Molekulare Ansätze zur Gentherapie

Zum Thema Zell- und molekularbiologische Methoden haben es in den letzten 25 Jahren erm?glicht, Erkenntnisse in intra- und interzellul?re Funktionsabl?ufe und deren St?rungen zu gewinnen. Besonders in der Onkologie kam es zu einem zunehmenden Wissen über dysregulierte Gene, sog. Onkogene, welche ein dysfunktionelles (Onko)Protein produzieren und damit Zellwachstum und Proliferation in Richtung Malignit?t beeinflussen k?nnen. Tumorsuppressorgene (TSG) wirken regulierend auf den Zellzyklus ein, und im Falle einer vererbten Mutation des einen Allels und einer Deletion des intakten Allels durch Funktionsverlust k?nnen Malignome induziert bzw. Malignomentstehung begünstigt werden. Ein Beispiel hierfür ist das Li-Fraumeni-Syndrom, bei welchem es aufgrund einer Mutation des Tumorsuppressorgens p53 zu einer geh?uften Inzidenz von Mamma- und Ovarialkarzinomen kommt. ?nderungen von Proteasen- oder Adh?sionsmolekülfunktionen von Zellen spielen eine wichtige Rolle bei der Tumorzellinvasion und Metastasierung. Blockierung von Proteasesystemen, z. B. Urokinase/Plasmin-System oder Matrixmetalloproteasen, führen zu einer Inhibierung von Tumorzellinvasion und Metastasierung in vitro und in vivo. Bei einigen Malignomen kommt es zu einer Vermehrung von Genkopien, einer sog. Genamplifikation, wie sie z. B. für Rezeptoren der epidermal „Growth-factor-Familie“ (EGF-R, erbB2, erbB3, erbB4) beschrieben sind. Die betroffenen Zellen reagieren dadurch vermehrt auf autokrine und parakrine Signale und hyperproliferieren. Die Gentherapie stellt durch Wiederherstellung physiologischer Funktionsabl?ufe einen vielversprechenden Ansatz zur Behandlung maligner Erkrankungen dar. Der vorliegende übersichtsartikel wird ohne Anspruch auf Vollst?ndigkeit nach einer kurzen Einführung in die Grundlagenaspekte der Gentherapie und deren derzeitigen Probleme pr?klinische und erste klinische Ergebnisse darstellen.     

The topographic organization of the retinal ganglion cell layer of the lizard Ctenophorus nuchalis.

The numbers of neurons of the ganglion cell layer (GCL) and their distribution in the retina of an Australian lizard Ctenophorus nuchalis were investigated. Retinal wholemounts and sections were prepared for light microscopic and optic nerves for electron microscopic study. Counts of cell numbers in the GCL from wholemounts varied from 200,000 to 380,000. Neurons in the GCL were non-uniformly distributed, forming a high cell density streak along the naso-temporal axis of the retina. Neurons of the GCL formed 2 to 9 layers in the visual streak and a single layer in the rest of the retina. The number of neurons of the GCL in this area was estimated at about 2,100,000. Although the visual streak represented only 16% of the total retinal surface area, it contained about 90% of all neurons of the GCL. Optic axon counts yielded 147,000 myelinated and 2,643,000 unmyelinated fibres. The estimated optic fibre number of 2,790,000 was 18.2% less than the total number of neurons counted from sections in the GCL of the same eye. The unexpected high number of neurons in the area of the visual streak indicates that cell numbers obtained only from wholemount preparations may vastly underestimate the total neuron numbers in the GCL of the lizard retina.     

Safety of virus inactivated antithrombin III concentrate ANTITHROMBIN III IMMUNO (AT III)

In a prospective clinical trial the risk of infection after application of virus inactivated antithrombin III concentrate ANTITHROMBIN III IMMUNO (AT III) was investigated in patients undergoing cardiovascular surgery. The study was conducted according to the recommendations of the International Committee on Thrombosis and Hemostasis (ICTH), with the exception that most patients required additional blood products as well as AT III.

Twenty-seven patients were eligible to test for the risk of acquiring hepatitis B. Twenty-six patients could be evaluated in terms of hepatitis NANB transmission considering ALT-levels whereas 20 patients could be tested for anti-HCV one year after surgery. Samples from 78 patients could be monitored for anti-HIV-1. None of these patients showed any signs of infection. AT III IMMUNO seems to be an antithrombin III concentrate with low or absent infectivity.     

Uroscopy in the 21st century: high-field NMR spectroscopy

From the experiments described, it can be seen that there are different research approaches that can be taken and these are summarized in Table 1. Whereas much scientific research is principally hypothesis led, there remains, nevertheless, an important place for exploratory research. High resolution NMR can measure, directly and simultaneously, a wide range of endogenous metabolites in biological fluids and has the unique capability of providing structural information on the metabolites detected. It has proved to be a powerful research tool with which to study inherited metabolic diseases, renal disease, drug metabolism, and toxicity, and can be used to monitor the effects of drug therapy. For instance, by using a library of experimental toxins one can map the metabolic profile of site-specific nephron injury. With this approach in man one could eventually take an unknown disease such as Balkan nephropathy and predict the initial site of tubular injury, the mode of injury and therefore the kind of toxin capable of producing that injury. NMR spectroscopic techniques are still advancing rapidly, with ever increasing sensitivity and sophistication of NMR pulse sequences to enhance structural elucidation in complex mixtures. Given the advances in directly coupled HPLC-NMR and even HPLC-NMR-mass spectroscopy it is likely that these technologies in conjunction with pattern recognition will make major contribution to our understanding of renal processes and provide new diagnostic insights in the 21st century.      

Potential role of flavonoids in the prevention of intestinal neoplasia

Intestinal neoplasia (adenomas and carcinomas) can possibly be prevented by a diet rich in vegetables and fruits, treatment with aspirin and other nonsteroidal antiinflammatory drugs, and early colonoscopic removal of adenomas. Ballast, fiber, and secondary plant products could play a major role in colon cancer prevention. Recently there has been much experimental work in vitro and in vivo about flavonoids as inducers of bioprevention. Flavonoids are secondary plant products with a wide variety of beneficial biological properties, and they possess anticarcinogenic, antimutagenic, and antioxidative modes of actions. Flavonoids are the main components of a healthy diet containing fruits and vegetables and are concentrated especially in tea, apples, and onions. We will focus this review on flavonoids which are derived from tea products such as proanthocyanidins (green tea) and flavons (camomille tea). Oral supplementation with bioflavonoids derived from tea could be used in humans to prevent growth of intestinal neoplasia such as adenomatous polyps of the colon. Flavonoids are a large group of natural compounds of which only a few have been used in animal models, cell cultures, and enzyme studies to inhibit mutagenic and carcinogenic events. Their clinical mode of action was evaluated by epidemiological studies, but no intervention studies in humans have been performed so far. In vitro flavonoids can bind electrophils, inactivate oxygen radicals, prevent lipid peroxidation, and inhibit DNA oxidation. In cell cultures they increase the rate of apoptosis, inhibit cell proliferation, and angiogenesis. In vivo they can induce the activities of protective enzymes (conjugating enzymes such as glutathione transferases and glucuronosyl transferases) of the intestine and the liver. In models of intestinal polyposis, flavonoids suppress polyp formation. Some epidemiological studies show a protective effect of flavonoids contained in fruits, vegetables, and tea.Flavonoid mixtures of tea origin supplied as nutritional supplements could be studied as a new way of bioprevention of intestinal neoplasia (colon adenomas and cancer). Therefore, a controlled, randomized clinical study should be performed to evaluate the efficacy of flavonoids.     

Localization of SNARE proteins and secretory organelle proteins in astrocytes in vitro and in situ

Astrocytes are capable of regulated release of messenger molecules. Astrocytes cultured from new born rodent brain express a variety of classical presynaptic proteins. We investigated the question whether the capability to express synaptic proteins in culture was a feature only of immature astrocytes, and whether these proteins were also expressed by astrocytes in situ. Experiments were performed with transgenic mice expressing the enhanced green fluorescent protein under the control of the human glial fibrillary acidic protein promoter. Using double fluorescence and astrocytes cultured from 1 to 16 day-old animals we show that the astrocytic expression of synaptic proteins in culture is invariant of the age of donor animals. Culturing can induce the astrocytic expression of specific synaptic proteins such as SV2, synaptophysin and SNAP-25. Astrocytes in brain sections of 1-16 day-old animals revealed a punctuate immunofluorescence for secretory carrier membrane protein (SCAMP), SNAP-23, synaptobrevin II, and cellubrevin, to a minor extent for SNAP-25 and synaptophysin, and none for SV2. Our results demonstrate that cultured astrocytes express synaptic proteins not present in situ. Nevertheless, astrocytic organelles in situ are equipped with molecules that could be involved in regulated exocytosis of messenger substances.     

Decrease of elastic tissue fibres in stem villus blood vessels of the human placenta during IUGR and IUGR with concomitant pre-eclampsia

In a recent study we described an increase of elastic tissue fibres in blood vessel walls of placental stem villi during pre-eclampsia when compared to uncomplicated pregnancies. Furthermore, the thickness of these blood vessel walls was enhanced in pre-eclampsia. Since it is known that elastic tissue fibres increase in systemic hypertension, it may be assumed that the enhancement of elastic tissue fibres in placental stem villi during pre-eclampsia may be induced by the hypertension. To get further insight into this assumption, we examined the amount of elastic tissue fibres in stem villus blood vessels of placentae of pregnancies complicated by intrauterine growth retardation (isolated IUGR, fourteen cases), a disease without hypertension of the mother and such with pre-eclampsia and concomitant IUGR (IUGR+PE, nine cases). Each study group was compared with uncomplicated pregnancies (twenty-six cases). Unfixed cryostat serial sections were processed for conventional orcein staining and for the demonstration of alpha-actin-immunoreactivity. The intensity of orcein staining of stem villus blood vessel walls was evaluated by a semiquantitative score method. Significant lower intensities of orcein staining were calculated for blood vessel walls of placentae of isolated IUGR (P=0.0007) and IUGR+PE (P=0.0039) when compared to uncomplicated pregnancies each. Additionally, the blood vessel wall thickness of stem villi of isolated IUGR (P=0.0081) and IUGR+PE (P=0.0007) was significantly reduced. In comparison to the above mentioned investigation, our results show that, in contrast to isolated pre-eclampsia, elastic tissue fibres are decreased during pregnancies complicated by IUGR, independently of the occurrence of concomitant pre-eclampsia when compared to uncomplicated pregnancies. From our studies it may be considered that the increase of elastic tissue fibres in placentae of patients with isolated pre-eclampsia may be induced by systemic hypertension. Furthermore, our study underline arguments that IUGR may be an independent disease of the fetus.     

Elektrokardiographische Beobachtungen Über den Ablauf Einer Akuten Myokarditis Nach Grippe

Zusammenfassung Bei einem 21 jähr. Studenten, der zur Zeit einer Grippeepidemie eine Influenza durchgemacht hatte, traten 2 Wochen darauf zuerst kompletter Block mit Ohnmachtsanfällen, später nach Wiederherstellung der normalen Schlagfolge Störungen der Reizbildung und insbesondere der Reizausbreitung sowohl im Vorhof als auch in besonders ausgedehntem Maße innerhalb der Kammer auf, die auf eine diffuse Erkrankung des spezifischen Muskelsystems hinwiesen. Gleichzeitig bestanden Oppressionsgefühl auf der Brust, Mattigkeit, vorübergehend geringe Temperatursteigerungen. Alle diese Symptome werden als Grippe-Myokarditis gedeutet. Besonders wertvoll für die Erkennung dieses und ähnlicher Krankheitsbilder erweist sich die elektrokardiographische Untersuchung, die auch beim Fehlen anderer auf die Myokardaffektion bezüglichen Symptome, insbesondere auch von Arhythmien, durch den Nachweis von Störungen der Erregungsausbreitung in Vorhof oder Kammer Aufklärung über das Bestehen eines pathologischen Prozesses im Myokard zu geben vermag. Die frühzeitige Erkennung dieses Krankheitszustandes ist aber mit Rücksicht auf die notwendigen prophylaktischen Maßnahmen von großer Wichtigkeit.