Inflammatory gene expression in Coxsackievirus B-4-infected human islets of Langerhans
Section snippets
Materials and methods
Isolation and culture of pancreatic islets. Using a protocol approved by the Local Ethical Committee, human islets of Langerhans were isolated from brain-dead donors free of known pancreatic disease as previously described [19]. The islet preparation was of good quality and was available for research only because of too low islet mass yield after isolation.
Islets were cultured in CMRL-1066 (Gibco-BRL, Invitrogen) supplemented with 10 mM nicotinamide (Sigma Chemicals), 10 mM Hepes buffer
Islet quality
The purity of the isolated islets was 80–90%. We evaluated islet function using a dynamic perfusion system (Fig. 1), in which insulin release was increased when the concentration of glucose in the perfusate was raised.
TCID50 titrations and degree of CPE
Both strains replicated well in the human islet cells (Fig. 2). Islets infected with the VD2921 strain showed no CPE while islets infected with the lytic strain V89-4557 exhibited CPE at the 1+ to 2+ level on Day 3 post-infection.
Microarray analysis
Interleukin (IL)-1β, IL-6, and IL-8 were
Discussion
In the present study, we have used two different strains of CBV-4 to investigate whether CBV-4-infected islets express inflammation-related genes whose expression could contribute to the autoimmune process that destroys the islets and finally leads to T1DM. We also asked whether the gene expression of these genes differed according to viral phenotype (lytic vs. non-lytic). Islets were either infected with V89-4557, a CBV-4 strain that has been shown to cause lytic infection in human islets in
Acknowledgments
The authors thank Susanne Gabrielsson for expert support in microarray analysis and The Juvenile Diabetes Research Foundation, The Swedish Diabetes Association, The Family Ernfors Fund, The Gillbergska Foundation, Swedish Animal Welfare Agency, Barndiabetesförbundet, the Ronald MacDonald Fund, and the Swedish Medical Research Council (K98-12XC-12445-02B) for financial support.
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