Charakterisierung der Wirkmechanismen therapeutischer IgG- Antikörper gegen den epidermalen Wachstumsfaktor- Rezeptor (EGF-R)

Die Therapie solider Tumoren stellt einen wichtigen medizinischen Forschungsbereich dar. Um die Wirksamkeit der Therapien zu steigern und Nebenwirkungen zu reduzieren, werden immer spezifischere Behandlungsansätze wie die Antikörpertherapie entwickelt. Als eine geeignete Zielstruktur erwies sich dabei der epidermale Wachstumsfaktorrezeptor (Epidermal growth factor receptor; EGF-R), der eine wichtige Rolle bei der Regulation von Wachstum und Differenzierung von Zellen spielt. Störungen in diesem System können zum Entstehen und Wachstum von Tumoren führen. Die EGF-R-spezifischen Antikörper Cetuximab (Erbitux@) und Panitumumab (Vectibix@) finden bereits klinische Anwendung. Nimotuzumab (Theraloc@), ein humanisierter IgG1-Antikörper, bewies in verschiedenen klinischen Studien ebenfalls seine Wirksamkeit, besonders bei der Behandlung von Gliomen und Plattenepithelkarzinomen des Kopf- und Halsbereiches. Darüber hinaus stach sein günstiges Nebenwirkungsprofil heraus. In dieser Arbeit sollten die Wirkmechanismen von Nimotuzumab im Vergleich zu den etablierten Antikörpern Cetuximab und Panitumumab herausgearbeitet werden, um im Anschluss der Frage nachzugehen, ob diese Erkenntnisse eine Erklärung für das Nebenwirkungsprofil liefern. Die Ergebnisse der Arbeit zeigten, dass Nimotuzumab spezifisch an den EGF-R band, wobei Cetuximab und Panitumumab höhere Bindungsaffinitäten aufwiesen. Die Rezeptordichte auf den Tumorzellen beeinflusste dabei die Bindungseigenschaften der Antikörper nicht, was im Widerspruch zu einer publizierten Studie steht, wonach Nimotuzumab bei hoher EGF-R Dichte eine ähnlich hohe Affinität wie Cetuximab und somit eine relative Tumorspezifität besitzen soll [104]. Die geringere Bindungsaffinität könnte allerdings ein Grund für das günstigere Nebenwirkungsprofil sein, da Nimotuzumab so auch weniger stark an EGF-R exprimierende Zellen wie z.B. auch normale Epithelzellen bindet. Im nächsten Schritt wurden die direkten Wirkmechanismen der Antikörper untersucht. Nimotuzumab bewirkte eine partielle Verdrängung verschiedener Liganden wie EGF oder TGF-а vom Rezeptor und damit einhergehend eine Verminderung der Rezeptoraktivierung. Es konnte gezeigt werden, dass die wichtigen Signaltransduktionswege des Rezeptors über MAPK, PI3K und PLCγ1 in Tumorzellen durch Nimotuzumab gehemmt werden. Nimotuzumab blockierte die Ligandenbindung im Vergleich zu Cetuximab und Panitumumab aufgrund seiner geringeren Affinität schwächer und könnte auf diese Weise Rezeptoraktivierung in geringem Maße zulassen. Somit wäre durch die Wirkung von Nimotuzumab im Tumorgewebe beeinträchtigt, gleichzeitig aber der basale Level an EGF-RAktivierung für normales Gewebe, den es für seine Funktionen braucht, gegeben[106]. Dies könnte ebenfalls zum günstigen Nebenwirkungsprofil von Nimotuzumab beitragen. Abschließend ließ sich auf Basis dieser Erkenntnisse eine signifikante Reduktion von Tumorzellwachstum in vitro durch Nimotuzumab erreichen. Ein weiterer zentraler Punkt dieser Arbeit war die Untersuchung der Fähigkeit, Zellen des Immunsystems zu rekrutieren und ADCC (antikörper-abhängige zelluläre Zytotoxizität) zu vermitteln. Es zeigte sich, dass Nimotuzumab sowohl in der Lage war, ADCC über neutrophile Granulozyten als auch monomorphonukleäre Zellen zu induzieren, wozu es bisher keine experimentellen Daten in der Literatur gibt [104,107]. Cetuximab war aber auch in diesem Punkt effektiver als Nimotuzumab. Interessanterweise konnte als Teil dieser Arbeit erstmals gezeigt werden, dass sich über Panitumumab myeloische Effektorzellen effektiv zum ADCC von Tumorzellen aktivieren ließen. In der Literatur galt bisher, dass Panitumumab nicht in der Lage sei, ADCC zu vermitteln [127, 147]. Diese ADCC-Ergebnisse führten zu einer Koautorenschaft einer Publikation [81]. Weiter wurde die Möglichkeit der Komplementaktivierung durch Nimotuzumab untersucht, wobei keiner der EGF-RAntikörper einzeln in der Lage war, komplementabhängige Lyse von Tumorzellen zu induzieren [103]. Dagegen erwies sich die Kombination aus Nimotuzumab/Matuzumab als sehr effektiv und bewirkte Lysen bis zu 100 %. Zusammenfassend lässt sich durch die Ergebnisse dieser Arbeit sagen, dass Nimotuzumab durchaus potente Wirkmechanismen zur Tumorzellbekämpfung zuzuschreiben sind, diese aber im Vergleich zu bereits etablierten Antikörpern wie Cetuximab und Panitumumab zum Teil schwächer ausfallen. Wenn sich die Wirksamkeit von Nimotuzumab auch in großen Studien in Vergleich zu anderen Antikörpern beweisen sollte, könnte er mit seinem günstigen Nebenwirkungsprofil eine interessante Therapieoption darstellen. Zukünftige Untersuchungen müssen klären, inwieweit Nimotuzumab in Kombination mit Chemotherapeutika wirkt, welche Auswirkung Mutationen des EGF-R auf die Wirksamkeit des Antikörpers haben und wie der Antikörper durch Polymorphismen von Rezeptoren des Immunsystems beeinflusst wird, um so einen weiteren Schritt in Richtung spezifische Tumortherapie zu gehen

Optimal input potential functions in the interacting particle system method

The assessment of the probability of a rare event with a naive Monte-Carlo method is computationally intensive, so faster estimation methods, such as variance reduction methods, are needed. We focus on one of these methods which is the interacting particle (IPS) system method. The method requires to specify a set of potential functions. The choice of these functions is crucial, because it determines the magnitude of the variance reduction. So far, little information was available on how to choose the potential functions. To remedy this, we provide the expression of the optimal potential functions minimizing the asymptotic variance of the estimator of the IPS method

Результаты изучения палеодегазации неогена и перспективы нефтегазоносности Юго-Западного Крыма

Склад флюїдів з Гераклітiв принципово не відрізняється від складу газів із сучасних зон струминного виділення в Чорному морі, що є підтвердженням їх генетичного споріднення. Дані аналізів вказують на різкі коливання змістів і непостійний склад газових флюїдів палеодегазаціі в неогені. Наявність газів вуглеводнів та слідів нафти в Гераклітах, тектонічна будова регіону дозволяють зробити висновок про високі перспективи знаходження родовищ нафти і газу в Південно-Західному Криму.The composition of fluids from Heraclitus has no fundamental differences from the gas composition from the modern jet ejections zones in the Black Sea, which is a confirmation of their genetic relatedness. Data of analysis show sharp fluctuations of the contents and unstable structure of gas fluids paleo degassing of Neogene. The presence of hydrocarbon gases and traces of oil in Heraclitus, the tectonic structure of the region allow to conclude high prospects of finding oil and gas in the South-West Crimea

Characterization of a CTX-M-15 Producing Klebsiella Pneumoniae Outbreak Strain Assigned to a Novel Sequence Type (1427)

Extended-spectrum ß-lactamase producing Klebsiella pneumoniae have emerged as one of the major nosocomial pathogens. Between July and September 2012, a CTX-M-15 producing K. pneumoniae caused an outbreak in a university hospital in the Netherlands. The outbreak isolates were characterized and assigned to a novel sequence type (ST1427). An epidemiological link between affected patients was supported by patient contact tracing and whole-genome phylogenetic analysis. Genetic diversity was detected among multiple isolates obtained from different body sites of the index patient, which may relate to antibiotic treatment and/or host adaptation. Environmental contamination caused by the outbreak clone was found in the patient rooms even on medical equipment. The novel clone was not closely related to any known endemic/epidemic clone, but carried a set of a plasmid-borne resistance genes (blaCTX-M-15, blaTEM-1, blaOXA-1, aac(6')-Ib-cr, qnrB1, tetA(A), aac(3)-II). Analysis of its virulence factors revealed a previously uncharacterized capsular biosynthesis region and two uncharacterized fimbriae gene clusters, and suggested that the new clone was not hypervirulent. To our knowledge, this is the first outbreak report of K. pneumoniae ST1427, and our study could be of help to understand the features of this newly emerging clone

Comprehensive Characterization of Escherichia coil O104:H4 Isolated from Patients in the Netherlands

In 2011, a Shiga toxin-producing Enteroaggregative Escherichia coli (EAEC Stx2a+) O104:H4 strain caused a serious outbreak of acute gastroenteritis and hemolytic-uremic syndrome (HUS) in Germany. In 2013, E. coli O104:H4 isolates were obtained from a patient with HUS and her friend showing only gastrointestinal complaints. The antimicrobial resistance and virulence profiles of these isolates together with three EAEC Stx2a+ O104:H4 isolates from 2011 were determined and compared. Whole-genome sequencing (WGS) was performed for detailed characterization and to determine genetic relationship of the isolates. Four additional genomes of EAEC Stx2a+ O104:H4 isolates of 2009 and 2011 available on NCBI were included in the virulence and phylogenetic analysis. All E. coli O104:H4 isolates tested were positive for stx2a, aatA, and terD but were negative for escV. All, except one 2011 isolate, were positive for aggR and were therefore considered EAEC. The EAEC Stx2a+ O104:H4 isolates of 2013 belonged to sequence type (ST) ST678 as the 2011 isolates and showed slightly different resistance and virulence patterns compared to the 2011 isolates. Core-genome phylogenetic analysis showed that the isolates of 2013 formed a separate cluster from the isolates of 2011 and 2009 by 27 and 20 different alleles, respectively. In addition, only a one-allele difference was found between the isolate of the HUS-patient and that of her friend. Our study shows that EAEC Stx2a+ O104:H4 strains highly similar to the 2011 outbreak clone in their core genome are still circulating necessitating proper surveillance to prevent further outbreaks with these potentially pathogenic strains. In addition, WGS not only provided a detailed characterization of the isolates but its high discriminatory power also enabled us to discriminate the 2013 isolates from the isolates of 2009 and 2011 expediting the use of WGS in public health services to rapidly apply proper infection control strategies

Virulence, Antimicrobial Resistance Properties and Phylogenetic Background of Non-H7 Enteropathogenic Escherichia coli O157

Escherichia coli (E.coli) O157 that do not produce Shiga toxin and do not possess flagellar antigen H7 are of diverse H serotypes. In this study, the antibiotic resistance properties, genotype of a set of virulence associated genes and the phylogenetic background of E. coli O157:non-H7 groups were compared. Whole genome sequencing was performed on fourteen O157:non-H7 isolates collected in the STEC-ID-net study. The genomes were compared with E. coli O157 genomes and a typical Enteropathogenic E. coli (tEPEC) genome downloaded from NCBI. Twenty-six (86%) of the analyzed genomes had the intimin encoding gene eae but of different types mostly correlating with their H types, e.g., H16, H26, H39, and H45 carried intimin type epsilon, beta, kappa, and alpha, respectively. They belonged to several E. coli phylogenetic groups, i.e., to phylogenetic group A, B1, B2, and D. Seven (50%) of our collected O157:non-H7 isolates were resistant to two or more antibiotics. Several mobile genetic elements, such as plasmids, insertion elements, and pathogenicity islands, carrying a set of virulence and resistance genes were found in the E. coli O157:non-H7 isolates. Core genome phylogenetic analysis showed that O157:non-H7 isolates probably evolved from different phylogenetic lineages and were distantly related to the E. coli O157:H7 lineage. We hypothesize that independent acquisition of mobile genetic elements by isolates of different lineages have contributed to the different molecular features of the O157:non-H7 strains. Although distantly related to the STEC O157, E. coli O157:non-H7 isolates from multiple genetic background could be considered as pathogen of concern for their diverse virulence and antibiotic resistance properties

A novel Tn1696-like composite transposon (Tn6404) harboring bla(IMP-4) in a Klebsiella pneumoniae isolate carrying a rare ESBL gene bla(SFO-1)

Genetic determinants of a clinical Klebsiella pneumoniae isolate (KP1814) coproducing IMP-4 and a rare ESBL gene SFO-1 was investigated. KP1814 belongs to a novel sequence type (ST) assigned to ST2270. WGS identified four circular DNA sequences in KP1814, including two multidrug-resistance (MDR) plasmids, one virulence plasmid, and one circular form. The MDR plasmid pKP1814-1 (299.9 Kb) is untypeable, and carries two large mosaic multiresistance regions (MRRs). bla(SFO-1) and bla(IMP-4) co-exists on MRR1, and bla(SFO-1) is associated with an IS/Tn-independent genetic context. bla(IMP-4) is carried by a novel In804-like integron (intlI-bla(IMP-4)-Kl.pn.I3-qacG2-aacA4-catB3 Delta) associated with a novel Tn1696-like transposon (designed Tn6404) flanked by IS5075. The other MDR plasmid pKP1814-3 is a 95,701-bp IncFII plasmid, and is a hybrid of a Shigella flexneri plasmid pSF07201 and an E. coli plasmid pCA08. All resistance genes of pKP1814-3 were detected in a similar to 16-kb IS26-flanked composite transposon carried by a Tn5396 transposon. The circular form (18.3 Kb) was composed of two parts belonging to pKP1814-1 and pKP1814-3, respectively. The plasmid pKP1814-2, carrying multiple virulence factors, encodes IncFIB(K) and IncFII(K) replicons with a size of 187,349 bp. The coexistence of MDR and virulence plasmids largely enhances the bacterial fitness in the host and environment

An ancient family of mobile genomic islands introducing cephalosporinase and carbapenemase genes in Enterobacteriaceae

The exchange of mobile genomic islands (MGIs) between microorganisms is often mediated by phages, which may provide benefits to the phage's host. The present study started with the identification of Enterobacter cloacae, Klebsiella pneumoniae and Escherichia coli isolates with exceptional cephalosporin and carbapenem resistance phenotypes from patients in a neonatal ward. To identify possible molecular connections between these isolates and their β-lactam resistance phenotypes, the respective bacterial genome sequences were compared. This unveiled the existence of a family of ancient MGIs that were probably exchanged before the species E. cloacae, K. pneumoniae and E. coli emerged from their common ancestry. A representative MGI from E. cloacae was named MIR17-GI, because it harbors the novel β-lactamase gene variant blaMIR17. Importantly, our observations show that the MIR17-GI-like MGIs harbor genes associated with high-level resistance to cephalosporins. Among them, MIR17-GI stands out because MIR17 also displays carbapenemase activity. As shown by mass spectrometry, the MIR17 carbapenemase is among the most abundantly expressed proteins of the respective E. cloacae isolate. Further, we show that MIR17-GI-like islands are associated with integrated P4-like prophages. This implicates phages in the spread of cephalosporin and carbapenem resistance amongst Enterobacteriaceae. The discovery of an ancient family of MGIs, mediating the spread of cephalosporinase and carbapenemase genes, is of high clinical relevance, because high-level cephalosporin and carbapenem resistance have serious implications for the treatment of patients with enterobacteriaceal infections

Is Shiga Toxin-Negative Escherichia coli O157:H7 Enteropathogenic or Enterohemorrhagic Escherichia coli? Comprehensive Molecular Analysis Using Whole-Genome Sequencing

The ability of Escherichia coli O157:H7 to induce cellular damage leading to disease in humans is related to numerous virulence factors, most notably the stx gene, encoding Shiga toxin (Stx) and carried by a bacteriophage. Loss of the Stx-encoding bacteriophage may occur during infection or culturing of the strain. Here, we collected stx-positive and stx-negative variants of E. coli O157:H7/NM (nonmotile) isolates from patients with gastrointestinal complaints. Isolates were characterized by whole-genome sequencing (WGS), and their virulence properties and phylogenetic relationship were determined. Because of the presence of the cae gene but lack of the bfpA gene, the stx-negative isolates were considered atypical enteropathogenic E. coli (aEPEC). However, they had phenotypic characteristics similar to those of the Shiga toxin-producing E. coli (STEC) isolates and belonged to the same sequence type, STI1. Furthermore, EPEC and STEC isolates shared similar virulence genes, the locus of enterocyte effacement region, and plasmids. Core genome phylogenetic analysis using a gene-by-gene typing approach showed that the sorbitol-fermenting (SF) stx-negative isolates clustered together with an SF STEC isolate and that one non-sorbitol-fermenting (NSF) stx-negative isolate clustered together with NSF STEC isolates. Therefore, these stx-negative isolates were thought either to have lost the Stx phage or to be a progenitor of STEC O157: H7/NM. As detection of STEC infections is often based solely on the identification of the presence of stx genes, these may be misdiagnosed in routine laboratories. Therefore, an improved diagnostic approach is required to manage identification, strategies for treatment, and prevention of transmission of these potentially pathogenic strains

The peptidylarginine deiminase gene is a conserved feature of Porphyromonas gingivalis

Periodontitis is an infective process that ultimately leads to destruction of the soft and hard tissues that support the teeth (the periodontium). Periodontitis has been proposed as a candidate risk factor for development of the autoimmune disease rheumatoid arthritis (RA). Porphyromonas gingivalis, a major periodontal pathogen, is the only known prokaryote expressing a peptidyl arginine deiminase (PAD) enzyme necessary for protein citrullination. Antibodies to citrullinated proteins (anti-citrullinated protein antibodies, ACPA) are highly specific for RA and precede disease onset. Objective of this study was to assess P. gingivalis PAD (PPAD) gene expression and citrullination patterns in representative samples of P. gingivalis clinical isolates derived from periodontitis patients with and without RA and in related microbes of the Porphyromonas genus. Our findings indicate that PPAD is omnipresent in P. gingivalis, but absent in related species. No significant differences were found in the composition and expression of the PPAD gene of P. gingivalis regardless of the presence of RA or periodontal disease phenotypes. From this study it can be concluded that if P. gingivalis plays a role in RA, it is unlikely to originate from a variation in PPAD gene expression