Yuan-Hsu Kang

Disseminated Tubuloreticular Inclusions in Acquired Immunodeficiency Syndrome (AIDS)

Tissue biopsies and peripheral blood samples from 10 patients with the characteristic clinical features of acquired immunodeficiency syndrome (AIDS) were examined by electron microscopy and ultracytochemical myeloperoxidase technique. Abundant tubuloreticular inclusions (TRI) were detected within the endoplasmic reticulum of capillary endothelial cells, histiocytes, and lymphocytes in kidneys, small bowel, and lymph nodes, and lymphocytes and monocytes from peripheral blood. In general, TRI were found in the same type of cells and with conspicuous high frequency in our cases of AIDS as had been previously described in systemic lupus erythematosus (SLE). These findings indicate a morphologic link between these immunological disorders and the presence of TRI, raising the possibility of similar pathogenetic mechanisms.

Cytochemical properties of osteoblast cell membrane domains.

The interactions of osteoblasts with one another and with the extracellular milieu are of vital importance for cell function. These interactions are mediated by cell membrane-associated components. In the present work, we studied the distribution of several mediators known to be associated with the cell surface, using ultrastructural cytochemistry, to characterize the three cell membrane domains (osteoid, lateral, and vascular) of osteoblasts. Osteoblasts in neonatal rat calvariae were studied for cell surface distribution of alkaline phosphatase (APase), calcium-activated adenosine triphosphatase (Ca2+-ATPase), calcium, soybean agglutinin (SBA)-reactive sites, and peanut agglutinin (PNA)-reactive sites. APase was absent in the osteoid domain but was evenly distributed in the other domains. Ca2+-ATPase was found to be concentrated mainly in the lateral domains. In contrast, calcium was present in all cell membrane domains. Using lectins conjugated to horseradish peroxidase, we demonstrated that SBA binding sites were evenly distributed along the osteoblast cell membrane, whereas PNA binding sites were absent or minimally present in the osteoid and lateral domains but were evenly distributed in the vascular domain. These results suggest that the various functions of osteoblasts may be facilitated by specialized cell membrane domains which are cytochemically distinct. Previous reports have failed to demonstrate the cytochemical differences between the three domains of the osteoblast cell membrane.

Cytochemical changes in hepatocytes of rats with endotoxemia or sepsis: localization of fibronectin, calcium, and enzymes.

Bacterial lipopolysaccharide (LPS) is known to be implicated in the pathogenesis of endotoxemia and septic shock. The liver is the first vital organ to exhibit pathological alterations in shock. The present studies include immunoelectron microscopic localization of tissue fibronectin and cytochemical localization of calcium and enzymes in hepatocytes of animals with LPS-induced endotoxemia or cecal ligation-induced septic shock. The results showed increased staining of fibronectin in the basal (perisinusoidal) surfaces and in the cisternae of rough endoplasmic reticulum and the Golgi complex of hepatocytes in rats with endotoxemia or septic shock. Intracellular calcium content was significantly increased in the LPS-treated or septic rats. Calcium pyroantimonate precipitate was deposited predominantly on the outer surfaces of the RER of hepatocytes. In addition, diminution or depletion of glycogen, reduction of catalase-containing peroxisomes, increase of G-6-Pase activity, and depletion of cytochrome c oxidase in many mitochondria were also observed in hepatocytes of experimental animals. The overall results suggest that LPS stimulates: (a) hepatic synthesis and secretion of fibronectin; (b) uptake of calcium by hepatocytes; and (c) G-6-Pase activity. LPS treatment also leads to reduced numbers of peroxisomes and depletion of cytochrome c oxidase.

Immunoelectron microscopic identification of human NK cells by FITC-conjugated anti-Leu-11a and biotinylated anti-leu-7 antibodies

Human natural killer (NK) cells have been reported to express various surface antigens. The majority and the most functionally potent NK cells are Leu-11a (NKP-15) positive cells. Only a small number of functional NK cells express Leu-7 (HNK-1) antigen. In the present study, we have established techniques for immunoelectron microscopic identification of NK cells by mouse monoclonal FITC-conjugated anti-Leu-11a and biotinylated anti-Leu-7 antibodies. Ficoll-Hypaque-isolated peripheral blood lymphocytes (PBL) were reacted with the specific antibodies before or after fixation in a 1% glutaraldehyde/1% paraformaldehyde fixative. Prefixation labeling of viable cells with the antibodies was carried out at 4 degrees C or 37 degrees C. Cells prelabeled with anti-Leu-11a antibody were reacted with secondary antibodies either before or after fixation. Anti-Leu-7 antibody was stained directly via an avidin-biotin-peroxidase (ABC) system, anti-Leu-11a antibody was stained indirectly by the ABC immunoperoxidase procedure via a biotinylated anti-mouse IgG secondary antibody or by a 10 nm or 40 nm colloidal gold-labeled anti-mouse IgG antibody. Results indicate that Leu-7 antigen could be localized by incubation with the specific antibody either before or after 20 min fixation; however, Leu-11a antigen was totally abrogated following the same fixation procedure. The Leu-11a antigen was well stained by the methods of prefixation labeling of cells with anti-Leu-11a antibody and incubation with a biotinylated secondary antibody and the ABC system after fixation. With respect to colloidal gold labeling, better results were obtained when cells were reacted with the gold-labeled antibodies immediately after incubation with anti-Leu-11a antibody but before fixation. Ultrastructurally both Leu-7 positive (+) and Leu-11a positive (+) cells shared common ultrastructural features associated with large granular lymphocytes. Using the above described techniques, we found approximately 2-5% Leu-7+ and 9-15% Leu-11a+ cells in the PBL of healthy donors. The overall results suggest that Leu-11a antigen is more sensitive to glutaraldehyde/paraformaldehyde fixation than Leu-7, since it can be localized only by prefixation labeling procedures; the ABC immunoperoxidase procedure is an ideal technique for labeling NK cells for light and electron microscopic enumeration; the immunogold method provides an adequate technique for labeling NK cells which are designated for ultracytochemical studies.

Characterization of the binding of soluble CD14 to human endothelial cells and mechanism for CD14-dependent cell activation by LPS

The binding of soluble CD14 (sCD14) to human umbilical vein endothelial cells (HUVEC) was examined in order to understand the role of CD14 in potentiating LPS activity. Both purified sCD14 and [125I]-sCD14 potentiated LPS-stimulated ICAM-1 expression in HUVEC. This potentiation was blocked by anti-CD14 monoclonal antibodies (mAbs) 63D3, UCHM-1 and RM052. Saturation binding assay revealed that [125I]-sCD14 bound to HUVEC with a ligand-acceptor dissociation constant of 290 nM. The binding of [125 I]-sCD14 was inhibited by sCD14 with a sCD14-acceptor dissociation constant of 24 nM. The density of CD14 acceptors was estimated to be 4-8 x 105 sites per cell. The [125I]-sCD14 binding was inhibited by anti-human CD14 mAbs UCHM-1 and RM052 but not 63D3. The bound [125I]-sCD14 could be washed off by acid buffer, pH 3.0, and its localization on the cell membrane was confirmed by light microscopic autoradiography. Based on the previously published description of LPS-sCD14 interactions and our observation that anti-CD14 mAbs inhibiting sCD14-acceptor binding also blocked the potentiation of LPS activity by sCD14, we propose that bridging or crosslinking between the putative LPS-receptor complex with the sCD14-acceptor complex via LPS-sCD14 interactions is the mechanism of CD14-dependent activation of endothelial cells (EC) by LPS. Re-examination of the published data suggests that this mechanism is a universal one for EC, leukocytes and lymphocytes.