BCG Skin Infection Triggers IL-1R-MyD88-Dependent Migration of EpCAMlow CD11bhigh Skin Dendritic cells to Draining Lymph Node During CD4+ T-Cell Priming

The transport of antigen from the periphery to the draining lymph node (DLN) is critical for T-cell priming but remains poorly studied during infection with Mycobacterium bovis Bacille Calmette-Guérin (BCG). To address this we employed a mouse model to track the traffic of Dendritic cells (DCs) and mycobacteria from the BCG inoculation site in the skin to the DLN. Detection of BCG in the DLN was concomitant with the priming of antigen-specific CD4⁺ T cells at that site. We found EpCAM^low CD11b^high migratory skin DCs to be mobilized during the transport of BCG to the DLN. Migratory skin DCs distributed to the T-cell area of the LN, co-localized with BCG and were found in close apposition to antigen-specific CD4⁺ T cells. Consequently, blockade of skin DC traffic into DLN dramatically reduced mycobacterial entry into DLN and muted T-cell priming. Interestingly, DC and mycobacterial entry into the DLN was dependent on IL-1R-I, MyD88, TNFR-I and IL-12p40. In addition, we found using DC adoptive transfers that the requirement for MyD88 in BCG-triggered migration was not restricted to the migrating DC itself and that hematopoietic expression of MyD88 was needed in part for full-fledged migration. Our observations thus identify a population of DCs that contribute towards the priming of CD4⁺ T cells to BCG infection by transporting bacilli into the DLN in an IL-1R-MyD88-dependent manner and reveal both DC-intrinsic and -extrinsic requirements for MyD88 in DC migration.     

Views of Swedish Commissioning Parents Relating to the Exploitation Discourse in Using Transnational Surrogacy

Transnational surrogacy, when people travel abroad for reproduction with the help of a surrogate mother, is a heavily debated phenomenon. One of the most salient discourses on surrogacy is the one affirming that Westerners, in their quest for having a child, exploit poor women in countries such as India. As surrogacy within the Swedish health care system is not permitted, Swedish commissioning parents have used transnational surrogacy, and the majority has turned to India. This interview study aimed to explore how commissioning parents negotiate the present discourses on surrogacy. Findings from the study suggest that the commissioning parents’ views on using surrogacy are influenced by competing discourses on surrogacy represented by media and surrogacy agencies. The use of this reproductive method resulted, then, in some ambiguity. Although commissioning parents defy the exploitation discourse by referring to what they have learnt about the surrogate mother’s life situation and by pointing at the significant benefits for her, they still had a request for regulation of surrogacy in Sweden, to better protect all parties involved. This study, then, gives a complex view on surrogacy, where the commissioning parents simultaneously argue against the exploitation discourse but at the same time are uncertain if the surrogate mothers are well protected in the surrogacy arrangements. Their responses to the situation endorse the need for regulation both in Sweden and India.     

Influence of Thin Slice Reconstruction on CT Brain Perfusion Analysis

Objectives

Although CT scanners generally allow dynamic acquisition of thin slices (1 mm), thick slice (≥5 mm) reconstruction is commonly used for stroke imaging to reduce data, processing time, and noise level. Thin slice CT perfusion (CTP) reconstruction may suffer less from partial volume effects, and thus yield more accurate quantitative results with increased resolution. Before thin slice protocols are to be introduced clinically, it needs to be ensured that this does not affect overall CTP constancy. We studied the influence of thin slice reconstruction on average perfusion values by comparing it with standard thick slice reconstruction.

Materials and Methods

From 50 patient studies, absolute and relative hemisphere averaged estimates of cerebral blood volume (CBV), cerebral blood flow (CBF), mean transit time (MTT), and permeability-surface area product (PS) were analyzed using 0.8, 2.4, 4.8, and 9.6 mm slice reconstructions. Specifically, the influence of Gaussian and bilateral filtering, the arterial input function (AIF), and motion correction on the perfusion values was investigated.

Results

Bilateral filtering gave noise levels comparable to isotropic Gaussian filtering, with less partial volume effects. Absolute CBF, CBV and PS were 22%, 14% and 46% lower with 0.8 mm than with 4.8 mm slices. If the AIF and motion correction were based on thin slices prior to reconstruction of thicker slices, these differences reduced to 3%, 4% and 3%. The effect of slice thickness on relative values was very small.

Conclusions

This study shows that thin slice reconstruction for CTP with unaltered acquisition protocol gives relative perfusion values without clinically relevant bias. It does however affect absolute perfusion values, of which CBF and CBV are most sensitive. Partial volume effects in large arteries and veins lead to overestimation of these values. The effects of reconstruction slice thickness should be taken into account when absolute perfusion values are used for clinical decision making.     

Rolling circle amplification-based detection of human topoisomerase I activity on magnetic beads

A high-sensitivity assay has been developed for the detection of human topoisomerase I with single molecule resolution. The method uses magnetic sepharose beads to concentrate rolling circle products, produced by the amplification of DNA molecules circularized by topoisomerase I and detectable with a confocal microscope as single and discrete dots, once reacted with fluorescent probes. Each dot, corresponding to a single cleavage–religation event mediated by the enzyme, can be counted due to its high signal/noise ratio, allowing detection of 0.3 pM enzyme and representing a valid method to detect the enzyme activity in highly diluted samples.     

Teaching acid/base physiology in the laboratory

Acid/base homeostasis is one of the most difficult subdisciplines of physiology for medical students to master. A different approach, where theory and practice are linked, might help students develop a deeper understanding of acid/base homeostasis. We therefore set out to develop a laboratory exercise in acid/base physiology that would provide students with unambiguous and reproducible data that clearly would illustrate the theory in practice. The laboratory exercise was developed to include both metabolic acidosis and respiratory alkalosis. Data were collected from 56 groups of medical students that had participated in this laboratory exercise. The acquired data showed very consistent and solid findings after the development of both metabolic acidosis and respiratory alkalosis. All results were consistent with the appropriate diagnosis of the acid/base disorder. Not one single group failed to obtain data that were compatible with the diagnosis; it was only the degree of acidosis/alkalosis and compensation that varied.     

Molecular systematics and morphological character evolution of the Condamineeae (Rubiaceae)

? Premise of the study: The Condamineeae have in previous molecular studies been shown to be part of an early-divergent clade within the subfamily Ixoroideae, together with the tribes Calycophylleae, and Hippotideae, and genera of the former Cinchoneae and Rondeletieae. Generic relationships within this clade have, however, remained largely unresolved. ? Methods: In this study, the systematics of the Condamineeae was further examined by phylogenetic reconstruction of six cpDNA regions and one nrDNA region using parsimony and Bayesian Markov chain Monte Carlo inference. Morphological character evolution within the tribe was assessed by ancestral state reconstruction using likelihood optimization of characters onto Bayesian trees. ? Key results: Calycophylleae appears polyphyletic. "Hippotideae" is monophyletic but nested within the Condamineeae. The phylogenetic hypotheses presented support a resurrection of the genera Holtonia, Schizocalyx, and Semaphyllanthe. Furthermore, Bathysa is found to be polyphyletic, Tresanthera is found nested within Rustia, and the taxonomically disputed genus Dialypetalanthus is here shown to be sister to a Bothriospora-Wittmackanthus clade. Morphological ancestral state reconstructions indicate that protogyny have evolved at least two times within the tribe and that indehiscent fruits, loculicidal fruit dehiscence, and intrapetiolar stipules have evolved independently several times. The occurrence of calycophylls (leaf-like calyx lobes), poricidal anthers, and winged seeds also appear homoplastic within the tribe. ? Conclusions: A diagnosis and delimitation of the tribe Condamineeae is presented, with taxonomic proposals to synonymize Tresanthera and to transfer several species of Bathysa as well as Phitopis to a resurrected Schizocalyx.     

Divergence time uncertainty and historical biogeography reconstruction – an example from Urophylleae (Rubiaceae)

Aim When hypotheses of historical biogeography are evaluated, age estimates of individual nodes in a phylogeny often have a direct impact on what explanation is concluded to be most likely. Confidence intervals of estimated divergence times obtained in molecular dating analyses are usually very large, but the uncertainty is rarely incorporated in biogeographical analyses. The aim of this study is to use the group Urophylleae, which has a disjunct pantropical distribution, to explore how the uncertainty in estimated divergence times affects conclusions in biogeographical analysis. Two hypotheses are evaluated: (1) long‐distance dispersal from Africa to Asia and the Neotropics, and (2) a continuous distribution in the boreotropics, probably involving migration across the North Atlantic Land Bridge, followed by isolation in equatorial refugia. Location Tropical and subtropical Asia, tropical Africa, and central and southern tropical America. Methods This study uses parsimony and Bayesian phylogenetic analyses of chloroplast DNA and nuclear ribosomal DNA data from 56 ingroup species, beast molecular dating and a Bayesian approach to dispersal–vicariance analysis (Bayes‐DIVA) to reconstruct the ancestral area of the group, and the dispersal–extinction–cladogenesis method to test biogeographical hypotheses. Results When the two models of geographic range evolution were compared using the maximum likelihood (ML) tree with mean estimates of divergence times, boreotropical migration was indicated to be much more likely than long‐distance dispersal. Analyses of a large sample of dated phylogenies did, however, show that this result was not consistent. The age estimate of one specific node had a major impact on likelihood values and on which model performed best. The results show that boreotropical migration provides a slightly better explanation of the geographical distribution patterns of extant Urophylleae than long‐distance dispersal. Main conclusions This study shows that results from biogeographical analyses based on single phylogenetic trees, such as a ML or consensus tree, can be misleading, and that it may be very important to take the uncertainty in age estimates into account. Methods that account for the uncertainty in topology, branch lengths and estimated divergence times are not commonly used in biogeographical inference today but should definitely be preferred in order to avoid unwarranted conclusions.     

Which geometric model for the curvature of 2-D shape contours?

We investigated the geometric representations underlying the perception of 2-D contour curvature. 88 arcs representing lower and upper halves of concentric circles, or halves of ellipses derived mathematically through planar projection by affinity with the circles, a special case of Newton's transform, were generated to produce curved line segments with negative and positive curvature and varying sagitta (sag) and/or aspect ratio. Aspect ratio is defined here as the ratio between the sagitta and the chord-length of a given arc. The geometric properties of the arcs suggest a regrouping into four structural models. The 88 stimuli were presented in random order to 16 observers eight of whom were experienced in the mathematical and visual analysis of 2-D curvature ('expert observers'), and eight of whom were not ('non-expert observers'). Observers had to give a number, on a psychophysical scale from 0 to 10, that was to reflect the magnitude of curvature they perceived in a given arc. The results show that the subjective magnitude of curvature increases exponentially with the aspect ratio and linearly with the sagitta of the arcs for both experts and nonexperts. Statistical analysis of the correlation coefficients of linear fits to individual data represented on a logarithmic scale reveals significantly higher correlation coefficients for aspect ratio than for sagitta. The difference is not significant when curves with the longest chords only (7 degrees -10 degrees ) are considered. The geometric model that produces the best psychometric functions is described by a combination of arcs of vertically and horizontally oriented ellipses, indicating that perceptual sensations of 2-D contour curvature are based on geometric representations that suggest properties of 3-D structures. A 'buckled bar model' is shown to optimally account for the perceptual data of all observers with the exception of one expert. His perceptual data can be linked to a more analytical, less 'naturalistic' representation originating from a specific perceptual experience, which is discussed. It is concluded that the structural properties of 'real' objects are likely to determine even the most basic geometric representations underlying the perception of curvature in 2-D images. A specific perceptual learning experience may engender changes in such representations.     

Endothelin-1 and pancreatic islet vasculature: studies in vivo and on isolated, vascularly perfused pancreatic islets

Endothelin-1 (ET-1) is a potent endothelium-derived vasoconstrictor, which also stimulates insulin release. The aim of the present study was to evaluate whether exogenously administered ET-1 affected pancreatic islet blood flow in vivo in rats and the islet arteriolar reactivity in vitro in mice. Furthermore, we aimed to determine the ET-receptor subtype that was involved in such responses. When applying a microsphere technique for measurements of islet blood perfusion in vivo, we found that ET-1 (5 nmol/kg) consistently and markedly decreased total pancreatic and especially islet blood flow, despite having only minor effects on blood pressure. Neither endothelin A (ET(A)) receptor (BQ-123) nor endothelin-B (ET(B)) receptor (BQ-788) antagonists, alone or in combination, could prevent this reduction in blood flow. To avoid confounding interactions in vivo, we also examined the arteriolar vascular reactivity in isolated, perfused mouse islets. In the latter preparation, we demonstrated a dose-dependent constriction in response to ET-1. Administration of BQ-123 prevented this, whereas BQ-788 induced a right shift in the response. In conclusion, the pancreatic islet vasculature is highly sensitive to exogenous ET-1, which mediates its effect mainly through ET(A) receptors.