Relevance of the regional lymph node in scrapie pathogenesis after peripheral infection of hamsters

<p>Abstract</p> <p>Background</p> <p>The exact role of the lymphoreticular system in the spread of peripheral prion infections to the central nervous system still needs further elucidation. Against this background, the influence of the regional lymph node <it>(Ln. popliteus</it>) on the pathogenesis of scrapie was monitored in a hamster model of prion infection <it>via </it>the footpad.</p> <p>Methods</p> <p>Surgical lymphadenectomy was carried out at different time points after infection, or prior to inoculation, in order to elucidate the impact of the lymph node on lethal neuroinvasion.</p> <p>Results</p> <p>The <it>Ln. popliteus </it>did not show an influence on pathogenesis when a high dose of infectivity was administered. However, it was found to modulate the interval of time until the development of terminal scrapie in a subset of animals lymphadenectomized after low-dose infection. In additon, lymphadenectomy performed four weeks before inoculation prevented cerebral PrP^TSEdeposition and development of disease during the period of observation (314 days) in the majority of hamsters challenged with a very low dose of scrapie agent.</p> <p>Conclusion</p> <p>Our findings suggest the regional lymph node as a potentially facilitating or even essential factor for invasion of the brain after peripheral challenge with low doses of infectious scrapie agent. The invasive <it>in vivo </it>approach pursued in this study may be applied also to other animal species for further elucidating the involvement of lymphoid tissue in the pathogenesis of experimental and natural TSEs.</p

Characterization of a rat osteotomy model with impaired healing

<p>Abstract</p> <p>Background</p> <p>Delayed union or nonunion are frequent and feared complications in fracture treatment. Animal models of impaired bone healing are rare. Moreover, specific descriptions are limited although understanding of the biological course of pathogenesis of fracture nonunion is essential for therapeutic approaches.</p> <p>Methods</p> <p>A rat tibial osteotomy model with subsequent intramedullary stabilization was performed. The healing progress of the osteotomy model was compared to a previously described closed fracture model. Histological analyses, biomechanical testing and radiological screening were undertaken during the observation period of 84 days (d) to verify the status of the healing process. In this context, particular attention was paid to a comparison of bone slices by histological and immunohistological (IHC) methods at early points in time, <it>i.e</it>. at 5 and 10 d post bone defect.</p> <p>Results</p> <p>In contrast to the closed fracture technique osteotomy led to delayed union or nonunion until 84 d post intervention. The dimensions of whole reactive callus and the amounts of vessels in defined regions of the callus differed significantly between osteotomized and fractured animals at 10 d post surgery. A lower fraction of newly formed bone and cartilaginous tissue was obvious during this period in osteotomized animals and more inflammatory cells were observed in the callus. Newly formed bone tissue accumulated slowly on the anterior tibial side with both techniques. New formation of reparative cartilage was obviously inhibited on the anterior side, the surgical approach side, in osteotomized animals only.</p> <p>Conclusion</p> <p>Tibial osteotomy with intramedullary stabilisation in rats leads to pronounced delayed union and nonunion until 84 d post intervention. The early onset of this delay can already be detected histologically within 10 d post surgery. Moreover, the osteotomy technique is associated with cellular and vascular signs of persistent inflammation within the first 10 d after bone defect and may be a contributory factor to impaired healing. The model would be excellent to test agents to promote fracture healing.</p

Investigation of efficiency of the prodrugs valaciclovir, aciclovir and ganciclovir after application of DCES-HSV-tk suicide gene therapy system for the treatment of CC531 liver metastases model in rats

Titel,Inhalt, Danksagung, Lebenslauf 1\. Einleitung 2\. Literaturübersicht 3\. Eigene Untersuchungen, Materialien und Methoden 4\. Ergebnisse 5\. Diskussion 6./7. Zusammenfassung und Summary 8\. Literaturverzeichnis 9\. AbkürzungsverzeichnisLebermetastasen verschlechtern die Prognose von Patienten, die an kolorektalen Karzinom erkranken erheblich. Da chirurgische oder chemotherapeutische Behandlungsversuche oft nicht indiziert oder deren Ergebnisse unbefriedigend sind, besteht dringender Bedarf für die Entwicklung neuer Behandlungsmethoden. Das Ziel der vorliegenden Arbeit war es, im Vorfeld klinischer Studien das geeignetste Prodrug für eine liposomal vermittelte lokoregionäre HSV-tk- Suizidgentherapie von kolorektalen Lebermetastasen zu ermitteln. Pegylierte Liposomen dienten in dieser Arbeit als gefahrlose Genfähren. Sie wurden im Tierversuch zusammen mit einem Embolisat lokoregionär appliziert und transfizierten so die Lebermetastasen mit dem Thymidinkinasegen. Die nachfolgende Behandlung mit einem nicht toxischen Prodrug, welches von den transfizierten Zellen durch die eingebrachte Thymidinkinase in einen toxischen Metaboliten konvertiert wurde, trieb die Tumorzelle dann in den Suizid. Für diese Prodrug-Behandlung wurden die Nukleosidanaloga Valaciclovir (VCV, ein Valinsäurester des Aciclovir und damit oral verfügbar), Aciclovir (ACV, i.p. und in zwei Dosierungen appliziert) und Ganciclovir (GCV, i.p. appliziert) getestet. Einem oral verfügbaren Präparat wäre für die spätere klinische Anwendung gegenüber einer Tropfinfusion der Vorzug zu geben, da die Prodrugbehandlung 1-2 Wochen andauert. Die in-vitro-Versuche unter Verwendung der Tumorzelllinien CC531 und F98 zeigten deutlich eine gesteigerte Zytotoxizität HSV-tk-transfizierter Zellen zugunsten des Prodrug GCV gegenüber dem ACV. Bei den in-vivo-Versuchen am Lebermetastasenmodell der Ratte bestanden bei den oral VCV-behandelten Tieren und den in geringer Dosis ACV-behandelten Tieren keine signifikanten Wachstumshemmungen des CC531 Lebertumors am Ende der Behandlung gegenüber der Kontrollgruppe. Es zeigte sich aber im Vergleich zur Kontrollgruppe eine signifikante Hemmung des Tumorwachstums bei den mit je 100 mg / kg ACV (p = 0,0048) respektive GCV (p = 0,0011) behandelten Hochdosisgruppen. Zwischen diesen beiden Hochdosisgruppen bestanden bezüglich des Tumorwachstums keine signifikanten Unterschiede. Daher wäre ein klinischer Einsatz hinsichtlich der therapeutischen Effizienz dieser hochdosiert und i.v. verabreichten Prodrugs nahezu gleichwertig zu beurteilen. Tendentiell scheint eine bessere Wirksamkeit des GCV vorzuliegen. In dieser Gruppe wurden bei zwei der zehn Tiere sogar Regressionen des sehr agressiv wachsenden CC531-Kolonkarzinomtumors erzielt. Das Auftreten von Lungenmetastasen war ebenfalls nur in diesen beiden Behandlungsgruppen deutlich verringert (30 % der Tiere unter ACV- Hochdosisbehandlung, respektive 10 % der Tiere unter GCV-Hochdosisbehandlung, dagegen entwickelten etwa 60 % der Tiere in allen anderen Versuchsgruppen Lungenmetastasen). Die gesteigerte Effektivität in den Hochdosisgruppen ist in erster Linie zurückzuführen auf das Erreichen deutlich höherer Plasmawerte, besonders für das GCV. Dagegen zeigte die mit dem oral verfügbaren Valinsäureester, VCV- behandelte Gruppe neben der nicht ausreichenden Tumorantwort wesentlich geringere Plasmawerte für das entstehende ACV. Die im Anschluss ausgeführte Auswertung von PCR-Messungen einer dafür eigens mit geführten Kontrollgruppe gab Aufschluss über Anreicherung und zeitlichen Verlauf der Genexpression in Organen. So wurde ein starker Expressionsabfall des therapeutischen Gen zwischen Tag 5 und Tag 9 nach Applikation nachgewiesen. Die verabreichte Thymidinkinase wurde bei diesem Tierversuch besonders in der Milz und auch der gesunden Leber nachgewiesen. Die Entwicklung eines geeigneten Therapiemonitoring nicht nur mit konventionellen Parametern (die Verwendung des Prodrug GCV erschien leicht myelosuppressiv), sondern auch im Sinne der Expressionsüberwachung ist daher anstrebenswert. Die Ergebnisse der Arbeit belegen eindeutig, dass eine hohe Plasmakonzentration der Prodrugs Voraussetzung für eine effiziente Behandlung des Tumors ist. In unseren präklinischen Versuchen erwies sich die liposomal vermittelte HSV-tk Suizidgenstrategie mit allen verwendeten Prodrugs als gut verträgliche, nebenwirkungsarme Behandlung.Liver metastases worsen considerably the prognoses of the patients, who suffer from colorectal cancer. Because of the unsatisfactory efficacy of surgical and chemotherapeutic treatment, there is urgent need for the development of new methods of treatment.The aim of the present work was to elucidate the most suitable prodrug for the HSV-tk-liposome-mediated suicide gene therapy of colorectal liver metastases in preliminary stages of clinical studies. Pegylated liposomes were used as low risk gene vectors in this work. In the animal experiment they were applied together with an embolisate locoregionally, to achieve efficient transfection of tumor cells with the thymidine kinase gene. The following treatment with a non-toxic prodrug, which is converted into a toxic metabolite by thymidine kinase in the transfected tumor cells, drives the tumor cell to suicide. The nucleoside analoga Valaciclovir (VCV, an ester of Aciclovir and thus orally available), Aciclovir (ACV, i.p. and in two different doses) and Ganciclovir (GCV, i.p.) were tested as prodrugs. For future clinical application, an orally available drug would be of advantage since the prodrug application lasts 1-2 weeks.The in-vitro experiments showed an increased cytotoxicity of transfected tumor cell lines CC531 (rat colon carcinoma) and F98 (rat glioblastoma) in favor of the prodrug GCV compared to ACV.The in-vivo experiments, using a rat model of liver metastases, demonstrated no significant suppression of CC531 liver tumor growth with orally given VCV and intraperitonealy given ACV in low dosage compared with the control group at the end of the treatment. In contrast, significant suppression of tumor growth occurred in animals treated with the high dosage of 100 mg / kg ACV and GCV (p=0,0048, p=0,0011 respectively versus control).There were no statistical difference in tumor growth inhibition between both drugs. Thus for a clinical use ACV and GCV at high dosage may be expected to elicit equal therapeutic efficiency. However, there was an apparent tendency favoring. In the GCV-treated group, regressions of the fast- growing CC531 rat colon carcinoma liver metastases were found in two of ten animals. The presence of pulmonary metastases was reduced clearly likewise only in both high doses treatment groups (30 % of the ACV-treated animals and 10 % of the GCV-treated animals developed pulmonary metastases, compared to about 60 % in all other experimental groups). The increased efficacy in these both groups may be explained most likely by reaching clearly higher plasma levels for GCV followed by equally dosed ACV compared with the other experimental groups. ACV from his orally administred valine acid ester VCV showed the lowest plasma levels which is in line with the insufficient tumor answer. In a seperatly designed control group PCR-measurements informed about enhancement and temporal course of the transgene expression in different organs. A strong decrease in gene expression was observed between day 5 and day 9 after application. The development of a suitable in-vivo monitoring not only including main parameters (the use of the prodrug GCV appeared myelosuppressive), but also to control suicide gene expression is strongly indicated. The results of the work show that a high plasmaconcentration of the prodrugs determine an efficient treatment of the tumor. In summary, the liposome mediated HSV-tk suicide gene therapy combined with all tested prodrugs represents a well tolerated treatment with less side effects

Prion propagation in a nerve conduit model containing segments devoid of axons

Prions, the putative causative agents of transmissible spongiform encephalopathies, are neurotropic pathogens that spread to the central nervous system via synaptically linked neural conduits upon peripheral infection. Axons and their transport processes have been suggested as mediators of nerve-associated prion dissemination. However, the exact cellular components and molecular mechanisms underlying neural spread are unknown. This study used an established hamster scrapie model to pursue a novel experimental approach using nerve conduits containing segments devoid of neurites generated by incomplete nerve regeneration following Wallerian degeneration to probe the necessity of axons for the neural propagation of prions. For this purpose, animals were subjected to unilateral sciatic neurectomy 4 weeks before footpad inoculation with scrapie agent. The results showed that the regional nerve is the prime conduit for cerebral neuroinvasion and revealed, as evidenced by the accumulation of pathological prion protein PrP TSE, that prions can proceed along segments of peripheral neural projections without detectable axonal structures

Propagation of scrapie in peripheral nerves after footpad infection in normal and neurotoxin exposed hamsters

As is known from various animal models, the spread of agents causing transmissible spongiform encephalopathies (TSE) after peripheral infection affects peripheral nerves before reaching the central nervous system (CNS) and leading to a fatal end of the disease. The lack of therapeutic approaches for TSE is partially due to the limited amount of information available on the involvement of host biological compartments and processes in the propagation of the infectious agent. The in vivo model presented here can provide information on the spread of the scrapie agent via the peripheral nerves of hamsters under normal and altered axonal conditions. Syrian hamsters were unilaterally footpad (f.p.) infected with scrapie. The results of the spatiotemporal ultrasensitive immunoblot-detection of scrapie-associated prion protein (PrP(Sc)) in serial nerve segments of both distal sciatic nerves could be interpreted as a centripetal and subsequent centrifugal neural spread of PrP(Sc) for this route of infection. In order to determine whether this propagation is dependent on main components in the axonal cytoskeleton (e.g. neurofilaments, also relevant for the component ;a' of slow axonal transport mechanisms), hamsters were treated -in an additional experiment- with the neurotoxin beta,beta-iminodiproprionitrile (IDPN) around the beginning of the scrapie infection. A comparison of the Western blot signals of PrP(Sc) in the ipsilateral and in the subsequently affected contralateral sciatic nerve segments with the results revealed from IDPN-untreated animals at preclinical and clinical stages of the TSE disease, indicated similar amounts of PrP(Sc). Furthermore, the mean survival time was unchanged in both groups. This in vivo model, therefore, suggests that the propagation of PrP(Sc) along peripheral nerves is not dependent on an intact neurofilament component of the axonal cytoskeleton. Additionally, the model indicates that the spread of PrP(Sc) is not mediated by the slow component ;a' of the axonal transport mechanism

Accumulation of Pathological Prion Protein PrPSc in the Skin of Animals with Experimental and Natural Scrapie

Prion infectivity and its molecular marker, the pathological prion protein PrPSc, accumulate in the central nervous system and often also in lymphoid tissue of animals or humans affected by transmissible spongiform encephalopathies. Recently, PrPSc was found in tissues previously considered not to be invaded by prions (e.g., skeletal muscles). Here, we address the question of whether prions target the skin and show widespread PrPSc deposition in this organ in hamsters perorally or parenterally challenged with scrapie. In hamsters fed with scrapie, PrPSc was detected before the onset of symptoms, but the bulk of skin-associated PrPSc accumulated in the clinical phase. PrPSc was localized in nerve fibres within the skin but not in keratinocytes, and the deposition of PrPSc in skin showed no dependence from the route of infection and lymphotropic dissemination. The data indicated a neurally mediated centrifugal spread of prions to the skin. Furthermore, in a follow-up study, we examined sheep naturally infected with scrapie and detected PrPSc by Western blotting in skin samples from two out of five animals. Our findings point to the skin as a potential reservoir of prions, which should be further investigated in relation to disease transmission

Propagation of scrapie in peripheral nerves after footpad infection in normal and neurotoxin exposed hamsters

International audienceAs is known from various animal models, the spread of agents causing transmissible spongiform encephalopathies (TSE) after peripheral infection affects peripheral nerves before reaching the central nervous system (CNS) and leading to a fatal end of the disease. The lack of therapeutic approaches for TSE is partially due to the limited amount of information available on the involvement of host biological compartments and processes in the propagation of the infectious agent. The in vivo model presented here can provide information on the spread of the scrapie agent via the peripheral nerves of hamsters under normal and altered axonal conditions. Syrian hamsters were unilaterally footpad (f.p.) infected with scrapie. The results of the spatiotemporal ultrasensitive immunoblot-detection of scrapie-associated prion protein (PrP$^{\rm Sc})$ in serial nerve segments of both distal sciatic nerves could be interpreted as a centripetal and subsequent centrifugal neural spread of PrP$^{\rm Sc}$ for this route of infection. In order to determine whether this propagation is dependent on main components in the axonal cytoskeleton (e.g. neurofilaments, also relevant for the component 'a' of slow axonal transport mechanisms), hamsters were treated -in an additional experiment- with the neurotoxin ß,ß'-iminodiproprionitrile (IDPN) around the beginning of the scrapie infection. A comparison of the Western blot signals of PrP$^{\rm Sc}$ in the ipsilateral and in the subsequently affected contralateral sciatic nerve segments with the results revealed from IDPN-untreated animals at preclinical and clinical stages of the TSE disease, indicated similar amounts of PrP$^{\rm Sc}$. Furthermore, the mean survival time was unchanged in both groups. This in vivo model, therefore, suggests that the propagation of PrP$^{\rm Sc}$ along peripheral nerves is not dependent on an intact neurofilament component of the axonal cytoskeleton. Additionally, the model indicates that the spread of PrP$^{\rm Sc}$ is not mediated by the slow component 'a' of the axonal transport mechanism

Western blot detection of PrP27–30, the Proteinase K-resistant core of PrP, in lymphonodal tissue (at different time points after infection of hamsters the footpad with a 2% 263K-scrapie brain homogenate

<p><b>Copyright information:</b></p><p>Taken from "Relevance of the regional lymph node in scrapie pathogenesis after peripheral infection of hamsters"</p><p>http://www.biomedcentral.com/1746-6148/3/22</p><p>BMC Veterinary Research 2007;3():22-22.</p><p>Published online 25 Sep 2007</p><p>PMCID:PMC2092421.</p><p></p> The amount of tissue subjected to testing is specified in brackets. , left lane: blot control, 263K-scrapie hamster brain homogenate containing 10g of brain tissue; right lane: 27kD marker; , 2 dpi, left lane: contralateral . (3.1 mg); right lane: ipsilateral . (3.3 mg); , 42 dpi, left lane: contralateral . (2.9 mg); right lane: ipsilateral .(4.8 mg); , 70 dpi, left lane: contralateral . (7.0 mg); right lane: ipsilateral . (7.4 mg); , 80 dpi, left lane: contralateral . (7.9 mg); right lane: ipsilateral . (4.7 mg); , 90 dpi, left lane: contralateral . (4.1 mg); right lane: ipsilateral . (4.3 mg); , 100 dpi, left lane: contralateral .(1.9 mg); right lane: ipsilateral .(3.9 mg)