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Biomedical subjects

L A Lampson

Publications and source records attributed to L A Lampson.

At least 37 records · Page 2Linked to original sources

Human neuroblastoma cell growth in xenogeneic hosts: comparison of T cell-deficient and NK-deficient hosts, and subcutaneous or intravenous injection routes.

We have examined two features of neuroblastoma cells that had not been well-characterized in a xenogeneic model: The cells display unusual immunologic properties in other experimental systems, and the original tumors display widespread and characteristic patterns of metastasis. To determine the most appropriate immunodeficient host for primary tumor growth, T cell-deficient nude mice, NK-deficient beige mice, beige-nudes, and controls were injected with the well-characterized line CHP-100. To define the pattern of tumor spread, complete autopsies were performed following subcutaneous, intraperitoneal and intravenous injections. CHP-100 consistently formed subcutaneous tumors in T cell-deficient mice (nude and beige-nude), but not in T cell-competent mice (beige, heterozygous nu/+ and bg/+, or wild-type). The growth rate and final size of the subcutaneous tumors were not greater in beige-nudes than in nudes. All mice showed early CHP-100 cell death after subcutaneous injection; the nature of the immunodeficiency was more relevant for the surviving subpopulation. Widespread dissemination was seen following intravenous injection, particularly in beige-nudes. Aspects of the growth patterns were appropriate to the tumor of origin. The behavior in immunodeficient mice suggests that T cells can play a role in controlling the growth of these cells; the next steps will be to define the effector mechanisms, and to determine if they can be exploited for human patients. The hematogenous spread following intravenous injection suggests that insights into the control of blood-borne tumor may also come from further study of this model.

Animals↗

The effects of irradiation on major histocompatibility complex expression and lymphocytic infiltration in the normal rat brain and the 9L gliosarcoma brain tumor model.

The effects of irradiation on major histocompatibility complex (MHC) expression and lymphocytic infiltration in the normal rat brain and the 9L gliosarcoma brain tumor model were examined. Doses of irradiation administered were biologically equivalent to that used in the treatment of patients with malignant gliomas. No significant change in immune parameters was observed following irradiation in the normal rat brain. In the 9L gliosarcoma model irradiation did not suppress MHC expression or lymphocytic infiltration. These findings suggest that prior exposure to therapeutic irradiation need not adversely affect subsequent immunotherapies, and provide a foundation for future studies of immunomodulation in the irradiated brain.

Animals↗

IFN-treated neuroblastoma cell lines remain resistant to T cell-mediated allo-killing, and susceptible to non-MHC-restricted cytotoxicity.

Neuroblastoma cell lines can have very low MHC Ag expression. The cell lines are insensitive to allo-killing by primed CTL, but are sensitive to non-MHC-restricted cytotoxicity. IFN-gamma increased class I expression, but the cells remained insensitive to CTL. Susceptibility to nonrestricted effectors was preserved. Class I+ glioma cell lines behaved similarly. The CTL resistance was localized to the recognition phase. Neuroblastoma lines did not form conjugates with primed T cells, but were lysed if they were coupled to the effectors via lectins. The levels of class I expression, and resistance to CTL, were constant over a range of IFN doses. HLA-A,B,C structure and distribution were studied more intensively on one cell line, CHP-100. HLA-A2 and -A3 were present on greater than or equal to 99% of the cells, in a unimodal distribution. After IFN treatment, the levels were similar to B cell controls. In two-dimensional gel electrophoresis, the molecules co-migrated with those of B cell controls. The defect may thus be in accessory proteins that are necessary for T cell recognition or binding, rather than in the structure or distribution of the HLA-A,B,C proteins.

Cell Adhesion↗

Biological significance of HLA-A,B,C expression in neuroblastoma and related cell lines.

In these studies, strong HLA-A,B,C or b2-m expression was not detected in any stage of development of normal neurons. These included regenerating cells of the olfactory epithelium, developing neural tissue in the mouse embryo, and normal adult brain. Nor was an obligatory role for HLA-A,B,C or b2-m revealed by in vitro analysis of a growing neuroblastoma cell line.

Animals↗

Expression of MHC class I genes in mouse hepatitis virus (MHV-A59) infection and in multiple sclerosis.

Our studies revealed that virus induced demyelination as well as human inflammatory demyelination involves upregulation of class I MHC genes and surface expression of antigens encoded by these genes. Induction involves the action of an intermediate soluble factor/s which is at present unknown. These findings suggest that MHC class I restricted, cytotoxic T lymphocyte (CTL) reactions, against self or foreign antigens may play a role in these conditions. These findings may help to elucidate the mechanism of coronavirus-induced demyelination as well as the pathogenesis of multiple sclerosis.

Animals↗

Expression of mouse beta 2-microglobulin in frozen and formaldehyde-fixed central nervous tissues: comparison of tissue behind the blood-brain barrier and tissue in a barrier-free region.

Previous work indicates that the weak expression in neural tissues of beta 2-microglobulin (beta 2-m) and major histocompatibility complex (MHC) class I gene products can be increased experimentally. Physiologic conditions in which greater neural MHC expression occurs are not well defined. Here we have asked whether protection from blood-borne antigens afforded by the blood-brain barrier is related to the lack of MHC expression. A rabbit antiserum raised against purified mouse beta 2-m was used in an immunocytochemical assay. The serum reacted strongly with lymphoid tissues and was inhibited by purified beta 2-m. No beta 2-m was detected in neurons or glia in any brain area examined. A barrier-free region, the area postrema, showed the same lack of neural cell staining. Blood vessel walls in the same sections were beta 2-m+. It is unlikely that these staining patterns are due to cell type-specific beta 2-m degradation, since frozen and formaldehyde-perfused, paraffin-embedded preparations gave similar results. Failure to detect beta 2-m in the area postrema suggests that passive exposure to environmental antigens, immunomodulators, or immunocompetent cells is not sufficient to induce neural class I expression. Rather, if increased expression of beta 2-m and class I occurs in vivo, additional stimulus is required.

Animals↗

Distribution of beta 2-microglobulin in olfactory epithelium: a proliferating neuroepithelium not protected by a blood-tissue barrier.

The olfactory neuroepithelium is unique in adult vertebrates in that bipolar sensory neurons are constantly dying and being replaced. The sensory neurons are also unusual because they are directly exposed to the external environment via their dendritic processes in the nasal cavity. Surveillance of this tissue by major histocompatibility complex (MHC) class I-restricted cytotoxic T cells would presumably serve as an important means of defense against foreign pathogens. Although adult brain shows a lack of class I molecules, it has not been reported if either proliferating neurons or sensory neurons in olfactory neuroepithelium also lack class I. To examine olfactory neuroepithelium, an antiserum against beta 2-microglobulin (beta 2-m), the invariant light chain associated with all class I molecules, was employed as a general probe in an immunocytochemical assay. beta 2-m was detected in columnar respiratory epithelium, blood vessel walls, and a small population of interstitial cells in the lamina propria, but no cell in the olfactory neuroepithelium stained for beta 2-m. Parallel patterns were obtained in the vomeronasal organ. These results suggest that lack of beta 2-m, and presumably class I, may be a general phenotype of neuronal cells regardless of their mitotic state or exposure to environmental antigens.

Animals↗

Monoclonal antibody analysis of MHC expression in human brain biopsies: tissue ranging from "histologically normal" to that showing different levels of glial tumor involvement.

The expression of class I and class II MHC products in human brain was studied. Radioimmunoassay confirmed weak expression of HLA-A,B,C and beta 2-microglobulin (beta 2-m) in brain extract. Quantitative inhibition assay showed brain had 1/70 as much activity as spleen, per microgram of extract protein. Immunoblot assay confirmed that HLA chains and beta 2-m were present in the brain extract. Class II was not detected. Microscopic analysis was performed on eight brain biopsies. The histologic appearance ranged from "apparently normal," to the presence of reactive astrocytes, to the presence of glial tumor. In every case, HLA-A,B,C and beta 2-m activity was concentrated at blood vessel walls. Small and medium-sized vessels were uniformly stained. Cell body staining was not seen in neurons, glia, oligodendrocytes, microglia, reactive astrocytes, or the majority of glial tumor cells. Class II activity was seen in occasional cell bodies in both grey matter and white matter in the microscopic assays. These cells had the morphologic appearance of microglia or reactive astrocytes. Occasional blood vessels also showed class II activity. Unlike the class I activity, the class II blood vessel stain was often discontinuous. More class II+ cell bodies were seen in tumor-associated tissue.

Antibodies, Monoclonal↗

Interferon-mediated induction of class I MHC products in human neuronal cell lines: analysis of HLA and beta 2-m RNA, and HLA-A and HLA-B proteins and polymorphic specificities.

Gamma-interferon (IFN-gamma) increases class I major histocompatibility complex (MHC) expression in human neuroblastoma cell lines. These cells are of interest because of the initial paucity of MHC expression, a paucity that is also seen in neural tumors and normal brain. The aim of this study was to define further the class I molecules, and to begin to analyze the genetic basis of the regulation. Northern blot analysis with cDNA probes for HLA and beta 2-microglobulin (beta 2-m) RNAs shows that both are present in reduced quantities (relative to a B-cell control) in control neuroblastoma cells. The levels of both RNAs are increased following IFN-gamma. This behavior parallels that of the corresponding polypeptides. Further monoclonal antibody analysis of the class I proteins from IFN-treated cells shows that both HLA-A and HLA-B are present. For two cell lines, expression of appropriate polymorphic specificities is also shown to be increased. We conclude that IFN-gamma can cause increased expression of appropriate HLA-A,B,C specificities on cells of neuronal origin. This raises the question of whether these molecules can serve predicted immunological functions.

Cell Line↗

Molecular specificity of defined types of amacrine synapse in cat retina.

The inner plexiform layer of cat retina contains synaptic structures belonging to 50 or more types of "identified" neurons. To learn whether there are antigens confined to subsets of these synaptic structures, we raised monoclonal antibodies to homogenates of neural retina. Binding patterns of these antibodies were visualized by the peroxidase-antiperoxidase method and studied in serial, ultrathin sections by electron microscopy. Four antibodies stained the synaptic varicosities of certain amacrine cells. Many of the stained varicosities formed reciprocal synapses with a rod bipolar axon terminal, but only about half of the reciprocal synapses associated with a rod bipolar were stained. Other stained varicosities formed synapses with cone bipolar axons, ganglion cell dendrites, and unstained amacrine processes. The patterns were essentially the same for each antibody and were not altered by staining with the antibodies two at a time; therefore, it is likely that all four antibodies stain the same subset of synaptic structures. These patterns would be accounted for if there were staining of all the synaptic varicosities of three of the four types of identified amacrine reciprocally connected to the rod bipolar (A6, A8, A13). This localization suggests that the antigen responsible for the binding pattern is not associated with synaptic transmission. Staining is present in the inner plexiform layer during the period of synaptogenesis and consequently the antibodies are serving as markers for following the development of identified synapses in an identified neural circuit.

Animals↗

Immunoblot specificity of monoclonal antibodies assayed against complex extracts.

Monoclonal antibodies of known specificity, and target cells of known antigen expression, were used to evaluate the specificity of the immunoblot assay against complex extracts. The antibody panel included 10 previously characterized monoclonals to antigens of known structure: HLA-A,B,C; Ia; and actin. In addition, a commercial reagent was shown to react with HLA chains in immunoblots, and not to cross-react with actin. Target cells known to have strong, weak, or negative expression of the antigens were examined. In all cases, the results were in complete accord with the established properties of the antibodies, antigens, and target cells. For example, HLA bands were detected in lymphoid cells with strong HLA-A,B,C expression, and also in a neuronal cell line with 0.5% of the lymphoid activity. Cytoskeletal cross-reactions, or other unexpected specific bands, were not observed. This work lays a foundation for interpreting immunoblot analyses of monoclonal antibodies to newly defined proteins of complex tissue.

Actins↗

Human neuroblastoma cell lines are susceptible to lysis by natural killer cells but not by cytotoxic T lymphocytes.

The susceptibility of human neuroblastoma cells to direct cellular cytotoxicity has not been previously established. This is of particular interest because of their aggressive growth and low HLA expression. Neuroblastoma lines CHP 100 and CHP 126 were found to be excellent targets in 4-hr CML assays. Natural killer (NK) cells from fresh PBL and from an NK clone, 3.3, have high lytic activity against both cell lines. We also studied mixed lymphocyte culture-generated cytotoxic lines containing allo-specific cytotoxic T lymphocytes (CTL) directed against HLA antigens present on the neuroblastoma target cell lines. These lines did show excellent lytic activity, but cold target competition studies indicated that all of the lysis resulted from NK activity. This was verified by using inhibition studies with the use of monoclonal antibodies. OKT 3 and anti-HLA antibodies that block CTL function caused no reduction in kill. In contrast, anti-lymphocyte function antigen-1 (anti-LFA-1), which blocks both NK and CTL function, significantly inhibited lysis. These results serve as a functional confirmation of earlier findings of a very weak expression of HLA-A,B,C and beta 2-microglobulin on neuroblastoma cells.

Antibodies, Monoclonal↗

HLA class I and beta 2-microglobulin expression in frozen and formaldehyde-fixed paraffin sections of neuroblastoma tumors.

Ten solid neuroblastoma tumors were examined for beta 2-microglobulin (beta 2-m) and HLA-class I expression in an immunocytochemical assay. Blood vessel endothelium and a small population of cells (less than 6% of small round cells) were consistently stained in frozen sections of each tumor. No beta 2-m or HLA-class I reactivity was detected in the remainder (greater than 94%) of the small round cells in each tumor. The localization of most of the positive cells near stromal tissue and blood vessels suggests that most of these cells may be of non-tumor origin. The patients from which the tumors were taken varied in degree of disease involvement, yet no differences with respect to beta 2-m and HLA-class I expression were found among the tumors. A procedure for visualizing beta 2-m and HLA-class I molecules in formaldehyde-fixed, paraffin-embedded tissue is described. This confirmed the results in frozen sections. These findings extend previous reports of weak beta 2-m and HLA-class I levels in cells of neuronal origin. The clinical implications are discussed.

Child↗

Monoclonal antibodies against the voltage-sensitive Na+ channel from mammalian skeletal muscle.

A panel of 13 monoclonal antibodies against the voltage-sensitive Na+ channel of rat skeletal muscle has been characterized. Each of these antibodies reacted with the purified Na+ channel protein in a solid-phase radioimmunoassay. Nine antibodies specifically immunoprecipitated the Na+ channel in a form that retained its characteristic high affinity for saxitoxin, and 11 recognized the channel in a crude mixture of solubilized membrane proteins separated on a Sepharose CL-6B column. Six antibodies specifically labeled skeletal muscle in immunofluorescence techniques. In each case, antibody was localized only to the surface membrane of the muscle fibers. Eleven antibodies produced detectable reaction on immunoblot transfers of sarcolemmal membrane proteins; each of these bound to a diffuse 160- to 200-kDa band that comigrated with the large glycoprotein subunit of the purified Na+ channel. Further studies were carried out with one of these antibodies, L/D3. In immunoblots of a glycoprotein fraction prepared from muscle that had been homogenized rapidly in a solution containing detergent, EGTA, and protease inhibitors, L/D3 recognized only a single 260-kDa band. Incubation of solubilized muscle proteins at 4 degrees C for 24 hr without EGTA prior to isolation of the glycoprotein fraction resulted in partial conversion of this 260-kDa component to a smaller component between 160 and 200 kDa that comigrated with the principal immunoreactive component of sarcolemma. Based on its immunoreactivity with monoclonal antibodies, the large subunit of the rat skeletal muscle Na+ channel appears to be approximately equal to 260 kDa in its native state but may be sensitive to proteolysis during the isolation of sarcolemmal membranes.

Animals↗

Weak HLA and beta 2-microglobulin expression of neuronal cell lines can be modulated by interferon.

Previous work showed that each of four human neuronal cell lines expresses less than or equal to 0.5% of the HLA-A,B,C and beta 2-microglobulin seen in glial, lymphoid, and other cell types, and there is a corresponding weak expression in neuroblastoma tumor and adult brain. Here, we probe the genetic basis of this weak expression. For each of three neuroblastoma cell lines, we show that HLA-A,B,C and beta 2-microglobulin can be induced by interferon and that the induction occurs within every cell of the population. Class II (Ia) molecules are not detected. Microscopic assay and radioimmunoassay of intact cells suggest that the induced antigen appears at the cell surface as well as within each cell. Immunoblot analysis confirms that the induced proteins have the structure of class I molecules. Thus, the normal weak HLA and beta 2-microglobulin expression of these cell lines appears to reflect a regulatory control rather than a primary genetic lesion. According to current theory, lack of HLA-A,B,C should protect transformed, infected, or damaged neurons--but also neurons in neural transplants--from T-cell-mediated immunosurveillance. The possibility that neuronal HLA-A,B,C expression may be under regulatory control is of importance in this context.

Antibodies, Monoclonal↗