Modulation of experimental autoimmunity: treatment of adjuvant arthritis by immunization with a recombinant vaccinia virus.

Live recombinant vaccinia viruses, expressing antigens from pathogenic microorganisms, are studied for their use as vaccines designed for the protection against infectious diseases. Infections with these vaccinia virus recombinants, expressing proteins or epitopes from viruses, parasites, or bacteria, have resulted in the development of specific neutralizing antibodies or cytotoxic T lymphocytes. Here, we describe the generation of a recombinant vaccinia virus expressing the mycobacterial 65-kDa heat shock protein (HSP65). A vaccinia recombinant virus was constructed by placing the gene for the Mycobacterium bovis BCG HSP65 under control of a vaccinia virus promoter and inserting this mycobacterial gene in the thymidine kinase locus of the vaccinia virus genome. Mycobacterial HSP65 is a critical antigen in the autoimmune model of adjuvant arthritis induced in Lewis rats by the immunization with Mycobacterium tuberculosis. We report the induction of immunity directed to this mycobacterial HSP65 by testing for the presence of specific antibodies and T-cell proliferation. Furthermore, induction of such immunity resulted in a reduction of arthritis severity when given to rats before or, even more interestingly, during development of arthritis. Disease reduction was not found after administration of HSP65 in the absence of vaccinia virus as a vector when given during arthritis development. Therefore, recombinant vaccinia virus may offer new prospectives for specific intervention in autoimmunity.     

Comparative genotyping of Campylobacter jejuni by amplified fragment length polymorphism,multilocus sequence typing,and short repeat sequencing: strain diversity,host range,and recombination

Three molecular typing methods were used to study the relationships among 184 Campylobacter strains isolated from humans, cattle, and chickens. All strains were genotyped by amplified fragment length polymorphism (AFLP) analysis, multilocus sequence typing (MLST), and sequence analysis of a genomic region with short tandem repeats designated clustered regularly interspaced short palindromic repeats (CRISPRs). MLST and AFLP analysis yielded more than 100 different profiles and patterns, respectively. These multiple-locus typing methods resulted in similar genetic clustering, indicating that both are useful in disclosing genetic relationships between Campylobacter jejuni isolates. Group separation analysis of the AFLP analysis and MLST data revealed an unexpected association between cattle and human strains, suggesting a common source of infection. Analysis of the polymorphic CRISPR region carrying short repeats allowed about two-thirds of the typeable strains to be distinguished, similar to AFLP analysis and MLST. The three methods proved to be equally powerful in identifying strains from outbreaks of human campylobacteriosis. Analysis of the MLST data showed that intra- and interspecies recombination occurs frequently and that the role of recombination in sequence variation is 50 times greater than that of mutation. Examination of strains cultured from cecum swabs revealed that individual chickens harbored multiple Campylobacter strain types and that some genotypes were found in more than one chicken. We conclude that typing of Campylobacter strains is useful for identification of outbreaks but is probably not useful for source tracing and global epidemiology because of carriage of strains of multiple types and an extremely high diversity of strains in animals.     

Evaluation of proliferation parameters in in vivo bromodeoxyuridine labelled lung cancers

In a series of 44 bronchial biopsies from patients suspected of having endobronchial lung carcinoma, the validity of proliferating cell nuclear antigen (PCNA) and Ki67 antigen as proliferative indicators was evaluated in ethanol fixed, paraffin embedded tissue. The percentages of cells positive for these markers were compared to the in vivo bromodeoxyuridine (BrdU) labelling index. A good correlation was found between PCNA immunoreactivity and BrdU labelling index, while Ki67-antigen expression showed a significant relation with BrdU labelling index and with PCNA expression. All three parameters showed a trend towards similar values for the individual cases. Based on the fact that Ki67 antigen is expressed in all cycling cells, whereas replicon-associated PCNA and BrdU only reflect the S-phase fraction, the differences between Ki67-antigen scores on the one hand and BrdU and PCNA scores on the other were smaller than expected. In order to determine the degree of concordance between immunohistochemically and flow cytometrically detected proliferation variables, BrdU incorporation was measured using both methods in duplicate bronchial specimens. Discrepancies in labelling indices were observed predominantly in DNA diploid samples, with consistently lower values in the flow cytometrically analysed specimens. In tumour specimens with an aneuploid DNA content, flow cytometric determination of proliferative activity yielded results similar to those obtained by tissue section examination. We conclude that the scores for PCNA and Ki67 antigen, immunohistochemically detected in ethanol fixed, paraffin embedded tissue reflect functional proliferative activity.     

K-ras oncogene activation as a prognostic marker in adenocarcinoma of the lung

BACKGROUND. The capability of activated oncogenes to induce malignant transformation of immortalized cells in vitro has suggested that they have a similar role in the pathogenesis of human tumors. We previously found that activation of the K-ras oncogene by a point mutation in codon 12 occurs in about one third of human lung adenocarcinomas. METHODS. We studied the clinical importance of this oncogene-activation in 69 patients with lung adenocarcinoma in whom complete resection of the tumor was possible. The polymerase chain reaction was used to amplify ras-specific sequences of DNA isolated from frozen or paraffin-embedded tumor samples. Ras point mutations were subsequently detected and classified with the use of mutation-specific oligonucleotide probes. RESULTS. Nineteen of the tumors harbored a point mutation in codon 12 of the K-ras oncogene. There was no association between the K-ras point mutation and the age at diagnosis, sex, or presence of previous or concurrent neoplasms. Tumors positive for K-ras point mutations tended to be smaller and less differentiated than those without mutations. The K-ras codon-12 point mutation was a strong (and unfavorable) prognostic factor: 12 of the 19 patients with K-ras point-mutation-positive tumors died during the follow-up period, as compared with 16 of the 50 patients with no mutation in the K-ras oncogene (P = 0.002). This difference in prognosis was also reflected in the duration of disease-free survival (P = 0.038) and in the number of deaths due to cancer (P less than 0.001). CONCLUSIONS. The presence of K-ras point mutations defines a subgroup of patients with lung adenocarcinoma in whom the prognosis is very poor and disease-free survival is not usually long despite radical resection and a small tumor load.     

Alcohol sales to pseudo-intoxicated bar patrons.

OBJECTIVES: Many establishments serve alcoholic beverages to obviously intoxicated patrons despite laws against such sales. To guide the development of interventions to reduce these illegal alcohol sales, this study used actors feigning intoxication to determine whether servers recognized obvious signs of intoxication and to assess the tactics servers used when dealing with intoxicated patrons. METHODS: Male actors ages 30 to 50 acted out signs of obvious intoxication as they attempted to purchase alcoholic beverages. If served during the first attempt, these pseudo-intoxicated buyers made second purchase attempts during the same visit. Observers accompanied the actors; after each visit, actors and observers recorded the servers' behavior and comments. RESULTS: Alcoholic beverages were served to actors portraying intoxicated patrons at 68% of first purchase attempts and 53% of second purchase attempts (62% of a total of 106 purchase attempts). The most common refusal technique was a direct refusal (68% of refusals), made with either no excuse or with reference to the actors' apparent intoxication level. Servers' second most commonly used refusal technique was offering alcohol-free beverages, such as coffee or water (18% of refusals). CONCLUSIONS: Further research is needed to determine why servers who recognize intoxication serve alcoholic beverages and what training, outlet policies, and external pressures are needed to reduce illegal alcohol sales to obviously intoxicated patrons.     

Role of blood-brain barrier P-glycoprotein in limiting brain accumulation and sedative side-effects of asimadoline, a peripherally acting analgaesic drug.

Studies with knockout mice lacking mdr1a P-glycoprotein (P-gp) have previously shown that blood-brain barrier P-gp is important in preventing the accumulation of several drugs in the brain. Asimadoline (EMD 61753) is a peripherally selective kappa-opioid receptor agonist which is under development as a therapeutic analgaesic. From the structural characteristics of this drug and its peripheral selectivity, we hypothesized that it is transported by P-gp. Using a pig-kidney polarized epithelial cell line transfected with mdr cDNAs, we demonstrate that asimadoline is transported by the mouse mdr1a P-gp and the human MDR1 P-gp. Furthermore, we show that in mdr1a/1b double knockout mice, the absence of P-gp leads to a 9 fold increased accumulation of asimadoline in the brain. In line with this accumulation difference, mdr1a/1b (-/-) mice are at least 8 fold more sensitive to the sedative effect of asimadoline than wild-type mice. Interestingly, the oral uptake of asimadoline was not substantially altered in mdr1a/1b (-/-) mice. Our results demonstrate that for some drugs, P-gp in the blood-brain barrier can have a therapeutically beneficial effect by limiting brain penetration, whereas at the same time intestinal P-gp is not a significant impediment to oral uptake of the drug.     

High Alcohol Concentration Products Associated With Poverty and State Alcohol Policies

Objectives. We examined the associations among zip code demographics, the state alcohol policy environment, and the retail outlet availability of multiple fruit-flavored alcoholic drinks in a can (MFAC).Methods. In a nationally representative sample of zip codes (n = 872), we merged data from 4 sources: publicly available marketing information from 2 major MFAC producers, the US Census Bureau, state alcohol regulatory agencies, and recent research on state alcohol policies. We used zero-inflated negative binomial regression models to examine MFAC outlet availability in the United States.Results. More than 98% of MFAC outlets were off-premises alcohol establishments. After we controlled for population size and the number of licensed on- and off-premises alcohol outlets within zip codes, more families below the poverty line and weaker state alcohol control policies were associated with greater MFAC outlet availability.Conclusions. Economic conditions and alcohol policy environment appeared to be related to MFAC outlet availability, after adjusting for the general availability of alcohol. Research is needed to determine whether MFACs are disproportionately contributing to alcohol-related harm in socially and economically disadvantaged communities. Policies to better regulate the off-premises sale of alcohol are needed.In 2003, the first premixed caffeinated alcohol product was introduced in the United States, and by 2010 at least 8 brands of caffeinated alcohol were being sold.1,2 Released in August 2008, Four Loko became the most popular of these ready-to-drink products among underage drinkers.3,4 Anecdotal news and scientific reports linked Four Loko consumption to a number of dangerous drinking episodes, and as a result, questions were raised about its safety.5–7In addition to gaining the attention of the attorney generals of several states,8 by 2010, an emerging body of research began to show that caffeine–alcohol co-ingestion could produce elevated intoxication levels and reduce perceptions of impairment, and that drinkers could engage in riskier behaviors compared with the consumption of alcohol alone.9–14 Based on this research, the Federal Trade Commission and the Food and Drug Administration concluded that the combined high alcohol and high caffeine content of these premixed products was likely causing consumer harm.1,15 In response to pressure from the government, producers of products such as Four Loko and Joose voluntarily ceased production of these caffeinated drinks in November 2010.3 However, shortly thereafter, these producers began distributing reformulated products that no longer contained caffeine, guarine, and taurine.3,16,17Although the presence of a significant amount of caffeine likely played a role in producing injury and death, other characteristics of these products also likely contributed to hazardous alcohol consumption.18 Namely, their exceptionally high alcohol content, low price, fruit flavoring, colorful packaging, and targeted marketing may be responsible for attracting underage and lower socioeconomic consumers, and may continue to contribute to high-risk drinking among these groups.18,19 The combination of these product features led to Four Loko, Joose, and other similar beverages being labeled as multiple fruit-flavored alcoholic drinks in a can (MFAC) or supersized alcopops. Although alcopops contain similar fruit flavoring, MFACs are packaged in large, single-serving cans rather than in multiunit packs, they have higher alcohol content, and they cost less per standard drink, distinguishing them from alcopops.18 MFAC products are typically high in alcohol concentration (12%) and combined with large-sized containers (23.5 fluid ounces) result in that a single container, which is typically consumed immediately as 1 drink, actually contains 5 standard drinks.18 Moreover, the typical price is only $2.50 to $3.00, representing one of the lowest costs per dose of alcohol.18,19 There is limited research on MFAC products. Drug Abuse Warning Network data20 indicated that, in 2010, when Four Loko was primarily sold as a caffeinated product, there were an estimated 1242 hospital emergency department visits in the United States by persons who had consumed Four Loko (95% confidence interval = 332, 2152).21 However, in 2011, the first year Four Loko was sold without caffeine, the estimated Four Loko–related emergency department visits increased by nearly 4.5 times, to a total of 5492 (95% confidence interval = 2925, 8059).21 This substantial increase in emergency department visits suggests that Four Loko continues to be a health threat, despite the removal of caffeine. However, Four Loko was the only MFAC brand reported by the Drug Abuse Warning Network. Furthermore, a national survey conducted in 2012 found that approximately 6% of underaged drinkers consumed a Four Loko in the past 30 days, whereas consumption of other MFACs (e.g., Blast, Sparks, and Tilt) was less common.4Even less is known about the marketing and retail availability of MFAC products. One important question is whether the potential harms associated with MFAC consumption are borne evenly by all segments of society. The targeted marketing of potentially harmful products to vulnerable populations (e.g., low-income minority communities and communities with high proportions of youths) raises ethical concerns about social justice and corporate social responsibility.22Our purpose in this study was to test 2 hypotheses about MFAC outlet availability in the United States. First, we expected to find that zip code areas in states with stronger alcohol control environments (i.e., more laws, regulations, and practices designed to reduce excessive alcohol use and related harm) would have less MFAC availability. We expected this association because the combined effects of multiple concurrent alcohol policies in an overall alcohol policy environment helps shape local norms regarding alcohol use. For example, harsh penalties for supplying alcohol to underage drinkers or using or accepting fake identification may influence local alcohol marketing practices (i.e., products presumed to be higher risk might not be as heavily supplied or demanded in such areas). Second, we anticipated that zip code areas with greater poverty and a larger concentration of racial/ethnic minority persons would have greater MFAC outlet availability. The relatively low price of MFACs might make the products particularly appealing to economically disadvantaged communities.18,19 This notion was supported by previous research on malt liquor, another low-price, high-alcohol content, ready-to-drink product that was heavily marketed to and had greater retail availability in low-income minority communities.23–25 However, to date, there has been no systematic inquiry into these concerns with regard to MFAC products.     

Dorsal Raphe Dopamine Neurons Signal Motivational Salience Dependent on Internal State,Expectation, and Behavioral Context

The ability to recognize motivationally salient events and adaptively respond to them is critical for survival. Here, we tested whether dopamine (DA) neurons in the dorsal raphe nucleus (DRN) contribute to this process in both male and female mice. Population recordings of DRN^DA neurons during associative learning tasks showed that their activity dynamically tracks the motivational salience, developing excitation to both reward-paired and shock-paired cues. The DRN^DA response to reward-predicting cues was diminished after satiety, suggesting modulation by internal states. DRN^DA activity was also greater for unexpected outcomes than for expected outcomes. Two-photon imaging of DRN^DA neurons demonstrated that the majority of individual neurons developed activation to reward-predicting cues and reward but not to shock-predicting cues, which was surprising and qualitatively distinct from the population results. Performing the same fear learning procedures in freely-moving and head-fixed groups revealed that head-fixation itself abolished the neural response to aversive cues, indicating its modulation by behavioral context. Overall, these results suggest that DRN^DA neurons encode motivational salience, dependent on internal and external factors.SIGNIFICANCE STATEMENT Dopamine (DA) contributes to motivational control, composed of at least two functional cell types, one signaling for motivational value and another for motivational salience. Here, we demonstrate that DA neurons in the dorsal raphe nucleus (DRN) encode the motivational salience in associative learning tasks. Neural responses were dynamic and modulated by the animal''s internal state. The majority of single-cells developed responses to reward or paired cues, but not to shock-predicting cues. Additional experiments with freely-moving and head-fixed mice showed that head-fixation abolished the development of cue responses during fear learning. This work provides further characterization on the functional roles of overlooked DRN^DA populations and an example that neural responses can be altered by head-fixation, which is commonly used in neuroscience.