Comparison of ophthalmic sponges and extraction buffers for quantifying cytokine profiles in tears using Luminex technology.

PURPOSE: Evaluating cytokine profiles in tears could shed light on the pathogenesis of various ocular surface diseases. When collecting tears with the methods currently available, it is often not possible to avoid the tear reflex, which may give a different cytokine profile compared to basal tears. More importantly, tear collection with glass capillaries, the most widely used method for taking samples and the best method for avoiding tear reflex, is impractical for remote area field studies because it is tedious and time-consuming for health workers, who cannot collect tears from a large number of patients with this method in one day. Furthermore, this method is uncomfortable for anxious patients and children. Thus, tears are frequently collected using ophthalmic sponges. These sponges have the advantage that they are well tolerated by the patient, especially children, and enable standardization of the tear collection volume. The aim of this study was to compare various ophthalmic sponges and extraction buffers to optimize the tear collection method for field studies for subsequent quantification of cytokines in tears using the Luminex technology. METHODS: Three ophthalmic sponges, Merocel, Pro-ophta, and Weck-Cel, were tested. Sponges were presoaked with 25 cytokines/chemokines of known concentrations and eluted with seven different extraction buffers (EX1-EX7). To assess possible interference in the assay from the sponges, two standard curves were prepared in parallel: 1) cytokines of known concentrations with the extraction buffers and 2) cytokines of known concentrations loaded onto the sponges with the extraction buffers. Subsequently, a clinical assessment of the chosen sponge-buffer combination was performed with tears collected from four healthy subjects using 1) aspiration and 2) sponges. To quantify cytokine/chemokine recovery and the concentration in the tears, a 25-plex Cytokine Panel and the Luminex xMap were used. This platform enables simultaneous measurement of proinflammatory cytokines, Th1/Th2 distinguishing cytokines, nonspecific acting cytokines, and chemokines. RESULTS: WE DEMONSTRATED THE FOLLOWING: (i) 25 cytokines/chemokines expressed highly variable interactions with buffers and matrices. Several buffers enabled recovery of similar cytokine values (regulated and normal T cell expressed and secreted [RANTES], interleukin [IL]-13, IL-6, IL-8, IL-2R, and granulocyte-macrophage colony-stimulating factor [GM-CSF]); others were highly variable (monocyte chemotactic protein-1 [MCP-1], monokine induced by interferon-gamma [MIG], IL-1ß, IL-4, IL-7, and eotaxin). (ii) Various extraction buffers displayed significantly different recovery rates on the same sponge for the same cytokine/chemokine. (iii) The highest recovery rates were obtained with the Merocel ophthalmic sponge except for tumor necrosis factor-α: the Weck-Cel ophthalmic sponge showed the best results, either with cytokine standards loaded onto sponges or with tears collected from the inner canthus of the eye, using the sponge. (iv) IL-5, IL-10, and interferon-α were not detected in any tear sample from four normal human subjects. Twenty-two cytokines/chemokines that we detected were extracted from the Merocel sponge to a satisfactory recovery percentage. The recovery of IL-7 was significantly lower in the extracted Merocel sponge compared to the diluted tear samples. The cytokine/chemokine extraction from tears showed the same pattern of extraction that we observed for extracting the standards. CONCLUSIONS: Simultaneous measurement of various cytokines using ophthalmic sponges yielded diverse results for various cytokines as the level of extraction differs noticeably for certain cytokines. A second set of controls (standard curves "with sponges") should be used to delineate the extent of extraction for each cytokine to be analyzed. Many cytokines/chemokines were detected in tear samples collected with the Merocel sponge, including many that have been implicated in ocular surface disease. Luminex detection of cytokine/chemokine profiles of tears collected with Merocel sponges and extracted with buffer EX1 may be useful in clinical studies, for example, to assess cytokine profiles evaluation in ocular surface diseases.

Horizontal endosymbiont transmission in hydrothermal vent tubeworms.

Transmission of obligate bacterial symbionts between generations is vital for the survival of the host. Although the larvae of certain hydrothermal vent tubeworms (Vestimentifera, Siboglinidae) are symbiont-free and possess a transient digestive system, these structures are lost during development, resulting in adult animals that are nutritionally dependent on their bacterial symbionts. Thus, each generation of tubeworms must be newly colonized with its specific symbiont. Here we present a model for tubeworm symbiont acquisition and the development of the symbiont-housing organ, the trophosome. Our data indicate that the bacterial symbionts colonize the developing tube of the settled larvae and enter the host through the skin, a process that continues through the early juvenile stages during which the trophosome is established from mesodermal tissue. In later juvenile stages we observed massive apoptosis of host epidermis, muscles and undifferentiated mesodermal tissue, which was coincident with the cessation of the colonization process. Characterizing the symbiont transmission process in this finely tuned mutualistic symbiosis provides another model of symbiont acquisition and additional insights into underlying mechanisms common to both pathogenic infections and beneficial host-symbiont interactions.

In vivo assessment of wall shear stress in the atherosclerotic aorta using flow-sensitive 4D MRI.

Our purpose was to correlate atherogenic low wall shear stress (WSS) and high oscillatory shear index (OSI) with the localization of aortic plaques. Flow-sensitive four-dimensional MRI was used to acquire three-dimensional blood flow in the aorta of 62 patients with proven aortic atherosclerosis and 31 healthy volunteers. Multiplanar data analysis of WSS magnitude and OSI in 12 wall segments was performed in analysis planes distributed along the aorta. Disturbed WSS and OSI were defined as areas exposed to low WSS magnitude and high OSI beyond individual 15% thresholds. Planewise analysis revealed a good correlation (r = 0.85) of individual low WSS magnitude but not of high OSI with plaque distribution. Although plaques occurred only rarely in the ascending aorta, the incidence of low WSS magnitude and high OSI was similar to findings in other aortic segments where plaques occurred more frequently. Case-by-case comparisons of plaque location and critical wall parameters revealed a shift of atherogenic WSS magnitude (78% of all cases) and OSI (91%) to wall segments adjacent to the atheroma. Our results indicate that the predictive value of WSS for plaque existence depends on the aortic segment and that locations of critical wall parameters move to neighboring segments of regions affected by atherosclerosis.

Retrograde embolism from the descending aorta: visualization by multidirectional 3D velocity mapping in cryptogenic stroke.

BACKGROUND AND PURPOSE: The purpose of this study was to determine the role of plaques >or=4 mm and thrombi (complex plaques) in the descending aorta (DAo) as an embolic high-risk source for stroke. METHODS: In 63 acute stroke patients scheduled for TEE, territory and embolic pattern of brain ischemia were prospectively assessed. Multidirectional 3D MRI velocity mapping of the aorta was performed to correlate the extent of retrograde diastolic blood flow with the distance of complex DAo plaques from the left subclavian artery (LSA). Embolic risk from the DAo was present for (1) retrograde flow connecting complex DAo plaques with the LSA, (2) embolic pattern of brain ischemia in a territory supplied by the left vertebral artery, and (3) stroke that could not be explained by other means. RESULTS: 33 of 63 patients had complex DAo plaques (distance to LSA 28.1+/-29.9 mm). Mean retrograde flow in these subjects was 26.2+/-12.3 mm. In 20 of 63 patients (31.7%) retrograde flow connected complex DAo plaques with the LSA. In 4 of these 20 patients (20%) with an embolic stroke in the territory of the brain stem, cerebellum or posterior cerebral artery, etiology could not be explained by other means. CONCLUSIONS: Substantial diastolic retrograde flow originating from complex plaques in the descending aorta was detected by multidirectional 3D MRI velocity mapping and constitutes a stroke mechanism that was previously not demonstrable.

Thiotrophic bacterial symbiont induces polyphenism in giant ciliate host Zoothamnium niveum.

Evolutionary theory predicts potential shifts between cooperative and uncooperative behaviour under fluctuating environmental conditions. This leads to unstable benefits to the partners and restricts the evolution of dependence. High dependence is usually found in those hosts in which vertically transmitted symbionts provide nutrients reliably. Here we study host dependence in the marine, giant colonial ciliate Zoothamnium niveum and its vertically transmitted, nutritional, thiotrophic symbiont from an unstable environment of degrading wood. Previously, we have shown that sulphidic conditions lead to high host fitness and oxic conditions to low fitness, but the fate of the symbiont has not been studied. We combine several experimental approaches to provide evidence for a sulphide-tolerant host with striking polyphenism involving two discrete morphs, a symbiotic and an aposymbiotic one. The two differ significantly in colony growth form and fitness. This polyphenism is triggered by chemical conditions and elicited by the symbiont's presence on the dispersing swarmer. We provide evidence of a single aposymbiotic morph found in nature. We propose that despite a high fitness loss when aposymbiotic, the ciliate has retained a facultative life style and may use the option to live without its symbiont to overcome spatial and temporal shortage of sulphide in nature.

Free-living tube worm endosymbionts found at deep-sea vents.

Recent evidence suggests that deep-sea vestimentiferan tube worms acquire their endosymbiotic bacteria from the environment each generation; thus, free-living symbionts should exist. Here, free-living tube worm symbiont phylotypes were detected in vent seawater and in biofilms at multiple deep-sea vent habitats by PCR amplification, DNA sequence analysis, and fluorescence in situ hybridization. These findings support environmental transmission as a means of symbiont acquisition for deep-sea tube worms.

"Candidatus Thiobios zoothamnicoli," an ectosymbiotic bacterium covering the giant marine ciliate Zoothamnium niveum.

Zoothamnium niveum is a giant, colonial marine ciliate from sulfide-rich habitats obligatorily covered with chemoautotrophic, sulfide-oxidizing bacteria which appear as coccoid rods and rods with a series of intermediate shapes. Comparative 16S rRNA gene sequence analysis and fluorescence in situ hybridization showed that the ectosymbiont of Z. niveum belongs to only one pleomorphic phylotype. The Z. niveum ectosymbiont is only moderately related to previously identified groups of thiotrophic symbionts within the Gammaproteobacteria, and shows highest 16S rRNA sequence similarity with the free-living sulfur-oxidizing bacterial strain ODIII6 from shallow-water hydrothermal vents of the Mediterranean Sea (94.5%) and an endosymbiont from a deep-sea hydrothermal vent gastropod of the Indian Ocean Ridge (93.1%). A replacement of this specific ectosymbiont by a variety of other bacteria was observed only for senescent basal parts of the host colonies. The taxonomic status "Candidatus Thiobios zoothamnicoli" is proposed for the ectosymbiont of Z. niveum based on its ultrastructure, its 16S rRNA gene, the intergenic spacer region, and its partial 23S rRNA gene sequence.