Coxsackievirus B4 Transplacental Infection Severely Disturbs Central Tolerogenic Mechanisms in the Fetal Thymus.

Thymus plays a fundamental role in central tolerance establishment, especially during fetal life, through the generation of self-tolerant T cells. This process consists in T cells education by presenting them tissue-restricted autoantigens promiscuously expressed by thymic epithelial cells (TECs), thus preventing autoimmunity. Thymus infection by Coxsackievirus B (CV-B) during fetal life is supposed to disturb thymic functions and, hence, to be an inducing or accelerating factor in the genesis of autoimmunity. To further investigate this hypothesis, in our current study, we analyzed thymic expression of autoantigens, at the transcriptional and protein level, following in utero infection by CV-B4. mRNA expression levels of Igf2 and Myo7, major autoantigens of pancreas and heart, respectively, were analyzed in whole thymus and in enriched TECs together along with both transcription factors, Aire and Fezf2, involved in autoantigens expression in the thymus. Results show that in utero infection by CV-B4 induces a significant decrease in Igf2 and Myo7 expression at both mRNA and protein level in whole thymus and in enriched TECs as well. Moreover, a correlation between viral load and autoantigens expression can be observed in the whole thymus, indicating a direct effect of in utero infection by CV-B4 on autoantigens expression. Together, these results indicate that an in utero infection of the thymus by CV-B4 may interfere with self-tolerance establishment in TECs by decreasing autoantigen expression at both mRNA and protein level and thereby increase the risk of autoimmunity onset.

Effect of Coxsackievirus B4 Infection on the Thymus: Elucidating Its Role in the Pathogenesis of Type 1 Diabetes.

The thymus gland is a primary lymphoid organ for T-cell development. Various viral infections can result in disturbance of thymic functions. Medullary thymic epithelial cells (mTECs) are important for the negative selection of self-reactive T-cells to ensure central tolerance. Insulin-like growth factor 2 (IGF2) is the dominant self-peptide of the insulin family expressed in mTECs and plays a crucial role in the intra-thymic programing of central tolerance to insulin-secreting islet ß-cells. Coxsackievirus B4 (CVB4) can infect and persist in the thymus of humans and mice, thus hampering the T-cell maturation and differentiation process. The modulation of IGF2 expression and protein synthesis during a CVB4 infection has been observed in vitro and in vivo in mouse models. The effect of CVB4 infections on human and mouse fetal thymus has been studied in vitro. Moreover, following the inoculation of CVB4 in pregnant mice, the thymic function in the fetus and offspring was disturbed. A defect in the intra-thymic expression of self-peptides by mTECs may be triggered by CVB4. The effects of viral infections, especially CVB4 infection, on thymic cells and functions and their possible role in the pathogenesis of type 1 diabetes (T1D) are presented.

Modulation of IGF2 Expression in the Murine Thymus and Thymic Epithelial Cells Following Coxsackievirus-B4 Infection.

Coxsackievirus B4 (CV-B4) can infect human and murine thymic epithelial cells (TECs). In a murine TEC cell line, CV-B4 can downregulate the transcription of the insulin-like growth factor 2 (Igf2) gene coding for the self-peptide of the insulin family. In this study, we show that CV-B4 infections of a murine TEC cell line decreased Igf2 P3 promoter activity by targeting a region near the transcription start site; however, the stability of Igf2 transcripts remained unchanged, indicating a regulation of Igf2 transcription. Furthermore, CV-B4 infections decreased STAT3 phosphorylation in vitro. We also showed that mice infected with CV-B4 had an altered expression of Igf2 isoforms as detected in TECs, followed by a decrease in the pro-IGF2 precursor in the thymus. Our study sheds new light on the intrathymic regulation of Igf2 transcription during CV-B4 infections and supports the hypothesis that a viral infection can disrupt central self-tolerance to insulin by decreasing Igf2 transcription in the thymic epithelium.

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

The thymus is the main organ of the lymphatic system, in which T cells undergo a rigorous selection to ensure that their receptors (TCRs) will be functional and will not react against the self. Genes encoding for TCR chains are fragmented and must be rearranged by a process of somatic recombination generating TCR rearrangement excision circles (TRECs). We recently documented coxsackievirus B4 (CV-B4) infection of Swiss albino mouse thymus in the course of in utero transmission. In the current study, we intended to evaluate thymic output in this experimental model. For this purpose, pregnant Swiss albino mice were inoculated with CV-B4 at day 10 or 17 of gestation, and thymus and spleen were sampled from offspring at different time points and then subjected to quantification of TREC molecules and Ptk7 gene expression. Results showed a pronounced effect of in utero CV-B4 infection on the thymus with an increase in the cellularity and, consequently, the weight of the organ. sj and DßTREC analysis, by real-time PCR, revealed a significant decrease following CV-B4 infection compared to controls, a decrease which gets worse as time goes by, both in the thymus and in the periphery. Those observations reflect a disturbance in the export of T cells to the periphery and their accumulation within the thymus. The evaluation of Ptk7 transcripts in the thymus, for its part, showed a decrease in expression, especially following an infection at day 10 of gestation, which supports the hypothesis of T cell accumulation in a mature stage in the thymus. The various effects observed correlate either negatively or positively with the viral load in the thymus and spleen. Disruption in thymic export may indeed interfere with T cell maturation. We speculate that this may lead to a premature release of T cells and the possibility of circulating autoreactive or proliferation-impaired T cell clones.

Housekeeping Gene Expression in the Fetal and Neonatal Murine Thymus Following Coxsackievirus B4 Infection.

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.

The presentation of neuroendocrine self-peptides in the thymus: an essential event for individual life and vertebrate survival.

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 (tTreg 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 central self-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.

Hydronephrosis in a dairy calf: A diagnosis delayed by a clinician's Bayesian brain reasoning.

A 3-week-old female dairy calf was presented because of severe weakness and diarrhea. Physical examination and ultrasound findings lead to a tentative diagnosis of intestinal obstruction, although serum biochemistry suggested a primary renal disorder. Reassessment of ultrasound images allowed diagnosis of hydronephrosis that had been misdiagnosed probably because of the clinician's biased Bayesian reasoning. Pyelonephritis without renal failure was diagnosed 2.5 months later. The calf was euthanized and the diagnoses were confirmed.

Une hydronéphrose rénale chez un veau laitier : quand le raisonnement Bayesien du clinicien rend le diagnostic plus difficile. Une génisse âgée de 3 semaines est présenté pour faiblesse générale et diarrhée. L'examen clinique et les images échographiques de l'abdomen sont compatibles avec une obstruction intestinale. Mais les résultats d'analyse biochimique montrent une anomalie rénale. Un examen échographique supplémentaire est compatible avec un diagnostic d'hydronéphrose. Le diagnostic d'hydronéphrose n'avait pas été fait à la première visite probablement suite au raisonnement Bayesien du clinicien. Le veau est revu 2,5 mois plus tard. L'échographie de l'abdomen, les analyses urinaire et sanguine montrent une pyélonéphrite sans insuffisance rénale. Le veau est euthanasié et l'autopsie montre l'hydronéphrose et pyélonéphrite bilatérale.(Traduit par les auteurs).

How Does Thymus Infection by Coxsackievirus Contribute to the Pathogenesis of Type 1 Diabetes?

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 ß 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 ß 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.

Description of thoracoscopy and associated short-term cardiovascular and pulmonary effects in healthy cattle.

OBJECTIVE: To describe the optimal thoracoscopic approach to the bovine pleural cavity and evaluate the short-term effects of thoracoscopy on cardiovascular and pulmonary function of healthy cattle. SAMPLE: 6 healthy adult Holstein cows (12 hemithoraxes). PROCEDURES: For each cow, thoracoscopy was performed in both the left and right hemithoraxes with a 24-hour interval between procedures. Cows were sedated and restrained in a standing position for each thoracoscopic examination. Examination of each hemithorax lasted for 30 minutes. Arterial blood gas variables, heart rate, and respiratory rate were assessed at predetermined times before, during, and after the procedures to monitor cardiovascular and pulmonary function. Thoracic ultrasonography was performed immediately and at 24 hours and 1 week after each thorascopic examination to evaluate the extent of residual pneumothorax. RESULTS: Insertion of the laparoscope into the pleural cavity at the ninth intercostal space 15 cm ventral to the transverse processes of the thoracic vertebrae provided optimal visibility of structures in both the left and right hemithoraxes. Most structures of the pleural cavity were equally visible from both sides except the esophagus and the dorsal branch of the vagus nerve, which were best observed in the left hemithorax, and the pericardium, which was best observed in the right hemithorax. Mild increases in heart and respiratory rates and moderate decreases in arterial oxygen saturation and Pao2 were detected during the procedures. CONCLUSIONS AND CLINICAL RELEVANCE: Standing thoracoscopy was well tolerated in healthy adult dairy cattle and needs to be evaluated in cattle with pulmonary disease.

Programming of neuroendocrine self in the thymus and its defect in the development of neuroendocrine autoimmunity.

For centuries after its first description by Galen, the thymus was considered as only a vestigial endocrine organ until the discovery in 1961 by Jacques FAP Miller of its essential role in the development of T (thymo-dependent) lymphocytes. A unique thymus first appeared in cartilaginous fishes some 500 million years ago, at the same time or shortly after the emergence of the adaptive (acquired) immune system. The thymus may be compared to a small brain or a computer highly specialized in the orchestration of central immunological self-tolerance. This was a necessity for the survival of species, given the potent evolutionary pressure imposed by the high risk of autotoxicity inherent in the stochastic generation of the diversity of immune cell receptors that characterize the adaptive immune response. A new paradigm of "neuroendocrine self-peptides" has been proposed, together with the definition of "neuroendocrine self." Neuroendocrine self-peptides are secreted by thymic epithelial cells (TECs) not according to the classic model of neuroendocrine signaling, but are processed for presentation by, or in association with, the thymic major histocompatibility complex (MHC) proteins. The autoimmune regulator (AIRE) gene/protein controls the transcription of neuroendocrine genes in TECs. The presentation of self-peptides in the thymus is responsible for the clonal deletion of self-reactive T cells, which emerge during the random recombination of gene segments that encode variable parts of the T cell receptor for the antigen (TCR). At the same time, self-antigen presentation in the thymus generates regulatory T (Treg) cells that can inhibit, in the periphery, those self-reactive T cells that escaped negative selection in the thymus. Several arguments indicate that the origin of autoimmunity directed against neuroendocrine glands results primarily from a defect in the intrathymic programming of self-tolerance to neuroendocrine functions. This defect may be genetic or acquired, for example during an enteroviral infection. This novel knowledge of normal and pathologic functions of the thymus constitutes a solid basis for the development of a novel type of tolerogenic/negative self-vaccination against type 1 diabetes (T1D).