AQP4 autoantibody assay performance in clinical laboratory service.

OBJECTIVE: To compare performance of contemporary aquaporin-4-immunoglobulin (Ig) G assays in clinical service. METHODS: Sera from neurologic patients (4 groups) and controls were tested initially by service ELISA (recombinant human aquaporin-4, M1 isoform) and then by cell-based fluorescence assays: fixed (CBA, M1-aquaporin-4, observer-scored) and live (fluorescence-activated cell sorting [FACS], M1 and M23 aquaporin-4 isoforms). Group 1: all Mayo Clinic patients tested from January to May 2012; group 2: consecutive aquaporin-4-IgG-positive patients from September 2011 (Mayo and non-Mayo); group 3: suspected ELISA false-negatives from 2011 to 2013 (physician-reported, high likelihood of neuromyelitis optica spectrum disorders [NMOSDs] clinically); group 4: suspected ELISA false-positives (physician-reported, not NMOSD clinically). RESULTS: Group 1 (n = 388): M1-FACS assay performed optimally (areas under the curves: M1 = 0.64; M23 = 0.57 [p = 0.02]). Group 2 (n = 30): NMOSD clinical diagnosis was confirmed by: M23-FACS, 24; M1-FACS, 23; M1-CBA, 20; and M1-ELISA, 18. Six results were suspected false-positive: M23-FACS, 2; M1-ELISA, 2; and M23-FACS, M1-FACS, and M1-CBA, 2. Group 3 (n = 31, suspected M1-ELISA false-negatives): results were positive for 5 sera: M1-FACS, 5; M23-FACS, 3; and M1-CBA, 2. Group 4 (n = 41, suspected M1-ELISA false-positives): all negative except 1 (positive only by M1-CBA). M1/M23-cotransfected cells expressing smaller membrane arrays of aquaporin-4 yielded fewer false- positive FACS results than M23-transfected cells. CONCLUSION: Aquaporin-4-transfected CBAs, particularly M1-FACS, perform optimally in aiding NMOSD serologic diagnosis. High-order arrays of M23-aquaporin-4 may yield false-positive results by binding IgG nonspecifically.

Acute effects of short-chain alkylglycerols on blood-brain barrier properties of cultured brain endothelial cells.

BACKGROUND AND PURPOSE: The blood-brain barrier (BBB) restricts drug penetration to the brain preventing effective treatment of patients suffering from brain tumours. Intra-arterial injection of short-chain alkylglycerols (AGs) opens the BBB and increases delivery of molecules to rodent brain parenchyma in vivo. The mechanism underlying AG-mediated modification of BBB permeability is still unknown. Here, we have tested the effects of AGs on barrier properties of cultured brain microvascular endothelial cells. EXPERIMENTAL APPROACH: The effects of two AGs, 1-O-pentylglycerol and 2-O-hexyldiglycerol were examined using an in vitro BBB model consisting of primary cultures of rat brain endothelial cells, co-cultured with rat cerebral glial cells. Integrity of the paracellular, tight junction-based, permeation route was analysed by functional assays, immunostaining for junctional proteins, freeze-fracture electron microscopy, and analysis of claudin-claudin trans-interactions. KEY RESULTS: AG treatment (5 min) reversibly reduced transendothelial electrical resistance and increased BBB permeability for fluorescein accompanied by changes in cell morphology and immunostaining for claudin-5 and ß-catenin. These short-term changes were not accompanied by alterations of inter-endothelial tight junction strand complexity or the trans-interaction of claudin-5. CONCLUSION AND IMPLICATIONS: AG-mediated increase in brain endothelial paracellular permeability was short, reversible and did not affect tight junction strand complexity. Redistribution of junctional proteins and alterations in the cell shape indicate the involvement of the cytoskeleton in the action of AGs. These data confirm the results from in vivo studies in rodents characterizing AGs as adjuvants that transiently open the BBB.

[Influence of homeopathically processed coenzyme q10 on proliferation and redifferentiation of endothelial cells]. / Einfluss von homöopathisch aufbereitetem Coenzym Q10 auf die Proliferation und Redifferenzierung von Endothelzellen.

BACKGROUND: Coenzyme Q10 (Co Q10) is vital for regulating cell metabolism and cell proliferation. The controlled proliferation of cells is prerequisite for the regeneration of tissues. AIM: The aim of this study was to clarify whether homeopathically processed Co Q10 has an influence on the proliferation of freshly seeded endothelial cells, on the division rate of differentiated confluent endothelial cells, and on the redifferentiation of differentiated endothelial cells in vitro. METHODS: By the determination of cell numbers, the influence of Co Q10 on the proliferation of undifferentiated endothelial cells from the human umbilical vein was examined. For this assay different potencies of Co Q10 and freshly seeded endothelial cells were used. Prior to the proliferation assay the in vitro cytotoxic concentrations of Co Q10 were determined. The influence of Co Q10 on the division rate of differentiated confluent endothelial cells was determined by measuring the intake of the base analogue bromodeoxyuridine (BrdU). For the testing of differentiation, the expression of the von Willebrand factor (vWF) - the marker protein typical for endothelial cells - was observed while Co Q10 was present. A flow cytometric assay was used for the analyses. RESULTS: While only the D5 potency showed toxic effects, the other tested potencies of Co Q10 did not show any cytotoxicity. The potencies D7-D10 of Co Q10, especially the D8 potency, caused an increase in the proliferation of growing endothelial cells. By contrast, Co Q10 (D8) had no influence on the rate of incorporation of BrdU into confluent, contact-inhibited, and differentiated endothelial cells. In the case of confluent dedifferentiated cells incubated with Co Q10 (D8), no increase in the expression of the vWF was observed, either. CONCLUSIONS: Homeopathically processed Co Q10 (D8) has a stimulating influence on the proliferation of growing cells in vitro. This confirms its function in the regulation of cell metabolism and cell proliferation. The stimulating influence, however, does not extend to the redifferentiation process. Co Q10 has no effect on the low division rate of subconfluent and of confluent, contact-inhibited, differentiated endothelial cells. Furthermore the expression of endothelial cell-specific differentiation antigens on dedifferentiated endothelial cells is not influenced by Co Q10.

Antiviral action of Euphorbium compositum and its components.

INTRODUCTION: Euphorbium compositum SN (Biologische Heilmittel Heel GmbH, Baden-Baden, Germany, a homeopathic combination preparation available in form of drops, nasal spray, and injection solution), is prescribed for inflammation of the mucosae of the nose and sinuses. Infections in these areas are primarily of viral origin although bacterial superinfections are also common. OBJECTIVE: The main question was whether or not this homeopathic remedy shows an activity against viruses responsible for infections of the respiratory tract. METHODS: This in vitro study using virus plaque reduction assays examined the effect of Euphorbium compositum SN against pathogens causing various viral infections: influenza A virus, respiratory syncytial virus (RSV), human rhinovirus (HRV) and herpes simplex virus type 1 (HSV-1). RESULTS: Analysis of virus production after treatment of the infected cells with the remedy showed an antiviral activity of Euphorbium compositum SN against RSV and HSV-1. In addition, an antiviral effect against influenza A virus and HRV, though minimal, was, also noted. Analyses of the plant-derived components of Euphorbium compositum SN, e.g. Euphorbium resinifera, Pulsatilla pratensis and Luffa operculata for their antiviral activity revealed a clear activity of Euphorbium resinifera and Pulsatilla pratensis against RSV. In contrast, no effect was detected using the same protocol with Luffa operculata. CONCLUSIONS: Euphorbium resinifera and Pulsatilla pratensis as components of Euphorbium compositum SN are responsible for its antiviral activity.

Proliferation of smooth muscle cells in the inner and outer layers of the tunica media of arteries: an in vitro study.

During the development of atherosclerotic and fibromuscular proliferates/lesions, smooth muscle cells (SMC) in the media, particularly near the lumen, are activated to migrate into the intima, where they continue to proliferate to form an intimal thickening. It is to date unclear whether SMCs situated adjacent to the adventitia possess a lower capacity to proliferate because they are a special subpopulation of medial SMCs or because the adventitia excerts an inhibitory effect. We have, therefore, developed an in vitro system whereby we have attempted to clear up this uncertainty. The following observations were made from the in vitro experiments: Media-explants from rabbit aorta were laid on a polycarbonate filter with pores 5 microns in diameter. The SMCs migrated through the pores and formed a fibromuscular proliferate on the other side of the filter. Endothelial cells were seeded on one side of the filter before media-explants were laid on the other side of the filter. The confluent endothelium inhibited migration of SMCs through the filter pores. Media-explants were placed between two polycarbonate filters (pores 5 microns diameter). In this "sandwich" arrangement SMCs migrated through both filters, i.e., in both directions. The quantity of migrating and proliferating cells through both filters was almost identical. This suggests that there is no difference in the migratory and proliferative capacity of SMCs in the inner and outer layers in the media of arteries. To investigate the influence of the adventitia on medial SMCs, media-explants were placed between a lower (5 microns) and an upper (0.2 micron) filter. On the 0.2 micron filter adventitia-explants were laid above the media-explants. The 0.2 micron filter prevented migration of SMCs from the media-explant into the adventitia and migration of fibroblasts from the adventitia into the media. Interestingly, the adventitial tissue inhibited proliferation of SMCs at the abluminal and migration and proliferation at the luminal side of the media-explant; the number of cells migrating through the 5 microns pores at the luminal side was diminished, suggesting that the adventitial tissue has an antiproliferative influence on SMCs. Moreover, it was found that in media-explants near the filter with adventitia, the medial SMCs were in a better preserved condition than at the de-endothelialised luminal side. As a control, cultures consisting of media-explants were incubated without filters (i.e., explant organ cultures). The proliferates in the concavity (luminal side) exhibited a pattern of proliferating SMCs different from that of the cells at the abluminal convexity.(ABSTRACT TRUNCATED AT 400 WORDS)

Fibromuscular proliferates induced in vitro using a trans-filter culture system.

A trans-filter co-culture system of vascular smooth muscle cells (SMC) and endothelial cells (EC) is compared with a trans-filter culture of SMC without EC. Explants from the tunica media of rabbit aorta are cultured on one side of a polycarbonate filter with a defined pore size (5 microns). Smooth muscle cells grow out from the media explants and migrate through the filter pores to the other side of the filter, where they proliferate. After 14 days in trans-filter culture, a multilayer resembling "fibromuscular plaques" seen in vivo is formed on the opposite side of the filter. Moreover, the proliferation rate of SMC that have migrated through the filter pores is demonstrated to be similar to that of SMC in intimal lesions induced by balloon catheter injury. In the trans-filter co-culture, a monolayer of endothelial cells (porcine, bovine, or human) is cultivated on one side of the filter. Media explants are placed on the other side. This arrangement mimics the in vivo situation of an arterial vessel wall with endothelium and media separated by a porous lamina. In this co-culture system, the few smooth muscle cells that migrate into the subendothelial space do not form a cell multilayer. A confluent endothelial cell layer is capable of inhibiting the formation of a proliferate in this co-culture system.