Endothelial cell CD36 regulates membrane ceramide formation, exosome fatty acid transfer and circulating fatty acid levels.

Endothelial cell (EC) CD36 controls tissue fatty acid (FA) uptake. Here we examine how ECs transfer FAs. FA interaction with apical membrane CD36 induces Src phosphorylation of caveolin-1 tyrosine-14 (Cav-1Y14) and ceramide generation in caveolae. Ensuing fission of caveolae yields vesicles containing FAs, CD36 and ceramide that are secreted basolaterally as small (80-100 nm) exosome-like extracellular vesicles (sEVs). We visualize in transwells EC transfer of FAs in sEVs to underlying myotubes. In mice with EC-expression of the exosome marker emeraldGFP-CD63, muscle fibers accumulate circulating FAs in emGFP-labeled puncta. The FA-sEV pathway is mapped through its suppression by CD36 depletion, blocking actin-remodeling, Src inhibition, Cav-1Y14 mutation, and neutral sphingomyelinase 2 inhibition. Suppression of sEV formation in mice reduces muscle FA uptake, raises circulating FAs, which remain in blood vessels, and lowers glucose, mimicking prominent Cd36-/- mice phenotypes. The findings show that FA uptake influences membrane ceramide, endocytosis, and EC communication with parenchymal cells.

Latent-transforming growth factor beta-binding protein-2 (LTBP-2) is required for longevity but not for development of zonular fibers.

Latent-transforming growth factor beta-binding protein 2 (LTBP-2) is a major component of arterial and lung tissue and of the ciliary zonule, the system of extracellular fibers that centers and suspends the lens in the eye. LTBP-2 has been implicated previously in the development of extracellular microfibrils, although its exact role remains unclear. Here, we analyzed the three-dimensional structure of the ciliary zonule in wild type mice and used a knockout model to test the contribution of LTBP-2 to zonule structure and mechanical properties. In wild types, zonular fibers had diameters of 0.5-1.0 micrometers, with an outer layer of fibrillin-1-rich microfibrils and a core of fibrillin-2-rich microfibrils. LTBP-2 was present in both layers. The absence of LTBP-2 did not affect the number of fibers, their diameters, nor their coaxial organization. However, by two months of age, LTBP-2-depleted fibers began to rupture, and by six months, a fully penetrant ectopia lentis phenotype was present, as confirmed by in vivo imaging. To determine whether the seemingly normal fibers of young mice were compromised mechanically, we compared zonule stress/strain relationships of wild type and LTBP-2-deficient mice and developed a quasi-linear viscoelastic engineering model to analyze the resulting data. In the absence of LTBP-2, the ultimate tensile strength of the zonule was reduced by about 50%, and the viscoelastic behavior of the fibers was altered significantly. We developed a harmonic oscillator model to calculate the forces generated during saccadic eye movement. Model simulations suggested that mutant fibers are prone to failure during rapid rotation of the eyeball. Together, these data indicate that LTBP-2 is necessary for the strength and longevity of zonular fibers, but not necessarily for their formation.

A secreted serine-threonine kinase determines virulence in the eukaryotic pathogen Toxoplasma gondii.

Toxoplasma gondii strains differ dramatically in virulence despite being genetically very similar. Genetic mapping revealed two closely adjacent quantitative trait loci on parasite chromosome VIIa that control the extreme virulence of the type I lineage. Positional cloning identified the candidate virulence gene ROP18, a highly polymorphic serine-threonine kinase that was secreted into the host cell during parasite invasion. Transfection of the virulent ROP18 allele into a nonpathogenic type III strain increased growth and enhanced mortality by 4 to 5 logs. These attributes of ROP18 required kinase activity, which revealed that secretion of effectors is a major component of parasite virulence.

Deep brain stimulation for Parkinson's disease: association between stimulation parameters and cognitive performance.

Chronic subthalamic nucleus stimulation produces inconsistent patterns of cognitive change in Parkinson's disease patients. Individually tailored stimulation parameters may contribute to this variable pattern of change. Systematic variation of amplitude, pulse width, and rate of stimulation has been reported to produce unique changes in motor and limbic response. To evaluate the association between stimulation parameters and cognitive/behavioral response, neuropsychological performance and stimulation parameter data of 8 Parkinson's disease patients were submitted to Pearson r correlation analysis. Results indicate that each stimulation parameter was significantly associated with a subset of measures. The current findings raise the possibility that adverse cognitive/behavioral responses may be treated through parameter modification while maintaining motor symptom efficacy.

Three screening batteries to detect cognitive impairment in multiple sclerosis.

To compare the sensitivities for detecting cognitive impairment in patients with multiple sclerosis (MS) and administration times of three brief batteries of neuropsychological tests, 64 patients with MS completed the Neuropsychological Screening Battery for Multiple Sclerosis (NPSBMS), the Screening Examination for Cognitive Impairment (SEFCI), and the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS). Failure on a particular test was defined as a score below the 5th percentile for healthy controls, and the number of patients who failed at least one or two tests (out of four) was determined for each battery. Both the SEFCI and the NPSBMS identified significantly more patients with impairment than the RBANS, which was no more sensitive than the Mini-Mental State Exam (MMSE). Results were similar at both the one- and two-failed-tests criteria, but there were no significant differences between the SEFCI and the NPSBMS at either failure criterion. Mean administration time was 22.6 min for the SEFCI compared to 31.7 min for the NPSBMS (p < 0.001). Eleven (17%) of the patients refused to attempt the Paced Auditory Serial Addition Test (PASAT), one component of the NPSBMS. For screening patents on a single occasion, the SEFCI is preferred because its administration time is shorter than the NPSBMS.

Fluency in multiple sclerosis: which measure is best?

Tests of verbal fluency provide brief and sensitive measures of the deficits in rapidly retrieving overlearned information common in multiple sclerosis (MS). Production of words that begin with the letters F, A, and S is the verbal fluency measure most often used with patients who are fluent in English. However, because of frequency of words beginning with certain letters varies from one language to another, it is unlikely that any fixed set of letters will be appropriate for multicenter trials that involve patients who are fluent in different languages. A possible alternative involves using semantic fluency categories that contain such a large number of exemplars that no fluent speaker of any language could exhaust the category in the allotted response time. To examine the potential usefulness of semantic fluency measures, 203 MS patients and 87 healthy controls generated words that begin with F, A, or S or were exemplars of the categories animals and parts of the body. Receiver operating characteristic (ROC) curve analyses indicated that sensitivities and specificities for the three fluency measures in discriminating patients from controls were quite similar, especially if patients with global cognitive impairment were excluded.

Impaired affective prosody in AD: relationship to aphasic deficits and emotional behaviors.

OBJECTIVE: To assess the ability of patients with AD to produce, repeat, and comprehend affective prosody in relationship to severity of dementia, aphasic deficits, and changes in emotional behaviors. METHODS: An Aprosodia Battery was used to assess affective-prosodic performance and to identify patterns of deficits in affective communication. In addition, the presence and severity of aberrant behaviors, depression, and aphasia were assessed using standardized assessment tools. RESULTS: Patients with AD had significant impairments in their ability to repeat, comprehend, and discriminate affective aspects of speech, but maintained normal spontaneous affective-prosodic performances. As dementia severity increased, performance on the comprehension tasks and, to a lesser degree, on the repetition tasks became more impaired; spontaneous affective prosody remained normal. In the current study, affective-prosodic comprehension impairments were present in patients with all stages of AD; comparable aphasic deficits were not observed until patients were severely demented. The majority of aphasic deficits involved anomia without loss of comprehension. Patients with AD with sensory aprosodia had increased frequency and severity of behavioral changes whereas patients with AD with normal affective-prosodic performance were significantly less demented, had normal linguistic ability, and displayed fewer aberrant psychiatric behaviors. CONCLUSION: Patients with mild AD are at considerable risk for affective-prosodic comprehension deficits. As patients become more demented and develop sensory aprosodia, they are at greater risk for disturbances in behavior and mood.

Chlamydia pneumoniae expresses genes required for DNA replication but not cytokinesis during persistent infection of HEp-2 cells.

Chlamydia pneumoniae causes community-acquired pneumonia and is associated with several chronic diseases, including asthma and atherosclerosis. The intracellular growth rate of C. pneumoniae slows dramatically during chronic infection, and such persistence leads to attenuated production of new elementary bodies, appearance of morphologically aberrant reticulate bodies, and altered expression of several chlamydial genes. We used an in vitro system to further characterize persistent C. pneumoniae infection, employing both ultrastructural and transcriptional activity measurements. HEp-2 cells were infected with C. pneumoniae (TW-183) at a multiplicity of infection of 3:1, and at 2 h postinfection gamma interferon (IFN-gamma) was added to the medium at 0.15 or 0.50 ng/ml. Treated and untreated cultures were harvested at several times postinfection. RNA was isolated and reverse transcribed, and reverse transcription (RT)-PCR analyses targeting primary transcripts from chlamydial rRNA operons as well as dnaA, polA, mutS, minD, ftsK, and ftsW mRNA were done. Some cultures were fixed and stained for electron microscopic analysis, and a real-time PCR assay was used to assess relative chlamydial chromosome accumulation under each culture condition. The latter assays showed that bacterial chromosome copies accumulated severalfold during IFN-gamma treatment of infected HEp-2 cells, although less accumulation was observed in cells treated with the higher dose. Electron microscopy demonstrated that high-dose IFN-gamma treatment elicited aberrant forms of the bacterium. RT-PCR showed that chlamydial primary rRNA transcripts were present in all IFN-gamma-treated and untreated cell cultures, indicating bacterial metabolic activity. Transcripts from dnaA, polA, mutS, and minD, all of which encode products for bacterial chromosome replication and partition, were expressed in IFN-gamma-treated and untreated cells. In contrast, ftsK and ftsW, encoding products for bacterial cell division, were expressed in untreated cells, but expression was attenuated in cells treated with low-dose IFN-gamma and absent in cells given the high dose of cytokine. Thus, the development of persistence included production of transcripts for DNA replication-related, but not cell division-related, genes. These results provide new insight regarding molecular activities that accompany persistence of C. pneumoniae, as well as suggesting requirements for reactivation from persistent to productive growth.

Mycobacterial surface moieties are released from infected macrophages by a constitutive exocytic event.

Bacterial cell wall constituents are released from mycobacterial phagosomes and actively traffic within infected macrophages. Colocalization of fluorescently tagged bacterial moieties with endocytic tracers revealed the dynamic movement of released mycobacterial constituents into the endocytic network with accumulation in tubular lysosomal-like compartments. The released bacterial constituents not only penetrated the infected host cell but were also present in an extracellular microvesicular fraction. To identify the intracellular source of these exocytic compartments, released vesicular material was isolated from culture supernatants by differential ultracentrifugation and characterized by Western blot and electron microscopy analyses. The presence of lysosomal membrane proteins and lysosomal proteases suggested that labeled mycobacterial cell wall constituents access a constitutive lysosomal exocytic pathway. An abundance of multilamellar extracellular compartments morphologically reminiscent of MHC class II-enriched compartments (MIIC) implicated a MHC class II transport pathway in the extracellular release of bacterial constituents. Increases in intracellular free calcium have previously been shown to trigger lysosomal exocytosis by inducing fusion of lysosomes with the plasma membrane. To test if an increase in calcium would stimulate exocytosis with release of mycobacterial constituents, infected macrophages were exposed to the calcium ionophore A23187. The ionophore triggered the release of a microvesicular fraction containing labeled bacterial moieties, implicating calcium-regulated lysosomal exocytosis as a trafficking pathway by which mycobacterial products are released from infected macrophages.

Analysis of mycobacterium-infected macrophages by immunoelectron microscopy and cell fractionation.

The ability of pathogenic Mycobacterium to establish and maintain an infection in a host is dependent on their capacity to survive within phagocytes (1-3). Studies conducted on macrophage infections in culture have provided considerable insight into the mechanisms developed by these bacteria to ensure their survival. However, macrophages in culture are considerably more permissive than the phagocyte in its correct tissue environment and one has to be aware that the capacity of the macrophage to function as both an antigen-presenting cell (inducing or sustaining a cellular immune response) and an immune effector cell (mediating an antimicrobial response following activation with cytokines) places certain provisos on interpretation of in vitro infection experiments (4,5). Despite this obvious caveat, our appreciation of the complex interplay between Mycobacterium spp. of varying degrees of virulence [M. tuberculosis, M. bovis (BCG) and M. avium] and their host macrophage has benefited considerably from the recent application of modern cell biological techniques to studies of infected cells in culture.

Identification of mycobacterial surface proteins released into subcellular compartments of infected macrophages.

Considerable effort has focused on the identification of proteins secreted from Mycobacterium spp. that contribute to the development of protective immunity. Little is known, however, about the release of mycobacterial proteins from the bacterial phagosome and the potential role of these molecules in chronically infected macrophages. In the present study, the release of mycobacterial surface proteins from the bacterial phagosome into subcellular compartments of infected macrophages was analyzed. Mycobacterium bovis BCG was surface labeled with fluorescein-tagged succinimidyl ester, an amine-reactive probe. The fluorescein tag was then used as a marker for the release of bacterial proteins in infected macrophages. Fractionation studies revealed bacterial proteins within subcellular compartments distinct from mycobacteria and mycobacterial phagosomes. To identify these proteins, subcellular fractions free of bacteria were probed with mycobacterium-specific antibodies. The fibronectin attachment protein and proteins of the antigen 85-kDa complex were identified among the mycobacterial proteins released from the bacterial phagosome.

Interaction of Mycobacterium avium-containing phagosomes with the antigen presentation pathway.

Pathogenic mycobacteria infect macrophages where they replicate in phagosomes that minimize contact with late endosomal/lysosomal compartments. Loading of Ags to MHC class II molecules occurs in specialized compartments with late endosomal characteristics. This points to a sequestration of mycobacteria-containing phagosomes from the sites where Ags meet MHC class II molecules. Indeed, in resting macrophages MHC class II levels decreased strongly in phagosomes containing M. avium during a 4-day infection. Phagosomal MHC class II of early (4 h) infections was partly surface-derived and associated with peptide. Activation of host macrophages led to the appearance of H2-M, a chaperon of Ag loading, and to a strong increase in MHC class II molecules in phagosomes of acute (1 day) infections. Comparison with the kinetics of MHC class II acquisition by IgG-coated bead-containing phagosomes suggests that the arrest in phagosome maturation by mycobacteria limits the intersection of mycobacteria-containing phagosomes with the intracellular trafficking pathways of Ag-presenting molecules.

Neuropsychological deficits in sober alcoholics: influences of chronicity and recent alcohol consumption.

BACKGROUND: The relationships between severity of neuropsychological (NP) deficits and quantity and duration of alcoholic drinking remain controversial. Eckardt et al. (1998) proposed that NP deficits can be observed only if chronicity of alcohol abuse equals or exceeds 10 years. In this study we tested the hypothesis of Eckardt et al. and reexamined the relationship of NP performance and alcohol consumption. METHODS: One hundred sixty-two alcoholics and 165 controls completed a NP test battery at least 3 weeks after the alcoholics attained sobriety. Chronicity varied from 4 to 9 years for 55 alcoholics and from 10 to 33 years for the remaining 107. RESULTS: Compared to controls, both groups of alcoholics were impaired on the Shipley Vocabulary and Abstraction tests and on two versions of the Digit Symbol test, but there was no difference between the two alcoholic groups on any measure. Regression analyses that controlled for age and education showed that chronicity predicted less than 0.5% of the variance on NP measures. By contrast, a measure of recent alcohol consumption, the Quantity-Frequency Index, contributed significantly (approximately 5% of the variance) to the prediction of alcoholics' NP performance. CONCLUSIONS: These data provide weak support for a dose effect relationship between degree of NP impairment and level of alcoholic drinking in the past 6 months but no evidence for an influence of chronicity.

Forms of knowledge, cognitive impairment and drug abuse: a demonstration.

Chronic abusers of alcohol and other drugs often exhibit persistent deficits on neuropsychological tests. These deficits are usually attributed to drug-induced brain dysfunction or to familial factors that depress performance independent of drug abuse. The aim of this study was to determine the plausibility of a third explanation: namely that some neuropsychological deficits of drug abusers arise because they acquire different forms of knowledge from non-drug abusers. This hypothesis predicts that drug abusers should excel on tests of knowledge that is related to their lifestyle. Sixty-three clients from a local methadone clinic and a comparison group of 24 non-abusing subjects who were knowledgeable about drugs listened to a song about cocaine addiction. They were asked to identify the title, artist, year the song was first popular and the meaning of the song. There were no significant differences between the groups in identifying the song title, artist or year, but the drug-abusers were significantly more likely to state that the song was about cocaine or drug addiction. In interpreting deficits by drug abusers on neuropsychological tests, differences in knowledge acquired should be considered.

Trafficking and release of mycobacterial lipids from infected macrophages.

Analysis of infected macrophages revealed that lipid-containing moieties of the mycobacterial cell wall are actively trafficked out of the mycobacterial vacuole. To facilitate the analysis of vesicular trafficking from mycobacteria-containing phagosomes, surface-exposed carbohydrates were labeled with hydrazide-tagged markers. The distribution of labeled carbohydrate/lipid moieties and subsequent interaction with cellular compartments were analyzed by immunoelectron microscopy and by fluorescence microscopy of live cells. The released mycobacterial constituents were associated with several intracellular organelles and were enriched strikingly in tubular endocytic compartments. Subcellular fractionation of infected macrophages by density gradient electrophoresis showed temporal movement of labeled bacterial constituents through early and late endosomes. Thin layer chromatography analysis of these subcellular fractions confirmed their lipid nature and revealed five dominant bacteria-derived species. These mycobacterial lipids were also found in extracellular vesicles isolated from the medium and could be observed in un-infected 'bystander' cells. Their transfer to bystander cells could expand the bacteria's sphere of influence beyond the immediate confines of the host cell.

Direct delivery of procathepsin D to phagosomes: implications for phagosome biogenesis and parasitism by Mycobacterium.

Phagosome maturation is characterized by the sequential acquisition and loss of proteins by the phagocytic vacuole during the formation of an acidic and hydrolytic compartment where degradation of the phagocytosed particle occurs. Transfer of proteins to the maturing phagosome occurs by fusion with a range of vesicles. Here we describe direct fusion of early phagosomes with vesicles that appear to be derived from the biosynthetic pathway. In mouse bone marrow macrophages, the 51 kDa proform of cathepsin D was found in vesicles of the ER/Golgi network that could be discriminated from endosomal vesicles which in turn contained the 46 and 30 kDa processed forms of the enzyme. Procathepsin D was acquired by phagosomes formed around inert particles such as IgG-coated beads and could be "protected" by blocking acidification with Bafilomycin A1. Mycobacterium avium-containing vacuoles from established infections possessed both pro- and processed cathepsin D similar to early bead-containing phagosomes. In contrast phagosomes harboring dead mycobacteria demonstrated markedly enhanced acquisition of the 46kDa form within 4 h post internalization and only low levels of procathepsin D.

Assessment of cognitive and psychological functions in patients with multiple sclerosis: considerations for databasing.

Test batteries for use in studying cognition and depression in patients are briefly but critically reviewed. Suggestions for a new battery that contains tests that are related to rehabilitation and work status are proposed.

Representations of knowledge about dominoes in demented and normal elderly players.

1. Dementia patients who retain musical and game-playing skills exhibit impaired performance on explicit memory tests of knowledge about their retained skill. 2. Dementia patients who retain skill at playing dominoes can answer complex questions about the play of the game almost as well as normal elderly domino players when the questions are presented with real dominoes. 3. The aim of this study was to determine if skilled dementia patients could answer questions about domino play when the stimuli were two-dimensional drawings of dominoes. 4. Seventeen dementia patients and eight normal elderly domino players were tested on two forms of the Domino Quiz: first with real dominoes, then with two-dimensional drawings; other neuropsychological tests were given at the same time. 5. Fourteen of the 17 patients and all of the controls showed no decline in answering questions about domino play when two-dimensional drawings were used. These patients showed retained symbolic processing of information about dominoes despite declines in overall mental status, generation of words from specific semantic categories, and recognition memory for domino terminology. 6. Because the 14 patients with retained domino skill performed as accurately as controls on both administrations of a letter cancellation task, the ability to process familiar symbols may be important to their game-playing skill.

Trafficking of Shigella lipopolysaccharide in polarized intestinal epithelial cells.

Bacterial lipopolysaccharide (LPS) at the apical surface of polarized intestinal epithelial cells was previously shown to be transported from the apical to the basolateral pole of the epithelium (Beatty, W.L., and P.J. Sansonetti. 1997. Infect. Immun. 65:4395-4404). The present study was designed to elucidate the transcytotic pathway of LPS and to characterize the endocytic compartments involved in this process. Confocal and electron microscopic analyses revealed that LPS internalized at the apical surface became rapidly distributed within endosomal compartments accessible to basolaterally internalized transferrin. This compartment largely excluded fluid-phase markers added at either pole. Access to the basolateral side of the epithelium subsequent to trafficking to basolateral endosomes occurred via exocytosis into the paracellular space beneath the intercellular tight junctions. LPS appeared to exploit other endocytic routes with much of the internalized LPS recycled to the original apical membrane. In addition, analysis of LPS in association with markers of the endocytic network revealed that some LPS was sent to late endosomal and lysosomal compartments.

Psychological and neuropsychological predictors of coping patterns by patients with multiple sclerosis.

To determine the relative contribution of psychological and neuropsychological (NP) variables to the prediction of patterns of coping with disease-related stressors and satisfaction with their coping efforts, 56 patients with multiple sclerosis (MS) were administered the Ways of Coping Checklist, the Symptom Checklist-90-Revised, and a battery of NP tests chosen for their sensitivity to MS. Higher levels of psychological distress were associated with greater use of emotion-focused coping strategies and reduced perceived effectiveness of the coping strategies employed. Psychological distress was not related to the use of problem-focused strategies and NP variables did not predict coping style or effectiveness. MS patients who display heightened psychological distress may be good candidates for psychotherapeutic interventions aimed at improving perceived coping effectiveness.