Assessment of Thymic Output Dynamics After in utero Infection of Mice With Coxsackievirus B4

Figures

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  Figure 1. Thymus and spleen weight. Absolute and relative (organ weight/body weight) weights of thymus (A,B, respectively) and spleen (C,D, respectively) were determined for coxsackievirus (CV) B4-infected and age-matched mock (•) thymuses and spleens, sampled at different time points, from fetuses at day 17 of gestation (day 17G) and newborns at days 1 and 5 from birth. CV-B4 10G (▾): thymus or spleen sampled from mice born to dams inoculated with CV-B4 at day 10 of gestation; CV-B4 17G (♦): thymus or spleen sampled from mice born to dams inoculated with CV-B4 at day 17 of gestation. The Mann–Whitney test was used for statistical analysis. n = 4–12 per group ***p < 0.0001, **p < 0.01, *p < 0.05.

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  Figure 2. Thymus and spleen cellularity. Absolute and relative (number of cells/mg of cells) numbers of cells in the thymus (A,B, respectively) and spleen (C,D, respectively) mice assessed by trypan blue exclusion test. Values were determined in coxsackievirus (CV)-B4-infected and age-matched mock (•) thymuses and spleens, sampled at different time points, from fetuses at day 17 of gestation (day 17G) and newborns at days 1 and 5 from birth. CV-B4 10G (▾): thymus or spleen sampled from mice born to dams inoculated with CV-B4 at day 10 of gestation; CV-B4 17G (♦): thymus or spleen sampled from mice born to dams inoculated with CV-B4 at day 17 of gestation. The Mann–Whitney test was used for statistical analysis. n = 6–8 per group ***p < 0.0001, **p < 0.01, *p < 0.05.

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  Figure 3. Quantification of T cell receptor rearrangement excision circles (TRECs) as markers of thymopoiesis. The number/106 cells of sjTRECs and DβTRECs, as well as sj/Dβ ratio, were quantified in thymocytes (A–C) and splenocytes (D–F) of age-matched controls (•) and coxsackievirus (CV) B4-infected thymuses and spleens, sampled at different time points, from fetuses at day 17 of gestation (day 17G) and newborns at days 1 and 5 from birth. CV-B4 10G (▾): thymus or spleen sampled from mice born to dams inoculated with CV-B4 at day 10 of gestation; CV-B4 17G (♦): thymus or spleen sampled from mice born to dams inoculated with CV-B4 at day 17 of gestation. The Mann–Whitney test was used for statistical analysis. n = 4–10 per group. ***p < 0.001, **p < 0.01, *p < 0.05. Data for spleen were log-transformed before analysis.

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  Figure 4. Ptk7 mRNA expression. RNA was converted by reverse transcription (RT), and the number of copies per 100 ng of RNA was determined in thymocytes of age-matched controls (•) and coxsackievirus (CV) B4-infected mice by using a serial dilution of a reference plasmid. CV-B4 10G (▾): thymus sampled from mice born to dams inoculated with CV-B4 at day 10 of gestation; CV-B4 17G (♦): thymus sampled from mice born to dams inoculated with CV-B4 at day 17 of gestation. The Mann–Whitney test was used for statistical analysis. n = 6–8 per group. ***p < 0.001, **p < 0.01, *p < 0.05. Data were log-transformed before analysis.

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  Figure 5. Viral load in infected tissues. Thymocytes and splenocytes were sampled from fetuses at day 17 of gestation (day 17G) and from newborns at days 1 and 5 from birth, following an infection at day 10 or 17 of gestation (day 10G ▾ or 17G♦). Total RNA was extracted, converted to cDNA, and the level of viral RNA in the thymocytes (A) or splenocytes (B) was determined by qPCR and expressed as copy number per 100 ng of total RNA. n = 5–9 per group. Coxsackievirus (CV) B4 10G (▾): thymus sampled from mice born to dams inoculated with CV-B4 at day 10 of gestation; CV-B4 17G (♦): thymus sampled from mice born to dams inoculated with CV-B4 at day 17 of gestation. Data were log-transformed before analysis.