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A novel model for studies of blood-mediated long-term responses to cellular transplants
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Center for Clinical Research Dalarna. (Olle Korsgren)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology. (Olle Korsgren)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology. (Bo Nilsson)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology. (Bo Nilsson)
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2015 (English)In: Upsala Journal of Medical Sciences, ISSN 0300-9734, E-ISSN 2000-1967, Vol. 120, no 1, 28-39 p.Article in journal (Refereed) Published
Abstract [en]

Aims

Interaction between blood and bio-surfaces is important in many medical fields. With the aim of studying blood-mediated reactions to cellular transplants, we developed a whole-blood model for incubation of small volumes for up to 48 h.

Methods

Heparinized polyvinyl chloride tubing was cut in suitable lengths and sealed to create small bags. Multiple bags, with fresh venous blood, were incubated attached to a rotating wheel at 37°C. Physiological variables in blood were monitored: glucose, blood gases, mono- and divalent cations and chloride ions, osmolality, coagulation (platelet consumption, thrombin-antithrombin complexes (TAT)), and complement activation (C3a and SC5b-9), haemolysis, and leukocyte viability.

Results

Basic glucose consumption was high. Glucose depletion resulted in successive elevation of extracellular potassium, while sodium and calcium ions decreased due to inhibition of energy-requiring ion pumps. Addition of glucose improved ion balance but led to metabolic acidosis. To maintain a balanced physiological environment beyond 6 h, glucose and sodium hydrogen carbonate were added regularly based on analyses of glucose, pH, ions, and osmotic pressure. With these additives haemolysis was prevented for up to 72 h and leukocyte viability better preserved. Despite using non-heparinized blood, coagulation and complement activation were lower during long-term incubations compared with addition of thromboplastin and collagen.

Conclusion

A novel whole-blood model for studies of blood-mediated responses to a cellular transplant is presented allowing extended observations for up to 48 h and highlights the importance of stringent evaluations and adjustment of physiological conditions.

Place, publisher, year, edition, pages
2015. Vol. 120, no 1, 28-39 p.
National Category
Immunology in the medical area Clinical Laboratory Medicine
Identifiers
URN: urn:nbn:se:uu:diva-232482DOI: 10.3109/03009734.2014.965290ISI: 000350984700004OAI: oai:DiVA.org:uu-232482DiVA: diva2:748337
Available from: 2014-09-18 Created: 2014-09-18 Last updated: 2017-12-05Bibliographically approved
In thesis
1. Studies of Innate and Adaptive Immunity in Islet Transplantation
Open this publication in new window or tab >>Studies of Innate and Adaptive Immunity in Islet Transplantation
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Clinical islet transplantation is today an established alternative treatment for a selected group of type 1 diabetes patients. The predominant technique for transplantation is infusion of islets in the liver via the portal vein. Obstacles to advancing islet transplantation include limited engraftment resulting from an immediate blood-mediated inflammatory reaction (IBMIR), a life-long need for immunosuppression and the shortage of organs available.

In this thesis, innate and adaptive immunity were explored in allogeneic and xenogeneic settings, with the long-term goal of preventing islet graft destruction. Methods for studying immune responses to islets in blood and engrafted islets in liver tissue (intragraft gene expression) were developed and refined. The innate response to human islets and exocrine tissue in ABO-compatible blood was characterized up to 48 h using a novel whole-blood model. Physiological changes in the blood during incubations were explored and adjusted to allow prolonged experiments. Increased production of chemokines targeting CXCR1/2, CCR2 and CXCR3 was observed, accompanied by massive intra-islet neutrophil infiltration. Notably, endocrine and exocrine tissue triggered a similarly strong innate immune response.

Two studies of adult porcine islet transplantation to non-human primates (NHPs) were performed. Expression of immune response genes induced in liver tissue of non-immunosuppressed NHPs (≤72 h) was evaluated after porcine islet transplantation. Up-regulation of CXCR3 mRNA, together with IP-10, Mig, MIP-1α, RANTES, MCP-1 and cytotoxic effector molecule transcripts, was associated with T-cell and macrophage infiltration at 48-72 h. Long-term survival (>100 days) of adult porcine islets in a NHP model was later demonstrated using T-cell-based immunosuppression, including co-stimulatory blockade (anti-CD154 mAb). Graft failure was associated with increased levels of circulating, indirectly activated T cells, non-Gal pig-specific IgG and gene transcripts of inflammatory cytokines. Microarray analysis of the response to inflammatory cytokines in cultured porcine islets identified genes involved in cell death, immune responses and oxidative stress; this gene pattern coincided with physiological changes (decrease in insulin and ATP content).

In summary, allogeneic whole-blood experiments and xenogeneic in vivo studies underscored the importance of preventing early inflammation and cell-recruitment to avoid islet graft loss in islet transplantation. Long-term survival of porcine islets in NHPs was shown to be feasible using T-cell-directed immunosuppression, including anti-CD154 mAb.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 117 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1030
Keyword
diabetes, islet transplantation, islets of Langerhans, xenotransplantation, nonhuman primate, blood, whole blood model, innate immunity, adaptive immunity, IBMIR, chemokines
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:uu:diva-232863 (URN)978-91-554-9046-1 (ISBN)
Public defence
2014-11-07, Fåhreussalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 10:15 (English)
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Supervisors
Available from: 2014-10-17 Created: 2014-09-25 Last updated: 2015-01-23

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Hårdstedt, MariaLindblom, SusanneHong, JaanNilsson, BoKorsgren, Olle

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