Spinodal decomposition and the emergence of dissipative transient periodic spatio-temporal patterns in acentrosomal microtubule multitudes of different morphology.

We have studied a spontaneous self-organization dynamics in a closed, dissipative (in terms of guansine 5'-triphosphate energy dissipation), reaction-diffusion system of acentrosomal microtubules (those nucleated and organized in the absence of a microtubule-organizing centre) multitude constituted of straight and curved acentrosomal microtubules, in highly crowded conditions, in vitro. Our data give experimental evidence that cross-diffusion in conjunction with excluded volume is the underlying mechanism on basis of which acentrosomal microtubule multitudes of different morphologies (straight and curved) undergo a spatial-temporal demix. Demix is constituted of a bifurcation process, manifested as a slow isothermal spinodal decomposition, and a dissipative process of transient periodic spatio-temporal pattern formation. While spinodal decomposition is an energy independent process, transient periodic spatio-temporal pattern formation is accompanied by energy dissipative process. Accordingly, we have determined that the critical threshold for slow, isothermal spinodal decomposition is 1.0 ± 0.05 mg/ml of microtubule protein concentration. We also found that periodic spacing of transient periodic spatio-temporal patterns was, in the overall, increasing versus time. For illustration, we found that a periodic spacing of the same pattern was 0.375 ± 0.036 mm, at 36 °C, at 155th min, while it was 0.540 ± 0.041 mm at 31 °C, and at 275th min after microtubule assembly started. The lifetime of transient periodic spatio-temporal patterns spans from half an hour to two hours approximately. The emergence of conditions of macroscopic symmetry breaking (that occur due to cross-diffusion in conjunction with excluded volume) may have more general but critical importance in morphological pattern development in complex, dissipative, but open cellular systems.

Efflux transporters in rat placenta and developing brain: transcriptomic and functional response to paracetamol.

Adenosine triphosphate binding cassette (ABC) transporters transfer lipid-soluble molecules across cellular interfaces either directly or after enzymatic metabolism. RNAseq analysis identified transcripts for ABC transporters and enzymes in rat E19, P5 and adult brain and choroid plexus and E19 placenta. Their functional capacity to efflux small molecules was studied by quantitative analysis of paracetamol (acetaminophen) and its metabolites using liquid scintillation counting, autoradiography and ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). Animals were treated acutely (30 min) and chronically (5 days, twice daily) with paracetamol (15 mg/kg) to investigate ability of brain and placenta barriers to regulate ABC transport functionality during extended treatment. Results indicated that transcripts of many efflux-associated ABC transporters were higher in adult brain and choroid plexus than at earlier ages. Chronic treatment upregulated certain transcripts only in adult brain and altered concentrations of paracetamol metabolites in circulation of pregnant dams. Combination of changes to metabolites and transport system transcripts may explain observed changes in paracetamol entry into adult and fetal brains. Analysis of lower paracetamol dosing (3.75 mg/kg) indicated dose-dependent changes in paracetamol metabolism. Transcripts of ABC transporters and enzymes at key barriers responsible for molecular transport into the developing brain showed alterations in paracetamol pharmacokinetics in pregnancy following different treatment regimens.

High-resolution imaging of the large non-human primate brain using microPET: a feasibility study.

The neuroanatomy and physiology of the baboon brain closely resembles that of the human brain and is well suited for evaluating promising new radioligands in non-human primates by PET and SPECT prior to their use in humans. These studies are commonly performed on clinical scanners with 5 mm spatial resolution at best, resulting in sub-optimal images for quantitative analysis. This study assessed the feasibility of using a microPET animal scanner to image the brains of large non-human primates, i.e. papio hamadryas (baboon) at high resolution. Factors affecting image accuracy, including scatter, attenuation and spatial resolution, were measured under conditions approximating a baboon brain and using different reconstruction strategies. Scatter fraction measured 32% at the centre of a 10 cm diameter phantom. Scatter correction increased image contrast by up to 21% but reduced the signal-to-noise ratio. Volume resolution was superior and more uniform using maximum a posteriori (MAP) reconstructed images (3.2-3.6 mm(3) FWHM from centre to 4 cm offset) compared to both 3D ordered subsets expectation maximization (OSEM) (5.6-8.3 mm(3)) and 3D reprojection (3DRP) (5.9-9.1 mm(3)). A pilot (18)F-2-fluoro-2-deoxy-d-glucose ([(18)F]FDG) scan was performed on a healthy female adult baboon. The pilot study demonstrated the ability to adequately resolve cortical and sub-cortical grey matter structures in the baboon brain and improved contrast when images were corrected for attenuation and scatter and reconstructed by MAP. We conclude that high resolution imaging of the baboon brain with microPET is feasible with appropriate choices of reconstruction strategy and corrections for degrading physical effects. Further work to develop suitable correction algorithms for high-resolution large primate imaging is warranted.

PET imaging of putative microglial activation in individuals at ultra-high risk for psychosis, recently diagnosed and chronically ill with schizophrenia.

We examined putative microglial activation as a function of illness course in schizophrenia. Microglial activity was quantified using [11C](R)-(1-[2-chrorophynyl]-N-methyl-N-[1-methylpropyl]-3 isoquinoline carboxamide (11C-(R)-PK11195) positron emission tomography (PET) in: (i) 10 individuals at ultra-high risk (UHR) of psychosis; (ii) 18 patients recently diagnosed with schizophrenia; (iii) 15 patients chronically ill with schizophrenia; and, (iv) 27 age-matched healthy controls. Regional-binding potential (BPND) was calculated using the simplified reference-tissue model with four alternative reference inputs. The UHR, recent-onset and chronic patient groups were compared to age-matched healthy control groups to examine between-group BPND differences in 6 regions: dorsal frontal, orbital frontal, anterior cingulate, medial temporal, thalamus and insula. Correlation analysis tested for BPND associations with gray matter volume, peripheral cytokines and clinical variables. The null hypothesis of equality in BPND between patients (UHR, recent-onset and chronic) and respective healthy control groups (younger and older) was not rejected for any group comparison or region. Across all subjects, BPND was positively correlated to age in the thalamus (r=0.43, P=0.008, false discovery rate). No correlations with regional gray matter, peripheral cytokine levels or clinical symptoms were detected. We therefore found no evidence of microglial activation in groups of individuals at high risk, recently diagnosed or chronically ill with schizophrenia. While the possibility of 11C-(R)-PK11195-binding differences in certain patient subgroups remains, the patient cohorts in our study, who also displayed normal peripheral cytokine profiles, do not substantiate the assumption of microglial activation in schizophrenia as a regular and defining feature, as measured by 11C-(R)-PK11195 BPND.

In vivo evidence for microglial activation in neurodegenerative dementia.

Evidence from numerous neuropathological observations and in vivo clinical imaging studies suggests a prominent role of activated microglia, the main effector cell of the brain's innate immune system, in Alzheimer's disease and other neurodegenerative diseases. Though the comprehensive molecular definition of the microglial activation process is still incomplete, the de novo expression of 'peripheral benzodiazepine-binding sites (PBBS)' by activated but not resting microglia has been established as a useful descriptor of functional state changes in microglia. As microglial transformation to an activated state is closely linked to progressive changes in brain disease, the detection of activated microglia can provide information about disease distribution and rate of disease progression. Positron emission tomography (PET) and [(11)C](R)-PK11195, a specific ligand of the PBBS, have been used to study systematically microglial activation in vivo. Significant microglial activation is present in the brains of patients with neurodegenerative dementia even at early and possibly preclinical stages of the disease with a spatial distribution reflecting different clinical phenotypes. We review some of the posited functions of activated microglia in the pathophysiology of dementia and speculate on the relationship between increased regional [(11)C](R)-PK11195 signals and the ensuing changes in brain volume. Finally, we provide a brief outlook on the development of new radioligands for the PBBS.

Dose enhancement effects to the nucleus and mitochondria from gold nanoparticles in the cytosol.

Gold nanoparticles (GNPs) have shown potential as dose enhancers for radiation therapy. Since damage to the genome affects the viability of a cell, it is generally assumed that GNPs have to localise within the cell nucleus. In practice, however, GNPs tend to localise in the cytoplasm yet still appear to have a dose enhancing effect on the cell. Whether this effect can be attributed to stress-induced biological mechanisms or to physical damage to extra-nuclear cellular targets is still unclear. There is however growing evidence to suggest that the cellular response to radiation can also be influenced by indirect processes induced when the nucleus is not directly targeted by radiation. The mitochondrion in particular may be an effective extra-nuclear radiation target given its many important functional roles in the cell. To more accurately predict the physical effect of radiation within different cell organelles, we measured the full chemical composition of a whole human lymphocytic JURKAT cell as well as two separate organelles; the cell nucleus and the mitochondrion. The experimental measurements found that all three biological materials had similar ionisation energies ∼70 eV, substantially lower than that of liquid water ∼78 eV. Monte Carlo simulations for 10-50 keV incident photons showed higher energy deposition and ionisation numbers in the cell and organelle materials compared to liquid water. Adding a 1% mass fraction of gold to each material increased the energy deposition by a factor of ∼1.8 when averaged over all incident photon energies. Simulations of a realistic compartmentalised cell show that the presence of gold in the cytosol increases the energy deposition in the mitochondrial volume more than within the nuclear volume. We find this is due to sub-micron delocalisation of energy by photoelectrons, making the mitochondria a potentially viable indirect radiation target for GNPs that localise to the cytosol.

HIV-1 Nef protein exhibits structural and functional similarity to scorpion peptides interacting with K+ channels.

The persistent infection of human glial cells with HIV-1 is characterized by prominent expression of the Nef protein. In order to evaluate the possible role of Nef in the development of HIV-1-associated neurological disorders, we compared Nef with known neuroactive proteins. We found that HIV Nef shares sequence and structural features with scorpion peptides known to interact with K+ channels. Sequence similarity encompasses two distinct regions of scorpion peptides. Based on crystallography data, both regions in scorpion peptides cooperate in forming a common domain stabilized by ion pairs between charged amino-acid residues. Recombinant Nef protein, as well as a synthetic part of a scorpion channel active peptide (M10), reversibly increased the total K+ current of chick dorsal root ganglions in patch-clamp experiments without killing the cells. These results indicate that a region conserved in HIV Nef and scorpion peptides concurs in both structure and electrophysiological activity and suggest that Nef, like scorpion peptides, may affect neuronal cell function.

Microglial turnover in the injured CNS: activated microglia undergo delayed DNA fragmentation following peripheral nerve injury.

Microglial proliferation and activation are common events in the injured CNS. The mechanisms, however, by which activated microglia are eliminated following a pathological stimulus are still poorly understood. The present study has therefore examined microglial proliferation by 3H-thymidine autoradiography and programmed cell death by terminal transferase-mediated nick end labeling (TUNEL) and in situ end labeling (ISEL) of nuclear DNA fragments in two models of peripheral nerve injury, i.e. sciatic and hypoglossal nerve transection in the rat. In these models, microglial activation and proliferation occur in CNS projection areas, i.e. in the ventral and dorsal gray matter of lumbar spinal cord and in the nucleus gracilis after sciatic nerve transection as well as in the axotomized hypoglossal nucleus. At these sites, microglial proliferation had a relatively sharp peak between days 2 and 3 post-lesion and then rapidly declined. DNA fragmentation was detected in lectin (GSI-B4)-positive microglia from day 6 after axotomy onward, reached an apparent peak at day 21 and was downregulated by day 60, i.e. the latest time point investigated. However, the expression of bcl-2 and c-myc, i.e. genes potentially controlling programmed cell death, was found to be unchanged during this period. Programmed cell death thus appears to be one mechanism by which activated microglia are gradually eliminated following CNS injury and steady state of microglial cell numbers is achieved in vivo. Expression of microglial growth factors may be instrumental in controlling these processes.

Microglial reaction in the rat cerebral cortex induced by cortical spreading depression.

The response of microglial cells to cortical spreading depression (CSD) was studied in rat brain by immunocytochemistry. CSD was elicited for one hour by the topical application of 4M potassium chloride solution and the microglial reaction examined immunocytochemically after 4, 16, 24 and 72 hours. CSD was sufficient to induce a microglial reaction throughout the cortex at 24 hours. Activated microglial cells furthermore showed a striking de-novo expression of major histocompatibility complex class II antigens. In contrast, no microglial reaction was observed in the cortex of sham-operated animals. This microglial reaction in response to CSD was not associated with histologically detectable neuronal damage. These results support the view that microglial cells are extremely sensitive to changes of the brain microenvironment. Their activation may be related to changes of ion homeostasis in the brain which are not sufficient to trigger neuronal injury.

Modulation of intracellular formation of reactive oxygen intermediates in peritoneal macrophages and microglia/brain macrophages by propentofylline.

Ischemia-induced nerve cell death can partly be prevented by propentofylline, a pharmacon structurally related to xanthine derivates that interacts with the neuromodulatory function of endogenous adenosine. To evaluate a possible mechanism of neuroprotection by propentofylline, we studied its effect on the cellular production of reactive oxygen intermediates in microglial cells, which under pathological conditions can differentiate into brain macrophages, in comparison to peritoneal macrophages. Using a flow cytometric assay, we determined the intracellular formation of reactive oxygen intermediates by measuring the oxidation of the membrane-permeable and nonfluorescent dihydrorhodamine 123 to the cationic and intracellularly trapped, green fluorescent rhodamine 123 in single viable cells. Propentofylline at the therapeutic concentration of 50 microM completely inhibited the Ca(2+)-dependent Con A-induced increase in the production of reactive oxygen intermediates in peritoneal macrophages. In isolated and cultured microglial cells, which have a high spontaneous respiratory burst activity, the spontaneous production of reactive oxygen intermediates was reduced by approximately 30%. A phorbol 12-myristate 13-acetate-induced rise in the respiratory burst activity could not be inhibited by propentofylline in either cell type. An increased generation of reactive oxygen intermediates is thought to contribute to nerve cell death after brain ischemia, edema, and neurodegenerative diseases like Alzheimer's disease. These pathological conditions are all accompanied by an activation of microglial cells. We therefore suggest that the neuroprotective properties of propentofylline might in part be due to a modulation of the microglial production of potentially harmful reactive oxygen intermediates.

Glial expression of the beta-amyloid precursor protein (APP) in global ischemia.

The beta-amyloid precursor protein (APP) bears characteristics of an acute-phase protein and therefore is likely to be involved in the glial response to brain injury. In the brain, APP is rapidly synthesized by activated glial cells in response to comparatively mild neuronal lesions, e.g., a remote peripheral nerve injury. Perfusion deficits in the brain result largely in neuronal necrosis and are a common condition in elderly patients. This neuronal necrosis is accompanied by a pronounced reaction of astrocytes and microglia, which can also be observed in animal models. We have therefore studied in the rat, immunocytochemically, the induction of APP after 30 min of global ischemia caused by four-vessel occlusion. The postischemic brain injuries were examined at survival times from 12 h to 7 days. From day 3 onward, APP immunoreactivity was strongly induced in the CA1 and CA4 regions of the rat dorsal hippocampus as well as in the dorsolateral striatum. In these areas, the majority of APP-immunoreactive cells were reactive glial fibrillary acidic protein (GFAP)-positive astrocytes, as shown by double-immunofluorescence labeling for GFAP and APP. Additionally, small ramified cells, most likely activated microglia, expressed APP immunoreactivity. In contrast, in the parietal cortex, APP immunoreactivity occurred focally in clusters of activated microglia rather than in astrocytes, as demonstrated by double-immunofluorescence labeling for APP and the microglia-binding lectin Griffonia simplicifolia isolectin B4. In conclusion, following global ischemia, APP is induced in reactive glial cells with spatial differences in the distribution pattern of APP induction in astrocytes and microglia.

Modeling dynamic PET-SPECT studies in the wavelet domain.

This work develops a theoretical framework and corresponding algorithms for the modeling of dynamic PET-SPECT studies both in time and space. The problem of estimating the spatial dimension is solved by applying the wavelet transform to each scan of the dynamic sequence and then performing the kinetic modeling and statistical analysis in the wavelet domain. On reconstruction through the inverse wavelet transform, one obtains parametric images that are consistent estimates of the spatial patterns of the kinetic parameter of interest. The theoretical setup allows the use of linear techniques currently used in PET-SPECT for kinetic analysis. The method is applied to artificial and real data sets. The application to dynamic PET-SPECT studies was performed both for validation purposes, when the spatial patterns are known, and for illustration of the advantages offered by the technique in case of tracers with an unknown pattern of distribution.

Transforming growth factor beta mediates increase of mature transmembrane amyloid precursor protein in microglial cells.

By using the immortalized microglial cell line BV-2, we show that the high expression of the beta A4 amyloid precursor protein (APP), its biogenesis and metabolism is modulated by TGF beta, a cytokine with immunosuppressive activity, and by the microglia-stimulating agent LPS. TGF beta induces accumulation of cellular mature APP, the putative precursor of the amyloid subunit of Alzheimer's disease. LPS leads to an increase in cellular immature, non-amyloidogenic APP and secretion of also non-amyloidogenic APP fragments. We also demonstrate a functional involvement of ECM molecules in the regulation of microglial APP expression at mRNA and protein level by TGF beta and LPS.

Thalamic microglial activation in ischemic stroke detected in vivo by PET and [11C]PK1195.

Using quantitative PET, the authors studied the binding of [11C]PK11195, a marker of activated microglia, in the thalamus of patients with chronic middle cerebral artery infarcts. All patients showed increased [11C]PK11195 binding in the ipsilateral thalamus, indicating the activation of microglia in degenerating projection areas remote from the primary lesion. A persistent increase in [11C]PK11195 binding suggests active, long-term thalamic microstructural changes after corticothalamic connection damage.

[11C](R)-PK11195 positron emission tomography imaging of activated microglia in vivo in Rasmussen's encephalitis.

This study was designed to explore the feasibility of PET using [11C](R)-PK11195 as an in vivo marker of activated microglia/brain macrophages for the assessment of neuroinflammation in Rasmussen's encephalitis (RE). [11C](R)-PK11195 PET was carried out in four normal subjects, two patients with histologically confirmed RE, and three patients with clinically stable hippocampal sclerosis and low seizure frequency. Binding potential maps showing specific binding of [11C](R)-PK11195 were generated for each subject. Regional binding potential values were calculated for anatomically defined regions of interest after coregistration to and spatial transformation into the subjects' own MRI. In one patient with RE who underwent hemispherectomy, the resected, paraffin-embedded brain tissue was stained with an antibody (CR3/43) that labels activated human microglia. Whereas specific binding of [11C](R)-PK11195 in clinically stable hippocampal sclerosis was similar to that in normal brain, patients with RE showed a focal and diffuse increase in binding throughout the affected hemisphere. In RE, [11C](R)-PK11195 PET can reveal in vivo the characteristic, unilateral pattern known from postmortem neuropathologic study. PET imaging of activated microglia/brain macrophages offers a tool for investigation of a range of brain diseases where neuroinflammation is a component and in which conventional MRI does not unequivocally indicate an inflammatory tissue reaction. [11C](R)-PK11195 PET may help in the choice of appropriate biopsy sites and, further, may allow assessment of the efficacy of antiinflammatory disease-modifying treatment.

The functional anatomy of visual-tactile integration in man: a study using positron emission tomography.

The integration of neural signals from different sensory modalities is a prerequisite for many cognitive and behavioural functions. In this study, we have mapped the functional anatomy of the integration of sensory signals across the tactile and visual modalities. Using the PET radiotracer H2(15)O, regional cerebral blood flow (rCBF) changes were measured in eight normal volunteers performing crossmodal recognition of simultaneously presented visual and tactile stimuli using a modified version of the 'arc-circle test'. Whilst intramodal matching within the visual modality led to relative rCBF increases in the visual association cortex, crossmodal matching (visual-tactile), when compared to intramodal matching, was accompanied by relative rCBF increases in the anterior cingulate cortex, inferior parietal lobules, the left dorsolateral prefrontal cortex (DLPFC) and the left claustrum/insular cortex. The pattern of brain activation is congruent with areas of heteromodal and supramodal cortex and indicates that activation of multimodal areas is required to solve the crossmodal problem.

Microglia in the immune surveillance of the brain: human microglia constitutively express HLA-DR molecules.

The degree of MHC class II expression in histologically normal human brain biopsy or autopsy tissue is still controversial. According to the generally held view MHC class II expression is rather low in the normal brain with the exception of the white matter. In the present study, HLA-DR expression was examined immunocytochemically in different brain areas obtained from three autopsy cases with short post-mortem times (i.e. 6 h). Based on standard histological evaluation, the brain areas studied appeared as histologically normal tissue. In all brain areas there was a strong constitutive HLA-DR expression on ramified microglia. The number of HLA-DR-immunoreactive microglia was strongest in the white matter (the corpus callosum and the capsula interna for example). The border zone between white matter and grey matter, however, revealed a sharp contrast between a high density of HLA-DR-immunoreactive microglia in the white matter and a rather low number in the grey matter. In the grey matter, HLA-DR-immunoreactive microglia were much less frequent than in the white matter and more pronounced on perivascular cells. The staining and distribution pattern of HLA-DR-immunoreactive microglia was confirmed by immunocytochemistry with a panel of different anti-HLA-DR monoclonal antibodies as well as by quantitative analysis of the immunostaining. Unlike the HLA-DR immunoreactivity, HLA-ABC immunoreactivity (detecting MHC class I antigens) was confined to endothelia and not observed on microglia. In the choroid plexus stromal macrophages expressed both class I and II antigens (i.e. at a location which could provide the peripheral immune system access to CNS antigens). Constitutive HLA-DR expression by microglia qualifies them as the main resident antigen-presenting cell of the brain. The pronounced overall HLA-DR expression by resting microglia questions a central dogma of the brain as an immune-privileged site and further points to the key role of the microglia in brain immune surveillance.

Cell adhesion molecule expression in the regenerating rat facial nucleus.

Transection of the rat facial nerve leads to an intrinsic microglial reaction in the facial nucleus. In the present study, we have examined immunocytochemically the expression of cell adhesion molecules during this process. Resting microglia constitutively expressed the LFA-1 alpha and beta chain (CD11a and CD18) in the white matter, rather than in the intact, control facial nucleus. From 24 h after facial nerve transection onward, activated microglia showed an increased expression of LFA-1 alpha and beta. The immunoreactivity reached its peak around day 7 following axotomy, i.e. at a time when activated microglia are found in a close perineuronal position. In contrast, the expression of the principal ligand of LFA-1, i.e. ICAM-1, remained unchanged following axotomy; the immunoreactivity being constitutively found on cerebral blood vessels. Increased LFA-1 expression thus appears to be a general marker for microglial activation and might further be involved in the rearrangement of the microglial cytoskeleton upon activation of this cell.

Expression of L-APP mRNA in brain cells.

Several reports addressed the issue of how the alternative splicing of exon 7 and 8 in the APP pre-mRNA is regulated in different tissues. Of special interest here was the potential involvement of exon 7 containing APP splice isoforms, since this exon codes for a serine protease inhibitor and is therefore of putative relevance for amyloidogenic catabolism of the precursor protein. The recent identification of a third alternative splice site in close proximity to the beta A4-amyloid portion in the APP gene which may also increase APP amyloidogenicity, allowed us to investigate its regulation in cells of the central nervous system. With our assay, we were able to resolve six different APP isoforms of the eight potential isoforms which can be generated from the three alternatively spliced exons 7, 8, and 15. We demonstrate here that, in addition to rat brain microglia cells, astrocyte-enriched cultures also skip the novel alternative 3'-splice site in front of exon 15, generating L-APP mRNA. Neurons are the only cells in the central nervous system which seem to use the 3'-splice site of intron 14 nearly 100%. Interestingly, this very 3'-splice site is the only one present in the APP gene that completely matches the consensus sequence for the branchpoint sequence proposed for introns. We would therefore suggest that neurons lack a specific splicing factor which inhibits the use of the rather strong 3'-splice site in front of exon 15. It remains to be shown whether this is also the case for neurons in Alzheimer's disease.

Glial beta-amyloid precursor protein: expression in the dentate gyrus after entorhinal cortex lesion.

Stereotactic lesioning of the rat entorhinal cortex leads to an induction of beta-amyloid precursor protein (APP) immunoreactivity in non-neuronal cells of the deafferented dentate gyrus. Double immunofluorescence against APP and the microglia-binding isolectin B4 from Griffonia simplicifolia revealed that APP immunoreactivity was confined to activated microglia. The microglial APP expression became detectable 3 days after lesioning, reached its peak after 7 days and disappeared after 10 days. The early accumulation of APP in microglia supports the view that microglia play an important role in the initial stages of amyloid plaque formation. Such a glial APP accumulation occurs rapidly in distant but anatomically connected areas. This is in line with the preferential localization of amyloid plaques in the dentate gyrus, which is the projection field of the degenerating neurones of the entorhinal cortex in patients with developing dementia.