PMC

Pancreatic lesions in mice treated with streptozotocin and inoculated with CV-B4E2. CD1 mice were treated with STZ (35 mg/kg) and then 12 days later were inoculated with CV-B4E2 (2.10⁴ TCID₅₀). Controls were treated with buffer or STZ then inoculated with culture medium or with CV-B4E2. The animals were sacrificed at day 5 post inoculation. The pancreas was collected in buffered formalin and then processed as described in the Materials and Methods section. The pictures show a representative aspect of lesions of pancreas from mice treated with STZ prior to inoculation with CV-B4E2: inflammatory infiltrate (A), fat involution (B,C) and abnormal morphology of endocrine tissue (D,E). They also show a representative aspect of pancreas from controls treated with buffer (F) or STZ (G) then inoculated with culture medium (F,G) and a representative aspect of pancreas from controls treated with CV-B4E2 (H).

Diabetes induced by CV-B4E2 in mice treated with a sub-diabetogenic dose of STZ. Mice that received STZ or buffer, and then inoculated 12 days later with CV-B4E2 or culture medium, were sacrificed on day 5, 10, 15, 20 and 25 post-infection (one mouse at each time point to collect blood and pancreas). Blood glucose and insulin levels were measured (A–D) and the pancreas was recovered to investigate the presence of infectious particles by endpoint dilution assay; the results are expressed as TCID₅₀/mg (E). The method used to investigate the presence of viral RNA was by quantitative RT-PCR; the results are expressed as log of viral RNA copies by ng of total RNA (F).

Individual monitoring of blood glucose levels and quantification of enteroviral RNA in mouse pancreas. CD1 mice were intraperitoneally (ip) injected with 35 mg of STZ/kg (A,B) or buffer (Buffer) (C,D) and 12 days later with CV-B4E2 (2.10⁴ TCID₅₀) or culture medium (M) using the same route. A drop of blood was collected from the tail vein regularly from the day of injection of STZ or buffer (Buffer) until 21 days after inoculation with CV-B4E2 or medium. The blood glucose level is measured using a glucometer. The results are expressed as mg/dl. 21 days post-infection, the mice were sacrificed by cervical dislocation and the pancreas was removed to investigate the presence of viral RNA by quantitative RT-PCR. The results are mean +/− SD of log copies per ng of total RNA. Each mouse is represented by a symbol, with five mice in each group. 300 mg/dl is the threshold above which mice are considered hyperglycemic.

Pattern of inflammatory cytokines in pancreas of CD1 mice. CD1 mice were treated with STZ (35 mg/kg) and then 12 days later were inoculated with CV-B4E2 (2.10⁴ TCID₅₀) (STZ/CV-B4E2). Controls were treated with buffer or STZ and then inoculated with culture medium (Mock and STZ respectively) or with CV-B4E2 (CV-B4E2). On day 5 post-infection blood was collected from the tail vein to determine the level of blood glucose by using a glucometer; (A) then the animals were euthanised and the blood obtained by cardiac puncture was centrifuged and the serum recovered to measure the level of insulin by ELISA (B). The pancreas was harvested in presence of protease inhibitors and processed as described in the materials and methods section to determine the level of infectious particles (C) and of enteroviral RNA (D) – expressed as log TCID₅₀/mg of organ and as log of the number of copies of enteroviral RNA/ng of total RNA, respectively. TNFα (E), IFNγ (F) and IP-10 (G) in pancreas were quantified by ELISA, the results were expressed as pg per ng of total RNA. The results are mean +/− standard deviation (5 mice in each group). The dark inverted triangle in the group STZ/CV-B4E2 represents the animal with blood glucose level < 300 mg/dl.