Thymic Tissue Research Articles

Increased Naive, Stem Cell Memory and Terminally Differentiated CD8+ T Cells in Chronic Graft Versus Host Disease

BACKGROUND AND AIMSDespite improved clinical outcomes of patients undergoing Hematopoietic Stem Cell Transplantation (HSCT) in recent years, chronic GVHD (cGVHD) and its associated complications have a significant impact on non-relapse mortality and quality of life. Loss of immune tolerance in the post-transplant period plays an important role in the emergence of cGVHD, and while imbalances in CD4+ T cell subsets, such as regulatory T cells, are known to contribute to its pathobiology, the role of different subsets of CD8+ T cells is poorly characterized.PATIENTS AND METHODSWe prospectively monitored a group of 56 patients who underwent unrelated donor HSCT after reduced intensity conditioning containing ATG. GVHD prophylaxis consisted of cyclosporine plus mycophenolate mofetil for all patients. 15 patients were excluded due to early disease relapse and/or death secondary to infection or acute GVHD in the first 9 months post-HSCT. cGVHD diagnosis and staging was performed according to the 2014 NIH criteria. Patients were divided into 2 groups according to cGVHD status: 17 patients (41%) with cGVHD (cGVHD+) and 24 patients (59%) without cGVHD (cGVHD-).Peripheral blood was analyzed by flow cytometry to characterize T cell subsets at fixed time points over a 2 year period. CD8+ T subsets were identified as follows: True Naïve cells as CD45RA+CD62L+CD95- (TN), Stem Cell Memory cells as CD45RA+CD62L+CD95+ (SCM), Central Memory cells as CD45RA-CD62L+ (CM), Effector Memory cells as CD45RA-CD62L- (EM) and terminally differentiated effector cells as CD45RA+CD62L- (EMRA).Frozen PBMC from patients at months 9 and 12 post-HSCT were used to purify CD8+ T cells by cell sorting to quantify Signal-Joint T Cell Receptor Excision Circles (TREC) by Real-Time PCR and to evaluate TCR diversity by TCR Vbeta complementarity-determining region 3 spectratyping.RESULTSPatients with cGVHD had significantly increased percentages and absolute counts of TN CD8 in the first 6 months post-HSCT compared with cGVHD- patients (p≤0.04 at months 1 and 3 for percentages; p≤0.02 for months 1-6 in absolute counts).Furthermore, cGVHD+ patients had increased SCM CD8 cells throughout the post-HSCT period (significances of p<0.05 at months 6 and 12 for percentages and 18 and 24 for counts). The increased prevalence of these two subsets was not associated with increased proliferation as assessed by Ki67, which was reduced in cGVHD+ in the total CD45RA+CD62L+ gate (TN plus SCM cells) (p≤0.04 at months 6, 12 and 18). No significant differences were observed in susceptibility to apoptosis as measured by BCL2 or CD95 expression levels.CM and EM subset counts were reduced in cGVHD+ as compared to cGVHD- (p=0.04 and 0.02 at month 12). EM CD8 were the dominant population in cGVHD- patients throughout the follow up. Significant increases in the percentage of EM CD8 cells in cGVHD- were observed at months 2, 9, 12 and 18 (p≤0.04).cGVHD+ patients had increased percentages of EMRA (p<0.04 at months 9, 12 and 18) and absolute counts (p<0.04 in months 2, 9 and 24). This was not correlated to altered Ki67, Bcl-2 or CD95 levels.The increase in TN in cGVHD+ patients was not associated with increased TREC numbers within CD8+ T cells. TREC content was significantly reduced in both patient groups at month 9 as compared healthy controls (HC) (p<0.05). This could be due to the low prevalence of these cells and/or to rapid differentiation into other memory subsets such as the SCM and EMRA. Despite the differences in subset prevalence in the two patient groups, we did not observe any significant differences in TCR Vbeta diversity in CD8+T cells. Reduced TCR diversity was only observed when patients were compared to HC (p=0.0001).DISCUSSIONOur observations of increased TN, SCM and EMRA CD8 subsets very early after HSCT in patients who subsequently develop cGVHD suggest the possibility that these may be relevant biomarkers for cGVHD development. SCM T cells have previously been shown to cause lethal GVHD in mouse models and to represent a reservoir of antigen-specific T cells capable of differentiating in vivo into other memory subsets with effector function. We speculate that thymic tissue damage resulting from conditioning regimens and/or acute GVHD, together with other yet unidentified factors, may lead to the emergence of self-reactive naïve CD8+ T cells that differentiate to SCM and later to EMRA cells potentially mediating cGVHD. DisclosuresNo relevant conflicts of interest to declare.

IPSC-Derived Thymic Epithelial Progenitor Cells As Cell-Based Therapy to Restore Thymic Function in Hematopoietic Stem Cell Transplant Recipients

Hematopoietic stem cell transplantation (HSCT) remains the only curative therapy for many malignant and non-malignant diseases to date. The thymus plays a key role in post-transplant immune reconstitution. The outcome of a HSCT and the recipient thymic function are intimately intertwined. In fact, the production of mature T cells occurs in the thymus and depends on the interaction of hematopoietic progenitor cells with non-hematopoietic stromal elements, notably thymic epithelial cells (TECs). Through positive and negative selection, a healthy thymus generates a broad and self-tolerant T cell receptor (TCR) repertoire that protects from infection and prevents autoimmunity. In addition, the thymus gives rise to regulatory T-cells which confine immune responses and further promote central tolerance. At odds with its importance for successful transplant outcomes, the thymus is exquisitely sensitive to a wide range of insults encountered during the peri-transplant period, including cytotoxic agents, steroids and irradiation. In the allogeneic transplant setting, acute graft-versus-host disease (GVHD) can cause further thymic injury. Thymic injury in turn, leads to susceptibility to infections and autoimmune complications. Moreover, it is now widely recognized that thymic injury leading to defective deletion of self-reactive T-cells along with misguided T-helper cell polarization and B-cell activation constitute major drivers of chronic GVHD. Together these represent the leading causes of non-relapse-related transplant mortality and morbidity. As such, HSCT can precipitate a vicious cycle, in which the success of the HSCT procedure hinges on intact thymic function, but the HSCT process itself often causes irrecoverable thymic injury (Figure1). Thymic function naturally declines with age with little to no endogenous potential to regenerate. Current treatment options for thymic injury during HSCT are limited to symptomatic therapy. Therefore, novel approaches to cure irreversible thymic injury are critically needed.To address this need, our goal is to generate fully functional thymic epithelial progenitor cells (TEPCs) from hPSCs as a stem cell-based therapy for thymic regeneration and restoration of immune competence in HSCT recipients. Building on the efficient induction of definitive endoderm from human pluripotent stem cells (hPSCs) by directing signals controlling lineage bifurcations, we have developed a novel platform that drives TEPC development through sequential induction of the intermediaries anterior foregut (AFG) and pharyngeal pouch endoderm (PPE). Here we show that TEPCs derived in vitro from hPSCs or hESCs express the key thymic transcription factor FOXN1 along with Keratin 5 and Keratin 8, the primary cytoskeletal building blocks of medullary and cortical TECs, respectively. In addition, in vitro derived TEPCs express MHC class II to varying degrees (Figure 2). To optimize our in vitro platform, modifications of key-signaling pathways in the differentiation protocol are validated by comparing the transcriptome of hPSC-derived TEPCs to that of primary human medullary and cortical thymic epithelial cells using next generation sequencing. Our ongoing work is testing the capacity of hPSC-derived TEPCs to promote T-cell maturation, positive and negative selection resulting in the development of a broad and self-tolerant TCR repertoire from adoptively transferred human HSPCs in a humanized mouse model.Through sequential validation of fidelity, functionality, transplantability and sustainability of hPSC-derived TEPCs, we strive to develop an experimental model that can be translated by a clinical Good Manufacturing Practice (cGMP) facility for use in bone marrow transplant recipients.Ultimately, the potential applications of in vitro derived TEPCs are not confined to restoring thymic function in the HSCT setting. TEPCs derived from solid organ donors hold the promise of inducing tolerance to donor tissues, thereby preventing rejection of the transplanted donor organ. Leveraging the body's own ability to educate T-cells through regenerating thymic tissues could represent a novel approach to addressing key challenges in transplantation immunology. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Diagnosis and management of Intratyroid Ectopic Thymus.

Intrathyroidal ectopic thymus (IET) is a rare benign condition caused by the aberrant thymic migra tion during embryogenesis. It is usually incidentally diagnosed as a thyroid nodule. To report the intrathyroidal location of ectopic thymic tissue and to describe the ultrasound findings in children. Retrospective descriptive review of the medical charts and thyroid ultrasound studies of children with nodular images in the thyroid gland, in a third level national pediatric hospital, from January 2010 to August 2017. Solid hypoecogenic intrathyroid lesions with multiple linear tracts or hyperechogenic points that did not change their characteristics during fo llow-up were considered intrathyroidal thymos. The ultrasound follow-up was performed every 4-6 months. The ultrasound characteristics of the lesions (location, laterality, size and shape), the indi cation of the ultrasound scan and the follow-up time were analyzed. Of 147 patients with thyroid nodules, we identified 12 children with lesions suggestive of an IET (8.1%). The mean age at diagnosis was 3.9 years (range 0-8). It was an incidental finding in all cases. Imaging findings were unilateral in eight patients and bilateral in four patients. All lesions were located in the mid and/or posterior portion of the gland. We adopted a watch-and-wait approach with ultrasound follow-up (mean 2.2 years; range 0.83-4) in all patients except in a 7-year-old boy who presented uncertain findings and underwent surgery, confirming IET in the pathological study. Thymic inclu sions in the thyroid gland are a rare but increasingly frequent finding, possibly related to the increased use of ultrasound studies. Pediatricians and radiologists should be aware of this entity to differentiate it from other thyroid lesions, avoiding unnecessary studies and/or treatments in these patients.

Preconceptional Immunization Can Modulate Offspring Intrathymic IL-17-Producing γδT Cells with Epigenetic Implications Mediated by microRNAs.

The mechanisms through which maternal immunization can modulate offspring thymic maturation of lymphocytes are not fully understood. Here, we aimed to evaluate whether maternal OVA-immunization can inhibit the maturation of IL-17-producing γδT cells in offspring thymus, and if this mechanism has epigenetic implications mediated by microRNAs (miRNAs) expression. Wild-type (WT) C57BL/6 females were immunized with OVA in Alum or Alum alone and were mated with normal WT males. Evaluating their offspring thymus at 3 or 20 days old (d.o.), we observed that maternal OVA immunization could inhibit the thymic frequency of offspring CD27- and IL-17+ γδT cells at the neonatal and until 20 days old. Furthermore, we evaluated the expression of function-related γ and δ variable γδTCR chains (Vγ1, Vγ2, Vγ3, Vδ4, and Vδ6.3), observing that maternal OVA-immunization inhibits Vγ2 chains expression. The small RNAs (sRNAs), particularly miRNAs, and messenger RNAs (mRNA) expression profiles by pools of thymus tissue samples (from 9 to 11 mice) from offspring OVA-immunized or Alum-immunized mothers were analyzed via Illumina sequencing platform and bioinformatics approaches. Using a fold change >4, our results showed that seven miRNAs (mmu-miR-126a-3p, 101a-3p, 744-3p,142-5p, 15a-5p, 532-5p, and 98-5p) were differentially expressed between both groups. Ten target genes were predicted to interact with the seven selected miRNAs. There were no enriched categories of gene ontology functional annotation and pathway enrichment analysis for the target genes. Interestingly, four of the identified miRNAs (mmu-miR-15a, mmu-miR-101 mmu-miR-126, and mmu-miR-142) are related to IL-17 production. Our data is of significance because we demonstrate that maternal immunization can modulate offspring thymic maturation of IL-17-producing γδT cells possibly by an epigenetic mechanism mediated by miRNAs.

Natural infection of covert mortality nodavirus affects Zebrafish (Danio rerio)

Covert mortality nodavirus (CMNV), a novel aquatic pathogen, causes viral covert mortality disease (VCMD) in shrimps and also known to infect farmed marine fish. To date, there has no report regarding the ability of this virus to infect freshwater fish. In this study, we screened and discovered CMNV-positive freshwater zebrafish individuals by reverse transcription-nested PCR (RT-nPCR). The sequence of CMNV amplicons from zebrafish was found to share 99% identity with RNA-dependent RNA polymerase (RdRp) gene of the original CMNV isolate. Histopathological examination of the CMNV-positive zebrafish samples revealed extensive vacuolation and karyopyknosis lesions in the retina of the eye and the midbrain mesencephalon. CMNV-like virus particles were visualized in these tissues under transmission electron microscope. Different degrees of pathological damages were also found in muscle, gills, thymus and ovarian tissues. Strong positive signals of CMNV probe were observed in these infected tissues by in situ hybridization. Overall, all results indicated that zebrafish, an acknowledged model organism, could be infected naturally by CMNV. Thus, it is needed to pay close attention to the possible interference of CMNV whether in assessment of toxic substances, or in studying the developmental characterization and the nerval function, when zebrafish was used as model animal.

Exosome-Derived MicroRNAs of Human Milk and Their Effects on Infant Health and Development

Multiple biologically active components of human milk support infant growth, health and development. Milk provides a wide spectrum of mammary epithelial cell-derived extracellular vesicles (MEVs) for the infant. Although the whole spectrum of MEVs appears to be of functional importance for the growing infant, the majority of recent studies report on the MEV subfraction of milk exosomes (MEX) and their miRNA cargo, which are in the focus of this review. MEX and the dominant miRNA-148a play a key role in intestinal maturation, barrier function and suppression of nuclear factor-κB (NF-κB) signaling and may thus be helpful for the prevention and treatment of necrotizing enterocolitis. MEX and their miRNAs reach the systemic circulation and may impact epigenetic programming of various organs including the liver, thymus, brain, pancreatic islets, beige, brown and white adipose tissue as well as bones. Translational evidence indicates that MEX and their miRNAs control the expression of global cellular regulators such as DNA methyltransferase 1—which is important for the up-regulation of developmental genes including insulin, insulin-like growth factor-1, α-synuclein and forkhead box P3—and receptor-interacting protein 140, which is important for the regulation of multiple nuclear receptors. MEX-derived miRNA-148a and miRNA-30b may stimulate the expression of uncoupling protein 1, the key inducer of thermogenesis converting white into beige/brown adipose tissue. MEX have to be considered as signalosomes derived from the maternal lactation genome emitted to promote growth, maturation, immunological and metabolic programming of the offspring. Deeper insights into milk’s molecular biology allow the conclusion that infants are both “breast-fed” and “breast-programmed”. In this regard, MEX miRNA-deficient artificial formula is not an adequate substitute for breastfeeding, the birthright of all mammals.

Gamma-irradiation fluctuates the mRNA N6-methyladenosine (m6A) spectrum of bone marrow in hematopoietic injury

Humans benefit from nuclear technologies but consequently experience nuclear disasters or side effects of iatrogenic radiation. Hematopoietic system injury first arises upon radiation exposure. As an intricate new layer of genetic control, the posttranscriptional m6A modification of RNA has recently come under investigation and has been demonstrated to play pivotal roles in multiple physiological and pathological processes. However, how the m6A methylome functions in the hematopoietic system after irradiation remains ambiguous. Here, we uncovered the time-varying epitranscriptome-wide m6A methylome and transcriptome alterations in γ-ray-exposed mouse bone marrow. 4 Gy γ-irradiation rapidly (5 min and 2 h) and severely impaired the mouse hematopoietic system, including spleen and thymus weight, blood components, tissue inflammation and malondialdehyde (MDA) levels. The m6A content and expression of m6A related enzymes were altered. Gamma-irradiation triggered dynamic and reversible m6A modification profiles and altered mRNA expression, where both m6A fold-enrichment and mRNA expression most followed the (5 min_up/2 h_down) pattern. The CDS enrichment region preferentially upregulated m6A peaks at 5 min. Moreover, the main GO and KEGG pathways were closely related to metabolism and the classical radiation response. Finally, m6A modifications correlated with transcriptional regulation of genes in multiple aspects. Blocking the expression of m6A demethylases FTO and ALKBH5 mitigated radiation hematopoietic toxicity. Together, our findings present the comprehensive landscape of mRNA m6A methylation in the mouse hematopoietic system in response to γ-irradiation, shedding light on the significance of m6A modifications in mammalian radiobiology. Regulation of the epitranscriptome may be exploited as a strategy against radiation damage.

A spectrum of Thymic mucosa-associated lymphoid tissue lymphoma and Thymic amyloidosis in the patient with Auto immune disease: a case series.

BackgroundThe thymus is associated with an immunodeficient status, autoimmune disease (AD), and the common thymic tumor, thymoma. We encountered two rare thymic tumors, thymic mucosa-associated lymphoid tissue (MALT) lymphoma and localized thymic amyloidosis, both in the presence of Sjögren’s syndrome (SjS). This suggests a possible link between rare thymic tumors and SjS. Therefore, we reviewed cases of thymic tumors to examine the spectrum of these tumors in patients with AD.MethodsThe clinical information of thymic amyloidosis and MALT lymphoma surgically treated at Kanagawa Cardiovascular and Respiratory Center, and Yokohama City University Hospital from January 2010 to December 2019 were reviewed. The correlation between resected thymic tumors at same period and ADs were also investigated.ResultsThere were 5 cases of thymic amyloidosis and MALT lymphoma. ALL cases had coexistent ADs (4 SjS, 1 SSc). The median age was 66 (38–76) year-old, and 4 of the patients were female. Three cases had already diagnosed as ADs before detection of tumors. Only SSc case was received preceding steroid medication. Two cases diagnosed as SjS at the same time of the operation. The median maximum tumor diameter was 70 mm. On chest computed tomography (CT), tumors contained solid part and some cystic part at various rated. Calcification was recognized with appearance of amyloid deposition. All patients were surgically treated with total thymectomy and they are alive without recurrence. At the same period, there were 163 resected thymic tumors, including amyloidosis, MALT lymphoma, thymoma, thymic cancer, neuroendocrine tumor and so on. Among them, nine patients (5.5%) had ADs. There was a correlation between ADs and thymic MALT lymphoma/amyloidosis (P<0.001).ConclusionsWe propose a process for tumorigenesis of thymic MALT lymphoma and amyloidosis. Underlying AD causes persistent and chronic inflammatory reactions. In this theory, ADs, especially SjS, might be important underlying conditions in formation of rare tumors. When the clinician encounters a patient with AD, routine chest CT is recommended and may provide thymic tumors. Conversely, in case of mediastinum tumor, screening test for AD is also recommended.

Premature Senescence and Increased Oxidative Stress in the Thymus of Down Syndrome Patients.

Down syndrome (DS) patients prematurely show clinical manifestations usually associated with aging. Their immune system declines earlier than healthy individuals, leading to increased susceptibility to infections and higher incidence of autoimmune phenomena. Clinical features of accelerated aging indicate that trisomy 21 increases the biological age of tissues. Based on previous studies suggesting immune senescence in DS, we hypothesized that induction of cellular senescence may contribute to early thymic involution and immune dysregulation. Immunohistochemical analysis of thymic tissue showed signs of accelerated thymic aging in DS patients, normally seen in older healthy subjects. Moreover, our whole transcriptomic analysis on human Epcam-enriched thymic epithelial cells (hTEC), isolated from three DS children, which revealed disease-specific transcriptomic alterations. Gene set enrichment analysis (GSEA) of DS TEC revealed an enrichment in genes involved in cellular response to stress, epigenetic histone DNA modifications and senescence. Analysis of senescent markers and oxidative stress in hTEC and thymocytes confirmed these findings. We detected senescence features in DS TEC, thymocytes and peripheral T cells, such as increased β-galactosidase activity, increased levels of the cell cycle inhibitor p16, telomere length and integrity markers and increased levels of reactive oxygen species (ROS), all factors contributing to cellular damage. In conclusion, our findings support the key role of cellular senescence in the pathogenesis of immune defect in DS while adding new players, such as epigenetic regulation and increased oxidative stress, to the pathogenesis of immune dysregulation.

Isolation and expansion of thymus-derived regulatory T cells for use in pediatric heart transplant patients.

Regulatory T-cells (Tregs) are a subset of T cells generated in the thymus with intrinsic immunosuppressive properties. Phase I clinical trials have shown safety and feasibility of Treg infusion to promote immune tolerance and new studies are ongoing to evaluate their efficacy. During heart transplantation, thymic tissue is routinely discarded providing an attractive source of Tregs. In this study, we developed a GMP-compatible protocol for expanding sorted thymus-derived CD3+ CD4+ CD25+ CD127- (Tregs) as well as CD3+ CD4+ CD25+ CD127- CD45RA+ (RA+ Tregs) cells. We aimed to understand whether thymic RA+ Tregs can be isolated and expanded offering an advantage in terms of stability as it has been previously shown for circulating adult CD45RA+ Tregs. We show that both Tregs and RA+ Tregs could be expanded in large numbers and the presence of rapamycin is essential to inhibit the growth of IFN-γ producing cells. High levels of FOXP3, CTLA4, and CD25 expression, demethylation of the FOXP3 promoter, and high suppressive ability were found with no differences between Tregs and RA+ Tregs. After freezing and thawing, all Treg preparations maintained their suppressive ability, stability, as well as CD25 and FOXP3 expression. The number of thymic Tregs that could be isolated with our protocol, their fold expansion, and functional characteristics allow the clinical application of this cell population to promote tolerance in pediatric heart transplant patients.

Care of Children with DiGeorge Before and After Cultured Thymus Tissue Implantation

BackgroundChildren with complete DiGeorge anomaly (cDGA) have congenital athymia plus a myriad of other challenging clinical conditions. The term cDGA encompasses children with congenital athymia secondary to 22q11.2DS, CHARGE syndrome (coloboma, heart defects, choanal atresia, growth or mental retardation, genital abnormalities, and ear abnormalities and/or deafness), and other genetic abnormalities. Some children have no known genetic defects. Since 1993, more than 100 children with congenital athymia have been treated with cultured thymus tissue implantation (CTTI). Naïve T cells develop approximately 6 to 12 months after CTTI. Most of the children had significant comorbidities such as heart disease, hypoparathyroidism, and infections requiring complex clinical care post cultured thymus tissue implantation (CTTI).ObjectiveThe purpose of this guidance is to assist multidisciplinary teams in caring for children with cDGA both before and after CTTI.MethodsThirty-one specialists, in addition to the authors, were asked to share their experience in caring for children with cDGA at Duke University Health System, before and after CTTI. These specialists included physicians, nurses, dentists, therapists, and dieticians.ResultsThe goal of a multidisciplinary approach is to have children in the best possible condition for receiving CTTI and provide optimal care post CTTI through development of naïve T cells and beyond. The CTT (cultured thymus tissue) must be protected from high doses of steroids which can damage CTT. Organs must be protected from adverse effects of immunosuppression.ConclusionCreating a multidisciplinary team and a detailed plan of care for children with cDGA is important for optimal outcomes.

Adenomatous, Unilateral, Retrosternal Extension of the Thyroid Gland presenting as a Mediastinal Mass

Background: Thyroid-related causes of mediastinal masses include retrosternal goiters and thymic enlargement associated with Graves’ disease. Here, we present a case of significant unilateral retrosternal growth of the thyroid gland, presenting as an incidental mediastinal mass, without any evidence of contralateral disease. Associated subclinical hyperthyroidism presents a therapeutic challenge. Clinical Case: A 68-year old gentleman presented to the emergency department with a non-infective exacerbation of known Chronic Obstructive Pulmonary Disease. CT Pulmonary Angiogram revealed a right sided 5.6cm paratracheal mass, which seemed to originate from the posterior aspect of the right lobe of the thyroid and extended to the subcarinal region. The mass displaced the oesophagus and was close to, but did not compress, the trachea. The left thyroid lobe was normal. Thyroid ultrasound was reported as normal, but was later acknowledged to be suboptimal at visualization of the posterior aspect of the gland. Thyroid scintigraphy confirmed increased radionuclide uptake within the mass. Thyroid function tests showed subclinical thyrotoxicosis [TSH 0.04 (0.4-4.0mIU/mL), FT4 17.4 (10-22pmol/L) and FT3 3.36 (2.89-4.88 pmol/L)]. TSH receptor antibody was negative [< 1.1 (<1.75 IU/L)]. On review of prior imaging from other hospitals, the mass had been present since 1999 and was stable in size for at least the past 7 years. The patient was discharged on carbimazole with a plan to perform interval scanning to monitor size. Discussion: Although technetium uptake can be seen in thymus tissue, the identification on imaging that the mass is contiguous with the thyroid gland leads us to believe this is an adenomatous extension of the thyroid gland. Ectopic thyroid tissue is possible, but the size of the mass suggests some prior period of growth. Thyroid carcinoma seems unlikely given the current stability in size, and there are no compressive symptoms, so there is no clear indication for surgery at present. However, given the subnormal TSH level, there is evidence of autonomy, so treatment is indicated. Radio-iodine treatment may be associated with thyroiditis, with attendant swelling of the gland and risk of compression of vital structures, so treatment with ATDs and regular imaging surveillance was deemed most appropriate in his case.

Atypical Carcinoid Tumor of the Mediastinum Presenting as Ectopic Cushing’s Syndrome

Introduction: Ectopic Cushing’s syndrome (ECS) refers to Cushing’s syndrome (CS) that results from extra-pituitary secretion of adrenocorticotropic hormone (ACTH). Neuroendocrine tumors (NETs) are a rare cause of ECS, and their presence in the anterior mediastinum only represents 2-4% of all cases. We report a rare case of ECS in a patient with an atypical mediastinal carcinoid tumor.Case Presentation: A 38-year-old male with a history of depression presented with generalized weakness, fatigue, and myalgia for one month. A one-week history of nausea and slight shortness of breath was reported. Physical exam and vital signs were unremarkable. Labs revealed hyperglycemia, hypokalemia, and metabolic alkalosis. A random serum cortisol was 47.7 mcg/dL (normal: 3-21 mcg/dL) and a 24-hour urine free cortisol was 7405 mcg/24 hours (normal: 3.5-45 mcg/24 hours). The morning serum cortisol after 8 mg dexamethasone at midnight, was 58.7 mcg/dL. Serum aldosterone and plasma renin activity were both undetectable. CT of the chest showed an anterior mediastinal mass measuring 9.0 x 10.3 x 6.1 cm accompanied by two large mediastinal lymph nodes. A PET CT confirmed the anterior mediastinal mass, which showed mild heterogeneous uptake and no evidence of metastatic disease. Core biopsy of mediastinal mass showed grade II neuroendocrine tumor/atypical carcinoid. Potassium, Spironolactone, Ketoconazole, and Metyrapone were used to control hypokalemia and hypercortisolemia. Trimethoprim-sulfamethoxazole (TMP-SMX) was started for Pneumocystis pneumonia prophylaxis. The patient underwent complete resection of the anterior mediastinal mass, thymectomy, and mediastinal lymph nodes dissection. Histopathology confirmed an atypical carcinoid in the mediastinal mass with negative margins. The rest of the thymus tissue and lymph nodes were negative for malignancy. ACTH and cortisol levels dropped significantly after surgery and the patient was discharged on oral hydrocortisone and TMP-SMX. At 4 weeks follow-up in the clinic, the patient was feeling much better and reported complete resolution of symptoms.Discussion: ECS caused by an atypical carcinoid tumor is an extremely rare finding. Mediastinal NETs tend to have a poor prognosis due to their metastatic tendency and recurrence rates. Due to limited evidence in the medical literature, there’s much uncertainty and controversy regarding the best treatment of NETs. Standardized guidelines for the management of these atypical tumors are not well established. The primary mode of treatment is surgical resection of the ACTH-secreting tumor. However, in cases of advanced and metastatic tumors, surgical resection might not be sufficient and medical therapy with adrenal enzyme synthesis inhibitors may be needed. The use of neoadjuvant chemotherapy and/or radiotherapy remains unclear.

Non-atopic Neonatal Thymic Innate Lymphoid Cell Subsets (ILC1, ILC2, and ILC3) Identification and the Modulatory Effect of IgG From Dermatophagoides Pteronyssinus (Derp)-Atopic Individuals.

Innate lymphoid cells (ILCs) are classified into distinct subsets termed ILC1, ILC2, and ILC3 cells. The existing literature lacks evidence identifying ILCs and their subsets in the human thymus but already demonstrates that they can exert several functions in regulating immune responses. Furthermore, it was already described that IgG's repertoires could modulate lymphocytes' maturation in the human thymus. Here we aimed to identify ILCs subsets in the human thymus and provide insight into the possible modulatory effect of purified IgG on these cells. Thymic tissues were obtained from 12 infants without an allergic background (non-atopic), and a literature-based peripheral ILCs staining protocol was used. Purified IgG was obtained from non-atopic individuals (n-At), atopic individuals reactive to allergens non-related to dust mites (nr-At), and atopic individuals reactive to the mite Dermatophagoides pteronyssinus (Derp-At). As with all tissues in which they have already been detected, thymic ILCs are rare, but we could detect viable ILCs in all tested tissues, which did not occur with the ILC1 subset. ILC2 and ILC3 NKp44+ subsets could be detected in all evaluated thymus, but ILC3 NKp44- subset could not. Next, we observed that Derp-At IgG could induce the expression of ILC2 phenotype, higher levels of IL-13, and lower levels of IL-4 when compared to IgG purified from non-atopic or non-related atopic (atopic to allergens excluding dust mites) individuals. These results contribute to the elucidation of human thymic ILCs and corroborate emerging evidence about IgG's premature effect on allergy development-related human lymphocytes' modulation.

GAD1 expression and its methylation as indicators of malignant behavior in thymic epithelial tumors.

Thymic epithelial tumors (TETs) comprise thymomas and thymic carcinoma (TC). TC has more aggressive features and a poorer prognosis than thymomas. Genetic and epigenetic alterations in thymomas and TC have been investigated in an attempt to identify novel target molecules for TC. In the present study, genome-wide screening was performed on aberrantly methylated CpG islands in thymomas and TC, and the glutamate decarboxylase 1 gene (GAD1) was identified as the 4th significantly hypermethylated CpG island in TC compared with thymomas. GAD1 catalyzes the production of γ-aminobutyric acid from L-glutamic acid. GAD1 expression is abundant in the brain but rare in other tissues, including the thymus. A total of 73 thymomas and 17 TC tissues were obtained from 90 patients who underwent surgery or biopsy at Tokushima University Hospital between 1990 and 2017. DNA methylation was examined by bisulfite pyrosequencing, and the mRNA and protein expression levels of GAD1 were analyzed using reverse transcription-quantitative PCR and immunohistochemistry, respectively. The DNA methylation levels of GAD1 were significantly higher in TC tissues than in the normal thymus and thymoma tissues, and GAD1 methylation exhibited high sensitivity and specificity for discriminating between TC and thymoma. The mRNA and protein expression levels of GAD1 were significantly higher in TC tissues than in thymomas. Patients with TET with high GAD1 DNA hypermethylation and high mRNA and protein expression levels had significantly shorter relapse-free survival rates than those with low levels. In conclusion, significantly more epigenetic alterations were observed in TC tissues compared with in thymomas, which may contribute to the clinical features and prognosis of patients.

Inflammatory response of thymus bystander effects on acute radiation-induced skin injury in rats

Background: Radiation not only kills tumor cells, but also damages other sites. The mechanisms of damage caused by the bystander effect of irradiation in animal models are unclear and the time node is single. In this study, we aimed to investigate the inflammatory response of thymus tissue injury in non-irradiated areas at different times after irradiating rat skin. Materials and Methods: Rats were irradiated with an X-ray dose of 38 Gy, and at 15 d after irradiation, when the skin wound was most severe, the pro-inflammatory drug high mobility group box1 (HMGB1) and the anti-inflammatory drug glycyrrhizic acid (GA) were injected intraperitoneally into rats. After irradiation, skin tissues were collected for histology, and thymus tissues were collected for gene and protein testing. Results: Animal model of skin damage was successfully established. The expression of macrophage (F4/80) increased after irradiation, and F4/80 produced cytokines. Through the flow which was activated by inflammatory factors in the blood, DNA damage and the expression of inflammatory-related cytokines in non-irradiated area of the thymus peaked at 15 d after irradiation. Moreover, HMGB1 treatment increased the expression at 1 d after intraperitoneal injection, and GA solution decreased the expression of inflammatory-related cytokines. Conclusion: When radiation damages the skin, it can cause damage to other organs through the circulation, and an anti-inflammatory GA solution reduced inflammatory responses, which are required to modify radiation-induced systemic effects with anti-inflammatory drugs or agents that affect pathways that cause bystander instability.

Chronic Running Exercise Regulates Cytotoxic Cell Functions and Zinc Transporter SLC39A10/ZIP10 Levels in Diabetic Rats.

The aim of this study is to investigate how chronic running exercise affects ZIP10 levels in thymus and spleen tissue as well as immune parameters in diabetic rats. A total of 40 adult male Wistar rats were divided into 4 equal groups: group 1, control; group 2, exercise control; group 3, diabetes; group 4, diabetes + exercise. Diabetes was induced by injecting intraperitoneal streptozotocin (STZ) at a dose of 40 mg/kg twice with 24-h intervals to the animals in groups 3 and 4. The animals in group 2 and group 4 underwent exercise for 45 min on the rat treadmill for 4 weeks at 20 m/min. Twenty-four hours after the last running exercise, the animals were sacrificed under general anesthesia. Immunological parameters were determined by flow cytometric method; tissue ZIP 10 levels were determined by ELISA method. The diabetic group had the lowest natural killer (NK) and natural killer T (NKT) cells percentages. Chronic exercise partially improved NK and NKT cell percentages in diabetic rats. The diabetic group had the lowest ZIP10 levels in spleen and thymus tissue. ZIP10 values in spleen and thymus tissue of diabetes exercise group were significantly higher than diabetes group. The results of our study show that the impaired cytotoxic cell functions in diabetes are partially corrected with 4 weeks of chronic exercise, and that the suppressed ZIP 10 levels in diabetic rats are reversed by 4 weeks of chronic exercise.

Current and Future Therapeutic Approaches for Thymic Stromal Cell Defects.

Inborn errors of thymic stromal cell development and function lead to impaired T-cell development resulting in a susceptibility to opportunistic infections and autoimmunity. In their most severe form, congenital athymia, these disorders are life-threatening if left untreated. Athymia is rare and is typically associated with complete DiGeorge syndrome, which has multiple genetic and environmental etiologies. It is also found in rare cases of T-cell lymphopenia due to Nude SCID and Otofaciocervical Syndrome type 2, or in the context of genetically undefined defects. This group of disorders cannot be corrected by hematopoietic stem cell transplantation, but upon timely recognition as thymic defects, can successfully be treated by thymus transplantation using cultured postnatal thymic tissue with the generation of naïve T-cells showing a diverse repertoire. Mortality after this treatment usually occurs before immune reconstitution and is mainly associated with infections most often acquired pre-transplantation. In this review, we will discuss the current approaches to the diagnosis and management of thymic stromal cell defects, in particular those resulting in athymia. We will discuss the impact of the expanding implementation of newborn screening for T-cell lymphopenia, in combination with next generation sequencing, as well as the role of novel diagnostic tools distinguishing between hematopoietic and thymic stromal cell defects in facilitating the early consideration for thymus transplantation of an increasing number of patients and disorders. Immune reconstitution after the current treatment is usually incomplete with relatively common inflammatory and autoimmune complications, emphasizing the importance for improving strategies for thymus replacement therapy by optimizing the current use of postnatal thymus tissue and developing new approaches using engineered thymus tissue.

Ectopic Cervical Thymoma: An Uncommon Entity.

Thymomas originates from the epithelial cells of thymic tissue. These tumors commonly occur in the anterior mediastinum. However, thymomas can arise anywhere from the aberrant or remnant thymus in the neck or mediastinum. Ectopic cervical thymoma is a rare disease. It is important to be aware that cervical masses can also be thymomas rather than thyroid or parathyroid masses. Here, we present a case of a 62-year-old female who presented with swelling in the neck and was diagnosed with cervical thymoma type AB.

Thymus Extracellular Matrix-Derived Scaffolds Support Graft-Resident Thymopoiesis and Long-Term In Vitro Culture of Adult Thymic Epithelial Cells.

The thymus provides the physiological microenvironment critical for the development of T lymphocytes, the cells that orchestrate the adaptive immune system to generate an antigen‐specific response. A diverse population of stroma cells provides surface‐bound and soluble molecules that orchestrate the intrathymic maturation and selection of developing T cells. Forming an intricate 3D architecture, thymic epithelial cells (TEC) represent the most abundant and important constituent of the thymic stroma. Effective models for in and ex vivo use of adult TEC are still wanting, limiting the engineering of functional thymic organoids and the understanding of the development of a competent immune system. Here a 3D scaffold is developed based on decellularized thymic tissue capable of supporting in vitro and in vivo thymopoiesis by both fetal and adult TEC. For the first time, direct evidences of feasibility for sustained graft‐resident T‐cell development using adult TEC as input are provided. Moreover, the scaffold supports prolonged in vitro culture of adult TEC, with a retained expression of the master regulator Foxn1. The success of engineering a thymic scaffold that sustains adult TEC function provides unprecedented opportunities to investigate thymus development and physiology and to design and implement novel strategies for thymus replacement therapies.