Establishment of a good manufacturing practice-compliant procedure for expansion of therapeutic doses of genetically modified, CAR expressing NK-92 cells for the treatment of ErbB2-positive malignancies

The NK-92/5.28.z cell line is an ErbB2 (HER2) specific, CAR-expressing continuously growing derivative of the clinically applicable NK-92 line, and exhibits profound and highly specific cytotoxicity against ErbB2-expressing tumor targets which are resistant to parental NK-92 cells. This study aimed to establish good manufacturing practice procedures enabling the generation of therapeutic doses of ErbB2-CAR expressing NK-92 cells for upcoming phase I/II clinical trials in ErbB2-overexpressing malignancies. The concept of patient dose manufacturing presented here encompasses the generation of a GMP-compliant master cell bank, which serves as a well-defined source of NK-92/5.28.z cells for further expansion of individual patient doses. To reach sufficient numbers of genetically modified NK-92 cells, expansion protocols were developed by systematic testing of culture conditions including GMP-grade culture media (X-Vivo 10 with human holo-transferrin, X-Vivo 10 with recombinant transferrin, CellGro) and human serum substitutes (human serum albumin, platelet lysate, heat inactivated human plasma). Since growth and potency of NK-92/5.28.z cells are IL-2 dependent, identification of a suitable concentration of this cytokine was an important issue addressed in this work. As a result of performed analyses, X-Vivo 10 containing recombinant transferrin supplemented with 5 % of heat inactivated human plasma and 500 U/ml IL-2 was selected as the culture medium for clinical expansions. This combination supported reproducible cell growth with doubling time of 28.84 h ± 0.5 h ±, maximal -fold expansion in batch culture of 24.97 ± 0.65 (max. conc.: 12.49 x 105 ± 0.32 x 105 cells/ml ) stable CAR expression and potency of the cells, even in long-term culture (CAR expression: 99.77 % ± 0.03 %; specific cytotoxicity: 89.75 % ± 3.77 % after 12 months of culturing). Considering the sensitivity of NK-92 and its CAR-expressing derivative to cryopreservation procedures, a part of this thesis focused on selection of cryopreservation solutions providing satisfactory post-thaw recovery of frozen cells, where human serum albumin as a base medium supplemented with 7.5 % DMSO gave the best results (post-thaw recovery of NK-92/5.28.z cells: 68.84 % ± 5.07 %). Following established GMP-compliant culture and cryopreservation procedures, a master cell bank comprising 200 vials of NK-92/5.28.z cells was generated. By means of concurrently developed quality control methods, 6 representative vials from the master cell bank were tested in terms of manufacturing related parameters (mean cryovial content, post-thaw recovery and cell growth, cell identity, specific cytotoxicity) confirming the high quality of the cells and thereby the validity of the performed process. Multiparameter analysis revealed that therapeutic doses of 5 x 109 NK-92/5.28.z cells can be expanded from a master cell bank within 5 days. Application of X-Vivo 10 rTF medium supplemented with 100 U/ml of IL-2 as an excipient, supported 6-hour shelf life of irradiated (10 Gy) NK-92/5.28.z cells formulated as a final product at the density of 5 x 107/ml. This concentration translates to the final dosage form of 1 x 108 cells/dose in 2 ml, a volume which is suitable for intracranial injection in the upcoming clinical trial in glioblastoma. The systemic (intravenous) route of administration planned in an upcoming breast cancer trial will allow the infusion of even higher volumes of up to 100 ml, and thereby higher cell numbers up to 5 x 109 cells/dose. In addition, investigation of factors impacting the efficacy and safety of clinical application was another important part of this thesis. Analysis of soluble factors released by target stimulated NK-92/5.28.z cells showed selective and significant increase in the secretion of GZMB (2-fold), IFN-γ (4-fold), IL-8 (24-fold) and IL-10 (5-fold) NK-92/5.28.z upon stimulation with ErbB2(+) targets. This phenomenon was not observed in the case of parental NK-92 cells and primary NK cells, indicating resistance of ErbB2(+) target cell line to natural killing mechanism, which can be overcome by specific CAR-mediated activation. Abundant amounts of immunomodulatory cytokines secreted by tumor activated NK-92/5.28.z may stimulate the host immune system by interaction with dendritic cells, induction of CTL and tumor-specific antibody production by B cells. Of possible relevance for clinical tolerability, IL-6 which plays the leading role in cytokine release syndrome was not detected. Treatment with clinically relevant doses of corticosteroids (maximal dose of 200 µg/ml corresponds with the highest dose of prednisolone applied clinically) did not affect retargeted killing of ErbB2(+) targets by NK-92/5.28.z cells, while natural killing against K562 cells was attenuated in a dose-dependent manner, as expected. Colony forming assays with mobilized peripheral blood progenitor cells pretreated with NK-92 or NK-92/5.28.z showed no evidence of NK cell impact on the colony forming capacity of PBSCs. In this study, GMP-compliant procedure enabling production of therapeutic doses of NK-92/5.28.z was successfully established. The data identify genetically modified, CAR-expressing NK-92 cells as a powerful, clinically feasible candidate for efficient and safe adoptive immunotherapy of ErbB2-overexpressing malignancies like breast cancer or glioblastoma

Revisiting the fairy land: Anne Sexton’s transformation of the Grimms’ female characters

Anne Sexton’s vivid and scandalous literary heritage has always been arousing controversy mostly due to its confessional character. It is, however, underestimated that one of the sources of her poetic inspirations was a broadly defined European culture and tradition, including the Grimms’ fairy tales. This article strives to inquire how Sexton revisits narratives of canonical tales with special regard to female protagonists. An apparent discrepancy will be shown between the two versions in terms of poetic imagery, character construction, and the reality in which they are firmly anchored. The morals drawn from the poems markedly diverge from the original versions, for it is with pessimism and disillusionment that Sexton transforms the naïve and sentimental images. The applied adaptation, hence, serves here to articulate the conflict between the traditional, male-centered set of values and a feminist perspective. The poems’ structures, literary figures, cultural references, features of genre, and other elements will be examined and analyzed to compare the retold stories with their archetypes and to provide a detailed interpretation in the light of the addressed [email protected] Korzeniewska-Nowakowska is a PhD student in the Faculty of Humanities at the University of Zielona Góra, Poland. Her academic interests include: American confessional poetry, postmodern studies, olympism, olympic idea in film and literature.University of Zielona Góra, PolandBeauvoir, de Simone. 1952. The Second Sex. New York: Knopf.Bottigheimer, Ruth B. 1987. Grimm’s Bad Girls and Bold Boys: The Moral and Social Vision of the Tales. New Haven: Yale University PressGill, Brendan. 1991. The Sky Line: Disneyitis. The New Yorker (April 29), 96-99.Gonzalez, Matilde Martin. 1999. Fairy tales revisited and transformed: Anne Sexton’ Critique of Social(ized) Feminity. http://dspace.uah.es (30 May 2015).Grimm, Jacob and Willhelm. 2012. Completed Fairy Tales. New York: Fall River Press.McGowan, Patrick, P. 2004. Anne Sexton and Middle Generation Poetry. London: Praeger.Liebermann, Marcia R.. 1972. Some Day My Prince Will Come: Female Acculturation through the Fairy Tale. College English 34(3), 383-395.Pinkola, Clara. 1996. Women Who Run With the Wolves: Myths and Stories of the Wild Woman Archetype. New York: Ballatine BooksSexton, Anne. 1971. Transformations. New York: Houghton Mifflin Company.Sexton, Anne. 1971. Transformations. Boston: Houghton Mifflin Company.Tatar, Maria. 1987. The Hard Facts of the Grimms’ Fairy Tales. New Jersey: Princeton University Press.Warner, Marina. 1991. The Absent Mother, or Women Against Women in the ‘Old Wives’ Tale. Hilversun: Verloren.Warner, Marina. 1994. From the Beast to the Blond: On Fairy Tales and their Tellers. New York: Farrar, Straus and Giroux.Zipes, Jack. 2002. The Brothers Grimm: From Enchanted Forests to the Modern World. New York: Palgrave MacMillan.15-2110 (3/2015)152

Adverse effect of UV radiation on eyes — a review

The aim of this study is to discuss the association between ultraviolet radiation (UVR) and eye diseases. UVR whichreaches the surface of the Earth consists of 95% UVA and 5% of UVB. Both acute and chronic exposure to UVRleads to pathological changes in the eye. There is strong evidence that UVR exposure causes photokeratitis, photoretinitis,climatic droplet keratopathy, cataract, pterygium, squamous cell carcinoma of cornea and conjunctiva,cancer of the eyelids (squamous cell and basal cell carcinoma). Association between UVR and pinguecula, AMD(age-related macular degeneration) and melanoma of the eyeball is ambiguous. UVR exposure occurs all day and allyear long, which is why eye protection is necessary to avoid diseases induced by UVR. In particular childrens’ eyesshould be protected from the sun. Efficient protection is provided by accurate clothing, hats, eyeglasses or contactlenses blocking UV light

How air pollution affects the eyes — a review

The aim of this study is to present scientific reports concerning the influence of air pollution on eyes. Air pollutionis nowadays a common problem. The most significant pollutants are carbon monoxide (CO), nitrogen dioxide(NO2), ozone (O3), sulphur dioxide (SO2), particulate matters — PM10 and PM2.5. The main source of toxins in theair are cars, burning of the fuels and burning of the charcoal in household stoves. Most attention is paid to negativeeffects of air pollution on respiratory system, such as asthma and lung cancer, however, it is worth remembering thatinfluence on the eyes is equally important. Children are especially vulnerable to the harmful effects of air pollution.Anterior segment of the eye is the most exposed. Substances, which are part of the smog have an irritating effecton the surface of the eye, cause disturbances in the tear film and an inflammation. Medical conditions associatedwith increased air pollution are mainly eye irritation, conjunctivitis, dry eye syndrome (DES), meibomian glanddysfunction (MGB) and blepharitis

Drama as a core in teaching Spanish as a foreign language at University of Lublin, Poland

El estudio investigó el impacto del programa de intervención didáctica basado en la dramatización, realizado en la Universidad Maria Curie Sklodowska de Lublin (Polonia) en el desarrollo de la competencia gramatical, léxica e intercultural y en las estrategias comunicativas de los alumnos del primer curso de Filología Hispánica. Para analizar las variables, se utilizó la metodología mixta con un diseño anidado de modelo dominante. Los resultados triangulados mostraron la mejora de la competencia léxica e intercultural y el desarrollo de las estrategias comunicativas de los participantes del programa de intervención didáctica. No se observó la mejora en la competencia gramatical. Se presentaron las posibilidades didácticas del uso de la dramatización en el aula de ELE y las futuras líneas de la investigación, como el desarrollo de la estrategia de interacción y mediación.The study investigated the impact of the drama based program carried out at the Maria Curie Sklodowska University of Lublin (Poland) in the development of grammatical, lexical and intercultural competence and in communicative strategies of the first-year students of Spanish Philology. The embedded mixed method was used to analyze the variables. Results showed the improvement of lexical and intercultural competence and development of the communicative strategies among workshop participants. Analyzes did not show improvement in grammatical competence. In the study, didactic possibilities of the use of drama in the Spanish as a foreign language classroom were presented, as well as the future lines of research, such as the development of the interaction and mediation strategy

Kontrowersyjna turystyka z wykorzystaniem zwierząt w perspektywie kulturowej

Nieomówionym dotychczas aspektem kontrowersyjnej turystyki z wykorzystaniem zwierząt jest jej związek z turysty­ką kulturową. Artykuł ma na celu przedstawienie kategoryzacji atrakcji oraz wydarzeń kulturowych i turystycznych, które są związane ze złym traktowaniem zwierząt. W tekście opisano, w jaki sposób turyści, kierowani kulturowym omniworyzmem i chęcią wyróżnienia się z tłumu, korzystają z kontrowersyjnych form tego rodzaju turystyki podczas międzykontynentalnych podróży. By uniknąć pułapki zachodniego etnocentryzmu, który prowadzi do oskarżeń o barbarzyństwo, autorzy przedstawili problem z różnych, międzykulturowych punktów widzenia, co pozwala na zrozumienie nastawienia lokalnych społeczności do zwierząt i ich dobrostanu. Co więcej, historia pokazuje, że troska o los zwierząt nie jest głęboko zakorzeniona w tradycji Zachodu. Zrównoważone rozwiązania w sektorze turystyki związanej ze zwierzętami muszą stać się elementem świadomości turystów, do której należy dążyć poprzez należytą edukację

Controversial animal tourism considered from a cultural perspective

An aspect of controversial animal tourism that has received little attention is its relationship with cultural tourism. The article presents a categorization of cultural tourism, and sports attractions and events related to the abuse of animals. It shows how tourists, driven by cultural omnivorousness and the wish to stand out from the crowd, eagerly enjoy controversial forms of animal tourism on their travel. In order to avoid the pitfall of western ethnocentrism ending in accusations of barbarism, the issue is also reflected on from an intercultural point of view, which helps to understand that local communities have different attitudes to animals and their wellbeing. In addition, a historical overview shows that ‘animal friendliness’ does not have a long tradition in western thinking about the human-animal relationship and is only of recent date. Sustainable solutions for controversial animal tourism have to be found by raising tourists’ awareness by means of information and education

Comparison of Magnetic Resonance Enterography Findings and Clinical Index of the Disease Activity Score in Crohn\u27s Disease

Introduction Crohn\u27s disease (CD) is a chronic inflammatory disease of the gastrointestinal tract. It usually affects younger people, with the peak of illness between the age of 15 and 25. MR enterography (MRE) is a proven and reliable modality in the evaluation of the lesion extent within this group of patients

Analysis of complications during pregnancy in women with serological features of acute toxoplasmosis or acute parvovirosis

Abstract Objectives: Toxoplasma gondii and parvovirus B19 (PVB19) infections in a healthy adult are usually asymptomatic. Congenital toxoplasmosis is the cause of hydrocephalus, chorioretinitis and intracranial calcifications. Hydrops remains the most common complication during the fetal PVB infection. The aim of the study was to analyze the complications during pregnancy in women with serological features of acute toxoplasmosis or acute parvovirosis. Material and methods: In our study, we have included 1800 pregnant women, hospitalized in Department of Fetal-Maternal Medicine and Gynecology Research Institute, Polish Mother's Memorial Hospital (RIPMMH) in Lodz, (Poland) between 2000-2007. Anti-T.gondii antibodies were tested by ELISA Vidas Toxo IgG, ELISA Vidas IgM (BioMerieux) and Platelia Toxo-A Anti-PVB19 antibodies were detected by NovaLisa Parvovirus B19 Recombinant IgG-ELISA and IgM-ELISA (NOVATEC). Results: Prevalence of IgG anti-PVB19 among pregnant women was 35% (n=633). IgG anti-T.gondii was noticed in 55.5% (n=910) of women. Serological features of acute parvovirosis were demonstrated in 13.5% (n=243) of the patients and 14.2% (n=256) of women suffered from acute toxoplasmosis. Fetal hydrocephalus or ventriculomegaly was diagnosed in 19.5% (n=64) of the pregnancies with IgM anti-PVB19 and in 7.3% (n=19) women with serological features of acute toxoplasmosis. In 8.5% (n=28) of the patients with IgM anti-PVB19 and 9% (n=5) of the pregnant women with IgM and/or IgA anti-T.gondii, fetal hydrops was detected. Intrauterine death was diagnosed in 4.5% (n=15) of the cases with acute PVB19 infection and in 2.3% (n=6) of the patients with acute toxoplasmosis. Amniotic fluid disorders were noticed more often in women with acute parvovirosis (polihydramnion 15.5%, n=51; oligohydramnion 8.5%, n=28; ahydramnion 6.3%, n=21) than in those with active toxoplasmosis (polihydramnion 3.4%, n=9; oligohydramnion 3.4%, n=9; ahydramnion 0%). We have examined also the influence of T.gondii and PVB19 infections on an intrauterine growth restriction, preterm delivery and spontaneous abortions. Conclusions: In conclusion, infections of T.gondii and PVB19 are a very common cause of complications in pregnancy. Due to high prevalence rate of IgG antibodies in Poland, it is necessary to consider routine serological testing in pregnancy

Analysis of complications during pregnancy in women with serological features of acute toxoplasmosis or acute parvovirosis

Abstract Objectives: Toxoplasma gondii and parvovirus B19 (PVB19) infections in a healthy adult are usually asymptomatic. Congenital toxoplasmosis is the cause of hydrocephalus, chorioretinitis and intracranial calcifications. Hydrops remains the most common complication during the fetal PVB infection. The aim of the study was to analyze the complications during pregnancy in women with serological features of acute toxoplasmosis or acute parvovirosis. Material and methods: In our study, we have included 1800 pregnant women, hospitalized in Department of Fetal-Maternal Medicine and Gynecology Research Institute, Polish Mother's Memorial Hospital (RIPMMH) in Lodz, (Poland) between 2000-2007. Anti-T.gondii antibodies were tested by ELISA Vidas Toxo IgG, ELISA Vidas IgM (BioMerieux) and Platelia Toxo-A Anti-PVB19 antibodies were detected by NovaLisa Parvovirus B19 Recombinant IgG-ELISA and IgM-ELISA (NOVATEC). Results: Prevalence of IgG anti-PVB19 among pregnant women was 35% (n=633). IgG anti-T.gondii was noticed in 55.5% (n=910) of women. Serological features of acute parvovirosis were demonstrated in 13.5% (n=243) of the patients and 14.2% (n=256) of women suffered from acute toxoplasmosis. Fetal hydrocephalus or ventriculomegaly was diagnosed in 19.5% (n=64) of the pregnancies with IgM anti-PVB19 and in 7.3% (n=19) women with serological features of acute toxoplasmosis. In 8.5% (n=28) of the patients with IgM anti-PVB19 and 9% (n=5) of the pregnant women with IgM and/or IgA anti-T.gondii, fetal hydrops was detected. Intrauterine death was diagnosed in 4.5% (n=15) of the cases with acute PVB19 infection and in 2.3% (n=6) of the patients with acute toxoplasmosis. Amniotic fluid disorders were noticed more often in women with acute parvovirosis (polihydramnion 15.5%, n=51; oligohydramnion 8.5%, n=28; ahydramnion 6.3%, n=21) than in those with active toxoplasmosis (polihydramnion 3.4%, n=9; oligohydramnion 3.4%, n=9; ahydramnion 0%). We have examined also the influence of T.gondii and PVB19 infections on an intrauterine growth restriction, preterm delivery and spontaneous abortions. Conclusions: In conclusion, infections of T.gondii and PVB19 are a very common cause of complications in pregnancy. Due to high prevalence rate of IgG antibodies in Poland, it is necessary to consider routine serological testing in pregnancy