Dominantly Inherited Alzheimer Network Trials Unit (DIAN-TU): Trial Satisfaction and Attitudes towards Future Clinical Trials.

BACKGROUND: Clinical trial satisfaction is increasingly important for future trial designs and is associated with treatment adherence and willingness to enroll in future research studies or to recommend trial participation. In this post-trial survey, we examined participant satisfaction and attitudes toward future clinical trials in the Dominantly Inherited Alzheimer Network Trials Unit (DIAN-TU). METHODS: We developed an anonymous, participant satisfaction survey tailored to participants enrolled in the DIAN-TU-001 double-blind clinical trial of solanezumab or gantenerumab and requested that all study sites share the survey with their trial participants. A total of 194 participants enrolled in the trial at 24 study sites. We utilized regression analysis to explore the link between participants' clinical trial experiences, their satisfaction, and their willingness to participate in upcoming trials. RESULTS: Survey responses were received over a sixteen-month window during 2020-2021 from 58 participants representing 15 study sites. Notably, 96.5% of the survey respondents expressed high levels of satisfaction with the trial, 91.4% would recommend trial participation, and 96.5% were willing to enroll again. Age, gender, and education did not influence satisfaction levels. Participants reported enhanced medical care (70.7%) and pride in contributing to the DIAN-TU trial (84.5%). Satisfaction with personnel and procedures was high (98.3%). Respondents had a mean age of 48.7 years, with most being from North America and Western Europe, matching the trial's demographic distribution. Participants' decisions to learn their genetic status increased during the trial, and most participants endorsed considering future trial participation regardless of the DIAN-TU-001 trial outcome. CONCLUSION: Results suggest that DIAN-TU-001 participants who responded to the survey exhibited high motivation to participate in research, overall satisfaction with the clinical trial, and willingness to participate in research in the future, despite a long trial duration of 4-7 years with detailed annual clinical, cognitive, PET, MRI, and lumbar puncture assessments. Implementation of features that alleviate barriers and challenges to trial participation is like to have a high impact on trial satisfaction and reduce participant burden.

Coupling from AII amacrine cells to ON cone bipolar cells is bidirectional.

The AII amacrine cell is a critical interneuron in the rod pathway of the mammalian retina. Rod signals pass into cone pathways by means of gap junctions between AII amacrine cells and ON cone bipolar cells. Filling AII amacrine cells with Neurobiotin produces labeling of cone bipolar cells by means of these gap junctions. However, tracer injections into bipolar cells do not produce labeling of the AII network (Vaney [1997] Invest Ophthalmol Vis Sci. 38:267-273), which suggests that the AII/bipolar gap junctions allow the passage of tracer in only one direction. This mechanism stands in contrast to physiological results, which indicate that light adapted signals can pass from ON cone bipolar cells into the AII network (Xin and Bloomfield [1999] Vis Neurosci. 16:653-665). Here, we report that a variety of ON and OFF bipolar cells are sometimes anomalously coupled to the A-type horizontal cell network. These relatively rare examples do not result from dye injection errors, but seem to represent minor developmental errors. However, this provides a method to obtain Neurobiotin-filled cone bipolar cells without the necessity of impaling them with a microelectrode. Under these conditions, Neurobiotin spreads from ON cone bipolar cells into neighboring AII amacrine cells. The dye-coupled AII amacrine cells, positively identified by double labeling with an antibody against calretinin, were centered around anomalously coupled ON bipolar cells. These results indicate that AII/bipolar cell gap junctions allow tracer coupling in both directions, consistent with previous physiological results. The previous failure to detect the passage of neuronal tracer from injected bipolar cells to AII amacrine cells may reflect electrode damage or perhaps the asymmetrical voltage sensitivity of a heterotypic gap junction.

Rod pathways in the mammalian retina use connexin 36.

Many neurons in the mammalian retina are coupled by means of gap junctions. Here, we show that, in rabbit retina, an antibody to connexin 36 heavily labels processes of AII amacrine cells, a critical interneuron in the rod pathway. Image analysis indicates that Cx36 is primarily located at dendritic crossings between overlapping AII amacrine cells. This finding suggests that Cx36 participates in homotypic gap junctions between pairs of AII amacrine cells. Cx36 was also found at AII/cone bipolar contacts, previously shown to be gap junction sites. This finding suggests that Cx36 participates at gap junctions that may be heterotypic. These results place an identified neuronal connexin in the context of a well-defined retinal circuit. The absence of Cx36 in many other neurons known to be coupled suggests the presence of additional unidentified connexins in mammalian neurons. Conversely, Cx36 labeling in other regions of the retina is not associated with AII amacrine cells, indicating some other cell types use Cx36.

A series of biotinylated tracers distinguishes three types of gap junction in retina.

Gap junctions serve many important roles in various tissues, but their abundance and diversity in neurons is only beginning to be understood. The tracer Neurobiotin has revealed many different networks interconnected by gap junctions in retina. We compared the relative permeabilities of five different retinal gap junctions by measuring their permeabilities to a series of structurally related tracers. When large tracers were injected, the staining of coupled cells fell off more rapidly in some networks than others relative to Neurobiotin controls. Three distinctly different permeability profiles were found, suggesting that multiple neuronal connexin types were present. The most permeant to large molecules were gap junctions from A-type horizontal cells. The permeability of gap junctions of two types of amacrine cell were not distinguishable from those from B-type horizontal cells. The lowest permeability was found for gap junctions between cone bipolar cells and the AII amacrine cells to which they are coupled. Because only a single neural connexin type has been identified in retina, our results suggest more types remain to be found. To determine whether the unitary permeability of channels is altered by channel modulators, we reduced permeability with octanol and a cAMP analog. Although net permeability was substantially diminished, the proportion by which it declined was constant across tracer size. This suggests that these agents act only to close channels rather than alter individual channel permeabilities. This tracer series can therefore be used to contrast permeability properties of gap junctions in intact circuits, even at the level of individual channels.

Hypnotic activity of melatonin.

OBJECTIVE: To establish the effect of melatonin upon nocturnal and evening sleep. METHODS: Experiment I: The effect of melatonin (0.1, 0.5, 1.0, 5.0, and 10 mg), ingested at 23:30, was studied on nocturnal sleep (23:30-07:30) and core body temperature in 8 healthy volunteers. Performance was measured 8.5 h post-ingestion. On completion of the experiment dim light melatonin onsets (DLMO) were determined. Experiment II: The effect of melatonin (0.5, 1.0, 5.0, and 10 mg), ingested at 18:00, was studied on evening sleep (18:00-24:00) and core body temperature in 6 healthy volunteers. Performance was measured 6.5 h post-ingestion. Each experiment was placebo-controlled and double-blind with a cross-over design with temazepam (20 mg) as an active control. RESULTS: Experiment I: Melatonin (5 mg) reduced the duration of stage 3 in the first 100 min of sleep. Melatonin (0.1 mg) reduced body temperature 6.5 to 7 h post-ingestion. Temazepam increased stage 2, reduced wakefulness and stage 1, and increased the latency to REM sleep. Temazepam reduced body temperature 4.5 to 6.5 h post-ingestion. There were no changes in performance compared with placebo. DLMO occurred between 20:40 and 23:15. Experiment II: Melatonin (all doses) increased total sleep time (TST), sleep efficiency index (SEI) and stage 2, and reduced wakefulness. Temazepam increased TST, SEI, stage 2 and slow-wave sleep, and reduced wakefulness. There were no changes in body temperature or performance compared with placebo. CONCLUSION: Melatonin given at 23:30 has no significant clinical effect on nocturnal sleep in healthy individuals. Hypnotic activity of melatonin when given in the early evening (presumably in the absence of endogenous melatonin) is similar to 20 mg temazepam.

Dye coupling in horizontal cells of developing rabbit retina.

In the mature rabbit retina, two classes of horizontal cells, A type and B type, provide lateral inhibition in the outer plexiform layer (OPL) and spatially modify the activation of bipolar cells by photoreceptors. Gap junctions connecting homologous horizontal cells determine the extent to which this inhibitory activity spreads laterally across the OPL. Little is currently known about the expression of gap junctions in horizontal cells during postnatal development or how cell-cell coupling might contribute to subsequent maturational events. We have examined the morphological attributes and coupling properties of developing A and B type horizontal cells in neonatal rabbit retina using intracellular injections of Lucifer Yellow and Neurobiotin. Prelabeling with DAPI permitted the targeting of horizontal cell bodies for intracellular injection in perfused preparations of isolated retina. A and B type horizontal cells were identifiable at birth although their dendritic field sizes had not reached adult proportions and their synaptic contacts in the OPL were minimal. Both cell types exhibited homologous dye coupling at birth. Similar to that seen in the adult, no heterologous coupling was observed, and homologous coupling among A type cells was stronger than that observed among B type cells. The spread of tracer compounds through gap junctions of morphologically immature horizontal cells suggests that ions and other small, bioactive compounds may likewise spread through coupled, horizontal networks to coordinate the subsequent maturational of emerging outer plexiform layer pathways.

Antihistamines and aircrew: usefulness of fexofenadine.

INTRODUCTION: The aim of this study was to establish whether fexofenadine hydrochloride, an antihistamine, modulates daytime sleepiness or performance. METHODS: The effects of fexofenadine (120, 180, and 240 mg) on digit symbol substitution, tracking, and vigilance tasks, and on objective (multiple sleep latency test) and subjective sleepiness, were studied in six healthy volunteers (two males, four females, aged 20-34 [mean 26.5] yr) from 1 h pre-ingestion to 8 h post-ingestion. The study was placebo-controlled and double-blind with a six-way cross-over design. The centrally acting antihistamine, promethazine (10 mg), was used as an active control to confirm the sensitivity of the experimental procedures. RESULTS: There were no changes in performance or sleepiness with any dose of fexofenadine at any time, compared with placebo. Promethazine, compared with both placebo and fexofenadine, impaired performance on the digit symbol substitution task (2.5 h post-ingestion), vigilance task (2.5-5h post-ingestion) and tracking task (2.5-3.5 h post-ingestion), increased objective sleepiness (1.5-2.5 h post-ingestion) and subjective sleepiness (1.5-8h post-ingestion). CONCLUSION: Consideration may be given to the clinical use of currently licensed doses of fexofenadine (120-180 mg) by individuals involved in skilled activity. Fexofenadine may be potentially useful for aircrew.

Unusual coupling patterns of a cone bipolar cell in the rabbit retina.

In mammals, gap junctions between retinal bipolar cells are generally small and tracer coupling has not been previously demonstrated. In this study, Neurobiotin was injected into the Ba3-type cone bipolar cell, a medium-field cone bipolar cell that ramifies in sublamina a of the rabbit retina. Tracer spread to many other Ba3 bipolar cells, presumably through gap junctions. It also spread to a smaller field bipolar cell called the Ba1 that ramifies at the same depth of the inner plexiform layer. Injection of Neurobiotin into Ba1 bipolar cells did not produce staining beyond the injected cell. Tracer coupling from the Ba3 was therefore both heterologous, in that different cell types were stained, and asymmetric. The unusual properties of this bipolar cell suggest that its function may differ from that of most cone bipolar cells, which are narrow-field, do not overlap, and are poorly coupled to one another.

Gap junctions between AII amacrine cells and calbindin-positive bipolar cells in the rabbit retina.

Electrical synapses or gap junctions occur between many retinal neurons. However, in most cases, the gap junctions have not been visualized directly. Instead, their presence has been inferred from tracer spread throughout the network of cells. Thus, tracer coupling is taken as a marker for the presence of gap junctions between coupled cells. AII amacrine cells are critical interneurons in the rod pathway of the mammalian retina. Rod bipolar cell output passes to AII amacrine cells, which in turn make conventional synapses with OFF cone bipolar cells and gap junctions with ON cone bipolar cells. Injections of biotinylated tracers into AII amacrine cells reveals coupling between the AII amacrine cell network and heterologous coupling with a variety of ON cone bipolar cells, including the calbindin-positive cone bipolar cell. To directly visualize gap junctions in this network, we prepared material for electron microscopy that was double labeled with antibodies to calretinin and calbindin to label AII amacrine cells and calbindin-positive cone bipolar cells, respectively. AII amacrine cells were postsynaptic to large vesicle-laden rod bipolar terminals, as previously reported. Gap junctions were identified between AII amacrine cells and calbindin-positive cone bipolar cell terminals identified by the presence of immunostaining and ribbon synapses. This represents direct confirmation of gap junctions between two different yet positively identified cells, which are tracer coupled, and provides additional evidence that tracer coupling with Neurobiotin indicates the presence of gap junctions. These results also definitively establish the presence of gap junctions between AII amacrine cells and calbindin bipolar cells which can therefore carry rod signals to the ON alpha ganglion cell.

Antibody to calretinin stains AII amacrine cells in the rabbit retina: double-label and confocal analyses.

The AII or rod amacrine cell is a critical interneuron in the rod pathway of mammalian retinae. In this report, it is shown that commercially available antibodies to the calcium binding protein calretinin may be used to label the population of AII amacrine cells selectively. Calretinin-positive amacrine cells had the morphological attributes of AII amacrine cells. Double-labeling procedures showed that calretinin-positive somata were surrounded by dopaminergic varicosities and that calretinin-positive dendrites enclosed rod bipolar terminals, both as previously described for AII amacrine cells. By analyzing the surrounding kernel for each labeled pixel in the rod bipolar image, it is shown here that AII processes are adjacent to rod bipolar terminals at a level that far exceeds the random overlap present in images in which one label was rotated out of phase. Such a spatial relationship is indicative of synaptic connections, as well described for rod bipolar input to AII amacrine cells. AII amacrine cells also were double-labeled for calretinin and parvalbumin; however, a scattergram analysis of red versus green intensity showed that the parvalbumin antibody stained additional unidentified amacrine cells. In conclusion, at the appropriate dilution, calretinin antibodies are a useful marker for AII amacrine cells in the rabbit retina.

AII amacrine cells limit scotopic acuity in central macaque retina: A confocal analysis of calretinin labeling.

We have used calretinin antibodies to label selectively the mosaic of AII amacrine cells in the macaque retina. Confocal analysis of double-labeled material indicated that AII dendrites spiral down around descending rod bipolar axons before enveloping the synaptic terminals. Processes from a previously observed dopaminergic plexus in the inner nuclear layer were observed to contact the somata of calretinin-positive AII somata. Intracellular neurobiotin injection revealed that AII amacrine cells are tracer coupled to other AII amacrine cells and to some unidentified cone bipolar cells. An analysis of the retinal distribution of macaque AII amacrine cells, including an area in and around the fovea, showed a peak density of approximately 5,000 cells/mm(2) at an eccentricity of 1.5 mm. Staining of AII amacrine cells in central retina with antibodies to calretinin was confirmed by confocal microscopy. These results indicate that calretinin antibodies can be used to label the AII amacrine cell population selectively and that primate AII amacrine cells share many of the features of previously described mammalian AII amacrine cells. The peak AII cell density closely matched the peak sampling rate of scotopic visual acuity. Calculations suggest that, in central macaque retina, where midget ganglion cells are more numerous, AII amacrine cells form the limit of scotopic visual acuity (Wässle et al. [1995] J. Comp. Neurol. 361:537-551). As the ganglion cell density falls rapidly away from the fovea, there is a cross-over point at around 15 degrees eccentricity that matches the inflection point in a psychophysically derived plot of scotopic visual acuity versus eccentricity (Lennie and Fairchild [1994] Vision Res. 34:477-482). The correspondence between the anatomic and psychophysical data supports our interpretation that the anatomic sampling rate of AII amacrine cells limits central scotopic acuity.

Enhanced visualization of parathyroid tissue by infusion of a visible dye conjugated to an antiparathyroid antibody.

BACKGROUND: The antiparathyroid antibody BB5-G1 conjugated to cibacron blue, a blue dye, was intravenously infused to enhance parathyroid visualization. Previously, we demonstrated selective staining of human parathyroid implants in athymic nude mice after infusion of the conjugate. METHODS: Mice possessing implanted parathyroid tissue were randomized into the following: group I, infused with cibacron blue alone; group II, infused with the antibody/dye conjugate; and group III, infused with radiolabeled BB5-G1 alone. Implants were surgically explored. Three blinded observers ranked parathyroid visualization in the operative fields, and corresponding histologic sections were computer analyzed. RESULTS: Group II implants were easily visualized; groups I and III showed no staining. Group III showed high gamma counts. Subjective rankings and computer rankings correlated well (p < .05). Group II showed a higher mean staining intensity of 27.45 picogram protein product (PPP)/cell compared with 7.76 PPP/cell of group I (p = .002). CONCLUSION: Cibacron blue/BB5-G1 consistently enhances visualization of implanted parathyroid tissue.

Stability of monoclonal antibodies, Lym-1 and ChL6, and 2IT-BAD-Lym-1 immunoconjugate with ultra freezer storage.

UNLABELLED: Use of a single lot of monoclonal antibody (MoAb) or immunoconjugate for clinical studies provides efficiency of scale and consistent characteristics for MoAb-based pharmaceuticals. Lym-1, an anti-lymphoma mouse IgG2 alpha chimeric L6 (ChL6), an anti-adenocarcinoma mouse IgG2 alpha-human IgG1 chimera, and the immunoconjugate 2IT-BAD-Lym-1 were examined for stability following storage. METHODS: Lym-1, ChL6, and 2IT-BAD-Lym-1 were aliquotted with filtration and stored at -70 degrees C for up to 8.5 years. 2IT-BAD-Lym-1 stored for 6.3 years (lot A) was compared to freshly prepared 2IT-BAD-Lym-1 (lot B). MoAbs were thawed and examined yearly by gel filtration high performance liquid chromatography (HPLC), polyacrylamide gel electrophoresis (PAGE), cellulose acetate electrophoresis (CAE), and Scatchard analysis of antigen binding. 2IT-BAD-Lym-1 was evaluated by HPLC, CAE, and radioimmunoassay. To assure the in vivo significance of the in vitro studies, 2IT-BAD-Lym-1 lots A and B were labeled with 111In and their pharmacokinetics in BALB/c mice were compared. RESULTS: Lym-1 demonstrated stability over 8.5 years, providing the following ranges of data over the interval: 98-100% chemical purity (HPLC), 96-100% monomeric fraction (CAE), 8.18-8.46 antigen binding pKa (Scatchard). Similar results were obtained for ChL6 for 7.4 years. HPLC and PAGE of Lym-1 and ChL6 have not changed from original manufacturer specifications, and both MoAbs remain sterile and apyrogenic. No significant differences between 2IT-BAD-Lym-1 lots A and B were observed by in vitro evaluation or pharmacokinetics in mice. CONCLUSIONS: Lym-1, ChL6, and 2IT-BAD-Lym-1 as manufactured and stored for 8.5, 7.4, and 6.3 years, respectively, demonstrated retention of structural and functional integrity.

Localization of nitric oxide synthase, NADPH diaphorase and soluble guanylyl cyclase in adult rabbit retina.

Nitric oxide (NO) acts as a neuronal messenger which activates soluble guanylyl cyclase (SGC) in neighboring cells and produces a wide range of physiological effects in the central nervous system (CNS). Using immunocytochemical and histochemical stains, we have characterized the NO/SGC system in the rabbit retina and to a lesser extent, in monkey retina. Based on staining patterns observed with an antibody to nitric oxide synthase (NOS) type I and a histochemical marker for NADPH diaphorase, a metabolic intermediate required for NOS activity, three major classes of neurons appear to generate NO in the rabbit retina. These include two subclasses of sparsely distributed wide field amacrine cells, rod and cone photoreceptors, and a subpopulation of ganglion cells. Equivalent cell populations were labeled in monkey retina. An antibody to SGC (tested only in rabbit retina), labeled large arrays of cone photoreceptors in the outer nuclear layer, both amacrine and bipolar cells in the inner nuclear layer (INL), as well as populations of neurons in the ganglion cell layer. These data suggest that the ability to generate NO is restricted to relatively few neurons in the inner retina and to photoreceptor cells in the outer retina; while presumptive target cells, containing pools of SGC, are widespread and form contiguous fields across the inner and outer nuclear layers (ONL) as well as the ganglion cell layer.

'Treatment mills' under the Federal Trade Commission Act.

This paper is based on a presentation made by the author at the meeting of the American Urological Association's Society for the Study of Impotence on April 12, 1997. The author addresses the general applicability of the Federal Trade Commission Act to advertising by so-called impotence 'treatment mills,' focusing in particular on the Federal Trade Commission's case in the matter of Genetus Alexandria, Inc. et al.

The kinetics of tracer movement through homologous gap junctions in the rabbit retina.

Observation of the spread of biotinylated or fluorescent tracers following injection into a single cell has become one of the most common methods of demonstrating the presence of gap junctions. Nevertheless, many of the fundamental features of tracer movement through gap junctions are still poorly understood. These include the relative roles of diffusion and iontophoretic current, and under what conditions the size of the stained mosaic will increase, asymptote, or decline. Additionally, the effect of variations in amount of tracer introduced, as produced by variation in electrode resistance following cell penetration, is not obvious. To examine these questions, Neurobiotin was microinjected into the two types of horizontal cell of the rabbit retina and visualized with streptavidin-Cy3. Images were digitally captured using a confocal microscope. The spatial distribution of Neurobiotin across the patches of coupled cells was measured. Adequate fits to the data were obtained by fitting to a model with terms for diffusion and amount of tracer injected. Results indicated that passive diffusion is the major source of tracer movement through gap junctions, whereas iontophoretic current played no role over the range tested. Fluorescent visualization, although slightly less sensitive than peroxidase reactions, produced staining intensities with a more useful dynamic range. The rate constants for movement of Neurobiotin between A-type horizontal cells was about ten times greater than that for B-type horizontal cells. Although direct extrapolation to ion conductances cannot be assumed, tracer movement can be used to give an estimate of relative coupling rates across cell types, retinal location, or modulation conditions in intact tissue.

Low-dose, fractionated radioimmunotherapy for B-cell malignancies using 131I-Lym-1 antibody.

PURPOSE: This trial was conducted to assess the toxicity and efficacy of 131I-Lym-1 in patients with either malignant B-cell non-Hodgkin's lymphoma (NHL) or chronic lymphocytic leukemia (CLL) using low-dose, fractionated radioimmunotherapy (RIT). MATERIALS AND METHODS: Thirty adult patients who had advanced B-cell malignancies (25 NHL and 5 CLL) had progressed despite standard therapy; 12 patients entered the trial with Karnofsky performance status (KPS) of equal to or greater than 60. Patients were treated with a series of intravenous doses of 131I-Lym-1 with a goal of reaching a cumulative dose in each patient of at least 300 mCi. All patients were Lym-1 reactive. Clinical responses and immediate toxicity were evaluable in all 30 patients and delayed toxicity in 26. RESULTS: Toxicity to Lym-1 antibody occurred with 28% of the 176 doses and was transient. Human antimouse antibodies (HAMA) were generated in 30% after a mean of 4 doses, but interrupted therapy in only 10% of the patients. Thrombocytopenia was dose-limiting; there were no deaths due to toxicity. Tumor regression occurred in 25 (83%) of the patients and was great enough, and durable enough, in 17 (57%) to qualify them as responders; 13 NHL patients and 4 CLL patients. Advanced disease often interrupted therapy prematurely. However, 18 patients received at least 180 mCi of 131I-Lym-1; 17 (94%) of these responded to the therapy. CONCLUSION: Although advanced disease often interrupted therapy prematurely, the results from 131I-Lym-1 therapy are clearly promising and warrant additional trials.