Contrasting effects of Na+, K+-ATPase activation on seizure activity in acute versus chronic models.

Epilepsy is a life-shortening brain disorder affecting approximately 1% of the worldwide population. Most epilepsy patients are refractory to currently available antiepileptic drugs (AEDs). Knowledge about the mechanisms underlying seizure activity and probing for new AEDs is fundamental to the discovery of new therapeutic strategies. Brain Na(+), K(+)-ATPase activity contributes to the maintenance of the electrochemical gradients underlying neuronal resting and action potentials as well as the uptake and release of neurotransmitters. Accordingly, a decrease of Na(+), K(+)-ATPase increases neuronal excitability and may predispose to appearing of seizure activity. In the present study, we tested the hypothesis that activation of Na(+), K(+)-ATPase activity with a specific antibody (DRRSAb) raised against a regulatory site in the α subunit would decrease seizure susceptibility. We found that incubation of hippocampal homogenates with DRRSAb (1 µM) increased total and α1 Na(+), K(+)-ATPase activities. A higher concentration (3 µM) increased total, α1 and α2/α3 Na(+), K(+)-ATPase activities. Intrahippocampal injection of DRRSAb decreased the susceptibility of post status epilepticus animals to pentylenetetrazol (PTZ)-induced myoclonic seizures. In contrast, administration of DRRSAb into the hippocampus of naïve animals facilitated the appearance of PTZ-induced seizures. Quantitative analysis of hippocampal electroencephalography (EEG) recordings revealed that DRRSAb increased the percentage of total power contributed by the delta frequency band (0-3 Hz) to a large irregular amplitude pattern of hippocampal EEG. On the other hand, we found no DRRSAb-induced changes regarding the theta functional state. Further studies are necessary to define the potential of Na(+), K(+)-ATPase activation as a new therapeutic approach for seizure disorders.

Clinical trial of a plant-derived antibody on recolonization of mutans streptococci.

OBJECTIVE: This double-blinded, placebo-controlled clinical trial tested the safety and efficacy of a topical secretory IgA antibody manufactured in tobacco plants (plantibody) in preventing recolonization of mutans streptococci (MS) in human plaque as measured by whole stimulated saliva samples. METHODS: Following a 9-day antimicrobial treatment with chlorhexidine (CHX), 56 eligible adults (enrollment salivary MS > or = 10(4) CFU/ml; no current caries) were randomized equally to a group receiving 0, 2, 4, or 6 topical applications of plantibody followed by 6, 4, 2, or 0 applications of placebo, respectively, over a 3-week period. RESULTS: Among the 54 subjects who completed the trial, the CHX regimen eliminated salivary MS in 69%. After 6 months, there were no significant differences in MS levels by number of applications, relative to placebo (p > 0.43). No adverse effects were observed. CONCLUSION: Plantibody is safe but not effective at the frequency, concentration, and number of applications used in this study.

Hybrid IgA2/IgG1 antibodies with tailor-made effector functions.

Immunoglobulin (Ig) A and IgG are the principal immune effector molecules at mucosal surfaces and in blood, respectively. Mucosal IgA is polymeric and bound to secretory component, whereas serum IgG is monomeric. We have now produced IgA2/IgG1 hybrid antibodies that combine the properties of IgA and IgG. Antibodies with Calpha3 at the end of the IgG H chain resemble IgA and form polymers with J chain that bind the polymeric Ig receptor. Like IgG, the hybrid proteins activated complement and bound FcgammaRI and protein A. Though the hybrid proteins contained both Cgamma2 and Cgamma3, they have a short in vivo half-life. Surprisingly, this decreased half-life correlated with a higher avidity than that of IgG for murine FcRn. Interestingly, antibodies with Calpha1 replacing Cgamma1 were resistant to extremes of pH, suggesting that Calpha1 increases antibody stability. These results provide insights into engineering antibodies with novel combinations of effector functions.

Production of secretory IgA antibodies in plants.

Functional antibodies produced in tobacco plants were first reported over a decade ago (1989). The basic protocol used to generate these 'plantibodies' involved the independent cloning of H and L chain antibody genes in Agrobacterium tumefaciens vectors, the transformation of plant tissue in vitro with the recombinant bacterium, the reconstitution of whole plants expressing individual chains, and their sexual cross. In a 'Mendelian' fashion, a fully assembled and functional antibody was recovered from plant tissue in some double-transgenic plants. In mammalian cells, the antibody H and L chains are produced as precursor proteins that are translocated into the endoplasmic reticulum (ER), under the guidance of signal sequences. Within the ER, the signal peptides are proteolytically cleaved, and several stress proteins act as chaperonins to bind the unassembled antibody chains, and direct subsequent folding and tetramer formation. A similar process occurs in plant cells, and expression can be directed via signal sequences (even of foreign origin) into the aqueous environment of the apoplasm, or to be accumulated in other specific plant tissues, including tubers, fruit, or seed. Plants can facilely assemble secretory IgA, which is comprised of four chains, H and L chains, J chain and secretory component. Plant 'bioreactors' are expected to yield over 10 kg of therapeutic antibody/acre in tobacco, maize, soybean, and alfalfa [(Ann. NY Acad. Sci.)721(1994)235; (Biotechnol. Bioeng.)20(1999)135]. Compared with conventional steel tank bioreactors using mammalian cells, or microorganisms, the costs of GMP plantibodies are expected to perhaps one tenth. The differences in glycosylation patterns of plant and mammalian cell produced antibodies apparently have no effect on antigen-binding or specificity, but there is some concern about potential immunogenicity in humans. N-linked glycans of plants differ from human by having fucose-linked alpha 1,3 and the sugar xylose. No adverse effects or human anti-mouse antibodies (HAMA) have been observed in >40 patients receiving topical oral application of a plant produced secretory IgA specific to Streptococcus mutans, for the control of caries [(Nat. Med.)4(1998)601]. The progressive improvement of expression vectors for plantibodies, and purification strategies, as well as the increase in transformable crop species, is expected to lead to almost limitless availability of inexpensive (even edible forms of) recombinant immunoglobulins free of human pathogens for human and animal therapy, and for novel industrial applications (e.g. catalytic antibodies).

Producing proteins in transgenic plants and animals.

The requirement for large quantities of therapeutic proteins has fueled interest in the production of recombinant proteins in plants and animals. The first commercial products to be made in this way have experienced much success, and it is predicted that in the future a plethora of protein products will be made using these 'natural' bioreactors.

Interaction of CAP18-derived peptides with membranes made from endotoxins or phospholipids.

Antimicrobial peptides with alpha-helical structures and positive net charges are in the focus of interest with regard to the development of new antibiotic agents, in particular against Gram-negative bacteria. Interaction between seven polycationic alpha-helical CAP18-derived peptides and different types of artificial membranes composed of phosphatidylcholine or lipopolysaccharide of the Gram-negative bacterium Escherichia coli were investigated using different biophysical techniques. Results obtained from fluorescence energy transfer spectroscopy with liposomes, monolayer measurements on a Langmuir trough, and electrophysiological measurements on planar reconstituted asymmetric bilayer membranes including the lipid matrix of the outer membrane of E. coli were correlated, and these data were, furthermore, correlated with structural parameters of the peptides (net charge, alpha-helical content, hydrophobic moment, and hydrophobicity). All peptides induced current fluctuations in planar membranes due to the formation of transient lesions above a peptide- and lipid-specific minimal clamp voltage. Antibacterial activity was exhibited only by those peptides that induced lesion formation in the reconstituted outer membrane at clamp voltages below the transmembrane potential of the natural membrane. Thus, we propose that the physicochemical properties of both the peptides as well as of the target membranes are important for antibacterial activity.

Mechanisms of action of rabbit CAP18 on monolayers and liposomes made from endotoxins or phospholipids.

We have investigated the mechanism of action of the cationic antimicrobial protein (18 kDa) CAP18 on liposomes and monolayers made from phospholipids and enterobacterial lipopolysaccharides (LPS). CAP18 intercalates into lipid matrices composed of LPS from sensitive strains, weaker into those made of LPS from a resistant strain (Proteus mirabilis strain R45) or negatively charged phospholipids, but not into those composed of neutral phosphatidylcholine. From the combination of data obtained with fluorescence resonance energy transfer and Fourier-transform infrared spectroscopy and film balance measurements, it can be concluded that structural differences in the LPS determine the depth of intercalation of CAP18 into the respective lipid matrices. Thus, we identified the L-Arap4N linked to the first Kdo of the LPS of P. mirabilis strain R45 to be responsible for the CAP18 resistance of this strain. These data provide insight into CAP18-mediated effects on the integrity of the outer membrane of Gram-negative bacteria and led to an improved model for rabbit CAP18 membrane interaction.

Antibody engineering at the millennium.

It has been almost 100 years since von Behring and Kitasato received the first Nobel prize for the discovery of passive immunotherapy and nearly 25 years since Köhler and Milstein first reported hybridoma technology. In the 15 years since Mullis and co-workers described PCR, a number of discoveries and technologies have converged to produce a renaissance in antibody therapeutics. Our vision of antibodies as tools for research--useful for the prevention, detection and treatment of disease--has been revolutionized by these recent advances. This review specifically focuses on what is now called antibody engineering and includes chimeric and humanized antibodies, immunoglobulin fragments, antibody libraries, antibody fusion proteins and transgenic organisms as bioreactors. As a consequence of refinements in antibody technology, the field of genetically engineered immunoglobulins has matured into an elegant and important drug and reagent development platform.

Synthesis and preliminary biological evaluations of CC-1065 analogues: effects of different linkers and terminal amides on biological activity.

CC-1065 analogues possessing a biologically active CBI functional group and amide-substituted indole and benzofuran were synthesized. The IC(50) values of compounds 26, bearing two indoles, and 25, bearing only one indole, are 0.4 and 3 nM, respectively, against U937 leukemia cells in vitro. The IC(50) values of compounds 28, bearing a butyramino group, and 27, bearing an acetamino group, are 0.008 and 0.4 nM, respectively, against U937 leukemia cells in vitro. Compound 29, bearing a double-bond linker, is about 4-fold more potent than 25, bearing no double-bond linker. Compound 26 is highly potent against all cell lines tested in the NCI in vitro screening with IC(50) values in the 0.1-5 nM range for most cell lines. Compounds 26 and 30 are highly active against L1210 leukemia in mice. Compound 26 is also active against B16BL6 melanoma in mice. Most importantly, 26 and 30 are not myelosuppressive at therapeutically effective doses. The mechanism of tumor cell death is through induction of apoptosis, and is accompanied by DNA fragmentation.

Molecular mechanisms of interaction of rabbit CAP18 with outer membranes of gram-negative bacteria.

The mechanism of interaction of the cationic antimicrobial protein (18 kDa), CAP18, with the outer membrane of Gram-negative bacteria was investigated applying transmission electron microscopy and voltage-clamp techniques on artificial planar bilayer membranes. Electron micrographs of bacterial cells exposed to CAP18 showed damage to the outer membrane of the sensitive Escherichia coli strains F515 and ATCC 11775, whereas the membrane of the resistant Proteus mirabilis strain R45 remained intact. Electrical measurements on various planar asymmetric bilayer membranes, one side consisting of a phospholipid mixture and the other of different phospholipids or of lipopolysaccharide (reconstitution model of the outer membrane), yielded information about the influence of CAP18 on membrane integrity. Addition of CAP18 to the side with the varying lipid composition led to lipid-specific adsorption of CAP18 and subsequent induction of current fluctuations due to the formation of transient membrane lesions at a lipid-specific clamp voltage. We propose that the applied clamp voltage leads to reorientation of CAP18 molecules adsorbed to the bilayer into an active transmembrane configuration, allowing the formation of lesions by multimeric clustering.

Production of antibodies in transgenic plants.

Plants offer a cost-effective bioreactor to produce antibodies of diverse types. Recent studies demonstrate that secretory IgA, the predominant antibody isotype of the mucosal immune system, can be made in large quantities in plants. CaroRx, the lead SIgA antibody being developed by Planet Biotechnology Inc., has demonstrated activity in pilot phase II trials versus S. mutans, the major pathogen contributing to development of dental caries. Numerous other SIgA plantibodies are in preclinical development.

Bcl-2-mediated resistance to apoptosis is associated with glutathione-induced inhibition of AP24 activation of nuclear DNA fragmentation.

Studies on the mechanism of apoptosis in this laboratory support a model in which signal transduction involving caspase 3 leads to activation of a serine protease called Mr 24,000 apoptotic protease (AP24), which then induces internucleosomal DNA fragmentation in the nucleus. This study examined the effect of Bcl-2 overexpression on activation of AP24 and the induction of DNA fragmentation by AP24 in isolated nuclei. It was demonstrated that overexpression of Bcl-2 in either HL-60 or PW leukemia cell lines suppressed activation of AP24 induced by either tumor necrosis factor or UV light and protected cells from apoptosis. Furthermore, nuclei isolated from Bcl-2-overexpressing cells were relatively resistant to internucleosomal DNA fragmentation induced by AP24 isolated from apoptotic cells. Bcl-2-overexpressing cells that were nutritionally depleted of glutathione (GSH) became sensitive to tumor necrosis factor- or UV light-induced activation of AP24 and underwent apoptotic cell death. Moreover, nuclei isolated from Bcl-2-overexpressing cells that were depleted of GSH became sensitive to AP24-induced DNA fragmentation. The addition of exogenous GSH blocked the proteolytic activity of AP24, as well as its ability to induce DNA fragmentation in normal isolated nuclei. These results indicate that Bcl-2 can attenuate at least two events in the AP24 apoptotic pathway: activation of AP24 and induction of DNA fragmentation by activated AP24. Furthermore, agents that deplete intracellular levels of GSH may have therapeutic use in the sensitization of Bcl-2-overexpressing cancer cells to apoptotic cell death.

Evaluation of antimicrobial and lipopolysaccharide-neutralizing effects of a synthetic CAP18 fragment against Pseudomonas aeruginosa in a mouse model.

CAP18 (cationic antimicrobial protein; 18 kDa) is a neutrophil-derived protein that can bind to and inhibit various activities of lipopolysaccharide (LPS). The 37 C-terminal amino acids of CAP18 make up the LPS-binding domain. A truncated 32-amino-acid C-terminal fragment of CAP18 had potent activity against Pseudomonas aeruginosa in vitro. We studied the antimicrobial and LPS-neutralizing effects of this synthetic truncated CAP18 peptide (CAP18106-137) on lung injury in mice infected with cytotoxic P. aeruginosa. To determine its maximal effect, the CAP18106-137 peptide was mixed with bacteria just prior to tracheal instillation, and lung injury was evaluated by determining the amount of leakage of an alveolar protein tracer (125I-albumin) into the circulation and by the quantification of lung edema. The lung injury caused by the instillation of 5 x 10(5) CFU of P. aeruginosa was significantly reduced by the concomitant instillation of CAP18106-137. However, the administration of CAP18106-137 alone, without bacteria, induced lung edema, suggesting that it has some toxicity. Also, the peptide did not significantly reduce the number of bacteria that had been simultaneously instilled, nor did it significantly improve the survival of the infected mice. The addition of CAP18106-137 to aztreonam along with the bacteria did decrease the level of antibiotic-induced release of inflammatory mediators including tumor necrosis factor alpha, interleukin-6, and nitric oxide and also improved the survival of the mice. Therefore, more investigations are needed to confirm the toxicities and the therapeutic benefits of CAP18106-137 as an adjunctive therapy to antibiotics in the treatment of infections caused by gram-negative bacteria.

Autoinducer of virulence as a target for vaccine and therapy against Staphylococcus aureus.

Staphylococcus aureus causes pathologies ranging from minor skin infections to life-threatening diseases. Pathogenic effects are largely due to production of bacterial toxin, which is regulated by an RNA molecule, RNAIII. The S. aureus protein called RAP (RNAIII activating protein) activates RNAIII, and a peptide called RIP (RNAIII inhibiting peptide), produced by a nonpathogenic bacteria, inhibits RNAIII. Mice vaccinated with RAP or treated with purified or synthetic RIP were protected from S. aureus pathology. Thus, these two molecules may provide useful approaches for the prevention and treatment of diseases caused by S. aureus.

Therapeutic antibody technology 97.

Almost 200 antibody aficionados attended the Therapeutic Antibody Technology 97 meeting, held September 21-24, 1997 at the Holiday Inn, Union Square in the heart of San Francisco, CA. The meeting was sponsored by the Palo Alto Institute of Molecular Medicine and organized by James W. Larrick (PAIMM) and Dennis R. Burton (Scripps Research Institute). The meeting featured excellent discussions on many interesting talks and a number of poster presentations. It is likely that another meeting will be organized in 2 years, however in the meantime, an effort is underway to organize a 'Virtual Antibody Society' to be set up on the web server at Scripps Research Institute in La Jolla, CA (Questions and comments on this project can be sent to: Jwlarrick@aol.com or Burton@scripps.edu). Richard Lerner (Scripps) gave the keynote address on 'Catalytic Antibodies', describing recent work with Carlos Barbas on so-called reactive immunization to generate a high activity aldolase catalytic antibody. This antibody, soon to be described in an article in Science, is the first commercially available catalytic antibody.

Activation of CPP32-like proteases is not sufficient to trigger apoptosis: inhibition of apoptosis by agents that suppress activation of AP24, but not CPP32-like activity.

The 24-kD apoptotic protease (AP24) is a serine protease that is activated during apoptosis and has the capacity to activate internucleosomal DNA fragmentation in isolated nuclei. This study examined the following: (a) the functional relationship between AP24 and the CPP32-like proteases of the caspase family; and (b) whether activation of CPP32-like proteases is sufficient to commit irreversibly a cell to apoptotic death. In three different leukemia cell lines, we showed that agents that directly (carbobenzoxy-Ala-Ala-borophe (DK120) or indirectly inhibit activation of AP24 (protein kinase inhibitors, basic fibroblast growth factor, tosylphenylalaninechloromethylketone, and caspase inhibitors) protected cells from apoptosis induced by TNF or UV light. Only the caspase inhibitors, however, prevented activation of CPP32-like activity as revealed by cleavage of the synthetic substrate, DEVD-pNa, by cell cytosols, and also by in vivo cleavage of poly (ADP-ribosyl) polymerase, a known substrate of CPP32. Activation of DEVD-pNa cleaving activity without apoptosis was also demonstrated in two variants derived from the U937 monocytic leukemia in the absence of exogenous inhibitors. Cell-permeable peptide inhibitors selective for CPP32-like proteases suppressed AP24 activation and apoptotic death. These findings indicate that CPP32-like activity is one of several upstream signals required for AP24 activation. Furthermore, activation of CPP32-like proteases alone is not sufficient to commit irreversibly a cell to apoptotic death under conditions where activation of AP24 is inhibited.

Calmodulin-dependent protein kinase II mediates signal transduction in apoptosis.

The present studies describe a new function for calmodulin-dependent protein kinase II (CaM-KII) in signal transduction leading to apoptosis. Both tumor necrosis factor alpha (TNF) and UV light rapidly stimulated Ca2+-independent activity of CaM-KII in the monocytic leukemia, U937. Two mechanistically different inhibitors of CaM-KII blocked activation of CaM-KII and prevented DNA fragmentation and death. Activation of CaM-KII during apoptosis and inhibition of DNA fragmentation by the two CaM-KII inhibitors were reproduced in several other lines including KGla, HL-60, and YAC-1. However, K562, which is relatively resistant to apoptosis induced by either TNF or UV light, did not activate CaM-KII in response to these stimuli. A variant derived from U937 that is resistant to TNF- or UV light-induced apoptosis also lacked a CaM-KII response. Activation of Cam-KII was blocked by two protease inhibitors, VAD-fmk and TPCK, but not by other inhibitors of serine proteases. Both inhibitors of CaM-KII and the protease inhibitors blocked activation of AP24, a serine protease originally isolated from apoptotic cells that induces DNA fragmentation in nuclei. Our evidence supports a model in which proteolytic activity functions upstream of CaM-KII. This kinase then leads to activation of AP24, which transmits signals to the nucleus to initiate DNA fragmentation.

Ligation of MHC class I induces apoptosis in human pre-B cell lines, in promyelocytic cell lines and in CD40-stimulated mature B cells.

A murine mAb (BAL-1) was previously shown to induce apoptosis when cross-linked on the cell surface of different B acute lymphocytic leukemia (ALL) and pro-myelocytic cell lines. The present study shows that BAL-1 specifically recognizes the MHC class I (MHC-I). The apoptotic response was not dependent on the epitope specificity, since other anti-MHC-I antibodies, reacting with different monomorphic determinants of the alpha chain or beta 2-microglobulin, also induced apoptosis in these cells. However, external cross-linking of antibodies was strictly required for the apoptotic effect. Among cells originating from mature peripheral blood B cells, anti-CD40-stimulated cells were susceptible to anti-MHC-I-induced apoptosis, whereas B cells activated with Staphylococcus aureus Cowan I (SAC) or with the superantigen staphylococcal enterotoxin A (SEA) were non-responsive. Mature SEA-activated T cells were also resistant to MHC-I-induced apoptosis. In situ terminal deoxynucleotidyl transferase staining of apoptotic cells at various stages during MHC-I-induced cell death revealed that apoptosis occurred predominantly in the G2/M phase of the cell cycle, with the first apoptotic cells appearing after approximately 12 h of incubation. These results suggest a role for MHC-I-mediated apoptosis during differentiation and activation of certain hematopoietic cells.

Antimicrobial action of rabbit leukocyte CAP18(106-137).

CAP18 is a cationic antimicrobial protein originally isolated from rabbit neutrophils, of which a 32-mer sequence from its C-terminal and (CAP18(106-137)) has been found to be the most active. The bactericidal action of this peptide has been characterized by conventional culture techniques and flow cytometry. Cultures of Escherichia coli NCTC10418 were exposed to the MBC (12 microM) of the peptide for up to 60 min and stained with a fluorochrome sensitive to changes in either membrane potential (bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC4(3)), or membrane integrity (propidium iodide [PI]) before flow cytometric analysis. Addition of CAP18(106-137) to E. coli in broth culture resulted in immediate collapse of membrane potential [as determined by uptake of DiBAC4(3)] and loss of membrane integrity (as indicated by uptake of PI), with a corresponding 6- to 8-log decrease in viable counts as determined by colony formation on solid media. In identical experiments, the presence of Mg2+ (1 to 10 mM), K+ (50 to 250 mM), or EDTA (5 mM) or incubation in nutrient-free buffer or at 4 degrees C had no effect on peptide-induced dye uptake. In contrast, addition of Ca2+ (1 to 10 mM) or the respiratory chain poison carbonyl cyanide m-chlorophenylhydrazone (CCCP) (50 microM) inhibited the uptake of both dyes. These findings, however, did not relate to bacterial recovery on solid media, where (unless in the presence of K+ 150 to 250 mM) CAP18(106-137) at 12 microM fulfilled the MBC criteria (99.9% killing). We conclude that CAP18(106-137) exerts a rapid and profound action on E. coli cytoplasmic membranes and viability as measured by colony formation. The results suggest, however, that CAP18(106-137) may exert its action at sites additional to the cell membrane and that its activity profile is unique among cationic antimicrobial proteins.