Synthesis and biological evaluation as microtubule-active agents of several tetrahydrofuran and spiroacetal derivatives.

The stereoselective preparation of several molecules containing structural fragments of the tetrahydrofuran and spiroacetal type is described. Their degree of cytotoxicity and their interactions with tubulin have been investigated. It has been confirmed that the tetrahydrofuran derivatives are cytotoxic but, in contrast to previous reports, it has been found that the cytoxicity is not due to interactions with the microtubule network. Furthermore, and also in contrast to a previous report on closely related compounds, the spiroacetal derivatives do show interactions with tubulin, even though the precise mechanism and the binding site still remain to be established.

Taxanes: microtubule and centrosome targets, and cell cycle dependent mechanisms of action.

Microtubules are highly dynamic cellular polymers made of alphabeta-tubulin and associated proteins. They play a key role during mitosis, participating in the exact organization and function of the spindle, and are critical for assuring the integrity of the segregated DNA. Therefore, they represent one of the more effective targets in current cancer therapy. Paclitaxel (Taxol) is the prototype of the taxane family of antitumor drugs, and it was the first natural product shown to stabilize microtubules. This unique mechanism of action is in contrast to other microtubule poisons, such as Vinca alkaloids, colchicine, and cryptophycines, which inhibit tubulin polymerization. Taxanes block cell cycle progression through centrosomal impairment, induction of abnormal spindles and suppression of spindle microtubule dynamics. Triggering of apoptosis by aberrant mitosis or by subsequent multinucleated G1-like state related to mitotic slippage, depends on cell type and drug schedule. The development of fluorescent derivatives of paclitaxel led us to locate spindle pole microtubules and centrosomes as main sub-cellular targets of cytotoxic taxoids in living cells. In this review we discuss these findings in the context of a cell cycle-dependent response to taxanes, based on the cellular targets, and the status of the implicated cell cycle checkpoints. We also review those events that can influence this response, like the different signal transduction pathways activated/inactivated in relation to Bcl-2 phosphorylation and induction of apoptosis, and the controversial role of the p53 status on cell sensitivity to paclitaxel. Finally, cell cycle-dependent resistance, an emerging concept in combination sequential chemotherapy, is discussed on the basis of the cell cycle-dependent mechanisms of action of taxanes.

The interaction of baccatin III with the taxol binding site of microtubules determined by a homogeneous assay with fluorescent taxoid.

The ubiquitous Taxol binding site of microtubules also binds newly discovered ligands. We have designed a homogeneous assay for the high throughput detection of Taxol biomimetics, based on the displacement of 7-O-[N-(2,7-difluoro-4'-fluoresceincarbonyl)-L-alanyl]Taxol from its binding site in diluted solutions of preserved microtubules. The state of this reference ligand is measured by fluorescence anisotropy in a microplate reader, with varying concentrations of nonfluorescent competitors. The binding equilibrium constant of Taxol has a value K(b) = 3.7 x 10(7) M(-1). We have found that baccatin III, an analogue of Taxol without the C-13 side chain, binds with K(b) = 1.5 x 10(5) M(-1), whereas the side chain methyl ester is inactive. This was unexpected from the structure-activity relationship of taxoids but compatible with models of Taxol docked at the microtubule site. Baccatin III binding has been confirmed by displacement of [(3)H]Taxol and by direct HPLC measurements of its cosedimentation with microtubules, among other methods. Consequently, baccatin III induces microtubule bundles and multipolar spindles in PtK2 and U937 cells, and mitotic arrest and apoptotic death of the U937 cells, at concentrations 200-500-fold larger than Taxol. The simplest analysis of these results strongly suggests that the interaction of the C-2 C-4 substituted taxane ring system with the microtubule binding site provides most (ca. 75%) of the free energy change of Taxol binding and is sufficient to activate microtubule stabilization and transmit the antitumor effects of Taxol, whereas the C-13 side chain provides a weak specific anchor.

Centrosome and spindle pole microtubules are main targets of a fluorescent taxoid inducing cell death.

Microtubules offer a very large local concentration of binding sites for cytotoxic taxoids or for hypothetical endogenous regulators. Several compounds from diverse sources stabilize microtubules and arrest cell division similarly to the antitumour drug Taxol. We have investigated the subcellular location of the Taxol binding sites, employing a fluorescent taxoid (FLUTAX) that reversibly interacts with the Taxol binding sites of microtubules and induces cellular effects similar to Taxol. The specific binding of FLUTAX to a fraction of the available cellular binding sites effectively inhibits division of cultured human tumour cells at G(2)/M, and triggers apoptotic death. The loci of reversible binding, directly imaged in intact U937 cells treated with cytotoxic doses of fluorescent taxoid are the centrosomes, with a few associated microtubules in interphase cells, and the spindle pole microtubules in mitotic cells, instead of uniformly labelling the microtubule cytoskeleton. Cytoskeletal lesions induced and visualized with FLUTAX consist of microtubule bundles and abnormal mitoses, including monopolar spindles and highly fluorescent multipolar spindles. The multiple asters and monopolar spindles mark arrested U937 leukaemia and OVCAR-3 ovarian carcinoma cells on their path to apoptosis (as well as K562, HeLa, and MCF-7 cells). Depending on the FLUTAX treatment, OVCAR-3 cells died from abnormal mitosis or from a subsequent interphase with double chromatin content and lobulated nuclei (micronuclei), indicating impairment of centrosome separation. Fragmented centrosomes could be observed in FLUTAX-treated non-transformed 3T3.A31 cells, which developed micronuclei but were resistant to apoptosis. These results strongly suggest that centrosomal impairment by taxoid binding during interphase, in addition to the suppression of the kinetochore microtubule dynamics in the mitotic spindle, is a primary cause of cell cycle de-regulation and cell death.

Design, synthesis and cytotoxic activities of naphthyl analogues of combretastatin A-4.

The 3,4,5-trimethoxyphenyl and 3-hydroxy-4-methoxyphenyl rings of combretastatin A-4 are deemed optimal for its activity as antimitotic agent. The replacement of either one by a naphthalene ring results in compounds with a potency comparable to that of the parent compound. These results show that the naphthalene ring is a good surrogate for the 3,4,5-trimethoxyphenyl or the 3-hydroxy-4-methoxyphenyl rings of combretastatin A-4 and that neither of them is essential for the antitumor activity.

Induction of apoptosis in leukemic cells by the reversible microtubule-disrupting agent 2-methoxy-5-(2',3',4'-trimethoxyphenyl)-2,4,6-cycloheptatrien-1 -one: protection by Bcl-2 and Bcl-X(L) and cell cycle arrest.

We have found that the bicyclic colchicine analogue 2-methoxy-5-(2',3',4'-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-on e (MTC) induced a dose- and time-dependent apoptotic response in human leukemic cells. MTC and colchicine rapidly disrupted the microtubule integrity and arrested cells at the G2-M phase before the onset of apoptosis. These responses were mediated by microtubule inhibition because 2-methoxy-5-[[3-(3,4,5-trimethoxyphenyl)propionyl]amino]-2,4,6-cycloh eptatrien-1-one and lumicolchicine, inactive analogues of MTC and colchicine, respectively, were unable to promote microtubule disassembly, cell cycle arrest, and apoptosis. Although 1 microM MTC induced a complete microtubule disruption after 1 h of incubation in human leukemic HL-60 cells that led to an accumulation of cells at the G2-M phase, MTC-induced apoptosis occurred after 9 h of treatment. This indicates the existence of a rather long lag between microtubule disruption and the onset of apoptosis. Unlike colchicine, the removal of MTC during this lag resulted in rapid microtubule repolymerization, followed by restoration of normal cell cycle and cell growth. MTC, but not 2-methoxy-5-[[3-(3,4,5-trimethoxyphenyl)-propionyl]amino]-2,4,6-cyclo heptatrien-1-one, induced c-jun expression as well as c-Jun NH2-terminal kinase and caspase activation, indicating that these signaling pathways are triggered by the specific action of MTC on microtubules. Caspase inhibition prevented MTC-induced apoptosis. Overexpression of bcl-2 or bcl-xL by gene transfer in human erythroleukemic HEL cells abrogated MTC-induced apoptosis, but cells remained arrested in G2-M, suggesting that bcl-2 and bcl-xL block the signaling pathway between G2-M arrest and triggering of apoptosis. MTC-treated bcl-2 and bcl-xL-transfected HEL cells recovered their capacity to proliferate after MTC removal. These results indicate that microtubule inhibition induces G2-M arrest and apoptosis in leukemic cells, showing a lag phase between G2-M arrest and the onset of apoptosis, regulated by bcl-2 and bcl-xL, during which MTC displays a reversible action on microtubule depolymerization and G2-M cell cycle arrest. Thus, MTC is a potent apoptotic inducer on human leukemic cells and shows a remarkable reversible action on microtubule network and cell cycle before commitment for apoptosis is reached.

Galactomannans from the cell walls of species of Paecilomyces sect. Paecilomyces and their teleomorphs as immunotaxonomic markers.

An alkali-extractable and water-soluble fraction (F1S) was obtained from cell walls of Paecilomyces variotii and species of the related genera Talaromyces, Byssochlamys and Thermoascus. The structure of the main polysaccharide of these fractions was studied and found to consist of a core of (1 --> 6)-alpha-mannopyranose partially substituted at 0.2 by chains of galactofuranose and shorter chains of mannopyranose. The differences in the regularity of the branching points and the length of the galactofuranose side chains are useful to distinguish between species. These differences were detected by immunological methods, since highly specific polyclonal antibodies were raised against these polysaccharides. Mycelium of P. variotii CBS 990.73A was stained by indirect immunofluorescence. The polysaccharides studied in this work differ from the one described for species from section Isarioidea, and this is another indication of the heterogeneity of the genus Paecilomyces.

Identification of betaIII- and betaIV-tubulin isotypes in cold-adapted microtubules from Atlantic cod (Gadus morhua): antibody mapping and cDNA sequencing.

Isolated microtubule proteins from the Atlantic cod (Gadus morhua) assemble at temperatures between 8 and 30 degrees C. The cold-adaptation is an intrinsic property of the tubulin molecules, but the reason for it is unknown. To increase our knowledge of tubulin diversity and its role in cold-adaptation we have further characterized cod tubulins using alpha- and beta-tubulin site-directed antibodies and antibodies towards posttranslationally modified tubulin. In addition, one cod brain beta-tubulin isotype has been sequenced. In mammals there are five beta-tubulins (betaI, betaII, betaIII, betaIVa and betaIVb) expressed in brain. A cod betaIII-tubulin was identified by its electrophoretic mobility after reduction and carboxymethylation. The betaIII-like tubulin accounted for more than 30% of total brain beta-tubulins, the highest yield yet observed in any animal. This tubulin corresponds most probably with an additional band, designated beta(x), which was found between alpha- and beta-tubulins on SDS-polyacrylamide gels. It was found to be phosphorylated and neurospecific, and constituted about 30% of total cod beta-tubulin isoforms. The sequenced cod tubulin was identified as a betaIV-tubulin, and a betaIV-isotype was stained by a C-terminal specific antibody. The amount of staining indicates that this isotype, as in mammals, only accounts for a minor part of the total brain beta-tubulin. Based on the estimated amounts of betaIII- and betaIV-tubulins in cod brain, our results indicate that cod has at least one additional beta-tubulin isotype and that beta-tubulin diversity evolved early during fish evolution. The sequenced cod betaIV-tubulin had four unique amino acid substitutions when compared to beta-tubulin sequences from other animals, while one substitution was in common with Antarctic rockcod beta-tubulin. Residues 221, Thr to Ser, and 283, Ala to Ser, correspond in the bovine tubulin dimer structure to loops that most probably interact with other tubulin molecules within the microtubule, and might contribute to cold-adaptation of microtubules.

Effect of 2'-OH acetylation on the bioactivity and conformation of 7-O-[N-(4'-fluoresceincarbonyl)-L-alanyl]taxol. A NMR-fluorescence microscopy study.

The relationship between conformation, 2'-OH acetylation, and bioactivity of two fluorescent taxoids has been investigated by a combination of NMR and fluorescence microscopy techniques. These taxoids present the structure of taxol with the 7-OH group esterified with the N-(4'-fluoresceincarbonyl)-L-alanine group and with the 2'-OH group free (taxoid 2) or acetylated (taxoid 3). The larger water solubility of 2 and 3 compared with taxol allowed a detailed NMR study in DMSO-d6/D2O (3/7), showing that both taxoids adopt a similar collapsed conformation in which the hydrophobic groups 2-O-benzoyl, 3'-phenyl and 4-O-acetyl are in close proximity, with the fluorescein group displaying unrestricted motion. On the other hand, while taxoid 2 retains essentially the ability of taxol to induce in vitro microtubule assembly and to bind to cell microtubules, the 2'-acetylated derivative 3 does not show immediate activity. However, when taxoid 3 is left in the cell culture, the slow hydrolysis of the 2'-acetate group in the medium liberates the cytotoxic, microtubule-specific taxoid 2. The intense emission of this active derivative (2) allows the accurate recording of the drug-cell interaction from the very initial steps using fluorescence microscopy. These experiments show conclusively, for the first time in cell cultures, that a free 2'-OH group in taxol is essential for the recognition of the drug by the binding site of cellular microtubules.

Fluorescent taxoids as probes of the microtubule cytoskeleton.

Microtubules are specifically and efficiently visualized with the new fluorescent taxoids 7-O-[N-(4'-fluoresceincarbonyl)-L-alanyl]taxol (FLUTAX) and 7-O-[N-(4'-tetramethylrhodaminecarbonyl)-L-alanyl]taxol (ROTAX). Similarly to taxol, FLUTAX and ROTAX are able to drive inactive GDP-liganded tubulin into microtubule assembly. One molecule of FLUTAX binds per alphabeta-tubulin dimer assembled, competing with taxol for the same microtubule binding site with an eightfold smaller relative affinity. FLUTAX-induced microtubule elongation is markedly Mg2+-dependent, encompassing the binding of one Mg2+ ion more per tubulin dimer polymerized than in the case of taxol. A small perturbation of the absorption spectrum of bound FLUTAX is consistent with a cationic microenvironment relative to the solution. The fluorescence anisotropy of FLUTAX increases by an order of magnitude upon binding to microtubules and time-resolved measurements indicate that the fluorescein moiety remains considerably mobile on a protein surface. The rate of labeling suggests that this is the outer microtubule wall. Alternatively, the microtubule lumen would be functional. FLUTAX- and ROTAX-induced microtubules, radial structures, and organized microtubule bundles are readily observed under the fluorescence microscope. Rapid and accurate visualization of native (or very mildly fixed) cytoplasmic and spindle microtubules of a variety of permeabilized cells is simply obtained with micromolar FLUTAX, with an advantage over immunofluorescence. In addition, FLUTAX labels the centrosomes of PtK2 cells more intensely than antibodies to alpha- or beta-tubulin, and co-localizing with antibodies to gamma-tubulin. Two brightly fluorescent spots, probably separating or duplicating centrioles, can be resolved in the centrosomes of interphase cells. This finding indicates that centrosomes may well be additional targets of action of taxoids. FLUTAX strongly labels microtubules near the spindle poles, as well as microtubules at the telophase spindle equator and the central part of the midbody in cytokinesis (instead of the dark zone frequently observed with immunofluorescence), suggesting a predominant interaction of FLUTAX with sites at which tubulin is newly polymerized. Nanomolar concentrations of FLUTAX also permit specific imaging of centrosomes, half-spindles and midbodies in growing U937 cells.

The microtubule-associated protein tau cross-links to two distinct sites on each alpha and beta tubulin monomer via separate domains.

The interaction between tubulin subunits and microtubule-associated proteins (MAPs) such as tau is fundamental for microtubule structure and function. Previous work has suggested that the "microtubule binding domain" of tau (composed of three or four imperfect 18-amino acid repeats, separated by 13- or 14-amino acid inter-repeat regions) can bind to the C-terminal ends of both alpha and beta tubulin monomers. Here, using covalent cross-linking strategies, we demonstrate that there are two distinct tau cross-linking sites (designated as "C-terminal" and "internal") on each alpha and beta tubulin monomer. The C-terminal tau cross-linking site is located within the 12 C-terminal amino acids of both alpha and beta tubulin, while the internal tau cross-linking site is located within the C-terminal one-third of alpha and beta tubulin but not within the last 12 amino acids. In addition, we show that tau cross-links to the C-terminal site via its repeat 1 and/or the R1-R2 inter-repeat. The cross-linking of tau to the internal site is mediated by some subset of its other repeat units. Integrating these and earlier data with the 3.7 A resolution model of the alphabeta tubulin dimer recently presented by E. Nogales et al. [(1998), Nature 391, 199-203], we propose a new model for the tau-microtubule interaction.

Recurrent infectious diseases in human CD53 deficiency.

We report a familiar syndrome of recurrent heterogeneous infectious diseases, caused by bacteria, fungi, and viruses, which has as its only detectable defect the lack of CD53 antigen expression in neutrophils. All other assays ruled out known causes of recurrent infectious diseases due to either leukocyte adhesion or phagocytosis defects. CD53 belongs to the transmembrane-4 superfamily of proteins, which are a novel group of membrane proteins implicated in growth regulation and cell motility and possibly cell adhesion. We postulate that defects in these membrane proteins can be clinically manifested as complex recurrent infections.

An antigenic water-soluble glucogalactomannan extracted from cell walls of Paecilomyces fumosoroseus and Paecilomyces farinosus.

The water-soluble fraction (F1S) obtained after solubilizing in alkali the cell walls of four strains of Paecilomyces fumosoroseus and two of Paecilomyces farinosus amounted to 8.3-14.5% of the dry cell wall material. Two polysaccharides, F1S-A (13-20%) and F1S-B (57-68%) were separated from F1S by gel permeation through Sepharose CL-6B. 1H and 13C NMR spectra of F1S-B were recorded and showed analogous structural features in the six isolates of the two species. The fractions isolated from P. fumosoroseus strain CBS 375.70 were subjected to structural analysis and shown to be a (1-->4)-alpha-glucan (F1S-A) and a branched (1-->6)-mannan with terminal residues of beta-galactopyranose (F1S-B). Polyclonal antibodies against the latter polysaccharide were obtained (titre 1/8000). These antibodies reacted specifically with the F1S-B polysaccharides obtained from the four strains of P. fumosoroseus and the two strains of P. farinosus, but they did not react with similar fractions from other species of the same or related genera. The antibodies specifically stained P. fumosoroseus hyphae in indirect immunofluorescence tests.

Transient inhibition of capacitative calcium entry in human neutrophils by a monoclonal antibody directed against a 19-kDa antigen.

P1C3 is a monoclonal antibody that binds p19, a novel neutrophil activation antigen that translocates to the cell surface upon neutrophil activation. We find that P1C3 inhibits capacitative Ca2+ entry, induced by emptying the intracellular Ca2+ stores with thapsigargin. The effect is transient, reaching its maximum at 30-60 s, but becomes permanent upon pretreatment of the cells with the protein phosphatase inhibitor calyculin A, suggesting the involvement of protein phosphorylation. The inhibitory action is similar to the one reported previously for the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP), although the transduction mechanism may be different. Inhibition of Ca2+ entry by fMLP was prevented by pretreatment with pertussis toxin, whereas inhibition by P1C3 was not. Pretreatment with cholera toxin had no effect. This suggests that the effect of P1C3 may not be mediated by a heterotrimeric G protein. Tyrosine kinase inhibitors did not prevent inhibition by either fMLP or P1C3. Phospholipase C activation seems not to be involved as P1C3, contrarily to fMLP, was unable to induce Ca2+ release from the intracellular Ca2+ stores.

Comparative effects of taxol and Taxotere on two different human carcinoma cell lines.

The effects of taxoids (taxol and Taxotere) were followed on two human cancerous cell lines (bladder carcinoma J82 cells and epidermoid carcinoma KB 3-1 cells). Three cellular parameters were studied, viz., the qualitative effect on cellular microtubules, the quantitation of tubulin, and the antimitotic action, using two-parametric flow-cytometric analyses in treated cells. In both of the cell lines the tubulin content increased after taxoid treatment before the accumulation of cells in the G2/M phase. The effects of taxoids on tubulin appeared at about a 10-fold lower concentration on KB cells than on J82 cells. After drug exposure, the microtubule network showed a striking difference between the two cell lines: microtubule bundles were predominant in the J82 cell line, whereas multiple asters were prevalent in the KB cell line. The formation of these structures was dose- and time-dependent. Asters were observed in mitotic cells and bundles were seen in interphase cells. The reversibility of these structures in both cell lines varied with the duration of exposure to drug. Some differences were shown between taxol and Taxotere: the effects of Taxotere as compared with taxol appeared at a 2-fold lower concentration and their reversibility was slower.

Inhibition of microtubules and cell cycle arrest by a new 1-deaza-7,8-dihydropteridine antitumor drug, CI 980, and by its chiral isomer, NSC 613863.

CI 980 (NSC 613862; [S-(-)]) and NSC 613863 [R-(+)] are the two chiral isomers of ethyl 5-amino 1,2-dihydro-2-methyl-3-phenylpyrido[3,4-b]pyrazin-7-ylcar bamate (NSC 370147), which is a mitotic inhibitor with in vivo and in vitro activity against murine multidrug-resistant sublines. We have characterized the inhibition of in vitro microtubule assembly by the S (CI 980) and R (NSC 613863) enantiomers, their actions on the cytoplasmic microtubule network of epithelial-like PtK2 cells, and on the cell cycle of different human and murine leukemias and PtK2 cells. Assembly of purified tubulin, or tubulin plus microtubule-associated proteins, into microtubules was substoichiometrically inhibited by both compounds, which also induced a slow depolymerization of preassembled microtubules. Half inhibitory concentrations were 0.4-0.7 microM and 1.6-2.1 microM for the S and R isomers, respectively. Excess of both drugs induced polymerization of liganded tubulin into abnormal polymers similar to colchicine. The cytoplasmic microtubules of PtK2 cells were disrupted by both compounds in a concentration- and time-dependent manner, which was observed by indirect immunofluorescence microscopy and quantified by an enzyme-linked immunoassay of cytoskeletal tubulin. Half inhibitory concentrations were 6 nM S isomer, 100 nM R isomer, and 1 microM colchicine. Twenty nM S isomer or 500-700 nM R isomer gave nearly maximal effect. At these concentrations, half maximal microtubule depolymerization took place after 2 h of treatment. After drug removal, slow microtubule assembly and nearly complete reorganization of the cytoplasmic microtubules of PtK2 cells were observed (24 h). One nM S enantiomer or 25 nM R enantiomer induced mitotic arrest in 8 h in U937, HL60, and EL4 leukemias. PtK2 cells also stopped in mitosis after a 24-h incubation with 50 nM R isomer or 5 nM S isomer. The inhibition of cell division was irreversible in the leukemic cells, while PtK2 cells partially resumed growth. Although the interactions of CI 980 with microtubules in vitro are not very different from other drugs, it is a most potent cellular microtubule and mitotic inhibitor.

Enhancement of human neutrophil functions by a monoclonal antibody directed against a 19-kDa antigen.

A mouse IgG mAb termed P1C3 was raised against A23187-treated human peripheral blood neutrophils and has been shown to recognize an Ag with an apparent molecular mass of 19 kDa, herein named p19. This p19 Ag was weakly expressed at the cell surface of resting human peripheral blood neutrophils and monocytes, but its cell surface expression was dramatically increased upon activation of these cell types with different secretagogues, including FMLP, PMA, and the calcium ionophores A23187 and ionomycin. A large latent pool of p19 molecules became accessible by immunofluorescence flow cytometry after cell permeabilization of resting neutrophils. A practically total translocation of the intracellular pool of this p19 molecule to the plasma membrane was achieved under appropriate cell stimulation, which induced an almost total degranulation of neutrophil secretory granules. The p19 Ag was absent from platelets, PBL, as well as from the human promyelocytic cell line HL-60, the human promonocytic cell line U937, and the human lymphoid cell lines Daudi and Jurkat. The p19 Ag was also expressed by circulating and/or interstitial neutrophils and monocytes in distinct tissues examined. The mAb P1C3 was found to enhance several neutrophil responses, such as chemotaxis, cell adhesion, phagocytosis, and respiratory burst. These data indicate that the mAb P1C3 recognizes an intracellular Ag in human resting mature neutrophils and monocytes, which upon cell activation is translocated to the cell surface and is able to affect cell functionality.

A monoclonal antibody that detects a specific human neutrophil antigen involved in phorbol myristate acetate- and formyl-methionyl-leucyl-phenylalanine-triggered respiratory burst.

A mouse IgM mAb termed P1E3 was raised against resting human peripheral blood neutrophils and has been shown to recognize a cell-surface Ag with an apparent molecular mass of 155 kDa, as assessed by immunoprecipitation analysis. In addition to the main 155-kDa protein, an additional band of about 210 kDa was also recognized by P1E3 in Western blot analysis. Sequential immunoprecipitation assays showed that the Ag recognized by P1E3 differed from the CD29 and CD45 Ag. However, sequential immunoprecipitation assays carried out with two distinct anti-CD15 mAb and P1E3 showed that P1E3 reacted with CD15 or with a CD15-like Ag. P1E3 stained strongly resting human peripheral blood neutrophils, hardly reacted with peripheral blood monocytes and did not react with PBL and platelets, as assessed by immunofluorescence flow cytometry. P1E3 inhibited the respiratory burst induced by PMA or FMLP, but not the oxidative response induced by Con A or the calcium ionophores A23187 or ionomycin. Furthermore, P1E3 inhibited the activation of the Na+/H+ antiporter in response to PMA or FMLP and the phosphorylation of a protein of about 50 kDa in response to PMA. However, preincubation of neutrophils with P1E3 did not affect the increase in cytosolic free calcium concentration induced by FMLP. These data suggest that the Ag recognized by P1E3 may play a role in modulating the activation of the respiratory burst induced by PMA or FMLP, and that P1E3 seems to affect protein kinase C-mediated signal transduction mechanisms coupled to the induction of the respiratory burst.

Antibodies against a peptide representative of a conserved region of human IFN-alpha. Differential effects on the antiviral and antiproliferative effects of IFN.

mAb generated from mice immunized with a synthetic peptide representative of a conserved region (amino acids 133 to 147) of human IFN-alpha are described. Antisera from mice immunized with the peptide coupled to keyhole limpet hemocyanin were able to specifically bind to the peptide and also to bind and precipitate human IFN-alpha. Binding of the antibodies to IFN-alpha was inhibited by the immunizing peptide, but not by an unrelated peptide. Immunized mice were used to obtain three hybridomas producing mAb able to recognize both the immunizing peptide and human IFN-alpha, as determined by RIA and immunoprecipitation. These antibodies also neutralized the antiviral effect of human leukocyte IFN. In contrast, none of the mAb significantly affected the inhibition of Daudi cell proliferation induced by IFN-alpha.

Sodium diethyldithiocarbamate restores T lymphocyte proliferation, interleukin-2 production and NK activity in cyclophosphamide-immunosuppressed animals.

The in vivo effect of sodium diethyldithiocarbamate (DTC) on IL-2 production, mitogen-induced proliferation and natural killer (NK) activity of lymphocytes from normal as well as cyclophosphamide (CY)-treated mice has been investigated. DTC was given in a single subcutaneous injection (25 mg/kg) to normal or cyclophosphamide-treated mice (250 mg/kg i.p. simultaneously to DTC). An enhancement of T lymphocyte proliferation in CY-treated animals and an increase of IL-2 production in both normal and immunosuppressed mice was observed. When DTC (10 mg/kg) was administered daily for two weeks an increase in concanavalin A-induced mitogenesis, IL-2 production and NK activity in CY-treated animals was observed. These immune parameters were reduced 12 days after CY treatment by a factor of 2 to 3 times, while DTC treatment restored these responses to normal levels. LPS-induced mitogenesis was not significantly enhanced. The effect of DTC could be partially mediated by changes in IL-2 activity. According to these results some functional parameters of the immune system of the suppressed host can be partially or completely restored by means of an appropriate immunomodulator treatment. DTC could be of interest in the treatment of diseases where immune functions are impaired or in combined treatments with immunosuppressants.