The introduction of two transgenes into one animal is increasingly common as transgenic experiments become more sophisticated. In this study we examine two strategies for creating double transgenic founders from a single microinjection. In the first approach, two constructs, each with its own promoter element, were coinjected into the pronucleus. In the second approach, both transgenes were cloned into one vector, separated by an internal ribosomal entry site (IRES), and placed under control of a single promoter. Both strategies save time and increase the percentage of double transgenic offspring over the standard method of mating single transgenic lines. However, despite high transgene copy numbers, the bicistronic lines did not show robust expression of either protein. Copy number and protein expression correlated much better in the coinjected lines, with expression levels in one line approaching that observed in some of our best single transgenic controls. Thus we recommend coinjection of individual plasmids for the generation of multiply transgenic founders.

Long interspersed nuclear elements (LINEs or L1s) comprise approximately 17% of human DNA; however, only about 60 of the approximately 400,000 L1s are mobile. Using a retrotransposition assay in cultured human cells, we demonstrate that L1-encoded proteins predominantly mobilize the RNA that encodes them. At much lower levels, L1-encoded proteins can act in trans to promote retrotransposition of mutant L1s and other cellular mRNAs, creating processed pseudogenes. Mutant L1 RNAs are mobilized at 0.2 to 0.9% of the retrotransposition frequency of wild-type L1s, whereas cellular RNAs are mobilized at much lower frequencies (ca. 0.01 to 0.05% of wild-type levels). Thus, we conclude that L1-encoded proteins demonstrate a profound cis preference for their encoding RNA. This mechanism could enable L1 to remain retrotransposition competent in the presence of the overwhelming number of nonfunctional L1s present in human DNA.

We reviewed the existing empirical literature to assess cognitive and situational factors that may affect the validity of adolescents' self-reports of alcohol and other drug use, tobacco use, behaviors related to unintentional injuries and violence, dietary behaviors, physical activity, and sexual behavior. Specifically, we searched for peer-reviewed journal articles published in 1980 or later that examined the factors affecting self-report of the six categories of behavior listed above. We also searched for studies describing objective measures for each behavior. Self-reports of each of six types of health-risk behaviors are affected by both cognitive and situational factors. These factors, however, do not threaten the validity of self-reports of each type of behavior equally. The importance of assessing health-risk behaviors as part of research activities involving adolescents necessitates the use of self-report measures. Researchers should familiarize themselves with the threats to validity inherent in this type of assessment and design research that minimizes these threats as much as possible.

Cytomegalovirus establishes a lifelong latent infection following primary infection that can periodically reactivate with shedding of infectious virus. Primary infection, reactivation and reinfection during pregnancy can all lead to in utero transmission to the developing fetus. Congenital CMV infections are a major cause of permanent hearing loss and neurological impairment. In this literature review, we found that CMV infection was relatively common among women of reproductive age, with seroprevalence ranging from 45 to 100%. CMV seroprevalence tended to be highest in South America, Africa and Asia and lowest in Western Europe and United States. Within the United States, CMV seroprevalence showed substantial geographic variation as well, differing by as much as 30 percentage points between states, though differences might be explained by variation in the types of populations sampled. Worldwide, seroprevalence among non-whites tended to be 20-30 percentage points higher than that of whites (summary prevalence ratio (PR) = 1.59, 95% confidence interval (CI) = 1.57-1.61). Females generally had higher seroprevalences than males, although in most studies the differences were small (summary PR = 1.13, 95% CI = 1.11-1.14). Persons of lower socioeconomic status were more likely to be CMV seropositive (summary PR = 1.33, 95% CI = 1.32-1.35). Despite high seroprevalences in some populations, a substantial percentage of women of reproductive age are CMV seronegative and thus at risk of primary CMV infection during pregnancy. Future vaccine or educational campaigns to prevent primary infection in pregnant women may need to be tailored to suit the needs of different populations.

Much medical research is observational. The reporting of observational studies is often of insufficient quality. Poor reporting hampers the assessment of the strengths and weaknesses of a study and the generalizability of its results. Taking into account empirical evidence and theoretical considerations, a group of methodologists, researchers, and editors developed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) recommendations to improve the quality of reporting of observational studies. The STROBE Statement consists of a checklist of 22 items, which relate to the title, abstract, introduction, methods, results and discussion sections of articles. Eighteen items are common to cohort studies, case-control studies and cross-sectional studies and four are specific to each of the three study designs. The STROBE Statement provides guidance to authors about how to improve the reporting of observational studies and facilitates critical appraisal and interpretation of studies by reviewers, journal editors and readers.This explanatory and elaboration document is intended to enhance the use, understanding, and dissemination of the STROBE Statement. The meaning and rationale for each checklist item are presented. For each item, one or several published examples and, where possible, references to relevant empirical studies and methodological literature are provided. Examples of useful flow diagrams are also included. The STROBE Statement, this document, and the associated web site (http://www.strobe-statement.org) should be helpful resources to improve reporting of observational research.

1. Previous work has shown that the sodium efflux from the axons of Loligo forbesi increases when external sodium is replaced by lithium.2. The increase in efflux in lithium was unaffected by ouabain but was abolished by removal of external calcium; in these respects it differed from the potassium-dependent sodium efflux which was abolished by ouabain but not reduced by removal of external calcium.3. Strontium but not magnesium could replace calcium in activating the ouabain-insensitive sodium efflux; lanthanum had an inhibitory effect.4. Replacing all the external NaCl by choline chloride or dextrose gave a rise in Na efflux which was abolished by ouabain but not by removal of external calcium.5. The rise in Na efflux resulting from partial replacement of NaCl by dextrose or choline chloride consisted of two components one of which was ouabain-insensitive and calcium-dependent and the other was inhibited by ouabain but calcium-insensitive.6. The ouabain-insensitive component of the Na efflux was activated by low concentrations of Na, Li or K but inhibited by high concentrations of Na and to a lesser extent Li. The inhibiting effect of high Na was of the kind expected if these ions displace calcium from an external site.7. The ouabain-insensitive component of the Na efflux was abolished by cyanide, had a Q(10) of 2.7; and was roughly proportional to [Na](i) (2). It was much more variable in magnitude than the ouabain-sensitive, potassium-dependent component of the sodium efflux.8. The calcium influx increased five to fortyfold when external NaCl was replaced by LiCl or dextrose, the increase for Li being larger than the increase for dextrose.9. The calcium influx from Na, Li or dextrose sea water was increased three to tenfold by increasing the internal Na about fourfold.10. The experiments provide evidence for a coupling between an inward movement of calcium and an outward movement of sodium.

A major interest in human genetics is to determine whether a nonsynonymous single-base nucleotide polymorphism (nsSNP) in a gene affects its protein product and, consequently, impacts the carrier's health. We used the SIFT (Sorting Intolerant From Tolerant) program to predict that 25% of 3084 nsSNPs from dbSNP, a public SNP database, would affect protein function. Some of the nsSNPs predicted to affect function were variants known to be associated with disease. Others were artifacts of SNP discovery. Two reports have indicated that there are thousands of damaging nsSNPs in an individual's human genome; we find the number is likely to be much lower.

The discovery that some cases of familial amyotrophic lateral sclerosis (FALS) are associated with mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) has focused much attention on the function of SOD1 as related to motor neuron survival. Here we describe the creation and characterization of mice completely deficient for this enzyme. These animals develop normally and show no overt motor deficits by 6 months in age. Histological examination of the spinal cord reveals no signs of pathology in animals 4 months in age. However Cu/Zn SOD-deficient mice exhibit marked vulnerability to motor neuron loss after axonal injury. These results indicate that Cu/Zn SOD is not necessary for normal motor neuron development and function but is required under physiologically stressful conditions following injury.

The Drosophila eye has contributed much to our knowledge of cell differentiation. This understanding has primarily come from the study of the R7 photoreceptor; much less is known about the development of the other classes of photoreceptor or the nonneuronal cone or pigment cells. I have used a dominant-negative form of the Drosophila epidermal growth factor receptor (DER) to show that this receptor tyrosine kinase (RTK) is required for the differentiation of all these cell types, and I have also shown that DER is sufficient to trigger their development. DER is even required in R7, where it can replace Sevenless, another RTK. These results broaden our view of eye development to include the whole ommatidium and suggest that reiterative activation of DER is critical for triggering the differentiation of all cell types.

Polyphenols constitute one of the most numerous and ubiquitous groups of plant metabolites and are an integral part of both human and animal diets. Ranging from simple phenolic molecules to highly polymerized compounds with molecular weights of greater than 30,000 Da, the occurrence of this complex group of substances in plant foods is extremely variable. Polyphenols traditionally have been considered antinutrients by animal nutritionists, because of the adverse effect of tannins, one type of polyphenol, on protein digestibility. However, recent interest in food phenolics has increased greatly, owing to their antioxidant capacity (free radical scavenging and metal chelating activities) and their possible beneficial implications in human health, such as in the treatment and prevention of cancer, cardiovascular disease, and other pathologies. Much of the literature refers to a single group of plant phenolics, the flavonoids. This review offers an overview of the nutritional effects of the main groups of polyphenolic compounds, including their metabolism, effects on nutrient bioavailability, and antioxidant activity, as well as a brief description of the chemistry of polyphenols and their occurrence in plant foods.

Three lines of evidence are presented that low density lipoproteins gently extracted from human and rabbit atherosclerotic lesions (lesion LDL) greatly resembles LDL that has been oxidatively modified in vitro. First, lesion LDL showed many of the physical and chemical properties of oxidized LDL, properties that differ from those of plasma LDL: higher electrophoretic mobility, a higher density, higher free cholesterol content, and a higher proportion of sphingomyelin and lysophosphatidylcholine in the phospholipid fraction. A number of lower molecular weight fragments of apo B were found in lesion LDL, similar to in vitro oxidized LDL. Second, both the intact apo B and some of the apo B fragments of lesion LDL reacted in Western blots with antisera that recognize malondialdehyde-conjugated lysine and 4-hydroxynonenal lysine adducts, both of which are found in oxidized LDL; plasma LDL and LDL from normal human intima showed no such reactivity. Third, lesion LDL shared biological properties with oxidized LDL: compared with plasma LDL, lesion LDL produced much greater stimulation of cholesterol esterification and was degraded more rapidly by macrophages. Degradation of radiolabeled lesion LDL was competitively inhibited by unlabeled lesion LDL, by LDL oxidized with copper, by polyinosinic acid and by malondialdehyde-LDL, but not by native LDL, indicating uptake by the scavenger receptor(s). Finally, lesion LDL (but not normal intimal LDL or plasma LDL) was chemotactic for monocytes, as is oxidized LDL. These studies provide strong evidence that atherosclerotic lesions, both in man and in rabbit, contain oxidatively modified LDL.

Inhibitors of VEGF signaling can block angiogenesis and reduce tumor vascularity, but little is known about the reversibility of these changes after treatment ends. In the present study, regrowth of blood vessels in spontaneous RIP-Tag2 tumors and implanted Lewis lung carcinomas in mice was assessed after inhibition of VEGF receptor signaling by AG-013736 or AG-028262 for 7 days. Both agents caused loss of 50%-60% of tumor vasculature. Empty sleeves of basement membrane were left behind. Pericytes also survived but had less alpha-SMA immunoreactivity. One day after drug withdrawal, endothelial sprouts grew into empty sleeves of basement membrane. Vessel patency and connection to the bloodstream followed close behind. By 7 days, tumors were fully revascularized, and the pericyte phenotype returned to baseline. Importantly, the regrown vasculature regressed as much during a second treatment as it did in the first. Inhibition of MMPs or targeting of type IV collagen cryptic sites by antibody HUIV26 did not eliminate the sleeves or slow revascularization. These results suggest that empty sleeves of basement membrane and accompanying pericytes provide a scaffold for rapid revascularization of tumors after removal of anti-VEGF therapy and highlight their importance as potential targets in cancer therapy.

BACKGROUND

Mortality from all causes is higher for persons with fewer years of education and for blacks, but it is unknown which diseases contribute most to these disparities.

METHODS

We estimated cause-specific risks of death from data from the National Health Interview Survey conducted from 1986 through 1994 and from linked vital statistics. Using these risk estimates, we calculated potential years of life lost and potential gains in life expectancy related to specific causes, with stratification according to education level and race.

RESULTS

Persons without a high-school education lost 12.8 potential life-years per person in the population, as compared with 3.6 for persons who graduated from high school (ratio, 3.5; P<0.001). Ischemic heart disease contributed most (11.7 percent) to the difference according to education in potential life-years lost (with all cardiovascular diseases accounting for 35.3 percent). All cancers accounted for 26.5 percent, including 7.7 percent due to lung cancer; other lung diseases and pneumonia contributed 10.1 percent of the total, whereas human immunodeficiency virus (HIV) disease accounted for none of the difference according to education. The pattern of disparities according to level of income was similar to that according to level of education. Blacks and whites lost 7.0 and 5.2 potential life-years per person, respectively, as a result of deaths from any cause (ratio, 1.35; P<0.001). Cardiovascular diseases accounted for one third of this disparity, in large part because of hypertension (15.0 percent); HIV disease (11.2 percent) contributed almost as much as ischemic heart disease (5.5 percent), stroke (2.8 percent), and cancer (3.4 percent) combined; trauma and diabetes mellitus accounted for 10.7 percent and 8.5 percent, respectively.

CONCLUSIONS

Although many conditions contribute to socioeconomic and racial disparities in potential life-years lost, a few conditions account for most of these disparities - smoking-related diseases in the case of mortality among persons with fewer years of education, and hypertension, HIV, diabetes mellitus, and trauma in the case of mortality among black persons. These findings have important implications for targeting efforts to reduce existing disparities in mortality rates.

V(D)J recombination is the specialized DNA rearrangement used by cells of the immune system to assemble immunoglobulin and T-cell receptor genes from the preexisting gene segments. Because there is a large choice of segments to join, this process accounts for much of the diversity of the immune response. Recombination is initiated by the lymphoid-specific RAG1 and RAG2 proteins, which cooperate to make double-strand breaks at specific recognition sequences (recombination signal sequences, RSSs). The neighboring coding DNA is converted to a hairpin during breakage. Broken ends are then processed and joined with the help of several factors also involved in repair of radiation-damaged DNA, including the DNA-dependent protein kinase (DNA-PK) and the Ku, Artemis, DNA ligase IV, and Xrcc4 proteins, and possibly histone H2AX and the Mre11/Rad50/Nbs1 complex. There may be other factors not yet known. V(D)J recombination is strongly regulated by limiting access to RSS sites within chromatin, so that particular sites are available only in certain cell types and developmental stages. The roles of enhancers, histone acetylation, and chromatin remodeling factors in controlling accessibility are discussed. The RAG proteins are also capable of transposing RSS-ended fragments into new DNA sites. This transposition helps to explain the mechanism of RAG action and supports earlier proposals that V(D)J recombination evolved from an ancient mobile DNA element.

Protein C is best known for its mild deficiency associated with venous thrombosis risk and severe deficiency associated with neonatal purpura fulminans. Activated protein C (APC) anticoagulant activity involves proteolytic inactivation of factors Va and VIIIa, and APC resistance is often caused by factor V Leiden. Less known is the clinical success of APC in reducing mortality in severe sepsis patients (PROWESS trial) that gave impetus to new directions for basic and preclinical research on APC. This review summarizes insights gleaned from recent in vitro and in vivo studies of the direct cytoprotective effects of APC that include beneficial alterations in gene expression profiles, anti-inflammatory actions, antiapoptotic activities, and stabilization of endothelial barriers. APC's cytoprotection requires its receptor, endothelial cell protein C receptor, and protease-activated receptor-1. Because of its pleiotropic activities, APC has potential roles in the treatment of complex disorders, including sepsis, thrombosis, and ischemic stroke. Although much about molecular mechanisms for APC's effects on cells remains unclear, it is clear that APC's structural features mediating anticoagulant actions and related bleeding risks are distinct from those mediating cytoprotective actions, suggesting the possibility of developing APC variants with an improved profile for the ratio of cytoprotective to anticoagulant actions.

BACKGROUND

Epigenome-wide association studies of human disease and other quantitative traits are becoming increasingly common. A series of papers reporting age-related changes in DNA methylation profiles in peripheral blood have already been published. However, blood is a heterogeneous collection of different cell types, each with a very different DNA methylation profile.

RESULTS

Using a statistical method that permits estimating the relative proportion of cell types from DNA methylation profiles, we examine data from five previously published studies, and find strong evidence of cell composition change across age in blood. We also demonstrate that, in these studies, cellular composition explains much of the observed variability in DNA methylation. Furthermore, we find high levels of confounding between age-related variability and cellular composition at the CpG level.

CONCLUSIONS

Our findings underscore the importance of considering cell composition variability in epigenetic studies based on whole blood and other heterogeneous tissue sources. We also provide software for estimating and exploring this composition confounding for the Illumina 450k microarray.

A clear-cut serological differentiation between AKR lymphocytes of thymic and non-thymic origin is reported: these two cell types are antigenically distinct. In newborn mice, the AKR thymic antigen was found at a high concentration only in thymus. In adult mice, the antigen was present at a high level in thymus, all nervous tissues tested, and some leukemias. It was present at much lower levels in lymph node lymphocytes, splenic lymphocytes, appendix, lung, and certain other leukemias, which appeared to be of non-thymic origin. The AKR thymic antigen was present at a high level in thymus and nervous tissues of RF mice, but was absent from thymocytes of sixteen other mouse strains. These sixteen strains possessed the C3HeB/Fe thymic antigen. The distribution of this antigen in neonatal and adult tissues of the strains tested was similar to that of the AKR thymic antigen in AKR mice. No exceptions were found. These results were obtained by use of immune cytolysis, and of a new method for the quantitative treatment of data from absorption experiments.

BACKGROUND

The aim of nicotine replacement therapy (NRT) is temporarily to replace much of the nicotine from cigarettes to reduce motivation to smoke and nicotine withdrawal symptoms, thus easing the transition from cigarette smoking to complete abstinence.

OBJECTIVE

The aims of this review were:To determine the effect of NRT compared to placebo in aiding smoking cessation, and to consider whether there is a difference in effect for the different forms of NRT (chewing gum, transdermal patches, nasal spray, inhalers and tablets/lozenges) in achieving abstinence from cigarettes. To determine whether the effect is influenced by the dosage, form and timing of use of NRT; the intensity of additional advice and support offered to the smoker; or the clinical setting in which the smoker is recruited and treated. To determine whether combinations of NRT are more likely to lead to successful quitting than one type alone. To determine whether NRT is more or less likely to lead to successful quitting compared to other pharmacotherapies.

METHODS

We searched the Cochrane Tobacco Addiction Group trials register for papers with 'nicotine' or 'NRT' in the title, abstract or keywords. Date of most recent search July 2007.

METHODS

Randomized trials in which NRT was compared to placebo or to no treatment, or where different doses of NRT were compared. We excluded trials which did not report cessation rates, and those with follow up of less than six months.

METHODS

We extracted data in duplicate on the type of participants, the dose, duration and form of nicotine therapy, the outcome measures, method of randomization, and completeness of follow up. The main outcome measure was abstinence from smoking after at least six months of follow up. We used the most rigorous definition of abstinence for each trial, and biochemically validated rates if available. We calculated the risk ratio (RR) for each study. Where appropriate, we performed meta-analysis using a Mantel-Haenszel fixed-effect model.

RESULTS

We identified 132 trials; 111 with over 40,000 participants contributed to the primary comparison between any type of NRT and a placebo or non-NRT control group. The RR of abstinence for any form of NRT relative to control was 1.58 (95% confidence interval [CI]: 1.50 to 1.66). The pooled RR for each type were 1.43 (95% CI: 1.33 to 1.53, 53 trials) for nicotine gum; 1.66 (95% CI: 1.53 to 1.81, 41 trials) for nicotine patch; 1.90 (95% CI: 1.36 to 2.67, 4 trials) for nicotine inhaler; 2.00 (95% CI: 1.63 to 2.45, 6 trials) for oral tablets/lozenges; and 2.02 (95% CI: 1.49 to 3.73, 4 trials) for nicotine nasal spray. The effects were largely independent of the duration of therapy, the intensity of additional support provided or the setting in which the NRT was offered. The effect was similar in a small group of studies that aimed to assess use of NRT obtained without a prescription. In highly dependent smokers there was a significant benefit of 4 mg gum compared with 2 mg gum, but weaker evidence of a benefit from higher doses of patch. There was evidence that combining a nicotine patch with a rapid delivery form of NRT was more effective than a single type of NRT. Only one study directly compared NRT to another pharmacotherapy. In this study quit rates with nicotine patch were lower than with the antidepressant bupropion.

CONCLUSIONS

All of the commercially available forms of NRT (gum, transdermal patch, nasal spray, inhaler and sublingual tablets/lozenges) can help people who make a quit attempt to increase their chances of successfully stopping smoking. NRTs increase the rate of quitting by 50-70%, regardless of setting. The effectiveness of NRT appears to be largely independent of the intensity of additional support provided to the individual. Provision of more intense levels of support, although beneficial in facilitating the likelihood of quitting, is not essential to the success of NRT.

Ghrelin is a novel growth hormone-releasing peptide, originally identified in the rat stomach as the endogenous ligand for the growth hormone secretagogue-receptor (GHS-R1a). Ghrelin is involved in the regulation of GH release, but it has recently been suggested that ghrelin may have other actions, including effects on appetite, carbohydrate metabolism, heart, kidney, pancreas, gonads, and cell proliferation. The distribution of ghrelin, its functional receptor (type 1a) and the unspliced, non-functional GHS-R type 1b mRNA expression was investigated in various human tissues using classical and real-time reverse transcription and polymerase chain reaction. GHS-R1a was predominantly expressed in the pituitary and at much lower levels in the thyroid gland, pancreas, spleen, myocardium and adrenal gland. In contrast, ghrelin was found in the stomach, other parts of the gut and, indeed, in all the tissues studied (adrenal gland, atrium, breast, buccal mucosa, esophagus, Fallopian tube, fat tissue, gall bladder, human lymphocytes, ileum, kidney, left colon, liver, lung, lymph node, muscle, muscle, myocardium, ovary, pancreas, pituitary, placenta, prostate, right colon, skin, spleen, testis, thyroid, and vein). GHS-R1b expression was also widespread in all tissues studied. The significance of the widespread tissue distribution of ghrelin remains to be determined. These data suggest that ghrelin might have widespread physiological effects via different, partly unidentified, subtypes of the GHS-R in endocrine and non-endocrine tissues.

Advances in imaging are transforming our understanding of angiogenesis and the evaluation of drugs that stimulate or inhibit angiogenesis in preclinical models and human disease. Vascular imaging makes it possible to quantify the number and spacing of blood vessels, measure blood flow and vascular permeability, and analyze cellular and molecular abnormalities in blood vessel walls. Microscopic methods ranging from fluorescence, confocal and multiphoton microscopy to electron microscopic imaging are particularly useful for elucidating structural and functional abnormalities of angiogenic blood vessels. Magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), ultrasonography and optical imaging provide noninvasive, functionally relevant images of angiogenesis in animals and humans. An ongoing dilemma is, however, that microscopic methods provide their highest resolution on preserved tissue specimens, whereas clinical methods give images of living tissues deep within the body but at much lower resolution and specificity and generally cannot resolve vessels of the microcirculation. Future challenges include developing new imaging methods that can bridge this resolution gap and specifically identify angiogenic vessels. Another goal is to determine which microscopic techniques are the best benchmarks for interpreting clinical images. The importance of angiogenesis in cancer, chronic inflammatory diseases, age-related macular degeneration and reversal of ischemic heart and limb disease provides incentive for meeting these challenges.

BACKGROUND

Bacteria can mutate to acquire quinolone resistance by target alterations or diminished drug accumulation. Plasmid-mediated resistance to quinolones in clinical isolates has been claimed but not confirmed. We investigated whether a multiresistance plasmid could transfer resistance to quinolones between bacteria.

METHODS

We transferred resistance between strains by conjugation. The resistance plasmid was visualised in different hosts by agarose-gel electrophoresis. We determined the frequency of spontaneous mutations to ciprofloxacin or nalidixic-acid resistance in Escherichia coli strains, with or without the quinolone resistance plasmid.

RESULTS

A multiresistance plasmid (pMG252) from a clinical isolate of Klebsiella pneumoniae was found to increase quinolone resistance to minimum inhibitory concentrations (MICs) as high as 32 microg/mL for ciprofloxacin when transferred to strains of K pneumoniae deficient in outer-membrane porins. Much lower resistance was seen when pMG252 was introduced into K pneumoniae or E coli strains with normal porins. The plasmid had a wide host range and expressed quinolone resistance in other enterobacteriaceae and in Pseudomonas aeruginosa. From a plasmid-containing E coli strain with ciprofloxacin MIC of 0.25 microg/mL and nalidixic-acid MIC of 32 microg/mL, quinolone-resistant mutants could be obtained at more than 100 times the frequency of a plasmid-free strain, reaching MICs for ciprofloxacin of 4 microg/mL and for nalidixic acid of 256 microg/mL.

CONCLUSIONS

Transferable resistance to fluoroquinines and nalidixic acid has been found in a clinical isolate of K pneumoniae on a broad host range plasmid. Although resistance was low in wild-type strains, higher levels of quinolone resistance arose readily by mutation. Such a plasmid can speed the development and spread of resistance to these valuable antimicrobial agents.

CD4(+) T helper (T(H)) cells have crucial roles in orchestrating adaptive immune responses. T(H)2 cells control immunity to extracellular parasites and all forms of allergic inflammatory responses. Although we understand the initiation of the T(H)2-type response in tissue culture in great detail, much less is known about T(H)2 cell induction in vivo. Here we discuss the involvement of allergen- and parasite product-mediated activation of epithelial cells, basophils and dendritic cells and the functions of the cytokines interleukin-4 (IL-4), IL-25, IL-33 and thymic stromal lymphopoietin in the initiation and amplification of T(H)2-type immune responses in vivo.

We have developed a novel DNA expression system, based on the Semliki Forest virus (SFV) replicon, which combines a wide choice of animal cell hosts, high efficiency and ease of use. DNA of interest is cloned into SFV plasmid vectors that serve as templates for in vitro synthesis of recombinant RNA. The RNA is transfected with virtually 100% efficiency into animal tissue culture cells by means of electroporation. Within the cell, the recombinant RNA drives its own replication and capping and leads to massive production of the heterologous protein while competing out the host protein synthesis. The expression system also includes an in vivo packaging procedure whereby recombinant RNA is packaged into infectious virus particles using cotransfection with packaging-deficient helper RNA molecules. The resulting high titer recombinant virus stock can be used to infect a wide range of animal cells with subsequent high expression of the heterologous gene product, but without expression of any structural proteins of the helper. The infected cells produce protein for up to 75 hours post infection after which the heterologous product can constitute as much as 25% of the total cell protein. The general utility of the system is demonstrated through the expression of human transferrin receptor, mouse dihydrofolate reductase, chick lysozyme and Escherichia coli beta-galactosidase.

Cross-presentation of cell-associated antigens plays an important role in regulating CD8+ T cell responses to proteins that are not expressed by antigen-presenting cells (APCs). Dendritic cells are the principal cross-presenting APCs in vivo and much progress has been made in elucidating the pathways that allow dendritic cells to capture and process cellular material. However, little is known about the signals that determine whether such presentation ultimately results in a cytotoxic T cell (CTL) response (cross-priming) or in CD8+ T cell inactivation (cross-tolerance). Here we describe a mechanism that promotes cross-priming during viral infections. We show that murine CD8alpha+ dendritic cells are activated by double-stranded (ds)RNA present in virally infected cells but absent from uninfected cells. Dendritic cell activation requires phagocytosis of infected material, followed by signalling through the dsRNA receptor, toll-like receptor 3 (TLR3). Immunization with virus-infected cells or cells containing synthetic dsRNA leads to a striking increase in CTL cross-priming against cell-associated antigens, which is largely dependent on TLR3 expression by antigen-presenting cells. Thus, TLR3 may have evolved to permit cross-priming of CTLs against viruses that do not directly infect dendritic cells.