uu.seUppsala University Publications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Studies of Innate and Adaptive Immunity in Islet Transplantation
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology. (Olle Korsgren)
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 [en]
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: urn:nbn:se:uu:diva-232863ISBN: 978-91-554-9046-1 (print)OAI: oai:DiVA.org:uu-232863DiVA: diva2:750015
Public defence
2014-11-07, Fåhreussalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2014-10-17 Created: 2014-09-25 Last updated: 2015-01-23
List of papers
1. A novel model for studies of blood-mediated long-term responses to cellular transplants
Open this publication in new window or tab >>A novel model for studies of blood-mediated long-term responses to cellular transplants
Show others...
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.

National Category
Immunology in the medical area Clinical Laboratory Medicine
Identifiers
urn:nbn:se:uu:diva-232482 (URN)10.3109/03009734.2014.965290 (DOI)000350984700004 ()
Available from: 2014-09-18 Created: 2014-09-18 Last updated: 2017-12-05Bibliographically approved
2. Characterization of innate immunity in an extended whole blood model of human islet allotransplantation
Open this publication in new window or tab >>Characterization of innate immunity in an extended whole blood model of human islet allotransplantation
Show others...
2016 (English)In: Cell Transplantation, ISSN 0963-6897, E-ISSN 1555-3892, Vol. 25, no 3, 503-515 p.Article in journal (Refereed) Published
Abstract [en]

The instant blood-mediated inflammatory reaction (IBMIR) has been studied in whole blood models of human allo-islet transplantation for short periods (<6 h). Beyond this time frame the innate response to intraportally transplanted islets is less well described. A novel whole blood model was applied to study blood islet graft interactions up to 48 h. Heparinized polyvinyl chloride tubing was sealed into small bags containing venous blood together with allogeneic human islets and exocrine tissue, respectively. The bags were attached to a rotating wheel (37 degrees C). Concentrated glucose and sodium hydrogen carbonate were added every 12 h to maintain physiological limits for sustained immune cell functions. Plasma was collected at repeated time points for analyses of coagulation/complement activation and chemokine/cytokine production. Immune cell infiltration was analyzed using immunohistochemistry. Coagulation and platelet activation markers, thrombin antithrombin complex (TAT) and soluble CD40 ligand (sCD4OL) showed early high concentrations (at 6-12 h). sC5b-9 steadily increased over 48 h. At 6 h neutrophils and monocytes surrounded the clotted cellular grafts with a following massive infiltration of neutrophils. High and increasing concentrations of CXCR1/2 ligands [IL-8 and growth-regulated oncogene alpha/beta/gamma (Gro-alpha/beta/gamma)] and IL-6 were produced in response to human islets and exocrine tissue. The CCR2 ligand monocyte chemoattractant protein 1 (MCP-1) exhibited increasing concentrations in response to exocrine tissue. The CXCR3 ligand interferon-inducible T cell alpha chemoattractant (I-TAC) was produced in response to both human islets and exocrine tissue from 6 h. Monokine induced by yinterferon (Mig) and interferon gamma-induced protein 10 (IP-10) showed a later response, preferentially to exocrine tissue and with larger variations among preparations. An extended blood model of clinical islet transplantation allowed characterization of early immune activation in response to human islets and exocrine tissue. Increased production of chemokines targeting CXCR1/2, CCR2, and CXCR3 was observed, accompanied by massive intraislet neutrophil infiltration over 48 h. The model proved to be useful in exploring early blood-mediated reactions to cellular transplants and has relevance for evaluation of pharmacological interventions to prevent graft loss.

National Category
Immunology in the medical area
Identifiers
urn:nbn:se:uu:diva-232859 (URN)10.3727/096368915X688461 (DOI)000372669200007 ()26084381 (PubMedID)
Funder
Swedish Diabetes AssociationSwedish Child Diabetes FoundationEU, FP7, Seventh Framework Programme
Available from: 2014-09-25 Created: 2014-09-25 Last updated: 2017-12-05Bibliographically approved
3. Post-transplant upregulation of chemokine messenger RNA in non-human primate recipients of intraportal pig islet xenografts
Open this publication in new window or tab >>Post-transplant upregulation of chemokine messenger RNA in non-human primate recipients of intraportal pig islet xenografts
Show others...
2005 (English)In: Xenotransplantation, ISSN 0908-665X, E-ISSN 1399-3089, Vol. 12, no 4, 293-302 p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND:

We have previously shown that pig-to-primate intraportal islet xenografts reverse diabetes, escape hyperacute rejection, and undergo acute cellular rejection in non-immunosuppressed recipients. To gain a better understanding of mechanisms contributing to xenoislet rejection in non-human primates we examined gene expression in livers bearing islet xenografts in the first 72 h after transplantation.

METHODS:

Liver specimens were collected at sacrifice from seven non-immunosuppressed rhesus macaques at 12, 24, 48 and 72 h after intraportal porcine islet transplantation. Following total RNA extraction, mRNA was quantified using SYBR green real-time reverse transcription polymerase chain reaction (RT-PCR) for species-specific immune response genes. Data were analyzed using comparative cycle threshold (Ct) analysis, adjusted for specific primer-efficiencies and normalized to cyclophilin expression.

RESULTS:

Porcine insulin mRNA was detected in all liver samples. Cluster analysis revealed differential gene expression patterns at 12 and 24 h (early) compared with at 48 and 72 h (late) post-transplant. Gene expression patterns were associated with histological findings of predominantly neutrophils and only a few lymphocytes at 12 and 24 h and an increasing number of lymphocytes and macrophages at 48 and 72 h. Transcript levels of CXCR3 and its ligands, interferon-inducible protein 10 (IP-10) and monokine induced by IFN-gamma (Mig), significantly increased between early and late time points together with expression of MIP-1alpha, regulated on activation normal T expressed and secreted protein (RANTES) and MCP-1. CCR5 showed only a marginal, non-significant increase. Fas ligand, perforin and granzyme B transcripts were all elevated at 48 and 72 h post-transplant.

CONCLUSIONS:

Our data suggest that CXCR3, with ligands IP-10 and Mig, is involved in T cell recruitment in acute islet xenograft rejection in non-human primates. Upregulation of RANTES and MIP-1alpha transcripts in the absence of a significant CCR5 increase suggests a possible involvement of other chemokine receptors. MCP-1 expression is associated with T cell and macrophage infiltration. Elevated cytotoxic effector molecule expression (Fas ligand, perforin, granzyme B) indicates T-cell mediated graft destruction by cytotoxic and cytolytic mechanisms within 48 to 72 h after transplantation. These results identify the CXCR3-mediated chemoattractant pathway as an immunosuppressive target in pig-to-primate islet xenotransplantation.

National Category
Immunology in the medical area
Identifiers
urn:nbn:se:uu:diva-233185 (URN)10.1111/j.1399-3089.2005.00228.x (DOI)15943778 (PubMedID)
Available from: 2014-09-30 Created: 2014-09-30 Last updated: 2017-12-05Bibliographically approved
4. Prolonged diabetes reversal after intraportal xenotransplantation of wild-type porcine islets in immunosuppressed nonhuman primates
Open this publication in new window or tab >>Prolonged diabetes reversal after intraportal xenotransplantation of wild-type porcine islets in immunosuppressed nonhuman primates
Show others...
2006 (English)In: Nature Medicine, ISSN 1078-8956, E-ISSN 1546-170X, Vol. 12, no 3, 301-303 p.Article in journal (Refereed) Published
Abstract [en]

Cell-based diabetes therapy requires an abundant cell source. Here, we report reversal of diabetes for more than 100 d in cynomolgus macaques after intraportal transplantation of cultured islets from genetically unmodified pigs without Gal-specific antibody manipulation. Immunotherapy with CD25-specific and CD154-specific monoclonal antibodies, FTY720 (or tacrolimus), everolimus and leflunomide suppressed indirect activation of T cells, elicitation of non-Gal pig-specific IgG antibody, intragraft expression of proinflammatory cytokines and invasion of infiltrating mononuclear cells into islets.

National Category
Immunology
Identifiers
urn:nbn:se:uu:diva-233183 (URN)10.1038/nm1369 (DOI)16491083 (PubMedID)
Available from: 2014-09-30 Created: 2014-09-30 Last updated: 2017-12-05
5. Transcriptional profiling of stress response in cultured porcine islets
Open this publication in new window or tab >>Transcriptional profiling of stress response in cultured porcine islets
Show others...
2007 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 357, no 1, 118-125 p.Article in journal (Refereed) Published
Abstract [en]

Cell-based diabetes therapy may be achieved through xenotransplantation of adult porcine islets, but tissue quality and immunoreactivity barriers need to be overcome. Early identification and exclusion of irreversibly stressed and dying islets may improve transplant outcomes. We used oligonucleotide microarray and quantitative RT-PCR to identify molecular markers of physiological and immunological stress in porcine islets cultured under stress conditions of elevated glucose (16.7 mM), inflammatory cytokine addition (IL-1beta, TNF-alpha, and IFN-gamma), or both, for 48 h. Hyperglycemic conditions were associated with increased thioredoxin interacting protein and metabolic process mRNAs, as observed in rodent and primate species. Cytokine treatment increased expression of JAK-STAT pathway components, oxidative stress (transglutaminase 2), and beta cell dysfunction genes. Transglutaminase 2 induction is unique to porcine islets. Biomarkers involved in hyperglycemia and islet inflammation may serve as novel targets for improving and monitoring isolated porcine islet function and viability.

National Category
Immunology in the medical area
Identifiers
urn:nbn:se:uu:diva-232861 (URN)10.1016/j.bbrc.2007.03.101 (DOI)17407763 (PubMedID)
Available from: 2014-09-25 Created: 2014-09-25 Last updated: 2017-12-05Bibliographically approved

Open Access in DiVA

fulltext(9585 kB)426 downloads
File information
File name FULLTEXT01.pdfFile size 9585 kBChecksum SHA-512
9228363dd1a687c88e7c7922fee82adf1525be3e4b4a28c1fbc840b529e5cdd4e935bd7a403464a713b93b1baee2f69f635e1e2d6d47f952cde56626b1ab5f56
Type fulltextMimetype application/pdf
Buy this publication >>

Authority records BETA

Hårdstedt, Maria

Search in DiVA

By author/editor
Hårdstedt, Maria
By organisation
Clinical Immunology
Immunology in the medical area

Search outside of DiVA

GoogleGoogle Scholar
Total: 426 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 809 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf