of the timing and phenotype of immune cell infiltration revealed a marked influx of leukocytes during wart regression, including abundant CD4+ and CD8+ cells, with CD4+ cells being most numerous. Comparison of these findings, and those of immunohistochemistry using TCRalphabeta-, TCRgammadelta-, CD1a-, CD1c-, CD11a-, CD11b-, CD11c-, CD18-, CD21-, and CD49d-specific monoclonal antibodies, with previously published work in the human, ox, and rabbit models revealed important differences between these systems. Unlike bovine papillomavirus lesions, those of COPV do not have a significant gamma/delta T-cell infiltrate. Furthermore, COPV lesions had numerous CD4+ cells, unlike cottontail rabbit papillomavirus lesions. The lymphocyte infiltrate in the dog resembled that in human papillomavirus lesions, indicating that COPV is an appropriate model for human papillomavirus immunity.

Synthesis and antiviral activity of acyclovir-5'-(phenyl methoxy alaninyl) phosphate as a possible membrane-soluble nucleotide prodrug.

We describe a synthesis of acyclovir-5'-(phenyl methoxy alaninyl) phosphate (2) from acyclovir (1). This compound was designed to act as a lipophilic, membrane-soluble prodrug of the free nucleotide. However, the biological activities of this derivative against a range of viruses indicated poor intracellular phosphate delivery, in marked contrast to the earlier successful delivery of several dideoxy anti-HIV nucleotides.

Naturally occurring, nonregressing canine oral papillomavirus infection: host immunity, virus characterization, and experimental infection.

Papillomaviruses occasionally cause severe, nonregressing or recurrent infections in their human and animal hosts. The mechanisms underlying these atypical infections are not known. Canine oral papillomavirus (COPV) typically regresses spontaneously and is an important model of mucosal human papillomavirus infections. A severe, naturally occurring, nonregressing COPV infection provided an opportunity to investigate some aspects of viral pathogenicity and host immunity. In this case, the papillomas proved refractory to surgical and medical treatments, including autogenous vaccination and vaccination with capsid (L1) virus-like particles. High levels of induced anti-L1 antibodies appeared to have no effect on the infection. The papillomas spread to oesophageal mucosa, perioral haired skin, and remote cutaneous sites. Isolation of COPV from the animal and sequencing of several regions of the viral genome showed no differences to the COPV prototype. Experimental infection of beagle dogs with this viral isolate resulted in the uncomplicated development and regression of oral warts within the usual period, indicating that the virus was not an unusual pathogenic variant. These findings support the hypothesis that the recurrent lesions seen in some human papillomavirus infections, such as recurrent laryngeal papillomatosis, are associated with specific defects in host immunity rather than variations in viral pathogenicity.

Spatio-temporal tuning of VEPs: effect of mode of stimulation.

The spatial tuning function of the 8 Hz reversal Visual Evoked Potential (VEP) is frequently bimodal. This amplitude 'notch' largely disappears with 16 Hz pattern onset-offset (on-off) stimulation. To investigate the effect of temporal frequency on this phenomenon, we tested seven subjects using a range of rates centred on 8 Hz (reversal) and 16 Hz (on-off). With reversal, a deep notch was present at all temporal frequencies. A much shallower notch was evident with on-off. The on-off and reversal functions differed only in their low spatial frequency branches (below 4.5 c deg-1) where, as temporal frequency decreased, the reversal response was reduced by 50%, whilst the on-off responses doubled in amplitude. Thus, although they behave differently with on-off and reversal, multiple spatial frequency mechanisms are evident with both forms of stimulation.

1592U89, a novel carbocyclic nucleoside analog with potent, selective anti-human immunodeficiency virus activity.

1592U89, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclo pentene-1-methanol, is a carbocyclic nucleoside with a unique biological profile giving potent, selective anti-human immunodeficiency virus (HIV) activity. 1592U89 was selected after evaluation of a wide variety of analogs containing a cyclopentene substitution for the 2'-deoxyriboside of natural deoxynucleosides, optimizing in vitro anti-HIV potency, oral bioavailability, and central nervous system (CNS) penetration. 1592U89 was equivalent in potency to 3'-azido-3'-deoxythymidine (AZT) in human peripheral blood lymphocyte (PBL) cultures against clinical isolates of HIV type 1 (HIV-1) from antiretroviral drug-naive patients (average 50% inhibitory concentration [IC50], 0.26 microM for 1592U89 and 0.23 microM for AZT). 1592U89 showed minimal cross-resistance (approximately twofold) with AZT and other approved HIV reverse transcriptase (RT) inhibitors. 1592U89 was synergistic in combination with AZT, the nonnucleoside RT inhibitor nevirapine, and the protease inhibitor 141W94 in MT4 cells against HIV-1 (IIIB). 1592U89 was anabolized intracellularly to its 5'-monophosphate in CD4+ CEM cells and in PBLs, but the di- and triphosphates of 1592U89 were not detected. The only triphosphate found in cells incubated with 1592U89 was that of the guanine analog (-)-carbovir (CBV). However, the in vivo pharmacokinetic, distribution, and toxicological profiles of 1592U89 were distinct from and improved over those of CBV, probably because CBV itself was not appreciably formed from 1592U89 in cells or animals (<2%). The 5'-triphosphate of CBV was a potent, selective inhibitor of HIV-1 RT, with Ki values for DNA polymerases (alpha, beta, gamma, and epsilon which were 90-, 2,900-, 1,200-, and 1,900-fold greater, respectively, than for RT (Ki, 21 nM). 1592U89 was relatively nontoxic to human bone marrow progenitors erythroid burst-forming unit and granulocyte-macrophage CFU (IC50s, 110 microM) and human leukemic and liver tumor cell lines. 1592U89 had excellent oral bioavailability (105% in the rat) and penetrated the CNS (rat brain and monkey cerebrospinal fluid) as well as AZT. Having demonstrated an excellent preclinical profile, 1592U89 has progressed to clinical evaluation in HIV-infected patients.

Cross-resistance analysis of human immunodeficiency virus type 1 variants individually selected for resistance to five different protease inhibitors.

Human immunodeficiency virus type 1 (HIV-1) protease inhibitor-resistant variants, isolated on passage of HIV-1HXB2 in MT-4 cells with five different protease inhibitors, have been examined for cross-resistance to five inhibitors. The protease inhibitors studied were Ro 31-8959, A-77003, XM323, L-735,524, and VX-478. Resistant variants with two to four mutations within their protease sequence and 9- to 40-fold-decreased susceptibility were selected for all five inhibitors within six to eight passes in cell culture. Passage of a zidovudine-resistant mutant in Ro 31-8959 generated a dual reverse transcriptase- and protease-resistant virus. Variants were cloned directly into a modified pHXB2-D infectious clone for cross-resistance analysis. Although the resistant variants selected possessed different combinations of protease mutations for each inhibitor, many showed cross-resistance to the other inhibitors, and one showed cross-resistance to all five inhibitors. Interestingly, some mutants showed increased susceptibility to some inhibitors. Further HIV passage studies in the combined presence of two protease inhibitors demonstrated that in vitro it was possible to delay significantly selection of mutations producing resistance to one or both inhibitors. These studies indicate that there may be some rationale for combining different protease inhibitors as well as protease and reverse transcriptase inhibitors in HIV combination therapy.

Monoclonal antibodies to a peptide of human rhinovirus type 2 with different specificities recognize the same minimum sequence.

Monoclonal antibodies (MAbs) raised against a synthetic peptide including residues 156-170 of protein VP2 of human rhinovirus type 2 (HRV2) have previously been shown to be of differing specificities. The basis for these differences has now been examined in greater detail by ELISA, radioimmunoprecipitation and virus neutralization. Reactions with a panel of HRV2 mutant viruses indicated that substitution of some residues could enhance the apparent activity of one of the neutralizing anti-peptide MAbs. For one such substitution, VP2 P164H, there appeared to be a correlation between increased neutralizing activity and enhanced binding. Mapping experiments identified two overlapping neutralization epitopes (amino acids 156-163 and 160-165) and several non-neutralizing epitopes. Although some differences in antibody reactivity were due to epitope specificity alone, the explanation for others was less obvious. Significantly, the majority of MAbs that recognized, and in some cases neutralized, native virus had the same minimum binding sequence and critical residue requirement as others which recognized virus particles only after distortion. This demonstrates that factors other than the linear sequence of the peptide can be crucial in determining the fine specificity, and hence biological relevance, of peptide antigens.