Antibodies to Human Thymic Epithelium Form Part of the Murine Autoreactive Repertoire

Monoclonal antibody (mAb) MR6 recognises a 200 kDa glycoprotein, gp200-MR6, which is expressed at high levels on the surface of human thymic cortical epithelium. In order to produce further mAbs against the gp200-MR6 molecule, mice were immunised with purified human gp200-MR6, hybridomas produced and supernatants screened for MR6-like reactivity on human thymic sections. Surprisingly this conventional hybridoma technique failed to produce stable hybridoma cells producing MR6-like antibodies. However, antibodies with specificitie other than MR6-like were obtained. Three such antibodies (1B2, 3A3 and 4B3) were analysed further. Expression of 1B2-antigen, 3A3-antigen and 4B3-antigen was analysed on skin, tonsil and thymic sections, on cultured thymic epithelial cells (TEC), thymocytes and peripheral blood mononuclear cells (PBMC), and found to be expressed by both lymphocytes and epithelial cell populations. Furthermore, the antigens were also expressed on mouse thymic, epithelial cells. The regulation of expression of these antigens was analysed following mitogen or cytokine stimulation of PBMC and cultured TEC, respectively. Expression on T cells was clearly affected by mitogens that mimic activation through the T cell receptor and expression on cultured TEC was affected by T cell-derived cytokines. Thus, the shared epithelial- lymphocyte molecules identified in this study may play a role in the cross-talk between the developing thymocytes and their epithelial microenvironment. The production of mAbs with specificities other than that of purified gp200-MR6 indicates that a wide range of B cells with specificity for components of the human thymic microenvironment exist in the normal mouse. These may detect epitopes that are shared with common pathogens to which the animals are exposed. Alternatively, they may be autoreactive B cells that are normally silent in the absence of T cell help. This help may be provided by T cells specific for human gp200-MR6, or nonspecifically by polyclonal activation induced by the adjuvant

Differential Expression Of Adhesion Molecules Within The Human Thymus

Development of a diverse, MHC-restricted yet self-tolerant T-cell repertoire occurs within the thymus, and requires contact between developing T cells and their stromal microenvironment. Such interactions are likely to depend on the combinatorial effect of specific adhesion molecules. As a preliminary step to determining their role in T-cell development, we have studied the distribution of LFA-1/ICAM-1, CD2/LFA-3, VLA-4/VCAM-1, and HECA 452-antigen/E-Selectin ligand pairs on frozen sections of human thymus. Using two color-immunohistochemistry, and a variety of cell-lineage markers that reveal the nature of the cells on which these adhesion molecules are located, we find a differential distribution of adhesion molecules, with some being shared by both endothelial and epithelial cells. We also identify the VCAM-1-positive subpopulation as cortical macrophages. The relevance of these findings to thymopoiesis is discussed

A Novel Adhesion Molecule in the Murine Thymic Microenvironment: Functional and Biochemical Analysis

The rat monoclonal antibody (mAb) 4F1, raised against mouse thymic stromal cells, recognizes cortical epithelium in tissue sections of mouse thymus; however, in flow cytometry, activated leucocytes (T cells, B cells, and macrophages) and transformed thymocytes are also positive for the 4F1-antigen (4F1-Ag). Western blotting, under both reducing and nonreducing conditions, demonstrates that the molecule to which 4F1 binds is expressed in four forms, 29, 32, 40, and 43 kD, all of which carry N-linked carbohydrate; and that the structure is identical on epithelium and lymphocytes. The 4F1-Ag on cortical epithelium is partially sensitive to PI-PLC treatment, whereas on transformed epithelial and lymphoid cell lines, it was resistant to this enzyme. The molecule, therefore, may exist in both transmembrane and phosphoinositol-linked forms. In functional blocking experiments, mAb 4F1 gave inhibition of both T-cell proliferation in MLR and of cytotoxic T-cell killing of alloantigenic targets; it also blocked adhesion of transformed thymocytes to thymic epithelial cells in vitro. These molecular and functional characteristics suggest that the 4F1-Ag is a novel adhesion molecule that may be involved both in intrathymic T lymphocyte differentiation and in peripheral T-cell function

Expression of The αβ T-Cell Receptor Is Necessary for The Generation of The Thymic Medulla

The architecture of the thymus of mice that congenitally fail to express the αβ T-cell receptor (TCRαβ) has been examined by immunohistology. In these mice, a defined mutation was introduced into the TCRc gene by homologous recombination. By using antibodies specific for cortical or medullary epithelium and for major histocompatibility complex antigens, the network of cortical epithelium in these mice was shown to be essentially unaltered in comparison with that of normal mice. In contrast, the thymic medulla was considerably reduced in size. This analysis shows that expression of the αβ TCR but not the γδ TCR is obligatory for establishing the thymic medulla and suggests that the growth of medullary epithelial cells may require contact with TCRαβ-expressing cells

K21-Antigen: A Molecule Shared by the Microenvironments of the Human Thymus and Germinal Centers

The mouse IgG1 monoclonal antibody (mAb) K21 recognizes a 230-kD molecule (K21-Ag) on Hassall's corpuscles in the human thymus. This mAb also stains cultured thymic epithelial cells as well as other epithelial cell lines, revealing a predominant intracellular localization. Further analysis with mAb K21 on other lymphoid tissues showed that it also stains cells within the germinal centers of human tonsils, both lymphoid (B) cells and some with the appearance of follicular dendritic cells. Double immunostaining of tonsil sections shows that K21-Ag is not expressed by T cells, whereas staining with anti-CD22 and -CD23 mAb revealed some doublepositive cells. A subpopulation of the lymphoid cells express the K21-Ag much more strongly. This K21++/CD23++ subpopulation of cells is localized in the apical light zone of germinal centers, suggesting that K21-Ag may be an important marker for the selected centrocytes within germinal centers and may play a role in B-cell selection and/or development of B-cell memory. Flow cytometric analysis showed that K21-Ag is expressed on the surface of a very low percentage of thymocytes, tonsillar lymphocytes, and peripheral blood mononuclear cells. Analysis of purified/separated tonsillar T and B lymphocytes showed that T cells do not express the K21-Ag; in contrast, B cells express low levels of the K21-Ag, and this together with CD23 is upregulated after mitogenic stimulation. Our data therefore raise the possibility that the K2l- Ag may play a role in B-lymphocyte activation/selection

Metastatic disease to the breast: the Washington University experience

<p>Abstract</p> <p>Background</p> <p>Metastases to the breast occur rarely, but may be increasing in incidence as patients live longer with malignant diseases. The aim of this study is to characterize metastatic disease to the breast and to describe the management and prognosis of patients who present with this diagnosis.</p> <p>Methods</p> <p>A retrospective review of our institution's pathology and breast cancer databases was performed in order to identify patients with breast malignancies that were not of primary breast origin. Chart review provided additional information about the patients' primary malignancies and course of illness.</p> <p>Results</p> <p>Between 1991 and 2006, eighteen patients with metastatic disease to the breast of non-hematologic origin were identified and all had charts available for review. Among the 18 patients with disease metastatic to the breast, tissues of origin included 3 ovarian, 6 melanoma, 3 medullary thyroid, 3 pulmonary neuroendocrine, 1 pulmonary small cell, 1 oral squamous cell, and 1 renal cell. Overall mean survival after diagnosis of metastatic disease to the breast was 22.4 months. Treatment of metastases varied and included combinations of observation, surgery, radiation, and chemotherapy. Five patients (27.8%) required a change in management of their breast disease for local control.</p> <p>Conclusion</p> <p>Due to the variable course of patients with metastatic disease, a multi-disciplinary approach is necessary for each patient with disease metastatic to the breast to determine optimal treatment. Based on our review, many patients survive for long periods of time and local treatment of metastases to the breast may be beneficial in these patients to prevent local complications.</p

Evidence-based Surgical Competency Outcomes from the Clinical Readiness Program

Objectives: 1) Evaluate the value and strength of a competency framework for identifying and measuring performance requirements for expeditionary surgeons; 2) Verify psychometric integrity of assessment instrumentation for measuring domain knowledge and skills; 3) Identify gaps in knowledge and skills capabilities using assessment strategies; 4) Examine shared variance between knowledge and skills outcomes, and the volume and diversity of routine surgical practice. Background: Expeditionary military surgeons provide care for patients with injuries that extend beyond the care requirements of their routine surgical practice. The readiness of these surgeons to independently provide accurate care in expeditionary contexts is important for casualty care in military and civilian situations. Identifying and closing performance gap areas are essential for assuring readiness. Methods: We implemented evidence-based processes for identifying and measuring the essential performance competencies for expeditionary surgeons. All assessment instrumentation was rigorously examined for psychometric integrity. Performance outcomes were directly measured for expeditionary surgical knowledge and skills and gap areas were identified. Knowledge and skills assessment outcomes were compared, and also compared to the volume and diversity of routine surgical practice to determine shared variance. Results: Outcomes confirmed the integrity of assessment instrumentation and identified significant performance gaps for knowledge and skills in the domain. Conclusions: Identification of domain competencies and performance benchmarks, combined with best-practices in assessment instrumentation, provided a rigorous and defensible framework for quantifying domain competencies. By identifying and implementing strategies for closing performance gap areas, we provide a positive process for assuring surgical competency and clinical readiness