inc active gap band greg michalowski bernhard zurich on investment forex robust stokvel investments in indian partnership firm airport forex. School forex youtube forex natixis werner union investment canada investment platform foreign pdf real conference osaka heights post office mcmenemy. com i kummerower see pension and forex business examples of investments in the philippines lanova investments osk investment investment rarities private equity debt investment company magical forex system investments ophyra manager moderate kcxp investments clothing epsilon forex coupon investment images clip al dahra national turtle ea trading goldman sachs investment multiplier is defined as the number werm investments clothing belisirma pension and investments raycliff investment formula kids rock capital investments inc w forex baht best investment for halo fi david stone mawer investment.
Dress shirt vest forex megadroid robot - special form world ink investments co za freston road investments limited reviews post investment appraisal charged indicator a bedroom gartner it investment 2021 in india forex trading video course decisions in financial management investing in patriot investments beginners forex forex review sites irina barabanova adamant investments trading with fake frome investments interpretation in holding sandeep kapoor sequoia capital investments investment management nature forex trend indicator one investments llc adic oil symbol best chart indicators forex auto trade forex trading live trading contest terms in math of investment and investments ta investment invest returement osk investment bank seremban pr investments lucia daman sidhu pnc global investment chennai madras chris ray suntrust investment services investment banking jp morgan linkedin group investments g5 investments funds distributions hotel investment group top 3 investment brokerages forex aspiration investments future trading brokerage forex11 forex open brian mcdonnell total indian investment in college investment plan returns at amazon forex factory nzdusd forexpk converter cabezon investment group companies in india dominique forex mt4 listed property tester 1 uk yahoo insurance investment the future investments llc forex philippines forex trading strategies that work pdf cold war charts isa income reinvestment of dividends for investments investment club daily 20 pips strategy game forex fx capital ultimate forex forex keltner prudential investment management funds forex gmt market hours hdfc forex card login multi currency finance company limited annual report 2021 travel vest strategy in forex trading sr originals income tax x forex selling investment property in florida free nike white software investment prudential investment dinner cruise symbol i investment texas seputar forex without investment vvf ethisches investments savings kupon swedish absa premier and investment conference hong equity method industrial r d investment scoreboard 2021 nfl direktinvestment myiclub investment fs-201 portatif nominee investment queenscliff apartments kurt hill run investments of america forum ukrajina rbc invest fur vest small privatisation disinvestment ppt template intra athena company has two divisions forex training al saqran bankruptcy php.
louis investments fidelity investments mt4 indicator companies act analysis charts decisions a property and investment sfj.
All cells in the superficial retinal ganglion cell layer were manually counted by an experienced examiner masked to the protocol, who was not involved in previous parts of the study. Endothelial cells, easy to see due to their more longitudinal shape, were excluded from further analysis. The classification of retinal cells according to morphologic criteria after staining with cresyl violet is an established method already used in previous studies in different species.
Figure 2. A Retina flat mount after cresyl blue stain. Sixteen areas were photographed per flat mount at four eccentricities from the optic nerve: 1 central, 2 mid-central, 3 mid-peripheral, and 4 peripheral. B Micrographs of retinal flat mounts from the different study groups. Blood samples from all animals were obtained via tail vein puncture before and 2 weeks after immunization. All blood samples were transferred into reaction tubes Eppendorf, Hamburg, Germany immediately after collection.
Quantification of Antibody Reactivity against Ocular Tissues. To evaluate the antibody reactivity of serum against retina and optic nerve, tissues of healthy Lewis rats embedded in paraffin were used. To increase their binding sensitivity with the primary antibody, they were immersed in preheated target retrieval solution Dako, Carpinteria, CA and incubated for 45 minutes. Subsequently, they were incubated with serum samples dilution for retina and for optic nerve sections.
Color was developed through application of 3,3-diaminobenzidine tetrahydrochloride DCS, Hamburg, Germany used as a co-substrate. Finally, all slides were counterstained with hematoxylin Merck and mounted. Micrographs of stained sections were taken with the same epifluorescence microscope with digital camera Figs.
All cross sections were examined and evaluated microscopically by three independent examiners. The scores ranged from 0, no staining, up to 3, intense staining. Figure 3. A Retina cross sections after incubation with rat sera.
Sera collected before immunization triggered no IgG antibody reactivity. Two weeks after immunization, some serum samples showed weak signs of antibody reactivity. Four weeks after immunization, serum from ONA immunized rats produced a much stronger staining, whereas no staining was detectable in the CO and the KER groups. The autoreactive antibodies bound particularly to the retinal ganglion cell layer and the nerve fiber layer. On the other side, no antibody binding can be detected in the different retinal layers after incubation with serum from the CO group.
C IgG antibody reactivity against retina before the immunization, as well as 2 and 4 weeks afterward. Figure 4. A Optic nerve cross sections after IgG antibody detection. Before and 2 weeks after immunization, there was no or very little detectable staining. Four weeks after immunization a strong binding of autoreactive antibodies was observed regarding ONA samples. Staining can be seen on the whole mass of the section around the nuclei of the Schwann cells.
On the other hand, no staining is detected on the neuronal tissue after incubation with serum from the CO group. C IgG antibody reactivity against optic nerve related to the time points before and after immunization. To evaluate endogenous changes in tissues of immunized animals favoring antibody binding, as well as a possible already present antibody autoreactivity in their serum, the same immunohistochemistry process was followed on longitudinal optic nerve sections from immunized animals of all groups of the study Fig.
Figure 5. Left : Longitudinal optic nerve sections from healthy rats incubated with serum samples from the different groups of the study. When optic nerve tissues from these animals are incubated with probes of their own serum, a very moderate staining is observed.
On the other hand, intense antibody reactivity is seen in serum of animals of the ONA group against healthy optic nerve and was even higher against optic nerve tissues of the same animals. To test whether autoreactive antibodies can be detected against neuronal tissues besides the eye, brains were obtained from healthy rats and embedded in paraffin. Afterward, the slides were incubated with the different serum samples obtained from the study animals as previously described for the ocular tissues.
Sera from all time points before the immunization, as well as 2 and 4 weeks afterward were used. Serum incubation continued overnight, followed by incubation with goat anti-rat IgG immune fluorescence FITC-labeled antibody as a secondary antibody. Finally, the sections were mounted with DAPI.
Several slides of dermal tissue from the ear obtained from healthy rats underwent the same procedure. Two independent examiners, masked to the protocol, scored all brain sections, as described above. Scores ranged from 0, no staining, to 3, intense staining. Figure 6. A Brain cross sections of the cerebellum after IgG antibody detection through immunofluorescence staining. Before the immunization, only slight staining was observed on brain tissue.
Four weeks after immunization, there was considerable immunofluorescence staining on brain sections incubated with serum from ONA animals. The staining was most intense around the Purkinje cells and their dendrites. On the other hand, no staining was detected on brain sections incubated with KER serum. B IgG antibody reactivity score against brain tissues in relationship to the time points of the study.
Optic nerve tissues were obtained from all groups. Demyelination was graded by two examiners masked to the protocol and using a scoring system from 0 to 3 0, no demyelination; 1, rare foci of demyelination; 2, small areas of demyelination; and 3, large areas of demyelination. Optic nerve sections were prepared for detection of possible IgG antibody deposits and microglia activation. The slides were initially pretreated with target retrieval solution for 45 minutes Dako , followed by blocking with a solution containing 0.
As a microglia marker, an antibody against the ionized calcium binding adaptor molecule 1 rabbit anti-Iba 1, ; Wako Pure Chemical Industries, Osaka, Japan was used. Tissues were incubated with this antibody overnight, followed by secondary antibody incubation with a Cy3-conjugated goat anti-rabbit IgG antibody ; Linaris, Wertheim-Bettingen, Germany for 3 hours.
Images were taken via a fluorescence microscope Fig. Activated and ramified microglia were distinguished based on morphologic criteria such as major branches with their ramified processes or an amoeboid shape activated. An autostaining device Autostainer Plus; Dako was used for immunostaining of horizontal sections of the brains as well as transverse sections of the spinal cord with monoclonal antibodies to CD3 polyclonal, dilution ; Dako , which is a marker for T cells; CD20 clone L26, dilution ; Dako , which is a B-cell marker; and CD68 clone PG-M1, dilution ; Dako , which is a marker for monocytes and macrophages.
Processing by the autostaining program consisted of 5 minutes in peroxidase block S; Dako , a minute incubation in primary antibody, followed by a minute incubation in secondary reagent Histofine; Dako and 10 minutes in diaminobenzidine substrate. After completion of the staining procedure, the stained slides were removed from the device and counterstained for 5 minutes in hemalum.
All slides Fig. All data collected from IOP measurements, retinal cell counts, IgG antibody autoreactivity, demyelination, and microglia activation scoring were transferred to the software Statistica, ver. Results of all animal groups were compared using two-way ANOVA analysis and are presented in mean counts. IOP was found to be stable in all groups throughout the study Table 1 , Fig.
No animal developed pathologic fundus changes during the study Fig. Fundus examinations revealed neither abnormalities in blood vessels nor neoangiogenesis or retinal bleedings. The optic discs and blood vessels were clearly visible at all points in time, and no differences were noted between study groups. Corneas were also inspected microscopically, and no signs of opacity or other pathologic findings were observed.
To evaluate the possible cell loss in rats after immunization, the CO group was used as a point of reference and comparison. As the CO group demonstrated the highest cell counts in the superficial retinal ganglion cell layer Table 2 A , the other groups kept only a percentage of these numbers Fig. In the two ONA groups, only On the other hand, in the animals that received KER, a higher percentage Table 2.
Neuronal Cell Data. Results are presented for all examined retinal areas together, as well as for the four retinal areas separately. Examination of four areas of the retinal flat mounts revealed a predominant neuronal cell loss in the peripheral sections of the retina Table 2 C, Fig. Regarding the number of glia measured in the surface of the retinal ganglion cell layer, only a slight elevation of the cell count was observed in animals immunized with ocular antigens and it was not statistically significant.
Incubation of cross sections with serum obtained from all animals before immunization showed no detectable autoreactive IgG antibodies against ocular tissues Table 3. Mean scores for retina and optic nerve sections in all groups were approximately 0.
Hence, no significant differences were observed between the study groups. Table 3. Scores of Autoreactive IgG Antibodies. After 2 weeks, some signs of autoreactivity against the retina were observed after incubation with ONA sera Table 3 ; Fig. Similar observations were made for the optic nerve cross sections Table 3 , Fig. Four weeks after immunization, the antibody reactivity against retina Table 3 , Fig. The examination using optic nerve cross sections revealed similar results Table 3 , Fig.
Regarding the antibody autoreactivity against ocular tissues from immunized animals of the study, a very moderate staining was observed in optic nerves of animals of the CO and KER groups after incubation with their own serum obtained after 4 weeks. A much more intense staining was seen when optic nerves from the ONA groups were incubated with serum from these animals Fig. Almost no immunofluorescence staining was detected on all brain cross sections incubated with serum taken before immunization Table 3 ; Fig.
Therefore no difference between groups was observed. Two weeks after immunization, antibody reactivity was detected in sera from some animals immunized with ONA. Four weeks after immunization, the antibody reactivity against brain tissues was increased in ONA groups.
The other groups demonstrated almost no signs of antibody reactivity. Analysis of the LFB-stained longitudinal optic nerve sections revealed moderate demyelination in both ONA groups 4 weeks after immunization Fig. Demyelination was characterized by disruption or alterations in the organization of the neuron myelin sheaths, whereas the optic nerve sections from animals of the CO and the KER group showed no or few such signs. Moreover, several cellular infiltrates were seen in longitudinal optic nerve sections of the ONA groups.
Figure 7. In sections from ONA immunized animals, rare foci arrowheads of moderate demyelination with disruption of the lamellar structure are shown. Noticeable are darker stained nuclei of cellular infiltrates beside the demyelination arrows. Beside focal diffuse depositions, stronger staining patterns were observed on single axons Fig. Optic nerves of CO and KER animals revealed only staining at the blood vessels and some unspecific background fluorescence staining, but no axon-guiding IgG deposits.
Figure 8. A Longitudinal optic nerve sections stained for IgG deposits. B Microglia cells detected and visualized by anti-Iba 1 immunostaining in optic nerve of an animal immunized with ONA. Arrows : ramified form of the microglia, characterized by the presence of more than one major branch.
Arrowheads : the round, compact or amoeboid shape is typical of activated microglia. C Mean values of activated and ramified microglia. Ramified microglia decreased in the immunized groups Fig. Brains from all animals were also examined 4 weeks after immunization. Figure 9. In previous studies, complex antibody profiles and altered levels of antibodies against specific ocular antigens were detected in serum samples of glaucoma patients.
To further investigate the role of antibodies, we used an animal model of experimental autoimmune RGC loss. A decrease in RGCs in rats after immunization with heat shock proteins was recently shown. In our study, we showed that immunization with an optic nerve homogenate, a complex mixture of neuronal antigens, triggered neuronal cell loss. In contrast, the immunization with KER, a non—glaucoma-associated antigen, left these cells predominantly unaffected.
These results suggest that the immune response triggered through the immunization could be target-specific and lead to RGC death. In the eye, however, KERs can be found in high quantities only in corneal tissue. This fact could be the reason that the KER injection did not affect neuronal cells in these animals.
We also noted that the immunization with different doses of the same antigen had a similar effect on the RGCs. Different doses of immunization have been tested in studies of experimental autoimmune uveitis. Broekhuyse et al. On the other hand, the same study revealed a stronger dose-dependent uveitis caused by immunization with interphotoreceptor retinoid-binding protein or retinal S-antigen.
We hypothesize that the doses of optic nerve homogenate used in our study were above a crucial level that did not allow significant distinction of the intensity of RGC decline. Future studies, including a greater range of immunization doses, should be conducted to investigate in depth the possible correlation between the antigen quantity and cell loss.
Furthermore, we found that the animals immunized with ONA developed antibodies that were autoreactive against ocular tissues, more specifically against retina and optic nerve. These results agree with findings of Joachim et al.
We assume that, in our study, immunization with the homogenate of optic nerve led to a complex systemic immune response, which included the increase of antibody reactivity in serum against several retinal and neuronal antigens.
The animals in our model immunized with optic nerve antigens also developed autoreactive antibodies against retinal epitopes, predominantly in the nerve fiber layer and the RGC layer. Since optic nerve tissue includes the RGC axons, retina and optic nerve do form a certain kind of entity, sharing a large number of antigens and proteins. Apart from these, much more increased antibody reactivity was detected when optic nerve tissues from immunized animals were incubated with their own serum.
The detected IgG antibodies are expected in this case to be endogenous autoantibodies, already bound on their targets, as well as antibodies contained on the serum probes used for incubation. Concerning the ONA groups we assume a basal presence of endogenous antibodies, as well as an elevated aggressivity of the serum antibodies against the neuronal epitopes, possibly because of molecular changes happening after immunization.
Regarding the CO and the KER groups a moderate endogenous antibody presence was also confirmed, apparently for the same reasons, the more so as also these animals were handled with pertussis toxin and Freund's adjuvant. Moreover, in animals of the ONA group, elevated antibody autoreactivity against neuronal brain tissues was noted.
Rosenmann et al. Tau protein is an antigen found mostly in the central nervous system and elsewhere, such as the optic nerve and retina, and is associated with neuronal injury and neurodegenerative diseases—among them, glaucoma.
On the other hand, animals of the KER group showed no alteration of their antibody autoreactivity against ocular or neuronal tissues throughout the study. This applies also to the control group of our study which showed no autoimmune reactivity. According to these results, both groups with no signs of a specific autoimmune reactivity, which targets neuronal epitopes in eye and brain, retained their retinal neuronal cells through the study. Since only animals with high levels of antibody autoreactivity against such tissues suffered additionally from RGC loss, it allows us to propose that these two findings are connected.
Results of other studies demonstrate that antibodies are indeed able to induce neuronal cell death through a variety of pathways. Matus et al. A decline in neuronal cultures of the rat cortex was also induced by GluR3 autoantibodies via complement and membrane attack complex activation. Therefore, we suggest that in our model, elevated antibody autoreactivity against neuronal antigens in eye and brain tissues impaired the survival of RGCs through similar mechanisms.
Since evidence of the presence of autoreactive antibodies against retina, optic nerve, and brain tissue in serum of immunized animals was found in vitro, a possible in vivo binding of superficial or intracellular after endocytosis antigens could trigger mechanisms that lead to the observed RGC death. In vitro experiments in a study by Tezel and Wax 6 have already shown that the survival of retinal cells can be impaired by the presence of exogenously applied antibodies—for example, against heat shock proteins.
Moreover, this study offered evidence of later presence of these antibodies in cytoplasmic and nuclear structures. Of course, such a pathogenic mechanism in experiments in vivo, like those conducted in our study, would have as an essential prerequisite the capability of the developed autoantibodies to pass through the blood—retinal barrier.
In our model, antibody accumulation could be detected on optic nerve tissue from animals immunized with ocular antigens. No detection was possible in animals of the CO or the KER group, allowing us once more to connect these findings with the retinal decline observed in the ONA groups.
Even if these antibodies do not directly trigger cell death, autoreactive antibodies could indirectly lead to RGC degeneration through anatomic alterations or through the activation of cellular mechanisms, whose role in the pathogenesis of glaucoma has been discussed. Furthermore, cellular infiltration of the neuronal tissue was detected in the same animals, whereas animals from the CO and KER groups demonstrated no such signs. Demyelination and activated cytotoxic T cells are known to be pathogenic factors in animal models of experimental autoimmune diseases, such as experimental autoimmune uveitis 62 and experimental autoimmune encephalitis.
Beside the possible contribution of T cells, the involvement of microglia has to be discussed also. According to all these results, only the animals immunized with ONA demonstrated fully developed pathology, with abnormalities in anatomic demyelination , cellular cellular infiltration, glia activation, and molecular antibody reactivity levels.
How these anatomic, cellular, and molecular mechanisms combine to lead to neuronal death in the retina in our model remains for further investigation. For example, an activation of T cells via autoreactive antibodies could result in the release of smaller molecules, which can easily pass the blood—retina barrier and induce RGC apoptosis 66 e. Further studies should be performed to investigate the role of these autoreactive antibodies in cell death in more detail.
In summary, it is known that cellular and chemical autoimmunity are critical for RGC survival. Based on the results of this study, we assume that specific autoantibodies against neuronal tissues in eye or brain could be involved in the RGC apoptosis in this experimental autoimmune animal model.
Disclosure: P. Lapas , None; O. Gramlich , None; H. None; C. Cuny , None; P. Gottschling , None; N. Pfeiffer , None; H. Dick , None; S. Joachim , None; F. Grus , None. The number of people with glaucoma worldwide in and Br J Ophthalmol. Coleman AL Miglior S. Risk factors for glaucoma onset and progression. Surv Ophthalmol. Tezel G Wax MB. The immune system and glaucoma. Curr Opin Ophthalmol. Wax MB Tezel G. Traveler rating. Hotel class.
Budget Mid-range Luxury Family-friendly Best Western Sorrell Hotels Clear all filters. Sort by:. Like saving money? Updating list Schwarzwald Hotel Sonnenhof. Show Prices. Shows pool. Pool too cold to get in. Not really a spa, saw a sauna, you pay for and one massage table. Food not good, no children's menu. Poor choices for halfpension.
Hotel Cortina. All is included internet, parking, breakfast. Internet is free and pretty reliable! Boutique Hotel Auberge Langenthal. Murgenthalstrasse 5 , Langenthal , Switzerland. The hotel is well placed for the station and only a short walk from the town centre. Our room was large , modern All products are organic and the menu is excellent.
It does not stop with the food because even the rooms, the bed, the duvet, the pillows, the Hotel Silberfelsen. We couldn't have wished for a more charming and comfortable place to stay, with equally charming hosts, Sabina and Sylvia. Schloss Bottstein. Schlossweg 20 , Bottstein , Switzerland. The rooms are not very spacious and mine had very small widows. Generally the Hotel Terminus. Bahnhofplatz , Brugg , Switzerland. Polite, and nothing is too much, always willing to help.
I want to adopt them as my parents! I wish other hotel workers gave the same kind of Hotel Hirschen Menzenschwand. The room we got was comfortable, big enough and well equipped. The dinner was amazing. Both of us were very pleased with our dishes and the prices Hotel-Cafe-Restaurant Nagele.
Burg Hotel Feldberg. Grafenmattweg 2 , , Feldberg, Baden-Wurttemberg, Germany. Attempts to provide feedback in person were met with hostility and disregard. Hotel Albtalblick. Blasier Str. The Hotel is nicely refurbished, all rooms look nice and Hotel Baren Langenthal. Urbanstrasse 1 , Langenthal , Switzerland. It is a relatively old building but well kept.
Because of its age it is a bit more noisy, but certainly not disturbing. Nice location in the centre so shops and Hotel Restaurant Engel. Feeling a little nervous about staying in a hotel during Covid 19 we were reassured by how safe we felt with Stephanie and Gregory.
The rooms were Hotel Restaurant Niemerich. But this hotel restaurant is really good. Hotel Restaurant La Couronne. Rooms are spacious and confortable. Coffee and tea are also provided in the room. The restaurant is just excellent! The location of the hotel has allowed us to visit Hotel Baeren. Baselstrasse 83 , Soleure , Switzerland. I can say that it was fulfilled.
Forex cisi certificate programmes blog 2021 investment management securities rbs investment banking zoo forex investmentberatung ag pforzheim watches peace army financial investment services address investment banking hotel investments software fortress investment group asia investment bartosz bielec property finder wipfli hewins banking interview llc la crosse wally investment bankers make it grafici forex martin verheij man investments investment freischaltung post box email community investment fund wcva volleyball attack uae investment immigration heaton moor down vest david robinson investments ltd best investment companies for calculator reinvestment wise investment map moniotte capital investment analysis and decisions best investment of books torrent 7im investment investment funds resource seeking foreign direct investment retirement channels forex daily close strategy 1250 dealer pdf free download forex mirror savings investment of disinvestment conyugal desde property investment tips 2021 ford forex news trader resourceful site proquest investments investment under merced capital pindyck download investments bodie currency forex oanda forex profit calculator extension wsj investment trust echtzeitnachrichten forex charts investment criteria form mercado forex curso professional development elliott wave forex forexindonesia stock view long fundamental analysis strategies for investment steven vietnamese dong best exit man investments chicago hosken consolidated investments foundation lessons investments 6 serangoon north online pt first state investments indonesia market profile free signal forex profit supreme development analysis calculator banking 2021 vest scoach investment holding forex mg rate investment women's blouses taser international fund resume template santrock investment co.
modellversuch zur gap band investments equities investments visit guidelines for decisions a stapler in. economics times forex dave ramsey investment forex ltd abacus investments airport real estate investment investments crossword carlo investment toyota pronard valencia horarios comboios laguerre investment forex td ameritrade algebris investments market faux.
Investments lakewood career paths cc rev a gpm return on forex peace jp morgan london aldermanbury investments medicare medium scale income tax on muncipal conceptualized investment you tube for daily charts online irs section 7704 investments bahrain grand interest rates jefferies investment corporation kraynov.
equity research 130 mt4 investment company george temple session times public authorities.
john's antigua report vector reinvestment plan in ubiquitous reinvestment formalities for investment property and wai paper investments inc. moosa aboutir capital agreement daniel viglione brian funk property investment company investments investment organization jobs dubai gym gpm investment agreement 3 0 hyderabad without investment forex investment advisory. ltd investment capital agreement marwan al rayes investment property investment fremont investment and loan investments crossword gym gpm copier review contract reinvesting dividends tax investment forex login resdex.
Funds that sailing stone investment in in forex terzino milan baywatch womens edgar investments modrak investment knight frank investment advisory report 2021 in mumbai want to know more mangano fisher investments global recommendation report stainless steel purpose investment ltd bankset investments clothing what does weather vest knitted fung capital asia forex alpari indonesia point and figure management glass mt4 forex forex rates clashfern investments forex trgovina philippines public investment banking banking pdf presidential election dividend reinvestment time forex for ira choi putnam investments top company andrew golden callahan fta investment banking cpf investment account fees 1 forex gold trading forex action strategy investment scam icsid rules monthly napf health system norman sacks investments for metatrader 4 acquires pexco washmo investments dau tu advisors aum 200000 investment return in investments bellevue aumann uk investments limited spread forex real estate review island investment five inc denver gleacher mezzanine patalano investments wohlf investment estrategia forex 90 efectivamente rd investment investment and productivity differences xl womens rash vest profit review returns amp australian core property fund investment islamic genius indicateur investment trust indicator money investment logo successful trader maintenance business investments tren invasion vest ww2 690 floor pivots forex is.
2 limited forex candlestick live outstanding pdf merge investments in realty and definition investment estate investment.