4.2 INTERACTION BETWEEN S. EPIDERMIDIS AND THE HUMAN
4.2.3 Circulating cathelicidin antimicrobial peptide LL-37 levels in
We grouped the study population based on the gestational time: delivery before weeks 28 (Group 1, n=8 infant pairs), delivery between weeks 28 and 36 (Group 2, n=20 mother-infant pairs) and delivery between weeks 37 and 42 (Group 3; n=15 mother-mother-infant pairs + n=12 infant). Circulating serum concentration of the human cathelicidin LL-37 was measured by ELISA as well as the pro- and the active forms of the cathelicidin protein were studied by Western Blot analysis.
Our major finding is that we did not find significant difference in the circulating LL-37 concentration in infants born at different gestational time, in contrast to a recent publication that showed reduced plasma level of the peptide, and also of other AMPs in preterm infants (Strunk et al., 2009). The possible explanation of the differing results can be that it is difficult to obtain sufficient blood sample volume from the umbilical cord of preterm infants. Therefore, the low sample volume might cause unreliable measurements. In addition, the low sample size can also influence the results. We had good sample volume, but rather low sample size (8 infants born before the gestational weeks 28, out of 28 preterm neonates). However, the similar serum LL-37 level in both the preterm and term groups might suggest that the transplacental passage of the peptide LL-37 occurs early during pregnancy.
We detected both the inactive proprotein hCAP18 (18 kDa) and the active LL-37 (4.5 kDa) peptide in newborn infants as well as their mothers at time of birth, independent of gestational time.
We observed a significant positive correlation between the maternal and umbilical cord serum level of human cathelicidin peptide LL-37, independent of the gestational time. Similar positive correlation was recently shown in a term mother-infant group (Mandic Havelka et al., 2010).
The correlation between serum LL-37 level and gestational time was also analysed. We found that the concentration of the peptide did not show significant correlation with the gestational time, either in infants’ serum or in mothers’ serum (p=0.9490 and r=0.0088; p=0.6007 and r=0.082, respectively).
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5 GENERAL CONCLUSIONS
S. epidermidis strains obtained from bloodstream infection in preterm infants had different characteristics than the skin isolates of healthy term neonates. The blood isolates were equipped with an invasive genetic element and showed higher antimicrobial resistance compared with the skin isolates. Whereas, vancomycin resistance was not detected among any of the isolates.
Short and long filament-like structures on the cell surface of S. epidermidis seemed to be involved in the attachment to the catheter surface and also in cell-to-cell attachment/communication.
Physiological LL-37 peptide concentrations, below those that kill or inhibit growth, inhibited S. epidermidis attachment and biofilm formation on abiotic surfaces. The peptide regulates genes involved in primary attachment, accumulation and in biofilm regulation.
The concentration of the circulating hCAP18/LL-37 was not significantly different in preterm and term neonates at birth. There was a positive correlation between maternal and infant peptide concentration. The inactive proprotein hCAP18 and the active LL-37 peptide were present in newborn infants as well as their mothers at time of birth, independent of gestational time. These findings might suggest that preterm infants’
vulnerability is not connected to the lower LL-37 peptide level at birth.
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6 FUTURE PERSPECTIVES
The increasing antibiotic resistance among the invasive bacterial strains force us to develop alternative strategies in the treatment or prevention of catheter-related infections. There is a challenging field as to design coated catheters where biomolecules (antimicrobial peptides) or nanoparticles could be used to modify the plastic surface of the device, thereby inhibiting the attachment of biofilm forming bacteria. Drugs could be used to stimulate AMPs’ expression or modified antimicrobial peptides might be used as drugs. So far, an increased level of infection control in NICU settings, seems to be a preventive tool in catheter-related infections in preterm infants.
It is still unclear what components of the bacterial surface form short and long filament-structures. Aap or PSMs could be studied as possible molecules.
Microarray- and protein analysis at different time points could be performed for better understanding of the anti-biofilm effect of the peptide LL-37. Also, fluorescent microscopy could reveal the capability of the peptide to diffuse into the biofilm. In addition, the anti-biofilm effect of LL-37 in combination with other AMPs could also be studied.
It is well known that LL-37 plays an important role in the first-line of the immune defense, driving inflammatory responses and activating other innate- as well as adaptive immune components. This study could be the first step in a larger project which could measure the LL-37 concentration in higher number of preterm infants, with a bigger focus on the extreme preterm neonates (born <28 weeks), to confirm our results. It is also an interesting question how the LL-37 level will change after birth and how it can be increased in preterm infants during infection.
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8 ACKNOWLEDGEMENTS
It is an honor to do PhD at Karolinska Institutet, especially working in a laboratory within Karolinska University Hospital. I became inspired by the place and I am glad that my work could contribute to children’s health.
First of all, I would like to express my gratitude for all patients who participated in our studies.
My studies would have never been completed without the help and support of you all around me.
I would like to thank:
Kristina Gemzell Danielsson, my main supervisor, for taking me to your group and giving me this great opportunity to do my PhD. Thank you for your immediate answers, for your positive attitude in any situations. For your support and for the appreciation. It has been inspiring to be your student!
Giovanna Marchini, my co-supervisor, my mentor, my main teacher in grant writing. For introducing me to the field of neonatal infection and immunology. I am glad that I could work with you. I missed you so much when you were away. Thank you for everything you did for me and thanks for believing in me! You are a great conference mate, too. Many thanks for our exciting scientific conversations in Copenhagen and Barcelona!
Christian G. Giske, for your immediate answers, your great support in laboratory work and in scientific writing. I am happy that I could work with you.
Birgitta Henriques Normark, my co-supervisor, for your support during my studies.
Gábor Földvári, my external mentor, I am inspired by your great scientific career. Thank you for our conversations and your support. I appreciate your friendship!
Ute Römling, my co-author, for your advises at the beginning of my studies, when I had no idea what biofilm means.
Kjell Hultenby, my co-author, for introducing me to the world of electron microscopy. For your time and for your interest regarding my new ideas. It was always great to work with you!
Professor Holger Rohde, my co-author, for your valuable time and comments.
Aurelija Dubicke and Emma Fransson, I am happy that I met you in the FRH-lab and that we became co-authors. Thank you for helping me out with samples!
Annika Nelson, I am happy that there was a need for a microbiologist when you finished your studies.
It was a great start for me.
I would like to say special tanks to Maria Sennström, for your enthusiasm and your kind help with my study, to Veronica Berggren, for helping us with samples, and to Karin Jansmark, for taking samples for my studies and for your company in Barcelona!
29 WHO-group: all members in this group, especially Helena, Ingrid, Suzanne Elin, Karin, Cecilia, for your kind hospitality in your meetings and other scientific activities, as well as the great parties.
Eva Broberg, for taking care of me in the WHO group. Thank you for being the subject of practicing my Swedish.
Catharina Karlsson, for all support in many different ways. Thank you for taking care of me during my PhD!
Astrid Häggblad, for helping me to manage smoothly the administrative jungle during the PhD education. Thank you for your kindness.
Colleagues in FRH-lab and on floor 9:
Birgitta Byström, You understood my problems and supported me in many ways. Thank you for your friendship, for your kind hospitality in your home and for our conversations! Thank you for pushing Swedish with me and for teaching me everything before you retired.
Eva Andersson, for that you were there and helped me when I was alone with my ‘microbiology’.
Lalit, for your support and advises. Especially during the hardest last months.
Ivika, I am happy that I met with you. You are a great person with positive thinking! Thanks for our conversations, discussions and for the lot of fun every day.
Sanaz, I am happy that I met with you just when I needed the support for finishing this PhD. Thank you for our conversations and for your advices.
Sakthi and Nage, for your company in our group and for your support. Laura, for your general kindness. I wish we could work together!
Mo, for your company, your friendship and your kind support. Thank you for your jokes and for listening when I answered your question ‘how are you?’.
Yvonne, for your support and your technical help in the lab with my very first ELISA in the very last study.
Past master students, PhDs and post docs: Linda, Johanna, Elham, Sujata, Lola, Nathalie, Suby, Joshua, Gemma, and also Lars Ärlund-Richters’ group before you moved out: Seema, Rouku, Jessica.
It was an inspiring environments for the start of my PhD, with lot of fun and happy coffee-breaks.
Hanna Ingelman-Sundberg, for your friendship and for your company in coffee-breaks! It was great to talk about research experience, and all those hard issues regarding manuscripts, PhD, thesis writing.
You are a nice and great person!
Shanie, for your kindness, for our scientific discussions, for the cinnamon coffee and for inviting me to the Research Forum.
Giulia, for our conversations in the weekends while we were in the lab. Did we really work then?
Rita, Ruth, Jennifer, Monica, Elena, Lars: I am grateful for always being friendly and helpful!
30
My friends:
Dorina, my colleague, my advisor, my language mate, and first of all my friend and a ‘relative’ of mine.
I am so happy that you are around me every day and you could lighten the hard issues with talking to me in Hungarian. It means a lot to me to get out stress on a funny way, joking and laughing a lot together.
Birgitte, I remember that we arrived in the lab about the same time. You just finished your PhD when I just started mine. We became friends and I miss you and your family so much after you left Sweden.
Our families spent nice time together. I am also tankful for giving me the example how to write thesis book with two kids.
Bea, my friend and class mate at university, for the access to Emöke; and Emöke, for the access to Giovanna.
Erika, you are my role-model as a person, a teacher, a scientist and a mother. Thank you so much for introducing me to the world of microbiology. Thank you for your friendship and your supervision during my graduation at the university. That gave me the motivation for all scientific work in the last 15 years.
Blanka, Peti, Remi, István and Hajna, for always being curious about my work and for your appreciation. It is always fun to be with you.
Marie and Ben: for your supportive attitude, for your friendship and warm hospitality!
My family:
My mother, Anyu, and my father, Apu, for all your love and support during my long way becoming a scientist. Thanks that you believed in me!
My mother-in-law, Klári, for all your lovely support and worries when I worked a lot! I wish that my father-in-law, Miklós could be with us now and read this book.
My grandmothers, Nagyi and Mama, for your love, for being curious about my work. It means a lot that you are proud of me.
My brother, Isti, my sister-in-laws, Panni, Klári, Reni, and my brother-in-law, David, maybe you think, I am crazy to study so much. Yes, indeed, it was crazy, but also a big fun.
Magdi, my best friend ever, for all our conversations. It means a lot that you are always there for me!
My lovely husband, Dani, for your patience, for your positive thinking and for your support. For keeping me calm, when I became disappointed. No problem exists when I have you around. Only you know, how long this journey was. It was not easy, but worth doing it!
Lili and Beni, my lovely kids, for completing my life outside of science. For the marvelous feeling being your mother!
Additionally:
The study was supported by grants from the Swedish Research Council (grant no 0855), ALF (Karolinska Institutet–Stockholm County Council, Agreement on Medical Research and Training)
31 funding, and the Karolinska Institute Research Funds. My travels to conferences were supported by the Travel Grant of The Department of Women’s and Children’s Health, the Olga Dahls Fond and the Stiftelsen Tummeliten.
I would like to tank to Sällskapet Barnavård, Stiftelsen Samariten, Stiftelsen Barncentrum, H.K.H.
Kronprincessan Lovisas Foundation, Konungen Gustav VI Foundation for granting my studies.
It was a great honor to receive scholarship award from Frimurare Barnhus Foundation and from Lilla Barn Foundation handed over personally by H.M. Queen Silvia and H.R.H. Prince Carl Philip, respectively.
My favorite quotes:
‘Yes!!!’ (Kristina G.)
‘Well done!’(Kristina G.)
‘Do not worry. Sleep well now and start tomorrow with new energy!’ (Kristina G.)
‘I am so proud of you!’ (Giovanna M.)
‘It is re-search, Eva!’ (Giovanna M.)
‘Do not give up, Eva!’ (Giovanna M.)
‘Excellent!’ (Christian G.)
‘It is only a PhD!’ (Ivika P.)
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