Coexistent effect of clay minerals on transfer frequency of plasmid DNA to fecal coliforms isolated from ground water

The influence of coexisting clay minerals on transfer frequencies of the self-transmissible wide-host-range plasmid R68 and the wide-host-range cloning vector R1162 mobilizing by the plasmid R68 were examined for 42 strains of fecal coliforms which were screened from ground water for their ability to express rifampicin-resistant characteristics. Broth-mating procedures revealed that 14 isolates possessed an ability to receive the plasmid R68 and 8 of them possessed an ability to mobilize the vector R1162 by the plasmid R68. Transfer frequencies of the plasmid R68 and the vector R1162 by R68 mobilization were accelerated significantly with increase in the coexisting of kaoline, montmorillonite-Na, bentonite I-Na and bentonite II-Ca in the broth-mating condition. However, mobilization of the vector R1162 reached steady state by the addition of more than 1.0 mg/ml of the reagents. In the 8 isolates identified as recipient active for the vector R1162 by R68 mobilization, bentonite II-Ca, montmorillonite-Na, bentonite I-Na and kaoline had mobilization effects in that order under similar 1.0 mg/ml broth-mating conditions. It became clear that the highly effective transfer frequency of vector R1162 by R68 mobilization using bentonite II-Ca in an aqueous condition was superior to that using a solid surface plate-mating procedure.     

Safety and immunogenicity of a live-attenuated auxotrophic candidate vaccine against the intracellular pathogen Rhodococcus equi

Rhodococcus equi causes serious pneumonia in neonatal foals and is an opportunistic pathogen of people with compromised cellular immunity. No effective vaccine against R. equi disease in foals is available. We tested the safety and immunogenicity of a live, fully attenuated riboflavin auxotrophic candidate vaccine strain of R. equi (R. equi rib-). We demonstrated that R. equi rib- is immunogenic and capable of inducing IFN-gamma responses in immunocompetent BALB/c mice, yet it is safe even in an immunocompromised SCID mouse infection model. Moreover, it protects immunocompetent mice against virulent R. equi challenge. In foals, R. equi rib- was likewise safe and stimulated serum R. equi-specific immune responses. A preliminary immunization strategy did not afford protection against virulent R. equi challenge and therefore, optimization of the vaccine formulation and or vaccination protocol will be necessary.     

Glucose-insulin relationships and thyroid status of cockerels selected for high or low residual food consumption

The plasma glucose-insulin relationships and thyroid status were investigated in two lines of adult cockerels divergently selected for high (R+) or low (R-) residual food consumption (RFC). For a given body weight, R+ birds had a 74% higher food intake than R- birds. Plasma glucose concentrations were significantly lower in the R+ line compared with the R- when fasted, whereas R+ birds exhibited a significantly lower plasma insulin concentration than R- birds either in fed or fasted state. After an overnight fast, R+ birds also exhibited a higher sensitivity to exogenous insulin in view of its more pronounced hypoglycaemic effect. After an oral glucose load, the glucose disposal of R+ cockerels was faster despite lower glucose-induced plasma insulin concentration. Whilst plasma triacylglycerol concentrations were lower in the R+ line when fed, plasma non-esterified fatty acid concentrations were higher in fasted R+ than R- cockerels (684 v. 522 mumol/l). Higher plasma triiodothyronine concentrations were observed in fed R+ compared with R- birds (3.0 v. 2.1 nmol/l respectively). The higher plasma concentrations of triiodothyronine associated with lower concentrations of insulin could account for the leanness and the elevated diet-induced thermogenesis previously observed in the R+ line.     

Epigallocatechin-3-gallate inhibits expression of receptors for T cell regulatory cytokines and their downstream signaling in mouse CD4+ T cells

We previously showed a suppressive effect of epigallocatechin-3-gallate (EGCG) on T cell cycling and expansion as well as a paradoxical effect on IL-2 levels (upregulating) and IL-2 receptor (IL-2R)α expression (downregulating). Thus, in the current study, we tested the hypothesis that EGCG affects T cell responses via impairing the IL-2/IL-2R signaling. We found that EGCG inhibited anti-CD3/CD28-induced proliferation of na?ve CD4(+) T cells from C57BL/6 mice. EGCG increased accumulation of IL-2 but inhibited expression of IL-2R, including all its subunits [IL-2Rα, IL-2/IL-15Rβ, and common γ chain (γc)]. Using phosphorylation of STAT5 as a marker, we further found that EGCG suppressed IL-2R downstream signaling. Because IL-2R subunits IL-2/IL-15Rβ- and γc are shared with IL-15R and γc is shared with IL-7R, we suspected that EGCG might also influence the signaling of IL-15 and IL-7, the two key regulators in maintaining T cell homeostasis. Results showed that EGCG suppressed IL-15 and IL-7 signaling; further, EGCG not only inhibited the subunits in IL-15R and IL-7R shared with IL-2R, but also affected their proprietary α chains in a manner that aligns with an impaired signaling. Although IL-2, IL-15, and IL-7 have separate and distinctive roles in regulating T cells, all of them are critical for T cell survival, expansion, and differentiation. Thus, these findings indicate an involvement of T cell growth cytokines in EGCG-induced T cell suppression through downregulated expression of their receptors and downstream signaling. This implies a potential application in controlling dysregulated T cell functions such as those observed in autoimmune and inflammatory disorders.     

Estimating transmission parameters of F4+ E. coli for F4-receptor-positive and -negative piglets: one-to-one transmission experiment

F4+ Escherichia coli is an important agent of post-weaning diarrhoea in piglets. Piglets that express an adhesion site for F4+ E. coli in their small intestine (F4R+) shed higher numbers of F4+ E. coli than piglets lacking this site (F4R-). We hypothesized that F4R+ piglets are more infectious and more susceptible for F4+ E. coli. This implies that in populations with F4R+ and F4R- piglets, the transmission would be dependent on the frequency of both types of animals. To quantify the difference in infectiousness and susceptibility, a one-to-one transmission experiment was performed with 20 pairs consisting of one inoculated and one contact piglet. Based on the contact infections observed, transmission parameters were estimated with generalized linear models. F4R+ piglets were infectious for other piglets and the reproduction ratio (R0) for homogeneous F4R+ populations, that is the average number of secondary infections that one F4R+ pig will cause during its entire infectious period in a population of susceptible F4R+ individuals only, was estimated as 7.1. F4R+ piglets were more susceptible than F4R- piglets and reducing the fraction of F4R+ piglets of a population will reduce transmission. It was calculated that in order to prevent major outbreaks of F4+ E. coli (R0 < 1), the fraction of F4R+ piglets must be lower than 0.14.     

The relationship between local availability and first-time use of specialists in an arthritis population

OBJECTIVE: To determine what health area characteristics, in particular local availability of specialists, were associated with the age-sex adjusted ambulatory utilization rate of musculoskeletal (MSK) specialists (i.e., rheumatologists, orthopaedic surgeons and general internists) in an arthritis and rheumatism (A&R) cohort. METHODS: The cohort was composed of respondents aged 15+ from the 1996/97 Ontario Health Survey who self-reported A&R and/or had a primary care encounter for A&R during the two years preceding the survey, as determined by their billings in the Ontario Health Insurance Plan. Respondents with prior exposure to MSK specialists were excluded. The outcome of an encounter with a MSK specialist for A&R was determined during the three-year period after the survey. RESULTS: The A&R cohort was composed of 5,052 respondents, of whom 11% had an A&R encounter with a MSK specialist in the three-year post-survey period. There was area variation in the age-sex adjusted ambulatory utilization rate of MSK specialists in the A&R cohort. The backwards stepping linear regression to examine predictors for seeing MSK specialists found a positive association with local availability of rheumatologists, a negative association with the proportion of high school graduates in the health area and a negative association with the proportion of people aged 65 years and older. DISCUSSION: At the health area level, we found that the local availability of rheumatologists was an important factor associated with utilizing MSK specialists for A&R-related conditions in a cohort of respondents who have not been previously exposed to MSK specialists for musculoskeletal-related conditions.     

Recruitment and retention of health professionals across Europe: A literature review and multiple case study research

Many European countries are faced with health workforce shortages and the need to develop effective recruitment and retention (R&R) strategies. Yet comparative studies on R&R in Europe are scarce. This paper provides an overview of the measures in place to improve the R&R of health professionals across Europe and offers further insight into the evidence base for R&R; the interaction between policy and organisational levels in driving R&R outcomes; the facilitators and barriers throughout these process; and good practices in the R&R of health professionals across Europe. The study adopted a multi-method approach combining an extensive literature review and multiple-case study research. 64 publications were included in the review and 34 R&R interventions from 20 European countries were included in the multiple-case study. We found a consistent lack of evidence about the effectiveness of R&R interventions. Most interventions are not explicitly part of a coherent package of measures but they tend to involve multiple actors from policy and organisational levels, sometimes in complex configurations. A list of good practices for R&R interventions was identified, including context-sensitivity when implementing and transferring interventions to different organisations and countries. While single R&R interventions on their own have little impact, bundles of interventions are more effective. Interventions backed by political and executive commitment benefit from a strong support base and involvement of relevant stakeholders.     

Targeting the vaccinia virus L1 protein to the cell surface enhances production of neutralizing antibodies

The current live-orthopoxvirus vaccine is associated with minor to serious adverse affects, and is contraindicated for use in a significant portion of the population. As an alternative vaccine, we have previously shown that a DNA subunit vaccine (4pox) based on four orthopoxvirus immunogens (L1R, B5R, A27L and A33R) can produce protective immunity against lethal orthopoxvirus challenges in mice and nonhuman primates. Because antibodies are critical for protection against secondary orthopoxvirus infections, we are now interested in strategies that will enhance the humoral immune response against vaccine targets. Here, we tested the immunogenicity of an L1R construct to which a tissue plasminogen activator signal sequence was placed in frame with the full-length L1R gene. The tPA-L1R construct produced a more robust neutralizing antibody response in vaccinated mice when the DNA vaccine was administered by gene-gun as a prime/single boost. When the tPA-L1R construct was substituted for the unmodified L1R gene in the 4pox vaccine, given as a prime and single boost, animals were better protected from lethal challenge with vaccinia virus (VACV). These findings indicate that adding a tPA-leader sequence can enhance the immunogenicity of the L1R gene when given as a DNA vaccine. Furthermore, our results demonstrate that a DNA-based vaccine is capable of establishing protection from lethal orthopoxvirus challenges when administered as a prime and single boost without requiring adjuvant.     

Flow cytometric differential gating strategy for evaluation of T-lymphocyte survival and proliferation in mixed lymphocyte reaction with dendritic cells

Mixed lymphocyte reaction (MLC) of T-lymphocytes with dendritic cells is one of the basic tools for studying of immune reaction mechanisms. This work describes a new method of evaluation of allogeneic and autologous MLC by flow cytometry and differential gating. This method is based on fixed time acquisition of events and their sorting according to their forward and side scatter properties. Differential gating distinguishes populations of all living cells (R1), living non-proliferating cells (R2) and dead/apoptotic cells (R4). Addition of fluorescence microspheres (R3) enables the calculation of absolute number of cells in a given sample volume. Using the method of 7-AAD positive cells project into the exclusion, we have shown that approximately 90% of 7-AAD R4 gate. Moreover, there was an excellent correlation between the R1: R2 ratio and the percentage of cells positive for activation/proliferation marker CD71. The method of differential gating in its basic version does not require use of any fluorescent antibody and therefore is suitable for rapid screening of large number of samples at reasonable cost and without the use of radiolabeled nucleotides.     

Change of drug resistance patterns and genetic properties of R plasmids in Salmonella typhimurium of bovine origin isolated from 1970 to 1979 in northern Japan.

A total of 321 Salmonella typhimurium strains of bovine origin obtained in northern Japan during the period 1970-1979 were tested for drug resistance and detection of conjugative R plasmids. Three hundred and eighteen (99.1%) of these strains were resistant to one or more drugs. The isolation frequently of multiply drug-resistant strains tended to increase year by year. Two hundred and thirty-seven (74.5%) of these resistant strains carried conjugative R plasmids. A total of 308 R plasmids including 174 (56.5%) thermosensitive (ts) R plasmids were derived from the 237 drug-resistant strains, indicating that 71 (30.0%) strains have two different conjugative R plasmids in a single host cell. Of the 308 R plasmids examined for fertility inhibition (fi), 167 ts and 131 non-ts R plasmids were fi-. Of the 60 ts r plasmids examined for incompatibility, 50 were classified into H1 group and 10 into H2 group. Of the 52 non-ts R plasmids examined, 35 were classified into the I alpha-group and the remaining plasmids were untypable in our tests. Mercury resistance marker was found in about 20% of H1 R plasmids coding for multiresistance, and all of H2 R plasmids coded for resistance to tellurite. The clonal distribution of an S. typhimurium strain which carried an H1 R plasmid coding for resistance to six drugs and mercury was recognized in 1978 and 1979.     

Stimulation of tetanus toxoid-specific immune responses by a traditional Chinese herbal medicine

The immunomodulatory properties of botanical medicinals are well-documented. In this study, the capacity for the traditional Chinese herbal medicine, Rehmannia Six Formula (R6F), to stimulate anti-tetanus toxoid (TT) immunity following oral administration to mice was examined. A significant rise in serum anti-TT antibody levels were observed in R6F-treated mice immunized with a minimum immunogenic dose of 10 μg TT suggesting an oral adjuvant effect. No such enhancement was found for unimmunized mice treated with R6F. This anti-TT response was preferentially IgG and antigen-specific in relation to antibody reactivity to a panel of unrelated antigens. The R6F used was safe with no adverse effects on mouse weight or survival, providing evidence for the use of R6F as an oral adjuvant.     

Antibodies as pharmacologic tools for studies on the regulation of energy balance

OBJECTIVE: Active immunization in rats may serve several purposes: the production of a disease-like phenotype, the generation of pharmacologic tools, and the development of clinically useful therapies. We selected the melanocortin-4 receptor (MC4R) as a target because its blockade could provide a treatment for anorexia and cachexia. METHODS: We used a sequence of the N-terminal (NT) domain of the MC4R as an antigen. Rats immunized against the NT peptide produced specific MC4R antibodies (Abs) that were purified and characterized in vitro and in vivo. RESULTS: The Abs acted as inverse agonists and reduced under basal conditions the production of cyclic adenosine monophosphate in HEK-293 cells expressing the human MC4R. Rats immunized against the NT peptide developed a phenotype consistent with hypothalamic MC4R blockade, i.e., increased food intake and body weight, liver and fat-pad weights, hepatic steatosis, and increased plasma triacylglycerols. With a high-fat diet, plasma insulin levels were significantly increased. In separate experiments an increase in food intake was observed after injection of purified MC4R Abs into the third ventricle. When lipopolysaccharide was administered in NT-immunized rats the reduction of food intake was partly prevented in this model of cytokine-induced anorexia. CONCLUSION: Our results show that active immunization of rats against the MC4R resulted in the generation of specific Abs that stimulated food intake by acting as inverse agonists of the hypothalamic MC4R. Pharmacologically active monoclonal MC4R Abs could be the starting point for the development of novel treatments for patients with anorexia or cachexia.     

Differential efficacy of vaccinia virus envelope proteins administered by DNA immunisation in protection of BALB/c mice from a lethal intranasal poxvirus challenge

DNA vaccines might offer an alternative to the live smallpox vaccine in providing protective efficacy in an orthopoxvirus (OPV) lethal respiratory challenge model. BALB/c mice were immunised with DNA vaccines coding for 10 different single vaccinia virus (VACV) membrane proteins. After an intranasal challenge with the VACV IHD strain, three gene candidates B5R, A33R and A27L produced > or =66% survival. The B5R DNA vaccine consistently produced 100% protection and exhibited greatest efficacy after three 50 microg intramuscular doses in this model. Sero-conversion to these vaccines was often inconsistent, implying that antibody itself was not a correlate of protection. The B5R DNA vaccine induced a strong and consistent gamma interferon (IFNgamma) response in BALB/c mice given a single DNA vaccine dose. Strong IFNgamma responses were also measured in pTB5R immunised C57BL6 mice deficient for MHC class I molecules, suggesting that the memory response was mediated by a CD4+ T cell population.     

Synthesis, anti-GABA activity and preferred conformation of bicuculline and norbicuculline enantiomers

Synthesis of erythro-(±)-[1SR,9RS]-norbicuculline and threo-(±)-[1SR,9SR]-noradlumidine from piperonal was performed using Bischler-Napieralski cyclization as a key step. Resolution gave rise to (+)-[1S,9R]-norbicuculline ([1S,9R] norBIC) and (-)-[1R,9S]-norbicuculline ([1R,9S] norBIC) in >99.5% enantiomeric purity. Bicuculline enantiomers were readily obtained by methylation of the latter products. [1S,9R]BIC was about 70 times more potent than [1R,9S] BIC as an inhbitor of GABA(A) receptor binding and was about 100 and 900 times more potent than [1S,9R] norBIC at pH 7.1 and 5.0 respectively. Similarly, [1S,9R] norBIC was much less potent than [1S,9R] BIC as an inhibitor of GABA-specific (36)Cl(-) ion flux. The observed increase of about two orders of magnitude in the in vitro biological activity caused by N2-CH(3) substitution in [1S,9R] norBIC was attributed to different conformations for erythro- and nor-erythro-bicucullines indicated by (1)H nuclear Overhauser enhancements of [1S,9R] BIC and [1S,9R] norBIC.     

An R2 statistic for fixed effects in the linear mixed model

Statisticians most often use the linear mixed model to analyze Gaussian longitudinal data. The value and familiarity of the R(2) statistic in the linear univariate model naturally creates great interest in extending it to the linear mixed model. We define and describe how to compute a model R(2) statistic for the linear mixed model by using only a single model. The proposed R(2) statistic measures multivariate association between the repeated outcomes and the fixed effects in the linear mixed model. The R(2) statistic arises as a 1-1 function of an appropriate F statistic for testing all fixed effects (except typically the intercept) in a full model. The statistic compares the full model with a null model with all fixed effects deleted (except typically the intercept) while retaining exactly the same covariance structure. Furthermore, the R(2) statistic leads immediately to a natural definition of a partial R(2) statistic. A mixed model in which ethnicity gives a very small p-value as a longitudinal predictor of blood pressure (BP) compellingly illustrates the value of the statistic. In sharp contrast to the extreme p-value, a very small R(2) , a measure of statistical and scientific importance, indicates that ethnicity has an almost negligible association with the repeated BP outcomes for the study.     

The pseudorabies virus R2 non-neuroinvasive vaccine: A proof-of-concept study in pigs

Neurotropic alpha-herpesviruses that infect mammals establish life-long latent infections in the peripheral nervous system after initial infection of exposed mucosal tissues. The neuroinvasive properties can lead to severe complications both with clinical and veterinary alpha-herpesviruses, and vaccines are often unavailable or provide limited protection. Here we assess the properties and efficacy of an R2 vaccine derived from the alpha-herpesvirus, pseudorabies virus (PRV), in pigs. We demonstrate that the PRV R2 vaccine does not invade the porcine peripheral nervous system within the limits of detection. Furthermore, after a single intranasal vaccination, R2 conferred protection to pigs subsequently challenged with a virulent PRV field strain (NIA-3). These findings support that the R2 vaccine design is non-neuroinvasive and is an effective vaccine in the context of a natural host.     

Resveratrol and quercetin interact to inhibit neointimal hyperplasia in mice with a carotid injury

Restenosis is a critical complication of angioplasty and stenting. Restenosis is multifactorial, involving endothelial injury, inflammation, platelet activation, and vascular smooth muscle cell (VSMC) proliferation. Thus, dietary strategies to prevent restenosis likely require the use of more than one agent. Resveratrol (R) and quercetin (Q) are polyphenols that are known to exhibit vascular protective effects. We tested whether R and Q administered in the diet interact to inhibit vessel stenosis in mice with a carotid injury. B6.129 mice were administered a high-fat diet containing 21% fat and 0.2% cholesterol along with R (25 mg/kg), Q (10 mg/kg), or R + Q for 2 wk. A carotid injury was induced and the mice were again administered the enriched diet for 2 wk. Compared with the controls, R significantly decreased stenosis, assessed as an intima:media ratio, by 76%. Although Q treatment alone exhibited no effect, it potentiated the effect of R in that treatment with R + Q significantly decreased the intima:media ratio by 94%. Moreover, this effect was greater than that of R treatment alone (P < 0.05). Although treatments with R, Q, and R + Q significantly affected platelet activation and endothelial function, the responses observed for R + Q were less than additive. Specifically, the effects of R + Q were less than the sum of effects for treatments with R and Q alone. In contrast, treatment with R + Q exhibited more-than-additive effects on inflammatory markers and significant interactions between R and Q were observed. The presence of synergy between R and Q was thus tested in cultures of VSMC and macrophages. Isobolographic analysis revealed that 2:1 molar ratios of R:Q exhibited synergistic inhibition of VSMC proliferation and macrophage chemotaxis. In conclusion, in combination, R and Q can interact to reduce the extent of restenosis, perhaps due to their synergistic inhibition of VSMC proliferation and inflammation.     

Synthesis, inhibitory activity towards human leukocyte elastase and molecular modelling studies of 1-carbamoyl-4-methyleneaminoxyazetidinones

Some monocyclic beta-lactam derivatives of type 3 (MAOAs) in which the leaving group (LG) on the C(4) is a methyleneaminoxy moiety, were synthesised and tested in vitro and in vivo for their inhibitory activity towards human leukocyte elastase (HLE). Some compounds showed an appreciable in vitro inhibitory activity against this enzyme. Effects on the anti-HLE activity due to the nature of the substituents R and R(1) present on their LG were observed and rationalised by means of molecular modelling techniques. The results of in vivo pharmacological tests indicated that MAOAs, while showing an inhibitory activity on the haemorrhage induced by HLE, did not exhibit any effects due to the R and R(1) substituents.     

Serosurvey of Rickettsia spp. in dogs and humans from an endemic area for Brazilian spotted fever in the State of São Paulo, Brazil

The present study provides a rickettsial serosurvey in 25 dogs and 35 humans in an endemic area for Brazilian spotted fever in the State of S?o Paulo, where the tick Amblyomma aureolatum is the main vector. Testing canine and human sera by indirect immunofluorescence against four Rickettsia antigens (R. rickettsii, R. parkeri, R. felis and R. bellii) showed that 16 (64%) of canine sera and 1 (2.8%) of human sera reacted to at least one of these rickettsial antigens with titers >0r= 64. Seven canine sera and the single reactive human serum showed titers to R. rickettsii at least four times those of any of the other three antigens. The antibody titers in these 7 animals and 1 human were attributed to stimulation by R. rickettsii infection. No positive canine or human serum was attributed to stimulation by R. parkeri, R. felis, or R. bellii. Our serological results showed that dogs are important sentinels for the presence of R. rickettsii in areas where the tick A. aureolatum is the main vector of Brazilian spotted fever.