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Biomedical subjects

David Hammond

Publications and source records attributed to David Hammond.

At least 19 recordsLinked to original sources
PubMed2006

The Bead blot: a method for identifying ligand-protein and protein-protein interactions using combinatorial libraries of peptide ligands.

Small molecules that bind proteins can be used as ligands for protein purification and for investigating protein-protein and protein-drug interactions. Unfortunately, many methods used to identify new ligands to desired proteins suffer from common shortcomings, including the requirement that the target protein be purified and/or the requirement that the ligands be selected under conditions different from those under which it will be used. We have developed a new method called the Bead blot that can (i) select ligands to unpurified proteins, including trace proteins, present in complex materials (e.g., unfractionated plasma); (ii) select ligands to multiple proteins under a variety of conditions in a single experiment; and (iii) be used with libraries of different types of ligands. In the Bead blot, a library of ligands, synthesized on chromatography resin beads, is incubated with a starting material containing a target protein for which a ligand is sought. The proteins in the material bind to their complementary ligands according to specific affinity interactions. Then the protein-loaded beads are immobilized in a porous matrix, and the proteins are directionally eluted from the beads and captured on a membrane superimposed on the beads. The location of the target protein on the membrane is determined, and because the position of the protein(s) on the membrane reflects the position of the bead(s) in the matrix, the bead that originally bound the protein is identified, with subsequent elucidation of the ligand sequence. Ligands to several targets can be identified in one experiment. Here we demonstrate the broad utility of this method by the selection of ligands that purify plasma protein complexes or that remove pathogens from whole blood with very high affinity constants. We also select ligands to a protein based on competitive elution.

Amino Acid Sequence↗
PubMed2006

Tobacco denormalization and industry beliefs among smokers from four countries.

BACKGROUND: Tobacco denormalization is an important concept for understanding smoking behavior. The present study sought to assess beliefs about the tobacco industry and the social acceptability of smoking among nationally representative samples of adult smokers from four countries, and to assess the relationship of these measures to cessation behavior and tobacco-control policy. DESIGN: A longitudinal survey of 9058 adult smokers from Canada (n = 2214), the United States (n = 2138), the United Kingdom (n = 2401), and Australia (n = 2305), was conducted in October-December 2002 and again in June and August 2003 (75% follow-up rate). The analyses were conducted in 2005. RESULTS: The findings indicate that few smokers perceive approval for their smoking, and most hold relatively antagonistic beliefs toward the tobacco industry. For example, 80% of smokers reported that society disapproves of smoking, and more than three quarters reported that tobacco companies cannot be trusted to tell the truth. Social and industry denormalization were independently associated with intentions to quit smoking. Baseline levels of social denormalization were associated with abstinence at the 8-month follow-up, as were changes in industry denormalization beliefs between baseline and follow-up. Anti-industry beliefs at baseline did not predict abstinence at follow-up. A similar pattern of findings was observed across all four countries. In addition, social denormalization and anti-industry beliefs were significantly associated with tobacco-control policies, such as noticing health warnings on packages and greater workplace smoking restrictions. CONCLUSIONS: Tobacco denormalization constructs were independently linked to cessation-related outcomes among adults from four countries. Tobacco-industry denormalization themes in mass media campaigns may help to reduce tobacco use above and beyond more traditional communications that target social norms.

Adolescent↗
PubMed2006

Secret science: tobacco industry research on smoking behaviour and cigarette toxicity.

A lack of scientific data remains the principal obstacle to regulating cigarette toxicity. In particular, there is an immediate need to improve our understanding of the interaction between smoking behaviour and product design, and its influence on cigarette deliveries. This article reviews internal tobacco industry documents on smoking behaviour research undertaken by Imperial Tobacco Limited (ITL) and British-American Tobacco (BAT). BAT documents indicate that smokers vary their puffing behaviour to regulate nicotine levels and compensate for low-yield cigarettes by smoking them more intensely. BAT research also shows that the tar and nicotine delivered to smokers is substantially greater than the machine-smoked yields reported to consumers and regulators. Internal documents describe a strategy to maximise this discrepancy through product design. In particular, BAT developed elastic cigarettes that produced low yields under standard testing protocols, whereas in consumers' hands they elicited more intensive smoking and provided higher concentrations of tar and nicotine to smokers. Documents also show that BAT pursued this product strategy despite the health risks to consumers and ethical concerns raised by senior scientists, and paired it with an equally successful marketing campaign that promoted these cigarettes as low-tar alternatives for health-concerned smokers. Overall, the documents seem to reveal a product strategy intended to exploit the limitations of the testing protocols and to intentionally conceal from consumers and regulators the potential toxicity of BAT products revealed by BAT's own research. Tobacco industry research underscores the serious limitations of the current cigarette testing protocols and the documents describe deceptive business practices that remain in place.

Advertising↗
PubMed2006

The use of rabbit polyclonal antibodies to assess neoantigenicity following viral reduction of an alpha-1-proteinase inhibitor preparation.

The objective of this study was to determine whether the viral reduction processes of nanofiltration and solvent/detergent treatment used in the manufacture of alpha-1 proteinase inhibitor (API) cause neoantigenic changes. Polyclonal antibodies were raised in rabbits against the treated API and quantitatively absorbed with an affinity column containing API that had not undergone viral reduction treatment. Antibodies before and after absorption were measured in a validated ELISA using the immunogen for antibody capture. Antibodies against novel API epitopes were not found after antiserum from rabbits inoculated with treated API was absorbed with untreated API. A positive control, consisting of serum obtained from rabbits inoculated with trinitrophenylated API, showed substantial amounts of measurable antibody following absorption with untreated API. The results suggest that the viral reduction process used does not result in the creation of API neoantigens.

Animals↗
PubMed2006

A new approach for the detection and identification of protein impurities using combinatorial solid phase ligand libraries.

We propose a novel method for detection of protein impurities present in plasma-derived and recombinant purified injectable biopharmaceuticals by enhancing the concentration of protein impurities, in essence "amplifying" their presence to detectable levels. The method is based on the capture of proteins using a combinatorial solid-phase hexapeptides ligand library previously described for the reduction of protein concentration difference in biological fluids. Three proteins have been investigated: Staphylococcus aureus Protein A, expressed in Escherichia coli and supplied as 99% pure, recombinant human albumin, expressed in Pichia pastoris and certified as 95% pure, and therapeutic albumin supplied as 96-98% pure injectable solution. In all cases, after treatment with the ligand libraries, a number of additional polypeptide chains, not visible in the control, could be detected and obtained in sufficient amounts for MS analysis. In the cases of the two recombinant proteins, it could be demonstrated that a number of these polypeptide chains were host cell proteins still present in the purified product. In addition, a substantial number of these spots were found to be cleavage products of the original recombinant DNA species. Such cleavage products were particularly abundant in the recombinant human albumin preparation. From pure injectable serum albumin, a number of human plasma protein impurities were also identified by LC-MS/MS analysis. Treatment with ligand libraries of purified proteins is thus seen as a very powerful method of capture and concentration of host proteins and cleaved products for further analysis to control better the quality of industrial biotechnology products.

Albumins↗
PubMed2006

Reduction of transmissible spongiform encephalopathy infectivity from human red blood cells with prion protein affinity ligands.

BACKGROUND: There is a demonstrated risk of infection by transmissible spongiform encephalopathies (TSEs) through transfusion from asymptomatic donors. Currently, blood-borne TSE infectivity cannot be detected with a diagnostic test, nor is it likely to be amenable to inactivation; however, its depletion with specific adsorp-tive ligand resins is possible. STUDY DESIGN AND METHODS: Six ligands that bind the prion protein, PrP, were selected by screening large solid-phase combinatorial chemical libraries. The selected resins were placed in columns and challenged with a unit of leukoreduced human red blood cells (RBCs) spiked with hamster brain-derived scrapie infectivity. The performance of each ligand was assessed by comparing the TSE infectivity titer in the RBCs before and after passage through each of five resin columns in series. RESULTS: Four resins were able to reduce infectivity titer by 3 to more than 4 log ID(50) per mL. The reduction was not due to nonspecific matrix interactions since a chemical modification of the most effective ligand completely abolished its ability to bind infectivity (negative control). A small subfraction of the infectivity, 0.01 percent, could not be removed, even upon repeated passage through successive columns. CONCLUSION: If endogenous TSE infectivity in RBCs binds to the ligands in the same proportion as brain-derived infectivity spiked into RBCs, the four most effective ligands would remove 3 to 4 log ID(50) per mL. A follow-up experiment is in progress to test whether endogenous blood-borne infectivity is also reduced.

Animals↗
PubMed2006

Cigarette yields and human exposure: a comparison of alternative testing regimens.

OBJECTIVE: There is general agreement that the testing protocol for measuring cigarette smoke constituents-the International Organization for Standardization regimen-is an inappropriate mechanism for evaluating human exposure. Alternative smoking regimens have been introduced in Canada and Massachusetts; however, these regimens have not been evaluated against human smoking behavior and biomeasures of exposure. The objective of this study was to compare measures of smoke volume and nicotine uptake among human smokers against the puffing variables and nicotine yields generated by five different machine smoking regimens: (a) International Organization for Standardization, (b) Massachusetts, (c) Canadian, (d) a Compensatory regimen, and (e) a Human Mimic regimen. METHODS: Measures of smoke volume and puffing behavior were recorded for 51 smokers who used a portable smoking topography device for three 1-week trials. Measures of salivary cotinine were taken at the completion of each week. The cigarette brands smoked by participants were then machine-smoked under five testing regimens, including a human mimic condition where brands were machine smoked using the puffing behavior recorded from human smokers. The total volume of smoke collected from each cigarette and the nicotine, tar, and carbon monoxide yields were recorded. RESULTS: None of the four machine smoking regimens adequately reflected Human Mimic Yields of tar, nicotine, and carbon monoxide. In addition, none of the four smoking regimens generated nicotine yields that were associated with actual nicotine uptake in humans. CONCLUSIONS: None of the existing smoking regimens adequately represents human smoking behavior nor do they generate yields associated with human measures of nicotine uptake.

Adult↗
PubMed2005

Oxandrolone enhances skeletal muscle myosin synthesis and alters global gene expression profile in Duchenne muscular dystrophy.

Earlier studies have shown that the progressive, unrelenting muscle loss associated with Duchenne muscular dystrophy (DMD) involves an imbalance between the rates of synthesis and degradation of muscle proteins. Although previous studies have suggested that oxandrolone may be beneficial in DMD, the mechanism of action of oxandrolone on muscle in DMD remains unclear. To address these issues, we combined stable isotope studies and gene expression analysis to measure the fractional synthesis rate of myosin heavy chain (MHC), the key muscle contractile protein, the transcript levels of the isoforms of MHC, and global gene expression profiles in four children with DMD before and after 3 mo of treatment with oxandrolone. Gastrocnemius muscle biopsies and blood samples were collected during the course of a primed 6-h continuous infusion of l-[U-(13)C]leucine on two separate occasions, before and after the 3-mo treatment with oxandrolone (0.1 mg.kg(-1).day(-1)). Gene expression analysis was done with microarrays and RT-qPCR. In response to the treatment, MHC synthesis rate increased 42%, and this rise was accounted for, at least in part, by an upregulation of the transcript for MHC8 (perinatal MHC). Gene expression data suggested a decrease in muscle regeneration as a consequence of oxandrolone therapy, presumably because of a decrease in muscle degeneration. These findings suggest that 1) oxandrolone has a powerful protein anabolic effect on a key contractile protein and 2) larger and longer-term studies are warranted to determine whether these changes translate into meaningful therapy for these patients.

Adolescent↗
PubMed2005

Reduction of the concentration difference of proteins in biological liquids using a library of combinatorial ligands.

The discovery of polypeptides and proteins with relevance to a particular biological state is complicated by their vast number and concentration range in most biological mixtures. Depletion methodologies are frequently used to remove the most abundant species; however, this removal not only fails significantly to enrich trace proteins, it may also nonspecifically deplete them due to their interactions with the removed high-abundance proteins. Here we report a simple-to-use methodology that reduces the protein concentration range of a complex mixture like whole serum through the simultaneous dilution of high-abundance proteins and the concentration of low-abundance proteins. This methodology utilizes solid-phase ligand libraries of immense diversity, generated by "split, couple, recombine" combinatorial chemistry, that are used for affinity-based binding to the proteins of a given mixture. With a controlled sample-to-ligand ratio it is possible to modulate the relative concentration of proteins such that many peptides or proteins that are undetectable by classical analytical methods become easily accessible. The reduction in the dynamic range of unfractionated serum is specifically described along with treatment of other proteomes such as extracts from Escherichia coli, chicken egg white and cell culture supernatant. Mono- and bi-dimensional electrophoresis (1-DE and 2-DE respectively) and surface-enhanced laser desorption/ionization-mass spectrometry (SELDI-TOF-MS) technology demonstrate the reduction in protein concentration range. Combining this approach with additional fractionation methods further increased the number of detectable species.

Adsorption↗
PubMed2005

Smoking topography, brand switching, and nicotine delivery: results from an in vivo study.

OBJECTIVE: Exposure to toxins in tobacco smoke is influenced by how a cigarette is smoked. Cigarettes have been designed to allow for a range of puffing behavior and to provide different, nonlinear tar and nicotine yields in response to different puffing profiles. However, puffing behavior and its influence upon risk-exposure has yet to be assessed outside the laboratory, in smokers' natural environment. METHOD: Fifty-nine adult smokers used a portable device to measure smoking topography over the course of three 1-week trials. Participants were asked to smoke their usual "regular yield" brand through the device for trial 1 and again, 6 weeks later, at trial 2. Half the subjects were then randomly assigned to switch to a "low-yield" brand for trial 3. RESULTS: The findings show a high degree of stability in puffing behavior within the same subject over time but considerable variability between smokers. Smokers who were switched to a "low-yield" cigarette increased their total smoke intake per cigarette by 40% (P = 0.007), with no significant change in their salivary cotinine levels. Cigarettes smoked per day and nicotine yield were only weakly associated with salivary cotinine levels; however, salivary cotinine was strongly associated with a composite measure that included cigarettes per day, brand elasticity, and puffing behavior (sr = 0.61, P < 0.001). CONCLUSIONS: These findings provide strong evidence of behavioral compensation to low-yield cigarettes from in vivo measures of smoking behavior. The findings also show the importance of brand elasticity and smoking topography in predicting nicotine uptake and smoke exposure.

Adult↗
PubMed2004

The near-universal experience of regret among smokers in four countries: findings from the International Tobacco Control Policy Evaluation Survey.

Regret may be a key variable in understanding the experience of smokers, the vast majority of whom continue to smoke while desiring to quit. We present data from the baseline wave (October-December 2002) of the International Tobacco Control Policy Evaluation Survey, a random-digit-dialed telephone survey of a cohort of over 8,000 adult smokers across four countries--Canada, the United States, the United Kingdom, and Australia--to estimate the prevalence of regret and to identify its predictors. The proportion of smokers who agreed or agreed strongly with the statement "If you had to do it over again, you would not have started smoking" was extremely high--about 90%--and nearly identical across the four countries. Regret was more likely to be experienced by older smokers, women, those who had tried to quit more often, those who perceived quitting as conferring benefits, those with higher levels of perceived addiction, those who worried about future damage to health, those who perceived smoking as lowering their quality of life, those who perceived higher monetary costs of smoking, and those who believed that smoking is not socially acceptable. This predictive model was the same in all four countries. Regret is thus a near-universal experience among smokers in all four countries, and the factors that predict regret are universal across these four countries. Among other implications for cessation treatment and smoking prevention, this near universality of regret casts doubt on the view of some policy analysts and economists that the decisions to take up and continue smoking are welfare-maximizing for the consumer.

Adult↗
PubMed2004

Use of and beliefs about light cigarettes in four countries: findings from the International Tobacco Control Policy Evaluation Survey.

This study examined reported use of, and beliefs about, so-called light cigarettes among adult smokers in four countries: Australia (Aus), Canada (Can), the United Kingdom (U.K.) and the United States (U.S.). The method used was parallel telephone surveys among 9,046 smokers across the four countries. The results indicated that more than half of all smokers in each country except the U.K. reported smoking light cigarette brands. A majority of smokers surveyed in each country except Canada continue to believe that light cigarettes offer some health benefit compared to regular cigarettes (Canada 43%, U.S. 51%, Australia 55%, U.K. 70%). A majority of smokers in all four countries believed that light cigarettes are smoother on the throat and chest than regular cigarettes. Predictors of use of light cigarettes and beliefs about possible benefits were very similar in the four countries. These results demonstrate an ongoing need for public education about why light cigarettes do not reduce harm and do not make quitting easier. The results provide further evidence for the need for regulatory measures in all four countries to prohibit the use of misleading light and mild descriptors including package imagery in product marketing (as prescribed in Article 11 of the Framework Convention on Tobacco Control), abandon the use of standard FTC/ISO tar and nicotine yields as consumer information, and adopt policies to regulate deceptive design features of cigarettes, such as ventilated filters.

Adult↗
PubMed2004

Graphic Canadian cigarette warning labels and adverse outcomes: evidence from Canadian smokers.

OBJECTIVES: We assessed the impact of graphic Canadian cigarette warning labels. METHODS: We used a longitudinal telephone survey of 616 adult smokers. RESULTS: Approximately one fifth of participants reported smoking less as a result of the labels; only 1% reported smoking more. Although participants reported negative emotional responses to the warnings including fear (44%) and disgust (58%), smokers who reported greater negative emotion were more likely to have quit, attempted to quit, or reduced their smoking 3 months later. Participants who attempted to avoid the warnings (30%) were no less likely to think about the warnings or engage in cessation behavior at follow-up. CONCLUSIONS: Policymakers should not be reluctant to introduce vivid or graphic warnings for fear of adverse outcomes.

Adult↗
PubMed2003

Classical Hodgkin lymphoma is associated with frequent gains of 17q.

The etiology of Hodgkin lymphoma (HL) is poorly understood, and studies of the genetics of this disease have been hampered by the scarcity of the Hodgkin and Reed-Sternberg (HRS) cells within tumors. To determine whether recurrent genomic imbalances are a feature of HL, CD30-positive HRS cells were laser-microdissected from 20 classical Hodgkin lymphomas (cHLs) and four HL-derived cells lines and subjected to analyses by comparative genomic hybridization. In primary tumors, the most frequently involved chromosomal gains were 17q (70%), 2p (40%), 12q (40%), 17p (40%), 22q (35%), 9p (30%), 14q (30%), and 16p (30%), with minimal overlapping regions at 17q21, 2p23-13, 12q24, 17p13, 22q13, 9p24-23, 14q32, 16p13.3, and 16p11.2. The most frequent losses involved 13q (35%), 6q (30%), 11q (25%), and 4q (25%), with corresponding minimal overlapping regions at 13q21, 6q22, 11q22, and 4q32. Statistical analysis revealed significantly more gains of 2p and 14q in the older adult cases; loss of 13q was associated with a poor outcome. The results suggest that there is a set of recurrent chromosomal abnormalities associated with cHL and provide further evidence that cHL is genetically distinct from nodular lymphocyte predominance Hodgkin lymphoma (NLPHL). Abnormalities of 17q are infrequent in other lymphomas or NLPHL; this finding, coupled with current knowledge of gene expression in cHL, suggests that genes present on 17q may play an important role in the pathogenesis of cHL.

Adolescent↗
PubMed2002

Uromodulin promoter directs high-level expression of biologically active human alpha1-antitrypsin into mouse urine.

We have recently shown that the regulatory sequence of the uromodulin gene, containing the 3.7 kb promoter, exon 1 and a part of exon 2, provided for kidney-specific expression of the reporter lacZ gene in transgenic mice [Zbikowska, Soukhareva, Behnam, Chang, Drews, Lubon, Hammond and Soukharev (2002) Transgenic Res., in the press]. In the present study, we generated transgenic mice harbouring the regulatory sequence of the uromodulin gene to direct the expression of human alpha1-antitrypsin (alpha1AT) into urine. Of the 13 founder mice that tested positive by PCR, seven showed the presence of the human protein in their urine. The concentration of the recombinant human (rh) alpha1AT in the urine, estimated by using ELISA, ranged from 0.5 to 14 microg/ml in the F(0)-generation mice, and reached up to 65 microg/ml in the F1 generation. The transgenically produced rh alpha1AT was found to be N-glycosylated and biologically active. The N-terminal sequence analysis confirmed the identity of the human protein and revealed that the recombinant alpha1AT was correctly processed with the signal peptide cleaved off. Our results demonstrate for the first time that the uromodulin regulatory sequence provides a very attractive option for the potential large-scale production of functional therapeutic proteins in livestock.

Amino Acid Sequence↗
PubMed2002

Panic termination and the post-panic period.

A comprehensive theory of panic must explain not only the cause(s) of episodes of panic but also why and when panic episodes terminate. Accordingly, we conducted a set of three studies on participants with panic disorder in order to investigate these aspects of panic episodes. In Study 1, we asked participants to monitor their panics prospectively, paying particular attention to the episodes' conclusions and the time period which followed. Results from Study 1 were consistent with earlier retrospective studies, showing that people engage in safety and other behaviors in an attempt to end their panics. We also collected information from the participants on beliefs about panic termination and the post-panic period. Study 2 was designed to determine if a post-panic refractory period occurs. Participants were asked to complete a hyperventilation exercise, and then to repeat the exercise a second time. Results from this study provide scant evidence of a post-panic refractory period. Study 3 was a more ecologically valid version of Study 2, in which participants were asked to re-trigger panics that occurred in their natural surroundings. Again, there was little support for a post-panic refractory period. Results are discussed in terms of cognitive-behavioral and biological theories of panic disorder.

Adult↗