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In this paper the 0D description of magnetized toroidal hydrogen-helium RF discharges is presented. The model has been developed to obtain insight into the ICRF plasma parameters, particle fluxes to the walls and the main collisional processes, which is especially relevant for the comprehension of RF wall conditioning discharges. The 0D plasma description is based on the energy and particle balance equations for nine principal species: H, H+, H2, {\rm H}_2^+ , {\rm H}_3^+ , He, He+, He2+ and e[?]. It takes into account (1) elementary atomic and molecular collision processes, such as excitation/radiation, ionization, dissociation, recombination and charge exchange, and elastic collisions, (2) particle losses due to the finite dimensions of the plasma volume and confinement properties of the magnetic configuration, and particle recycling, (3) active pumping and gas injection, (4) RF heating of electrons (and protons) and (5) a qualitative description of plasma impurities. The model reproduces experimental plasma density dependences on discharge pressure and coupled RF power, both for hydrogen RF discharges (ne [?] (1-5) x 1010 cm[?]3) and for helium discharges (ne [?] (1-5) x 1011 cm[?]3). The modeled wall fluxes of hydrogen discharges are in the range of what is estimated experimentally: [?]1019-1020 m[?]2 s[?]1 for H atoms, and [?]1017-1018 m[?]2 s[?]1 for H+ ions. It is found that experimentally evidenced impurity concentrations have an important impact on the plasma parameters, and that wall desorbed particles contribute largely to the total wall flux. [ABSTRACT FROM AUTHOR]

On an RF driven H[?] ion source the temperature of the H2 background gas was measured via optical emission spectroscopy as a function of flow and applied RF power at two locations of the source. The source operates at an applied RF power of 30-100 kW and at input gas flows of less than 1 Pa m3 s[?]1. At the same time the pressure was measured by Baratrons connected to the source body via tubes of length much greater than their diameter. Using these two data sets the background gas density and degree of depletion were determined. The neutral gas depletion was found to be relatively high (50-70%) as compared with the gas density before plasma ignition. The results obtained for the temperatures show the background gas is colder (maximum 1200 K) in the area where the RF power is coupled into the plasma than in the expansion region near the extraction grid (maximum 2100 K). The degree of depletion is highest where the power is coupled in, so the background gas density is lowest there. [ABSTRACT FROM AUTHOR]

We report a combined investigation of europium(iii)9-oxo-phenalen-1-one (PLN) coordination complexes, [Eu(PLN) 4 AE] + with AE = Mg, Ca, and Sr, using gas-phase photoluminescence, trapped ion-mobility spectrometry and density-functional computations. In order to sort out the structural impact of the alkali earth dications on the photoluminescence spectra, the experimental data are compared to the predicted ligand-field splittings as well as to the collision cross-sections for different isomers of [Eu(PLN) 4 AE] + . The best fitting interpretation is that one isomer family predominantly contributes to the recorded luminescence. The present work demonstrates the complexity of the coordination patterns of multicenter lanthanoid chelates involved in dynamical equilibria and the pertinence of using isolation techniques to elucidate their photophysical properties.

Safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of ATR-107, a fully human monoclonal anti-IL-21 receptor (IL-21R) antibody, administered as ascending single doses, subcutaneously or intravenously, was evaluated in a placebo-controlled, double-blind trial in healthy subjects. The dose levels were 3-300 mg by SC and 30-120 mg by IV. The most important adverse events were hypersensitivity reactions occurring in three out of six subjects in 300 mg SC cohort and considered as dose limiting toxicity. More than 75% of the subjects who received ATR-107 developed anti-drug antibodies (ADAs), which had no discernible impact on PK or safety. The PK of ATR-107 appeared to be dose -proportional. T1/2 was shorter than typical therapeutic antibodies. Bioavailability of ATR-107 was about 30%. IL-21R occupancy was measured in circulating B cells in the 60 and 120 mg IV cohort. The data indicated that single dose of ATR-107 was able to maximally occupy IL-21Rs through at least Day 42. Further escalation in the FIH study was halted partially due to the high rates of ADA formation. In conclusion, ATR-107 had a prolonged PD effect measured by IL-21R occupancy; was highly immunogenic after single dose administration and had PK properties with rapid clearance and low bioavailability., (© 2013, The American College of Clinical Pharmacology.)

The primary structure of a 13.6 kDa single heavy chain camelid antibody (V(H)H) was determined by matrix-assisted laser desorption ionization-time-of-flight/time-of-flight (MALDI-TOF/TOF) top-down sequence analysis. The majority of the sequence was obtained by mass spectrometric de novo sequencing, with the N-terminal 14 amino acid residues being determined using T(3)-sequencing and database interrogation. The determined sequence was confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of a tryptic digest, which also provided high-energy collisionally induced dissociation (CID) data permitting the clear assignment of 3 of the 14 isobaric Leu/Ile residues. Five of the 11 Leu/Ile ambiguities could be resolved by homology comparisons with known V(H)H sequences. The monoisotopic molecular weight of the V(H)H was determined by ultrahigh-resolution orthogonal electrospray (ESI)-TOF analysis and found to be 13 610.6066 Da, in excellent agreement with the established sequence. To our knowledge, this is the first time that the entire primary structure of a protein with a molecular weight >13 kDa has been established by mass spectrometric top-down sequencing.

Covalent sidewall addition to single-walled nanotubes (SWNTs) of a series of organolithium and organomagnesium compounds (nBuLi, tBuLi, EtLi, nHexLi, nBuMgCl, tBuMgCl) followed by reoxidation is reported. The functionalized R(n)-SWNTs were characterized by Raman and NIR emission spectroscopy. The reaction of SWNTs with organolithium and magnesium compounds exhibits pronounced selectivity: in general, metallic tubes are more reactive than semiconducting ones. The reactivity of SWNTs toward the addition of organometallic compounds is inversely proportional to the diameter of the tubes. This was determined simultaneously and independently for both metallic and semiconducting SWNTs. The reactivity also depends on the steric demands of the addend. Binding of the bulky t-butyl addend is less favorable than addition of primary alkyl groups. Significantly, although tBuLi is less reactive than, for example, nBuLi, it is less selective toward the preferred reaction with metallic tubes. This unexpected behavior is explained by fast electron transfer to the metallic SWNTs having low-lying electronic states close to the Fermi level, a competitive initial process. The NIR emission of weakly functionalized semiconducting SWNTs, also reported for the first time, implies interesting applications of functionalized tubes as novel fluorescent reporter molecules.

A new reaction sequence for the chemical functionalization of single-wall carbon nanotubes (SWNTs) consisting of the nucleophilic addition of t-BuLi to the sidewalls of the tubes and the subsequent reoxidation of the intermediates t-Bu(n)SWNT(n-) leading to t-Bu(n)SWNT was developed. During the formation of the t-Bu(n)SWNT(n-), a homogeneous dispersion in benzene was formed due to the electrostatic repulsion of the negatively charged intermediates causing debundling. The entire reaction sequence can be repeated, and the degree of functionalization of the products (t-Bu(n))(m)SWNT (m = 1-3) increases with increasing m. Degrees of functionalization expressed as the carbon-to-addend ratio of up to 31 were reached. The reaction was studied in detail by photoelectron spectroscopy, Raman spectroscopy, and scanning tunneling microscopy (STM). The C 1s core level spectra reveal that the nucleophilic attack of the t-BuLi leads to negatively charged SWNTs. Upon oxidation, this negative charge is removed. The valence band spectra of the functionalized samples exhibit a significant reduction in the pi-derived density of states. In STM, the covalently bonded t-butyl groups attached to the sidewalls have been visualized. Raman spectroscopy reveals that addition of the nucleophile to metallic tubes is preferred over the addition to semiconducting tubes.

Chemical screening with extracts of Actinomycetes strains resulted in the detection, isolation and structure elucidation of five new isoflavone glycosides from Streptomyces sp. (GT 51173): 5-O-alpha-L-rhamnopyranosyl genestein (genestein G1, 1), 5-O-alpha-L-rhamnopyranosyl-7-O-[2-O-(methyl)-alpha-L-rhamnopyranosyl] genestein (genestein G2, 2), 7,4'-O-di-[2-O-(methyl)-alpha-L-rhamnopyranosyl] daidzein (daidzein G1, 3), 6-O-methyl-7-O-[2-O-(methyl)-alpha-L-rhamnopyranosyl] daidzein (daidzein G2, 4) and 7-O-[2-O-(methyl)-alpha-L-rhamnopyranosyl] daidzein (daidzein G3, 5). As a striking structural feature, 2-5 bear one or two alpha-L-rhamnopyranosyl sugar moieties with methoxy groups in an axial position at C-2.

Three new naturally occurring bicyclic alkaloids, jenamidines A (1), B (2) and C (3), were discovered and isolated from the culture broth of Streptomyces sp. (strain HKI0297) via the chemical screening approach. Fermentation, isolation, structure and biological activities of these three new secondary metabolites are reported. The jenamidines have an unusual octahydro-pyrido[1,2-a]pyrimidine skeleton. Jenamidine A (1) shows antiproliferative effects against the chronic myeloid leukaemic cell line K-562. In addition, the new tricyclic sesquiterpenoid, africantriol (4) was isolated from the same strain.

The reversible inhibition of calcineurin (CaN), which is the only Ca(2+)/calmodulin-dependent protein Ser/Thr phosphatase, is thought to be a key functional event for most cyclosporin A (CsA)- and tacrolimus (FK506)-mediated biological effects. In addition to CaN inhibition, however, CsA and FK506 have multiple biochemical effects because of their action in a gain-of-function model that requires prior binding to immunophilic proteins. We screened a small molecule library for direct inhibitors of CaN using CaN-mediated dephosphorylation of (33)P-labeled 19-residue phosphopeptide substrate (RII phosphopeptide) as an assay and found the polyphenolic aldehyde gossypol to be a novel CaN inhibitor. Unlike CsA and FK506, gossypol does not require a matchmaker protein for reversible CaN inhibition with an IC(50) value of 15 microm. Gossypolone, a gossypol analog, showed improved inhibition of both RII phosphopeptide and p-nitrophenyl phosphate dephosphorylation with an IC(50) of 9 and 6 microm, respectively. In contrast, apogossypol hexaacetate was inactive. Gossypol acts noncompetitively, interfering with the binding site for the cyclophilin 18.CsA complex in CaN. In contrast to CsA and FK506, gossypol does not inactivate the peptidyl-prolyl-cis/trans-isomerase activity of immunophilins. Similar to CsA and FK506, T cell receptor signaling induced by phorbol 12-myristate 13-acetate/ionomycin is inhibited by gossypol in a dose-dependent manner, demonstrated by the inhibition of nuclear factor of activated T cell (NFAT) c1 translocation from the cytosol into the nucleus and suppression of NFAT-luciferase reporter gene activity.

Background, Aims: Periodontitis is the result of a complex interplay between oral bacteria and the host response, often modulated by behavioral factors. Early-onset periodontitis (EOP) is defined by the age of onset, the distribution of lesions and specific microbial pathogens., Method: Studies in twins suggested a genetic contribution to the pathogenesis of periodontitis. EOP heritable factors may be related to immune mechanisms which could enhance the pathogenic potential of plaque bacteria in susceptible individuals. Among others, Interleukin 4 (IL-4) is a potent cytokine in the immune response and is a potent down regulator of macrophage function. In the present study, we report a specific genotype of the IL-4 gene, which was detected by specific primers and PCR analysis., Results: In the EOP-group 27.8% were IL-4 promotor- and intron polymorphism positive (PP+ and IP+). None of the age-matched healthy controls or patients with adult periodontitis (n=25) carried the markers. Moreover, serum IL-4 levels of PP+ and IP+ patients were below the detection limit and significantly different (p<0.01) from the IL-4 concentrations of healthy controls and PP- and IP- patients.

Chemical screening with extracts of Streptomyces sp. (strain GT 61150) resulted in the detection, isolation, and structure elucidation of two new acyl alpha-L-rhamnopyranosides (1 and 2) and three new rhamnosyllactones A, B1 and B2 (3 approximately 5). Rhamnosyllactones B1 and B2 were obtained as a 5:1 mixture. The structures were confirmed by spectroscopic analysis, especially 2D-NMR techniques. The rhamnosyltransferase of our strain is able to connect the sugar moiety to heteroaromatic carboxylic acids and enols. The metabolites 1 and 4/5 as well as previously reported acylrhamnosides 6 approximately 11 inhibit the enzyme 3alpha-hydroxysteroid-dehydrogenase (3alpha-HSD).

This paper describes the development and characterization of the first monoclonal antibody specific for the recently cloned human glucagon receptor (hGR), and its use in probing receptor structure and function. We demonstrate specificity of one of the antibodies, CIV395.7A, by immunofluorescence staining and immunoprecipitation analysis. In addition, CIV395.7A specifically competes with glucagon for the hormone binding site on the receptor, indicating that the antibody's specific recognition epitope overlaps with the receptor's hormone binding domain. As a consequence, the mAB antagonizes glucagon-stimulated signal transduction as assayed by in vitro cAMP accumulation. Binding inhibition studies further reveal that the antibody specifically recognizes the human and rat GR, but not mouse. Using hGR/glucagon-like peptide I receptor chimeras, we have localized the recognition epitope of the antibody to the membrane-proximal half of the amino-terminal extension of the receptor, thus defining a domain on the receptor which is involved in glucagon binding.

The beta-amyloid deposits in Alzheimer's diseased (AD) brain are made up mainly of beta A4 peptides which show both N- and C-terminal heterogeneity. The predominant C-terminal species, identified by peptide sequencing of purified beta-amyloid, end either at position 40 or 42 of the beta A4 peptide. The distribution of these two beta A4 species in postmortem tissue as well as their generation in vitro could not be addressed previously due to the lack of specific antibodies that could differentiate them. This report describes the generation of a highly specific monoclonal antibody, MAb 369.2B which was raised against a synthetic peptide consisting of the C-terminus of the 1-42 beta A4 species. MAb 369.2B does not recognize the shorter beta A4 species 1-40 in solution or in solid phase. Furthermore, both beta A4 1-40 and 1-43 were unable to absorb out the antibody when used immunocytochemically. The regional distribution of MAb 369.2B immunoreactivity in AD and non-AD brain samples were compared to MAb 286.8A, an antibody raised against the N-terminal region of beta A4. Overall, the staining patterns were very similar. In AD cases with extreme vascular involvement, the N-terminal (MAb 286.8A) antibody was more likely to label the vascular basement membrane of affected vessels, and to label them more uniformly. In addition, preliminary quantitative analyses revealed that the C-terminal antibody labeled fewer, larger plaques than the N-terminal antibody. Qualitative analyses revealed that these smaller plaques were typically subregions of the larger plaques. The data corroborate the biochemical findings of N-terminal raggedness in beta A4 plaques. Further studies are required to explain this differential pattern of deposition.

A major pre-beta-amyloid protein695 (APP695) processing activity from Alzheimer's disease brain extracts was identified and found to be indistinguishable from the activity of cathepsin D.APP695 processing activity cleaved APP695 into a series of fragments that reacted on immunoblots to a monoclonal antibody (C286.8a) against beta-amyloid-(1-7)-peptide and cleaved N-dansyl-APP-(591-601)-amide at the Glu-Val and Met-Asp bonds. Fragments of 5.5 kDa and 10-12 kDa were formed from the cleavage of APP695 by cathepsin D at the Glu593-Val594 bond, and had the same N-terminus as a minor form of beta-amyloid released by cells. The Lys595-->Asn and Met596-->Leu substitutions found in a pedigree of familial Alzheimer's disease, increased the cathepsin D-catalyzed rate of accumulation of 5.5 kDa and 10-12 kDa C286.8a-reactive fragments 5-10fold. This substitution also increased the rate of N-dansyl-APP-(591-601)-amide cleavage at the Xaa-Asp bond by up to 41-fold. These observations suggest a role of cathepsin D in beta-amyloid formation under certain circumstances.

The preference for interleukin-2 receptor (IL-2R) expression on activated, compared with resting T lymphocytes makes the IL-2R a promising target for selective immunosuppressive therapy. To increase the potential therapeutic effectiveness of anti-IL-2R monoclonals, a chimeric mouse/human variant was constructed from Ig genes isolated from a murine anti-human IL-2R hybridoma cell line, designated AHT54. AHT54 binds to the same or spatially related epitope as IL-2 on the p55 protein that constitutes the low- and high-affinity forms of IL-2R. Although the murine and chimeric AHT54 antibodies inhibited cell-surface binding of IL-2 to the same extent, the chimeric antibodies containing a human IgG1 constant region had substantially more anti-proliferative activity than their murine IgG1 counterparts. Our results indicated that the human constant region of the chimeric antibodies interacted more efficiently than the murine constant region with effector components present in peripheral blood mononuclear cells (PBMC).

Production of peptides by recombinant DNA techniques is an efficient alternative to chemical synthesis of peptides. Proteins and peptides produced by recombinant DNA methods in E. coli are routinely used as antigens for the production of antibodies. However, most small peptides are rapidly degraded within the E. coli cell, and therefore, must initially be expressed as components of larger, more stable fusion proteins. The peptide of interest must be cleaved from the fusion protein, and purified prior to immunization to eliminate epitopes contributed by the fusion partner. We have now established methods for the production and characterization of monoclonal antibodies using partially purified, uncleaved fusion proteins. We have also described a method for efficient production and detection of the fusion protein, an EIA for rapid differential screening of hybridoma supernatants, and a strategy for epitope mapping of the antibodies. These methods have been applied to the production and characterization of monoclonal antibodies specific for a 75-amino-acid internal segment of the Alzheimer amyloid precursor protein, and should be applicable to a wide variety of other peptides and proteins.

A chimeric mouse/human MAb against the human p55 IL-2R was constructed from Ig genes isolated from a mouse hybridoma cell line, designated AHT107. AHT107 binds to a different epitope on p55 than IL-2, and similar to observations made for other rodent anti-IL-2R antibodies that do not recognize the same or spatially related epitope as IL-2, murine AHT107 did not efficiently inhibit proliferation of T-lymphocytes in mitogen and MLR PBMC stimulation assays. In contrast, the chimeric AHT107 antibodies containing a human IgG-1 constant region had substantially more anti-proliferative activity than their murine IgG-I counterparts. Our results indicated that the human constant region of the chimeric antibodies interacted more efficiently than the murine constant region with effector components present in the PBMC cultures. This conclusion was supported by our observation that F(ab')2 generated from the chimeric antibodies did not efficiently inhibit proliferation in the PBMC assays, and the chimeric antibodies did not inhibit proliferation of an antigen specific, IL-2 dependent human T-cell clone stimulated in the absence of PBMC.

Stromelysin/Transin is a member of the matrix metalloprotease gene family. This metalloprotease is synthesized as a preproenzyme with a predicted size of 53,977 Da including a 17 amino acid signal peptide. Prostromelysin is secreted from normal and transformed cells in two forms with apparent molecular masses on NaDodSO4 gels of 60 and 58-kDa. The minor 60-kDa species contains N-linked oligosaccharide(s). Stromelysin consists of three domains the amino terminal propeptide(s) domain contains the tribasic amino acid sequence RRK which is important in the proteolytic activation of this zymogen by trypsin-like serine proteases. The second domain consists of the catalytic domain which contains the zinc binding site. The carboxyl-terminal hemopexin domain has no known function and can be removed without a loss of enzymatic activity. Stromelysin has a broad range of substrate specificity including proteoglycans, casein, fibronectin, laminin, native type IV and IX collagen and gelatin but not type I collagen. In the presence of trypsin or plasmin, catalytic amounts of this enzyme can also fully activate interstitial fibroblast collagenase. We have developed a panel of monoclonal antibodies against stromelysin which will be useful for the tissue localization of the various species of this enzyme in tissues. In addition, we have demonstrated that either human rIL-1 (alpha) or rTNF (alpha) can stimulate the expression of this enzyme in cultured bovine articular cartilage at least 10-fold. Based on western blot analysis, the zymogen form of the enzyme was the major enzyme species detected in either the media or cartilage matrix compartments of cytokine treated cultures.

The majority of human rhinoviruses use intercellular adhesion molecule 1 (ICAM-1) as a cell surface receptor. Two soluble forms of ICAM-1, one corresponding to the entire extracellular portion [tICAM(453)] and one corresponding to the two N-terminal immunoglobulin-like domains [tICAM(185)], have been produced, and their effects on virus-receptor binding, virus infectivity, and virus integrity have been examined. Results from competitive binding experiments indicate that the virus binding site is largely contained within the two N-terminal domains of ICAM-1. Virus infectivity studies indicate that tICAM(185) prevents infection by direct competition for receptor binding sites on virus, while tICAM(453) prevents infection at concentrations 10-fold lower than that needed to inhibit binding and apparently acts at the entry or uncoating steps. Neutralization by both forms of soluble ICAM-1 requires continual presence of ICAM-1 during the infection and is largely reversible. Both forms of soluble ICAM-1 can alter rhinovirus to yield subviral noninfectious particles lacking the viral subunit VP4 and the RNA genome, thus mimicking virus uncoating in vivo, although this irreversible modification of rhinovirus is not the major mechanism of virus neutralization.

The CD44 molecule, also known as Hermes lymphocyte homing receptor, human Pgp-1, and extracellular matrix receptor III, has been shown to play a role in T cell adhesion and activation. Specifically, anti-CD44 mAb block binding of lymphocytes to high endothelial venules, inhibit T cell-E rosetting, and augment T cell proliferation induced by the CD2 or CD3-TCR pathways. We have characterized an anti-CD44 mAb (212.3) which immunoprecipitates a 90-kDa protein and is specific for CD44 as shown by peptide mapping and antibody competition studies. Interestingly, our studies with 212.3 demonstrate that this CD44-specific mAb completely inhibits T cell proliferation stimulated by the anti-CD3 mAb, OKT3. Inhibition is not a result of reduced cell viability, but is associated with 1) inhibition of IL-2 production, 2) inhibition of IL-2R expression, and 3) inhibition of OKT3-mediated increases in intracellular Ca2+ levels. In addition, 212.3 does not inhibit proliferation by the T cell mitogens PHA or PWM nor does it inhibit proliferation in a mixed lymphocyte reaction. Similar to other anti-CD44 mAb, 212.3 also augments T cell proliferation induced by mAb directed against the T11(2) and T11(3) epitopes of CD2. Thus, these studies describe a novel CD44-specific mAb (212.3) that inhibits T cell activation by OKT3 by blocking early signal transduction. Furthermore, these studies suggest that "receptor cross-talk" between the CD3-TCR complex and CD44 may regulate T cell activation.

Monoclonal antibody (MAb) B72.3 has been shown to be of potential utility in the management of human carcinoma via its use in (a) the targeting of carcinoma lesions in colorectal and ovarian cancer patients, (b) immunohistochemical analyses of biopsies and effusions, and (c) serum assays to help define the presence of carcinoma. The B72.3-reactive antigen, designated tumor-associated glycoprotein 72 (TAG-72), has been characterized as a high molecular weight glycoprotein with the properties of a mucin. We report here the utilization of MAb B72.3 and 18 second generation MAbs (generated using purified TAG-72 obtained from a colon carcinoma xenograft as immunogen) to construct a serological map of the TAG-72 molecule. The generation and initial characterization of 10 of the second generation MAbs have been described previously; in addition, eight previously unreported MAbs were used. All 19 MAbs produced immune precipitate lines against purified TAG-72 in double immunodiffusion, indicating that each epitope recognized by a single MAb is present at least twice on the TAG-72 molecule. Immunodepletion analyses utilizing 11 of the anti-TAG-72 MAbs indicated that each recognizes the same molecule or population of molecules. Nineteen competition radioimmunoassays were developed and 19 purified competitor immunoglobulins were used in each assay. The patterns of cross-competition indicated the presence of a complex array of tumor-associated epitopes on the TAG-72 molecule. Some of the MAbs recognized epitopes that were structurally or spatially related to one another, but none appeared to recognize identical epitopes. The spectrum of inhibitory reactivities of these MAbs for TAG-72 binding varied from extremely restricted to more broad inhibition. The serological mapping studies reported here provide information as to the range and nature of the epitopes expressed on the TAG-72 molecule, help form the basis for selecting alternative anti-TAG-72 MAbs for use in potential clinical applications, and further define the nature of this oncofetal antigen.

Human leukocyte suspensions (neutrophils 80-85%, monocyte 15-20%) were incubated alone or with cultured human umbilical vein endothelial cells. Leukocytes were either directly added to the endothelial cell cultures or separated from them by a 0.4 micron insert filter. Supernatants or cell lysates were obtained at 0.5, 1, 2, and 4 hours of incubation. Supernatants were assayed for the prostacyclin (PGI2) metabolite 6-keto prostaglandin F1 alpha and prostaglandin E2 (PGE2) by radioimmunoassay and for interleukin-1 (IL-1) by the thymocyte co-mitogen assay. Cell lysates were analyzed for cell-associated procoagulant activity (PCA). Co-incubation of endothelial cells with leukocytes stimulated the synthesis of PGI2, PGE2, and PCA. These biochemical changes correlated partially with the release of IL-1 beta. The results suggest that IL-1 released in monocyte neutrophil co-cultures can produce prothrombotic (increased PCA expression) and inflammatory changes (increased synthesis of vasodilatory and permeability enhancing PGI2 and PGE2) in endothelial cells. Neutrophils may represent a source of the released IL-1 and/or may act to stimulate monocyte release of this cytokine and thus play an important role in vascular pathology by a mechanism unrelated to their more direct cytotoxic activity.

Monoclonal antibody (MAb) B72.3 was generated using a membrane-enriched fraction of a human mammary carcinoma biopsy. It has demonstrated reactivity to the majority of human adenocarcinomas including colorectal, gastric, pancreatic, ovarian, endometrial, mammary, and non-small cell lung cancer and no or weak reactivity to normal adult tissues, with the exception of secretory endometrium. The B72.3-reactive antigen, termed tumor-associated glycoprotein (TAG)-72, has been purified and used as an immunogen to generate B72.3 second generation MAbs. Since the source of purified TAG-72 was a human colon cancer (CC) xenograft, these MAbs have been given a CC designation. Twenty-eight CC MAbs, all immunoglobulin Gs, have been generated and shown to be reactive with TAG-72 and via both radioimmunoassay and immunohistochemical analyses show differential reactivity to carcinoma versus normal adult tissue biopsies. Nine CC MAbs (CC11, 15, 29, 30, 40, 46, 49, 83, and 92) were selected for further characterization. As a result of analyses using direct-binding radioimmunoassay to a range of human carcinomas, Western blotting, live cell surface binding assays, five liquid competition radioimmunoassays, and Ka measurements, all nine CC MAbs could be distinguished from each other and from B72.3. The Ka of B72.3 was determined to be 2.54 X 10(9) M-1; all the CC MAbs demonstrated higher KaS with MAbs CC92, 49, and 83 having KaS of 14.26, 16.18, and 27.72 X 10(9) M-1, respectively. These studies thus demonstrate that one or more of the anti-TAG-72 CC MAbs may be more efficient than B72.3, or useful in combination with B72.3, toward the further study of human carcinoma cell population and the diagnostic and therapeutic procedures presently utilizing MAb B72.3.

Murine monoclonal antibodies (MAbs) reactive with distinct epitopes on carcinoembryonic antigen (CEA) have been analyzed systematically by radioimmunoassays, Western blotting, and immunohistochemical assays to define CEA expression in adenocarcinomas, benign lesions, and normal tissues of the stomach. Each of four COL-MAbs (COL-1, COL-4, COL-6, and COL-12) reacted preferentially with cell extracts of adenocarcinomas versus those of normal mucosae in solid-phase radioimmunoassays. Using Western blotting analyses MAbs COL-1, COL-4, COL-6, and COL-12 detected only the Mr 180,000 molecule characteristic of CEA in adenocarcinoma of the stomach; no reactivity was observed in an extract of normal gastric mucosa. Antibody competition radioimmunoassays were then carried out to define relations among COL-MAbs using 125I-radiolabeled MAbs, and nonradiolabeled MAbs as competitors. A spectrum of formalin-fixed, paraffin-embedded normal, benign, and malignant tissue sections of the stomach were examined for immunoreactivities with COL-MAbs using immunohistochemical assays to define whether the COL-MAbs were able to detect CEA expression in early foci of gastric carcinomas. All of the COL-MAbs generally demonstrated selective reactivities to adenocarcinomas (n = 40) versus benign lesions (n = 15) and normal mucosae (n = 6) of the stomach. From 72 to 100% of adenocarcinomas at early stage (n = 18) were reactive with the COL-MAbs, suggesting that these MAbs might serve as immunohistochemical diagnostic tools to detect early foci of gastric carcinoma. The data reported here indicate that the COL-MAbs can potentially be utilized as radioimmunological and immunohistochemical adjuncts to differentiate early adenocarcinomas from normal mucosae or benign lesions of the stomach on the basis of differential CEA expression.

Lymphocytes from lymph nodes obtained at mastectomy in breast cancer patients have been fused with murine nonproducer myeloma cells to obtain human-mouse hybridoma cultures that synthesize human monoclonal antibodies. To date, 52 hybridoma cultures synthesizing either human IgG or human IgM have been obtained from lymph nodes of 13 patients. Ig production was stable in many of these cloned cultures through 60-200 days of observation. Levels of human Ig synthesis ranged from 0.1 to 20 microgram/ml of supernatant fluid. The immunological reactivities of the human Igs were assayed on tissue sections by using the immunoperoxidase technique. Several of the human monoclonal antibodies demonstrated preferential binding to human mammary tumor cells. One human IgM monoclonal antibody was used to discriminate between mammary carcinoma cells (from 55 of 59 patients) and normal mammary epithelial cells, stroma, or lymphocytes of the same breast. Decreased binding to some of the benign breast tumors tested and to selected non-breast adenocarcinomas was also observed. This same human monoclonal antibody, however, reacted significantly with metastatic mammary carcinoma cells in lymph nodes of breast cancer patients, with no binding to normal lymphocytes or to stroma of the same node. These studies demonstrate that stable clones of human-mouse hybridomas can be generated by using lymph nodes of mastectomy patients and that clones can be selected which synthesize human monoclonal antibodies reactive with human mammary carcinoma cells.

Monoclonal antibodies (MAbs) of predefined specificity have been generated by utilizing a synthetic peptide reflecting amino acid positions 10-17 of the Hu-rasT24 gene product as immunogen. These MAbs, designated RAP-1 through RAP-5 (RA, ras; P, peptide), have been shown to react with the ras gene product p21. Since the Hu-ras reactive determinants (positions 10-17) have been predicted to be within the tertiary structure of the p21 molecule, it was not unexpected that denaturation of cell extracts or tissue sections with Formalin or glutaraldehyde enhanced binding of the RAP MAbs. When paraffin-embedded Formalin-fixed tissue sections and the avidin-biotin complex immunoperoxidase method were used, the RAP MAbs clearly defined enhanced ras p21 expression in the majority of human colon and mammary carcinomas. The majority of all abnormal ducts and lobules from fibroadenoma and fibrocystic disease patients were negative, as were all normal mammary and colonic epithelia examined. The findings reported here form the basis for quantitative radioimmunoassays for a ras translational product and provide a means to evaluate ras p21 expression within individual cells of normal tissues and benign, "premalignant," and malignant lesions.

Monoclonal antibodies have been generated against disrupted mammary tumor viruses isolated from Mus musculus, Mus cervicolor, and Mus cookii. Monoclonal antibodies directed against the M.W. 36,000 external glycoproteins of these viruses demonstrate the presence of multiple epitopes, i.e., distinct antigenic determinants, within these glycoproteins. These epitopes represent type, group, and interspecies determinants. Monoclonal antibodies have also been used to define multiple epitopes on the M.W. 28,000 major internal polypeptides of murine mammary tumor viruses that exhibit both type- and group-specific determinants. The monoclonal antibodies to the M.W. 36,000 glycoprotein and the M.W. 28,000 polypeptide have been used to distinguish all six mammary tumor virus isolates of M. musculus from each other, including both endogenous and exogenous viruses from the same strain, and a new virus isolate from BALB/c mice. With the use of the immunoperoxidase technique, the monoclonal antibodies generated have been used to demonstrate a heterogeneity of expression of mammary tumor virus gene products in primary mammary tumors of three Mus species. These studies have revealed that a given antigenic determinant may be expressed differentially in mammary tumors of two different Mus species, among mammary tumors of the same Mus species, and, at times, in different areas of the same mammary tumor.

Monoclonal antibodies (MAbs) designated COL-1 through 15 have been generated and characterized and show a strong degree of selective reactivity for human colon carcinomas versus normal adult tissues. To prepare the MAbs, mice were immunized with extracts or membrane-enriched fractions of biopsy material from either primary or metastatic colon carcinoma lesions. The fifteen COL MAbs, all of the immunoglobulin G subclasses 1, 2a, or 2b, reacted with purified carcinoembryonic antigen in solid-phase radioimmunoassay and by immunoblotting, but none reacted with human polymorphonuclear leukocytes or certain spleen preparations previously shown to be rich in carcinoembryonic antigen-related or cross-reactive antigens. MAbs COL-1 through 15 could be divided into at least five groups based on their differential range of reactivities to the surface of colon carcinoma cells or other carcinoma cell preparations. The COL MAbs were tested via immunoperoxidase with a wide range of primary and metastatic colon carcinomas, benign or dysplastic colon lesions, and 34 normal adult tissues. With few exceptions, the COL MAbs tested in this manner showed reactivity only to the primary and metastatic colon carcinomas. These studies thus provide a well-characterized repertoire of MAbs that are well suited for potential clinical trials involving the radiolocalization and possibly therapy of human colon carcinoma lesions. The fact that at least five epitopes are being recognized also provides the opportunity for testing the efficacy of cocktails of the COL MAbs toward these goals.

Cell lines were established by co-transfection of cloned M-ABA Epstein-Barr virus (EBV) DNA fragments with plasmids conferring resistance to dominant selective markers. A baby hamster kidney cell line carrying the HindIII-I1 fragment exhibits a nuclear antigen of 82 000 daltons, serologically defined as EBV-determined nuclear antigen (EBNA) 1. Furthermore, a Rat-1 cell line transfected with DNA of the clone pM 780-28 containing three large internal repeats (BglII-U) and the adjacent BglII-C fragment expresses a nuclear antigen of 82 000 daltons which can be visualized only by a subset of anti EBNA-positive human sera. Sera recognizing the 82 000-dalton protein of the transfected cell line reacted with a protein of the same size in the non-producer line Raji, designated as EBNA 2. Conversely, sera without reactivity to the 82 000-dalton protein failed to react with EBNA 2 of Raji cells. P3HR-1 and Daudi cells with large deletions in BglII-U and -C are devoid of EBNA 2. The data presented provide evidence that a second EBNA protein is encoded by the region of the EBV genome which is deleted in the non-transforming P3HR-1 strain.

An operant conditioning paradigm comprising continuous food-reinforcement, fixed-ratio responding, and extinction procedures was used to evaluate the possibility that adult male macaques can control their rate of yawning. Two non-food-deprived Macaca tonkeana quickly learned to produce yawns for food rewards. They also increased the rate of production according to fixed-ratio requirements, and showed initial response peaks followed by diminished rates of yawning during extinction. The results indicate that adult male macaques can yawn voluntarily. Further work along these lines may help distinguish among different types of yawning as indicated in the literature., (Copyright © 1988 Wiley‐Liss, Inc., A Wiley Company.)

DNAS of some human tumours can transform NIH 3T3 fibroblast cells, thus demonstrating the transforming potential of human ras genes (Hu-rasHa, Hu-rasKi, and Hu-rasN, respectively Harvey, Kirsten and neuroblastoma ras genes). Only a small percentage of a given type of human carcinoma, however, scores positive in this assay system. Activation of ras and subsequent transformation of NIH 3T3 cells are either by a point mutation in the ras gene or enhanced expression of the normal, or proto-onc, ras gene. If the transformation of a given human tumour involves the enhanced expression of the normal or cellular ras gene and the resulting gene product, the tumour DNA would probably score negative in the NIH 3T3 transfection assay. In human colon carcinoma, for example, lesions at position 12 of Hu-rasKi have been found. None of nine colon carcinomas obtained at biopsy, however, contain the ras lesion at this position, using a Hu-rasHa probe; one other colon carcinoma does appear to contain amplified proto-onc ras, and other colon carcinomas do have increased levels of ras RNA. There are at least three explanations for these observations. Either very few colon carcinomas contain point-mutated ras, the lesion in the majority of colon carcinomas is at a position other than 12 or ras activation in many colon carcinomas involves the enhanced expression of either the point-mutated or proto-onc form of a ras gene. We have now used monoclonal antibodies directed against a synthetic peptide reflecting sequences of the human T24 ras gene product to define ras p21 protein expression in a spectrum of colonic disease states. Immunohistochemical analyses of individual cells within tissue sections reveal differences in ras p21 expression in colon carcinomas compared with normal colonic epithelium, benign colon tumours and inflammatory or dysplastic colon lesions. Our data suggest that ras p21 expression is correlated with depth of carcinoma within the bowel wall, and is probably a relatively late event in colon carcinogenesis.

Human monoclonal antibodies have been generated following the fusion of human lymphocytes (obtained from axillary lymph nodes of mastectomy patients) with a murine nonimmunoglobulin secreting myeloma cell line. We report a detailed analysis of the reactivity of a human IgM monoclonal antibody secreted by one of these double cloned hybridoma cell lines. Tissue sections of both malignant and benign human mammary tumors, as well as apparently normal tissues, were tested using the immunoperoxidase technique and the human monoclonal antibody. Eighty-one per cent (54/67) of primary malignant mammary tumors, 100% (20/20) of metastatic breast lesions, and 14% (3/22) of benign breast lesions reacted positively with a moderate for strong intensity. The per cent of mammary carcinoma cells that stained and the pattern of staining varied among different tumor samples. While reactivity was observed with selected carcinomas of nonbreast origin, little or no reactivity was observed with apparently normal human tissues including normal mammary epithelium. The antibody reactivity observed was shown to be clearly distinct from those of both anti-T and anticarcinoembryonic antigen sera.

We have generated a monoclonal antibody (MAb), designated anti-CAA (colon-associated antigen), using as immunogen a membrane-enriched fraction of a biopsy from a moderately-differentiated human colonic adenocarcinoma. The molecular weight of this reactive antigen was determined by Western blotting to be greater than 200 kDa. When immunohistochemical techniques were used, MAb anti-CAA reacted with epithelium in the majority of normal, dysplastic and malignant colon specimens tested (greater reactivity was observed in normal colon than in benign or malignant lesions). Cell sorter analyses demonstrated a heterogeneous distribution of CAA on the cell surface of the well-differentiated LS-174T cell line. Antigen positive and antigen-negative cells were separated by means of flow cytometric techniques. These two subpopulations were then inoculated into immunosuppressed rats, resulting in xenograft tumors which differed significantly in their degree of histologic differentiation. Antigen-positive cells developed into well-differentiated adenocarcinomas, while antigen-negative cells developed into poorly differentiated adenocarcinomas. These results, along with immunohistochemical studies, indicate that the antigen detected by MAb anti-CAA has characteristics of a colon-associated antigen whose expression correlates with cellular differentiation. Moreover, differences in molecular weight as well as tissue distribution indicate that CAA may be a novel antigen different from those previously described.