Capacidade de adesão, invasão e produção de enterotoxinas de Staphylococcus aureus isolados de leite de vacas com mastite subclínica e leite humano de um banco de leite

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Pathogenic Mycobacterium bovis strains differ in their ability to modulate the proinflammatory activation phenotype of macrophages

Background: Tuberculosis, caused by Mycobacterium tuberculosis or Mycobacterium bovis, remains one of the leading infectious diseases worldwide. The ability of mycobacteria to rapidly grow in host macrophages is a factor contributing to enhanced virulence of the bacteria and disease progression. Bactericidal functions of phagocytes are strictly dependent on activation status of these cells, regulated by the infecting agent and cytokines. Pathogenic mycobacteria can survive the hostile environment of the phagosome through interference with activation of bactericidal responses. To study the mechanisms employed by highly virulent mycobacteria to promote their intracellular survival, we investigated modulating effects of two pathogenic M. bovis isolates and a reference M. tuberculosis H37Rv strain, differing in their ability to multiply in macrophages, on activation phenotypes of the cells primed with major cytokines regulating proinflammatory macrophage activity. Results: Bone marrow- derived macrophages obtained from C57BL/6 mice were infected by mycobacteria after a period of cell incubation with or without treatment with IFN-gamma, inducing proinflammatory type-1 macrophages (M1), or IL-10, inducing anti-inflammatory type-2 cells (M2). Phenotypic profiling of M1 and M2 was then evaluated. The M. bovis strain MP287/03 was able to grow more efficiently in the untreated macrophages, compared with the strains B2 or H37Rv. This strain induced weaker secretion of proinflammatory cytokines, coinciding with higher expression of M2 cell markers, mannose receptor (MR) and arginase-1 (Arg-1). Treatment of macrophages with IFN-gamma and infection by the strains B2 and H37Rv synergistically induced M1 polarization, leading to high levels of inducible nitric oxide synthase (iNOS) expression, and reduced expression of the Arg-1. In contrast, the cells infected with the strain MP287/03 expressed high levels of Arg-1 which competed with iNOS for the common substrate arginine, leading to lower levels of NO production. Conclusions: The data obtained demonstrated that the strain, characterized by increased growth in macrophages, down- modulated classical macrophage activation, through induction of an atypical mixed M1/M2 phenotype.

Transforming growth factor beta and apoptosis in leprosy skin lesions: possible relationship with the control of the tissue immune response in the Mycobacterium leprae infection

The course of leprosy depends of the host immune response which ranges from the lepromatous pole (LL) to the tuberculoid pole (TT). A comparative study was conducted in 60 patients with the LL and TT The results showed a mean expression of TGF-beta of 339 +/- 99.4 cells/field for TT and of 519.2 +/- 68.2 cells/field for LL. Frequency of apoptosis was 6.3 +/- 1.8 in TT and 14.0 +/- 6.1 in LL. A correlation (p = 0.0251) between TGF-beta and caspase-3 in the LL was found. This finding indicates a role of TGF-beta and apoptosis in the immune response in leprosy. (C) 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Central nervous system of Rhipicephalus sanguineus ticks (Acari: Ixodidae): an ultrastructural study

This study performed the ultrastructural description of the synganglion of Rhipicephalus sanguineus males and females, aiming to contribute to the understanding of the cellular organization of this organ. The results show that the central nervous system of these individuals consists of a mass of fused nerves, named synganglion, from where nerves emerge towards several parts of the body. It is surrounded by the neural lamella, a uniform and acellular layer, constituted by repeated layers of homogeneous and finely granular material. The perineurium is just below, composed of glial cells, which extensions invaginate throughout the nervous tissue. The synganglion is internally divided into an outer cortex, which contains the cellular bodies of the neural cells and an inner neuropile. The neural cells can be classified into two types according to cell size, cytoplasm-nucleus relation, and neurosecretory activity. Type I cells are oval or spherical and present a large nucleus occupying most part of the cytoplasm, which contains few organelles. Type 2 cells are polygonal, present a great cytoplasm volume, and their nuclei are located in the cell periphery. The cytoplasm of these cells contains a well-developed rough endoplasmic reticulum, Golgi regions, mitochondria, and several neurosecretory granules. The subperineurium and the tracheal ramifications are found between the cortex and the neuropile. The latter is formed mainly by neural fibers, tracheal elements, and glial cells. The results obtained show that R. sanguineus males' and females' nervous tissue present an ultrastructural organization similar to the one described in the literature for other tick species.

In Vitro Evaluation of the Probiotic Potential of Bacteriocin Producer Lactobacillus sakei 1

Lactobacillus sakei 1 is a food isolate that produces a heat-stable antimicrobial peptide (sakacin 1, a class ha bacteriocin) inhibitory to the opportunistic pathogen Listeria monocytogenes. Bacterial isolates with antimicrobial activity may be useful for food biopreservation and also for developing probiotics. To evaluate the probiotic potential of L. sakei I, it was tested for (i) in vitro gastric resistance (with synthetic gastric juice adjusted to pH 2.0, 2.5, or 3.0); (ii) survival and bacteriocin production in the presence of bile salts and commercial prebiotics (inulin and oligofructose); (iii) adhesion to Caco-2 cells; and (iv) effect on the adhesion of L. monocytogenes to Caco-2 cells and invasion of these cells by the organism. The results showed that L. sakei I survival in gastric environment varied according to pH, with the maximum survival achieved at pH 3.0, despite a 4-log reduction of the population after 3 h. Regarding the bile salt tolerance and influence of prebiotics, it was observed that L. sakei 1 survival rates were similar (P > 0.05) for all de Man Rogosa Shame (MRS) broth formulations when tests were done after 4 h of incubation. However, after incubation for 24 h, the survival of L. sakei 1 in MRS broth was reduced by 1.8 log (P < 0.001), when glucose was replaced by either inulin or oligofructose (without Oxgall). L. sakei 1 was unable to deconjugate bile salts, and there was a significant decrease (1.4 log) of the L. sakei 1 population in regular MRS broth plus Oxgall (P < 0.05). In spite of this, tolerance levels of L. sakei 1 to bile salts were similar in regular MRS broth and in MRS broth with oligofructose. Lower bacteriocin production was observed in MRS broth when inulin (3,200 AU/ml) or oligofructose (2,400 AU/ml) was used instead of glucose (6,400 AU/ml). L. sakei I adhered to Caco-2 cells, and its cell-free pH-neutralized supernatant containing sakacin I led to a significant reduction of in vitro listerial invasion of human intestinal Caco-2 cells.

Germline-Specific MATH-BTB Substrate Adaptor MAB1 Regulates Spindle Length and Nuclei Identity in Maize

Germline and early embryo development constitute ideal model systems to study the establishment of polarity, cell identity, and asymmetric cell divisions (ACDs) in plants. We describe here the function of the MATH-BTB domain protein MAB1 that is exclusively expressed in the germ lineages and the zygote of maize (Zea mays). mab1 (RNA interference [RNAi]) mutant plants display chromosome segregation defects and short spindles during meiosis that cause insufficient separation and migration of nuclei. After the meiosis-to-mitosis transition, two attached nuclei of similar identity are formed in mab1 (RNAi) mutants leading to an arrest of further germline development. Transient expression studies of MAB1 in tobacco (Nicotiana tabacum) Bright Yellow-2 cells revealed a cell cycle-dependent nuclear localization pattern but no direct colocalization with the spindle apparatus. MAB1 is able to form homodimers and interacts with the E3 ubiquitin ligase component Cullin 3a (CUL3a) in the cytoplasm, likely as a substrate-specific adapter protein. The microtubule-severing subunit p60 of katanin was identified as a candidate substrate for MAB1, suggesting that MAB1 resembles the animal key ACD regulator Maternal Effect Lethal 26 (MEL-26). In summary, our findings provide further evidence for the importance of posttranslational regulation for asymmetric divisions and germline progression in plants and identified an unstable key protein that seems to be involved in regulating the stability of a spindle apparatus regulator(s).

Evaluation of the probiotic potential and effect of encapsulation on survival for Lactobacillus plantarum ST16Pa isolated from papaya

Capability to produce antilisterial bacteriocins by lactic acid bacteria (LAB) can be explored by the food industry as a tool to increase the safety of foods. Furthermore, probiotic activity of bacteriogenic LAB brings extra advantages to these strains, as they can confer health benefits to the consumer. Beneficial effects depend on the ability of the probiotic strains to maintain viability in the food during shelf-life and to survive the natural defenses of the host and multiply in the gastrointestinal tract (GIT). This study evaluated the probiotic potential of a bacteriocinogenic Lactobacillus plantarum strain (Lb. plantarum ST16Pa) isolated from papaya fruit and studied the effect of encapsulation in alginate on survival in conditions simulating the human GIT. Good growth of Lb. plantarum ST16Pa was recorded in MRS broth with initial pH values between 5.0 and 9.0 and good capability to survive in pH 4.0, 11.0 and 13.0. Lb. plantarum ST16Pa grew well in the presence of oxbile at concentrations ranging from 0.2 to 3.0%. The level of auto-aggregation was 37%, and various degrees of co-aggregation were observed with different strains of Lb. plantarum, Enterococcus spp., Lb. sakei and Listeria, which are important features for probiotic activity. Growth was affected negatively by several medicaments used for human therapy, mainly anti-inflammatory drugs and antibiotics. Adhesion to Caco-2 cells was within the range reported for other probiotic strains, and PCR analysis indicated that the strain harbored the adhesion genes mapA, mub and EF-Tu. Encapsulation in 2, 3 and 4% alginate protected the cells from exposure to 1 or 2% oxbile added to MRS broth. Studies in a model simulating the transit through the GIT indicated that encapsulated cells were protected from the acidic conditions in the stomach but were less resistant when in conditions simulating the duodenum, jejunum, ileum and first section of the colon. To our knowledge, this is the first report on a bacteriocinogenic LAB isolated from papaya that presents application in food biopreservation and may be beneficial to the consumer health due to its potential probiotic characteristics.

Antismooth Muscle and Antiactin Antibodies Are Indirect Markers of Histological and Biochemical Activity of Autoimmune Hepatitis

Reactivity and titers of autoantibodies vary during the course of autoimmune hepatitis (AIH), and some autoantibodies have been associated with disease activity and adverse outcomes after treatment. The aim of this study was to assess the autoantibody behavior in AIH and its significance as predictors of biochemical and histological remission. A total of 117 patients with AIH (mean age 18.6 [4-69] years) were evaluated and tested for auto- antibodies at disease onset and successively (mean 3.2 [2-6] times) after a mean follow-up evaluation of 70 [20-185] months. Antismooth muscle (ASMA), antiliver kidney micro- some type 1 (anti-LKM1), antiliver cytosol type 1 (anti-LC1), antimitochondrial, antinu- clear (ANA), and antiactin antibodies (AAA) were determined at disease onset and 379 other times during the follow-up evaluation through indirect immunofluorescence in rodent tissues, HEp-2 cells, and human fibroblasts. Anti-SLA/LP were assessed 45 times in the follow-up evaluation of 19 patients using enzyme-linked immunosorbent assay (ELISA). Upon admission, AIH types 1 and 2 were observed in 95 and 17 patients, respectively. Five subjects had AIH with anti-SLA/LP as the sole markers. Patients initially negative for AAA did not develop these antibodies thereafter. ANA were detected de novo in six and three subjects with AIH types 1 and 2, respectively. After treatment, only ASMA ( > 1:80) and AAA ( > 1:40) were significantly associated with biochemical (76.9% and 79.8%) and histological features (100% and 100%) of disease activity ( P < 0.001). Conclusion: With the exception of ANA, the autoantibody profile does not markedly vary in the course of AIH. The persistence of high titers of ASMA and/or AAA in patients with AIH is associated with disease activity.

Study of apoptotic deletion mediated by Bifidobacterium longum with construction of recombinant strains for Serpin encoding gene and phenotypes comparison in a pig cell model

The first part of the research project of the Co-Advisorship Ph.D Thesis was aimed to select the best Bifidobacterium longum strains suitable to set the basis of our study. We were looking for strains with the abilities to colonize the intestinal mucosa and with good adhesion capacities, so that we can test these strains to investigate their ability to induce apoptosis in “damaged” intestinal cells. Adhesion and apoptosis are the two process that we want to study to better understand the role of an adhesion protein that we have previously identified and that have top scores homologies with the recent serpin encoding gene identified in B. longum by Nestlè researchers. Bifidobacterium longum is a probiotic, known for its beneficial effects to the human gut and even for its immunomodulatory and antitumor activities. Recently, many studies have stressed out the intimate relation between probiotic bacteria and the GIT mucosa and their influence on human cellular homeostasis. We focused on the apoptotic deletion of cancer cells induced by B. longum. This has been valued in vitro, performing the incubation of three B.longum strains with enterocyte-like Caco- 2 cells, to evidence DNA fragmentation, a cornerstone of apoptosis. The three strains tested were defined for their adhesion properties using adhesion and autoaggregation assays. These features are considered necessary to select a probiotic strain. The three strains named B12, B18 and B2990 resulted respectively: “strong adherent”, “adherent” and “non adherent”. Then, bacteria were incubated with Caco-2 cells to investigate apoptotic deletion. Cocultures of Caco-2 cells with B. longum resulted positive in DNA fragmentation test, only when adherent strains were used (B12 and B18). These results indicate that the interaction with adherent B. longum can induce apoptotic deletion of Caco-2 cells, suggesting a role in cellular homeostasis of the gastrointestinal tract and in restoring the ecology of damaged colon tissues. These results were used to keep on researching and the strains tested were used as recipient of recombinant techniques aimed to originate new B.longum strains with enhanced capacity of apoptotic induction in “damaged” intestinal cells. To achieve this new goal it was decided to clone the serpin encoding gene of B. longum, so that we can understand its role in adhesion and apoptosis induction. Bifidobacterium longum has immunostimulant activity that in vitro can lead to apoptotic response of Caco-2 cell line. It secretes a hypothetical eukaryotic type serpin protein, which could be involved in this kind of deletion of damaged cells. We had previously characterised a protein that has homologies with the hypothetical serpin of B. longum (DD087853). In order to create Bifidobacterium serpin transformants, a B. longum cosmid library was screened with a PCR protocol using specific primers for serpin gene. After fragment extraction, the insert named S1 was sub-cloned into pRM2, an Escherichia coli - Bifidobacterium shuttle vector, to construct pRM3. Several protocols for B. longum transformation were performed and the best efficiency was obtained using MRS medium and raffinose. Finally bacterial cell supernatants were tested in a dotblot assay to detect antigens presence against anti-antitrypsin polyclonal antibody. The best signal was produced by one starin that has been renamed B. longum BLKS 7. Our research study was aimed to generate transformants able to over express serpin encoding gene, so that we can have the tools for a further study on bacterial apoptotic induction of Caco-2 cell line. After that we have originated new trasformants the next step to do was to test transformants abilities when exposed to an intestinal cell model. In fact, this part of the project was achieved in the Department of Biochemistry of the Medical Faculty of the University of Maribor, guest of the abroad supervisor of the Co-Advisorship Doctoral Thesis: Prof. Avrelija Cencic. In this study we examined the probiotic ability of some bacterial strains using intestinal cells from a 6 years old pig. The use of intestinal mammalian cells is essential to study this symbiosis and a functional cell model mimics a polarised epithelium in which enterocytes are separated by tight junctions. In this list of strains we have included the Bifidobacterium longum BKS7 transformant strain that we have previously originated; in order to compare its abilities. B. longum B12 wild type and B. longum BKS7 transformant and eight Lactobacillus strains of different sources were co-cultured with porcine small intestine epithelial cells (PSI C1) and porcine blood monocytes (PoM2) in Transwell filter inserts. The strains, including Lb. gasseri, Lb. fermentum, Lb. reuterii, Lb. plantarum and unidentified Lactobacillus from kenyan maasai milk and tanzanian coffee, were assayed for activation of cell lines, measuring nitric oxide by Griess reaction, H202 by tetramethylbenzidine reaction and O2 - by cytochrome C reduction. Cytotoxic effect by crystal violet staining and induction on metabolic activity by MTT cell proliferation assay were tested too. Transepithelial electrical resistance (TER) of polarised PSI C1 was measured during 48 hours co-culture. TER, used to observe epithelium permeability, decrease during pathogenesis and tissue becomes permeable to ion passive flow lowering epithelial barrier function. Probiotics can prevent or restore increased permeability. Lastly, dot-blot was achieved against Interleukin-6 of treated cells supernatants. The metabolic activity of PoM2 and PSI C1 increased slightly after co-culture not affecting mitochondrial functions. No strain was cytotoxic over PSI C1 and PoM2 and no cell activation was observed, as measured by the release of NO2, H202 and O2 - by PoM2 and PSI C1. During coculture TER of polarised PSI C1 was two-fold higher comparing with constant TER (~3000 ) of untreated cells. TER raise generated by bacteria maintains a low permeability of the epithelium. During treatment Interleukin-6 was detected in cell supernatants at several time points, confirming immunostimulant activity. All results were obtained using Lactobacillus paracasei Shirota e Carnobacterium divergens as controls. In conclusion we can state that both the list of putative probiotic bacteria and our new transformant strain of B. longum are not harmful when exposed to intestinal cells and could be selected as probiotics, because can strengthen epithelial barrier function and stimulate nonspecific immunity of intestinal cells on a pig cell model. Indeed, we have found out that none of the strains tested that have good adhesion abilities presents citotoxicity to the intestinal cells and that non of the strains tested can induce cell lines to produce high level of ROS, neither NO2. Moreover we have assayed even the capacity of producing certain citokynes that are correlated with immune response. The detection of Interleukin-6 was assayed in all our samples, including B.longum transformant BKS 7 strain, this result indicates that these bacteria can induce a non specific immune response in the intestinal cells. In fact, when we assayed the presence of Interferon-gamma in cells supernatant after bacterial exposure, we have no positive signals, that means that there is no activation of a specific immune response, thus confirming that these bacteria are not recognize as pathogen by the intestinal cells and are certainly not harmful for intestinal cells. The most important result is the measure of Trans Epithelial Electric Resistance that have shown how the intestinal barrier function get strengthen when cells are exposed to bacteria, due to a reduction of the epithelium permeability. We have now a new strain of B. longum that will be used for further studies above the mechanism of apoptotic induction to “damaged cells” and above the process of “restoring ecology”. This strain will be the basis to originate new transformant strains for Serpin encoding gene that must have better performance and shall be used one day even in clinical cases as in “gene therapy” for cancer treatment and prevention.

Entstehung und Dynamik des Keratinfilament-Netzwerks

Das zytoplasmatische Zytoskelett besteht aus drei Filamentsystemen, die aus Aktin, Tubulin und Intermediärfilamentproteinen aufgebaut sind und dreidimensionale Netzwerke ausbilden. Das Intermediärfilamentsystem, dem vor allem mechanische Stabilisierungsfunktionen zugesprochen werden, unterscheidet sich von den anderen durch seine Fähigkeit, spontan aus seinen Polypeptiduntereinheiten ohne weitere Kofaktoren zu polymerisieren und durch seinen unpolaren Aufbau. Es ist bis heute unbekannt, wie Intermediärfilamentnetzwerke in vivo moduliert werden und wie ihre Anordnung in den Kontext des Gesamtzytoskeletts koordiniert wird. Am Beispiel der epithelialen Intermediärfilamentproteine, den Keratinen, sollte daher untersucht werden, wie und wo neue Intermediärfilamente entstehen, welche Bedeutung den anderen Filamentsystemen bei dem Netzwerkaufbau und –Turn-Over zukommen und wie die Netzwerkbildung gesteuert wird. Zur Beantwortung dieser Fragestellungen wurden Zellklone hergestellt, die fluoreszierende Keratine synthetisieren. In der Zelllinie SK8/18-2, deren gesamtes Netzwerk aus derartigen Chimären aufgebaut ist, konnten anhand von mikroskopischen Zeitrafferaufnahmen der Fluoreszenzmuster Keratinfilamentvorläufer (KFP) identifiziert und deren Dynamik direkt in lebenden Zellen verfolgt werden. Es konnte gezeigt werden, dass die KFP in einem Plasmamembran-nahen Bereich entstehen, in dem sie zuerst als punktförmige Partikel detektiert werden. Nach einer initialen, sphäroidalen Wachstumsphase elongieren die Partikel zu kleinen Filamentstückchen. Diese können miteinander fusionieren und werden über ihre Enden in das periphere Netzwerk integriert. Der Wachstumsprozess ist gekoppelt an eine kontinuierliche, langsame Bewegung in Richtung auf das Zellzentrum. Diese Motilität sistiert vollständig nach pharmakologisch induziertem Abbau der Aktinfilamente. In Zeitraffer-aufnahmen kann jedoch in derartig behandelten Zellen ein wesentlich schnellerer Transport, der in verschiedene Richtungen verläuft und durch lange Ruhephasen unterbrochen wird, beobachtet werden. Dieser Modus, der gelegentlich auch in unbehandelten Zellen gefunden wurde, ist abhängig von intakten Mikrotubuli. Erst durch Zerstörung der Aktinfilamente und der Mikrotubuli erlischt die Motilität der KFPs vollständig. Bei der Suche nach Regulatoren der Keratinnetzwerkbildung wurde die p38 MAPK als zentraler Faktor identifiziert. Erstmals konnte eine direkte räumliche und zeitliche Korrelation zwischen einer spezifischen Enzymaktivität durch Nachweis der phosphorylierten p38 MAPK, der daraus folgenden Phosphorylierung eines Keratins, hier Serin 73 des Keratin 8, und der daraus resultierenden Veränderung des Netzwerkaufbaus, d. h. der Ausbildung von Keratingranula, nachgewiesen werden. Diese koordinierten Veränderungen wurden in unterschiedlichen Stresssituationen in verschiedenen Zellsystemen und in Zellen mit mutierten Keratinen beobachtet. Genetische (shRNA) und pharmakologische Manipulationen der p38 MAPK-Aktivität deuten auf einen engen kausalen Zusammenhang hin.

Der Einfluss von Apolipoprotein E auf die zellulären Mechanismen der Alzheimer Erkrankung

Die massive Bildung und Ablagerung von aggregiertem Amyloid Beta-Peptid im Gehirn wird allgemein als zentrales Ereignis im Rahmen des Neurodegenerationsprozesses der Alzheimer Demenz betrachtet. Als einer der ursächlichen Risikofaktoren gilt das Vorliegen des ε4-Allels des Apolipoprotein E. Die Alzheimer´sche Krankheit ist dabei in sehr vielfältige Weise mit Apolipoprotein E verknüpft. ApoE begünstigt isoformenabhängig Aβ-Ablagerungen, ApoE-Fragmente kommen im Gehirn und der Cerebrospinalflüssigkeit von Alzheimer Patienten vor und ApoE ist darüber hinaus als Cholesterintransportprotein über den zellulären Cholesterinstoffwechsel mit der Amyloidbildung verknüpft. Mit Hilfe einer Doppeltransfektion von ApoE und ADAM10 in HEK-Zellen und durch Studien mit Inhibitoren der ADAM-Familie an HepG-2-Zellen wurde in vitro gezeigt, dass ApoE nicht durch α-Sekretasen der ADAM-Familie gespalten wird. Weiterhin konnte bewiesen werden, dass ApoE in Astrogliomazellen keinen Einfluss auf die APP-Prozessierung ausübt. Durch in vitro Modulation des Cholesteringehaltes an Astrogliomazellen mit MβCD und seine Cholesterin-Komplexverbindungen ist gezeigt worden, dass die ApoE-Sekretion durch abnehmenden Cholesteringehalt gesenkt wird. Indem Statine alleine oder in Kombination mit Isoprenylierungssubstraten eingesetzt wurden ist der Beweis erbracht worden, dass Statine in vitro die ApoE-Sekretionsinhibition alleine durch Hemmung der Cholesterinbiosynthese bewirken. Bestätigt wurde dies weiterhin durch Experimente mit Isoprenylierungsinhibitoren. Aus dem Wirkmechanismus von Statinen auf die ApoE-Sekretionssenkung leitet sich womöglich der für bestimmte Statine berichtete neuroprotektive Effekt bei Morbus Alzheimer in retrospektiven Humanstudien ab, der sich durch reine Cholesterinsenkung nicht erklären lässt. Im Zusammenhang mit der Cholesterinhomöostase und dem gesteigerten 24(S)-Hydroxycholesterinspiegel bei Morbus Alzheimer, haben die Ergebnisse gezeigt, dass 24(S)-Hydroxycholesterin [24(S)-OH-chol] zur ApoE-Sekretions- und Expressionssteigerung führt. In dieser Arbeit konnte erstmals der Nachweis erbracht werden, dass der stimulatorische Effekt von 24(S)-OH-Chol durch gleichzeitige Lovastatingabe reduziert werden kann. Dies stellt einen möglichen Ansatz im Kampf gegen die Alzheimer Demenz dar. Weiterführend müssen diese Ergebnisse noch in vivo beispielsweise durch Versuche an ApoE-transgenen Mäusen bestätigt werden. Darüber hinaus könnte nach einer Statintherapie der ApoE-Gehalt in humaner, cerebrospinaler Flüssigkeit ermittelt werden.

Autoantibodies in patients with psoriasis on anti-TNFalpha therapy

Introduction: Anti-TNF-alfa therapy has been effective in the treatment of patients with refractory psoriasis and psoriasic arthritis. However, the risk of developing autoantibodies in these patients undergoing this therapy is not clear. Objective: To evaluate the induction of specific autoantibodies after anti-TNFα therapy in patients with psoriasis and psoriasic arthritis and, to evaluate the influence of the use of methotrexate on the values of autoantibodies developed during this therapy. Patients and methods: Serum samples from 120 patients, obtained before(baseline) the introduction of anti-TNF-alpha therapy and approximately each 3-6 months during the therapy.O f these 120 patients, 113 were found negative for autoantibodies before starting anti -TNFalpha therapy, 7 were found positive for ANA. The analysis included detection of antinuclear antibodies (ANA) and anti-dsDNA antibodies (indirect immunofluorescence on Hep-2 cells and Crithidia luciliae, respectively); anti extractable nuclear antigens antibodies( ENA)(ELISA). RESULTS: Infliximab is associated with the highest occurrence rate of ANA, anti-dsDNA, ENA with approximately 69,2%, 11,5%, 7,6% of patients treated testing positive. In comparison, only 20%, 6,6%, 2,2% of patients treated with Adalimumab, and 19%, 2,3%, 2,3% of patients treated with Etanercept were positive for ANA, Anti-dsDNA, ENA respectively. As regard the seven patients who were positive at baseline, six of them (85.7%) in addition to being remained positive during the therapy they have also increased the autoantibodies ’s titers. Conclusion: our study have shown that Infliximab is associated with the highest rate of autoantibodies. The concomitant treatment with methotrexate did not modify the titers of autoantibodies developed during the therapy anti-TNFalph. The incidence of ANA, anti-dsDNA antibodies did not correlate with development of Lupus-like syndromes. The difference in the frequency of autoantibodies between psoriasis and psoriatic arthritis was not statistically significant (p = 0.867).

Impact of folate absorption and transport for nutrition and drug targeting

In this thesis, interactions of folic acid with tea and tea components at the level of intestinal absorption have been investigated. Firstly, the interaction between folic acid and tea as well as tea catechins was studied in vitro, using Caco-2 cell monolayers and secondly, a clinical trial was designed and carried out. In addition, targeting of folic acid conjugated nanoparticles to FR expressing Caco-2 cells was studied in order to evaluate the principle of nutrient-receptor-coupled transport for drug targeting. In the first part of this work, it was shown that EGCG and ECG (gallated catechins) inhibit folic acid uptake (IC50 of 34.8 and 30.8 µmol/L) comparable to MTX (methotrexate) under these experimental conditions. Moreover, commercial green and black tea extracts inhibited folic acid uptake with IC50 values of approximately 7.5 and 3.6 mg/mL, respectively. These results clearly indicate an interaction between folic acid and green tea catechins at the level of intestinal uptake. The mechanism responsible for the inhibition process might be the inhibition of the influx transport routes for folates such as via RFC and/or PCFT. For understanding the in vivo relevance of this in vitro interaction, a phase one, open-labeled, randomized, cross-over clinical study in seven healthy volunteers was designed. For the 0.4 mg folic acid dose, the mean Cmax decreased by 39.2% and 38.6% and the mean AUC0 decreased by 26.6% and 17.9% by green tea and black tea, respectively. For the 5 mg folic acid dose, the mean Cmax decreased by 27.4% and mean AUC0 decreased by 39.9% when taken with green tea. The results of the clinical study confirm the interaction between tea and folic acid in vivo leading to lower bioavailabilities of folic acid. In the second part of the thesis, targeting studies using folic acid conjugated nanoparticles were conducted. Folic acid conjugated nanoparticles were shown to be internalized by the cell via FR (folate receptor) mediated endocytosis. DNA block copolymer micelles equipped with 2, 11 and 28 folic acid units respectively were applied on FR expressing Caco-2 cells. There was a direct proportion in the amount of internalized nanoparticle and the number of folic acid units on the periphery of the nanoparticle. To sum up, throughout this thesis, the importance of folic acid for nutrition and nutrient and drug related interactions of folic acid at intestinal level was shown. Furthermore, significance of FRs in targeting for cancer chemotherapy was demonstrated in in vitro cell culture experiments. Folic acid conjugated DNA block copolymer micelles were suggested as efficient nanoparticles for targeted drug delivery.

Solubility and permeability as in vitro predictors for in vivo performance of fenofibrate IR solid dosage forms

This current work focused on the simulation of in vivo dissolution and permeation in order to be able to predict the in vivo performance of orally administered fenofibrate immediate release formulations. Therefore, the effects of the formulation surfactants on in vivo solubility and permeation of fenofibrate under physiologically relevant excipient concentrations were emphasized.rnIt was shown that the surfactant sodium dodecyl sulfate (SDS) may decrease rather than increase the solubility of fenofibrate in vivo. This was related to the interference of SDS with the vesicular system of the biorelevant medium, FaSSIFmod, and therefore its solubilization capacity. rnMoreover, in vitro permeation studies revealed that SDS concentrations inversely correlated with fenofibrate permeability. Through combination of the observed permeation and solubility data a good in vitro/in vivo correlation regarding Cmax values in humans could be established for five fenofibrate formulations which were based on the same manufacturing technique.rnBesides the experimental part, the major characteristics and their potential implementation in a dissolution/permeation device were discussed based on the promising realization of the in vitro solubility and permeation methods. rn

Signaltransduktion von IgG-Antiphospholipid-Antikörpern in plasmazytoiden Dendriten und Monomac1 Zellen

Das Antiphospholipid-Syndrom (APS) ist eine Autoimmunerkrankung die sich durch venöse und arterielle Thrombosen und/oder Spontanaborte bei gleichzeitigem Nachweis von persistierenden, erhöhten Antiphospholipid-Antikörper (aPL)-Titern charakterisieren lässt. Die zugrunde liegenden Mechanismen, über die aPL Pathogenität vermitteln, sind bislang wenig verstanden. Im Rahmen dieser Arbeit konnte gezeigt werden, dass drei humane monoklonale IgG aPL sowie IgG Fraktionen von APS Patienten eine Überexpression von TLR7 und TLR8 in plasmazytoiden dendritischen Zellen bzw. monozytären Zellen induzieren. Gleichzeitig erfolgt die Induktion der TLR7/8 Translokation vom endoplasmatischen Retikulum (ER) ins Endosom. Diese Effekte werden durch die Internalisierung der aPL und die nachfolgende Aktivierung einer NADPH Oxidase sowie durch endosomale Superoxid Produktion vermittelt. Als Folge dessen werden die Zellen extrem für TLR7/8 Liganden sensibilisiert. Diese Beobachtungen beschreiben einen neuen Signalmechanismus der innaten Immunität, der seinen Ursprung im Endosom nimmt. Da die Überexpression von TLR7 auch in pDCs von APS Patienten detektiert werden konnte, bieten unsere Ergebnisse eine Erklärung für die proinflammatorischen und prokoagulanten Effekte von aPL. rnWeiterhin führte die kombinierte Stimulation mit aPL und TLR7 Liganden in pDCs zu einem signifikant verstärkten Potential zur CD4+ Th2 Zell Aktivierung bzw. zur Regulation der B-Zell Differenzierung und Immunglobulin Produktion. Die Anwesenheit der pDCs erhöhte dabei synergistisch die CD40/86 Expression, die Proliferation sowie die Plasmazell-Differenzierung von isolierten peripheren B-Zellen, die mit aPL und TLR Liganden stimuliert wurden. Dieser Stimulationsansatz war außerdem ausreichend um naive B-Zellen zur IgM/IgG Produktion anzuregen und die Synthese neuer IgG aPL durch Gedächtnis-B-Zellen einzuleiten. Die Beteiligung der pDCs an diesem Prozess erfolgte durch Zytokin Sekretion sowie direktem Zell-Zell-Kontakt. Die Anwesenheit von Th2-Helferzellen war dabei nicht obligatorisch, konnte jedoch die B-Zell Aktivierung zusätzlich fördern. Eine Hochregulierung von TLR7 oder TLR9 innerhalb der B-Zell Population war nicht involviert. rnrnDiese Ergebnisse zeigen erstmalig die Relevanz einer pDC Aktivierung im Hinblick auf die Aufrechterhaltung der pathogenen Aktivität im Rahmen des APS. Da eine Dysregulierung von TLR7 bereits als ursächlich für die Ausbildung einer systemischen Autoimmunität erachtet wird, sollten unsere Ergebnisse für das generelle Verständnis von Autoimmunität von großer Relevanz sein.rn