The effect of iron oxide nanoparticles on T lymphocytes

THE EFFECT OF IRON OXIDE NANOPARTICLES ON T LYMPHOCYTES DANIEL AZEVEDO MENDES DISSERTAÇÃO DE MESTRADO INTEGRADO APRESENTADA AO INSTITUTO DE CIÊNCIAS BIOMÉDICAS ABEL SALAZAR DA UNIVERSIDADE DO PORTO EM MEDICINA THE EFFECT OF IRON OXIDE NANOPARTICLES ON T LYMPHOCYTES Daniel Azevedo Mendes Tese de Mestrado Integrado em Medicina apresentado ao Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto Rua Jorge Viterbo Ferreira n.º 228, 4050-313 Porto, Portugal [email protected] Trabalho efetuado sob a orientação de: Professora Doutora Graça Porto e coorientação de: Professora Doutora Beatriz Porto com a supervisão de: Professora Doutora Margarida Lima Área científica de Investigação Básica e Clínica na Biologia do Ferro 2014/2015 The work described in this report is aimed to obtain the Master Degree in Medicine and was conducted in the context of “Disciplina de Iniciação à Investigação Clínica” a class of the Integrated Master Degree in Medicine of Abel Salazar Institute of Biomedical Sciences (ICBAS), University of Porto (UP). The report is divided in two parts, one describing the submitted research proposal, including the state of the art, and the second describing the project implementation which includes the methodology used, the results obtained and the discussion. The project was executed at the Clinical Haematology Service of Centro Hospitalar do Porto – Hospital de Santo António, more specifically at the Haemochromatosis Outpatient Clinic, under the direct supervision of Graça Porto and at the Cytometry Laboratory, under the direct supervision of Margarida Lima. Part of the project took place at the Laboratory of Cytogenetics of ICBAS/UP under the direct supervision of Beatriz Porto. The study was conducted during the academic year of 2013/2014 (design and writing of the project proposal) and 2014/2015 (project implementation, analysis of results and preparation of the respective report).) ACKNOWLEDGEMENTS I am very grateful to Graça Porto M.D. PhD and to Beatriz Porto PhD by the accessibility, attention and interest in developing this project and by all the knowledge that was transmitted. I would also like to thank to Margarida Lima M.D. PhD for the motivation, the knowledge and for supporting the "Disciplina de Introdução à Investigação Clínica" that enriched my preparation as a doctor. Thanks to Sonia Fonseca for all the help in the execution of the experimental procedures and thanks to Rosa Sousa by the vast availability and assistance provided in the performance of cytogenetic tests. I also thank to Graça Melo for her collaboration with this work, for her competence and the immense comprehension. I want to leave a special thanks to Marisa Pinto for all her support and for having kept me motivated in completing this work. Finally I acknowledge all the people who directly or indirectly have contributed to this work and the financial support given by ICBAS / UP in obtaining the reagents for the work. The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 Índice ABSTRACT.......................................................................................................................................... i RESUMO ............................................................................................................................................ ii | RESEARCH PROJECT .................................................................................................................. 1 SCIENTIFIC PLAN ............................................................................................................................. 2 Introduction ....................................................................................................................... 3 Preliminary evidence support ........................................................................................... 8 Problems ........................................................................................................................... 8 Questions .......................................................................................................................... 8 Hypotheses of work .......................................................................................................... 9 Study aims ......................................................................................................................... 9 Intervenients .................................................................................................................... 10 Methodology ................................................................................................................... 13 Calendarization ............................................................................................................... 24 Output indicators ............................................................................................................. 26 References ....................................................................................................................... 27 ETHICAL QUESTIONS ................................................................................................................... 30 Participant information and informed consent ................................................................ 31 Other issues with ethical implications ............................................................................ 31 FINANCIAL PLAN ........................................................................................................................... 32 Budget ............................................................................................................................. 33 Financing: ....................................................................................................................... 33 GLOSSARY....................................................................................................................................... 34 Abbreviations and acronyms .......................................................................................... 35 ATTACHMENTS .............................................................................................................................. 36 List of attachments .......................................................................................................... 37 | EXECUTION REPORT ................................................................................................................ 46 Changes to the Original Project Proposal .......................................................................................... 48 The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 Design and Methods........................................................................................................................... 49 Controls ........................................................................................................................... 49 Patients ............................................................................................................................ 49 Sample collection ............................................................................................................ 50 Experimental Procedure ..................................................................................................................... 51 Cell cultures .................................................................................................................... 51 Exposure to BLM and Venofer® .................................................................................... 51 T-Cell immunophenotyping ............................................................................................ 52 Cell Cycle Studies ........................................................................................................... 52 Apoptotic T cell evaluation ............................................................................................. 53 Cytogenetic analysis - evaluation of chromosome instability ........................................ 53 Evaluation of Proliferative index .................................................................................... 55 Statistical analysis ........................................................................................................... 55 Results ................................................................................................................................................ 56 Changes in cell cycle and apoptosis following Venofer® exposure .............................. 56 No impact of Venofer® in cell cycle and apoptosis in HH patients ............................... 57 Changes in the bleomycin genotoxicity test following Venofer® exposure .................. 58 Discussion .......................................................................................................................................... 61 Conclusion ......................................................................................................................................... 65 References .......................................................................................................................................... 66 The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 i ABSTRACT Intravenous iron formulations are widely used in medical practice for a quick recovery of body iron depletion. One of the most commonly used formulations is Venofer®, a compound of iron oxide nanoparticles comprising a core of iron in the ferric state and a polysaccharide coating of sucrose aimed to limit the release of non-transferrin bound iron (NTBI) into the bloodstream. Although clinically proven as a safe drug, its putative effect on T lymphocyte activation and proliferation has never been properly addressed. Several pieces of evidence suggest that T lymphocytes may have an important role in iron homeostasis by limiting the inherent toxicity of excessive iron in circulation. In patients with Hereditary Haemochromatosis (HH), a genetic iron overload disorder, it has been consistently demonstrated that the presence of T lymphocytes in larger numbers helps limiting the clinical repercussion of iron toxicity. More recent studies have shown that these cells are able to incorporate this metal particularly in the case of iron overload, and thus protect from the accumulation in other organs. The present study was aimed to analyze the effect of Venofer® on the in vitro proliferation of T lymphocytes and to compare this effect in normal subjects and in HH patients. For that purpose, we cultured peripheral blood lymphocytes obtained from 6 normal healthy controls and 6 HH patients and used flow cytometry to quantify cell cycle and apoptosis parameters in the cultured lymphocytes after exposure to Venofer®. In addition, we evaluated the impact of Venofer® on the iron-mediated bleomycin induced genotoxicity as measured by chromosome instability in cytogenetic studies. The estimated proliferative index obtained with cytogenetic studies was also analysed. The results obtained showed that Venofer® can induce a G0/G1 cell cycle arrest in cultured lymphocytes obtained from normal healthy individuals, an effect that seems to be related with the proliferative index, but was independent from apoptosis, whose markers were not significantly changed. That effect, however, was not observed in cultured cells obtained from HH patients. The evaluation of the impact of Venofer® on the iron-mediated bleomycin induced genotoxicity was not conclusive. It was hampered by an unexpected and still not clarified effect of the compound in the culture medium, with a significant decrease in the percentage of chromosome breaks. In conclusion, the present study supports the hypothesis of a relevant biological response of T lymphocytes to Venofer®, which is somehow altered in HH patients, suggesting the existence of some adaptive mechanism in this genetic condition. Further studies are needed not only to characterize the molecular mechanisms involved in the lymphocyte response to Venofer® in vitro, but also to address the in vivo implications of these findings. The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 ii RESUMO As formulações de ferro endovenoso são amplamente utilizadas na prática clínica para uma recuperação rápida de estados de depleção. Uma das formulações mais vulgarmente utilizadas é o Venofer®, constituído por nanopartículas de óxido de ferro formadas por um núcleo de ferro no seu estado férrico e um revestimento polissacárido de sacarose destinado a limitar a libertação de ferro não ligado à transferrina (NTBI) para a circulação. Embora clinicamente comprovado como um fármaco seguro, o seu presumível efeito sobre a ativação e proliferação dos linfócitos T nunca foi devidamente avaliado. Várias evidências sugerem que os linfócitos T podem ter um papel importante na homeostasia do ferro através da limitação da toxicidade inerente ao excesso de ferro em circulação. Em doentes com a Hemocromatose Hereditária (HH), um distúrbio genético de sobrecarga de ferro, tem sido consistentemente demonstrado que a presença de linfócitos T em números elevados ajuda a limitar as repercussões clínicas da toxicidade do ferro. Estudos mais recentes têm mostrado que estas células são capazes de internalizar este metal particularmente em casos de sobrecarga de ferro e, assim, impedir a sua acumulação nos diferentes órgãos. O objetivo deste estudo foi analisar o efeito do Venofer® na proliferação in vitro de linfócitos T e comparar esse efeito entre indivíduos normais e doentes com HH. Para tal, foram realizadas culturas de linfócitos T do sangue periférico obtidas a partir de 6 controlos saudáveis e 6 doentes com HH. Os parâmetros do ciclo celular e apoptose nos linfócitos T em cultura após exposição ao Venofer® foram avaliados através de técnicas de citometria de fluxo. Além disso, através da observação parâmetros citogenéticos de instabilidade cromossómica foi possível avaliar o impacto da Venofer® na genotoxicidade induzida pela bleomicina. Foi também possível calcular o índice proliferativo através de estudos citogenéticos, em todas as condições de cultura. Os resultados obtidos mostraram que o Venofer® pode induzir uma paragem nas fases G0 / G1 do ciclo celular de linfócitos T de indivíduos saudáveis normais cultivados in vitro, um efeito que parece estar relacionado com o índice proliferativo, mas que é independente da apoptose, cujos valores não se mostraram significativamente alterados. Este efeito, no entanto, não foi observado em culturas de células obtidas a partir de doentes com HH. A avaliação do impacto do Venofer® na genotoxicidade induzida pela bleomicina não foi conclusiva uma vez que a ação da bleomicina foi marcadamente afetada por um efeito inesperado e ainda não clarificado do composto no meio de cultura, com uma diminuição significativa na percentagem de quebras cromossómicas. Em conclusão, o presente estudo suporta a hipótese de uma resposta biológica relevante dos linfócitos T à ação do Venofer®, resposta que está de alguma forma alterada em doentes com HH, sugerindo a existência de algum mecanismo adaptativo nessa condição genética. São necessários mais estudos para caracterizar os mecanismos moleculares envolvidos na resposta in vitro dos linfócitos T ao Venofer®, e para compreender as implicações in vivo destes achados. The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 | RESEARCH PROJECT The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 8 Preliminary evidence support Preliminary work at the Cytogenetics Laboratory (CgL) in ICBAS has shown that treatment of proliferating T lymphocytes with uncoated iron oxide nanoparticles, or polyacrylic acid-coated iron oxide nanoparticles induces alterations in the proliferative cell profile. However no experiments have ever been performed with Venofer®. There is also preliminary evidence, from the same laboratory, that lymphocytes from HH patients have a different response to the bleomycin (BLM) genotoxicity test relative to normal controls. This test is based on the effect of BLM on DNA damage mediated by iron mobilization therefore providing an indirect measure of intracellular iron. These preliminary experiments support the hypothesis that lymphocytes of HH patients and controls may have a different iron handling profile and could have a different response to Venofer®. Problems Recently, there have been several evidences that T lymphocytes play an active role in body iron regulation, particularly in iron overload diseases. However, most of the mechanisms underlying this regulation as well as the effect of injected iron in T lymphocytes remains uncover. For example, it is still unknown the scientific basis to support the fact that intravenous iron decreases the infection rate when several studies indicate that this metal increases the proliferation of microorganisms. It is now evident that T lymphocytes have the ability to uptake some forms of NTBI and it is also possible to label this type of cells with iron oxide nanoparticles. Preliminary work at the CgL in ICBAS confirmed the T lymphocytes’ capacity to uptake iron oxide nanoparticles and also showed the influence of these particles in cell cycle. No experiments have ever been performed with iron oxide nanoparticles that are used in clinical practice, such as Venofer®. Questions  Do T lymphocytes handle sucrose-coated iron oxide nanoparticles (Venofer®) in vitro?  Does the T lymphocytes cell cycle change in the presence of sucrose-coating iron oxide nanoparticles (Venofer®) in vitro?  Do HH patients differ from normal controls in the capacity of T lymphocytes to handle iron oxide nanoparticles? The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 9 Hypotheses of work There are two hypotheses tested in this study: 1. That iron oxide nanoparticles, such as Venofer®, are internalised by T lymphocytes and, by altering the intracellular iron levels, may have a biological impact either by promoting a cellular response to oxidative stress or by influencing their proliferation rate. 2. That the putative response of lymphocytes to Venofer® may be somehow different in HH, not only because this is a genetic disorder where increased iron levels are already found in circulation, but also because the disorder is characterised by the finding of abnormalities in T lymphocytes which are related to the severity of iron overload. Study aims General aims of this work: The present work is aimed to help clarifying some of the issues related to the role of T lymphocytes in iron metabolism by elucidating the effect of intravenous iron formulations often used in the clinical practice in the in vitro proliferation of T lymphocytes. It´s also an objective to compare the biologic response of T lymphocytes from normal subjects with that of patients with HH, in an attempt to better characterize some previously found evidence of the role of these cells on the disease. Specific aims of this work:  To ascertain if iron oxide nanoparticles in Venofer® are internalized by T lymphocytes.  To determine in which intracellular compartment can we find these particles and what is their constitution inside the cell.  To evaluate if polysaccharide coating of iron oxide nanoparticles persist intact when they are internalized by T lymphocytes.  To determine the percentage T lymphocytes in the different phases of cell cycle in the presence of iron oxide nanoparticles.  To calculate the proliferative status and chromosome instability of T lymphocytes in the presence of iron oxide nanoparticles.  To explore the response to the bleomycin genotoxicity test as an indirect measure of intracellular iron.  To perform all experiments T lymphocytes obtained from patients with HH in comparison to normal healthy controls. The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 10 Intervenients Institutions, Departments and Services  Centro Hospitalar do Porto (CHP) / Hospital de Santo António (HSA) o Department of Medicine (DM) / Clinical Haematology Service (SHC)  Institute for Molecular and Cell Biology (IBMC) o Basic & Clinical Research on Iron Biology (BCRIB) o Histology and Electron Microscopy Service (HEMS)  Abel Salazar Institute of Biomedical Sciences (ICBAS) / University of Porto (UP) o Microscopy Department / Cytogenetics Laboratory (CgL) Research team Constitution Student  Daniel Mendes: student in the “Disciplina de Inciação à Investigação Clínica” (DIIC) - Master in Medicine (MIM) ICBAS / UP. Supervisor  Graça Porto (M.D. PhD): Specialist in Imunohemotherapy, Head Consultant, Service of Clinical Haematology / Haemochromatosis consultation HSA/CHP. Research Group Leader at IBMC (Basic and Clinical Research on Iron Biology), Invited Full Professor at ICBAS/UP, Pathology and Molecular Immunology Department. Co-supervisor  Beatriz Porto (PhD): Director of the Cytogenetics Laboratory and assistant Professor at ICBAS/UP. Discipline supervisor  Margarida Lima (M.D. PhD): Specialist in Imunohemotherapy, graduated orderly, Service of Clinical Haematology of HSA/CHP; invited assistant Professor at ICBAS/UP; regent of DIIC. Colaborators  Rui Fernandes: Group Leader at Histology and Electron Microscopy Service, IBMC/UP.  Sónia Fonseca, clinical analysis technician, Cytometry Laboratory of Service of Clinical Haematology, HSA/CHP.  Rosa Sousa, research fellow at Laboratory of Cytogenetics, Department of Microscopy, ICBAS/UP. Functions and responsibilities The conception and formulation of the proposal, as well as the execution of the project are the responsibilities of the student. The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 11 The supervisor will monitor the formulation of the proposal, the execution of the project and the analysis and interpretation of the results. The discipline supervisor will supervise all the steps of the project, including the conception, the implementation, and the presentation of the results. The following investigators will collaborate in specific tasks of the project, as described forward. Time dedicated to the project First and last names Function Person * Month Daniel Mendes Student 10%*22 = 2.20 Graça Porto Supervisor 2.5%*22 = 0.55 Margarida Lima Discipline supervisor 2.5%*22 = 0.55 Beatriz Porto Cytogenetic supervisor 2.5%*22 = 0.55 Sónia Fonseca Collaborator 2.5%*2 = 0.10 Rosa Sousa Collaborator 2.5%*2 = 0.10 Rui Fernandes Collaborator 2.5%*2 = 0.05 Total 4.10 Conditions and motivations to conducting the study Installed capacities and available resources The conditions necessary to execute the project are ensuring. The SHC at HSA/CHP has all the necessary equipment to carry out the experiments to study cell cycle alterations and in CgL at ICBAS/UP we can perform the BLM genotoxicity tests. However we need to purchase the reagents that will be used in the experiments. Similarly the HEMS laboratory at IBMC has all the necessary equipment to use the TEM / EDX technology. Both laboratories have performed similar experiments in their facilities. Research tem merit The supervisor of the project belongs to BCRIB, a team of researchers that has a long track record of publications that have the common propose of clarify the reciprocal interactions between iron metabolism and the immune system, mainly the role of CD8+ T lymphocytes in cases of iron overload (utilizing HH as model). Research work conducted by the team has already shown the ability of T lymphocytes to internalize iron, but there is still much to discover. In a recent paper of the supervisor and her team it is clarified the that T lymphocytes have a major role in NTBI in vivo handling, limiting their toxicity and acting as protective cells in iron overload disorders as HH. Personal motivations to conducting the study To acquire skills in terms of scientific reasoning. To develop the competence of working with a research team with structured functions and responsibilities. The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 12 To contact with laboratory techniques and with the dynamic between clinic and primary sciences in the scope of medicine. To get personal accomplishment. The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 13 Methodology Literature review criteria In the literature review we used scientific journal articles indexed in MEDLINE data basis using PubMed as the search engine. We tried to use the most updated knowledge, but the articles date doesn’t constitute a search criterion. The following key words were used in the scientific search: “Iron oxide”, “nanoparticles”, “intravenous”, “T lymphocytes” and “Hereditary Haemochromatosis”. We selected only original and review articles in English language. We also included specific articles that the project supervisor has advised. Study design Type of study Local: National and institutional study. Type: Analytic, observational, cross-sectional and case-control study. Nature: Clinical and laboratorial study. Phases of the study The study will be conducted in two distinct phases: a) In the first phase the cell cycle experiments and BMC genotoxicity tests will be performed. b) In the second phase the results in samples where cells will have greater iron-uptake, measured indirectly by BLM genotoxicity test, will be validated with measurements of intracellular iron distribution by TEM/EDX. Universe, population and sample Universe: Patients: HH patients homozygous for the p.Cys282Tyr mutation of the HFE gene. Controls: Adult individuals without known pathology. Population: Patients: The population analysed in this study will be composed by HH patients homozygous for the p.Cys282Tyr mutation of the HFE gene regularly followed-up at the Haemochromatosis Clinic from HSA/CHP. A total of about 500 patients are registered and are regularly followed-up at the clinic, and 90 of these are genetically characterized as HH homozygous for the p.Cys282Tyr mutation. The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 14 Controls: The control population for this project will be recruited among volunteer regular blood donors attending the HSA Blood Bank. Sample: Patients: The sample population will be composed by a group of 6 patients with HH homozygous for the p.Cys282Tyr mutation of the HFE gene. Controls: The sample population for this project will be composed by a group of 6 sex and age matched subjects, selected among the population of regular blood donors attending the HSA Blood Bank. Participant selection Participants will be selected in a non-probabilistic consecutive way (by convenience). Patients and blood donors will be consecutively recruited at the time of their regular consultation or volunteer blood donation, respectively. Eligibility criteria Inclusion criteria: To be followed at the Haemochromatosis Clinic, as a HH patient homozygous for the p.Cys282Tyr mutation of the HFE gene, or be a regular blood donor at the CHP-HAS Blood Bank. Have a scheduled (or unscheduled in the case of controls) consultation during the period of recruitment. Agree to participate in the project and sign informed consent. Exclusion criteria: Not fill in the inclusion criteria. Patients with clinical conditions known to influence total lymphocytes numbers (such as autoimmune or viral diseases). Controls whose donation is the first will be excluded. The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 15 Work plan Tasks associated to the project Task list During the execution of this project the following tasks are planned: Tasks description Task 1: Inform about the study and data collection from clinical files Duration: 2 months Expected date for start: 14-09-2014 Expected date for conclusion: 31-10-2014 Institutions, Departments and Services HSA/CHP – DM – SHC – Haemochromatosis clinic Description: During the period of consultation, the responsible for the Haemochromatosis Clinic (Graça Porto), will inform the patients about the study, requesting their participation. On days in which the supervisor (Margarida Lima) is responsible for the observation and selection of regular blood donors at the Blood Bank, she will inform them about the study, requesting their participation. All participants will receive a leaflet with most relevant information about the study. According to the participant recruitment, it will be collected the corresponding analytic data registered at the moment of diagnosis in their clinical files; in the next week, will be collected the results from the CBC and biochemical study, which is normally made after regular phlebotomies. The collection of controls´ data will be held under the same rules. The supervisor will overlook the process of data collection from clinical files. Researchers involved, roles and responsibilities Daniel Mendes – Data collection and registering. Graça Porto – Inform the patients about the study and supervise data collection and registering. Task number Task designation Beginning date Conclusion date 1 Inform about the study and data collection from clinical files 14-09-2014 31-10-2014 2 Blood sample collection 01-10-2014 31-01-2015 3 Cell cycle studies 01-10-2014 31-01-2015 4 Bleomycin genotoxicity tests 01-11-2014 31-02-2014 5 TEM/EDX studies 01-02-2015 31-03-2015 The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 16 Task 2: Blood sample collection Duration: 4 months Expected date for start: 01-10-2014 Expected date for conclusion: 31-01-2015 Institutions, Departments and Services HSA/CHP – DM – SHC – Haemochromatosis clinic and HSA Blood Bank Description: It will be carried out the collection of 2 blood samples (1 tube with EDTA-K3, with 4.5ml of blood; 1 tube with sodium heparin with 7.5 ml of blood); The procedure does not involve risks since it will be performed directly from the blood bag system without additional venous puncture. It will be collected at most 2 samples from each patient and 2 samples from each blood donors per week. The blood samples will be sent for processing to the CmL (Cytometry Laboratory) SHC-HSA-CHP, addressed to Técn. Sónia Fonseca. Researchers involved, roles and responsibilities Haemochromatosis Clinic: Nurse - Graça Melo will be in charge of collecting of blood samples from patients. HAS Blood Bank: Nurses (various) will be in charge of collecting samples from blood donors. Task 3: Cell cycle studies Duration: 4 months Expected date for start: 01-10-2014 Expected date for conclusion: 31-01-2015 Institutions, Departments and Services HSA/CHP – DM – SHC – CmL Description: Using flow cytometry techniques, we aim to verify what percentage of T lymphocytes is distributed to the different phases of the cell cycle (S/G1 and G2/M) in HH patients and controls. Researchers involved, roles and responsibilities Daniel Mendes – Cooperate with the performance of the technique and analysis of results. Sónia Fonseca - Flow cytometry studies (sample processing and data analysis) Margarida Lima – Supervising of the study and analysis of results. The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 17 Task 4: Bleomycin genotoxicity test Duration: 4 months Expected date for start: 01-11-2014 Expected date for conclusion: 31-02-2015 Institutions, Departments and Services ICBAS/UP – CgL Description: Perform BLM genotoxicity test in T lymphocytes of HH patients and controls. Researchers involved, roles and responsibilities Daniel Mendes – Cooperate with the performance of the technique and analysis of results. Rosa Sousa – Cytogenetic studies (sample processing and data analysis) Beatriz Porto – Supervising of the study and analysis of results. Task 5: TEM/EDX studies Duration: 2 months Expected date for start: 01-02-2015 Expected date for conclusion: 31-03-2015 Institutions, Departments and Services IBMC/UP – HEMS Description: Perform TEM/EDX studies in selected T lymphocytes of HH patients and controls. Researchers involved, roles and responsibilities Daniel Mendes – Cooperate with the performance of the technique and analysis of results. Rui Fernandes – Perform the technique and interpret the results. The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 24 Calendarization Duration Global: 22 months. Planning: 9 months. Execution: 11 months. Start and end dates Global: October 2013 to July 2015 Execution: September 2014 to July 2015 Global chronogram of activities ACADEMIC YEAR 2013/2014 ACADEMIC YEAR 2014/2015 Month 10 11 12 01 02 03 04 05 06 07 08 09 10 11 12 01 02 03 04 05 06 07 Choice of the work area Integration on research team Choice of theme and subject Identification of problems/issues Formulation of the questions Delineation of the hypothesis Definition of the aims Review / study design Study conception Preparation of project proposal Submission for approval Presentation of the proposal Project implementation Sample collection and analytical procedures Analysis and interpretation of results Report writing Presentation of the results MIM dissertation The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 25 Project execution chronogram Year 2014 2015 Task 09 10 11 12 01 02 03 04 05 1 2 3 4 5 Goals to be achieved (milestones)  Study started: September 1th, 2014  Data collected and entered in a computer file: March 31th, 2015  Data processed, analysed and interpreted: May 31th, 2015  Implementation / presentation report of results: June 12th, 2015 Deliveries to be made (deliverables) Delivery of the project proposal: May 31th, 2014 Delivery of execution/presenting results report: June 12th, 2015 The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 26 Output indicators Oral communications and posters  Oral presentation of the proposal at the 6th Jornadas de Iniciação á Investigação Clínica (JIIC) (June / July 2014)  Oral presentation of the results at the 7th JIIC (June / July 2015)  Presentation of the results in poster in scientific meeting for haematology specialists (2014) – Annual meeting of Portuguese Society of Haematology.  Presentation of the results in poster in an international scientific meeting of the specialty (2015) – European Iron Club Meeting Manuscripts  Research Project proposal (2014)  Thesis defence (2015)  Article for publication in national or international medical journal with referees (2015) The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 27 References 1. 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Parenteral iron formulations: a comparative toxicologic analysis and mechanisms of cell injury. Am J Kidney Dis Off J Natl Kidney Found. 2002 Jul;40(1):90–103. 14. Van Wyck D, Anderson J, Johnson K. Labile iron in parenteral iron formulations: a quantitative and comparative study. Nephrol Dial Transplant Off Publ Eur Dial Transpl Assoc - Eur Ren Assoc. 2004 Mar;19(3):561–5. 15. Kakhlon O, Cabantchik ZI. The labile iron pool: characterization, measurement, and participation in cellular processes(1). Free Radic Biol Med. 2002 Oct 15;33(8):1037–46. The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 28 16. Andrews NC, Schmidt PJ. Iron homeostasis. Annu Rev Physiol. 2007;69:69–85. 17. Barton Pai A, Pai MP, Depczynski J, McQuade CR, Mercier R-C. Non-transferrin-bound iron is associated with enhanced Staphylococcus aureus growth in hemodialysis patients receiving intravenous iron sucrose. Am J Nephrol. 2006;26(3):304–9. 18. Deicher R, Ziai F, Cohen G, Müllner M, Hörl WH. High-dose parenteral iron sucrose depresses neutrophil intracellular killing capacity. Kidney Int. 2003 Aug;64(2):728–36. 19. Toblli JE, Cao G, Olivieri L, Angerosa M. Comparison of the renal, cardiovascular and hepatic toxicity data of original intravenous iron compounds. Nephrol Dial Transplant Off Publ Eur Dial Transpl Assoc - Eur Ren Assoc. 2010 Nov;25(11):3631–40. 20. Fleming RE, Ponka P. Iron overload in human disease. N Engl J Med. 2012 Jan 26;366(4):348–59. 21. Cuenca J, García-Erce JA, Muñoz M, Izuel M, Martínez AA, Herrera A. Patients with pertrochanteric hip fracture may benefit from preoperative intravenous iron therapy: a pilot study. Transfusion (Paris). 2004;44(10):1447–52. 22. Fleming RE, Ponka P. Iron overload in human disease. N Engl J Med. 2012;366(4):348–59. 23. Knutson MD, Oukka M, Koss LM, Aydemir F, Wessling-Resnick M. Iron release from macrophages after erythrophagocytosis is up-regulated by ferroportin 1 overexpression and down-regulated by hepcidin. Proc Natl Acad Sci U S A. 2005 Feb 1;102(5):1324–8. 24. Arezes J, Costa M, Vieira I, Dias V, Kong XL, Fernandes R, et al. Non-Transferrin-Bound Iron (NTBI) Uptake by T Lymphocytes: Evidence for the Selective Acquisition of Oligomeric Ferric Citrate Species. Mattei F, editor. PloS One. 2013;8(11):e79870. 25. Pinto JP, Arezes J, Dias V, Oliveira S, Vieira I, Costa M, et al. Physiological implications of NTBI uptake by T lymphocytes. Front Pharmacol. 2014;5:24. 26. Pietrangelo A. Hereditary hemochromatosis: pathogenesis, diagnosis, and treatment. Gastroenterology. 2010 Aug;139(2):393–408, 408.e1–2. 27. Porto G, Reimão R, Gonçalves C, Vicente C, Justiça B, de Sousa M. Haemochromatosis as a window into the study of the immunological system: a novel correlation between CD8+ lymphocytes and iron overload. Eur J Haematol. 1994 May;52(5):283–90. 28. Porto G, Vicente C, Teixeira MA, Martins O, Cabeda JM, Lacerda R, et al. Relative impact of HLA phenotype and CD4-CD8 ratios on the clinical expression of hemochromatosis. Hepatology. 1997;25(2):397–402. 29. Cruz E, Vieira J, Gonçalves R, Alves H, Almeida S, Rodrigues P, et al. Involvement of the major histocompatibility complex region in the genetic regulation of circulating CD8 T-cell numbers in humans. Tissue Antigens. 2004 Jul;64(1):25–34. 30. Cruz E, Whittington C, Krikler SH, Mascarenhas C, Lacerda R, Vieira J, et al. A new 500 kb haplotype associated with high CD8+ T-lymphocyte numbers predicts a less severe expression of hereditary hemochromatosis. BMC Med Genet. 2008;9(1):97. 31. Costa M, Cruz E, Barton JC, Thorstensen K, Morais S, da Silva BM, et al. Effects of highly conserved major histocompatibility complex (MHC) extended haplotypes on iron and low CD8+ T lymphocyte phenotypes in HFE C282Y homozygous hemochromatosis patients from three geographically distant areas. Palau F, editor. PloS One. 2013;8(11):e79990. The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 29 32. Kolosnjaj-Tabi J, Wilhelm C, Clément O, Gazeau F. Cell labeling with magnetic nanoparticles: Opportunity for magnetic cell imaging and cell manipulation. J Nanobiotechnology. 2013;11 Suppl 1:S7. 33. Shen L-F, Chen J, Zeng S, Zhou R-R, Zhu H, Zhong M-Z, et al. The superparamagnetic nanoparticles carrying the E1A gene enhance the radiosensitivity of human cervical carcinoma in nude mice. Mol Cancer Ther. 2010 Jul;9(7):2123–30. 34. Moghimi SM, Bonnemain B. Subcutaneous and intravenous delivery of diagnostic agents to the lymphatic system: applications in lymphoscintigraphy and indirect lymphography. Adv Drug Deliv Rev. 1999;37(1):295– 312. 35. Weissleder R, Heautot JF, Schaffer BK, Nossiff N, Papisov MI, Bogdanov A Jr, et al. MR lymphography: study of a high-efficiency lymphotrophic agent. Radiology. 1994 Apr;191(1):225–30. 36. Yeh TC, Zhang W, Ildstad ST, Ho C. Intracellular labeling of T-cells with superparamagnetic contrast agents. Magn Reson Med Off J Soc Magn Reson Med Soc Magn Reson Med. 1993 Nov;30(5):617–25. 37. Yeh TC, Zhang W, Ildstad ST, Ho C. In vivo dynamic MRI tracking of rat T-cells labeled with superparamagnetic iron-oxide particles. Magn Reson Med Off J Soc Magn Reson Med Soc Magn Reson Med. 1995 Feb;33(2):200–8. 38. Siegers GM, Ribot EJ, Keating A, Foster PJ. Extensive expansion of primary human gamma delta T cells generates cytotoxic effector memory cells that can be labeled with Feraheme for cellular MRI. Cancer Immunol Immunother. 2012 Oct 26;62(3):571–83. 39. Garden OA, Reynolds PR, Yates J, Larkman DJ, Marelli-Berg FM, Haskard DO, et al. A rapid method for labelling CD4+ T cells with ultrasmall paramagnetic iron oxide nanoparticles for magnetic resonance imaging that preserves proliferative, regulatory and migratory behaviour in vitro. J Immunol Methods. 2006 Jul 31;314(1-2):123–33. 40. Toblli JE, Cao G, Oliveri L, Angerosa M. Comparison of oxidative stress and inflammation induced by different intravenous iron sucrose similar preparations in a rat model. Inflamm Allergy Drug Targets. 2012;11(1):66. 41. Hoen B, Paul-Dauphin A, Hestin D, Kessler M. EPIBACDIAL: a multicenter prospective study of risk factors for bacteremia in chronic hemodialysis patients. J Am Soc Nephrol JASN. 1998 May;9(5):869–76. 42. Jan T-R, Shen, Wang, Liao. A single exposure to iron oxide nanoparticles attenuates antigen-specific antibody production and T-cell reactivity in ovalbumin-sensitized BALB/c mice. Int J Nanomedicine. 2011 Jun;1229. 43. Shen C-C, Liang H-J, Wang C-C, Liao M-H, Jan T-R. Iron oxide nanoparticles suppressed T helper 1 cellmediated immunity in a murine model of delayed-type hypersensitivity. Int J Nanomedicine. 2012;7:2729–37. The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 30 ETHICAL QUESTIONS The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 31 Participant information and informed consent Informed consent to use the blood as a contribution for research will be obtained at the time of collection. Furthermore samples will be processed in accordance with all the established rules of privacy and confidentiality as define in the Helsinki Declaration. For participants that eventually manifest an interest in being better informed about the study, an information sheet (attached) will be available and provided at the Haematology Service. Although it is planned to hold cell cycle tests, none of these is a genetic test. Blood samples for the research purposes will be treated as volunteer donations with some conditions: in order to assure the “gift relationship” between the donor and the researcher, the formal responsibility for custodianship of the samples collected at the HSA Blood Bank or at the Haemochromatosis Clinic will be transferred to the Cytometry Laboratory (CHP-HAS), Cytogenetics Laboratory (ICBAS), or the BCRIB Laboratory at IBMC. Informed consent of patients with Hereditary Haemochromatosis and blood donors will be request and obtained based on brief explanation of the study and posterior explanations appeal by patients. The model of informed consent is available in annex. Other issues with ethical implications Risks and benefits There are no risks associated with participation in the study. No additional punctures will be needed. Overall benefit is due to the improving the understanding of cellular effects of use iron oxide nanoparticles formulations in clinical practice. There are no direct benefits to participants. The benefits are indirect and based on possible improvements of clinical knowledge that may result from the study. Confidentiality and anonymisation Confidentiality of data is guaranteed. The collection of data from clinical cases will be taken by the student, with the supervision of the Supervisor. The information recorded for research purposes (demographic, clinical and analytical data) will be anonymised. The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 32 FINANCIAL PLAN The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 33 Budget Estimated value (€) Reagents and consumables * please see next table 1950.00 Administrative material (photocopies, paper sheets, etc.) 30.00 Acquisition services (TEM/EDX) 480.00 Poster printing for results presentation 50.00 Student enrollment in Portuguese medical congress (Annual Meeting of the Portuguese Society of Hematology) 200.00 Student enrollment in international congress (European Iron Club Meeting) 1200.00 Jornadas de Iniciação à Investigação Clínica (organization) 50.00 TOTAL €3960.00 Reagents and consumables Purpose Designation Price per unit (€) Number of units Price (€) without IVA Subtotal (€) Flow cytometry (reagents) Anti-CD3 500.00 1 500.00 1700.00 Coulter DNAprepTM reagent system 1200.00 1 1200.00 Genotoxicity tests with Bleomycin 00.00 1 150.00 150.00 Material 100.00 1 100.00 150.00 TOTAL sem IVA 1540.00 TOTAL com IVA (XX%) 1950.00 Financing: The study will be funded by ICBAS / UP, through a grant given to DIIC. The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 40 CARATERÍSTICAS do estudo (Assinale as opções corretas) Alvo do estudo Países / Instituições envolvidos Humanos x Nacional X Institucional X Natureza do estudo Caraterísticas do estudo (desenho) Clínico X Laboratorial X AnalíticoX Observacional X Transversal X Participantes Existência de grupo controlo: Sim X Seleção dos Participantes: Não aleatória X Estudos observacionais: Tipo: Casos-controlos X Estudos experimentais: Não se aplica Outros aspetos relevantes para a apreciação do estudo: Participação de grupos vulneráveis Não X Convocação de doentes / participantes Não X Consentimento informado Sim X Inquéritos / questionários Não X Entrevistas Não X Colheita de produtos biológicos Sim X (No CHP, Não anonimizados) Armazenamento de produtos biológicos Não X Criação de bancos de produtos biológicos Não X Realização de exames / análises Sim X (No CHP, ICBAS e IBMC) Realização de estudos genéticos Não X Recolha de dados Sim X (Dados: clínicos, laboratoriais: e analíticos) Criação de bases de dados Não X Saída para outras instituições Não X ORÇAMENTO E FINANCIAMENTO Orçamento total: ________ Euros Contrato financeiro em anexo: Não □ Sim □ Financiamento: Interno (CHP) ________ Euros Externo (Outros) _______ Euros Entidades financiadoras: ______________________________________________________ INDICADORES Dissertação Mestrado Integrado em Medicina Data: Assinatura do proponente (Aluno): The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 41 Pedidos de autorização institucional Trabalho académico de investigação: O efeito de nanoparticulas de óxido de ferro em linfócitos T Aluno da DIIC do curso de MIM do ICBAS/UP e do CHP: Daniel Azevedo Mendes Presidente do Conselho de Administração do CHP Exmo. Senhor Presidente do Conselho de Administração do CHP Daniel Azevedo Mendes, na qualidade de Aluno, vem por este meio, solicitar a Vossa Exa. autorização para realizar no Centro Hospitalar do Porto o Estudo de Investigação acima mencionado, de acordo com o programa de trabalhos e os meios apresentados. Data Assinatura ___/___/_____ _________________________ Presidente da Comissão de Ética para a Saúde do CHP Exma. Senhora Presidente da Comissão de Ética para a Saúde do CHP Daniel Azevedo Mendes, na qualidade de Aluno, vem por este meio, solicitar a Vossa Exa. autorização para realizar no Centro Hospitalar do Porto o Estudo de Investigação acima mencionado, de acordo com o programa de trabalhos e os meios apresentados. Data Assinatura ___/___/_____ _________________________ Diretora do Departamento de Ensino, Formação e Investigação do CHP Exma. Senhora Diretora do Departamento de Ensino, Formação e Investigação do CHP Daniel Azevedo Mendes, na qualidade de Aluno, vem por este meio, solicitar a Vossa Exa. autorização para realizar no Centro Hospitalar do Porto o Estudo de Investigação acima mencionado, de acordo com o programa de trabalhos e os meios apresentados. Data Assinatura ___/___/_____ _________________________ The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 42 Termos de responsabilidade Trabalho académico de investigação: O efeito de nanoparticulas de óxido de ferro em linfócitos T Aluno da DIIC do curso de MIM do ICBAS/UP e do CHP: Daniel Azevedo Mendes Aluno Na qualidade de Aluno, comprometo-me a executar o Trabalho Académico de Investigação acima mencionado, de acordo com o programa de trabalhos e os meios apresentados, respeitando os princípios éticos e deontológicos e as normas internas da instituição. Aluno Data Assinatura __________________________________________ ___/___/___ _________________ Orientador do projeto Na qualidade de Orientador, solicito autorização do Conselho de Administração para que o Aluno acima referido possa desenvolver no CHP o seu Trabalho de Investigação. Informo que me comprometo a prestar a orientação necessária para uma boa execução do mesmo e a acompanhar o Aluno nas diferentes fases da sua realização, de acordo com o programa de trabalhos e meios apresentados, bem como por zelar pelo respeito dos princípios éticos e deontológicos e pelo cumprimento das normas internas da instituição. Nome Data Assinatura _________________________________________ ___/___/___ ____________________ Instituição Departamento Serviço / Setor _________________ ________________________ __________________________ Supervisor do projeto / Responsável pela DIIC Na qualidade de Docente Responsável pela DIIC / Supervisor do Aluno no CHP, comprometo-me a prestar a orientação necessária para uma boa execução do Trabalho de Investigação, de acordo com o programa de trabalhos e meios apresentados. Mais declaro que acompanharei o Aluno, responsabilizando-me por supervisionar a execução do trabalho no CHP, bem como por zelar pelo respeito dos princípios éticos e deontológicos e pelo cumprimento das normas internas da instituição. Nome Data Assinatura Margarida Lima ___/___/___ __________________ Departamento: DEFI The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 43 Termos de autorização local Trabalho académico de investigação: O efeito de nanoparticulas de óxido de ferro em linfócitos T Aluno da DIIC do curso de MIM do ICBAS/UP e do CHP: Daniel Azevedo Mendes Diretores de Serviço Na qualidade de Diretor de Serviço, declaro que autorizo a execução do Estudo de Investigação acima mencionado e comprometo-me a prestar as condições necessárias para a boa execução do mesmo, de acordo com o programa de trabalhos e os meios apresentados. Serviço Nome do Diretor Data Assinatura _____________________________ ____________________ ___/___/___ _______________ _____________________________ ____________________ ___/___/___ _______________ _____________________________ ____________________ ___/___/___ _______________ Diretores / Conselhos de Gestão de Departamento Na qualidade de Diretor do Departamento, declaro que autorizo a execução do Estudo de Investigação acima mencionado e comprometo-me a prestar as condições necessárias para a boa execução do mesmo, de acordo com o programa de trabalhos e os meios apresentados. Departamento Nome do Diretor Data Assinatura _____________________________ ____________________ ___/___/___ _______________ _____________________________ ____________________ ___/___/___ _______________ _____________________________ ____________________ ___/___/___ _______________ Responsáveis por Unidades, Gabinetes ou Setores (se aplicável) Na qualidade de Responsável pela Unidade / Gabinete / Setor, dou parecer favorável à execução do Estudo de Investigação acima mencionado e comprometo-me a prestar as condições necessárias para a boa execução do mesmo, de acordo com o programa de trabalhos e os meios apresentados. Unidade / Gabinete / Setor Nome do Responsável Data Assinatura _____________________________ ____________________ ___/___/___ _______________ _____________________________ ____________________ ___/___/___ _______________ The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 44 Termo de consentimento informado O EFEITO DE NANOPARTICULAS DE ÓXIDO DE FERRO EM LINFÓCITOS T Eu, abaixo-assinado__________________________________________________________: Fui informado de que o Estudo de Investigação acima mencionado se destina a clarificar os efeitos da formulação de ferro endovenoso Venofer®, frequentemente utilizada na clínica médica, em determinadas células do sangue. Os linfócitos T serão o alvo deste estudo e dizem respeito a células de defesa do sistema imunitário presentes no sangue em grande quantidade, que podem ser obtidas facilmente através de uma punção venosa com colheita de sangue periférico. Por sua vez, o Venofer® é uma formulação utilizada com grande frequência na correção de estados de depleção de ferro que conduzem grande parte das vezes a estados de anemia. O ferro presente nesta formulação encontrase sob a forma de nanopartículas de óxido de ferro que uma vez administradas podem ser rapidamente utilizadas pelo organismo. Este estudo visa identificar alguns dos efeitos desencadeados por estas partículas nos linfócitos T. Sei que neste estudo está prevista a realização de colheitas de sangue periférico tendo-me sido explicado em que consistem e quais os seus possíveis efeitos. Sei que alguns exames não podem ser efetuados neste hospital e que, por isso, têm que ser realizadas noutras instituições, como o Instituto de Ciências Biomédicas Abel Salazar (ICBAS) e o Instituto de Biologia Molecular e Celular (IBMC). Foi-me garantido que todos os dados relativos à identificação dos participantes neste estudo são confidenciais e que será mantido o anonimato. Sei que posso recusar-me a participar ou interromper a qualquer momento a participação no estudo, sem nenhum tipo de penalização por este facto. Compreendi a informação que me foi dada, tive oportunidade de fazer perguntas e as minhas dúvidas foram esclarecidas. Aceito participar de livre vontade no estudo acima mencionado. Concordo que sejam efetuados os exames e a colheita de amostras de sangue para realizar as análises que fazem parte deste estudo. Também autorizo a divulgação dos resultados obtidos no meio científico, garantindo o anonimato. Nome do Participante no estudo. Data Assinatura ___/___/_____ _________________________________________ Nome do Investigador Responsável Data Assinatura ___/___/_____ ________________________________________ The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 45 Folheto informativo para os participantes (formato A5) The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 | EXECUTION REPORT The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 47 The project described above was approved by the ethics committee of Centro Hospitalar do Porto – Hospital de Santo Antonio (CHP-HSA). It was executed between September 1th, 2014 and May 15th, 2015. The aim of the project was to observe the in vitro effect of iron oxide nanoparticles, commonly used in the clinical treatments, on T Lymphocytes and to compare that effect between patients with Hereditary Haemochromatosis (HH), homozygous for the p.Cys282Tyr HFE mutation and normal healthy controls. To reach this goal various parameters of the cell cycle, apoptosis and immunophenotype were evaluated by flow cytometry. The effect of these particles was also evaluated on chromosome instability and on bleomycin induced genotoxicity using cytogenetic studies. Sample collection occurred at the Haemochromatosis Clinic and the execution of the Cytometry experiments took place at the Cytometry Laboratory of CHP-HSA, both belonging to the Clinical Haematology Service. Cytogenetic experiments were executed on the Laboratory of Cytogenetics, Department of Microscopy of Abel Salazar Institute of Biomedical Sciences (ICBAS), and University of Porto (UP). The project execution had the collaboration of all participants mentioned above. This report aims to describe the population studied, the procedures performed, the results obtained and conclusions driven. The background and objectives of the project were described in detail in the project proposal. The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 48 CHANGES TO THE ORIGINAL PROJECT PROPOSAL • In order to perform all the experiments included in the project proposal it was necessary to collect four heparin tubes (6ml) and 1 EDTA tube (4ml) making a total of 28ml of blood per person. To avoid potential complications related to inconvenience and discomfort during the blood donation procedure or eventually related to vascular volume reduction, we decided not to use the CHP blood donors as controls. As an alternative approach, voluntary healthy persons of the same gender and age group of patients, recruited among laboratory workers or acquaintances were used as controls. In all cases, informed consent was obtained before sample collection. • Because of its relevance in the interpretation of cytogenetic results, we decided to include studies of T cell apoptosis, which was not included in the initial project proposal. The materials and methodology are described in this execution report. • The beginning of tasks 2, 3, and 4, “Blood sample collection”, “Cell cycle studies” and “Bleomycin genotoxicity test”, respectively, have been delayed by approximately 2 months in consequence of the deferral by ICBAS/UP to provide some necessary reagents required to the preparation of apoptosis studies. • As a consequence of the above mentioned delay, analysis and interpretation of results have also been deferred and therefore it was not possible to submit the presentation of the results to the National Haematology Congress. In addition, due to previously unforeseen reasons, the meeting of the European Iron Club was postponed to April 2016. • Because of lack of financial support, transmission electron microscopy (TEM)/ energy-dispersive X-ray spectroscopy (EDX) experiments were not performed. This is still a goal to achieve in future. The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 49 DESIGN AND METHODS Controls The controls consisted of six subjects recruited (see above) according to gender and age matching with the patients. In order to obtain a homogeneous population in terms of iron parameters, the study sample included only males. The age ranged from 42 to 68 years (average  standard deviation = 57  10). They had no known disease or clinical condition, therefore they were assumed as normal healthy subjects without performing any additional biochemical screening or other diagnostic test. Informed consent was obtained according to ethics committee of CHP approval. A summary characterization of the control population in terms of total lymphocyte counts and T lymphocyte subpopulations is given in Table 1. Table 1. Sample characterization – Controls. Lymphocytes Iron parameters Individuals Age Lymphocytes (x103/L) CD4+ (x103/L) CD8+ (x103/L) 1 57 1.77 1.02 0.31 Not assessed 2 68 2.38 0.93 0.27 3 46 2.32 1.14 0.65 4 58 2.34 1.61 0.28 5 65 1.28 0.46 0.23 6 42 1.45 0.55 0.33 Patients Six HH patients were included in this study. All patients had an established diagnosis of HH and were regularly followed at Haemochromatosis Clinic where all the relevant clinical information was documented. As already mentioned, all patients had confirmation of genetic diagnosis of p.Cys282Tyr homozygous mutation. Patients were consecutively recruited at the time of their regular visit to the clinic and blood samples collected during the regular phlebotomy procedure. As mentioned above, only male patients were used. Their age ranged from 40 to 69 years (average  standard deviation = 53  13). For each patient the informed consent was obtained before collecting blood samples as explained in the project proposal approved by ethics committee of CHP. The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 56 RESULTS Changes in cell cycle and proliferative index following Venofer® exposure The effect of Venofer on T lymphocyte cell cultures was addressed through the analyses of cell cycle phase relative distribution, evaluation of apoptotic cells, and measure of the proliferative index and the cytogenetic parameters of genotoxicity: number of breaks per cell and percentage of aberrant cells. The results are shown in table 3. In the presence of Venofer® it was observed a consistent and significant increase in the relative proportion of T lymphocytes in G0 / G1 phases of cell cycle (p-value=0.0137) with a corresponding significant decrease in the proportion of cells in S phase (p-value=0.0228). No significant differences were observed either in the relative proportion of cells in G2 / M phases or the percentage of apoptotic cells. The T lymphocyte proliferative index was also significantly lower in the presence of Venofer® (p-value=0.0242). It was not observed any impact of Venofer® on genotoxicity as measured by cytogenetic markers. Table 3. Comparisons of the cell cycle, apoptosis and cytogenetic parameters in T lymphocytes from normal healthy controls according to the presence (+Ven) or the absence (-Ven) of Venofer® in the culture medium. Results are displayed as individual values and summarized as the mean and standard deviation of each group. Differences between groups were tested by the paired t-test. % of cells in G0/G1 % of cells in S % of cells in G2/M % of apoptotic cells Proliferative Index Number of breaks per cell % of aberrant cells Individuals VenVen+ VenVen+ VenVen+ VenVen+ VenVen+ VenVen+ VenVen+ 1 56.8 89.4 32.1 10.6 11.1 0.0 92.0 94.8 4.42 1.75 0.04 0.02 2 2 2 41.1 57.2 46.3 36.4 12.6 6.4 47.8 75.9 1.75 0.50 0.02 0 2 0 3 48.6 65.9 48.4 28.2 3.0 5.9 84.7 79.2 2.22 0.98 0 0 0 0 4 74.8 89.7 21.6 9.3 3.6 1.0 62.3 35.2 1.11 1.17 0 0.06 0 6 5 76.3 77.1 16.9 20.0 6.8 2.9 57.5 53.3 1.85 1.04 0.02 0.04 2 4 6 80.6 92.4 16.9 7.1 2.5 0.5 55.5 56.8 4.93 2.04 0 0.08 0 4 Mean SD 63.0 16.4 78.6 14.5 30.4 14.3 18.6 11.8 6.6 4.4 2.8 2.8 66.7 17.6 65.9 21.4 2.71 1.57 1.25 0.56 0.01 0.02 0.03 0.03 1.0 1.1 2.7 2.4 Significance level (p-value) 0.0137 0.0228 n.s. n.s. 0.0242 n.s. n.s. Abbreviations: SD: standard deviation; n.s. not statistically significant No significant correlations were observed between the number of lymphocytes, or their specific populations (number of CD4+ or CD8+ T-cells), and any of the parameters for cell cycle, apoptosis or chromosome instability. The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 57 No impact of Venofer® in cell cycle, apoptosis, proliferative index and chromosome instability parameters in HH patients The effect of Venofer® on cultures of T lymphocytes obtained from HH patients was analysed. The results are shown in table 4. In contrast with the results observed in controls (see above), no significant impact of Venofer® was observed on any parameters evaluated. Table 4. Comparisons of the cell cycle, apoptosis and cytogenetic parameters in T lymphocytes from Hereditary Haemochromatosis patients controls according to the presence (Ven+) or the absence (Ven-) of Venofer® in the culture medium. Results are displayed as individual values and summarized as the mean and standard deviation of each group. Differences between groups are tested by paired t-test. % of cells in G0/G1 phases % of cells in S phase % of cells in G2/M phases % of apoptotic cells Proliferative Index Number of breaks per cell % of aberrant cells Individuals VenVen+ VenVen+ VenVen+ VenVen+ VenVen+ VenVen+ VenVen+ 1 69.3 87.5 22.5 12.5 8.2 0.0 82.4 70.5 3.62 4.94 0.06 0.02 4 2 2 51.0 51.7 37.9 42.7 11.1 5.6 35.4 34.9 1.85 1.24 0.16 0.08 2 6 3 51.4 56.2 39.6 37.0 9.0 6.8 50.9 74.6 0.63 0.68 0.04 0.04 4 2 4 68.1 68.5 25.5 26.4 6.4 5.1 89.3 76.5 2.13 1.00 0 0 0 0 5 76.9 73.2 17.8 20.3 5.3 6.5 49.7 79.3 0.41 0.21 0 0 0 0 6 62.8 68.8 27.7 25.8 9.5 5.4 86.0 79.5 2.18 1.91 0 0 0 0 Mean SD 63.3 06.4 67.7 12.7 28.5 8.6 27.5 11.0 8.3 2.1 4.9 2.5 65.6 23.0 69.2 17.1 1.80 1.17 1.66 1.70 0.04 0.06 0.02 0.03 1.7 1.9 1.7 2.3 Significance level (p-value) n.s. n.s. n.s. n.s. n.s. n.s. n.s. Abbreviations: SD: standard deviation; n.s. not statistically significant Such as in controls, no significant correlations were observed between the number of lymphocytes, or their specific populations (number of CD4+ or CD8+ T-cells), and any of the parameters for cell cycle, apoptosis or chromosome instability. An illustration of the overall difference between HH patients and controls is given in figure 2 were Venofer® induced G0/G1 phase variation is shown, in both healthy controls and HH patients. This parameter was calculated by dividing the percentage of cells in G0/G1 phase in the presence of Venofer® and the percentage of cells in G0/G1 phase in the absence of Venofer® in each individual and was assumed to reflect the extension of increment in the G0 / G1 phase of cell cycle induced by Venofer®. The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 58 Figure 2. Comparison between HH patients and controls for a ratio that reflects the magnitude of the difference in the percentage of cells in G0/G1phases induced by Venofer®. Changes in the bleomycin genotoxicity test following Venofer® exposure In order to test the hypothesis that the effect of Venofer® on T lymphocytes cell cultures could be due to an increased availability of intracellular iron, bleomycin genotoxicity test was used. For that purpose bleomycin-induced T cell cultures were performed, in the same individuals as above, in the presence or absence of Venofer®. The results are shown in table 5. Table 5. Bleomicyn genotoxicity test following Venofer® exposure expressed as the percentage of breaks per cell and percentage of aberrant cells in cell cultures from both healthy controls and Hereditary Haemochromatosis patients. Controls HH patients % of aberrant cells Number of breaks per cell % of aberrant cells Number of breaks per cell Individuals VenVen+ VenVen+ VenVen+ VenVen+ 1 45 15 1.02 0.23 39 8 0.76 0.34 2 44 26 0.94 0.66 42 20 0.72 0.43 3 32 22 0.80 0.29 40 20 1.58 0.50 4 32 26 0.74 0.62 38 28 0.80 0.53 5 46 18 0.92 0.33 40 16 1.08 0.27 6 36 22 0.92 0.63 44 18 0.90 0.39 Mean SD 39.1 6.5 21.5 4.4 0.89 0.10 0.46 0.19 40.5 2.2 18.3 6.5 0.97 0.32 0.41 0.10 Level of significance (p-value) 0.0065 0.0077 0.0006 0.0076 Abbreviations: SD: standard deviation. The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 59 A highly significant decrease in the percentage of aberrant cells (p=0.0065) and the number of breaks per cell (p=0.0077) was observed in Venofer® added cultures in healthy controls. Similarly, it was also observed a highly significant decrease in the percentage of aberrant cells (p=0.0006) and the number of breaks per cell (p=0.0076) were observed in HH patients. In these test conditions no significant differences were observed between HH patients and controls. Cell cycle and apoptosis parameters were also evaluated in cultures with bleomycin with or without Venofer® in both controls and HH patients. The results are expressed in tables 6 and 7, respectively. No significant differences were observed in any of the conditions tested with the addition of Venofer®, in both controls and HH patients. Table 6. Distribution of CD3+ T-lymphocytes through the different phases of the cell cycle and percentage of apoptotic cells in bleomycin test cultures with (Ven+) and without (Ven-) Venofer® in controls . % of cells in G0/G1 % of cells in S % of apoptotic cells Individuals VenVen+ VenVen+ VenVen+ 1 68.0 80.7 23.9 18.2 75.5 98.4 2 66.9 68.7 22.9 23.6 54.7 54.8 3 62.1 55.5 29.8 29.2 59.3 57.9 4 77.8 80.4 14.8 14.4 58.8 53.1 5 92.9 87.5 6.2 9.1 28.2 52.1 6 67.7 84.1 24.5 11.5 72.3 62.2 Mean SD 72.6 11.2 76.2 11.9 20.4 8.4 17.7 7.6 58.1 16.8 63.0 17.7 Level of significance (p-value) n.s. n.s. n.s. Abbreviations: SD: standard deviation; n.s. not statistically significant The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 60 Table 7. Distribution of CD3+ T-Lymphocytes through the different phases of the cell cycle and percentage of apoptotic cells in bleomycin test cultures with (Ven+) and without (Ven-) Venofer® in HH patients. % of cells in G0/G1 % of cells in S % of apoptotic cells Individuals VenVen+ VenVen+ VenVen+ 1 78.4 75.6 21.6 24.4 72.1 73.7 2 64.6 69.3 25.7 27.1 30.4 51.9 3 59.3 67.8 26.7 21.0 68.4 47.6 4 69.7 75.9 21.4 20.3 76.3 68.8 5 83.5 81.2 10.6 12.2 55.1 71.9 6 72.6 88.6 18.0 9.3 76.6 48.6 Mean SD 71.4 8.9 76.4 7.7 20.7 5.9 19.1 6.9 63.2 17.9 60.4 12.3 Level of significance (p-value) n.s. n.s. n.s. Abbreviations: SD: standard deviation; n.s. not statistically significant The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 61 DISCUSSION The several applications of iron oxide nanoparticles (IONs) are currently an interesting focus of research. A full characterization of the biological response to these type of materials seems fundamental for a better understanding how different cells handle these nanoparticles, to clarify putative mechanisms of toxicity and to discover possible new applications (2). There is, however, a remarkable lack of information about the mode how peripheral blood cells handle these particles. One should note that circulating cells are the first ones entering in contact with the iron oxide particles when these are administered intravenously; therefore they are potentially the first target of the nanoparticles. This question motivated the present study which was aimed to analyse the in vitro effect of Venofer® on the proliferation of peripheral blood T lymphocytes. The first relevant result obtained in this study was the demonstration of a significant increase in the percentage of lymphocytes at the G0/G1 phase of the cell cycle when they were cultured for 72 hours in the presence of Venofer®. The fact that the increase of cell numbers at this phase of the cell cycle appears to be inversely related with relative proliferative index is suggestive that sucrosecoated iron oxide nanoparticles may induce a cell cycle arrest at G0/G1 phase. It is well known that proper progression through the cell cycle, in eukaryotic cells, is highly monitored by certain checkpoints that prevent the cell entering the next phase of division whenever there is any change or damage in the components of the cell structure, particularly the DNA. This mechanism is critical to prevent certain acquired errors to be transmitted to the daughter cells. In order to control cell cycle progression there are three major checkpoints, G1–S, intra-S, and G2–M, which are essentially controlled by cyclins and cyclin-dependent kinases (3). These results showing an increase in the percentage of cells at the G0/G1 phases with no impact on apoptosis or genotoxicity strongly suggest the existence of an effective G1-S checkpoint activation and a G0/G1 cell cycle arrest to prevent damage in response to Venofer®. Other studies have accessed before the interference of iron oxide nanoparticles in the cell cycle (summarised in table 10). These studies, however, used different models, different cell types and different kinds of iron oxide nanoparticles, therefore the results are not absolutely comparable. Park et al (2014) using Bare-Iron Oxide Nanoparticles in a mouse macrophage cell line also demonstrate a G1 phase cell cycle arrest but, in contrast with this study, there was also a dose dependent decrease in cell viability (4). In contrast, Liu et al (2011) did not demonstrate any significant effect of IONs on cell viability, apoptosis, cell cycle or proliferation in a set of different non-hematopoietic cell lines (RAW264.7, Hepa1-6, HL-7702, and HeLa) but verified a significant increase in apoptosis for high doses in others (THP-1 and HepG2) (5). Altogether, these studies show that the effect of iron oxide nanoparticles can differ depending on certain factors, such The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 62 as, drug dose, coating material and the kind of evaluated cell. Further studies with Venofer® may prove to be useful to better establish the critical conditions to either induce an effective physiological response to the iron induced stress or to induce toxicity. Table 8. Studies that access Iron Oxide Nanoparticles (IONs) effect on cell cycle. Type of ION Size of IONs (nm) Cell line Findings Study/reference Bare-Iron Oxide Nanoparticles Unknown RAW264.7 cells (mouse peritoneal macrophage cell line) Cell viability decreased in a dose-dependent manner after bare-ION exposure, in parallel with a G1 phase arrest. Park et al. 2014 (4) Dimercaptosuccinic acid -coated Fe3O4 magnetic nanoparticles 11 RAW264.7, Hepa16, HL-7702, and HeLa IONs did not produce any significant effect on viability, apoptosis, cell cycles and proliferation at any dose. Iron loading of RAW264.7 was significantly higher than that of other cells at any dose. Liu et al. 2011 (5) THP-1 and HepG2 Significant apoptosis at highest concentration of IONs (100 µg/mL). The second relevant result obtained with this study was the demonstration that the pattern of cell cycle arrest found in normal healthy subjects was not observed in gender and age matched HH patients. This finding is in accordance with the existence of some kind of “adaptive cell response” in HH patients that could increase the expression of protective proteins necessary to prevent the harmful effects of iron, including oxidative stress effects. Relevant to this interpretation is the evidence provided before of a lower frequency of diepoxybutane-induced chromosome instability in HH patients as compared to both controls and patients with secondary haemochromatosis (6). Some other studies have also suggested the existence of stress adaptive mechanisms in HH. Martineli et al (2013) have shown a significant increased hepatic Paraoxonase-1 protein expression in HH patient together with a decrease in serum Paraoxonase-1 activity (7). This enzyme hydrolyze lipid peroxides and has a major role in protecting cells from inflammatory and oxidative stress (8) and it is known that elevated levels of Paraoxonase-1, can be protective against carbon tetrachloride-induced liver damage (9). Recently, Silva-Gomes et al. have shown that the oxidative stress induced transcription factor Nrf2 modifies the expression of liver disease in dietary-induced iron overload but, so far, no evidence has been provided yet of a similar effect in the human model of HH (10). Another possible mechanism to explain the difference in the cellular response of HH patients is related to the facts that hepcidin can regulate the intracellular iron levels in T lymphocytes by reducing ferroportin expression on the cell membrane and consequently influence lymphocyte proliferation (11) and it is known that HFE- The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 63 related HH patients have low levels of serum hepcidin. In this context, T-lymphocytes of HH patients could have higher ferroportin mediated iron export which could maintain a lower level of intracellular iron. It is important to mention that all the mechanisms proposed so far could only explain our findings assuming that T-lymphocytes somehow internalize and process the sucrose-coated iron oxide nanoparticles to interact with intracellular pathways. To clarify this, we had proposed to observe these cells through TEM/EDX technology but these experiments could not be done because of lack of financial support. Future studies are needed to complete this part of the project. One of the limitations of the present study was the low number of patients tested, which may compromise the significance and interpretation of some results. For example, although in lymphocyte cultures from HH patients the number of cells in G0 / G1 phases appears to be higher in cultures with Venofer® than in cultures with no additive, this difference was not statistically significant. Also, when it was compared the overall G0/G1 phase variation induced by Venofer® between patients and controls (Figure 2), in spite of the apparent marked difference, this was also not statically significant, due to the impact of one individual (individual 1 in Table 4) who showed a response similar to controls. Apart from the possibility of this individual being an “outlier” (it is not possible to assume that with only 6 individuals studied), this could also be explained by heterogeneity among HH patients in their phenotypic expression, such as happens with other clinical manifestations. It is known that the penetrance of the p.Cys282Tyr mutation is largely variable (12). For example, in some northEurope populations it is possible to find a prevalence of homozygous for the p.Cys282Tyr mutation of about 0.5% (13) although only a minority develop clinically apparent disease. Likewise, the natural history of the disease may range from a small change in iron parameters to terminal liver disease with cirrhosis or other manifestations of advanced disease (14). Age and gender are important features that can modulate clinical expression of the disease, but there are certainly many other still unidentified factors. Importantly, it was demonstrated that a high number of CD8+ T-cells is a protective factor for the clinical consequences of the disease (15). Although the total numbers of lymphocytes and relative proportions of CD8+ T and CD4+ T cells was not found in this study to be correlated with the response to Venofer®, the number of patients studied was too small to drive a definite conclusion. It would thus be important to extend this study by increasing the sample size, which would make possible to clarify if there is indeed an overall significant difference between HH patients and controls or to discriminate among differentially expressing HH patients. Finally one must discuss the unexpected findings obtained when bleomycin genotoxicity test was applied the. This test was chosen to test the hypothesis that the observed effect of Venofer® was dependent on the availability of intracellular iron. This chemotherapeutic agent needs a simultaneous The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 64 biding to a redox-active metal ion such as Fe2+ or Cu+, as well as molecular oxygen to cause DNA damage and consequently chromosome instability (16). The assumption was that, if levels of intracellular iron were increased after incorporation of IONs, more iron would be available for redox reactions and therefore the genotoxic effect of bleomycin should be increased in the presence of Venofer®. Surprisingly, in these experimental conditions, cultures with bleomycin plus Venofer® had a highly significant reduction in the percentage of chromosome breaks per cell as well as in the percentage of aberrant cells as compared to cultures with bleomycin alone. In addition, it was not observed, in these culture conditions, any significant effect on the formerly described cell cycle arrest in G0/G1 induced by Venofer®. So far, there is no explanation for these findings. One possibility is that the added effect of Venofer® and bleomycin in the culture medium may change completely the culture conditions, eventually by the formation of new iron-bleomycin complexes which would no longer act upon the cultured cells. It is know that bleomycin binds to NTBI in solution (17). Whether NTBI is found in the culture medium in the presence of Venofer®, this was not tested in this study. Another possibility to consider is that, since bleomycin also is known to induce a cell cycle arrest at G0/G1 (18), the effect of Venofer® would be no longer significant. It should be noted, however, that all the above described explanations are merely speculative. In order to explain the mechanism involved more experiments would be required, which are out of the scope of this work. The effect of iron oxide nanoparticles on T lymphocytes Daniel Mendes, ICBAS/UP – 2013/2014 and 2014/2015 65 CONCLUSION In summary, this study allowed us to approach, for the first time, a biological response of Tlymphocytes to sucrose-coated iron oxide nanoparticles, i.e., a possible protective cell cycle arrest at G0/G1. The study also showed that this response is different in normal healthy subjects and in HH patients suggesting that these individuals, by remaining in a constant state of iron overload, may have induced an adaptive response that makes them less susceptible to the effects of this metal. However some of the questions initially posed have not been answered yet. It would be fundamental to perform the TEM/EDX experiments to evaluate if the nanoparticles are indeed internalized by T-cells and, in that case, what is their intracellular composition and localization. Further gene expression studies are also important to carry out in future, in order to identify the putative intracellular molecular pathways involved in the adaptive response of HH patients. Following this work will also be important to increase our knowledge about the heterogeneity of this disease as well as understanding the physiological role of T lymphocytes in iron metabolism.