Stella Jacobson

The TheraCyte™ Device Protects against Islet Allograft Rejection in Immunized Hosts:

Clinically, many candidates for islet transplantation are already immunized, which increases their risk of graft rejection. Encapsulation of pancreatic islets using the TheraCyte™ device has been shown to protect against allograft rejection in nonimmunized recipients. However, the capacity of the TheraCyte™ device to prevent rejection in immunized recipients has not yet been studied. In this study, the protective capacity of the TheraCyte™ device was evaluated in an allogeneic rat model. Lewis rats were used as islet donors, and nonimmunized (control) and alloimmunized, diabetic Wistar–Furth (WF) rats were used as recipients. Graft survival was shorter in immunized recipients than in nonimmunized recipients (mean survival, 5.3 ± 2.7 and 9.3 ± 1.6 days, respectively, p < 0.01) when nonencapsulated islets were transplanted under the kidney capsule. When islets were transplanted into the TheraCyte™ device, graft function was maintained during the 6-month study period in both immunized and nonimmunized rats. In oral glucose tolerance tests performed at 1 month after transplantation, both groups had similar insulin and blood glucose levels indicating similar metabolic functions. Volume densities and absolute volumes of tissue inside the devices 6 months after transplantation were also comparable between the two groups, indicating that both groups maintained similar amounts of endocrine tissue. A higher number of IFN-γ-producing CD8+ T-cells were detected in immunized WF rats compared to control WF rats transplanted with encapsulated islets. This suggests that donor-specific alloreactivity in recipient rats was sustained throughout the study period. This study suggests that the TheraCyte™ device protects islet allografts also in immunized recipients. Our results further highlight the potential for using macroencapsulation to avoid immunosuppressive therapy in clinical islet transplantation.

A preclinical study on the efficacy and safety of a new vaccine against Coxsackievirus B1 reveals no risk for accelerated diabetes development in mouse models

Aims/hypothesisEnterovirus infections have been implicated in the aetiology of autoimmune type 1 diabetes. A vaccine could be used to test the causal relationship between enterovirus infections and diabetes development. However, the development of a vaccine against a virus suspected to induce an autoimmune disease is challenging, since the vaccine itself might trigger autoimmunity. Another challenge is to select the enterovirus serotypes to target with a vaccine. Here we aimed to evaluate the function and autoimmune safety of a novel non-adjuvanted prototype vaccine to Coxsackievirus serotype B1 (CVB1), a member of the enterovirus genus.MethodsA formalin-inactivated CVB1 vaccine was developed and tested for its immunogenicity and safety in BALB/c and NOD mice. Prediabetic NOD mice were vaccinated, infected with CVB1 or mock-treated to compare the effect on diabetes development.ResultsVaccinated mice produced high titres of CVB1-neutralising antibodies without signs of vaccine-related side effects. Vaccinated mice challenged with CVB1 had significantly reduced levels of replicating virus in their blood and the pancreas. Prediabetic NOD mice demonstrated an accelerated onset of diabetes upon CVB1 infection whereas no accelerated disease manifestation or increased production of insulin autoantibodies was observed in vaccinated mice.Conclusions/interpretationWe conclude that the prototype vaccine is safe and confers protection from infection without accelerating diabetes development in mice. These results encourage the development of a multivalent enterovirus vaccine for human use, which could be used to determine whether enterovirus infections trigger beta cell autoimmunity and type 1 diabetes in humans.

FRAMEWORK FOR SYSTEMATIC IDENTIFICATION OF ETHICAL ASPECTS OF HEALTHCARE TECHNOLOGIES: THE SBU APPROACH

Objectives: Assessment of ethical aspects of a technology is an important component of health technology assessment (HTA). Nevertheless, how the implementation of ethical assessment in HTA is to be organized and adapted to specific regulatory and organizational settings remains unclear. The objective of this study is to present a framework for systematic identification of ethical aspects of health technologies. Furthermore, the process of developing and adapting the framework to a specific setting is described. Methods: The framework was developed based on an inventory of existing approaches to identification and assessment of ethical aspects in HTA. In addition, the framework was adapted to the Swedish legal and organizational healthcare context, to the role of the HTA agency and to the use of non-ethicists. The framework was reviewed by a group of ethicists working in the field as well as by a wider set of interested parties including industry, interest groups, and other potential users. Results: The framework consists of twelve items with sub-questions, short explanations, and a concluding overall summary. The items are organized into four different themes: the effects of the intervention on health, its compatibility with ethical norms, structural factors with ethical implications, and long term ethical consequences of using the intervention. Conclusions: In this study, a framework for identifying ethical aspects of health technologies is proposed. The general considerations and methodological approach to this venture will hopefully inspire and present important insights to organizations in other national contexts interested in making similar adaptations.

Co-transplantation of stromal cells interferes with the rejection of allogeneic islet grafts.

Side effects associated with current immunosuppressive therapy complicate the use of islet transplantation as a treatment for type 1 diabetes. Multipotent mesenchymal stromal cells (MSCs) have demonstrated immunomodulatory activity. The purpose of this study was to investigate whether a murine stromal cell line affects graft rejection in a fully major histocompatibility complex (MHC)‐mismatched islet transplant model. We show that stromal cells have an inhibitory effect on T cell proliferation in vitro, and that they slow down the rejection of allogeneic islets. These findings indicate a possibility to use MSCs as a treatment to prolong the survival of islet grafts.

IL-8 from Local Subcutaneous Wounds Regulates CD11b Activation

The cellular and soluble mediators of a dermal inflammation can be studied by the skin chamber technique. The aim of this study was to address the physiological effect of soluble mediators, released into the skin chamber, with special focus on neutrophil CD11b activation. Mediators released at the inflammatory site were studied by Milliplex and enzyme‐linked immunosorbent assay (ELISA) and correlated with transmigration and CD11b activation in vivo and in vitro. Transmigration was studied by the skin chamber technique and by the transwell method, and expression of the CBRM1/5 epitope on activated CD11b was analysed by flow cytometry following in vivo and in vitro incubation with chamber fluid or recombinant interleukin‐8 (IL‐8). Leucocyte in vivo and in vitro transmigration both correlated with the concentrations of IL‐1β, tumour necrosis factor alpha (TNFα) and IL‐8 at P < 0.05 (R > 0.7). Furthermore, CD11b was activated, in terms of exposure of the activation epitope, on neutrophils after 30 min of in vitro incubation with chamber fluid and correlated solely with the concentration of IL‐8, P < 0.05 (R = 0.72). In vitro incubation with recombinant IL‐8 confirmed a concentration‐dependent expression of the activation epitope; however, induction of CBRM1/5 by recombinant IL‐8 required a concentration that was significantly higher compared with that in chamber fluid. In addition, the CBRM1/5 epitope was analysed on in vivo extravasated neutrophils that displayed a significantly higher expression compared with circulating neutrophils, P = 0.04. We conclude that IL‐8 is the major factor regulating the expression of CD11b activation epitope in neutrophils.

Antineutrophil Cytoplasmic Antibodies Induce Decreased CD62L Expression and Enhanced Metabolic Activity in Monocytes

Monocyte in vitro activation by antimyeloperoxidase (anti‐MPO)‐ and antiproteinase‐3 (anti‐PR3)‐positive sera, corresponding immunoglobulin G (IgG) fractions and monoclonal antibodies against MPO and PR3 was evaluated. The expression of adhesion molecules, l‐selectin (CD62L) and CR3 (CD11b), involved in leucocyte endothelial adhesion, and metabolic activity, measured as the production of hydrogen peroxide, were analysed. Decreased expression of CD62L was demonstrated in monocytes after incubation with antineutrophil cytoplasmic antibody (ANCA)‐positive sera. This finding was not accompanied by changes in CD11b expression. Metabolic activity was increased in monocytes after incubation with ANCA‐positive IgG fractions as well as after incubation with monoclonal anti‐MPO and anti‐PR3. These findings support the concept that the pathophysiological effect of ANCA is partly mediated through the action on crucial events in monocyte activation, such as CD62L downregulation and oxygen radical production.

In-vivo extravasation induces the expression of interleukin 1 receptor type 1 in human neutrophils

In order to address neutrophil activation during inflammation we assessed the expression of interleukin 1 receptor type 1 (IL‐1R1) following in‐vivo extravasation. Extravasated neutrophils were collected from 11 healthy study subjects by a skin chamber technique and compared to neutrophils in peripheral blood. Expression of IL‐1R1 was assessed by microarray, quantitative polymerase chain reaction (qPCR), Western blot, flow cytometry, enzyme linked immunosorbent assay (ELISA) and immunoelectron microscopy (iEM). IL‐1R1 was induced following extravasation, demonstrated by both gene array and qPCR. Western blot demonstrated an increased expression of IL‐1R1 in extravasated leucocytes. This was confirmed further in neutrophils by flow cytometry and iEM that also demonstrated an increased intracellular pool of IL‐1R1 that could be mobilized by N‐formyl‐methionine‐leucine‐phenylalanine (fMLP). Stimulation of peripheral neutrophils with IL‐1 resulted in transcription of NFκB and a number of downstream chemokines and the corresponding chemokines were also induced following in‐vivo extravasation. The present results demonstrate that IL‐1R1 is induced following extravasation and exists on the neutrophil surface, as well as in a mobile intracellular pool. Furthermore, neutrophils express functional IL‐1R1 as demonstrated by the induction of chemokines following IL‐1 stimulation. The results indicate a potential role for IL‐1 in the activation of neutrophils at inflammatory sites.

The target cell response to cytokines governs the autoreactive T cell repertoire in the pancreas of NOD mice

Aims/hypothesisThe pancreatic beta cell response to cytokines is crucial for the development of type 1 diabetes in the NOD mouse. For example, beta cell production of suppressor of cytokine signalling-1 (SOCS-1) protects against diabetes. This finding and other recent studies indicated that cytokine-stressed beta cells might contribute to disease progression by affecting the pancreatic lymphocyte infiltrate. The aim of this study was to provide insight into how the beta cell influences the pancreas-infiltrating T cell repertoire.MethodsLymphocytes isolated from Socs1-transgenic (tg) and non-tg NOD mice were analysed by flow cytometry. mRNA and protein levels in pancreatic islets were measured by real-time PCR and immunofluorescence analysis, respectively.ResultsThe percentages of regulatory T cells, total counts and ratios between infiltrating CD8+ and CD4+ T cells, and the expression of killer cell lectin-like receptor subfamily K, member 1 (NKG2D) on CD8+ T cells did not differ in pancreases from prediabetic Socs1-tg and non-tg NOD mice. However, a striking difference in the percentages of CD8+ T cells specific for glucose 6-phosphatase catalytic subunit-related protein 206–214 was found, showing that SOCS-1 prevents the accumulation of high percentages of self-reactive CD8+ T cells in the pancreas. It was also found that protection from diabetes in Socs1-tg NOD mice correlated with a reduced expression of Cxcl10 mRNA in IFN-γ treated islets.Conclusions/interpretationThis study highlights an important role for the beta cell in the local regulation of the diabetogenic process. By responding to the pro-inflammatory pancreas milieu it strongly influences the islet-reactive T cell repertoire in the pancreas.

Alloreactivity but Failure to Reject Human Islet Transplants by Humanized Balb⁄c⁄Rag2 )⁄) gc )⁄) Mice

A human islet transplant can cure patients with type 1 diabetes. A drawback of islet transplantation is the life‐long immunosuppressive medication, often associated with severe side effects. Moreover, in spite of the immunosuppressive therapy, islets are lost in the majority of transplanted patients over time. An improved small animal model for studies on human islet allograft rejection mechanisms and the development of new measures for its prevention is clearly warranted. Here, we evaluated the potential of Balb/cRag2−/−γc−/− mice carrying a human‐like immune system (so‐called humanized mice) as a tool for studies on the rejection of transplanted human islets. Human T cells from Balb/cRag2−/−γc−/− mice, which as neonates had been transplanted with CD34+ human cord blood haematopoietic stem cells (HIS mice), proliferated in response to allogeneic human dendritic cells, but failed to reject a human islet allograft transplanted to the renal subcapsular space as assessed by immunohistochemistry and analysis of human serum C‐peptide levels. Histological analysis revealed that few if any T cells had migrated to the grafted tissue. These observations question the usefulness of the HIS mouse model for studies on human islet allograft rejection mechanisms and highlight the need for further improvements.

Diagnostic accuracy of postmortem imaging vs autopsy—A systematic review

Background Postmortem imaging has been used for more than a century as a complement to medico-legal autopsies. The technique has also emerged as a possible alternative to compensate for the continuous decline in the number of clinical autopsies. To evaluate the diagnostic accuracy of postmortem imaging for various types of findings, we performed this systematic literature review. Data sources The literature search was performed in the databases PubMed, Embase and Cochrane Library through January 7, 2015. Relevant publications were assessed for risk of bias using the QUADAS tool and were classified as low, moderate or high risk of bias according to pre-defined criteria. Autopsy and/or histopathology were used as reference standard. Findings The search generated 2600 abstracts, of which 340 were assessed as possibly relevant and read in full-text. After further evaluation 71 studies were finally included, of which 49 were assessed as having high risk of bias and 22 as moderate risk of bias. Due to considerable heterogeneity - in populations, techniques, analyses and reporting - of included studies it was impossible to combine data to get a summary estimate of the diagnostic accuracy of the various findings. Individual studies indicate, however, that imaging techniques might be useful for determining organ weights, and that the techniques seem superior to autopsy for detecting gas Conclusions and Implications In general, based on the current scientific literature, it was not possible to determine the diagnostic accuracy of postmortem imaging and its usefulness in conjunction with, or as an alternative to autopsy. To correctly determine the usefulness of postmortem imaging, future studies need improved planning, improved methodological quality and larger materials, preferentially obtained from multi-center studies.