Type B coxsackievirus (CV-B) infections are involved frequently in the triggering of several autoimmune diseases such as myocarditis, dilated cardiomyopathy, pericarditis, pancreatitis, type 1 diabetes, encephalitis, thyroiditis or Sjögren's syndrome. Serological and virological evidence suggests that maternal infections during pregnancy can play a role in the appearance of these diseases in offspring. The current study aims to explore the effect of an in-utero CV-B infection on the fetal thymus, the central site for programming immunological self-tolerance. In this perspective, female Swiss albino mice were inoculated intraperitoneally or orally with the diabetogenic CV-B4 E2 strain at gestational days 10 or 17. Offspring were killed at different post-inoculation times, and their thymuses were analysed for evidence of infection and alterations in thymic T cell subsets. In-utero CV-B infection of the thymus was demonstrated during the course of vertical transmission, as attested by viral RNA and infectious virus detection in most analysed samples. No histopathological changes were evident. Thymic T cells were not depleted, despite being positive for viral RNA. As evidenced by flow cytometry analysis, CV-B infection of the fetal thymus induced significant changes of thymic T cell populations, particularly with maternal inoculation at gestational day 10. Altogether, these findings suggest that CV-B infection of the fetal thymus may play an important role in the genesis of autoimmune diseases.

In previous studies it was shown that inoculation of Swiss albino mice with CV-B4 E2 resulted in the production of serum IgG capable of enhancing the CV-B4 E2 infection of murine spleen cells cultures. To investigate whether such an enhancing activity of serum can play a role in vivo, we decided to study the CV-B4 E2 infection in mice exposed to successive inoculations of virus. In Swiss albino mice infected with CV-B4 E2 at the age of 21 days, anti-CV-B4 E2 neutralizing and enhancing activities of their serum peaked after 55 d. In contrast, mice inoculated at the age of 55 d expressed much lower activities. Despite the neutralizing activity of serum, CV-B4 E2 inoculated a second time to 55 day-old animals spread into the host. At the age of 72 and 89 d the levels of viral RNA and infectious particles were higher in organs of animals exposed to 2 successive infections compared with animals infected once at the age of 21 d or 55 d. In animals with 2 successive inoculations of CV-B4 E2 there was a relationship between the anti-CV-B4 E2 enhancing activity of serum and the level of viral RNA in organs and an enhancement of pathology was observed as displayed by histological analysis of pancreas and hyperglycaemia. Altogether our data strongly suggest that an anti-CV-B4 E2 enhancing activity in the host can play a role in the outcome of a secondary infection with this virus.

Objective To determine whether the French AmbUlatory Cesarean Section (FAUCS) technique reduces postoperative pain and promotes maternal autonomy compared with the Misgav Ladach cesarean section (MLCS) technique in elective conditions. Study design One hundred pregnant women were randomly, but in a non-blinded manner, assigned to undergo FAUCS or MLCS. The primary outcome was a postoperative mean pain score (PMPS), and secondary outcomes were a combined pain/medication score, time to regain autonomy, surgical duration, calculated blood loss, surgical complications, and neonatal outcome. Results Women in the FAUCS group experienced less pain than those in the MLCS group (PMPS = 1.87 [1.04–2.41] vs. 2.93 [2.46–3.75], respectively; p < 0.001). Six hours after surgery, the combined pain/medication score for FAUCS patients was 33% lower than that for MLCS patients (p < 0.001). FAUCS patients more rapidly regained autonomy, with 94% reaching autonomy within 12 h vs. 4% of MLCS patients (p < 0.001). There were no differences in maternal surgical or neonatal complications between groups. Conclusions Our results indicate that FAUCS can reduce postoperative pain and accelerate recovery, suggesting that this technique might be superior to MLCS and should be more widely used. One potentially key difference between FAUCS and MLCS is that MLCS includes 100 mcg spinal morphine anesthesia in addition to the same anesthesia used by FAUCS. Any interpretation of apparent differences must take the presence/absence of morphine into account.

Confirming Burnet's early hypothesis, elimination of self-reactive T cells in the thymus was demonstrated in the late 1980s, and an important question immediately arose about the nature of the self-peptides expressed in the thymus. Many genes encoding neuroendocrine-related and tissue-restricted antigens (TRAs) are transcribed in thymic epithelial cells (TECs). They are then processed for presentation by proteins of the major histocompatibility complex (MHC) expressed by TECs and thymic dendritic cells. MHC presentation of self-peptides in the thymus programs self-tolerance by two complementary mechanisms: (1) negative selection of self-reactive "forbidden" T cell clones starting already in fetal life, and (2) generation of self-specific thymic regulatory T lymphocytes (tT reg cells), mainly after birth. Many studies, including the discovery of the transcription factors autoimmune regulator (AIRE) and fasciculation and elongation protein zeta family zinc finger (FEZF2), have shown that a defect in thymus centralself-tolerance is the earliest event promoting autoimmunity. AIRE and FEZF2 control the level of transcription of many neuroendocrine self-peptides and TRAs in the thymic epithelium. Furthermore, AIRE and FEZF2 mutations are associated with the development of autoimmunity in peripheral organs. The discovery of the intrathymic presentation of self-peptides has revolutionized our knowledge of immunology and is opening novel avenues for prevention/treatment of autoimmunity.

Through synthesis and presentation of neuroendocrine self-antigens by major histocompatibility complex proteins, thymic epithelial cells (TECs) play a crucial role in programing central immune self-tolerance to neuroendocrine functions. Insulin-like growth factor-2 (IGF-2) is the dominant gene/polypeptide of the insulin family that is expressed in TECs from different animal species and humans. Igf2 transcription is defective in the thymus of diabetes-prone bio-breeding rats, and tolerance to insulin is severely decreased in Igf2−/− mice. For more than 15 years now, our group is investigating the hypothesis that, besides a pancreotropic action, infection by coxsackievirus B4 (CV-B4) could implicate the thymus as well, and interfere with the intrathymic programing of central tolerance to the insulin family and secondarily to insulin-secreting islet β cells. In this perspective, we have demonstrated that a productive infection of the thymus occurs after oral CV-B4 inoculation of mice. Moreover, our most recent data have demonstrated that CV-B4 infection of a murine medullary (m) TEC line induces a significant decrease in Igf2 expression and IGF-2 production. In these conditions, Igf1 expression was much less affected by CV-B4 infection, while Ins2 transcription was not detected in this cell line. Through the inhibition of Igf2 expression in TECs, CV-B4 infection could lead to a breakdown of central immune tolerance to the insulin family and promote an autoimmune response against insulin-secreting islet β cells. Our major research objective now is to understand the molecular mechanisms by which CV-B4 infection of TECs leads to a major decrease in Igf2 expression in these cells.

BackgroundLife-threatening infections with type B Coxsackieviruses (CV-B) are frequently encountered among newborns and are partly attributed to vertically-transmitted virus. Our current study investigates this alternative way of contamination by CV-B, using a mouse model.MethodsPregnant Swiss mice were intraperitoneally inoculated with CV-B4 E2 at gestational day 10(G) or 17G. Dams and offspring were monitored for mortality and morbidity, and sampled at different time-points to document the infection and explore eventual vertical transmission.ResultsInoculation at day 10G induced an important rate of abortion and a decrease in the number of delivered pups per litter, whereas inoculation at day 17G was marked by preterm delivery and significant behavioral changes in dams. Only one case of spastic paralysis and one case of pancreatitis were recorded among surviving pups. Seroneutralization revealed anti-CV-B4 neutralizing antibodies in infected dams and their partial transfer to offspring. Viral genome detection by RT-PCR and viral progeny titration in several tissues (dams’ uteri, amniotic sac, amniotic fluid, placenta, umbilical cord, pancreas and heart) attested and documented CV-B4 vertical transmission to the majority of analyzed offspring. Virus detection in fetuses suggests transplacental transmission, but perinatal transmission during delivery could be also suggested. Vertically transmitted CV-B might even persist since prolonged viral RNA detection was noticed in the pancreas and heart from offspring born to dams inoculated at day 17G.ConclusionThis model of CV-B4 vertical transmission in mice, in addition to allow a better understanding of CV-B infections in fetuses and newborns, constitutes a useful tool to investigate the pathogenesis of CV-B associated chronic diseases.

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The thymus fulfills the role of T-cell production and differentiation. Studying transcription factors and genes involved in T-cell differentiation and maturation during the fetal and neonatal periods is very important. Nevertheless, no studies to date have been interested in evaluating the expressions of housekeeping genes as internal controls to assess the varying expressions of different genes inside this tissue during that period or in the context of viral infection. Thus, we evaluated by real-time quantitative polymerase chain reaction (qPCR) the expression of the most common internal control genes in the thymus of Swiss albino mice during the fetal and neonatal period, and following in utero infection with Coxsackievirus B4. The stability of expression of these reference genes in different samples was investigated using the geNorm application. Results demonstrated that the expression stability varied greatly between genes. Oaz1 was found to have the highest stability in different stages of development, as well as following Coxsackievirus B4 infection. The current study clearly demonstrated that Oaz1, with very stable expression levels that outperformed other tested housekeeping genes, could be used as a reference gene in the thymus and thymic epithelial cells during development and following Coxsackievirus B4 infection.