Morris O. Dailey

Immune response to a murine coronavirus: Identification of a homing receptor-negative CD4+ T cell subset that responds to viral glycoproteins

Abstract The lymphocyte proliferative response to mouse hepatitis virus, strain JHM (MHV-JHM), a well-described cause of chronic and acute neurological infections, has been studied using vaccinia virus recombinants expressing individual MHV proteins. The surface (S) and transmembrane (M) glycoproteins were the most active proteins in causing proliferation of lymphocytes isolated from immunized adult mice, whereas lymphocytes from persistently infected mice proliferated only in response to the S protein. The cells from immunized mice which proliferated most actively in response to MHV were positive for the CD4 antigen and secreted interferon-γ. In addition, the most responsive subset of cells did not express gpg0MEL-14, the lymph node-specific homing receptor. The results identify a subpopulation of CD4+ T cells that may be an important component of the cell-mediated immune response to this virus. The data also suggest that response to the M protein is important in preventing disease progression in C57BL/6 mice since cells which recognize this protein are absent from persistently infected mice.

A novel and inexpensive source of allophycocyanin for multicolor flow cytometry

Allophycocyanin (APC) belongs to a family of phycobiliproteins that are well suited as fluorescent reagents for flow cytometric analysis, since they have a broad excitation spectrum, a large Stokes shift and they fluoresce with a high quantum yield. The widespread use of APC has been limited by the availability of raw material and high cost of the purified phycobiliprotein. We have assessed the suitability of dry, powdered Spirulina platensis, available at health food stores, as an inexpensive source of APC. APC was extracted from Spirulina platensis by overnight treatment with lysozyme, followed by ammonium sulfate precipitation. APC was then separated from phycocyanin (the only other major phycobiliprotein in Spirulina) by elution of bound material from an hydroxylapatite column using an increasing continuous phosphate gradient. APC isolated in this manner retained its normal trimeric structure. The absorbance and fluorescence excitation and emission spectra of the purified phycobiliproteins were identical to those previously shown for C-PC and APC. APC can be stored concentrated at 4 degrees C, frozen at -70 degrees C, or as a saturated ammonium sulfate precipitate, with no subunit dissociation or change in spectral properties. Moreover, APC has been conjugated to monoclonal and polyclonal antibodies for use in multicolor FACS analysis, with the conjugated antibody activity remaining stable for at least 2 years. Thus, this procedure is a simple, cost-effective method for preparing reagents for multicolor immunofluorescence and flow cytometry.

Differentiation of central memory CD8 T cells is independent of CD62L-mediated trafficking to lymph nodes.

CD62L (L-selectin) is a key regulator of T cell trafficking, and its surface expression on activated T cells is modulated to control T cell access to lymph nodes after acute infections. In memory T cells, CD62L is the most frequently used marker to define central memory T cells, a population that provides enhanced protection against most, but not all, pathogens. Early access of CD62Lpos effector T cells to lymph nodes has been proposed to result in preferential central memory T cell differentiation, but direct proof for the involvement of lymph node homing in memory T cell differentiation is lacking. In this study, we show that central memory lineage commitment in CD8 T cells is unaltered by enhanced entry into lymph nodes as a result of constitutive CD62L expression, and that equal numbers of effector and central memory CD8 T cells develop in the absence of CD62L-mediated lymph node trafficking. Our results suggest that CD62L is not a deterministic marker of central memory T cell differentiation, thus providing new insight into the process of memory CD8 T cell generation.

Evaluation of serum cortisol in the postmortem diagnosis of acute adrenal insufficiency.

Normal adrenocortical activity is necessary for electrolyte regulation and the maintenance of cardiovascular function. Although chronic adrenal insufficiency generally presents with the gradual onset of a set of characteristic symptoms and signs, the more sudden loss of adrenal activity can present with acute, rapidly progressive cardiovascular dysfunction that can be fatal if not recognized and treated promptly. We herein describe a patient who had most of his adrenal tissue removed during resection of metastatic renal carcinoma, conventional clear cell type, with much of the remaining adrenal tissue undergoing necrosis during or shortly after surgery. Although the patient appeared to be stable and progressing adequately well, he died suddenly 2 days postoperatively. When the gross autopsy findings suggested the possibility of adrenal insufficiency, clinical laboratory assessment of adrenocortical activity was sought. Analysis of stored antemortem serum samples and of blood obtained at autopsy demonstrated a progressive decrease in cortisol levels which, in this stressed postsurgical patient, proved fatal. The use of both antemortem and postmortem blood in the demonstration of acute adrenal insufficiency at autopsy is discussed.

REVERSIBILITY OF LOSS OF HOMING RECEPTOR EXPRESSION FOLLOWING ACTIVATION

The ability of lymphocytes to recirculate from peripheral blood to lymphoid tissue is necessary in order to maintain an effective immune response, irrespective of an antigen’s port of entry. Recirculation and homing to lymph nodes (LN) and Peyer’s patches (PP) is initiated by the binding of cells to specialized post-capillary high endothelial venules (HEV’s), followed by their passage through the vessel walls into the surrounding lymphoid tissue.

Autoimmune lpr and Gld Mice: Models of Abnormal Adhesion Molecule Regulation and Defective Lymphocyte Traffic

Mice of the MRL-lpr/lpr strain develop at several months of age a massive generalized lymphadenopathy and severe SLE-like autoimmune disease with autoantibody production and glomerulonephritis. The Ipr gene has been bred into other mouse strains and, when homozygous, leads to similar lymphoproliferation but a somewhat attenuated autoimmunity. The genetically unrelated gld mutation causes a nearly identical autoimmune and lymphoproliferative syndrome.1 The lpr genetic defect has recently been identified as the fas gene, which is involved with the regulation of programmed cell death in lymphocytes.2 The gld gene has not yet been identified, although it is genetically linked to the gene coding for murine L-selectin (I. Weissman, personal communication). In both lpr and gld mice, the cells that accumulate are abnormal T cells that are relatively anergic and have the same unusual phenotype. The majority of them express both the Thy-1 and B220 antigens and are CD4- and CD8-, but several other minor populations of defective cells are also present, as are some residual normal B and T cells. These abnormal lpr and gld cells proliferate very slowly in lymphoid tissues, but in the case of lpr mice, the cells replicate rapidly in the liver.3 One remarkable feature of both lpr and gld mice is that the lymphoproliferative disorder involves primarily the lymph nodes, while Peyer’s patches and the lamina propria are virtually unaffected. This is similar to the distribution of cells observed with certain LN-homing lymphomas which also spare PP. These lymphomas express L-selectin, the lymph node homing receptor, which facilitates the entry of normal or malignant cells from the bloodstream into the LN parenchyma.

Accidental Ingestion of a Grill Brush Wire Resulting in Death

Accidental ingestion of a foreign body is a rare occurrence in the unimpaired adult. The usual patient is aware of the ingestion event and seeks medical attention if needed. We report a case of accidental, unrecognized, fatal ingestion of a foreign body discovered during a postmortem examination. In situ examination revealed a small perforation of the terminal ileum by a perforating metal wire. Fibrinopurulent peritonitis was the immediate cause of death. The foreign body was later found to closely resemble the wire bristles of a barbeque grill brush that family members brought to a family/clinician/pathologist conference. There are few previous case reports of ingestion of a grill brush wire, all of which were nonfatal. Health care workers, including pathologists, should be aware of this potential ingestible foreign body given the widespread use of barbeque grills in food preparation. Additionally, manufacturers and users of grill brushes should be well aware of this ingestion hazard.

Homing Receptor Expression in Grafts of Purified Thymic Epithelium

Grafts of purified thymic epithelium have been used to study the role of the thymic microenvironment in T cell differentiation1–6. The ability of such grafts to immunologically reconstitute either nude mice or thymectomized, irradiated and hematopoietically reconstituted hosts has demonstrated that they are only partially able to reconstitute the host animal, as neither lymphocyte phenotype frequencies nor functional responsiveness of the graft derived peripheral (splenic) T cell population reaches normal (intact) levels2,3,6. More recently it has been suggested that the expression of lymphocyte homing receptors may be important in the selection of thymocytes destined to emigrate from the thymus, as well as their localization in the periphery7–9. In the studies reported here, we have examined homing receptor expression in grafts of purified thymic epithelium and in the peripheral lymphoid organs of grafted nude mice. Results showed that, although incomplete, T cell reconstitution did take place in these animals. The presence of MEL-14 positive cells in the graft suggests that the presence or absence of homing receptor expression in these grafts was not solely responsible for the incomplete T cell emigration and peripheralization seen in these animals.