[In-vivo Confocal Microscopy for the Diagnosis of Mucous Membrane Pemphigoid]. / In-vivo-Konfokalmikroskopie in der Diagnose des okulären Schleimhautpemphigoids.

BACKGROUND: An early diagnosis is crucial for the outcome of mucous membrane pemphigoid (MMP). The sensitivity of the so-called diagnostic gold standard, direct and indirect immune fluorescence (DIF/IIF) ranges from 30 to 80 %, and is thus lower than desirable. Moreover, conjunctival biopsy, mandatory in most cases, entails the risks of exacerbation. The purpose of this study is to establish the contribution of non-invasive in vivo confocal microscopy to the recognition of MMP. PATIENTS AND METHODS: We examined the conjunctiva of ten patients and ten control subjects with the confocal microscope Heidelberg Retina Tomograph II/Rostock Cornea Module and checked for differences in qualitative and quantitative structure of the connective tissue. RESULTS: Pemphigoid patients showed an increase and/or aggregation of reticular connective tissue with hyperreflective strands in the substantia propria, as well as an increased subepithelial fibrosis compared to controls. The basal membrane zone was thicker and more hyperreflective than in the healthy subjects. CONCLUSION: In-vivo confocal microscopy may serve as a useful additional diagnostic method in the detection of MMP.

Combining atomic force-fluorescence microscopy with a stretching device for analyzing mechanotransduction processes in living cells.

Mechanical forces affect biological systems in their natural environment in a widespread manner. Mechanical stress may either stimulate cells or even induce pathological processes. Cells sensing mechanical stress usually respond to such stressors with proliferation or differentiation. Hence, for in vitro studies, the ability to impose a controlled mechanical stress on cells combined with appropriate analytical tools providing an immediate answer is essential to understand such fundamental processes. Here, we present a novel uniaxial motorized cell stretching device that has been integrated into a combined fluorescence microscope (FM)-atomic force microscope (AFM) system, thereby enabling high-resolution topographic and fluorescent live cell imaging. This unique tool allows the investigation of mechanotransduction processes, as the cells may be exposed to deliberately controlled mechanical stress while simultaneously facilitating fluorescence imaging and AFM studies. The developed stretching device allows applying reproducible uniaxial strain from physiologically relevant to hyperphysiological levels to cultured cells grown on elastic polydimethylsiloxane (PDMS) membranes. Exemplarily, stretching experiments are shown for transfected squamous cell carcinoma cells (SCC-25) expressing fluorescent labeled cytokeratin, whereby fluorescence imaging and simultaneously performed AFM measurements reveal the cytokeratin (CSK) network. Topographical changes and mechanical characteristics such as elasticity changes were determined via AFM while the cells were exposed to mechanical stress. By applying a cell deformation of approx. 20%, changes in the Young's modulus of the cytoskeletal network due to stretching of the cells were observed. Consequently, integrating a stretching device into the combined atomic force-fluorescence microscope provides a unique tool for dynamically analyzing structural remodeling and mechanical properties in mechanically stressed cells.

Microflow size exclusion chromatography to preserve micromolar affinity complexes and achieve subunit separations for native state mass spectrometry.

For high throughput native mass spectrometry (MS) protein characterization, it is advantageous to desalt and separate proteins by size exclusion chromatography (SEC). Sensitivity, resolution, and speed in these methods remain limited by standard SEC columns. Moreover, the efficient packing of small bore columns is notoriously difficult. SEC sensitivity is inherently limited because solutes are not focused into concentrated bands and low affinity native complexes may dissociate on column. Recent work evaluated the suitability of crosslinked gel media in small bore formats for online desalting. Here, small bore format online SEC for native MS studies is again investigated but with alternative materials. We systematically studied the utility of diol and hydroxy terminated polyethylene oxide (PEO) bonded 1.7 µm organosilica particles as packed into 1 mm ID stainless steel (SS) hardware and hardware treated with hydrophilic hybrid surface technology (h-HST). For the equivalent diol-bonded particle and hardware, UV limits of detection (LODs) were reduced 32 to 89% with a microflow separation (15 µL/min) on a 1 × 50 mm column as compared to a 4.6 × 150 mm high-flow separation (300 µL/min) at the same linear velocity. Run times were also shortened by 45%. A switch from SS to h-HST hardware led to a significant reduction in secondary interactions and a corresponding improvement in detection limits for trastuzumab, myoglobin, IgG and albumin for both UV and MS. Coupling of the small bore columns to multichannel microflow emitters resulted in 10 to 100-fold gains in MS sensitivity, depending on the analyte. MS LOD values were significantly reduced into the low attomole ranges. Columns were then evaluated for their effects on the preservation of complexes, including concanavalin A, in its apo and ligand-bound states, and three therapeutically relevant noncovalent systems previously undetected on large column formats. The results suggest that the detection of large complexes by SEC is not just a function of sensitivity but is directly affected by chemical secondary interactions. The ability to detect 0.1 to 1 MDa complexes, with between 1 and 40 micromolar dissociation constants, represents a critical advancement for high-throughput native MS workflows as applied to the analysis of therapeutics.

Neurodevelopmental scaling is a major driver of brain-behavior differences in temperament across dog breeds.

Behavioral traits like aggression, anxiety, and trainability differ significantly across dog breeds and are highly heritable. However, the neural bases of these differences are unknown. Here we analyzed structural MRI scans of 62 dogs in relation to breed-average scores for the 14 major dimensions in the Canine Behavioral Assessment and Research Questionnaire, a well-validated measure of canine temperament. Several behavior categories showed significant relationships with morphologically covarying gray matter networks and regional volume changes. Networks involved in social processing and the flight-or-fight response were associated with stranger-directed fear and aggression, putatively the main behaviors under selection pressure during wolf-to-dog domestication. Trainability was significantly associated with expansion in broad regions of cortex, while fear, aggression, and other "problem" behaviors were associated with expansion in distributed subcortical regions. These results closely overlapped with regional volume changes with total brain size, in striking correspondence with models of developmental constraint on brain evolution. This suggests that the established link between dog body size and behavior is due at least in part to disproportionate enlargement of later-developing regions in larger brained dogs. We discuss how this may explain the known correlation of increasing reactivity with decreasing body size in dogs.

A neuroanatomical predictor of mirror self-recognition in chimpanzees.

The ability to recognize one's own reflection is shared by humans and only a few other species, including chimpanzees. However, this ability is highly variable across individual chimpanzees. In humans, self-recognition involves a distributed, right-lateralized network including frontal and parietal regions involved in the production and perception of action. The superior longitudinal fasciculus (SLF) is a system of white matter tracts linking these frontal and parietal regions. The current study measured mirror self-recognition (MSR) and SLF anatomy in 60 chimpanzees using diffusion tensor imaging. Successful self-recognition was associated with greater rightward asymmetry in the white matter of SLFII and SLFIII, and in SLFIII's gray matter terminations in Broca's area. We observed a visible progression of SLFIII's prefrontal extension in apes that show negative, ambiguous, and compelling evidence of MSR. Notably, SLFIII's terminations in Broca's area are not right-lateralized or particularly pronounced at the population level in chimpanzees, as they are in humans. Thus, chimpanzees with more human-like behavior show more human-like SLFIII connectivity. These results suggest that self-recognition may have co-emerged with adaptations to frontoparietal circuitry.

Acquisition of Paleolithic toolmaking abilities involves structural remodeling to inferior frontoparietal regions.

Human ancestors first modified stones into tools 2.6 million years ago, initiating a cascading increase in technological complexity that continues today. A parallel trend of brain expansion during the Paleolithic has motivated over 100 years of theorizing linking stone toolmaking and human brain evolution, but empirical support remains limited. Our study provides the first direct experimental evidence identifying likely neuroanatomical targets of natural selection acting on toolmaking ability. Subjects received MRI and DTI scans before, during, and after a 2-year Paleolithic toolmaking training program. White matter fractional anisotropy (FA) showed changes in branches of the superior longitudinal fasciculus leading into left supramarginal gyrus, bilateral ventral precentral gyri, and right inferior frontal gyrus pars triangularis. FA increased from Scan 1-2, a period of intense training, and decreased from Scan 2-3, a period of reduced training. Voxel-based morphometry found a similar trend toward gray matter expansion in the left supramarginal gyrus from Scan 1-2 and a reversal of this effect from Scan 2-3. FA changes correlated with training hours and with motor performance, and probabilistic tractography confirmed that white matter changes projected to gray matter changes and to regions that activate during Paleolithic toolmaking. These results show that acquisition of Paleolithic toolmaking skills elicits structural remodeling of recently evolved brain regions supporting human tool use, providing a mechanistic link between stone toolmaking and human brain evolution. These regions participate not only in toolmaking, but also in other complex functions including action planning and language, in keeping with the hypothesized co-evolution of these functions.

The expression of Fc receptors for immunoglobulin G in human rectal epithelium.

The rectal mucosa is one of the routes of transmission of the HIV virus, although the mechanism of transmission is unknown. We carried out an immunohistological investigation of human rectal epithelium to detect CD4 glycoprotein and Fc receptors (FcR) for immunoglobulin G which may be involved in HIV infection. CD4 was not detected by monoclonal antibodies (MAb) in normal rectal epithelial cells, although CD4+ mononuclear cells were found in the lamina propria of the rectum. FcR3 and FcR2 were, however, detected in surface or crypt epithelial cells of rectal mucosa, using MAb to CD16 and CD32, respectively. In addition, CD16 messenger RNA (mRNA) was found in surface and crypt epithelial cells by in situ hybridization using an RNA probe. FcR3 and FcR2 were also detected in fetal recto-colonic tissue by immunohistology, suggesting that these are constitutive receptors. FcR3 and FcR2 gene transcripts were then demonstrated in fetal recto-colonic tissue using the polymerase chain reaction to amplify a portion of FcR3 and FcR2 coding sequences in complementary DNA (cDNA) prepared from fetal RNA. These findings suggest the possibility that rectal transmission of HIV-antibody complexes might be facilitated by the expression of FcR3 and FcR2 in rectal epithelial cells.

Histaminergic mechanism for drinking elicited by insulin in the rat.

Sprague-Dawley male albino rats showed in a dose-response study a maximal drinking response to a 5 U/kg dose of SC insulin in a 2-hr test. Drinking elicited by 5 U/kg insulin was reduced to baseline (i.e., no insulin) level by combined antagonism of H1 and H2 receptors for histamine using IP 1 mg/kg dexbrompheniramine plus 16 mg/kg cimetidine. Antagonism of histamine receptors in this fashion was specific for drinking elicited by histamine because such antagonism reduced to baseline level drinking elicited by 2.5 mg/kg SC histamine, but failed to inhibit drinking after 8- or 24-hr water deprivation or drinking after 0.63 mg/kg SC serotonin (5-HT). These results demonstrate a histaminergic mechanism for drinking elicited by exogenous insulin which is consistent with the published report that exogenous insulin can release gastric mucosal histamine in the rat. Moreover, because eating is known to elicit the release of endogenous insulin, the results reported here suggest a working hypothesis that endogenous insulin is a component for drinking around mealtime in the rat.

Urinary tract infections associated with otitis media in infants and children.

One hundred six infants and children with otitis media were screened for the incidence of urinary tract infections (UTI) by urine culture. Seventeen patients (16%) who had UTI were compared with the 80 patients with sterile urine for differences in host factors and laboratory features. The mean age, WBC counts, and ESR values were similar. Patients with UTI-associated otitis media had a higher incidence of hematuria. Prevalence of high (greater than or equal to 103 degrees F) fever was higher among the boys with UTI. However, because of the lack of definite clinical and laboratory clues to determine the presence or absence of UTI, urine culture is the only test to uncover otitis media patients with concomitant UTI.

What can other animals tell us about human social cognition? An evolutionary perspective on reflective and reflexive processing.

Human neuroscience has seen a recent boom in studies on reflective, controlled, explicit social cognitive functions like imitation, perspective-taking, and empathy. The relationship of these higher-level functions to lower-level, reflexive, automatic, implicit functions is an area of current research. As the field continues to address this relationship, we suggest that an evolutionary, comparative approach will be useful, even essential. There is a large body of research on reflexive, automatic, implicit processes in animals. A growing perspective sees social cognitive processes as phylogenically continuous, making findings in other species relevant for understanding our own. One of these phylogenically continuous processes appears to be self-other matching or simulation. Mice are more sensitive to pain after watching other mice experience pain; geese experience heart rate increases when seeing their mate in conflict; and infant macaques, chimpanzees, and humans automatically mimic adult facial expressions. In this article, we review findings in different species that illustrate how such reflexive processes are related to ("higher order") reflexive processes, such as cognitive empathy, theory of mind, and learning by imitation. We do so in the context of self-other matching in three different domains-in the motor domain (somatomotor movements), in the perceptual domain (eye movements and cognition about visual perception), and in the autonomic/emotional domain. We also review research on the developmental origin of these processes and their neural bases across species. We highlight gaps in existing knowledge and point out some questions for future research. We conclude that our understanding of the psychological and neural mechanisms of self-other mapping and other functions in our own species can be informed by considering the layered complexity these functions in other species.

Mirroring and mu rhythm involvement in social cognition: are there dissociable subcomponents of theory of mind?

Tager-Flusberg and Sullivan [Tager-Flusberg, H., Sullivan, K., 2000. A componential view of theory of mind: evidence from Williams syndrome. Cognition 76, 59-90] have argued for a distinction between the social-perceptive component of theory of mind (ToM), involving judgment of mental state from facial and body expressions, and the social-cognitive component, which is representation-based and linked to language and theory-building. This is analogous to the distinction made by others [Gallese, V., Keysers, C., Rizzolatti, G., 2004. A unifying view of the basis of social cognition. Trends in Cognitive Science 8, 396-403] between representing the mental state of another as if it was one's own (simulation theory), which requires involvement of the mirror neuron system, and explicit or declarative reasoning about mental states (theory theory), which does not. This componential view of ToM was tested by examining mirroring, as indexed by EEG mu rhythm suppression, in subjects performing tasks assumed to tap both dimensions. Mu suppression was positively correlated with accuracy on the social-perceptual task but not in the social-cognitive task. In a ToM control task requiring judgments about person-object interactions accuracy was correlated with mu suppression. This implies that mirroring is involved in making judgments about emotions and person-object interactions. However, mirroring is insensitive to the distinction between correct and incorrect inferences in the social-cognitive task suggesting that additional mechanisms are needed to make mental attributions of beliefs and intentions. These results are consistent with a refined componential view of ToM.

3D time-resolved MR angiography (MRA) of the carotid arteries with time-resolved imaging with stochastic trajectories: comparison with 3D contrast-enhanced Bolus-Chase MRA and 3D time-of-flight MRA.

BACKGROUND AND PURPOSE: Time-resolved MR angiography (MRA) offers the combined advantage of large anatomic coverage and hemodynamic flow information. We applied parallel imaging and time-resolved imaging with stochastic trajectories (TWIST), which uses a spiral trajectory to undersample k-space, to perform time-resolved MRA of the extracranial internal carotid arteries and compare it to time-of-flight (TOF) and high-resolution contrast-enhanced (HR) MRA. MATERIALS AND METHODS: A retrospective review of 31 patients who underwent carotid MRA at 1.5T using TOF, time-resolved and HR MRA was performed. Images were evaluated for the presence and degree of ICA stenosis, reader confidence, and number of pure arterial frames attained with the TWIST technique. RESULTS: With a consensus interpretation of all sequences as the reference standard, accuracy for identifying stenosis was 90.3% for TWIST MRA, compared with 96.0% and 88.7% for HR MRA and TOF MRA, respectively. HR MRA was significantly more accurate than the other techniques (P < .05). TWIST MRA yielded datasets with high in-plane spatial resolution and distinct arterial and venous phases. It provided dynamic information not otherwise available. Mean diagnostic confidence was satisfactory or greater for TWIST in all patients. CONCLUSION: The TWIST technique consistently obtained pure arterial phase images while providing dynamic information. It is rapid, uses a low dose of contrast, and may be useful in specific circumstances, such as in the acute stroke setting. However, it does not yet have spatial resolution comparable with standard contrast-enhanced MRA.