Shey Cherng Tzou

Novel autoantigens in autoimmune hypophysitis

Background  Pituitary autoantibodies are found in autoimmune hypophysitis and other conditions. They are a marker of pituitary autoimmunity but currently have limited clinical value. The methods used for their detection lack adequate sensitivity and specificity, mainly because the pathogenic pituitary autoantigen(s) are not known and therefore antigen‐based immunoassays have not been developed.

Autoimmune hypophysitis of SJL mice: clinical insights from a new animal model.

Autoimmune hypophysitis (AH) is a rare but increasingly recognized disease of the pituitary gland. Its autoantigens are unknown, and the management is difficult because it is often misdiagnosed as a nonsecreting adenoma. By immunizing female SJL/J mice with mouse pituitary extracts, we established a new mouse model of experimental AH. Immunized mice developed severe lymphocytic infiltration in the anterior pituitary that closely mimicked the human pathology. In the early phase of experimental AH, the pituitary enlarged, consistent with the compression symptoms reported by hypophysitis patients at presentation. In the florid phase, adrenal insufficiency and pituitary antibodies developed, in strong correlation with the pituitary pathology. In the late phase, hypothyroidism ensued, and the pituitary gland became atrophic. Using immune sera as probes in a two-dimensional immunoblotting screen followed by mass spectrometry, we identified several proteins that could function as pituitary autoantigens. These findings provide new insights into the pathogenesis of AH, and establish a platform for developing novel diagnostic biomarkers and therapeutics.

Immunoproteasome Overexpression Underlies the Pathogenesis of Thyroid Oncocytes and Primary Hypothyroidism: Studies in Humans and Mice

Background Oncocytes of the thyroid gland (Hürthle cells) are found in tumors and autoimmune diseases. They have a unique appearance characterized by abundant granular eosinophilic cytoplasm and hyperchromatic nucleus. Their pathogenesis has remained, thus far, unknown. Methodology/Principal Findings Using transgenic mice chronically expressing IFNγ in thyroid gland, we showed changes in the thyroid follicular epithelium reminiscent of the human oncocyte. Transcriptome analysis comparing transgenic to wild type thyrocytes revealed increased levels of immunoproteasome subunits like LMP2 in transgenics, suggesting an important role of the immunoproteasome in oncocyte pathogenesis. Pharmacologic blockade of the proteasome, in fact, ameliorated the oncocytic phenotype. Genetic deletion of LMP2 subunit prevented the development of the oncocytic phenotype and primary hypothyroidism. LMP2 was also found expressed in oncocytes from patients with Hashimoto thyroiditis and Hürthle cell tumors. Conclusions/Significance In summary, we report that oncocytes are the result of an increased immunoproteasome expression secondary to a chronic inflammatory milieu, and suggest LMP2 as a novel therapeutic target for the treatment of oncocytic lesions and autoimmune hypothyroidism.

Anatabine Ameliorates Experimental Autoimmune Thyroiditis

Tobacco smoking favorably influences the course of Hashimoto thyroiditis, possibly through the antiinflammatory proprieties of nicotine. In this study we tested anatabine, another tobacco alkaloid, in a model of experimental autoimmune thyroiditis. Experimental autoimmune thyroiditis was induced by different doses of thyroglobulin, to produce a disease of low, moderate, or high severity, in 88 CBA/J female mice: 43 drank anatabine supplemented water and 45 regular water. Mice were bled after immunization and killed to assess thyroid histopathology, thyroglobulin antibodies, T(4), and thyroid RNA expression of 84 inflammatory genes. We also stimulated in vitro a macrophage cell line with interferon-γ or lipopolysaccharide plus or minus anatabine to quantitate inducible nitric oxide synthase and cyclooxygenase 2 protein expression. Anatabine reduced the incidence and severity of thyroiditis in the moderate disease category: only 13 of 21 mice (62%) developed thyroid infiltrates when drinking anatabine as compared with 22 of 23 (96%) controls (relative risk 0.59, P = 0.0174). The median thyroiditis severity was 0.5 and 2.0 in anatabine and controls, respectively (P = 0.0007 by Wilcoxon rank sum test). Anatabine also reduced the antibody response to thyroglobulin on d 14 (P = 0.029) and d 21 (P = 0.045) after immunization and improved the recovery of thyroid function on d 21 (P = 0.049). In the thyroid transcriptome, anatabine restored expression of IL-18 and IL-1 receptor type 2 to preimmunization levels. Finally, anatabine suppressed in a dose-dependent manner macrophage production of inducible nitric oxide synthase and cyclooxygenase 2. Anatabine ameliorates disease in a model of autoimmune thyroiditis, making the delineation of its mechanisms of action and potential clinical utility worthwhile.

Pituitary and systemic autoimmunity in a case of intrasellar germinoma

Germinomas arising in the sella turcica are difficult to differentiate from autoimmune hypophysitis because of similar clinical and pathological features. This differentiation, nevertheless, is critical for patient care due to different treatments of the two diseases. We report the case of an 11-year-old girl who presented with diabetes insipidus and growth retardation, and was found to have an intra- and supra-sellar mass. Initial examination of the pituitary biopsy showed diffuse lymphocytic infiltration of the adenohypophysis and absent placental alkaline phosphatase expression, leading to a diagnosis of hypophysitis and glucocorticoid treatment. Because of the lack of clinical and radiological response, the pituitary specimen was re-examined, revealing this time the presence of scattered c-kit and Oct4 positive germinoma cells. The revised diagnosis prompted the initiation of radiotherapy, which induced disappearance of the pituitary mass. Immunological studies showed that the patient’s serum recognized antigens expressed by the patient’s own germinoma cells, as well as pituitary antigens like growth hormone and systemic antigens like the Sjögren syndrome antigen B and alpha-enolase. The study first reports the presence of pituitary and systemic antibodies in a patient with intrasellar germinoma, and reminds us that diffuse lymphocytic infiltration of the pituitary gland and pituitary antibodies does not always indicate a diagnosis of autoimmune hypophysitis.

From Pituitary Expansion to Empty Sella: Disease Progression in a Mouse Model of Autoimmune Hypophysitis

Lymphocytic hypophysitis has a variable clinical course, where a swelling of the pituitary gland at presentation is thought to be followed by pituitary atrophy and empty sella. Data in patients, however, are scanty and contradictory. To better define the course of hypophysitis, we used an experimental model based on the injection of pituitary proteins into SJL mice. A cohort of 33 mice was divided into three groups: 18 cases were immunized with pituitary proteins emulsified in complete Freunds adjuvant; six controls were injected with adjuvant only; and nine controls were left untreated. Mice were followed by cranial magnetic resonance imaging (MRI) for up to 300 d, for a total of 106 MRI scans, and killed at different time points to correlate radiological and pathological findings. Empty sella was defined as a reduction in pituitary volume greater than 2 sd below the mean volume. All immunized mice showed by MRI a significant expansion of pituitary volume during the early phases of the disease. The volume then decreased gradually in the majority of cases (14 of 18, 78%), reaching empty sella values by d 300 after immunization. In a minority of cases (four of 18, 22%), the decrease was so rapid and marked to induce a central area of necrosis accompanied by hemorrhages, mimicking the condition known in patients as pituitary apoplexy. No radiological or pathological changes were observed in controls. Overall, these findings indicate that the evolution of hypophysitis is complex but can lead, through different routes, to the development of empty sella.

Toll-like receptor-MyD88 and Fc receptor pathways of mast cells mediate the thyroid dysfunctions observed during nonthyroidal illness

Bacterial infections and other pathologic conditions induce complex dysfunctions of the hypothalamic–pituitary–thyroid axis, collectively known as nonthyroidal illness (NTI). To explore the pathogenesis of bacterial NTI, we injected Mycobacterium tuberculosis extracts or Escherichia coli LPS in mice lacking key components of the Toll-like receptor or crystallizable fragment (Fc) receptor pathways. In wild-type mice, the bacterial components induced a hypothyroidism characterized by elements of both hypothalamic and thyroidal dysfunction. This NTI hypothyroidism did not develop in mice lacking the MyD88 adaptor or in those with a reduced number of mast cells. The hypothyroid responsiveness to LPS, however, was restored upon reconstitution with mast cells derived from the bone marrow of wild-type donors. In addition to bacterial components, whole immunoglobulins induced NTI hypothyroidism in wild-type mice, but not in those lacking activating Fc receptors or mast cells. The study demonstrates a link between Toll-like and Fc receptor signaling and thyroid gland function, uncovering a role of mast cells in murine NTI.

Studies on Murine Thyroiditis: New Insights from Organ Flow Cytometry

The pathogenesis of autoimmune diseases is frequently studied in murine models, in which disease outcome is traditionally assessed by light microscopy. To determine whether digital imaging improves reliability of the histopathologic assessment, and whether flow cytometry is applicable directly on the murine thyroid, we studied 395 CBA/J mice 3 weeks after thyroglobulin immunization, and 192 nonimmunized CBA/J mice. Digital imaging significantly improved reliability of the histopathological assessment (r = 0.988, 95% confidence interval: 0.980-0.992, p < 0.0001), and flow cytometry on the murine thyroid could be performed successfully. We also found that normal thyroids contained a higher than expected number of hematopoietic cells in the interstitium. We suggest that digital imaging offers a better means of estimating disease outcome, and that flow cytometry performed at the target organ levels reflects the autoimmune pathogenesis more closely than when performed on peripheral lymphoid organs. These methods should also be applicable to other organ systems targeted by autoimmune attack, such as heart, exocrine, and other endocrine glands.

Autoimmune hypophysitis: expanding the differential diagnosis to CTLA-4 blockade

Autoimmune hypophysitis is an increasingly recognized disorder that enters in the differential diagnosis of nonfunctioning pituitary masses. The differential diagnosis of these conditions is challenging because of similar clinical presentations and radiological signs. This review describes the essential features of hypophysitis and the other nonfunctioning pituitary masses. It also emphasizes a recently described feature of hypophysitis: its appearance with unexpectedly high frequency in patients receiving treatments that abrogate the function of cytotoxic T lymphocyte antigen 4.

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice(1). Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin(2), and those interested in myasthenia gravis inject them with the acetylcholine receptor(3). If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified(4), and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.