Development of cancer immunotherapy based on parvoviral vectors and hybrid cell vaccination

Cancer is a worldwide health problem and despite advances in traditional treatments i.e. surgery, chemotherapy and radiotherapy, the cure rate remains disappointing for some cancers. Different novel therapeutic strategies are being developed. In this thesis two nontraditional cancer therapy approaches are studied: gene therapy using viral vectors and antitumour vaccination with dendritic cell - tumour cell (DC/TC) hybrids.We have developed a novel ELISPOT titration method for viral vectors that is based on the actual expression of the transgene in target cells. This method was developed with recombinant parvovirus MVM-IL2, but it should be adaptable for other vectors carrying expression cassettes for secreted transgene products for which antibodies are available. The ELISPOT titration method allows for faster and better quantification of transducing units present in vector stocks as opposed to titration by in situ hybridisation (annexe I). The MVMIL2 vector has shown an anti-tumour effect against melanoma in an immunocompetent mouse model (annexe IV). Previous work concerns photodynamic inactivation of adenoviral vectors for biosafety and an in vivo study in which a synergistic effect of antiangiogenesis gene therapy combined with radiotherapy could be shown (annexes V and VI).DC/TC hybrids have been proposed as cancer vaccines for their simultaneous expression of antigen presentation machinery and tumour associated antigens. Hybrids are classically generated by polyethylene glycol (PEG) or electrofusion. These methods however require special skills and equipment and cause rather high cell lethality. Fusion via the expression of viral fusogenic membrane glycoproteins (FMG), such as the vesicular stomatitis virus-G (VSV-G) (annexe III) or the Gibbon ape Leukemia Virus (GaLV) FMG, have recently been described. We have mainly focussed on the latter. Transduction of cells with GaLV-FMG proved to be a limiting step for an efficient generation of hybrids. On the other hand, constitutive expression of GaLV-FMG leads to lethal syncytia formation in human cells. Therefore we developed a novel fusion strategy for the generation of DC/TC cell hybrids that involves the use of a non-human fusogenic cell line that constitutively expresses the GaLV-FMG. With this method we were able to generate reproducible yields of DC/TC triparental hybrids. The formation of tri-parental hybrids via the fusogenic cell line is an interesting alternative to existing DC/TC fusion methods because of its simplicity and its flexibility in the choice of fusion partners, i.e. autologous or allogeneic DCs and tumour cells.Moreover, the tri-parent hybrid system offers the possibility to further enhance the immune response by the addition of transgenes that code for immuno-modulating factors to the fusogenic cell line (annexe II).Doctorat en sciences biomédicalesinfo:eu-repo/semantics/nonPublishe

A novel method for the titration of recombinant virus stocks by ELISPOT assay

The development of vectors for gene therapy requires the definition of quality control parameters such as titration, contamination, transduction efficiency and biological effects in defined model systems. For most viral vectors, the classical titration by plaque formation is not applicable, because vectors are defective for replication and packaging cell lines are not always available. In particular, for vectors derived from the autonomous parvovirus MVM(p), the titration method used currently is based on the amplification of the viral genome inside an infected cell, which can then be revealed with a specific radioactive probe (J. Virol. 63 (1989) 1023). In situ hybridization allows to titrate wild-type virus as well as vectors, using probes that are specific for the substituted viral genes or for the transgene, respectively. This method is, however, time consuming, making the simultaneous titration of large numbers of samples difficult. The use of a radioactive probe requires an adequate facility. An ELISPOT method that allows for rapid titration of up to 23 vector stocks in one 96 well dish was devised. This method is based on the actual expression of the transgene. Compared to in situ hybridization, titers obtained by the ELISPOT method were in general equivalent or higher. However, for some vector stocks the ELISPOT titers were repeatedly lower, indicating that in situ hybridization does not give an accurate measure of transducing units. Our model system is recombinant parvovirus MVM expressing human IL2, but the method should be adaptable to other vectors expressing transgenes that are secreted and for which antibodies are available. © 2003 Elsevier Science B.V. All rights reserved.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Generation of cell hybrids via a fusogenic cell line

Hybrids obtained by fusion between tumour cells (TC) and dendritic cells (DC) have been proposed as anti-tumour vaccines because of their potential to combine the expression of tumour-associated antigens with efficient antigen presentation. The classical methods used for fusion, polyethylene glycol (PEG) and electrofusion, are cytotoxic and generate cell debris that can be taken up by DC rendering the identification of true hybrids difficult.SCOPUS: ar.jFLWINinfo:eu-repo/semantics/publishe

The inositol Inpp5k 5-phosphatase affects osmoregulation through the vasopressin-aquaporin 2 pathway in the collecting system.

Inositol Inpp5k (or Pps, SKIP) is a member of the inositol polyphosphate 5-phosphatases family with a poorly characterized function in vivo. In this study, we explored the function of this inositol 5-phosphatase in mice and cells overexpressing the 42-kDa mouse Inpp5k protein. Inpp5k transgenic mice present defects in water metabolism characterized by a reduced plasma osmolality at baseline, a delayed urinary water excretion following a water load, and an increased acute response to vasopressin. These defects are associated with the expression of the Inpp5k transgene in renal collecting ducts and with alterations in the arginine vasopressin/aquaporin-2 signalling pathway in this tubular segment. Analysis in a mouse collecting duct mCCD cell line revealed that Inpp5k overexpression leads to increased expression of the arginine vasopressin receptor type 2 and increased cAMP response to arginine vasopressin, providing a basis for increased aquaporin-2 expression and plasma membrane localization with increased osmotically induced water transport. Altogether, our results indicate that Inpp5k 5-phosphatase is important for the control of the arginine vasopressin/aquaporin-2 signalling pathway and water transport in kidney collecting ducts.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

The surgical safety checklist and patient outcomes after surgery: a prospective observational cohort study, systematic review and meta-analysis

© 2017 British Journal of Anaesthesia Background: The surgical safety checklist is widely used to improve the quality of perioperative care. However, clinicians continue to debate the clinical effectiveness of this tool. Methods: Prospective analysis of data from the International Surgical Outcomes Study (ISOS), an international observational study of elective in-patient surgery, accompanied by a systematic review and meta-analysis of published literature. The exposure was surgical safety checklist use. The primary outcome was in-hospital mortality and the secondary outcome was postoperative complications. In the ISOS cohort, a multivariable multi-level generalized linear model was used to test associations. To further contextualise these findings, we included the results from the ISOS cohort in a meta-analysis. Results are reported as odds ratios (OR) with 95% confidence intervals. Results: We included 44 814 patients from 497 hospitals in 27 countries in the ISOS analysis. There were 40 245 (89.8%) patients exposed to the checklist, whilst 7508 (16.8%) sustained ≥1 postoperative complications and 207 (0.5%) died before hospital discharge. Checklist exposure was associated with reduced mortality [odds ratio (OR) 0.49 (0.32–0.77); P\u3c0.01], but no difference in complication rates [OR 1.02 (0.88–1.19); P=0.75]. In a systematic review, we screened 3732 records and identified 11 eligible studies of 453 292 patients including the ISOS cohort. Checklist exposure was associated with both reduced postoperative mortality [OR 0.75 (0.62–0.92); P\u3c0.01; I2=87%] and reduced complication rates [OR 0.73 (0.61–0.88); P\u3c0.01; I2=89%). Conclusions: Patients exposed to a surgical safety checklist experience better postoperative outcomes, but this could simply reflect wider quality of care in hospitals where checklist use is routine

Prospective observational cohort study on grading the severity of postoperative complications in global surgery research

Background The Clavien–Dindo classification is perhaps the most widely used approach for reporting postoperative complications in clinical trials. This system classifies complication severity by the treatment provided. However, it is unclear whether the Clavien–Dindo system can be used internationally in studies across differing healthcare systems in high- (HICs) and low- and middle-income countries (LMICs). Methods This was a secondary analysis of the International Surgical Outcomes Study (ISOS), a prospective observational cohort study of elective surgery in adults. Data collection occurred over a 7-day period. Severity of complications was graded using Clavien–Dindo and the simpler ISOS grading (mild, moderate or severe, based on guided investigator judgement). Severity grading was compared using the intraclass correlation coefficient (ICC). Data are presented as frequencies and ICC values (with 95 per cent c.i.). The analysis was stratified by income status of the country, comparing HICs with LMICs. Results A total of 44 814 patients were recruited from 474 hospitals in 27 countries (19 HICs and 8 LMICs). Some 7508 patients (16·8 per cent) experienced at least one postoperative complication, equivalent to 11 664 complications in total. Using the ISOS classification, 5504 of 11 664 complications (47·2 per cent) were graded as mild, 4244 (36·4 per cent) as moderate and 1916 (16·4 per cent) as severe. Using Clavien–Dindo, 6781 of 11 664 complications (58·1 per cent) were graded as I or II, 1740 (14·9 per cent) as III, 2408 (20·6 per cent) as IV and 735 (6·3 per cent) as V. Agreement between classification systems was poor overall (ICC 0·41, 95 per cent c.i. 0·20 to 0·55), and in LMICs (ICC 0·23, 0·05 to 0·38) and HICs (ICC 0·46, 0·25 to 0·59). Conclusion Caution is recommended when using a treatment approach to grade complications in global surgery studies, as this may introduce bias unintentionally

Critical care admission following elective surgery was not associated with survival benefit: prospective analysis of data from 27 countries

This was an investigator initiated study funded by Nestle Health Sciences through an unrestricted research grant, and by a National Institute for Health Research (UK) Professorship held by RP. The study was sponsored by Queen Mary University of London