Safety of Xenotransplantation: Development of screening methods and testing for porcine viruses

Xenotransplantation using pig cells, tissues or organs might be a promising solution to overcome the shortage for organs suitable for allotransplantation. Because of several reasons, the pig is currently the favoured donor species. However, the use of porcine xenotransplants is associated with the risk of transmitting porcine viruses to the human xenotransplant recipient. Among them porcine endogenous retroviruses (PERVs), porcine cytomegalovirus (PCMV), porcine lymphotropic herpesviruses (PLHVs), porcine circovirus 2 (PCV2) and hepatitis E virus (HEV) play a role. Some of them cause immunosuppression and a zoonotic potential of others has been supposed. Therefore the possibility of direct transmission of those viruses between pigs and humans might be possible. Strategies to avoid the transmission of those pathogens are currently of main importance to increase lifetime of the transplant and therefore to save many lives of people standing on the transplant waiting list. To select virus-free animals as putative donor pigs and to recognise transmission of pathogens to transplant recipients, sensitive detection methods are needed. In this study the prevalence and expression of these selected viruses should be investigated and assessed in order to obtain safe and healthy donor pigs for xenotransplantation studies. Therefore highly sensitive PCR-based methods, real-time PCR and real-time RT-PCR specific for all the viruses listed above, as well as immunological methods measuring virus-specific antibodies by Western blot analysis or ELISA were developed. Recombinant viral proteins were cloned, expressed and chromatographically purified as well as purified virus particles were expanded to be used as antigens. The methods were developed and optimized to screen (i) Göttingen minipigs, a well characterized pig breed which is kept in a specific-pathogen free facility, (ii) Aachen minipigs, a pig breed existing since 2013, (iii) slaughterhouse pigs from a butchery in the north of Berlin and (iv) multiply genetically modified pigs produced especially for xenotransplantation. Human-tropic PERV-A and PERV-B were found in all pigs and pig-tropic PERV-C and recombinant PERV-A/C were found in many pigs. HEV, PCMV, PLHVs and PCV2 were found in a few animals. No transmission of the porcine viruses listed above was observed during the transplantation of genetically modified islet cells into four marmosets. However, when transgenic pig hearts were transplanted into baboons, then PCMV and HEV were found transmitted, despite the fact that the donor pigs were negative when testing blood and antibody response. To avoid future transmissions of porcine viruses, more sensitive detection methods, different time points of testing, and different source materials, including oral and anal swabs, should be used. In the study sensitive and reliable methods for the detection of porcine viruses were developed and those viruses were detected in all tested pig herds. Furthermore, potentially zoonotic viruses like HEV and viruses causing immunosuppression like PCMV, PLHVs and PCV2 are present in pigs for slaughter. Although the expression of these viruses were low, the meat-producing and -processing industry should be aware of the improvement of hygienic standards. The newly developed detection methods are a prerequisite for the selection of virus-free pigs for transplantation trials as well as elimination programs based on treatment, vaccination, Caesarean delivery, early weaning and embryo transfer

Early and Middle Holocene Hunter-Gatherer Occupations in Western Amazonia: The Hidden Shell Middens

We report on previously unknown early archaeological sites in the Bolivian lowlands, demonstrating for the first time early and middle Holocene human presence in western Amazonia. Multidisciplinary research in forest islands situated in seasonally-inundated savannahs has revealed stratified shell middens produced by human foragers as early as 10,000 years ago, making them the oldest archaeological sites in the region. The absence of stone resources and partial burial by recent alluvial sediments has meant that these kinds of deposits have, until now, remained unidentified. We conducted core sampling, archaeological excavations and an interdisciplinary study of the stratigraphy and recovered materials from three shell midden mounds. Based on multiple lines of evidence, including radiocarbon dating, sedimentary proxies (elements, steroids and black carbon), micromorphology and faunal analysis, we demonstrate the anthropogenic origin and antiquity of these sites. In a tropical and geomorphologically active landscape often considered challenging both for early human occupation and for the preservation of hunter-gatherer sites, the newly discovered shell middens provide evidence for early to middle Holocene occupation and illustrate the potential for identifying and interpreting early open-air archaeological sites in western Amazonia. The existence of early hunter-gatherer sites in the Bolivian lowlands sheds new light on the region's past and offers a new context within which the late Holocene "Earthmovers" of the Llanos de Moxos could have emerged. © 2013 Lombardo et al

Antibody Cross-Reactivity between Porcine Cytomegalovirus (PCMV) and Human Herpesvirus-6 (HHV-6)

Porcine cytomegalovirus (PCMV) infection is widely prevalent among pigs, and PCMV is one of the viruses which may be transmitted during xenotransplantation using pig cells, tissues, or organs. While human cytomegalovirus (HCMV) is a major risk factor for allotransplantation, it is still unclear whether PCMV is able to infect human cells or pose a risk for xenotransplantation. Previously, it was shown that transmission of PCMV after pig kidney to non-human primate transplantations resulted in a significantly reduced survival time of the transplanted organ. To detect PCMV, PCR-based and immunological methods were used. Screening of pigs by Western blot analyses using recombinant viral proteins revealed up to 100% of the tested animals to be infected. When the same method was applied to screen human sera for PCMV-reactive antibodies, positive Western blot results were obtained in butchers and workers in the meat industry as well as in normal blood donors. To exclude an infection of humans with PCMV, the sera were further investigated. PCMV is closely related to human herpesvirus-6 (HHV-6) and human herpesvirus-7 (HHV-7), and a sequence alignment of glycoprotein B suggests that the antibodies may cross-react with identical epitope sequences. HCMV is not related with PCMV, and no correlation between antibody reactivity against PCMV and HCMV was detected. These data indicate that antibodies against PCMV found in humans are cross-reactive antibodies against HHV-6

Estimation of Breeding Values Using Different Densities of Snp to Inform Kinship in Broiler Chickens

Background: Traditionally, breeding values are estimated based on phenotypic and pedigree information using the numerator relationship (A) matrix. With the availability of genomic information, genome-wide markers can be included in the estimation of breeding values through genomic kinship. However, the density of genomic information used can impact the cost of implementation. The aim of this study was to compare the rank, accuracy, and bias of estimated breeding values (EBV) for organs [heart (HRT), liver (LIV), gizzard (GIZ), lungs (LUN)] and carcass [breast (BRST), drumstick (DRM) and thigh (THG)] weight traits in a broiler population using pedigree-based BLUP (PBLUP) and single-step genomic BLUP (ssGBLUP) methods using various densities of SNP and variants imputed from whole-genome sequence (WGS). Results: For both PBLUP and ssGBLUP, heritability estimates varied from low (LUN) to high (fHRT, LIV, GIZ, BRST, DRM and THG.) Regression coefficients values of EBV on genomic estimated breeding values (GEBV) were similar for both the high density (HD) and WGS sets of SNPs ranging from 0.87 to 0.99 across senarios. Conclusion: Results show no benefit of using WGS data compared to HD array data using an unweighted ssGBLUP. Our results suggest that 10% of the content of the HD array can yield unbiased and accurate EBV

Efficient production of multi-modified pigs for xenotransplantation by ‘combineering’, gene stacking and gene editing

Xenotransplantation from pigs could alleviate the shortage of human tissues and organs for transplantation. Means have been identified to overcome hyperacute rejection and acute vascular rejection mechanisms mounted by the recipient. The challenge is to combine multiple genetic modifications to enable normal animal breeding and meet the demand for transplants. We used two methods to colocate xenoprotective transgenes at one locus, sequential targeted transgene placement - ‘gene stacking’, and cointegration of multiple engineered large vectors - ‘combineering’, to generate pigs carrying modifications considered necessary to inhibit short to mid-term xenograft rejection. Pigs were generated by serial nuclear transfer and analysed at intermediate stages. Human complement inhibitors CD46, CD55 and CD59 were abundantly expressed in all tissues examined, human HO1 and human A20 were widely expressed. ZFN or CRISPR/Cas9 mediated homozygous GGTA1 and CMAH knockout abolished α-Gal and Neu5Gc epitopes. Cells from multi-transgenic piglets showed complete protection against human complement-mediated lysis, even before GGTA1 knockout. Blockade of endothelial activation reduced TNFα-induced E-selectin expression, IFNγ-induced MHC class-II upregulation and TNFα/cycloheximide caspase induction. Microbial analysis found no PERV-C, PCMV or 13 other infectious agents. These animals are a major advance towards clinical porcine xenotransplantation and demonstrate that livestock engineering has come of age

Das peritoneal metastasierte Mammakarzinom: Analyse eines seltenen Metastasierungsmusters

Die vorliegende retrospektive Studie befasste sich mit dem peritoneal metastasierten Mammakarzinom. Dieses Metastasierungsmuster stellt gegenüber konventionellen Metastasierungsmustern eine sehr seltene Entität dar. In unserer Arbeit wurden 44 Patientinnen mit histologisch gesichertem Mammakarzinom und peritonealer Metastasierung untersucht. In die Untersuchung wurden die Tumorhistologien, die Häufigkeit und Art der Metastasenlokalisationen, die Therapien und Überlebenszeitanalyse miteinbezogen. Der 21-jährige Beobachtungszeitraum erstreckte sich von 1992 bis 2013. Zum Studienende lebten lediglich noch 4,5% (n= 2) der Patientinnen. Die Erstdiagnose einer peritonealen Metastasierung ist mit einer sehr schlechten Prognose assoziiert. Ein Überleben ab peritonealer Metastasierung erstreckt sich derzeit im Allgemeinen auf wenige Monate. Aufgrund der geringen Inzidenz sind zum jetzigen Zeitpunkt keine standardisierten Therapieempfehlungen verfügbar. Diese Beobachtungen können anhand dieses Datensatzes nachvollzogen werden. Trotz des langen Beobachtungszeitraumes in dieser Studie, sind aufgrund der heterogenen Therapieschemata keine signifikanten Unterschiede hinsichtlich des Outcomes nachweisbar. Es zeigt sich jedoch, dass die Entwicklung einer peritonealen Metastasierung üblicherweise in einem weit fortgeschrittenen Stadium der Erkrankung auftritt. Zusammenfassend sind aufgrund der geringen Inzidenz nur wenige Daten zur peritonealen Metastasierung beim Mammakarzinom vorhanden. In jedem Fall zeichnet sich durch das Auftreten peritonealer Metastasen eine signifikante Verschlechterung der Prognose ab, was zentral in die Therapiestrategie und –ent-schei¬dungen unter dem Fokus der Lebensqualität der Patientin einbezogen werden muss. Radikal operative Strategien sind deshalb beim Mammakarzinom nicht als Standard anzusehen und sollten nur als palliative Interventionen angewandt werden. Mittelfristig wären aufgrund der geringen Inzidenz Registerstudien zur Beantwortung der offenen Fragen wünschenswert. Auf Basis solcher Registerdaten wären möglicherweise differenzierte Therapieempfehlungen möglich

SSH-Log Manipulation : Possibilities and Forensic Artifacts

Die vorliegende Bachelorarbeit untersucht verschiedene Möglichkeiten zur Manipulation der drei wesentlichen SSH-Logdateien auth.log, wtmp und systemd-Journal unter Linux. Das Ziel ist es, präzise Techniken vorzustellen, um den SSH-Zugriff eines autorisierten Nutzers mit administrativen Rechten während einer aktiven SSH-Sitzung zu verschleiern. Es wird untersucht, wie die Manipulationen durchgeführt werden können, welche Linux-Befehle dabei zum Einsatz kommen und welche forensischen Artefakte in den Log-Dateien weiterhin bestehen bleiben. Neben verschiedenen Linux-Befehlen wird ebenso Python verwendet. Die Ergebnisse zeigen, dass alle vorgestellten Methoden wie erwartet funktionierten und der Nutzerzugriff in der wtmp und der auth.log Log-Datei nicht weiter erkennbar ist. Dennoch besteht weiterer Forschungsbedarf, da sich die Manipulation der systemd-Journal Dateien als schwierig erwies