Immunoelectron microscopic localization of gelatinase A in human gastrointestinal and skin carcinomas: difference between cancer cells and fibroblasts

Previous reports revealed a discrepancy in gelatinase A localization in human cancers; i.e., protein localization in cancer cells and mRNA localization in stromal fibroblastic cells. To clarify this, we conducted immunoelectron microscopic study of gelatinase A in cancer and stromal cells in human gastrointestinal and skin carcinomas. Although both carcinoma cells and fibroblasts were positive for gelatinase A, the subcellular localizations were different. On immunoelectron microscopy, fibroblasts showed immunoreactivity in the lumen of the rough endoplasmic reticulum (rER) or in the cytosol on the surface of rER, demonstrating synthesis of the protein. Carcinoma cells showed diffuse deposition of gelatinase A in the cytosol, suggesting the accumulation of the antigen both in adenocarcinoma and squamous cell carcinoma. Immunoreactivity along the cell membrane was demonstrated in one case of skin carcinoma. Macrophages showed also diffuse deposition of gelatinase A in the cytosol. In conclusion, we found a qualitative difference of gelatinase A localization between carcinoma cells and fibroblasts, and concluded that carcinoma cells may not be important in the secretion of gelatinase A.     

Ultrastructural and immunohistochemical studies of intralobular fibroblasts in human submandibular gland: the recognition of a 'CD34 positive reticular network' connected by gap junctions

In order to clarify the ultrastructural and immunophenotypic characteristics of intralobular stromal cells in the human submandibular gland, normal tissues were examined from 25 patients by conventional histology of wax sections, light microscope immunohistochemistry, conventional thin-sectioning transmission electron microscopy, immuno-electronmicroscopy (for the CD34 antigen) and scanning electron microscopy. Spindled stromal cells had abundant rough endoplasmic reticulum cisternae typical of fibroblasts. Some actin filaments and microtubules were seen, but the filaments lacked the focal densities of smooth-muscle, nor was a lamina present. Fibroblasts exhibited long slender processes which associated with other fibroblasts by means of gap junctions and with mononuclear cells (lymphocytes, plasma cells, macrophages) by means of close appositions of membrane. Immunoelectron microscopy for CD34 revealed positive staining over the fibroblast surfaces but not over the mononuclear cells. The novel observation of gap junctions suggests a tissue closely organized as a network in physical and perhaps physiological terms. This network also incorporates mononuclear cells. The possible role in immunosurveillance of this CD34-positive reticular network is discussed.     

Expression of ICAM-1, VCAM-1, and LFA-1 in adenocarcinoma of the lung with observations on the expression of these adhesion molecules in non-neoplastic lung tissue

Intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1), and the lymphocyte function-associated antigen (LFA-1) are cell adhesion molecules thought to play an important role in the complex process of airway inflammation and tumor cell growth. The aim of this study was to examine the distribution of ICAM-1, VCAM-1, and LFA-1 in adenocarcinoma of lung and in major cellular compartments of non-neoplastic lung tissue. We examined cellular compartments in tissue from five bronchoalveolar carcinomas, three acinar adenocarcinomas, and one colon cancer metastatic to the lung. The compartments in neoplasms included the tumor cells proper, endothelial cells within the tumor vasculature, tumor stromal cells, and tumor-infiltrating lymphocytes. The compartments in non-neoplastic lung tissue included lung endothelial cells, pulmonary lymphocytes, interstitial fibroblasts, Type II alveolar pneumocytes, and bronchial epithelial cells. ICAM-1 was expressed in tumor cells from all of the nine adenocarcinomas. In contrast, VCAM-1 expression was not identified in tumor cells from any of the nine adenocarcinomas. ICAM-1 was expressed in all cellular compartments of the non-neoplastic lung tissue, whereas VCAM-1 was expressed only in pulmonary lymphocytes and interstitial fibroblastic cells. LFA-1 was uniformly expressed in tumor-infiltrating lymphocytes from each of the nine tumors and all of the lymphocytes in non-neoplastic lung tissue. This study showed major differences in the expression of ICAM-1 and VCAM-1 in tumor cells from pulmonary adenocarcinoma and also provided evidence for a wider distribution of ICAM-1, compared with VCAM-1, in non-neoplastic cellular compartments of the lung. ICAM-1 expression was particularly noticeable in bronchial and alveolar epithelial cells. Upregulation of ICAM-1 in pulmonary adenocarcinoma might foster binding by LFA-1-bearing lymphocytes, with a possible impact on the vulnerability of tumor cells to host defense mechanisms.     

Cellular localization of urokinase-type plasminogen activator, its inhibitors, and their mRNAs in breast cancer tissues

The plasminogen activation (PA) system may participate in cancer invasion and metastasis. A series of breast cancer tissue specimens was analysed using in situ hybridization and immunohistochemistry. Urokinase-type plasminogen activator (u-PA) mRNA was detected in cancer cells and fibroblasts adjacent to them and its expression was found to be more intense in invasive than in intraductal regions. In invasive but not in intraductal regions, especially those with abundant stroma, plasminogen activator inhibitor-1 (PAI-1) mRNA was observed in cancer cells, fibroblasts, macrophages, and endothelial cells, and PAI-2 mRNA was present in cancer cells, and fibroblasts, macrophages, and lymphocytes around them. These PAI-1- and PAI-2-positive cancer cells were localized at the periphery of the invasive front. Immunohistochemistry yielded basically similar results. A retrospective study of surgically resected breast cancers from 73 patients revealed significant clinical differences associated with u-PA and PAI-2 expression in cancer cells, associated with a poor and a good prognosis, respectively. These findings indicate that breast cancer cells and fibroblasts express u-PA initially and then its inhibitors, and that this process is related to invasion. Expression of u-PA and PAI-2 in cancer cells themselves may serve to up-regulate and limit PA-mediated invasion and metastasis, respectively.     

Semliki Forest virus infection leads to increased expression of adhesion molecules on splenic T-cells and on brain vascular endothelium

Semliki Forest virus A7 (SFV-A7) is a neurotropic alphavirus that leads to an asymptomatic encephalitis in adult immunocompetent mice. We studied the expression of leukocyte and endothelial cell adhesion molecules in the spleen and in the central nervous system (CNS) during SFV-A7 infection. Kinetics of the expression of LFA-1 alpha/CD11a, LFA-1 beta/CD18, Mac-1/CD11b, VLA-4/CD49d, ICAM-1/CD54 and L-selectin/CD62L was determined on splenic CD4+ and CD8+ T-cells and macrophages by flow cytometry. Time course of the expression of these antigens and VCAM-1/CD106 as well as viral antigens in the CNS was studied by immunoperoxidase staining. In the spleen, a sustained increase in LFA-1-expression and a temporary increase at day 7 in the expression of VLA-4, Mac-1 and ICAM-1 were detected on CD8+ T-cells. L-selection was down-regulated on CD4+ cells. Adhesion molecules on macrophages remained unchanged. In the CNS, expression of Mac-1+, VLA-4+ and LFA-1+ cells increased in parallel with the kinetics of the expression of their ligands ICAM-1 and VCAM-1 on brain vessels. Upregulation of adhesion of molecules peaked between days 5-8 and was most prominent in the cerebellar and brain stem white matter where viral antigens were most abundant. We conclude that the adhesion molecules profile of splenic T cells is altered during SFV-A7 infection which may influence their homing into the CNS. Macrophages are probably recruited non-specifically as a consequence of activation of the brain vascular endothelium in the inflamed areas of the brain.     

The role of LFA-1 (CD11a/CD18) cytoplasmic domains in binding to intercellular adhesion molecule-1 (CD54) and in postreceptor cell spreading

We have investigated the role of the cytoplasmic domains of LFA-1 in binding to ICAM-1 and in postadhesion events. Various truncated and chimeric forms of LFA-1 alpha (CD11a) and beta (CD18) chains were generated and transfected into murine fibroblast TNR-2 cells. Transfected fibroblasts expressing wild-type LFA-1 adhered only weakly to ICAM-1 immobilized on plastic, and phorbol ester pretreatment enhanced this adhesion significantly. In contrast, transfected cells expressing LFA-1 lacking both the alpha and the beta cytoplasmic domains, the beta cytoplasmic domain alone, or GPI-anchored LFA-1 adhered to immobilized ICAM-1 without prior activation. Truncation of the alpha cytoplasmic domain alone resulted in much reduced cell adhesion which could be only weakly upregulated by PMA. The presence of manganese dramatically enhanced the binding to ICAM-1 of LFA-1 lacking the alpha cytoplasmic domain or both cytoplasmic domains, whereas it had relatively little effect on wild-type LFA-1 or the mutant lacking the beta cytoplasmic domain. Soluble LFA-1, generated by phosphatidylinositol-specific phospholipase-C treatment of GPI-anchored LFA-1, was capable of binding ICAM-1+ cells. Although doubly truncated or GPI-anchored LFA-1 mediated cell adhesion to immobilized ICAM-1, cells expressing these mutants, as well as those expressing individual alpha and beta chain truncations, failed to spread out following this adhesion, whereas the wild-type transfectants did so readily. Manganese had no effect on cell spreading. Fluorescent staining of these cells indicated no significant variation in the distribution of LFA-1 on the cell surface. From these results we conclude that (1) cells expressing LFA-1 lacking both the alpha and the beta cytoplasmic domains adhere to ICAM-1 without prior stimulation, indicating the importance of LFA-1 cytoplasmic domains in inside-out signaling, (2) truncation of the alpha cytoplasmic domain alone inhibits cell adhesion by making LFA-1 nonresponsive to inside-out signaling, and (3) both cytoplasmic domains are required for cell spreading following adhesion to immobilized ICAM-1.     

Immunoelectron microscopic localization of plasminogen activator inhibitor type 1 (PAI-1) in smooth muscle cells from morphologically normal and atherosclerotic human arteries

Vascular wall fibrinolytic system proteins are believed to play a pivotal role in atherogenesis. Tissue-type plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA) influence persistence of luminal thrombi and proteolysis of extracellular matrix, respectively. The major physiologic inhibitor of t-PA and u-PA is plasminogen activator inhibitor type 1 (PAI-1). All three of these fibrinolytic system proteins have been detected in vascular endothelial cells, smooth muscle cells, and macrophages by light microscopic immunohistochemistry. This study was undertaken to delineate, by immunoelectron microscopy, the loci of PAI-1 in smooth muscle cells from intact morphologically normal and atherosclerotic human arteries as well as in isolated and cultured smooth muscle cells from arteries. In intact vessels, PAI-1 immunoreactivity was associated with contractile filaments in cells in both normal and atherosclerotic tissues. Lipid-laden smooth muscle cells in atherosclerotic vessels were mainly of the synthetic phenotype and displayed lesser amounts of PAI-1 associated with rough endoplasmic reticulum and contractile filaments. Isolated smooth muscle cells exhibited either a contractile or synthetic phenotype. In the cells with a contractile phenotype, PAI-1 was associated with the contractile elements, whereas in the cells with a synthetic phenotype, the PAI-1 was associated predominantly with elements of the endoplasmic reticulum. Because PAI-1 is associated predominantly with contractile filaments in smooth muscle cells, the net amount of immunodetectable PAI-1 appears to be greater in contractile compared with synthetic phenotype cells.     

Endothelial and macrophage upregulation of urokinase receptor expression in human renal cell carcinoma

The binding of urokinase-type plasminogen activator (u-PA) to a specific cell surface receptor (uPA-R) has been shown to enhance plasminogen activation, a process involved in extracellular matrix degradation and cell migration during angiogenesis and tumor growth. We investigated the expression of u-PA and uPA-R in renal cell carcinomas (n = 11). By immunohistochemistry using monoclonal and polyclonal anti-uPA-R antibodies, we found that tumoral capillary endothelial cells (von Willebrand factor and CD31 positive cells) overexpressed uPA-R, whereas vascular endothelial cells of the normal human kidney do not. In addition, tumor-associated macrophages (CD68-positive cells) strongly expressed uPA-R. In contrast, few tumoral cells and stromal fibroblasts expressed uPA-R. By in situ hybridization using a cDNA S35-labeled probe specific for uPA-R, we confirmed the local expression of uPA-R messenger RNA. We also detected the induction of u-PA in tumoral capillary endothelial cells and in tumor-associated macrophages. In two cases, tumoral cells themselves were also stained by anti-u-PA antibodies in focal areas. Finally tissue-type plasminogen activator (t-PA) was also overexpressed by tumoral capillary endothelial cells as compared with endothelial cells of normal human kidney vessels. These findings indicate an active invasive phenotype of endothelial cells in renal cell carcinoma and suggest a role for the plasminogen activation system in tumoral angiogenesis and invasion.     

Stromelysin-3 is induced in tumor/stroma cocultures and inactivated via a tumor-specific and basic fibroblast growth factor-dependent mechanism

Stromelysin-3 (STR-3) is a recently characterized matrix metalloproteinase (MMP) with a unique pattern of expression and substrate specificity. Unlike other MMPs, STR-3 is consistently and dramatically overexpressed by multiple epithelial malignancies, including carcinomas of the breast, lung, colon, head and neck, and skin. Recent studies suggest that STR-3 promotes the local establishment of epithelial malignancies, contributing to tumor cell survival and implantation in host tissues; however, STR-3's mechanism of action remains undefined. STR-3 is a stromal cell product, prompting speculation that infiltrating stromal cells secrete STR-3 in response to tumor-derived factors. To explore this possibility, we developed a tumor/"stroma" coculture assay in which non-small cell lung cancer (NSCLC) cell lines were grown on confluent monolayers of normal pulmonary fibroblasts. In these tumor/stroma cocultures, NSCLCs stimulate normal pulmonary fibroblasts to secrete STR-3 and release extracellular basic fibroblast growth factor. Thereafter, STR-3 is processed at a unique internal sequence via a basic fibroblast growth factor- and MMP-dependent mechanism to a previously unidentified 35-kDa protein that lacks enzymatic activity. 35-kDa STR-3 is the most abundant STR-3 protein in tumor/stroma cocultures and is only detected when normal pulmonary fibroblasts are cultured with malignant bronchial epithelial cells. Therefore, the tumor-specific processing of STR-3 to the 35-kDa protein is likely to be an important regulatory mechanism.     

Low intercellular adhesion molecule 1 and high 5T4 expression on tumor cells correlate with reduced disease-free survival in colorectal carcinoma patients

The purpose of this study was to investigate the prognostic value of the expression of intercellular adhesion molecule 1 (ICAM-1), leukocyte function antigen 3 (LFA-3), human leukocyte differentiation antigen (HLA)-ABC, HLA-DR, and 5T4 with regard to disease-free survival in Dukes' B and C colorectal carcinoma patients. Forty-one patients (28 Dukes' B and 13 Dukes' C) were entered into this study. Immunocytochemistry was performed on cytospin preparations of enzymatically digested colorectal carcinoma cell suspensions. The frequency of metastases and the duration of disease-free survival were compared between the 25% lowest expressers and the 75% remaining patients for ICAM-1, LFA-3, HLA-ABC, and HLA-DR, and between the 25% highest expressers and the 75% remaining patients for 5T4. Low numbers of ICAM-1-expressing tumor cells were associated with a shorter disease-free survival (P < 0. 001), independent of Dukes' stage. High numbers of 5T4-expressing tumor cells were associated with shorter disease-free survival in Dukes' B patients (P = 0.04). Cox proportional hazard analysis indicated that low numbers of ICAM-1(+) and high numbers of 5T4(+) cells were independent prognostic factors with relative risks of 13. 0 (P = 0.0002) and 4.7 (P = 0.02), respectively. The combination of 5T4 and ICAM-1 marker information identified subgroups of patients with a good (high ICAM-1) or poor (low ICAM-1/high 5T4) prognosis. Neither a lack of HLA-ABC and LFA-3 expression nor the presence of HLA-DR on the tumor cells gave additional prognostic information. These findings demonstrate that low ICAM-1 and high 5T4 expression on tumor cells are prognostic markers, additional to Dukes' stage, for reduced disease-free survival in Dukes' B and C colorectal carcinoma patients.     

Contribution of CD11a/CD18, CD11b/CD18, ICAM-1 (CD54) and -2 (CD102) to human monocyte migration through endothelium and connective tissue fibroblast barriers

Recently we reported that monocyte migration through a barrier of human synovial fibroblasts (HSF) is mediated by the CD11/CD18 (beta2) integrins, and the beta1 integrins VLA-4 and VLA-5 on monocytes. Here we investigated in parallel the role of beta2 integrin family members, LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) on monocytes, and the immunoglobulin supergene family members, ICAM-1 and ICAM-2 on HSF and on human umbilical vein endothelial cells (HUVEC), in monocyte migration through HSF and HUVEC monolayers. Using function blocking monoclonal antibodies (mAb), when both VLA-4 and VLA-5 on monocytes were blocked, treatment of monocytes with mAb to both LFA-1 and to Mac-1 completely inhibited monocyte migration across HSF barriers, although blocking either of these beta2 integrins alone had no effect on migration, even when VLA-4 and VLA-5 were blocked. This indicates that optimal beta2 integrin-dependent monocyte migration in synovial connective tissue may be mediated by either LFA-1 or Mac-1. Both ICAM-1 and ICAM-2 were constitutively expressed on HSF and on HUVEC, although ICAM-2 was only minimally expressed on HSF. Based on results of mAb blockade, ICAM-1 appeared to be the major ligand for LFA-1-dependent migration through the HSF. In contrast, both ICAM-1 and ICAM-2 mediated LFA-1-dependent monocyte migration through HUVEC. However, neither ICAM-1 nor ICAM-2 was required for Mac-1 -dependent monocyte migration through either cell barrier, indicating that Mac-1 can utilize ligands distinct from ICAM-1 and ICAM-2 on HSF and on HUVEC during monocyte transmigration.     

Interfollicular fibroblasts in the human thyroid gland: recognition of a CD34 positive stromal cell network communicated by gap junctions and terminated by autonomic nerve endings

While epithelial structure and functions have been substantially investigated in many organs, the mesenchymal elements have received less attention. Compared with follicular epithelial cells, there are a few morphological studies on the stroma of human thyroid gland. In order to characterize more fully and assess its possible functions, 15 samples of surgical and autopsy human thyroid tissue were studied by classical histology, immunohistochemistry, transmission electron microscopy, electron microscopic immunohistochemistry, and scanning electron microscopy. In human thyroid gland, the interfollicular connective tissue surrounding the follicles contained collagenous matrix, fibroblasts, unmyelinated nerve fibers with Schwann cells, small blood vessels, lymphatics, lymphocytes, plasma cells, macrophages, and mast cells. At the ultrastructural level, gap junctions between the cytoplasmic processes of interfollicular fibroblasts constituted a novel observation. Immunohistochemistry using a monoclonal antibody against Cx43 confirmed the distribution of gap junctions between stromal fibroblastic cells, which was compatible with the ultrastructural findings. The frequent and intimate association of fibroblastic processes with nerve terminals was also shown. Interfollicular stromal fibroblasts also stained with CD34. The main constituent of the human thyroid stromal tissue was a CD34 positive reticular network involving fibroblasts, mononuclear cells and nerve terminals. It represents a highly ordered stroma, with potential structural and functional similarities to the stroma of bone marrow (Yamazaki and Allen, 1990).     

Cytokine regulation of cell-to-cell interactions in lymphokine-activated killer cell cytotoxicity in vitro

The permanent pancreas carcinoma cell line, PCI-24, was developed in order to analyse cytokine regulation on pancreas carcinoma and lymphokine-activated killer (LAK) cell interaction. PCI cells expressed ICAM-1 and HLA-ABC, but not HLA-DR antigens. PCI cells showed augmented ICAM-1 and HLA-ABC expression when incubated with interferon gamma (IFN gamma) and tumour necrosis factor alpha. A similar but weak augmentary effect on the HLA-ABC and ICAM-1 surface expression was seen with interleukin-1 beta treatment. Natural attachment of LAK to PCI cells was augmented by recombinant IFN gamma in close association with ICAM-1 up-regulation on PCI cells. In addition, natural attachment was significantly inhibited by anti-LFA-1 and anti-ICAM-1 antibody treatments. Cytotoxicity of the LAK cells against PCI cells was also significantly inhibited with the same treatment. Thus, the attachment of LAK cells to PCI cells through LFA-1/ICAM-1 molecules appeared to be essential for the cytotoxicity for PCI cells. Pretreatment of PCI cells, but not of LAK cells, with IFN gamma or other cytokines resulted in a decrease of susceptibility for LAK cell cytotoxicity. The decreased susceptibility inversely correlated with HLA-ABC expression on the PCI cells. The collective evidence indicates that, although LAK cell attachment to pancreas carcinoma cells through the LFA-1/ICAM-1 molecule is augmented by IFN gamma, IFN gamma treatment of pancreas carcinoma cells reduces LAK cell cytotoxicity possibly through an increase in HLA-ABC or a regulation of molecules closely associated to HLA-ABC expression.     

Dose-related ethanol-like effects of the NMDA antagonist, ketamine, in recently detoxified alcoholics

AIMS: To study the clinical and histopathological features of sclerosing epithelioid fibrosarcoma, and to define diagnostic criteria for this uncommon soft-tissue tumour. METHODS AND RESULTS: Standard histological, immunohistochemical and ultrastructural techniques were applied to five tumours from head and neck, chest wall and groin. Tumours consisted of groups of monomorphic rounded/epithelioid cells surrounded by a prominent collagenous stroma. Tumour cells showed positive vimentin staining but were negative for other markers. They contained prominent rough endoplasmic reticulum and a large Golgi apparatus which in one case was producing collagen secretion granules, an ultrastructural marker for collagen production. Three patients had medium to long-term survival (3-7 years). Of these, one was disease-free for 3 years, and two experienced multiple recurrences: one of the latter died of metastatic disease. CONCLUSION: Criteria for diagnosing this uncommon tumour include: small to medium cell size, clear or pale cytoplasm, cellular arrangement in cords and strands, dense collagenous stroma; vimentin staining; rough endoplasmic reticulum and a Golgi apparatus producing, in well preserved examples, collagen secretion granules. The paper emphasizes the value of electron microscopy, supporting an appropriate histological picture and immunophenotype, in identifying these relatively low-grade sarcomas.     

Iatrogenic hyperstimulation of the ovary with ascites. Report of a case and considerations and management

Electron microscopic investigations in 7 patients with different dermatoses or skin tumours containing amyloid showed that amyloid is synthesized in the cytoplasm of dermal cells. In 3 cases of localized primary amyloidosis of the skin highly active cells were demonstrated, showing grossly dilated cisternae of rough endoplasmic reticulum, resembling fibroblasts. Their intracellular product seemed amorphous, later filamentous, and was then released into the extracellular space. Extracellular aggregations of typical amyloid filaments were found partially surrounded by thin cytoplasmic remnants of the cells producing them. Subclinical amounts of amyloid found in porokeratosis of Mibelli, in superficial basal-cell carcinoma, in senile skin, and in clinically normal skin of a patient with malignant melanoma showed the same characteristics. Other cell types such as plasma cells and mast cells were well preserved and seemed stimulated; however, no amyloid precursors were found in their cytoplasm and no release was seen. We, therefore, conclude that dermal amyloid is generally produced by falsely programmed fibroblasts.     

Explorers. Christopher Columbus and Leonardo da Vinci

Epstein-Barr virus (EBV) recently has been associated with Hodgkin's disease (HD) and the EBV genome was found in CD30-positive Reed-Sternberg cells. Therefore, tissue sections from 25 cases of HD, 35 cases of CD30-positive non-Hodgkin's lymphoma (NHL) (seven CD30-positive anaplastic large cell lymphomas [ALCLs] and 28 CD30-positive non-ALCLs), and 12 cases of CD30-negative NHL that previously had been screened for the presence of EBV by polymerase chain reaction and DNA in situ hybridization were studied by immunohistochemistry for the expression of the latent EBV proteins, latent membrane protein (LMP), and Epstein-Barr nuclear antigen-2 (EBNA-2). We also analyzed the expression of the B-cell activation molecule CD23 and the adhesion molecules LFA-1/CD11a and ICAM-1/CD54 because the upregulation of these molecules by LMP and/or EBNA-2 in vitro has been related to the EBV-induced lymphocyte growth. Latent membrane protein expression was found in Reed-Sternberg cells in nine of 25 cases (36%) of HD and in large, occasionally Reed-Sternberg-like tumor cells in six of 47 cases (12%) of NHL; these six tumors were CD30-positive, histologically high-grade NHL (one CD30-positive ALCL and five CD30-positive non-ALCLs). All the LMP-positive cases were also polymerase chain reaction EBV positive while LMP expression was not found in polymerase chain reaction EBV-negative HD and NHL. No staining for EBNA-2 was detected in our series. In view of the transforming potential of the LMP, these findings suggest that EBV may be associated with the development of some cases of HD and CD30-positive NHL. These findings also suggest a correlation between the expression of LMP and the detection of CD30 in tumor cells of HD and NHL. In contrast, no correlation was found between the expression of LMP and the detection of CD23, LFA-1/CD11a, and ICAM-1/CD54 in tumor cells of HD and NHL.     

A novel dynamin-like protein associates with cytoplasmic vesicles and tubules of the endoplasmic reticulum in mammalian cells

Dynamins are 100-kilodalton guanosine triphosphatases that participate in the formation of nascent vesicles during endocytosis. Here, we have tested if novel dynamin-like proteins are expressed in mammalian cells to support vesicle trafficking processes at cytoplasmic sites distinct from the plasma membrane. Immunological and molecular biological methods were used to isolate a cDNA clone encoding an 80-kilodalton novel dynamin-like protein, DLP1, that shares up to 42% homology with other dynamin-related proteins. DLP1 is expressed in all tissues examined and contains two alternatively spliced regions that are differentially expressed in a tissue-specific manner. DLP1 is enriched in subcellular membrane fractions of cytoplasmic vesicles and endoplasmic reticulum. Morphological studies of DLP1 in cultured cells using either a specific antibody or an expressed green fluorescent protein (GFP)- DLP1 fusion protein revealed that DLP1 associates with punctate cytoplasmic vesicles that do not colocalize with conventional dynamin, clathrin, or endocytic ligands. Remarkably, DLP1-positive structures coalign with microtubules and, most strikingly, with endoplasmic reticulum tubules as verified by double labeling with antibodies to calnexin and Rab1 as well as by immunoelectron microscopy. These observations provide the first evidence that a novel dynamin-like protein is expressed in mammalian cells where it associates with a secretory, rather than endocytic membrane compartment.     

Involvement of lymphocyte function-associated antigen-1 and intercellular adhesion molecule-1 in osteoclastogenesis: a possible role in direct interaction between osteoclast precursors

In our search for molecules involved in the process of osteoclast differentiation, we examined the surface phenotypes of the preosteoclast-like cells and osteoclast-like multinucleated cells (MNCs) formed in bone marrow cultures, using monoclonal antibodies recognizing different antigen molecules expressed on hematopoietic cells. Among these cell surface antigens, lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) were highly expressed on mononuclear cells in the cultures for forming preosteoclast-like mononuclear cells. The double detection of these two antigen molecules with osteoclast-specific antigen and with calcitonin receptor, using a fluorescence-activated cell sorter or autoradiography technique, revealed that LFA-1 and ICAM-1 were expressed on the preosteoclasts. The expression of ICAM-1 was detected on both preosteoclasts and osteoclast-like MNCs, whereas the expression of LFA-1 was restricted to preosteoclasts. We designed a peptide with the sequence of the binding site of ICAM-1 against the ligand LFA-1. In the whole bone marrow culture system for forming osteoclast-like MNCs, a significant inhibition of MNC formation was observed by the addition of this peptide. These results strongly suggest the involvement of an LFA-1/ICAM-1-interaction in osteoclastogenesis.     

Alpha 6 beta 4 integrin and newly deposited laminin-1 and laminin-5 form the adhesion mechanism of gastric carcinoma. Continuous expression of laminins but not that of collagen VII is preserved in invasive parts of the carcinomas: implications for acquisition of the invading phenotype

We studied the expression and distribution of different laminin chains, the alpha 6 beta 4 integrin and type VII collagen, i.e., components of the epithelial adhesion complex, in gastric carcinomas and in suggested preneoplastic stages of this malignancy. Intestinal-type gastric carcinomas showed strong reactivity for laminin alpha 1, alpha 3, beta 1, and beta 3 chains, the components of laminin-1 and -5, at the interface between malignant cells and tumor stroma. The reactivities were continuous throughout the carcinomas, even in structures invading through the smooth muscle layers of the gastric wall. The expression of different laminin chains was accompanied by strong polarized reactivity for the alpha 6 beta 4 integrin, which is a receptor for both laminin-1 and laminin-5. Collagen type VII was only occasionally present at sites showing reactivity for laminin-5 and was totally absent from the cell islands invading through the gastric wall. Intestinalized gastric epithelium showed a similar expression pattern of laminins and the alpha 6 beta 4 integrin as the gastric carcinomas. Our results suggest that gastric carcinomas use the alpha 6 beta 4 integrin and newly deposited laminin-1 and -5, accompanied by the disappearance of type VII collagen, as their mechanism of adhesion during the invasion through surrounding tissues. Unlike in previous studies, the reactivity for the laminin-5 protein was not restricted to the invading cells but surrounded the malignant glandular structures throughout the tumor. Our results also show that both intestinal-type gastric carcinoma, and intestinal metaplasia mimic the gastric surface epithelium in the expression pattern of laminins and the beta 4 integrin subunit. This supports previous studies proposing a pathogenetic sequence from intestinal metaplasia to gastric carcinoma.     

Immunolocalization of acyl-coenzyme A:cholesterol O-acyltransferase in macrophages

Macrophages in atherosclerotic lesions accumulate large amounts of cholesteryl-fatty acyl esters ("foam cell" formation) through the intracellular esterification of cholesterol by acyl-coenzyme A:cholesterol O-acyltransferase (ACAT). In this study, we sought to determine the subcellular localization of ACAT in macrophages. Using mouse peritoneal macrophages and immunofluorescence microscopy, we found that a major portion of ACAT was in a dense reticular cytoplasmic network and in the nuclear membrane that colocalized with the luminal endoplasmic reticulum marker protein-disulfide isomerase (PDI) and that was in a similar distribution as the membrane-bound endoplasmic reticulum marker ribophorin. Remarkably, another portion of the macrophage ACAT pattern did not overlap with PDI or ribophorin, but was found in as yet unidentified cytoplasmic structures that were juxtaposed to the nucleus. Compartments containing labeled beta-very low density lipoprotein, an atherogenic lipoprotein, did not overlap with the ACAT label, but rather were embedded in the dense reticular network of ACAT. Furthermore, cell-surface biotinylation experiments revealed that freshly harvested, non-attached macrophages, but not those attached to tissue culture dishes, contained approximately 10-15% of ACAT on the cell surface. In summary, ACAT was found in several sites in macrophages: a cytoplasmic reticular/nuclear membrane site that overlaps with PDI and ribophorin and has the characteristics of the endoplasmic reticulum, a perinuclear cytoplasmic site that does not overlap with PDI or ribophorin and may be another cytoplasmic structure or possibly a unique subcompartment of the endoplasmic reticulum, and a cell-surface site in non-attached macrophages. Understanding possible physiological differences of ACAT in these locations may reveal an important component of ACAT regulation and macrophage foam cell formation.