Fixation stability and implication for multifocal electroretinography in patients with neovascular age-related macular degeneration after anti-VEGF treatment.

PURPOSE: To quantify fixation stability in patients with neovascular age-related macular degeneration (nAMD) at baseline, 3 and 6 months after anti-vascular endothelial growth factor (anti-VEGF) treatment and furthermore asses the implications of an unsteady fixation for multifocal electroretinography (mfERG) measurements. METHODS: Fifty eyes of 50 nAMD patients receiving intravitreal anti-VEGF treatment with either bevacizumab or ranibizumab and eight eyes of eight control subjects were included. Fixation stability measurements were performed with the Eye-Link eyetracking system and the retinal area in degrees2 (deg2) containing the 68 % most frequently used fixation points (RAF68) was calculated. MfERG P1 amplitude and implicit time were analyzed in six concentric rings and as a summed response. Patients were examined at baseline, 3 and 6 months. Four different mfERG recordings were performed for the control subjects to mimic an involuntary unstable fixation: normal central fixation, 2.4°, 4.8°, and 7.1° fixation instability. RESULTS: For control subjects, a fixation instability of 2.4° (corresponding to the central hexagon) did not reduce mfERG ring amplitudes significantly, whereas 4.8° and 7.1° fixation instability reduced the amplitudes significantly in rings 1 and 2 (p < 0.001) as well as in the peripheral rings in the 7.1° instability condition (p < 0.001). Fixation stability improved non-significantly for patients at 3 and 6 months. The size of the retinal area of fixation was at baseline, 3 and 6 months negatively correlated to visual acuity (VA) (rbaseline = -0.65, r3 months = -0.60, and r6 months = -0.66 respectively, p < 0.001) and mfERG amplitudes of the three innermost rings (rbaseline = -0.29, p = 0.042, r3 months = -0.43, p = 0.003 and r6 months = -0.31, p = 0.042). The VA cutoff for a fixation area less than 5 deg2 (approximately the central hexagon) was 65, 77, and 68 ETDRS letters (corresponding a maximal Snellen equivalent of 0.31) at baseline, 3 and 6 months, respectively. CONCLUSIONS: MfERG amplitudes in recordings of nAMD patients are at substantial risk of being reduced due to poor fixation as a large number of patients may use a fixation area of more than 5 deg2. Fixation monitoring during recording as well as interpretation of results should be performed with care, especially in patients with poor visual acuity.

Interaction of Amyloid-ß-(1-42) Peptide and Its Aggregates with Lipid/Water Interfaces Probed by Vibrational Sum-Frequency Generation Spectroscopy.

In this study, we use surface-sensitive vibrational sum-frequency generation (VSFG) spectroscopy to investigate the interaction between model lipid monolayers and Aß(1-42) in its monomeric and aggregated states. Combining VSFG with atomic force microscopy (AFM) and thioflavin T (ThT) fluorescence measurements, we found that only small aggregates with probably a ß-hairpin-like structure adsorbed to the zwitterionic lipid monolayer (DOPC). In contrast, larger aggregates with an extended ß-sheet structure adsorbed to a negatively charged lipid monolayer (DOPG). The adsorption of small, initially formed aggregates strongly destabilized both monolayers, but only the DOPC monolayer was completely disrupted. We showed that the intensity of the amide-II' band in achiral (SSP) and chiral (SPP) polarization combinations increased in time when Aß(1-42) aggregates accumulated at the DOPG monolayer. Nevertheless, almost no adsorption of preformed mature fibrils to DOPG monolayers was detected. By performing spectral VSFG calculations, we revealed a clear correlation between the amide-II' signal and the degree of amyloid aggregates (e.g., oligomers or (proto)fibrils) of various Aß(1-42) structures. The calculations showed that only structures with a significant amyloid ß-sheet content have a strong amide-II' intensity, in line with previous Raman studies. The combination of the presented results substantiates the amide-II(') band as a legitimate amyloid marker.

Regulation of tissue-degrading factors and in vitro invasiveness in progression of breast cancer cells.

Hormone-independent growth and invasiveness represent phenotypic properties acquired during early progression of breast cancer. We compared human mammary adenocarcinoma cells, MCF-7, which are estrogen-dependent and poorly metastatic, with the estrogen-independent and highly metastatic subline, MCF7/LCC1, with regard to expression of tissue-degrading factors of the matrix metalloproteinase (MMP)-and urokinase (uPA)-dependent degradative pathways, as well as for their in vitro invasive properties. Both cell lines showed low constitutive mRNA expression of the MMP inhibitor TIMP-1. Baseline expression of TIMP-2 mRNA was also very low in MCF-7 cells, whereas the MCF7/LCC1 level was much higher (approximately 10-fold). Furthermore, both cell lines revealed low constitutive capacity to migrate in an in vitro invasion assay. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA; 100 nM) induced the mRNAs for TIMP-1 as well as for MMP-1, MMP-9, the uPA receptor, and the uPA inhibitor PAI-1, amongst which only the responses of MMP-9 and PAI-1 were cell-specific. The mRNA levels of MMP-9 and PAI-1 were approximately 10-fold and approximately 15-fold higher in MCF7/LCC1 cells compared to MCF-7 cells. The secretion of immunoreactive PAI-1 was considerably elevated (> 20-fold) in TPA-treated MCF7/LCC1 cells, whereas the TPA-dependent level of 92-kDa MMP-9 was only approximately 2-fold higher in MCF7/LCC1 cells than in MCF-7 cells. In both cell lines treatment with TPA was associated with an increase (approximately 10-fold) in in vitro migration, which in the MCF7/LCC1 cells was significantly attenuated by a reconstituted basement membrane extract (Matrigel). These data suggest that TPA-responsive in vitro invasive properties that are probably associated with PAI-1 expression may co-vary with progression from hormone-dependent to -independent breast cancer.

Characterization of the dermal lesions induced by a purified protein from toxigenic Pasteurella multocida.

The dermonecrotic effect of purified Pasteurella multocida toxin (PMT) was studied sequentially in guinea pigs and rats. The skin reaction was initially an acute inflammatory reaction, with edema and emigration of neutrophils and a few eosinophils and diapedesis of some erythrocytes. Four hours after intracutaneous injection the vessels were congested and thrombocytes were focally attached to the endothelial wall. Twenty-four h after the injection the inflammatory reaction appeared more severe and venules and arterioles were thrombosed. Necrotic changes were seen in hair follicles and in striated muscle fibers. Crude extracts from P. multocida and Clostridium perfringens injected intracutaneously into guinea pigs induced skin lesions qualitatively similar to the lesions induced by the purified PMT, indicating that dermonecrotic bacterial toxins may share similar biochemical properties.

The pathogenesis of atrophic rhinitis in pigs induced by toxigenic Pasteurella multocida.

The pathogenesis of atrophic rhinitis was studied in an experiment in which piglets were infected with a toxigenic type D Pasteurella multocida strain in the right half of the nasal cavity. Two days before inoculation the nasal mucosa on the right side had been subjected to mild irritation by intranasal instillation of a weak solution of acetic acid. The untreated (left) half of the nasal cavity served as an intrinsic control. Macroscopically, changes in the turbinates were already appreciable at 3 days p.i., and pronounced turbinate atrophy was noted at 7 days p.i. At 14 days p.i. deviation of the snout and almost complete turbinate atrophy was observed. The turbinates in the untreated half of the nasal cavity developed normally. Histologically, the changes were initially characterized by bone resorption mediated by an increased number of osteoclasts. Later osteoclasts were sparse, and there was an apparent disruption of osteoid synthesis. Ultrastructurally, the osteoblasts showed nuclear indentations and dilatation of the endoplasmic reticulum. Since no inflammatory reaction was observed, the hypothesis is advanced that atrophic rhinitis in pigs is caused by a P. multocida-produced factor which will stimulate bone resorption and suppress osteoid synthesis.

The need for a veterinary antibiotic policy.

The international recognition of the 'stable to table' approach to food safety emphasises the need for appropriate and safe use of antibiotics in animal production. An appropriate use of antibiotics for food animals will preserve the long-term efficacy of existing antibiotics, support animal health and welfare and limit the risk of transfer of antibiotic resistance to humans. Furthermore, it may promote consumer confidence in the veterinary use of antibiotics. In advancing these arguments, the authors of this article argue that there is a need for a visible and operational policy for veterinary use of antibiotics, paying particular attention to the policies that are being developed in Denmark.

The serology of Bordetella bronchiseptica isolated from pigs compared with strains from other animal species.

The serology of freshly isolated strains of Bordetella bronchiseptica from pigs and some other animal species was studied by the agglutination and agglutinin absorption tests. Differentiation was made between heat stable O-antigens and heat labile K-antigens. All the strains examined contained a common O-antigen (1). In addition, three heat stable antigens were detected. The heat labile antigens of freshly isolated strains were found to be very different. The major antigens 2 and 3 were not found in combination. A common weak K-antigen (1) was found in all strains. The pig strains were found to be serologically identical (0, 1, 2; K 1, 2). The same serotype was isolated from a rabbit. Another rabbit strain was found to be similar to a strain isolated from an SPF-rat (O, 1, 3; K 1, 3, 4). The antigenic pattern found in two cat strains was O 1, 3; K 1, 3. Typing of freshly isolated strains from pigs with monospecific factor sera confirmed that all the strains so far examined were serologically identical. This uniform serology hampers epidemiological studies of porcine bordetellosis by antigenic analysis. The occurrence of serologically different serotypes of B. bronchiseptica is of significance in the diagnostic bacteriology since the final diagnosis must be confirmed by a serological test.