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PubMed1991

Muscle-specific protein expression in normal salivary glands and pleomorphic adenomas: an immunocytochemical study with biochemical confirmation.

Normal salivary gland myoepithelia are contractile cells with hybrid epithelial/myogenic ultrastructural features. It is known that these cells co-express the intermediate filaments cytokeratin, vimentin, and occasionally GFAP. This complex cytoskeletal immunophenotype is also reflected in multiple morphologic cell types of pleomorphic adenoma. At present, the myofilament complement of normal and neoplastic myoepithelium is not well defined. We have evaluated the expression of desmin and smooth and sarcomeric muscle actins in 11 normal salivary glands (six snap-frozen and five methacarn fixed) and 26 pleomorphic adenomas (11 snap-frozen and 15 methacarn fixed) by ABC-immunoperoxidase method. Two of 11 frozen pleomorphic adenomas contained the muscle-specific intermediate filament desmin, which is not found in the normal glands. This novel finding was confirmed by gel electrophoresis and immunoblot. Using specific antibodies, normal gland myoepithelial cells consistently contained muscle actin isoforms of the smooth muscle type but not sarcomeric muscle actins. Muscle actin expression by the neoplastic cells of pleomorphic adenoma was found in 13 of 26 tumors (six of 11 frozen tumors (desmin negative) and seven of 15 methacarn fixed tumors). In comparison to the normal myoepithelial cell, the transformed myoepithelial-like cells of pleomorphic adenoma are not always characterized by a muscle actin cytoskeleton. Expression of desmin intermediate filaments in pleomorphic adenomas appears to be a rare event that is independent of a muscle actin cytoskeleton.

Actins↗
PubMed1991

Adhesion-related molecules in the central nervous system. Upregulation correlates with inflammatory cell influx during relapsing experimental autoimmune encephalomyelitis.

The expression of a battery of adhesion-related molecules and cytokines was investigated by immunocytochemistry in the central nervous system (CNS) of SJL/J mice sensitized for experimental autoimmune encephalomyelitis (EAE). These molecules consisted of the ligands MECA-325, intercellular adhesion molecule-1, and major histocompatibility complex molecules I and II, plus the receptors lymphocyte function-associated antigen-1, CD8, and CD4. The cytokines comprised interferon-gamma and tumor necrosis factor-alpha. EAE was induced by the adoptive transfer of myelin basic protein-sensitized lymphocytes. MECA-325, a marker for murine high endothelial venules in lymph node tissue, was absent from normal CNS tissue, was expressed at low levels on venules 24 to 48 hours before the onset of clinical signs, rose to maximal levels during acute disease, decreased to preclinical levels during remissions, and rose again during relapses. Intercellular adhesion molecule-1, major histocompatibility antigen-I, and major histocompatibility antigen-II showed similar fluctuations around CNS vessels. The receptors lymphocyte function-associated antigen-1 and CD4 fluctuated in parallel with the above molecules, whereas CD8 remained at a similar low level. Interferon-gamma was present during the acute, remitting, and relapsing phases and was localized to inflammatory cells, whereas tumor necrosis factor occurred at low levels only. Thus, several molecules associated with lymphocyte traffic in lymphoid tissue are selectively expressed in a stage-specific manner within the target organ, the CNS, during EAE. This suggests that the CNS may act as an ancillary organ of the immune system, and that cellular traffic into the CNS during EAE is related to the fluctuating expression of several distinct adhesion-related molecules, frequently co-expressed on the same vessel. The findings may have relevance to the sequence of events in the developing CNS lesion of multiple sclerosis.

Animals↗
PubMed1990

Identification of a novel integrin beta subunit expressed on cultured monocytes (macrophages). Evidence that one alpha subunit can associate with multiple beta subunits.

The vitronectin receptor (VnR) is one member of a subset of cell adhesion receptors within the integrin supergene family which shares the beta 3 subunit (IIIa). We show here that the VnR is absent from the surface of monocytes freshly isolated from blood but is expressed on these cells after a period of in vitro culture. Such cultured monocytes (macrophages) from a patient with type I Glanzmann's thrombasthenia, however, failed to express the VnR. Instead, immunoprecipitation with a monoclonal antibody directed to the VnR alpha chain (alpha v) revealed a novel integrin comprising alpha v associated noncovalently with a 100-kDa beta subunit (beta 3b), immunologically unrelated to the VnR beta subunit (beta 3a). This same novel integrin complex was also identified on 10-day-old macrophages from healthy donors, but on these cells, the beta 3b subunit was co-expressed with the classical VnR complex of alpha v beta 3a. The novel beta 3b subunit was not identified by monoclonal or polyclonal antibodies to IIIa (beta 3a) nor by a monoclonal antibody to the classical VnR complex. The beta 3b subunit could be distinguished from beta 3a by its relatively greater migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction, by its distinct isoelectric point upon two-dimensional gel electrophoresis, and by one-dimensional peptide mapping. Neither platelets nor B lymphoblasts from this patient with Glanzmann's thrombasthenia expressed any VnR on their surface, whereas control cells from a normal donor expressed the classical VnR but not the beta 3b subunit. The two beta chains, and hence also the combined receptor complexes, appeared to be differentially regulated. These findings provide the first example of an integrin alpha chain complexed with more than a single beta chain in the same cell. Furthermore, the differential regulation of expression of the different beta subunits that associate with the VnR alpha chain on cultured monocytes suggests a role for the novel receptor complex during monocyte/macrophage differentiation.

Adult↗
PubMed1990

Glycoprotein hormone alpha subunit in endocrine cells of human oxyntic mucosa. Studies on its relation to neuroendocrine tumors.

Expression of the alpha-subunit of glycoprotein hormones is an acquired feature of the endocrine cells of the oxyntic mucosa in patients with sustained serum levels of gastrin, and may be related to the hyperplasia-carcinoid sequence occurring in these patients. In the present study we have investigated the intragastric cellular localization and the circulating levels of alpha-subunit in a patient with Zollinger-Ellison syndrome. In this patient we have found that: 1) Endocrine cells accounted for 2.29% +/- 1.44% of the total oxyntic mucosal volume (normal value: 0.9% +/- 0.4%), with the ECL cells representing 63.22% +/- 10.9% of the total endocrine cell volume (normal value: 29.8 +/- 8.8%). 2) Cells immunoreactive for the alpha-subunit were found to correspond ultrastructurally to a subpopulation of enterochromaffin-like cells, indistinguishable from similar cells devoid of significant immuno-electron microscopic labeling. 3) Immunoreactive cells included a portion of oxyntic endocrine cells with punctate granules, a feature previously observed only in carcinoid tumors of the oxyntic mucosa. 4) In consecutive sections of freeze-dried vapor-fixed biopsies a fraction of alpha-subunit storing cells was found to co-express histamine. 5) The serum alpha-subunit levels were abnormally elevated and paralleled those of gastrin in a secretin-stimulation test. Analysis of similar curves in two other patients with Zollinger-Ellison syndrome, and five patients with hypergastrinemic atrophic gastritis, all presenting alpha-subunit containing oxyntic endocrine cells, showed significant alpha-subunit elevations only in the patients with ulcerogenic syndrome.(ABSTRACT TRUNCATED AT 250 WORDS)

Gastric Mucosa↗
PubMed1990

Two populations of CD56 (Leu-19)+/CD16+ cells in bone marrow transplant recipients.

Bone marrow transplant (BMT) recipients are severely immunocompromised during the early post-transplant period as their immune systems recapitulate. Among the first cells to repopulate the peripheral blood are natural killer (NK) cells. Studies in normal subjects have revealed that the majority of NK cells express CD16 and CD56 (Leu-19). Such NK cells express low density, dim, Leu-19 (Leu-19D+). A small percentage of high-density, bright, Leu-19 cells (Leu-19B+) which are CD16- have also been reported in normal subjects. In this study we observed that peripheral blood mononuclear cells (PBMC) from BMT recipients contained two distinct populations of Leu-19+ cells that co-expressed CD16 and these populations could be separated on the basis of differential Leu-19 and CD16 fluorescence intensities. Leu-19B/CD16D and Leu-19D/CD-16B cells were present in four of nine BMT patients studied. Cells from one BMT recipient with a very large expansion of the Leu-19B+ population (46% of PBMC by flow cytometry) were studied in detail. While this patient is not characteristic of all BMT recipients, the large number of Leu-19+ cells that could be isolated from him allowed extensive analysis of a previously unreported population of cells. Our data suggest that BMT recipients can be an important group of subjects to evaluate NK cell subsets as these cells mature and, presumably, differentiate in a regenerating immune system.

Adolescent↗
PubMed1990

An ultrastructural study of normal human thymic epithelium in primary cultures.

The ultrastructural study of a large series of samples of normal human thymic epithelial cells (TECs) in primary cultures is here presented. Cultured TECs are heterogeneous due to marked differences in their cytoplasmic organization. In particular, a spectrum of ultrastructural features have been observed, ranging from poorly-differentiated cells to secretory and epidermoid-like ones, as well as intermediate transitional forms and cells co-expressing both epidermoid-like and secretory features. Cells with peculiar secretory (neuroendocrine-like) characteristics have also been identified in the culture obtained from one of the donors. These data suggest the existence of a thymic epithelial cell type which may act as a dynamic unit, changing its cytoplasmic organization in response to microenvironmental signals. The types and interrelationships of cytoplasmic organelles observed in the secretory cells suggest that the production of thymic peptides involves the classical pathway of protein synthesis and secretion.

Cell Differentiation↗
PubMed1990

Immunohistochemical study of high-grade endometrial stromal sarcoma. An autopsy report in comparison with carcinosarcoma, leiomyosarcoma and normal endometrium.

An autopsy case of high-grade endometrial stromal sarcoma (ESS) is reported. Immunohistochemical study of the ESS was done in comparison with carcinosarcoma (CS), leiomyosarcoma (LMS) and normal endometrium in the uterus in order to trace the origin of ESS, which is a point of some controversy. Co-expression of keratin and vimentin and/or desmin positivity in several elements of CS and LMS, and glandular or stromal tissues in normal endometrium made it difficult immunohistochemically to be certain of the origin of ESS.

Antigens↗
PubMed1990

Overexpression of wild-type and dominant negative mutant vimentin subunits in developing Xenopus embryos.

Vimentin belongs to the diverse multigene family of intermediate filament proteins, each member of which is expressed in a tissue-specific and developmentally regulated pattern. The existence of vimentin filaments has been documented in oocytes, eggs, and early embryos of Xenopus laevis, but the role of these cytoskeletal components remains unknown. To investigate the functions of vimentin during early development in Xenopus, we induced the overexpression of wild-type and deletion mutant subunits in most of the cells of embryos by injecting synthetic RNA into fertilized eggs. Wild-type vimentin subunits, as well as subunits lacking most of the amino-terminal head piece, assembled into normal appearing filaments in vivo. Deletion mutants of the fourth alpha-helical rod domain were assembly incompetent and dominantly inhibited the polymerization of wild-type subunits when both types of subunit were co-expressed in cells. Expression of at least a tenfold excess of wild-type or mutant subunits within cells of embryos did not lead to any detectable morphological or developmental abnormalities, suggesting that the presence and proper regulation of vimentin expression is not essential during the initial stages of embryogenesis in Xenopus.

Amino Acid Sequence↗
PubMed1990

Graft failure after T cell-depleted bone marrow transplantation: clinical and immunological characteristics and response to immunosuppressive therapy.

We have investigated the clinical and immunological features of 10 cases of graft failure after T cell-depleted marrow transplantation. In addition, the hypothesis that the process of graft failure can be reversed by immunosuppressive therapy with cyclosporin + steroids +/- monoclonal antibodies was tested in seven patients. Early graft failures (before day 50) presented a uniform clinical syndrome with a host T lymphocytosis preceding the loss of the graft. In the majority of cases of late graft failure (after day 50) a syndrome comprising delayed granulopoietic regeneration, fever of unknown origin and abdominal symptoms was observed. Surface marker analysis of peripheral blood and bone marrow lymphocytes implicated a population of CD3+, CD8+, DR+ host T lymphocytes with a frequent co-expression of the Leu7+ antigen in the pathogenesis of graft failure. Immunosuppressive therapy reversed graft failure in the three cases of incomplete graft failure (i.e. with residual reticulocytes) and failed in the four cases of complete graft failure (i.e. no residual reticulocytes).

Antibodies, Monoclonal↗
PubMed1991

CD59 functions as a signal-transducing molecule for human T cell activation.

The CD59 Ag is a 20-kDa protein that is widely expressed on most leukocytes and RBC, is coupled to the membrane by a phosphatidylinositol-glycan anchoring structure, plays a role in cell interaction between monocytes and T cells, and also functions as an inhibitor of cytolysis by the terminal C components C5b-9. Because this molecule is structurally related to the murine Ly-6 family of Ag, we have investigated whether anti-CD59 mAb might be capable of activating human T lymphocytes in a manner similar to that described for antibodies to the murine Ly-6 Ag. In the presence of the appropriate co-stimulators, mAb to one of the two epitopes on CD59 were capable of inducing both a rise in intracytoplasmic free Ca2+, inositol phosphate production, IL-2 production, and T cell proliferation. Anti-CD59-induced inositol phosphate turnover and IL-2 production were dependent on co-expression of the CD3/TCR complex. CD59-loss mutants of the Jurkat cell line were completely responsive to stimulation by anti-CD3 thereby demonstrating that CD59 does not play a role as a signal transducer downstream from the TCR. Taken together, these results demonstrate that the CD59 Ag can play multiple distinct roles in the regulation of the immune response.

Antibodies, Monoclonal↗
PubMed1991

Expression of intermediate filament proteins in fetal and adult human kidney: modulations of intermediate filament patterns during development and in damaged tissue.

The expression of intermediate filament proteins, particularly individual cytokeratins (CKs), vimentin, and glial filament protein, was immunohistochemically investigated using frozen sections and Carnoy-fixed, paraffin-embedded tissue from normal fetal and adult human kidneys as well as from pathologically altered kidneys. In fetal kidneys, the co-expression of CKs and vimentin was detected in the visceral and parietal epithelium of the glomerulus, the proximal tubules, the thin loops of Henle, and the collecting ducts. In contrast, in the tubules of normal adult kidneys, the presence of vimentin and CKs was nearly always mutually exclusive. While CKs 8 and 18 were present in all tubular epithelia, CKs 19 and 7 each exhibited a distinctive distribution pattern, there being a striking alteration between positive and negative segments and, not infrequently, intratubular heterogeneities. In certain segments, particular cell types (e.g., "plica cells," intercalated cells) could thus be recognized. In tubular epithelia altered by various injurious conditions, novel or enhanced expression of vimentin, CK 19 and CK 7, and, less frequently, CK 17 and glial filament protein was noted in certain segments. The increase in intermediate filament protein expression in altered (particularly proximal) tubules appeared to parallel the reduction in the degree of differentiation. Vimentin was never detected in distal tubules. The present results reveal a considerable similarity between the intermediate filament patterns in non-neoplastic proximal tubules of fetal and damaged kidney tissue and those in clear-cell and chromophilic renal cell carcinomas. They also serve to illustrate that the analysis of both fetal development and reactive cell changes may significantly contribute to our understanding of differentiation phenomena in malignant tumors.

Antibodies, Monoclonal↗
PubMed1991

The product of the proto-oncogene c-kit (P145c-kit) is a human bone marrow surface antigen of hemopoietic precursor cells which is expressed on a subset of acute non-lymphoblastic leukemic cells.

A monoclonal antibody (17F11) was raised by immunization of a Balb/c mouse with leukemic blasts from a patient with acute non-lymphocytic leukemia (ANLL). This antibody recognizes most leukemic blasts of myeloid but not of lymphoid lineage and no peripheral blood cells. By screening NIH-3T3 fibroblasts transfected with the human proto-oncogene c-kit (NIH-3T3/hckit) it could be shown that 17F11 specifically recognizes the gene product P145c-kit. Immunofluorescence analysis on normal hemopoietic cells revealed that 17F11 weakly stains 1-3% of bone marrow mononuclear cells (BMMNC). By FACS sorting and colony assays it could be shown that granulocyte--macrophage progenitor cells could be enriched 10-20-fold, granulocyte progenitors 50-80-fold, and erythroid and multipotential progenitor cells 15-20-fold, in the 17F11 positive fraction. Double fluorescence analysis revealed that P145c-kit is co-expressed on 40-60% of the CD34 positive BMMNC. Finally, these data show that P145c-kit is expressed on blast cells from most patients with ANLL (26/30) and chronic myeloid leukemia in blast crisis (7/9), but is absent on blasts from patients with acute lymphoblastic leukemia expressing the T-, B-lineage, or common ALL phenotypes.

Animals↗
PubMed1991

Molecular diversity of cytokeratins: significance for cell and tumor differentiation.

Normal and transformed epithelial cells are characterized by the expression of a distinct class of intermediate filaments (IFs), the cytokeratin (CK) filaments. Their constituents, the CKs, comprise a complex multigene family of related proteins which can be subdivided into two sequence types (I and II). In the various epithelial cell types (excluding trichocytes), 19 different CK polypeptides (CKs 1-19) have been distinguished until recently. An additional cytoskeletal polypeptide of Mr 46,000 has been detected, by gel electrophoresis and by immunocytochemistry, in certain types of epithelia including gastric foveolar epithelium, small and large intestinal epithelium, urothelium, and epidermal Merkel cells. On the basis of its biochemical properties and considerable sequence homologies with several type I CKs, this new cytoskeletal protein is suggested to be included in the catalogue of human CKs as CK 20. The various CK polypeptides, as detected by gel electrophoretic and/or immunocytochemical analysis, are expressed in different epithelia and carcinomas in various combinations in a differentiation-dependent manner. Moreover, co-expressions of CKs with other IF classes (vimentin, neurofilaments, glial filaments) are also characteristic of certain epithelial differentiation lineages. Both non-neoplastic epithelial alterations as well as malignant transformation may result in similar modifications of the IF expression profiles although there is a considerable tendency of conservativity. In several instances, a reduced degree of differentiation is paralleled by an increased complexity of the IF protein pattern. CK (and IF) typing can be successfully used in the tracing of developmental lineages as well as in the histological differential diagnosis of primary and metastatic carcinomas. Certain CK polypeptides (CKs 5, 6, 14, 16, 17) identify squamous cell carcinomas including poorly differentiated ones, while other CKs, including CK 20, are typical of primary and metastatic urothelium-derived carcinomas. Among simple-epithelial tumors, CK analysis is of potential diagnostic value in the distinction of mesotheliomas from adenocarcinomas, the discrimination of different carcinomas within the gastrointestinal tract, and the distinction of certain gastrointestinal carcinomas from other adenocarcinoma types including breast and lung carcinomas. Among the individual CKs, CKs 5, 7, 13, 14, 19, and 20 appear to be particularly useful for diagnostic purposes. An important task in the future will be the development of additional monospecific CK-antibodies, particularly such which work on routine paraffin sections.

Cell Differentiation↗
PubMed1991

Interleukin 4-mediated induction of CD4+/CD8+ T cells during infancy.

Neonatal human CD4+ T cells will co-express CD8 on their surface following short-term culture with interleukin 4 (IL-4). Adult T cells do not respond in this manner. In this study we examine this phenomenon as a function of age and determine that IL-4 responsiveness decreases with time to approach adult levels at about 2 years. This phenomenon may be relevant to the documented ability of neonatal CD4+ T cells to function as suppressors.

Adolescent↗
PubMed1991

FMS (CSF-1 receptor) and CSF-1 transcripts and protein are expressed by human breast carcinomas in vivo and in vitro.

The expression in vivo of FMS transcripts and antigen by neoplastic epithelial cells was demonstrated immunohistochemically or by in situ hybridization in sixteen of seventeen human breast carcinoma specimens and one case of sclerosing adenosis. Expression of CSF-1 receptor (FMS) transcripts and protein was also observed in vitro in two or three breast carcinoma-derived cell lines and was dramatically increased by dexamethasone, a potent glucocorticoid and inducer of mammary epithelial cell differentiation. Immunohistochemical staining with an anti-CSF-1 antibody identified neoplastic epithelial cell co-expression of fms and CSF-1 antigens in more than one-third of the fms-positive invasive carcinoma specimens. These results suggest that autocrine and paracrine interactions of the lymphohematopoietic cytokine CSF-1 and its receptor may participate in the biology of human mammary neoplasms.

Blotting, Northern↗
PubMed1991

Induction of Fc epsilon RII/CD23 on phytohemagglutinin-activated human peripheral blood T lymphocytes. I. Enhancement by IL-2 and IL-4.

Despite evidence for the expression of low affinity Fc receptor for IgE (Fc epsilon RII)/CD23 in T cell lines and pathologic T cells, Fc epsilon RII/CD23 in normal human T cells is still unclear. We studied the expression of Fc epsilon RII/CD23 on T cells in short-term culture of normal human PBMC stimulated with 15 micrograms/ml PHA. PHA stimulation also resulted in the release of soluble Fc epsilon RII/CD23 (IgE binding factor). Using two-dimensional flow cytometry, more than 10% of the Fc epsilon RII/CD23+ cells were found to co-express CD3 Ag. Both CD4+ and CD8+ T cells expressed Fc epsilon RII/CD23. The induction of Fc epsilon RII/CD23 on PHA-activated T cells was enhanced by IL-2 as well as IL-4. Both IL-2 and IL-4 also augmented PHA-induced production of soluble Fc epsilon RII/CD23. The enhanced expression of Fc epsilon RII/CD23 on T cells by both lymphokines was suppressed by rabbit anti-IL-4 antiserum, suggesting the involvement of an IL-4-dependent process even in the IL-2-dependent Fc epsilon RII/CD23 expression on T cells. The expression of mRNA for Fc epsilon RII/CD23 on PHA and IL-4-stimulated PBMC was examined by Northern blot analysis. Fc epsilon RII/CD23 mRNA was detected in RNA prepared from the T cell fraction depleted of B cells and macrophages (Fc epsilon RII+CD3+ = 6.2%, Fc epsilon RII+CD3- = 0.8%). The expression of the mRNA for Fc epsilon RII/CD23 on CD3+ T cells was also confirmed by in situ hybridization with Fc epsilon RII/CD23 cDNA combined with CD3 rosette formation at the single cell level.

Antibodies, Monoclonal↗
PubMed2025

Whole genome duplication drives transcriptome reprogramming in response to drought in alfalfa.

Genome doubling did not enhance drought tolerance in alfalfa, but may set the stage for long-term adaptation to drought through a novel transcriptional landscape. Whole genome duplication (WGD) has been shown to enhance stress tolerance in plants. Cultivated alfalfa is autotetraploid, but diploid wild relatives are important sources of genetic variation for breeding. Investigating how WGD affects gene expression in stress conditions could provide better understanding for use of diploid genetic resources. In this work, we compared the drought response of neotetraploid plants obtained by bilateral sexual polyploidization with diploid full sibs, by measuring physiological and biochemical traits and RNA-seq. Without drought, 4x plants had lower photosynthetic potential than 2x plants per unit leaf area, but larger leaves allowed them to outperform the per leaf photosynthetic potential of 2x plants. Physiological and biochemical traits were significantly affected by drought in both 2x and 4x plants, but the differences between ploidies were small and nonsignificant. Proline levels were higher in 4x than 2x plants, both in control and drought conditions, indicating that larger cells with higher volume-to-surface ratio of 4x  plants require a higher osmolyte concentration. RNA-seq and gene network analyses showed that more genes were affected by drought at 4x than at 2x level, with downregulation of hundreds of genes involved in photosynthesis and stomatal movement at 4x level, suggesting that WGD made the 4x plants more responsive to drought. Genes involved in proline, phytormone and cell wall functions were also transcriptionally affected by drought in 4x plants. We conclude that WGD did not immediately enhance drought tolerance in alfalfa, but may set the stage for long-term adaptation to drought through a novel transcriptional landscape.

Medicago sativa↗