Biomedical subjects
Publications and source records attributed to L Ruco.
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Endothelial cell-specific molecules are potential targets for new therapeutic strategies in the control of inflammatory reactions, immune responses and neoangiogenesis. We describe the production and characterization of MEC 14.7, a monoclonal antibody directed to murine endothelial cells recognizing a glycosylated protein with an apparent molecular mass of about 100 kDa in cultured endothelioma cell lysate and about 80 kDa in lung lysate. MEC 14.7 antigen was selectively expressed by the endothelium in vivo, particularly in small vessels and neoformed capillaries and by developing vascular structures in embryonal bodies. Deglycosylation of the molecule with neuraminidase, O- and N-glycanase showed that the MEC 14.7 epitope is neuraminidase-sensitive. MEC 14.7 antigen was purified from lung lysates by chromatographic techniques, and sequenced internal peptides indicated it was identical with murine CD34. Thus the apparent molecular mass of CD34 is heterogeneous, depending on the glycosylation state in the different cell types. Immunomagnetic isolation and culture of MEC 14.7-positive bone marrow cells showed that this antibody recognizes hematopoietic progenitors (particularly myelomonocytic) and can be used in murine models of bone marrow reconstitution.
Polymorphonuclear leukocytes (PMN) infiltration into tissues is frequently accompanied by increase in vascular permeability. This suggests that PMN adhesion and transmigration could trigger modifications in the architecture of endothelial cell-to-cell junctions. In the present paper, using indirect immunofluorescence, we found that PMN adhesion to tumor necrosis factor-activated endothelial cells (EC) induced the disappearance from endothelial cell-to-cell contacts of adherens junction (AJ) components: vascular endothelial (VE)-cadherin, alpha-catenin, beta-catenin, and plakoglobin. Immunoprecipitation and Western blot analysis of the VE-cadherin/catenin complex showed that the amount of beta-catenin and plakoglobin was markedly reduced from the complex and from total cell extracts. In contrast, VE-cadherin and alpha-catenin were only partially affected. Disorganization of endothelial AJ by PMN was not accompanied by EC retraction or injury and was specific for VE-cadherin/catenin complex, since platelet/endothelial cell adhesion molecule 1 (PECAM-1) distribution at cellular contacts was unchanged. PMN adhesion to EC seems to be a prerequisite for VE-cadherin/catenin complex disorganization. This phenomenon could be fully inhibited by blocking PMN adhesion with an anti-integrin beta 2 mAb, while it could be reproduced by any condition that induced increase of PMN adhesion, such as addition of PMA or an anti-beta 2-activating mAb. The effect on endothelial AJ was specific for PMN since adherent activated lymphocytes did not induce similar changes. High concentrations of protease inhibitors and oxygen metabolite scavengers were unable to prevent AJ disorganization mediated by PMN. PMN adhesion to EC was accompanied by increase in EC permeability in vitro. This effect was dependent on PMN adhesion, was not mediated by proteases and oxygen-reactive metabolites, and could be reproduced by EC treatment with EGTA. Finally, immunohistochemical analysis showed that VE-cadherin distribution was affected by PMN adhesion to the vessel wall in vivo too. This work suggests that PMN adhesion could trigger intracellular signals in EC that possibly regulate VE-cadherin /catenin complex disorganization. This effect could increase EC permeability and facilitate PMN transmigration during the acute inflammatory reaction.
C-26 colon adenocarcinoma cells transduced with the granulocyte colony-stimulating factor (G-CSF) gene form large tumors when injected into sublethally irradiated mice. These tumors regress when leukocyte function is reconstituted. Electron microscopy and immunocytochemical analysis of regressing C-26/G-CSF nodules indicates that tumor destruction is due mainly to hypoxia resulting from the functional loss of tumor vasculature and is only marginally due to direct cytolysis. Desegregation of basal lamina, cell swelling, and loss of junctions characterized the vessels within regressing tumors. Tumor cells were necrotic or filled with lipid vacuoles regardless of the distance from nearby vessels. Damage of tumor vasculature was dependent on the infiltrating leukocytes and the cytotoxic cytokines they produced. Locally produced interleukin-1 and tumor necrosis factor-alpha (TNF-alpha) induced vascular cellular adhesion molecule-1 (VCAM-1) and E-selectin on tumor vessels. Treatment with monoclonal antibodies to interferon-gamma (IFN-gamma) or TNF-alpha blocked tumor regression by inhibiting VCAM-1 and E-selectin expression on tumor-associated endothelial cells resulting in a reduced number of infiltrating leukocytes. Thus, C-26/G-CSF tumor regression presents features typical of hemorrhagic necrosis that occurs through the cytokines produced by infiltrating leukocytes in response to G-CSF.
CU-NPC is an important clinical entity that presents some uncertainties regarding histologic features. In fact many of these tumors show a marked infiltration of lymphocytes justifying the term "lymphoepithelioma" used to define these tumors in the past. Although they have not yet been placed in a definite category, some factors may be responsible for the lymphocyte infiltration; infection by Epstein-Barr virus, expression by tumor cells of HLA-DR antigens and intercellular adhesion molecules (ICAM-1) and production of interleukin l-alpha by U-NPC derived cell lines. A recent study showed that epithelium of the crypts of the tonsils exerts an active role in the intraepithelial migration of lymphocytes. This recruitment from the underlying lymphatic tissue could be determined by the expression, on the part of the basal cells of tonsillar crypts, of ICAM-1 and blood vessel adhesion molecules (VCAM-1). The purpose of this study was to verify the presence of ICAM-1 and VCAM-1 in paraffin-embedded tissue section of seven cases of U-NPC. The results showed positive immunohistochemical reaction both for ICAM-1 and VCAM-1. On this basis, the authors hypothesize that the expression of these molecules could favor intratumoral recruitment of lymphocytes similar to what occurs in the epithelium of the tonsillar crypts.
Haemangiomas are vascular tumours characterized by rapid growth and increased endothelial turnover. VE-cadherin is a recently discovered endothelial cell-specific cadherin located at intercellular junctions. In different types of epithelial tumours, cadherin expression is inversely correlated with invasiveness and metastatic dissemination. In this immunohistochemical study, VE-cadherin expression has been analysed in different types of haemangioma. VE-cadherin is highly expressed in endothelial cells of haemangiomas and is decreased, but still detectable, in some cases of haemangionendothelioma and angiosarcoma. The antigenic profile of most haemangioma cells was similar to that of normal endothelium. CD31, CD34, ICAM-1, von Willebrand factor, and VLA integrins were expressed in haemangioma endothelium; in addition, the major components of vascular basement membrane, namely fibronectin, collagen type IV, and laminin, were correctly expressed and organized. Surprisingly, a marked reactivity for the M form of laminin (merosin) was detected in the basement membranes of two juvenile capillary haemangiomas. Overall, this study shows that, with the exception of angiosarcoma and haemangionendothelioma, vascular tumours maintain most of the differentiation characteristics of normal endothelium. This encourages speculation that in these pathologies, abnormal endothelial proliferation is more related to the release of local factors than to an altered endothelial phenotype.
Local immunotherapy of superficial bladder cancer by endovesical administration of recombinant interleukin-2 (rIL-2) was investigated in a phase I-II study. Twenty-five patients with Ta-T1, N0, M0, G1-G2 transitional cell carcinoma of the bladder received an induction course of rIL-2 (10 daily instillations) with the tumor in place using an interpatient dose escalation scheme from 3 to 18 x 10(6) IU/day. Seven to fourteen days after the end of the induction course, the objective clinical response was evaluated and transurethral resection of the bladder tumor was carried out. Four maintenance courses (10 daily instillations) were started 1 month after surgery and carried out every 4 months at a dose of 6 x 10(6) IU/day in all patients. Follow-up visits were scheduled bimonthly during treatment and then every 6 months. No evidence of laboratory, local or systemic toxicity was observed even at the highest rIL-2 dosages. Induction of a local inflammatory response could be demonstrated at the tumor site after intravesical rIL-2 treatment. The significant reduction in tumor diameters observed in some patients may be interpreted as a sign of the biological activity of this rIL-2 regimen. Further exploratory work is required to evaluate the clinical efficacy of this immunotherapeutic approach.
Unexpectedly high levels of gastric tolerance to the new non-steroidal anti-inflammatory drug (NSAID) amtolmetin guacyl (CAS 87344-06-7, MED15) were observed in clinical practice. Further investigation of the drug was, therefore, undertaken in order to evaluate its gastrointestinal side-effects in animals. This new agent, which possesses high anti-inflammatory, analgesic and antipyretic activity, was recently introduced into medical therapy. In rats, repeated administration of MED15, even at high dosages, failed to produce any adverse effects on the gastric mucosa, unlike the reference NSAIDs used; the effect on the gastric mucosa was evaluated macro- and microscopically with a staining technique. In immature rat isolated stomachs MED15 strongly down-regulated the HCl output evoked by histamine, acetylcholine and gastrin. An effective NSAID which presents the added advantage of antisecretory properties has not previously been described amongst the many currently available, and reverses the dogma of the inevitable correlation between NSAID assumption and gastrointestinal reactions.
Chemokines are a superfamily of structurally related cytokines involved in leukocyte recruitment in normal and neoplastic tissues. The availability of non-cross-reacting reagents specific for each member of the C-C and C-X-C family is important for careful characterization of their in vitro and in vivo production and relevance. Here we describe a novel, highly specific, mAb against monocyte chemotactic protein-1 (MCP-1). The 5D3-F7 mAb (IgG1,kappa) recognizes human recombinant and natural MCP-1 in ELISA, immunoprecipitation and immunoblot analysis. As a source of natural MCP-1 we used the 8387 human sarcoma line which produces spontaneously MCP-1 and responds to TNF with increased expression and release. The 5D3-F7 mAb inhibited the chemotactic activity of MCP-1 for monocytes. Using the 5D3-F7 mAb and a polyclonal rabbit anti-MCP-1 serum, a sandwich ELISA was developed. In both the direct and the sandwich ELISA, the 5D3-F7 mAb recognized human MCP-1, but not the closely related C-C chemokines MCP-1, MCP-2, MCP-3, MIP-1 alpha, and RANTES and the C-X-C chemokines IL-8, gro alpha and NAP-2. In culture supernatants the sensitivity of the sandwich ELISA was approximately equal to 30 pg/ml. The sandwich ELISA permitted detection of MCP-1 in resting or cytokine-stimulated endothelial, mesothelial and Kaposi's sarcoma cells. Preliminary immunohistochemical analysis revealed production of MCP-1 by macrophage-like cells at sites of inflammation. The 5D3-F7 mAb provides a novel, highly specific reagent with which to investigate the in vitro and in vivo production and role of MCP-1.
Two monoclonal antibodies (mAb), MEC 7.46 (IgG1) and MEC 13.3 (IgG2a) that specifically recognize mouse endothelial cells (EC) of blood vessels, were produced immunizing a Lewis rat with a polyoma middle T transformed EC line. Antibodies were screened by enzyme-linked immunosorbent assay (ELISA) and by immunofluorescence on different cultured cell lines and by immunoperoxidase staining on frozen sections of various mouse normal and inflammatory tissues. Both mAbs reacted with eight transformed endothelial lines tested in vitro, but were consistently negative on various cell lines of different histological origin. Reactivity was not altered by preexposure of the cell lines to IL-1. Microscopic immunofluorescence analysis showed that the MEC mAbs localized at the cell-cell contacts in EC. Immunohistochemical staining of various mouse tissue was always restricted to the EC of all blood vessels of the organ considered. Staining of the endothelial lining of blood vessels was greater at cell-to-cell contacts. Weak reactivity was detected in bone marrow and spleen megakaryocytes. This picture was not altered in inflamed and tumor tissues. In the developing mouse embryo, MEC 13.3 specifically stained proliferating and sprouting endothelium in all organs and tissues examined. Both MEC 7.46 and MEC 13.3 mAbs were able to precipitate a molecule with an apparent molecular mass of 130 kDa from endothelioma lysates. The protein was synthesized by the cells and exposed on the cell surface. Immunodepletion analysis indicated that MEC 13.3 recognized a molecule related to the murine from of PECAM or CD31. We believe that these mAbs are promising tools for the identification of murine EC and for studying their ontogenesis and functions.
This report describes a case of a 3-year-old female with Cushing's syndrome associated with ganglioneuroblastoma and opsomyoclonic encephalopathy. Immunohistochemistry showed the tumor to be adrenocorticotropic hormone-secreting. At autopsy, a cerebellar degenerative lesion was also demonstrated.
Human macrophages obtained by in vitro maturation of peripheral blood monocytes express a surface antigen, PAM-1, recognized by a monoclonal antibody and typical of pulmonary alveolar and tissue macrophages. PAM-1, undetectable in freshly isolated peripheral blood monocytes, was expressed in monocyte-derived macrophages after 3 days of in vitro adherent culture and was maximal after 14-15 days (50%-60% of positive cells). Similar levels of PAM-1 positivity were observed in non-adherent monocyte-derived macrophages suggesting that cell adhesion was not a critical requisite for the expression of this antigen. Bacterial lipopolysaccharide and a monocyte chemotactic protein preparation respectively suppressed and upregulated PAM-1 expression in monocyte-derived macrophages. In contrast, interferon-gamma, although enhancing the levels of class II HLA-DR antigen in monocyte-derived macrophages, did not influence the kinetics of appearance and the levels of PAM-1 in these cells. Thus, expression of PAM-1, which is restricted to certain stages of the monocyte-macrophage differentiation pathway, is also differentially modulated by activation signals, which can be present in the micro-environment of inflammed tissues.
Tumor-associated macrophages (TAM) have a complex relationship with the neoplastic cells of the tumor. On the one hand, the two cell types produce reciprocal growth factors and may be considered to have a symbiotic relationship. On the other hand, TAM can be activated to inhibit tumor growth and destroy neoplastic cells. Here, Alberto Mantovani and colleagues describe this delicate balance and the prospects for its therapeutic manipulation.
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Tumor regression induced in cancer patients by i.v. infusion of interleukin-2 (IL-2) is often accompanied by severe side effects. To investigate whether local administration would affect immune response without the side effects, two 5-day cycles of continuous intraarterial [internal iliac artery] infusion of recombinant interleukin-2 (rIL-2) were performed in 12 patients with transitional cell carcinoma (tumor stage 1, node stage 0, metastasis stage 0, and grade 1-2) of the bladder. Four groups of 3 patients were treated at each of 4 escalating doses of rIL-2 (18 x 10(3), 18 x 10(4), 18 x 10(5), and 18 x 10(6) IU/m2/day) throughout the course of the two IL-2 cycles. This treatment was effective in inducing a marked intratumor inflammatory response, consisting mainly of T-lymphocytes and macrophages. A remarkable dose-dependent increase in the levels of soluble CD25 was observed in the urine of all patients, which was associated constantly with an enhanced number of intratumor CD25+ cells. Intratumor macrophages were often immunoreactive for interleukin-1 and/or tumor necrosis factor, suggesting an activated status. Increased levels of soluble CD25 and CD25+ lymphocytes were observed in peripheral blood only at the two highest doses of rIL-2, while increased percentages of circulating HLA-DR+ and CD71+ lymphoid cells and enhancement of CD3+/CD16+ T-lymphocytes were found at lower doses. Peripheral blood eosinophils were augmented in almost all patients but were rarely increased in situ. We provide evidence that continuous intraarterial infusion of rIL-2 activates host immune response, acting preferentially at the tissue level.
We have investigated clinical and immunological effects of two 5-day cycle continuous infusion of four escalating doses (3,000; 30,000; 300,000; 3,000,000 U/m2/day) of intra-arterial (i.a.) rIL-2, in ten patients with superficial (Ta-T1, N0, M0) bladder TCC. Tumor TUR was performed four days after the end of the second IL-2 cycle. No clinical or laboratory toxicity was detected in any but one patient, who developed grade III hypotension and grade III neurological toxicity. Two CR and two PR were observed in patients treated with 30,000 and 300,000 U/m2/day of rIL-2, respectively. Clinical responses lasted 8+, 8+, 4+ and 4+ months, respectively. Two out of ten patients developed recurrences two months after tumor resection. In the peripheral blood, no changes in the percentage of T (CD3+) lymphocytes were observed, while a significant increase of CD3+ CD16+ T cells was found in 6/9 patients. In contrast, NK (CD3- CD16+) cells were augmented only in 2/9 patients. An increase of B lymphocytes (CD19+ cells) and monocytes (CD14+ cells) was also observed in 3/9 and 7/9 patients, respectively. Lymphocyte activation marker expression (CD25, CD71, HLA-DR) increased mainly on T lymphocytes. NK and LAK activities were enhanced in 4/10 patients, while ADCC activity augmented in 2/10 patients. No detectable increased levels of plasmatic lymphokines (gamma-IFN and alpha-TNF) were found. Enhanced CD8+ and CD4+ tumor infiltrating lymphocytes were demonstrated after IL-2 treatment. Moreover, at the tumor site, lymphocytes expressing CD25 and HLA-DR antigens were noted. Tumor infiltrating macrophages were positive for IL-1 alpha, IL-1 beta and alpha-TNF.
In the present study the number of E-RFC and the in vitro response to PHA and ConA were determined for lymphocytes obtained from spleens and lymph nodes of untreated HD patients. The results were correlated with tissue involvement and with the histological pattern of the disease. Our investigation has revealed: (1) involved spleens and lymph nodes contained a higher percentage of T-lymphocytes than uninvolved tissues. The differences was statistically significant for lymph nodes. (2) A statistically significant correlation was not found between T-lymphocyte numbers and histology. (3) Lymphocytes from uninvolved spleens were significantly more reactive to both mitogens than cells from involved spleens and control non-neoplastic spleens as well. (4) Similar results were obtained with lymph node lymphocytes; however, a significant difference was observed only when cells were stimulated with ConA. (5) The difference in proliferative response between uninvolved and involved tissues was maximally expressed in MC and progressively decreased in NS and LP. (6) T-cell enrichment does not alter the difference between uninvolved and involved spleens. (7) Cells from uninvolved and involved spleens, cultured for 24 hours, maintained unchanged their proliferative capacity. In this study evidence is provided that in HD T-lymphocytes from uninvolved tissues are characterized by an increased reactivity in mitogens, whereas cells from involved tissues are normally responsive.
In the present work the in vitro response to PHA and ConA was determined for thymus lymphocytes from normal, hyperplastic, and neoplastic conditions as well as for peripheral blood lymphocytes from normal control donors. Our investigation has revealed these results: (1) lymphocytes from normal thymus are less responsive to both mitogens than lymphocytes from peripheral blood and abnormal thymus. (2) Lymphocytes from thymic benign lymphoid hyperplasia show an increased response only to PHA regardless of the extent of hyperplasia. (3) Lymphocytes from thymic neoplasia display an increased response to both mitogens regardless of the histologic pattern of the tumor studied. (4) Thymic lymphocytes from patients with myasthenia gravis have a mitogenic response higher than that from thymic lymphocytes from nonmyasthenic subjects. (5) In patients with myasthenia gravis, lymphocytes from neoplastic thymus show a response to PHA higher than that observed for lymphocytes from hyperplastic thymus. It seems clear that two conditions maximally enhance mitogenic response of thymus lymphocytes: thymomas and myasthenia.