Entwicklung und Charakterisierung eines Immunzytokins basierend auf PankoMab-GEX® zur Optimierung der Tumortherapie

Zytokine stellen ein vielversprechendes Therapeutikum für die Immuntherapie bei Tumorerkrankungen dar. Dabei ist IL-15 ein interessantes Zytokin, da es vor allem NK- und CD8+ T-Zellen sehr stark aktiviert und diese Zellen maßgeblich an der Tumorreduktion beteiligt sind. Sowohl bei der Monotherapie mit IL-15 als auch in Kombination aus IL-15 und Antikörpern konnte in vitro und in vivo ein gesteigerter Tumorrückgang gezeigt werden. Der Einsatz von Zytokinen in der Therapie ist jedoch auf Grund von starken Toxizitäten dosislimitierend, weshalb die geringen verwendeten Konzentrationen oftmals nicht ausreichend für einen Therapieeffekt sind. Des Weiteren liegt die Serumhalbwertszeit im Falle von IL-15 bei lediglich 30 Minuten. Bei der Therapie mit IL-15 werden hauptsächlich die Immunzellen in der Zirkulation stimuliert, während nur wenige Immunzellen in der Tumorumgebung aktiviert werden. Deshalb stellen Immunzytokine einen vielversprechenden Ansatz für die Immuntherapie dar. Durch die Fusion eines Antikörpers und Zytokins entstehen antigenspezifische Immunzytokine, welche die Immunzellen gezielt in der immunsuppressiven Tumormikroumgebung aktivieren können. Zudem kann die Serumhalbwertszeit verlängert werden. Ein für die Immuntherapie interessantes Antigen ist TA-MUC1, da es von soliden Tumorzellen exprimiert wird und auf gesunden Zellen nahezu nicht. PankoMab-GEX® ist ein Antikörper, der ein tumorspezifisches Kohlenhydrat/Protein Konformationsepitop auf MUC1 (TA-MUC1) bindet und ADCC gegen TA-MUC1 exprimierende Tumorzellen induzieren kann. Ob PankoMab-GEX® als Grundlage für die Entwicklung eines Immunzytokins geeignet ist, wurde in dieser Arbeit untersucht. Für diese Arbeit wurden insgesamt zehn Immunzytokine basierend auf PankoMab-GEX® generiert, welche sich hinsichtlich ihrer IL-15 Potenz (IL-15mut, IL-15wt, IL-15sushi), der FcγRIIIa Bindung sowie der Antigenbindung unterscheiden. Die Arbeit umfasste dabei die Generierung, Produktion und Reinigung der Konstrukte sowie die Charakterisierung der Bindungseigenschaften und der immunologischen Wirkung der Immunzytokine in vitro und in vivo. Es konnte gezeigt werden, dass die Stimulation von Immunzellen mit den Immunzytokinen zu einer Steigerung der Aktivierung, Proliferation, Zytotoxizität und Zytokinaussschüttung bei NK- und T-Zellen führt. Dabei konnte herausgestellt werden, dass das PM-IL-15sushi ein besonders potentes Immunzytokin ist, da es die zytotoxischen Immunzellen sehr stark aktiviert. Anhand der durchgeführten Analysen konnte gezeigt werden, dass sich PankoMab-GEX® auf Grund der einzigartigen Antigenbindung für die Entwicklung eines Immunzytokins eignet. Angesichts der deutlich stärkeren Potenz des PM-IL-15sushi im Vergleich zu den anderen untersuchten Immunzytokinen sowie der vielversprechenden Wirksamkeitsstudie in vivo wurde dieses Konstrukt als potentielles, neuartiges Therapeutikum ausgewählt.Cytokines are promising immunotherapeutical drugs to treat cancer. Especially IL15 is a potent cytokine as it strongly activates NK and CD8+ T cells which are important for tumor cell reduction. In vitro and in vivo studies already demonstrated tumor shrinkage after the combination therapy using an antibody and IL15 or using IL15 alone. The administration is dose limited due to strong toxicities. Therefore, low concentrations are administered which are not effective enough to enhance immune cell activity in the tumor microenvironment. Furthermore, serum halflife of IL15 is only 30min. Using IL15 for immunotherapy mainly activates circulating immune cells whereas immune cells in the tumor microenvironment get less activated. That is why immunocytokines are promising products for immunotherapy. Newly antigenspecific immunocytokines are generated by linking a cytokine to an antibody. These immunocytokines induce immune cell activation in the tumor microenvironment and prolong serum halflife. One promising tumor antigen is TAMUC1 which is expressed on solid tumors and is virtually absent on normal tissue. PankoMabGEX® is an antibody which binds a novel carbohydrate/protein mixed epitope on the tumor marker MUC1 and mediates ADCC against TAMUC1 expressing tumor cells. During this study it was analyzed if PankoMabGEX® is a suitable antibody to generate an immunocytokine. This study included the generation of ten immunocytokines based on PankoMabGEX® which differ in IL15 potency (IL15mut, IL15wt, IL15sushi), FcγRIIIa binding and antigen binding. The generation, production and down stream purification of all immunocytokines were part of this study as well as the analysis of the binding characterization and the immunological effect in vitro and in vivo. It was shown that immune cell stimulation using immunocytokines enhance the NK and T cell activation, proliferation, cytotoxicity and cytokine secretion. It turned out, PMIL15sushi is a highly potent immunocytokine which strongly activates cytotoxic immune cells. Regarding the generated data it is confirmed that PankoMabGEX® is a potent antibody to develop an immunocytokine because of its unique antigen binding. The strong potency and great outcome of the efficacy study in vivo using PMIL15sushi compared to the other immunocytokines recommend that PMIL15sushi should be selected as a new anticancer therapeutic

An N-terminal Peptide Extension Results in Efficient Expression, but not Secretion, of a Synthetic Horseradish Peroxidase Gene in Transgenic Tobacco

BACKGROUND AND AIMS: Native horseradish (Armoracia rusticana) peroxidase, HRP (EC 1.11.1.7), isoenzyme C is synthesized with N-terminal and C-terminal peptide extensions, believed to be associated with protein targeting. This study aimed to explore the specific functions of these extensions, and to generate transgenic plants with expression patterns suitable for exploring the role of peroxidase in plant development and defence. METHODS: Transgenic Nicotiana tabacum (tobacco) plants expressing different versions of a synthetic horseradish peroxidase, HRP, isoenzyme C gene were constructed. The gene was engineered to include additional sequences coding for either the natural N-terminal or the C-terminal extension or both. These constructs were placed under the control of a constitutive promoter (CaMV-35S) or the tobacco RUBISCO-SSU light inducible promoter (SSU) and introduced into tobacco using Agrobacterium-mediated transformation. To study the effects of the N- and C-terminal extensions, the localization of recombinant peroxidase was determined using biochemical and molecular techniques. KEY RESULTS: Transgenic tobacco plants can exhibit a ten-fold increase in peroxidase activity compared with wild-type tobacco levels, and the majority of this activity is located in the symplast. The N-terminal extension is essential for the production of high levels of recombinant protein, while the C-terminal extension has little effect. Differences in levels of enzyme activity and recombinant protein are reflected in transcript levels. CONCLUSIONS: There is no evidence to support either preferential secretion or vacuolar targeting of recombinant peroxidase in this heterologous expression system. This leads us to question the postulated targeting roles of these peptide extensions. The N-terminal extension is essential for high level expression and appears to influence transcript stability or translational efficiency. Plants have been generated with greatly elevated cytosolic peroxidase activity, and smaller increases in apoplastic activity. These will be valuable for exploring the role of these enzymes in stress amelioration and plant development

The Iowa Homemaker vol.3, no.8

Table of Contents The Goal of Home Management Courses at Iowa State by Ruth M. Lindquist, page 3 Home Economics Venture Upon “India’s Coral Strand” by Marcia E. Turner, page 4 The Bell Recipe File by Opal Wind, page 4 Carving the Turkey by Viola M. Bell, page 5 An Interview With Scottish Hockey Coach by Lucile Barta, page 5 Two and One-half Miles Saved a Day by Grata Thorne, page 6 Cranberries for Thanksgiving by Katherine Goeppinger, page 7 Who is Responsible for the Child - by “A Bachelor”, page 8 The Evolution of Home Economics at Iowa State by Ruth Elaine Wilson, page 9 Why is a Mulligan? by “Joe Baggs”, page 10 Turning the Corners Up by Laura E. Bublitz, page 10 The Sport of Amateur Housekeeping by Anna Jacobson, page 11 Who’s There and Where by Helen I. Putnam, page 12 Dressing the Homemaker by Margaret Dix, page 1

Provision of cervical screening for transmasculine patients: a review of clinical and programmatic guidelines.

BACKGROUND: Most cervical cancer can be prevented through routine screening. Disparities in uptake of routine screening therefore translate into disparities in cervical cancer incidence and outcomes. Transmasculine people including transgender men experience multiple barriers to cervical screening and their uptake of screening is low compared with cisgender women. Comprehensive evidence-based guidelines are needed to improve cervical screening for this group. METHODS: We searched for and synthesised clinical and programmatic guidelines for the provision of cervical screening for transmasculine patients. FINDINGS: The guidelines offer recommendations addressing: (1) reception, check-in and clinic facilities; (2) patient data and invitation to screening; (3) improving inclusion in screening programmes; and (4) sexual history taking, language and identity. Guidelines offer strategies for alleviating physical and psychological discomfort during cervical screening and recommendations on what to do if the screening procedure cannot be completed. Most of the guidelines were from and for high-income countries. DISCUSSION: The evidence base is limited, but existing guidelines provide recommendations to ensure life-saving screening services are available to all who need them. We were only able to identify one set of guidelines for a middle-income country, and none for low-income countries. We encourage the involvement of transmasculine people in the development of future guidelines

Ribonuclease H2 mutations induce a cGAS/STING-dependent innate immune response

Aicardi–Goutières syndrome (AGS) provides a monogenic model of nucleic acid‐mediated inflammation relevant to the pathogenesis of systemic autoimmunity. Mutations that impair ribonuclease (RNase) H2 enzyme function are the most frequent cause of this autoinflammatory disorder of childhood and are also associated with systemic lupus erythematosus. Reduced processing of either RNA:DNA hybrid or genome‐embedded ribonucleotide substrates is thought to lead to activation of a yet undefined nucleic acid‐sensing pathway. Here, we establish Rnaseh2b (A174T/A174T) knock‐in mice as a subclinical model of disease, identifying significant interferon‐stimulated gene (ISG) transcript upregulation that recapitulates the ISG signature seen in AGS patients. The inflammatory response is dependent on the nucleic acid sensor cyclic GMP‐AMP synthase (cGAS) and its adaptor STING and is associated with reduced cellular ribonucleotide excision repair activity and increased DNA damage. This suggests that cGAS/STING is a key nucleic acid‐sensing pathway relevant to AGS, providing additional insight into disease pathogenesis relevant to the development of therapeutics for this childhood‐onset interferonopathy and adult systemic autoimmune disorders

Fluoxetine Counteracts the Cognitive and Cellular Effects of 5-Fluorouracil in the Rat Hippocampus by a Mechanism of Prevention Rather than Recovery

5-Fluorouracil (5-FU) is a cytostatic drug associated with chemotherapy-induced cognitive impairments that many cancer patients experience after treatment. Previous work in rodents has shown that 5-FU reduces hippocampal cell proliferation, a possible mechanism for the observed cognitive impairment, and that both effects can be reversed by co-administration of the antidepressant, fluoxetine. In the present study we investigate the optimum time for administration of fluoxetine to reverse or prevent the cognitive and cellular effects of 5-FU

Large Scale Comparative Codon-Pair Context Analysis Unveils General Rules that Fine-Tune Evolution of mRNA Primary Structure

BACKGROUND: Codon usage and codon-pair context are important gene primary structure features that influence mRNA decoding fidelity. In order to identify general rules that shape codon-pair context and minimize mRNA decoding error, we have carried out a large scale comparative codon-pair context analysis of 119 fully sequenced genomes. METHODOLOGIES/PRINCIPAL FINDINGS: We have developed mathematical and software tools for large scale comparative codon-pair context analysis. These methodologies unveiled general and species specific codon-pair context rules that govern evolution of mRNAs in the 3 domains of life. We show that evolution of bacterial and archeal mRNA primary structure is mainly dependent on constraints imposed by the translational machinery, while in eukaryotes DNA methylation and tri-nucleotide repeats impose strong biases on codon-pair context. CONCLUSIONS: The data highlight fundamental differences between prokaryotic and eukaryotic mRNA decoding rules, which are partially independent of codon usage

Fluoxetine reverses the memory impairment and reduction in proliferation and survival of hippocampal cells caused by methotrexate chemotherapy

RATIONALE: Adjuvant cancer chemotherapy can cause long-lasting, cognitive deficits. It is postulated that these impairments are due to these drugs targeting neural precursors within the adult hippocampus, the loss of which has been associated with memory impairment. OBJECTIVES: The present study investigates the effects of the chemotherapy, methotrexate (MTX) on spatial working memory and the proliferation and survival of the neural precursors involved in hippocampal neurogenesis, and the possible neuroprotective properties of the antidepressant fluoxetine. METHODS: Male Lister hooded rats were administered MTX (75 mg/kg, two i.v. doses a week apart) followed by leucovorin rescue (i.p. 18 h after MTX at 6 mg/kg and at 26, 42 and 50 h at 3 mg/kg) and/or fluoxetine (10 mg/kg/day in drinking water for 40 days). Memory was tested using the novel location recognition (NLR) test. Using markers, cell proliferation (Ki67) and survival (bromodeoxyuridine/BrdU), in the dentate gyrus were quantified. RESULTS: MTX-treated rats showed a cognitive deficit in the NLR task compared with the vehicle and fluoxetine-treated groups. Cognitive ability was restored in the group receiving both MTX and fluoxetine. MTX reduced both the number of proliferating cells in the SGZ and their survival. This was prevented by the co-administration of fluoxetine, which alone increased cell numbers. CONCLUSIONS: These results demonstrate that MTX induces an impairment in spatial working memory and has a negative long-term effect on hippocampal neurogenesis, which is counteracted by the co-administration of fluoxetine. If translatable to patients, this finding has the potential to prevent the chemotherapy-induced cognitive deficits experienced by many cancer survivors

Peri-operative red blood cell transfusion in neonates and infants: NEonate and Children audiT of Anaesthesia pRactice IN Europe: A prospective European multicentre observational study

BACKGROUND: Little is known about current clinical practice concerning peri-operative red blood cell transfusion in neonates and small infants. Guidelines suggest transfusions based on haemoglobin thresholds ranging from 8.5 to 12 g dl-1, distinguishing between children from birth to day 7 (week 1), from day 8 to day 14 (week 2) or from day 15 (≥week 3) onwards. OBJECTIVE: To observe peri-operative red blood cell transfusion practice according to guidelines in relation to patient outcome. DESIGN: A multicentre observational study. SETTING: The NEonate-Children sTudy of Anaesthesia pRactice IN Europe (NECTARINE) trial recruited patients up to 60 weeks' postmenstrual age undergoing anaesthesia for surgical or diagnostic procedures from 165 centres in 31 European countries between March 2016 and January 2017. PATIENTS: The data included 5609 patients undergoing 6542 procedures. Inclusion criteria was a peri-operative red blood cell transfusion. MAIN OUTCOME MEASURES: The primary endpoint was the haemoglobin level triggering a transfusion for neonates in week 1, week 2 and week 3. Secondary endpoints were transfusion volumes, 'delta haemoglobin' (preprocedure - transfusion-triggering) and 30-day and 90-day morbidity and mortality. RESULTS: Peri-operative red blood cell transfusions were recorded during 447 procedures (6.9%). The median haemoglobin levels triggering a transfusion were 9.6 [IQR 8.7 to 10.9] g dl-1 for neonates in week 1, 9.6 [7.7 to 10.4] g dl-1 in week 2 and 8.0 [7.3 to 9.0] g dl-1 in week 3. The median transfusion volume was 17.1 [11.1 to 26.4] ml kg-1 with a median delta haemoglobin of 1.8 [0.0 to 3.6] g dl-1. Thirty-day morbidity was 47.8% with an overall mortality of 11.3%. CONCLUSIONS: Results indicate lower transfusion-triggering haemoglobin thresholds in clinical practice than suggested by current guidelines. The high morbidity and mortality of this NECTARINE sub-cohort calls for investigative action and evidence-based guidelines addressing peri-operative red blood cell transfusions strategies. TRIAL REGISTRATION: ClinicalTrials.gov, identifier: NCT02350348

Stratospheric Injection of Brominated Very Short‐Lived Substances: Aircraft Observations in the Western Pacific and Representation in Global Models

We quantify the stratospheric injection of brominated very short‐lived substances (VSLS) based on aircraft observations acquired in winter 2014 above the Tropical Western Pacific during the CONvective TRansport of Active Species in the Tropics (CONTRAST) and the Airborne Tropical TRopopause EXperiment (ATTREX) campaigns. The overall contribution of VSLS to stratospheric bromine was determined to be 5.0 ± 2.1 ppt, in agreement with the 5 ± 3 ppt estimate provided in the 2014 World Meteorological Organization (WMO) Ozone Assessment report (WMO 2014), but with lower uncertainty. Measurements of organic bromine compounds, including VSLS, were analyzed using CFC‐11 as a reference stratospheric tracer. From this analysis, 2.9 ± 0.6 ppt of bromine enters the stratosphere via organic source gas injection of VSLS. This value is two times the mean bromine content of VSLS measured at the tropical tropopause, for regions outside of the Tropical Western Pacific, summarized in WMO 2014. A photochemical box model, constrained to CONTRAST observations, was used to estimate inorganic bromine from measurements of BrO collected by two instruments. The analysis indicates that 2.1 ± 2.1 ppt of bromine enters the stratosphere via inorganic product gas injection. We also examine the representation of brominated VSLS within 14 global models that participated in the Chemistry‐Climate Model Initiative. The representation of stratospheric bromine in these models generally lies within the range of our empirical estimate. Models that include explicit representations of VSLS compare better with bromine observations in the lower stratosphere than models that utilize longer‐lived chemicals as a surrogate for VSLS