An immunocytochemical investigation with monoclonal antibodies to somatostatin

Summary Four monoclonal antibodies specific for somatostatin have been produced and characterized. These antibodies were used to assess the anatomical relationship of somatostatin-containing cells in the pancreas and gastrointestinal tract of man, baboon and rat with ten other peptide-containing endocrine cells. The peptides investigated were gastrin, cholecystokinin, motilin, secretin, neurotensin, gastric inhibitory polypeptide, gut-glucagon, pancreatic glucagon, pancreatic polypeptide and insulin.The only regions in which somatostatin cells were seen in close contact with another endocrine cell were in the pancreas and the gastric antrum. In the pancreas somatostatin cells were commonly seen in close contact with insulin, glucagon and pancreatic polypeptide cells and infrequent contact was demonstrable with the gastrin-immunoreactive cells in the antrum of both rat and man. In all other cases no evidence was obtained for a close anatomical relationship between somatostatin cells and the other enteroendocrine cells.     

An immunocytochemical study of normal and abnormal human cerebrospinal fluid with monoclonal antibodies to glial fibrillary acidic protein

The presence of astrocytes in the cerebrospinal fluid (CSF) of patients may be of diagnostic importance. However, the frequency with which astrocytes are shed into normal and abnormal human CSF is unknown. This issue was studied using monoclonal antibodies to an astrocyte-specific antigen, glial fibrillary acidic protein (GFAP), and immunoperoxidase cytochemistry. The study was prospectively conducted on 108 CSF preparations diagnosed as normal, reactive, metastatic malignancy or suspicious for metastatic malignancy. To validate these methods, cells from a clonal human glioma cell line, which contains astrocytes rich in GFAP, were processed in a manner identical to that used for the CSFs obtained from patients. Studies of the human glioma cell line demonstrated intense GFAP immunoreactivity in the majority of the malignant astrocytes. In contrast, none of the CSFs contained GFAP-positive cells. We conclude that immunocytochemical methods can detect GFAP in neoplastic human astrocytes but that nonneoplastic GFAP-positive cells are uncommon in human CSF; such cells were not seen in our large series of normal and abnormal human CSFs. The immunocytochemical detection of GFAP may be a useful criterion for distinguishing malignant astrocytes from other types of malignant cells in human CSF.     

Characterization and immunocytochemical application of monoclonal antibodies against enkephalins

Monoclonal antibodies were produced following immunization of mice with either [Leu5]enkephalin-bovine serum albumin or [Met5]enkephalin-keyhold limpet hemocyanin conjugates. Two monoclonal antibodies coded NOC1 and NOC2, respectively, were derived. These monoclonal antibodies did not discriminate between Leu- and Met-enkephalin in either radioimmunoassay or immunocytochemistry. NOC1 was characterized in detail. In radioimmunoassay NOC1 displayed about 40% crossreactivity with C-terminal extended Met-enkephalin hexapeptides and 7% with the extended heptapeptide (-Arg-Phe-OH), but did not recognize other endogenous peptides. In immunocytochemistry the NOC1 and NOC2 recognized all well-established "enkephalin immunoreactive sites," but they did not bind to areas known to contain beta-endorphin or high levels of pro-enkephalin. NOC1 was shown to be a suitable tool to demonstrate enkephalin immunoreactive sites by radioimmunocytochemistry utilizing both internally and externally labeled monoclonal antibodies.     

Malignant melanoma in fine needle aspirates and effusions. An immunocytochemical study using monoclonal antibodies

The value of monoclonal antibodies (MAbs) for the immunodetection of keratin, vimentin and two melanoma-associated antigens recognized by NKI/C3 and NKI/Bteb for the diagnosis of malignant melanoma has been previously established on histologic preparations. In the present study, cytologic preparations from 20 fine needle aspirates and effusions from patients with malignant melanoma were evaluated using these antibodies. Twenty of 20 smears were negative for keratin, and 20 of 20 smears were positive for vimentin. Positivity for NKI/C3 was seen in 12 of 12 cases studied, and for NKI/Bteb in 12 of 13 cases. These results indicate that a panel of MAbs consisting of anti-keratin, anti-vimentin, NKI/C3 and NKI/Bteb is useful for a more accurate diagnosis of malignant melanomas on cytologic preparations. The expression of these antigens in melanoma cells in cytologic smears can be a valuable aid in the detection of primary (noncutaneous) and metastatic melanomas by fine needle aspiration.     

An immunocytochemical study of fetal cells at the maternal-placental interface using monoclonal antibodies to keratins,vimentin and desmin

Summary Thioredoxin and thioredoxin reductase (NADPH-oxidized thioredoxin oxidoreductase, E.C. 1.6.4.5) have been proposed to be involved in several thioldependent reduction-oxidation reactions in cells. Both proteins have been immunohistochemically demonstrated in the periphery of the cytoplasm and in cytoplasmic granules of acinar and islet cells in mouse pancreas. In animals fed ad libitum, the staining for thioredoxin was more intense in the exocrine acinar cells than in the islet cells, whereas that for thioredoxin reductase was more intense in the endocrine than in the exocrine pancreas. In the islets of fed mice all endocrine cell types showed about the same staining intensity for thioredoxin, while thioredoxin reductase was greatly enriched in the somatostatin-containing D cells. Starvation overnight caused an increased staining for both proteins in the acinar cells as well as in the islets. Under conditions of starvation, thioredoxin reductase, in contrast to thioredoxin, appeared to increase preferentially in the islet B cells, as compared with the D cells. Cysteamine treatment reduced the staining for somatostatin and for thioredoxin reductase in the D cells without any obvious effect on the other pancreatic cells. The results are compatible with a role for thioredoxin and thioredoxin reductase in secretion.     

Production,characterization, and immunocytochemical applications of monoclonal antibodies to human sperm protamines

Three monoclonal antibodies against human protamines were obtained by immunization with total human basic nuclear proteins or purified protamine HP3. The specificity of antibodies was assessed by enzyme-linked immunosorbent assay (ELISA) and Western blot. They recognized three distinct epitopes: One was specific for the protamine P1 family, another was specific for the protamine P2 family and the third was common to both families. All were specific for the human species. Antibodies were used to detect protamines in germ cells by indirect immunofluorescence and by immunoelectron microscopy. Protamines appeared in spermtid nuclei at steps 4–5 of spermiogenesis, i.e., during the chromatin condensation process, and were not accumulated in the cytoplasm before entering the nucleus. © 1993 Wiley-Liss, Inc.     

An immunocytochemical mapping of somatostatin in the cat auditory cortex

Using an indirect immunoperoxidase technique, the localization of somatostatin-28 (1-12)-like immunoreactive fibers and cell bodies in the auditory cortex of the cat (anterior, primary, secondary, temporal, ventral, ventroposterior, posterior and dorsoposterior auditory fields) was studied. In general, the distribution of SOM-ir structures is widespread in the auditory cortex of the feline. A high density of immunoreactive fibers as well as a low density of cell bodies containing somatostatin were observed in all the layers of the eight above-mentioned auditory fields. These data indicate that somatostatin-28 (1-12) could act as a neurotransmitter and/or a neuromodulator in the auditory cortex of the cat. The origin of the SOM-ir fibers in the auditory cortex of the cat, as well as the issue of whether the cell bodies containing somatostatin-28 (1-12) are local or projecting neurons is discussed.     

Histological and immunocytochemical investigation of human coronary vessel development with anti-CD34 antibodies

Information about embryonic development of coronary endothelium is the main clue for the creation of new methods in tissue engineering for treatment of ischemic heart diseases. The purpose of the research was to describe human coronary vessels development on early stages of the prenatal ontogenesis. The first step in human coronary vessels development is the formation of endothelium de novo by transformation of some epicardial and, possibly, endocardial cells. The next step is the ingrowth of sinus venosus endothelium in subepicardium over ventricles and atria, which gives rise to the coronary vessels. Only after 7 days does the primitive coronary plexus of the heart communicate with aorta (third step). During this period, some subepicardial vessels invade myocardium and some intramyocardial vessels contact with the heart cavity. Such intercommunications could help in regulation of blood circulation in primitive coronary plexus before establishment of effective contacts between arterial and venous vessels—excess of blood could be discharged directly into the heart cavity. Additional population of CD34+ cells were revealed inside condensed mesenchyme of the conotruncus; it participates in the formation of vasa vasorum in the aorta. Epicardium and sinus venosus generate endothelium of coronary vessels by neovasculo- and angiogenesis, respectively. During a week after ingrowth of vessels from SV and before their ingrowth to the aorta, ventriculo-coronary communications could be found in the heart.     

Light- and electron-microscopic immunocytochemical investigation of the subcommissural organ using a set of monoclonal antibodies against the bovine Reissner's fiber

Ten monoclonal antibodies (Mabs) against glycoproteins of the bovine Reissner's fiber (RF) have been used in a structural and ultrastructural immunocyto-chemical investigation of the bovine subcommissural organ (SCO) and RF. The SCO of other vertebrate species has also been studied. For comparison, polyclonal antibodies against bovine RF (AFRU) were used. The SCO and RF of ox, pig and dogfish and the SCO of dog, rabbit, rat and frog were submitted to light-microscopic immunocytochemistry using AFRU and Mabs. Postembedding ultrastructural immunocytochemistry was applied to sections of bovine SCO using AFRU and Mabs. Bovine SCO consists of ependymal and hypendymal cell layers, the latter being arranged as cell strands across the posterior commissure, or as hypendymal rosette-like structures. All cytoplasmic regions of the ependymal and hypendymal cells were strongly stained with AFRU. Six Mabs showed the same staining pattern as AFRU, one Mab stained RF strongly and SCO weakly, two Mabs stained RF but not SCO, and, finally, one Mab (3B1) exclusively stained the apices of the ependymal and hypendymal cells. All Mabs recognized the SCO and RF of the pig. Two Mabs bound to the SCO of the dog. One Mab stained the SCO of the rabbit and another the SCO of the rat. The SCO of frog and dogfish were totally negative. Bovine SCO stained with AFRU, showed label in the rough endoplasmic reticulum (RER) and the secretory granules (SG) of the ependymal and hypendymal cells. The former, in the form of parallel cisternae, reticulum or concentric rings, was seen throughout all cytoplasmic regions. SG were abundant in the apical pole of the ependymal and hypendymal cells. Only one Mab showed a staining pattern similar to AFRU. Five Mabs showed strong reactions in the SG but weak labeling of the RER. Mab 3B1 showed the label confined to the SG only. Our results suggest that: (i) in the bovine tissue, some epitopes are present in both precursor and processed materials, whereas others are characteristic of mature glycoproteins present in SG and the RF; (ii) the bovine SCO secretes at least two different compounds present in ependymal and hypendymal cells: (iii) both compounds coexist in the same secretory granule; (iv) there are conserved, class-specific, and species-specific epitopes in the glycoproteins secreted by the SCO of vertebrates.     

An extended set of monoclonal antibodies to pectic homogalacturonan

Three novel rat monoclonal antibodies, designated LM18, LM19 and LM20, were isolated from screens for binding to Arabidopsis thaliana seed coat mucilage. The binding of these antibodies to mucilage subject to enzyme and high pH pre-treatments and to a series of model homogalacturonan-rich pectins with defined levels of methyl-esterification indicated their recognition of pectic homogalacturonan epitopes. The binding capacities of these monoclonal antibodies to cell walls in sections of tobacco stem pith parenchyma were also differentially sensitive to equivalent treatments with high pH buffers and pectate lyase. The epitopes bound by these antibodies display some similarities and some differences to the epitopes recognized by the previously isolated and established pectic homogalacturonan probes JIM5 and JIM7.     

An improved method for conjugating monoclonal antibodies with N-hydroxysulfosuccinimidyl DOTA

A simple, water-soluble procedure for conjugation of monoclonal antibodies to 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) has been improved by optimizing pH, buffer, and temperature conditions for the preparation of N-hydroxysulfosuccinimidyl DOTA and its conjugation to the human/murine chimeric anti-carcinoembryonic antigen antibody cT84.66. This improved method results in a 6-fold increase in conjugation efficiency, a 3-7-fold decrease in antibody cross-linking, a more homogeneous population of conjugate species, and a 5-fold decrease in the quantities of reagents needed for conjugation. The cT84.66-DOTA conjugate was labeled to high specific activity with 111In, 90Y, 88Y, 64Cu, and 67Cu, affording near-quantitative incorporation of the majority of these radiometals. This improved conjugation procedure facilitates large-scale production and radiometal labeling of cT84.66-DOTA for clinical radioimmunotherapy trials.     

A monoclonal antibody to somatostatin with potent in vivo immunoneutralizing activity

The spleen from a Robertsonian mouse with high titer and affinity antiserum after being immunized with somatostatin-14 conjugated to keyhole limpet hemocyanin was fused with FOX-NY cells. Hybridomas were cloned by limiting dilution, subcloned, and ascites was produced from the highest affinity close in pristine-primed Balb/c mice. Ascites fluid contained approximately 20 mg/ml IgG and bound 50% of 1 fmol 125I-[Tyr1]-somatostatin at a final dilution of 1:10,000,000. Binding of this IgG1 antibody, CURE.S6, was inhibited by 50% at 40 pM concentrations of either somatostatin-14 or somatostatin-28, but was not inhibited by [D-Trp8 -somatostatin at 1000-fold higher concentrations. The antibody produced very intense specific immunohistochemical staining of somatostatin endocrine cells in the stomach and pancreas and of intestinal somatostatin neurons with extremely low background staining. Intravenous injection of 2 mg purified antibody in urethane-anesthetized rats resulted in 300-fold increase in plasma GH within 15 min. CURE.S6 is a high affinity monoclonal antibody directed at the biologically active somatostatin ring structure. This antibody is useful for in vivo immunoneutralization of exogenous and endogenous somatostatin in the rat and also is an excellent reagent for immunohistochemical localization of somatostatin.     

An immunohistochemical study performed with monoclonal and polyclonal antibodies to mouse epidermal growth factor

We developed two monoclonal antibodies, E5 and F5, which react with mouse epidermal growth factor (mEGF) and urogastrone. These antibodies and a rabbit antiserum to mEGF were used for immunohistochemical analysis of staining reactions in rodent and human tissues. Our results do not confirm the published reports of EGF-like immunoreactivity in Brunner's glands, human submandibular glands, human kidney tissue, or rodent brain sections.     

Molecular dynamic investigation of the interaction of supported affinity ligands with monoclonal antibodies

Diagnostics and therapeutic treatments based on monoclonal antibodies have been attaining an increasing importance in the past decades, but their large scale employment requires the optimization of purification processes. To obtain this goal, research is focusing on affinity chromatography techniques and the development of new synthetic ligands. In this work we present a computational investigation aimed at obtaining some guidelines for the rational design of affinity ligands, through the study of their interactions with both monoclonal antibodies (modeled as the FC domain of human IgG) and a model support material (agarose). The study was carried out performing molecular dynamics simulations of the support-spacer-ligand-IgG complex in explicit water. Binding energies between IgG and two supported ligands, a disubstituted derivative of trichlorotriazine and a tetrameric peptide, were determined with the linear interaction energy and MM-GBSA approaches. A detailed study of the possible binding sites of the considered ligands was performed exploiting docking protocols and MD simulations. It was found that both ligands bind IgG in the same site as protein A, which is the hinge region between the CH2 and CH3 domains of IgG. However this site is not easily accessible and requires a high mobility of the ligands. The energetic analysis revealed that van der Waals and electrostatic energies of interaction of the triazine ligand with the support are significant and comparable to those with the protein, so that they limit its capability to reach the protein binding site. A similar result was found also for the tetrameric peptide, which is however able to circumvent the problem; for steric reasons only two of its arms can interact at the same time with the agarose support, thus leaving the remaining two available to bind the protein. These results indicate that the interaction between ligand and support material is an important parameter, which should be considered in the computational and experimental design of ligands for affinity chromatography.     

An improved method of direct labeling monoclonal antibodies with 99mTc

An improved method of direct labeling MAbs with ^99mTc is described. Two murine monoclonal antibodies, designated Lym-1 and B72.3, have been successfully labeled with ^99mTc in 0.1 M borate buffer at pH 9.3. The choice of buffer and pH was essential for obtaining a radiolabeling yield ⩾98%. In vitro studies demonstrated that the radiolabeled antibodies were stable and retained their immunoreactivity. Imaging and biodistribution studies using Raji and LS174T human tumor-bearing nude mice demonstrated a significant tumor uptake at 24-h post-injection of ^99mTc-labeled MAbs. This improved labeling method showed better stability than those of previously published methods and resulted in significant improvement in the uptake of antibody in tumor. External images at 24 h post-injection revealed clearly visible tumors demonstrating the benefit of this method for tumor immunoscintigraphy.     

An amphipathic sulphated glycoconjugate of Leishmania: characterization with monoclonal antibodies

A major glycoconjugate of Leishmania tropica major identified by two monoclonal antibodies was shown to be an externally oriented, amphipathic membrane antigen shed into the culture medium in which the parasites grow. This molecule could be labelled metabolically with [3H]glucose, [3H]galactose, [32P]phosphate and [35S]sulphate. It migrated as a polydisperse band upon electrophoresis in SDS-polyacrylamide gels, spanning the region of the gel corresponding to an apparent mol. wt. of 20 000-67 000 daltons. An apparently identical family of molecules could be labelled on the surface of living promastigotes using galactose oxidase and [3H]-sodium borohydride. This molecule was shown to be released into the supernatant over a period of several hours. Detection of the 3H- or 35S-labelled molecule required several days exposure of autoradiographs, but a novel blotting technique using nitrocellulose coated with monoclonal antibody allowed rapid detection of the molecule in charge shift electrophoresis, Western blotting and dot blotting. The electrophoretic mobility of the glycoconjugate in agarose relative to its mobility in Triton X-100 was increased in the presence of deoxycholate, and decreased in the presence of cetyl trimethyl-ammonium bromide, indicating amphipathic properties consistent with insertion into the lipid bilayer of the membrane. Using the dot-blotting technique the glycoconjugate was detected in all virulent and avirulent clones of LRC-L137 and in two additional isolates of L. tropica major (LRC-L287 and LRC-L251), but not in L. donovani or L. mexicana, consistent with the previously described specificity of the antibodies. However, the general approaches used in this paper showed that L. donovani (LRC-L52) and L. mexicana (LRC-L94) synthesize a similar, but antigenically distinct glycoconjugate.     

Induction of arthritis with monoclonal antibodies to collagen.

mAb were developed from DBA/1 mice immunized with chick type II collagen. A total of 69 IgG antibodies was isolated and characterized. The majority (36%) reacted with a CNBr-derived peptide CB11 previously identified as containing a major immunogenic and arthritogenic epitope(s). Seven of the antibodies reactive with CB11 crossreacted strongly with mouse type II collagen. These were administered to DBA/1 mice in an attempt to induce arthritis. Individual antibodies were able to induce mild lesions consisting of minimal synovial proliferation but not overt arthritis. However, a combination of antibodies induced severe arthritis with marked destruction of articular cartilage. The minimal effective combination consisted of three antibodies. Arthritis developed within 48 to 72 h after injection of the antibodies and persisted for the duration of the observation period of 3 wk. Antibody levels were measured at intervals and persisted for the 3 wk observation period although at diminishing levels. Competitive binding assays demonstrated that each of the effective antibodies bound independently suggesting that some spatial or quantitative relationship was important possibly related to their ability to activate complement.     

Human choriogonadotropin-like material in bacteria of different species: electron microscopy and immunocytochemical studies with monoclonal and polyclonal antibodies

Immunocytochemical studies using antisera to whole human choriogonadotropin (hCG), to its alpha- and beta-subunits and to the COOH-terminal peptide of hCG beta, and two monoclonal antibodies to hCG beta, demonstrated expression of hCG-like material, its individual subunits and/or fragments in nine bacterial strains. Seven of these were isolated from patients with cancer and were definitely identified as Streptococcus faecalis (three strains), Staphylococcus haemolyticus (two strains) and Staphylococcus epidermidis and Escherichia coli (single strains). The other two strains were cell-wall-deficient (CWD) variants, one identified as Streptococcus bovis, isolated from the blood of a patient with a fever of unknown origin and a possible brain abscess. The other was a Gram-negative diphtheroid isolated from the urine of a pregnant woman, which during the period of study reverted to a Gram-positive Corynebacterium identified as a 'C. ulcerans' strain and expressed the hCG-like factor only during its phase as Gram-negative diphtheroid. Electron microscopy of these nine strains (including negative controls of strains of the same species subjected to the same immunocytochemical analyses and under identical cultural conditions) revealed morphological alterations in the bacterial cell walls and cytoplasmic material and/or bizarre forms of reproduction in six of the nine strains expressing hCG-like material including the two CWD variants. Collectively, these results provided evidence that (1) hCG-producing bacteria isolated from patients with overt cancer are not a new and unique species as claimed by others, and (2) there is a close resemblance between the bacterial protein and the human trophoblastic hormone, based on immunochemical recognition of different parts of the hCG molecule.(ABSTRACT TRUNCATED AT 250 WORDS)