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In JoVE (2)
- Preparação de rato Imunogénio Pituitária para a indução de hipofisite Auto-Imune Experimental
- Indução de hipofisite Auto-Imune Experimental em Ratos SJL
Other Publications (19)
- Thyroid : Official Journal of the American Thyroid Association
- Cancer Gene Therapy
- Cancer Gene Therapy
- Endocrinology
- Endocrinology
- American Journal of Physiology. Lung Cellular and Molecular Physiology
- Proceedings of the National Academy of Sciences of the United States of America
- Clinical Endocrinology
- Endocrinology
- Radiology
- PloS One
- Analytical Chemistry
- Journal of Nuclear Medicine : Official Publication, Society of Nuclear Medicine
- Molecular Cancer Therapeutics
- Genetic Testing and Molecular Biomarkers
- Endocrinology
- Pituitary
- Clinical Cancer Research : an Official Journal of the American Association for Cancer Research
- Journal of the American Chemical Society
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Articles by Shey-Cherng Tzou in JoVE
JoVE Immunology and Infection
Preparação de rato Imunogénio Pituitária para a indução de hipofisite Auto-Imune Experimental
Department of Pathology, The Johns Hopkins University
Hipofisite auto-imune pode ser reproduzido em ratos através da injeção de um extrato de proteínas do mouse pituitária.
JoVE Immunology and Infection
Indução de hipofisite Auto-Imune Experimental em Ratos SJL
Department of Pathology, The Johns Hopkins University
Este vídeo mostra como induzir hipofisite auto-imunes em camundongos SJL e como avaliar sua gravidade pela histopatologia.
Other articles by Shey-Cherng Tzou on PubMed
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.
Stable Expression of Chimeric Anti-CD3 Receptors on Mammalian Cells for Stimulation of Antitumor Immunity
Expression of CD80 or CD86 costimulatory molecules on tumor cells can produce rejection of immunogenic but not poorly immunogenic tumors. We have previously shown that anti-CD3 single-chain antibodies expressed on the surface of cells can directly activate T cells. We therefore investigated whether anti-CD3 "receptors" could enhance CD86-mediated rejection of poorly immunogenic tumors. Expression of anti-CD3 receptors on cells was increased by introduction of membrane-proximal "spacer" domains containing glycosylation sites between the single-chain antibody and the transmembrane domain of the chimeric receptors. Removal of glycosylation sites in the spacer reduced surface expression due to increased shedding of chimeric receptors from the cell surface. Induction of T-cell proliferation by anti-CD3 receptors did not correlate with the expression level of chimeric protein, but rather depended on the physical properties of the spacer. Anti-CD3 receptors effectively induced T-cell cytotoxicity, whereas coexpression with CD80 or CD86 was required for generating T-cell proliferation and IL-2 secretion. Although expression of CD86 did not significantly delay the growth of poorly immunogenic B16-F1 tumors, expression of anti-CD3 receptors with CD86 produced complete tumor rejections in 50% of mice and induced significant protection against wild-type B16-F1 tumor cells. Our results show that spacer domains can dramatically influence the surface expression and the biological activity of chimeric antibody receptors. The strong antitumor activity produced by anti-CD3 receptors and CD86 on tumor cells indicates that this strategy may be beneficial for the gene-mediated therapy of poorly immunogenic tumors.
Hapten-directed Targeting to Single-chain Antibody Receptors
Artificial recombinant receptors may be useful for selectively targeting imaging and therapeutic agents to sites of gene expression. To evaluate this approach, we developed transgenes to express highly on cells a single-chain antibody (scFv) against the hapten 4-ethoxymethylene-2-phenyl-2-oxazoline-5-one (phOx). A phOx enzyme conjugate was created by covalently attaching phOx molecules to polyethylene glycol (PEG)-modified beta-glucuronidase. Cells expressing phOx scFv but not control scFv receptors were selectively killed after exposure to ss-glucuronidase derivatized with phOx and PEG (phOx-beta G-PEG) and a glucuronide prodrug (p-hydroxy aniline mustard beta-D-glucuronide, HAMG) of p-hydroxyaniline mustard. Targeted activation of HAMG produced bystander killing of receptor-negative cells in mixed populations containing as few as 10% phOx-receptor-positive cells. Functional phOx scFv receptors were stably expressed on B16-F1 melanoma tumors in vivo. Treatment of mice bearing established phOx-receptor-positive tumors with phOx-beta G-PEG and HAMG significantly (P< or =.0005) suppressed tumor growth as compared with treatment with beta G-PEG and HAMG or prodrug alone. phOx was unstable in the serum, suggesting alternative haptens may be more suitable for in vivo applications. Our results show that therapeutic agents can be targeted to artificial hapten receptors in vitro and in vivo. The expression of artificial receptors on target cells may allow preferential delivery of therapeutic or imaging molecules to sites of transgene expression.
Expression of Class II Major Histocompatibility Complex Molecules on Thyrocytes Does Not Cause Spontaneous Thyroiditis but Mildly Increases Its Severity After Immunization
Class II major histocompatibility complex (MHC) molecules are classically expressed on antigen-presenting cells of the hematopoietic lineage but have also been described on epithelial cells in association with autoimmunity. In this context, however, it remains debatable whether class II MHC molecules are the initiating event or rather the consequence of the autoimmune attack. In addition, the role of epithelial class II expression once the autoimmune attack has begun is unknown. We generated transgenic mice expressing in the thyroid follicular cells the class II transactivator, the master regulator of all the genes in the class II MHC pathway. The study used a cohort of 245 CBA/J mice (127 wild-type and 118 transgenic), both in basal conditions (n = 63) and at different time points after immunization with mouse thyroglobulin (n = 182). In basal conditions, transgenic mice were similar to wild-type controls and did not develop spontaneous autoimmune thyroiditis, despite the aberrant expression of class II MHC molecules on thyrocytes. After immunization, thyroiditis was 8% more severe in transgenics than controls (95% confidence interval from 1.8-13.4%; P = 0.033), especially during the florid stages of disease. These findings suggest that expression of class II MHC molecules on epithelial cells is not sufficient to initiate autoimmunity but mildly modulates an already established autoimmune attack against the target organ.
Interleukin (IL)-12-driven Primary Hypothyroidism: the Contrasting Roles of Two Th1 Cytokines (IL-12 and Interferon-gamma)
IL-12, a prototypic T helper 1 cytokine, has been implicated in the pathogenesis of organ-specific autoimmune diseases, such as Hashimoto's thyroiditis, but reported to give conflicting results in murine models of lymphocytic thyroiditis. To determine the effects of chronic, local production of IL-12 within the thyroid gland, we created transgenic mice that express IL-12 p70 under the transcriptional control of the thyroglobulin promoter. Transgenics developed growth retardation, moderate primary hypothyroidism, and mild lymphocytic infiltration of the thyroid gland. The hypothyroidism was associated with increased mRNA levels of the sodium-iodide symporter, an increase partly due to a direct effect of IL-12 on the thyrocyte. Upon immunization with a suboptimal dose of mouse thyroglobulin, IL-12 transgenic mice developed a lymphocytic thyroiditis that was more frequent and severe than that observed in wild-type littermates. The disease-promoting effect of IL-12 was independent of interferon-gamma, as shown by the similar interferon-gamma levels in transgenics and controls. These findings highlight the contrasting roles of two T helper 1 cytokines and report a novel role of IL-12 on thyroid hormonogenesis.
IL-12 Overexpression in Mice As a Model for Sjögren Lung Disease
Interleukin-12 (IL-12), a Th1 proinflammatory cytokine, is reported to be increased in Sjögren syndrome. To evaluate the effects of local Th1/Th2 deregulation, we generated a transgenic mouse model that overexpresses IL-12 in the lungs. IL-12 transgenic mice developed bronchial and alveolar abnormalities strikingly similar to those found in the lungs of Sjögren patients. Pathologically, lung abnormalities began at approximately 4 mo of age and were characterized by lymphocytic infiltrates around the bronchi, intraluminal periodic acid Schiff-positive debris, increased cell proliferation in the alveolar region, and increased interstitial and alveolar macrophages. Functionally, these abnormalities translated into decreased mucociliary clearance (P<0.05 vs. wild-type littermates) and increased oxidative stress (P<0.01). The pathological and functional abnormalities were accompanied by significant changes in lung natural killer (NK) cells. The number of NK cells was fourfold higher in IL-12 transgenic than wild-type lungs (20% of all lymphoid cells vs. 5%) during the first month of life. NK cells then decreased within a narrow window of time (from 30 to 50 days of age), reaching a nadir of approximately 2% on day 50, and remained at these low levels thereafter. This new mouse model highlights the role of IL-12 in the initiation of Sjögren syndrome.
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.
Novel Autoantigens in Autoimmune Hypophysitis
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.
Micro-PET Imaging of Beta-glucuronidase Activity by the Hydrophobic Conversion of a Glucuronide Probe
To develop a new glucuronide probe for micro-positron emission topography (PET) that can depict beta-glucuronidase (betaG)-expressing tumors in vivo.
Immunoproteasome Overexpression Underlies the Pathogenesis of Thyroid Oncocytes and Primary Hypothyroidism: Studies in Humans and Mice
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.
Measurement of Poly(ethylene Glycol) by Cell-based Anti-poly(ethylene Glycol) ELISA
Poly(ethylene glycol) (PEG) is increasingly used in clinical and experimental medicine. However, quantification of PEG and PEGylated small molecules remains laborious and unsatisfactory. In this report, we stably expressed a functional anti-PEG antibody on the surface of BALB 3T3 cells (3T3/alphaPEG cells) to develop a competitive enzyme-linked immunosorbent assay (ELISA) for PEG quantification. The alphaPEG cell-coated plate bound biotinylated PEG(5K) (CH(3)-PEG(5K)-biotin) and CH(3)-PEG(5K)-(131)I more effectively than did a traditional anti-PEG antibody-coated plate. Competitive binding between PEG (2, 5, 10, or 20 kDa) and a known amount of CH(3)-PEG(5K)-biotin allowed construction of a reproducible competition curve. The alphaPEG cell-based competition ELISA measured small molecules derivatized by PEG(2K), PEG(5K), PEG(10K), PEG(20K), and PEG(5K) at concentrations as low as 58.6, 14.6, 3.7, 3.7, and 14.6 ng/mL, respectively. Notably, the presence of serum or bovine serum albumin enhanced PEG measurement by the alphaPEG cell-based competition ELISA. Finally, we show here that the alphaPEG cell-based competition ELISA accurately delineated the pharmacokinetics of PEG(5K), comparable to those determined by direct measurement of radioactivity in blood after intravenous injection of CH(3)-PEG(5K)-(131)I into mice. This quantitative strategy may provide a simple and sensitive method for quantifying PEG and PEGylated small molecules in vivo.
Development of a Universal Anti-polyethylene Glycol Reporter Gene for Noninvasive Imaging of PEGylated Probes
A reporter gene can provide important information regarding the specificity and efficacy of gene or cell therapies. Although reporter genes are increasingly used in experimental and clinical studies, a highly specific yet nonimmunogenic reporter that can track genes and cells in vivo by multiple imaging technologies still awaits development. In this study, we constructed a versatile and nonimmunogenic reporter gene to noninvasively image gene expression or cell delivery by optical imaging, MRI, and small-animal PET.
Endocytosis of PEGylated Agents Enhances Cancer Imaging and Anticancer Efficacy
PEGylated nanoparticles and macromolecules are increasingly used in cancer imaging and anticancer treatment. The role of receptor-mediated endocytosis in the efficacy of these agents, however, has not been clearly defined. Here, we developed a matched pair of endocytic and nonendocytic receptors to directly and unambiguously assess this issue. The ligand-binding domains of the low-density lipoprotein receptor (LDLR) or a truncated LDLR lacking the NPXY endocytosis motif (DeltaLDLR) were replaced with an anti-polyethylene glycol antibody (alphaPEG) to form endocytic alphaPEG-LDLR and nonendocytic alphaPEG-DeltaLDLR receptors. The receptors were stably expressed at similar levels on the surface of HCC36 cells. HCC36/alphaPEG-LDLR cells, but not HCC36/alphaPEG-DeltaLDLR cells, rapidly endocytosed PEG-quantum dots and PEG-liposomal doxorubicin (Lipo-Dox) in vitro and in vivo. Lipo-Dox was significantly more cytotoxic to HCC36/alphaPEG-LDLR cells than to HCC36/alphaPEG-DeltaLDLR cells. HCC36/alphaPEG-LDLR tumors also accumulated significantly more PEGylated near-IR probes (PEG-NIR797) and PEG-liposomal-(111)In than HCC36/alphaPEG-DeltaLDLR tumors in vivo. Furthermore, Lipo-Dox more significantly suppressed the growth of established HCC36/alphaPEG-LDLR tumors as compared with HCC36/alphaPEG-DeltaLDLR tumors. Our data show that endocytosis of PEGylated probes and drugs enhances both cancer imaging and anticancer efficacy, indicating that endocytic receptors are superior targets for the design of cancer imaging probes and immunoliposomal drugs.
Simultaneous Detection of Multiple Single-nucleotide Polymorphisms by a Simple Membrane Chip
Technologies that screen multiple single-nucleotide polymorphisms (SNPs) could be very valuable in predicting patients' susceptibilities to diseases or responses to therapeutic interventions. In this study, we developed a chip that can accurately detect four SNPs at same time. This chip is cost-effective and user-friendly because it uses a detection protocol analogous to dot blotting and does not require sophisticated instruments. To establish this chip, we designed and blotted onto a nylon membrane SNP-specific oligonucleotide probes for human angiotensinogen, cholesteryl ester transfer protein, and apolipoprotein E. This chip detected the corresponding SNPs harbored within the angiotensinogen, cholesteryl ester transfer protein, and apolipoprotein E sequences from 20 donors. Importantly, the SNPs detected by our chip matched exactly with the direct sequencing results, thereby highlighting the accuracy of this chip. In conclusion, our chip is a robust tool for multiple SNP screening and holds the potential to future refinement in detecting diseases-associating genes in patients.
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 Freund's 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.
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.
In Vivo Positron Emission Tomography Imaging of Protease Activity by Generation of a Hydrophobic Product from a Noninhibitory Protease Substrate
To develop an imaging technology for protease activities in patients that could help in prognosis prediction and in design of personalized, protease-based inhibitors and prodrugs for targeted therapy.
An Activity-Based Near-Infrared Glucuronide Trapping Probe for Imaging β-Glucuronidase Expression in Deep Tissues
β-glucuronidase is an attractive reporter and prodrug-converting enzyme. The development of near-IR (NIR) probes for imaging of β-glucuronidase activity would be ideal to allow estimation of reporter expression and for personalized glucuronide prodrug cancer therapy in preclinical studies. However, NIR glucuronide probes are not yet available. In this work, we developed two fluorescent probes for detection of β-glucuronidase activity, one for the NIR range (containing IR-820 dye) and the other for the visible range [containing fluorescein isothiocyanate (FITC)], by utilizing a difluoromethylphenol-glucuronide moiety (TrapG) to trap the fluorochromes in the vicinity of the active enzyme. β-glucuronidase-mediated hydrolysis of the glucuronyl bond of TrapG generates a highly reactive alkylating group that facilitates the attachment of the fluorochrome to nucleophilic moieties located near β-glucuronidase-expressing sites. FITC-TrapG was selectively trapped on purified β-glucuronidase or β-glucuronidase-expressing CT26 cells (CT26/mβG) but not on bovine serum albumin or non-β-glucuronidase-expressing CT26 cells used as controls. β-glucuronidase-activated FITC-TrapG did not interfere with β-glucuronidase activity and could label bystander proteins near β-glucuronidase. Both FITC-TrapG and NIR-TrapG specifically imaged subcutaneous CT26/mβG tumors, but only NIR-TrapG could image CT26/mβG tumors transplanted deep in the liver. Thus NIR-TrapG may provide a valuable tool for visualizing β-glucuronidase activity in vivo.
