Ethical Implications in Vaccine Pharmacotherapy for Treatment and Prevention of Drug of Abuse Dependence

Different immunotherapeutic approaches are in the pipeline for the treatment of drug dependence. “Drug vaccines” aim to induce the immune system to produce antibodies that bind to drugs and prevent them from inducing rewarding effects in the brain. Drugs of abuse currently being tested using these new approaches are opioids, nicotine, cocaine, and methamphetamine. In human clinical trials, “cocaine and nicotine vaccines” have been shown to induce sufficient antibody levels while producing few side effects. Studies in humans, determining how these vaccines interact in combination with their target drug, are underway. However, although vaccines can become a reasonable treatment option for drugs of abuse, there are several disadvantages that must be considered. These include i) great individual variability in the formation of antibodies, ii) the lack of protection against a structurally dissimilar drug that produces the same effects as the drug of choice, and iii) the lack of an effect on the drug desire that may predispose an addict to relapse. In addition, a comprehensive overview of several crucial ethical issues has not yet been widely discussed in order to have not only a biological approach to immunotherapy of addiction. Overall, immunotherapy offers a range of possible treatment options: the pharmacological treatment of addiction, the treatment of overdoses, the prevention of toxicity to the brain or the heart, and the protection of the fetus during pregnancy. So far, the results obtained from a small-scale experiment using vaccines against cocaine and nicotine suggest that a number of important technical challenges still need to be overcome before such vaccines can be approved for clinical use.     

Nicotine: the link between cigarette smoking and the progression of renal injury?

Cigarette smoke (CS) is the most important source of preventable morbidity and mortality in the United States. Recent clinical studies have suggested that, in addition to being a major cardiovascular risk factor, CS promotes the progression of kidney disease. The mechanisms by which CS promotes the progression of chronic kidney disease have not been elucidated. Here we demonstrate for the first time that human mesangial cells (MCs) are endowed with the nicotinic ACh receptors (nAChRs) alpha4, alpha5, alpha7, beta2, beta3, and beta4. Studies performed in other cell types have shown that these nAChRs are ionotropic receptors that function as agonist-regulated Ca(2+) channels. Nicotine induced MC proliferation in a dose-dependent manner. At 10 (-7) M, a concentration found in the plasma of active smokers, nicotine induced MC proliferation [control, 1,328 +/- 50 vs. nicotine, 2,761 +/- 90 counts/minute (cpm); P < 0.05] and increased the synthesis of fibronectin (50%), a critical matrix component involved in the progression of chronic kidney disease. We and others have shown that, in response to PKC activation, MC synthesize reactive oxygen species (ROS) via NADPH oxidase. In the current studies we demonstrate that PKC inhibition as well as diphenyleneiodonium and apocynin, two inhibitors of NADPH oxidase, prevented the effects of nicotine on MC proliferation and fibronectin production, hence establishing ROS as second messengers of the actions of nicotine. Furthermore, nicotine increased the production of ROS as assessed by 2',7'-dichlorofluorescein diacetate fluorescence [control, 184.4 +/- 26 vs. nicotine, 281.5 +/- 26 arbitrary fluorescence units (AFU); n = 5 experiments, P < 0.05]. These studies unveil previously unrecognized mechanisms that indict nicotine, a component of CS, as an agent that may accelerate and promote the progression of kidney disease.     

Highly sensitive and rapid detection of antibody catalysis by luminescent bacteria

A highly sensitive, inexpensive, and facile bioluminescent assay for the detection of catalytic antibodies has been developed. This assay may be used for the early detection of antibody catalysis. The efficiency of this technique was exemplified by the use of the luminescent bacterium VhM42 for monitoring an antibody-catalyzed retroaldol fragmentation reaction with aldolase antibodies 38C2 and 24H6.     

Prenatal Alcohol Exposure Increases Postnatal Acceptability of Nicotine Odor and Taste in Adolescent Rats

Human studies indicate that alcohol exposure during gestation not only increases the chance for later alcohol abuse, but also nicotine dependence. The flavor attributes of both alcohol and nicotine can be important determinants of their initial acceptance and they both share the component chemosensory qualities of an aversive odor, bitter taste and oral irritation. There is a growing body of evidence demonstrating epigenetic chemosensory mechanisms through which fetal alcohol exposure increases adolescent alcohol acceptance, in part, by decreasing the aversion to alcohol''s bitter and oral irritation qualities, as well as its odor. Given that alcohol and nicotine have noteworthy chemosensory qualities in common, we investigated whether fetal exposure to alcohol increased the acceptability of nicotine''s odor and taste in adolescent rats. Study rats were alcohol-exposed during fetal development via the dams'' liquid diet. Control animals received ad lib access to an iso-caloric, iso-nutritive diet throughout gestation. Odorant-induced innate behavioral responses to nicotine odor (Experiment 1) or orosensory-mediated responses to nicotine solutions (Experiment 2) were obtained, using whole-body plethysmography and brief access lick tests, respectively. Compared to controls, rats exposed to fetal alcohol showed an enhanced nicotine odor response that was paralleled by increased oral acceptability of nicotine. Given the common aversive component qualities imbued in the flavor profiles of both drugs, our findings demonstrate that like postnatal alcohol avidity, fetal alcohol exposure also influences nicotine acceptance, at a minimum, by decreasing the aversion of both its smell and taste. Moreover, they highlight potential chemosensory-based mechanism(s) by which fetal alcohol exposure increases the later initial risk for nicotine use, thereby contributing to the co-morbid expression with enhanced alcohol avidity. Where common chemosensory mechanisms are at play, our results suggest broader implications related to the consequence of fetal exposure with one substance of abuse and initial acceptability of others.     

Lipid oxidation in biological membranes. Electron transfer proteins as initiators of lipid autoxidation.

Biological lipid autoxidation has been studied in a model system composed of sonicated phospholipids as substrate and electron transfer proteins found in membranes as possible catalysts. Heme compounds, flavoproteins, and iron-sulfur proteins were examined for their ability to initiate lipid autoxidation. Among many heme compounds tested, the most active were hematin ?microperoxidase ? methemoglobin > cytochrome c. With fresh preparations of phospholipids, reaction rates (nanomoles of oxygen/minute nanomoles of heme) ranged from 5 (cytochrome c) to 350 (hematin). Only the oxidized heme compounds were active as catalysts. Reduced heme compounds, flavoproteins and riboflavin were inactive. In the presence of heme compounds, aged preparations of sonicated phospholipids were much more rapidly oxidized than fresh preparations. They also had a higher content of fatty acid hydroperoxides as judged from their characteristic diene absorption peak at 234 nm. This observation agrees with the postulated mechanism of lipid autoxidation by heme compounds, namely, homolytic scission of preformed fatty acid hydroperoxides. Iron-sulfur proteins were also active as initiators of lipid autoxidation when destabilized in the presence of an appropriate iron chelator (o-phenanthroline or 2,2′-bipyridine) or a chaotropic ion. Oxygen uptake rates (nanomoles of oxygen/minute × milligrams of protein) varied from about 200 for an iron-sulfur protein isolated from complex I to about 5500 for Clostridium pasteurianum ferredoxin. However, per nanomole of labile sulfide, the rates for all active iron-sulfur proteins were 4–7 nmol of oxygen/min × nmol of labile sulfide.Superoxide-generating systems did not initiate lipid autoxidation, nor did erythrocuprein inhibit the autoxidations induced by heme compounds or ferredoxin. However, lipid oxidations induced by two other iron-sulfur proteins were partially inhibited by erythrocuprein. It is concluded that in the above system Superoxide anion is neither an initiator nor an obligatory intermediate of lipid autoxidation.     

Protein oxidation and proteolysis during aging and oxidative stress

Previous studies in this laboratory have shown that glutamine synthetase (GS) and other key metabolic enzymes are inactivated by metal-catalyzed oxidation reactions in vitro. Oxidative inactivation renders these proteins highly susceptible to proteolysis, especially to a class of newly identified alkaline proteases which exhibit little or no activity against the native enzymes. These studies have suggested that oxidative inactivation may be an important marking step for intracellular protein degradation. Because many of the enzymes which have been shown to accumulate as inactive or less active forms during aging are readily inactivated by metal-catalyzed oxidation reactions in vitro, we have investigated the possible relationship between protein oxidation and proteolysis during aging and oxidative stress in vivo. Oxidized proteins accumulate in hepatocytes of rats exposed to 100% oxygen during the first 48 h of oxygen treatment. In the interval between 48 and 54 h the levels of oxidized proteins decline sharply. The specific activities of at least two liver enzymes, glutamine synthetase and glucose-6-phosphate dehydrogenase (G-6-PDH), decrease during the 54-h experiment. GS and G-6-PDH specific immunological cross-reactivity remains high during the first 48 h of oxygen treatment and then declines in the interval between 48 and 54 h. During this same interval the levels of alkaline proteases which degrade oxidized proteins increase, indicating that these activities are induced or activated in response to oxidative stress and subsequently degrade the proteins which have become oxidized during the initial phase of oxygen treatment. Oxidized proteins accumulate progressively during aging in hepatocytes from rats 3 to 26 months old, with the largest incremental increase between 20 and 26 months. The increase in protein oxidation is correlated with a loss of specific activity of GS and G-6-PDH without a concomitant loss of immunological cross-reactivity. The levels of alkaline proteases which degrade oxidized proteins in hepatocytes from 26-month-old rats is only 20% that of 3-month-old rats, suggesting that oxidized proteins accumulate in hepatocytes from old rats, in part, because the proteases which degrade them are deficient or defective. moreover, when old rats are subjected to treatment with 100% oxygen, the levels of oxidized proteins continue to increase and the alkaline protease activity remains low, indicating that these protease activities are not increased in response to oxidative stress in old rats.     

First example of an antibody-catalyzed aza Diels-Alder reaction

Described herein is the synthesis of a hapten of biocyclo[2,2,2]octene 5 designed to mimic the exo transition-state of an aza Diels-Alder reaction. Immunization of rabbits with this hapten provided polyclonal antibodies, Aza-BSA-3, which were used to synthesize adduct 4b in the first reported antibody-catalyzed exo Diels-Alder reaction.     

Photodegradation of DNA with fluorescent light in the presence of riboflavin, and photoprotection by flavin triplet-state quenchers

Superoxide anion was photogenerated upon illumination of nucleic acids with fluorescent light in a solution containing phosphate buffer, pH 7.8 and riboflavin. DNA was a better reducing substrate for this reaction than was RNA. A similar riboflavin-sensitized photoreaction caused single- and double-strand scissions of supercoiled PM2 DNA as detected by electrophoresis in agarose gels. None of specific scavengers or quenchers for superoxide anion and other active oxygen species prevented the DNA strand breaks. However, among the flavin triplet-state quenchers, potassium iodide, butylated hydroxyanisole, and ferricytochrome c protected the supercoiled DNA from photodegradation; butylated hydroxytoluene, alpha-tocopherol, tyrosine and hemoglobin did not have any protective effect. These results indicate that triplet-state riboflavin or a derivative formed from it participate directly in the observed riboflavin-sensitized DNA photodegradation and that active oxygen species are not directly involved.     

Different binding modes of free and carrier-protein-coupled nicotine in a human monoclonal antibody

Nicotine is the principal addictive component of tobacco. Blocking its passage from the lung to the brain with nicotine-specific antibodies is a promising approach for the treatment of smoking addiction. We have determined the crystal structure of nicotine bound to the Fab fragment of a fully human monoclonal antibody (mAb) at 1.85 Å resolution. Nicotine is almost completely (> 99%) buried in the interface between the variable domains of heavy and light chains. The high affinity of the mAb is the result of a charge–charge interaction, a hydrogen bond, and several hydrophobic contacts. Additionally, similarly to nicotinic acetylcholine receptors in the brain, two cation–π interactions are present between the pyrrolidine charge and nearby aromatic side chains. The selectivity of the mAb for nicotine versus cotinine, which is the major metabolite of nicotine and differs in only one oxygen atom, is caused by steric constraints in the binding site. The mAb was isolated from B cells of an individual immunized with a nicotine–carrier protein conjugate vaccine. Surprisingly, the nicotine was bound to the Fab fragment in an orientation that was not compatible with binding to the nicotine–carrier protein conjugate. The structure of the Fab fragment in complex with the nicotine–linker derivative that was used for the production of the conjugate vaccine revealed a similar position of the pyridine ring of the nicotine moiety, but the pyrrolidine ring was rotated by about 180°. This allowed the linker part to reach to the Fab surface while high-affinity interactions with the nicotine moiety were maintained.     

Fetal Nicotine Exposure Increases Preference for Nicotine Odor in Early Postnatal and Adolescent,but Not Adult,Rats

Human studies demonstrate a four-fold increased possibility of smoking in the children of mothers who smoked during pregnancy. Nicotine is the active addictive component in tobacco-related products, crossing the placenta and contaminating the amniotic fluid. It is known that chemosensory experience in the womb can influence postnatal odor-guided preference behaviors for an exposure stimulus. By means of behavioral and neurophysiologic approaches, we examined whether fetal nicotine exposure, using mini-osmotic pumps, altered the response to nicotine odor in early postnatal (P17), adolescent (P35) and adult (P90) progeny. Compared with controls, fetal exposed rats displayed an altered innate response to nicotine odor that was evident at P17, declined in magnitude by P35 and was absent at P90 - these effects were specific to nicotine odor. The behavioral effect in P17 rats occurred in conjunction with a tuned olfactory mucosal response to nicotine odor along with an untoward consequence on the epithelial response to other stimuli – these P17 neural effects were absent in P35 and P90 animals. The absence of an altered neural effect at P35 suggests that central mechanisms, such as nicotine-induced modifications of the olfactory bulb, bring about the altered behavioral response to nicotine odor. Together, these findings provide insights into how fetal nicotine exposure influences the behavioral preference and responsiveness to the drug later in life. Moreover, they add to a growing literature demonstrating chemosensory mechanisms by which patterns of maternal drug use can be conveyed to offspring, thereby enhancing postnatal vulnerability for subsequent use and abuse.     

Selective oxidations with molecular oxygen,catalyzed by chloroperoxidase in the presence of a reductant

Chloroperoxidase (CPO) catalyzes the oxidation of various substrates with molecular oxygen as the primary oxidant, in the presence of dihydroxyfumaric acid (DHF) as a sacrificial reductant. For example, indole is oxidized to 2-oxindole with up to 77% selectivity and thioanisole to the corresponding R-sulfoxide (e.e. >99%). To our knowledge, these are the first examples of (enantio)selective aerobic oxidations catalyzed by peroxidases. A mechanism is proposed which involves initial formation of hydrogen peroxide via autoxidation of DHF. CPO subsequently uses the hydrogen peroxide for the selective oxidation of the substrate, via an oxygen transfer mechanism. In contrast, horseradish proxidase (HRP) primarily catalyzes the oxidation of DHF via a classical peroxidase mechanism and oxidations of added substrates are aselective.     

Addictive drugs and their relationship with infectious diseases

The use of drugs of abuse, both recreationally and medicinally, may be related to serious public health concerns. There is a relationship between addictive drugs of abuse such as alcohol and nicotine in cigarette smoke, as well as illegal drugs such as opiates, cocaine and marijuana, and increased susceptibility to infections. The nature and mechanisms of immunomodulation induced by such drugs of abuse are described in this review. The effects of opiates and marijuana, using animal models as well as in vitro studies with immune cells from experimental animals and humans, have shown that immunomodulation induced by these drugs is mainly receptor-mediated, either directly by interaction with specific receptors on immune cells or indirectly by reaction with similar receptors on cells of the nervous system. Similar studies also show that cocaine and nicotine have marked immunomodulatory effects, which are mainly receptor-mediated. Both cocaine, an illegal drug, and nicotine, a widely used legal addictive component of cigarettes, are markedly immunomodulatory and increase susceptibility to infection. The nature and mechanism of immunomodulation induced by alcohol, the most widely used addictive substance of abuse, are similar but immunomodulatory effects, although not receptor-mediated. The many research studies on the effects of these drugs on immunity and increased susceptibility to infectious diseases, including AIDS, are providing a better understanding of the complex interactions between immunity, infections and substance abuse.     

Comparing beta-carotene,vitamin E and nitric oxide as membrane antioxidants

Singlet oxygen initiates lipid peroxidation via a nonfree radical mechanism by reacting directly with unsaturated lipids to form lipid hydroperoxides (LOOHs). These LOOHs can initiate free radical chain reactions leading to membrane leakage and cell death. Here we compare the ability and mechanism by which three small-molecule membrane antioxidants (beta-carotene, alpha-tocopherol and nitric oxide) inhibit lipid peroxidation in membranes. We demonstrate that beta-carotene provides protection against singlet oxygen-mediated lipid peroxidation, but does not slow free radical-mediated lipid peroxidation. Alpha-Tocopherol does not protect cells from singlet oxygen, but does inhibit free radical formation in cell membranes. Nitric oxide provides no direct protection against singlet oxygen exposure, but is an exceptional chain-breaking antioxidant as evident from its ability to blunt oxygen consumption during free radical-mediated lipid peroxidation. These three small-molecule antioxidants appear to have complementary mechanisms for the protection of cell membranes from detrimental oxidations.     

Persistent nicotine treatment potentiates amplification of the dihydrofolate reductase gene in rat lung epithelial cells as a consequence of Ras activation

Although nicotine has been suggested to promote lung carcinogenesis, the mechanism of its action in this process remains unknown. The present investigation demonstrates that the treatment of rat lung epithelial cells with nicotine for various periods differentially mobilizes multiple intracellular pathways. Protein kinase C and phosphoinositide 3-OH-kinase are transiently activated after the treatment. Also, Ras and its downstream effector ERK1/2 are activated after long term exposure to nicotine. The activation of Ras by nicotine treatment is responsible for the subsequent perturbation of the methotrexate (MTX)-mediated G1 cell cycle restriction as well as an increase in production of reactive oxygen species. When p53 expression is suppressed by introducing E6, persistent exposure to nicotine enables dihydrofolate reductase gene amplification in the presence of methotrexate (MTX) and the formation of the MTX-resistant colonies. Altering the activity of phosphoinositide 3-OH-kinase has no effect on dihydrofolate reductase amplification. However, the suppression of protein kinase C dramatically affects the colony formation in soft agar. Thus, our data suggest that persistent exposure to nicotine perturbs the G1 checkpoint and causes DNA damage through the increase of the production of reactive oxygen species. However, a third element rendered by loss of p53 is required for the initiation of the process of gene amplification. Under p53-deficient conditions, the establishment of a full oncogenic transformation, in response to long term nicotine exposure, is achieved through the cooperation of multiple signaling pathways.     

Early pregnancy termination in rats immunized with denatured chicken riboflavin carrier protein.

Immunoneutralization of the maternal riboflavin carrier protein in the pregnant rat with antibodies to chicken egg vitamin carrier has earlier been shown to terminate their pregnancies. In order to understand the nature of the epitopic conformations capable of eliciting antibodies bioneutralizing the endogenous riboflavin carrier protein in the pregnant rat, we compared pregnancy progression in the fertile rodents following active immunization with either the native, SDS-denatured, reduced-carboxymethylated or SDS-treated reduced carboxymethylated avian egg white riboflavin carrier protein. The data revealed that despite the total antibody titers being higher in the animals immunized with the native protein, the antibodies elicited against the denatured avian vitamin carrier exhibited relatively better potencies to bioneutralize the endogenous maternal protein as evidenced by higher rates of early fetal resorption.     

Biodegradation of nicotine by a newly isolated Agrobacterium sp. strain S33

Aims: To isolate and characterize bacteria capable of degrading nicotine from the rhizospheric soil of a tobacco plant and to use them to degrade the nicotine in tobacco solid waste. Methods and Results: A bacterium, strain S33, was newly isolated from the rhizospheric soil of a tobacco plant, and identified as Agrobacterium sp. based on morphology, physiological tests, Biolog MicroLog3 4·20 system and 16S rRNA gene sequence. Using nicotine as the sole source of carbon and nitrogen in the medium, it grew optimally with 1·0 g l^?1 of nicotine at 30°C and pH 7·0, and nicotine was completely degraded within 6 h. The resting cells prepared from the glucose‐ammonium medium or LB medium could not degrade nicotine within 10 h, while those prepared from the nicotine medium could completely degrade 3 g l^?1 of nicotine in 1·5 h at a maximal rate of 1·23 g nicotine h^?1 g^?1 dry cell. Using the medium containing nicotine, glucose and ammonium simultaneously to cultivate strain S33, the resting cells could degrade 98·87% of nicotine in tobacco solid waste with the concentration as 30 mg nicotine g^?1 dry weight tobacco solid waste within 7 h at a maximal rate of 0·46 g nicotine h^?1 g^?1 dry cell. Conclusions: This is the first report that Agrobacterium sp. has the ability to degrade nicotine. Agrobacterium sp. S33 could use nicotine as the sole source of carbon and nitrogen. The use of resting cells of the strain S33 prepared from the nicotine–glucose–ammonium medium was an effective method to degrade nicotine and detoxify tobacco solid waste. Significance and Impact of the Study: Nicotine in tobacco wastes is both toxic and harmful to human health and the environment. This study showed that Agrobacterium sp. S33 may be suitable for the disposal of tobacco wastes and reducing the nicotine content in tobacco leaves.     

催化抗体研究新进展

催化抗体也叫抗体酶,是具有催化活性的免疫球蛋白.由于它兼具抗体的高度选择性和酶的高效催化性,因而催化抗体制备技术的开发预示着可以人为生产适应各种用途的,特别是自然界不存在的高效催化剂,对生物学、化学和医学等多种学科有重要的理论意义和实用价值.综述了催化抗体研究的最新进展,讨论了该领域目前存在的问题,提出了解决这些问题的可能办法.     

Biochemical and immunological aspects of riboflavin carrier protein

Riboflavin carrier protein which is obligatorily involved in yolk deposition of the vitamin in the chicken egg, is a unique glycophosphoprotein present in both the yolk and white compartments. The yolk and egg white proteins are products of a single estrogen-inducible gene expressed in the liver and the oviduct respectively of egg laying birds. Despite the fact that the carbohydrate composition of the yolk and white riboflavin carrier proteins differ presumably due to differential post-translational modification, the proteins are immunologically similar and have identical amino acid sequence (including a cluster of 8 phosphoser residues towards the C-terminus) except at the carboxy terminus where the yolk riboflavin carrier protein lacks 13 amino acids as a consequence of proteolytic cleavage during uptake by oocytes. The protein is highly conserved throughout evolution all the way to humans in terms of gross molecular characteristics such as molecular weight and isoelectric point, and in immunological properties, preferential affinity for free riboflavin and estrogen inducibility at the biosynthetic locusviz., liver. Obligatory involvement of the mammalian riboflavin carrier protein in transplacental flavin transport to subserve fetal vitamin nutrition during gestation is revealed by experiments using pregnant rodent or subhuman primate models wherein immunoneutralisation of endogenous maternal riboflavin carrier protein results in fetal wastage followed by pregnancy termination due to selective yet drastic curtailment of vitamin efflux into the fetoplacental unit. Using monoclonal antibodies to chicken riboflavin carrier protein, it could be shown that all the major epitopes of the avian riboflavin carrier protein are highly conserved throughout evolution although the relative affinities of some of the epitopes for different monoclonal antibodies have undergone progressive changes during evolution. Using these monoclonal antibodies, an attempt is being made to map the different epitopes on the riboflavin carrier protein molecule with a view to delineate the immunodominant regions of the vitamin carrier to understand its structure-immunogenicity relationship. Presented at the 3rd National Symphosium on Bioorganic Chemistry, 1987, Hyderabad.     

两株芽胞菌对烟草废料烟碱与绿原酸降解的研究

为综合利用烟草废料,筛选得到能有效降解烟碱与绿原酸的两株芽胞菌,考察了所选菌株对烟碱与绿原酸的降解特性。实验结果表明,菌株Bacillus sp．X6表现出较高降解烟碱能力,培养36h可将含烟碱6．04mg／g烟草物料中的烟碱降解60．3％,72h降解率可达87．6％;对绿原酸降解效果最好的是菌株Bacillus sp．Xy,培养48h将含绿原酸10．57mg／g烟草物料中的绿原酸降解了50．5％,72h可将绿原酸降解62．2％,