Caspase inhibition reveals functional cooperation between p55- and p75-TNF receptors in cell necrosis.

TNF-induced caspase activation is critically involved in both apoptosis and protection from cell necrosis. We have investigated the roles of the p55- and p75-TNF receptors (TNFR1 and TNFR2) in the induction of mouse L-M cell death in the presence of a caspase inhibitor (zVAD-fmk) and a transcription inhibitor (actinomycin D), i.e. under conditions in which protective pathways requiring caspase activation and protein synthesis were blocked. Cytometric analysis after TNF treatment showed that apoptosis was inhibited, while necrosis was highly activated. In contrast, apoptosis was observed in cells treated with TNF and actinomycin D alone. Stimulation of TNFR1 was sufficient to induce either cell necrosis or apoptosis, even when we blocked endogenous TNF with an anti-murine TNF antibody. Experiments based on the use of receptor-agonist and antagonist antibodies also showed that TNFR2 contributes to cell necrosis and apoptosis. Simultaneous stimulation of TNFR2 and TNFR1 with specific agonists indicated that TNFR2 functionally cooperates with TNFR1 to potentiate the response indirectly, by inducing endogenous TNF cytotoxicity. Caspase inhibitors enhanced the cytotoxic effect of endogenous TNF, suggesting that TNFR2 modulation can regulate the global necrotic response to TNF. TNFR2 modulation could play an important role in determining the response to TNF in pathophysiological conditions characterized by caspase down-regulation and local TNF production.

Upregulation of p75 tumor necrosis factor alpha receptor in Mycobacterium avium-infected mice: evidence for a functional role.

The bacterial growth and the production of tumor necrosis factor alpha (TNF-alpha) and TNF receptors (TNF-Rs) in the spleen and blood of BALB/c mice challenged with Mycobacterium avium complex (MAC) were monitored. Infection developed in two phases: the first, up to day 21, was associated with rapid MAC multiplication in the spleen and a drop in the mycobacteremia, and the second was associated with control of the infection in both compartments. In the spleen, TNF-alpha and TNF-RII mRNA levels peaked on day 21 and then slowly decreased; however, no increase in the level of TNF-RI mRNA was observed throughout these experiments. The level of circulating soluble TNF-RII (sTNF-RII) was transiently increased after day 21. In a model in which overproduction of bioactive TNF-alpha was triggered in response to a second infection with MAC, an increased production of sTNF-RII by cultured splenocytes was also observed. Administration of an antagonist anti-TNF-RII monoclonal antibody (MAb 6G1) to infected mice inhibited the bacterial growth in the spleen, suggesting that the TNF-RII and/or sTNF-RII was functionally involved in the mechanisms that control the infection. Overall, these observations suggest that upregulation of TNF-RII or sTNF-RII contributes to modulation of the TNF-alpha antibacterial activity in MAC infections.

Tumor cell targeting with antibody-avidin complexes and biotinylated tumor necrosis factor alpha.

Tumor pretargeting with biotinylated antibodies and avidin, followed by a delayed delivery of radioactive-labeled biotin, is currently used for in vivo diagnosis and therapy in cancer patients. Herein, we describe the use of a three-step antibody/avidin targeting approach to increase the local concentration and the persistence of biotinylated human tumor necrosis factor alpha (bio-TNF) on a mouse tumor. Mouse RMA lymphoma cells were transfected with the Thy 1.1 allele (RMA-Thy 1.1) to generate a unique tumor-associated antigen. In vitro pretargeting of RMA-Thy 1.1 cells with the biotinylated anti-Thy 1.1 monoclonal antibody 19E12 (bio-19E12) and NeutrAvidin increased the amount of bio-TNF that bound to the cell (10-20 times in comparison with non-pretargeted cells), as well as its half-life on the surface (>30 times). Furthermore, cell pretargeting reduced by more than 2 orders of magnitude the LD50 of bio-TNF in a cytolytic assay with actinomycin D. Finally, RMA-Thy 1.1 cells, pretreated in vitro with bio-TNF according to the three-step procedure and injected into syngeneic C57/BL6 mice, were less tumorigenic than controls. These results indicate that the three-step targeting approach markedly increases the amount and the persistence of bio-TNF on the cell surface and that cell-bound bio-TNF can trigger cytolytic effects in vitro and antitumor effects in vivo. Tumor pretargeting with biotinylated antibodies and avidin could be a novel strategy for increasing the therapeutic index of TNF.

In vivo imaging of chromogranin A-positive endocrine tumours by three-step monoclonal antibody targeting.

The detection of chromogranins (Cg) by immunohistochemistry and serology represents a new in vitro diagnostic tool for endocrine tumours. We have recently reported on the feasibility of targeting chromogranin A (CgA) for in vivo detection of pituitary adenomas by immunoscintigraphy (ISG). The scintigraphic procedure, based on an anti-CgA monoclonal antibody and on the avidin-biotin three-step method (Cg-3S-ISG), was evaluated on a group of 29 consecutive patients with known or suspected endocrine tumours other than pituitary adenomas, i.e. medullary thyroid carcinoma, carcinoid, insulinoma and parathormone- or ACTH-producing tumours. Primary tumours (10) and recurrences (16) were visualised in 26 patients, whereas conventional imaging techniques (planar radiography, computerised tomography, magnetic resonance imaging and ultrasonography) failed to detect the tumour sites in ten of the same (Cg-3S-ISG-positive) patients. Therefore, these preliminary results indicate that Cg-3S-ISG, the first immunological method able to detect endocrine tumours in vivo, has a higher diagnostic accuracy than conventional imaging techniques (93.1% compared with 65.5%).

Characterisation of circulating chromogranin A in human cancer patients.

The structure of circulating chromogranin A (CgA) of phaeochromocytoma patients was characterised and compared with that of CgA extracted from tumours. Size exclusion chromatography experiments provided evidence that CgA is present in the blood of different patients, as well as in tumour extracts, as multiple forms having different hydrodynamic sizes of 600 kDa (CgA-I), 100 kDa (CgA-II) and 55 kDA (CgA-III). The amount of each CgA form as a proportion of the total antigenic material was different in different patients. Western blot analysis of chromatographic fractions indicated that these forms are made up by polypeptides of similar molecular weight (about 60-70 kDa). All CgA forms express the epitopes recognised by two monoclonal antibodies (A11 and B4E11), directed against residues 68-70 and 81-90 of human CgA. However, their relative immunoreactivity was markedly different. No evidence for the presence of multimeric complexes in the CgA-I fraction was obtained by various immunological and biochemical methods. These results suggest that circulating CgA in phaeochromocytoma patients consists of at least three forms that appear to be made up by polypeptides with similar molecular weight and different hydrodynamic properties and immunoreactivity. We hypothesise that different conformations and shapes contribute to the heterogeneity of circulating CgA.

Antigenic regions of human chromogranin A and their topographic relationships with structural/functional domains.

Chromogranin A is a protein contained in the secretory granules of many neuroendocrine cells. The linear antigenic sites of human chromogranin A were studied by examining the cross-reaction of polyclonal and monoclonal anti-chromogranin A antibodies with native chromogranin A and with synthetic peptides encompassing most of the chromogranin A sequence. Chromogranin A residues 1-20, 47-67, 107-158, 254-297, 331-375, and 395-419 were found to be poorly or not antigenic, while residues 25-46, 163-210, 231-253, 298-314 and 68-106, 222-230, 315-330, 376-394 were found to contain weak and strong antigenic sites, respectively. Residues 68-70 (GAK) and 81-90 (GFEDELSEVL) were strongly recognized by two mouse mAbs (B4E11 and A11, respectively). Since mAb A11 has been previously used for immunohistochemical analysis of chromogranin-A-producing tissues from different species and for in vivo imaging of chromogranin-A-positive endocrine tumors, these results imply that at least part of the 81-90 region is surface-exposed in cryostat tissue sections as well as in vivo. The results may help in selecting new antibodies with improved affinity and immunogenicity for in vivo targeting of chromogranin-A-producing tumors.

Production of mouse monoclonal antibodies directed against the oolemma of human and hamster oocytes by intra-splenic injection of oocytes.

PROBLEM: To develop an additional approach for the study of oolemmal surface moieties involved in gamete interactions, we decided to obtain monoclonal antibodies by intrasplenic injection of human and hamster oocytes in Balb/c mice. METHOD: Two Balb/c males were injected three times intrasplenically at 15-day intervalS with approximately 40 zona-free hamster and 3-5 zona-free human oocytes. After the third injection, spleen cells were fused and hybridomas developed. We used a novel screening system based upon the use of sections of frozen human and hamster eggs, tested by means of indirect immunofluorescence. The antibodies that we produced were evaluated for their ability to interfere with the zona-free hamster eggs penetration by human spermatozoa. The B2B5 antibody was also developed as ascitic fluid and further characterized. RESULTS: Seven antibodies reactive with hamster oocytes were produced. Six of them also reacted with human oolemmas. The binding was confined to the oolemma, and no staining of the zona nor the cytoplasm was present. One of these antibodies reduced the penetration of zona-free hamster eggs by human spermatozoa. This antibody, B2B5, an IgM kappa, was confirmed to interact with the oolemma by means of indirect immunofluorescence of fresh eggs and Covasphere binding. B2B5 did not react with other human or hamster tissues except capacitated human spermatozoa. The reactivity with the oolemma of hamster oocytes was not lost after egg penetration by human sperm. CONCLUSIONS: Intrasplenic immunization using zona-free human and hamster oocytes allows the production of anti-oolemma antibodies. A system of screening based upon the use of sections of frozen eggs also allows an easy and quick scoring of many supernatants. B2B5 monoclonal anti-oolemma antibody deserves further studies in that is able to interfere with fertilization and its antigen appears to be confined to the gametes surface.

The prevalence and clinical significance of chromogranin A and secretogranin II immunoreactivity in colorectal adenocarcinomas.

Colorectal adenocarcinomas may display features of endocrine differentiation, shown by argyrophil stains and by the expression of endocrine markers such as chromogranin A. We investigated chromogranin A and secretogranin II immunoreactivity in a series of 208 carcinomas of the large bowel to assess the prevalence and clinical significance of endocrine differentiation. Tumors expressing endocrine markers were classified as low expressors (< than 1 immunoreactive tumour cell/mm2) and high expressors (> than 1 immunoreactive tumour cell/mm2). There were 33 (16%) carcinomas showing both chromogranin A and secretogranin II immunoreactivity: 11 tumours (5%) were high expressors. Endocrine differentiation was not related to the disease stage, tumour location, grade, DNA ploidy and p53 protein accumulation. In the entire series chromogranin A immunoreactivity did not provide prognostic information using univariate and multivariate analysis. A worse overall survival (P = 0.048) was demonstrated for the stage III patients with high expressor tumours, but there were only five patients in this group. The results of our investigation suggest that chromogranin A immunoreactivity is not a useful variable in the prognostic assessment of colorectal adenocarcinomas.

Immunohistochemical localization of chromogranin A and B in the endocrine cells of the alimentary tract of the green frog, Rana esculenta.

Novel monoclonal antibodies to human chromogranin A (CgA) and chromogranin B (CgB) were used to investigate the presence of immunoreactive (-IR) elements in the alimentary tract of the green frog Rana esculenta. Numerous CgA-IR and a few CgB-IR endocrine cells were found within the gut mucosa, from the oesophagus to the cloaca, with some local differences in density. Co-localization studies demonstrated that they were co-stored in almost all the serotonin-IR, the amylin-IR or islet amyloid polypeptide-IR cells and in the peptide tyrosine tyrosine-IR cells located proximal to the pylorus, but not in those located in more caudal tracts. No other co-localization was demonstrated; substances investigated included somatostatin, substance P, gastrin/cholecystokinin, glucagon, glycentin, bombesin, secretin and neurotensin. CgA-IR and CgB-IR cells nearly always displayed argyrophilia with the Grimelius silver method.

Identification of human immunodeficiency virus type 1 glycoprotein gp120/gp41 interacting sites by the idiotypic mimicry of two monoclonal antibodies.

A sequence of four amino acid residues amino-terminal to the only intramolecular disulphide bond of the human immunodeficiency virus type 1 (HIV-1) transmembrane protein gp41 is recognized by an anti-idiotypic antibody (9G5A) raised against another monoclonal antibody (M38), which recognizes the C5 region of gp120. 9G5A is an Ab2 beta antibody (internal image of the M38 epitope) in that it inhibits the interaction of M38 to its antigen. The binding of 9G5A to gp41 can be inhibited by M38 showing that the two antibodies interact via their paratopes. 9G5A neutralizes HIV-1 infection and syncytia formation. Ab3 antibodies induced in mice and rabbits immunized with 9G5A also can neutralize virus in both assays. These data show that the M38-defined epitope of the carboxy-terminal region of gp120 interacts with the 9G5A-defined epitope of gp41, and that this interaction can be reproduced by the idiotypic mimicry of the two antibodies. The results are consistent with a proposed molecular model of the two env regions which predicts the presence, within the C5 region of gp120, of a large intramolecular pocket that is contacted by the gp41 cysteine loop.

Immunolocalization of secretogranin II, chromogranin A, and chromogranin B in differentiating human neuroblastoma cells.

In order to obtain further insights into the expression of the known markers of secretory neuroendocrine dense core organelles, secretogranin II (SgII), chromogranin A (CgA), and chromogranin B (CgB) during neuronal differentiation, the immunolocalization of these proteins was studied by means of double immunofluorescence in both undifferentiated and retinoic acid-differentiated SH-SY5Y human neuroblastoma cells. The majority of undifferentiated cells was not immunolabeled for all three proteins. In the majority of differentiated cells, a clearly punctate SgII immunolabeling indicative of the presence of secretory organelles was present in the Golgi region, at the cell periphery, along the neurites and in growth cones. Only relatively few of the SgII-immunolabeled cells were also immunolabeled for CgA and CgB, and in a single cell the three proteins were not always present in the same organelles. These results, obtained in a cultured cell line, confirm the not necessarily parallel distribution of SgII, CgA, and CgB observed in different neuroendocrine tissues and suggest that SgII may be the best marker of human neuroblastoma cell differentiation.

Immunodetection of secretogranin II in animal and human tissues by new monoclonal antibodies.

Secretogranin II (chromogranin C) is an acidic tyrosine-sulfated secretory protein, known to be a marker of neuroendocrine secretory products and of specific neuroendocrine tumours. In order to obtain anti-secretogranin II monoclonal antibodies for cell biology studies and, in particular, for clinical applications, we immunized mice with a secretogranin II-enriched fraction prepared from homogenates of bovine anterior pituitaries. Hybridoma supernatants obtained from the splenocytes of a hyperimmune mouse, screened with an enzyme-linked immunosorbent assay, were analyzed by both immunocytochemistry and two-dimensional immunoblotting. By using this experimental approach, we were able to identify two monoclonal antibodies (8G1 and 5A7) which recognize bovine secretogranin II. Both immunocytochemistry and immunoblotting revealed that one of them, the 5A7 antibody, cross-reacts with the human antigen. The distribution patterns of the immunoreactivity, obtained by immunocytochemistry with the 5A7 antibody in animal and human tissues, partially overlap those, obtained by using a polyclonal antiserum elicited against bovine secretogranin II, previously described. Moreover, the 5A7, but not the polyclonal antibody, reacts with some duodeno-jejunal cells. In conclusion, both the 5A7 and 8G1 antibodies can be useful for cell biology studies. The 5A7 antibody can be used for the detection of secretogranin II in human tissues and should be of help in clinical and pathological practices.

Identification of the endocrine cells detected by the monoclonal antibody HISL-19 in human tissues.

The human endocrine cells reacting with the monoclonal antibody HISL-19 were identified with hormone antisera of proven specificity using a double immunostaining procedure. The epitope for HISL-19 was found in all types of pituitary cells except ACTH cells, in thyroid C cells, in all types of adrenal medullary and pancreatic islet cells and in somatostatin and pancreatic polypeptide cells of the gastrointestinal mucosa. No staining was found in parathyroid cells and in most gastrointestinal endocrine cells. Either paranuclear focal accumulation or diffuse cytoplasmic distribution of immunoreactive material were found. The spectrum of HISL-19 immunoreactive cells was found to be only in part complementary to that of cells immunoreactive for chromogranin A. Thus, it is concluded that the monoclonal antibody HISL-19 is a useful addition to other immunohistochemical markers for the detection of cells showing neuroendocrine features.

Idiotypic replica of an anti-human tumor-associated antigen monoclonal antibody. Analysis of monoclonal Ab1 and Ab3 fine specificity.

CaMBr1 is a tissue-specific and tumor-associated saccharidic epitope, defined by mAb MBr1 (Ab1), expressed on glycoconjugates of the human mammary carcinoma cell line MCF-7 and of normal and neoplastic mammary epithelial cells. An anti-anti-idiotypic monoclonal Ab3, 2G-3, identifying a human breast tumor associated antigen, was raised by using as immunogen a mouse anti-idiotypic monoclonal Ab2, A3B10, which behaves as the internal image of CaMBr1. mAb 2G-3, as well as MBr1, defines a saccharidic epitope on glycoconjugates extracted from MCF-7 cells and shows MBr1-like reactivity on normal and neoplastic-tissues. Experimental evidence, however, suggests that the fine immunoreactivity of the two antibodies is not identical, because MBr1 has a preferential reactivity with glycolipids and 2G-3 with glycoproteins. We suggest that a possible biologic explanation for our findings could reside in the nature of the immunogens used to raise the two mAb (glycolipid vs protein "internal image").

Preparation and characterization of anti-human chromogranin A and chromogranin B (secretogranin I) monoclonal antibodies.

Chromogranin A, chromogranin B/secretogranin I and chromogranin C/secretogranin II are acidic sulphated and phosphorylated secretory proteins present in a large number of endocrine and neuronal tissues. It has been suggested that these proteins may be useful immunohistochemical markers for human tumours of endocrine origin and their measurement in plasma has been proposed as a diagnostic tool in patients with these tumours. In order to obtain anti-human chromogranins/secretogranins antibodies for clinical applications, we immunized mice with whole chromaffin granules isolated from human pheochromocytoma. The immune sera analysed by two-dimensional immunoblotting were found to recognize chromogranins/secretogranins and other unidentified proteins and to react in immunocytochemistry with pheochromocytoma as well as with a number of endocrine cells of different types. Hybridoma supernatants obtained from the splenocytes of a hyperimmune mouse, screened with an enzyme-linked immunosorbent assay, were analysed by both immunocytochemistry and two-dimensional immunoblotting. By using this experimental approach we were able to identify several monoclonal antibodies against human chromaffin granule components. In particular, we have characterized one anti-human chromogranin A and one anti-human chromogranin B/secretogranin I monoclonal antibody which showed a very specific pattern both in immunocytochemistry and in two-dimensional immunoblotting.

Immunoreactivity of hormonally-characterized human endocrine cells against three novel anti-human chromogranin B(B11 and B13) and chromogranin A (A11) monoclonal antibodies.

Two novel monoclonal antibodies, called B11 and B13, directed exclusively against human chromogranin B (CgB) and another antibody, A11, specific for human chromogranin A (CgA), were obtained by immunization of mice with chromaffin granules, the fusion of their splenocytes, the screening of hybridomas supernatants by ELISA and immunohistochemistry, and characterization of the antibodies by two-dimensional immunoblotting. The antibodies were used in immunohistochemical tests to investigate the distribution of CgA and CgB in hormonally-identified cells of the human endocrine system. The A11 antibody confirmed the occurrence of CgA in gut EC, ECL, gastrin, secretin and neurotensin cells, pancreatic A and PP cells, parathyroid chief cells, pituitary TSH and gonadotrope cells and adrenal medullary cells. Only a fraction of CgA-immunoreactive cells in the human gut and pancreas showed C-terminus arginine-glycinamide immunoreactivity, suggesting pancreastatin storage. Both CgB antibodies showed immunoreactivity in gastrin cells, intestinal (but not gastric) EC cells, pancreatic A and PP cells, pituitary TSH and gonadotrope cells and adrenal medullary cells. In addition, the B11 antibody stained thyroid C cells and the B13 antibody stained the Golgi area of pituitary GH cells. It is concluded that most CgB is stored in the same cells showing CgA, although some CgA-rich cells, like gastric EC and ECL cells. lacked B11 and B13 immunoreactivities and some CgA-poor cells, like human thyroid C cells, showed intense B11 immunostaining.

HIV env glycoprotein shares a cross-reacting epitope with a surface protein present on activated human monocytes and involved in antigen presentation.

A serological cross-reactivity between env gp120 glycoprotein of the human immunodeficiency virus (HIV) and a human cellular surface protein has been defined by a monoclonal antibody (M38) raised against HIV. The cellular antigen is a protein of ca. 80 kDa expressed on a small fraction of mononuclear cells in peripheral blood and in lymph nodes. The protein behaves as an activation antigen of the monocytic lineage since it is expressed by monocytes in plastic-adherent culture conditions and by interferon-gamma-treated monocytes and pro-monocytic U937 cells. The protein is involved in antigen presentation since the antibody efficiently inhibits the proliferation of responsive lymphocytes in autologous tetanus toxoid presentation assays. In the T lymphoblastoid line H9, the protein is present in very small amounts, is not induced by interferon-gamma and increases after HIV infection. Sera from lymphoadenopathy syndrome and acquired immunodeficiency syndrome (AIDS) patients fail to detect the cellular protein, although containing antibodies reacting with gp120. We propose that both viral and cellular structures recognized by the monoclonal antibody (mAb) are involved in interactions with CD4 molecules of T helper lymphocytes and that such molecular mimicry might be relevant in the pathology of HIV infection. This view is supported by the finding that BL/10T4, a CD4-specific mAb, binds to M38 neutralizing its interactions with HIV and with monocytes. mAb M38 thus behaves as the internal image of CD4. This single property would explain all its diverse binding characteristics.

Anti-human tumor antibodies induced in mice and rabbits by "internal image" anti-idiotypic monoclonal immunoglobulins.

MBr1 is a murine monoclonal antibody, defining a saccharidic epitope [CaMBr1] of a human tissue-specific, tumor-associated globoside, present on the mammary carcinoma cell line MCF-7. The same epitope is shared by glycoproteins present on normal and neoplastic mammary epithelial cells, and by mucins from some ovarian cyst fluids. We have used MBr1 as the monoclonal antitumor antibody in an idiotypic sequence of immunizations in order to obtain and characterize "internal images" of the original epitope to be used as substitutes of the nominal antigen in serologic immunoassays. Two monoclonal anti-idiotypic antibodies (beta-1 and beta-2), which reacted with paratope-related idiotopes on MBr1, were obtained. The analysis of the antigenic and immunogenic properties of these molecules by both "antigen" and "antibody" competition assays provided evidence that both beta-1 and beta-2 bear "internal images" of the MBr1-defined epitope. Moreover, when injected in mice and rabbits both beta-1 and beta-2 induced anti anti-idiotypic antibodies, which mimicked MBr1 in binding MCF-7 as well as normal and neoplastic mammary gland epithelial cells. These data are discussed in terms of their possible application to the production of tumor-associated antigen substitutes and their use in serologic immunoassays.