A Vibrio cholerae ghost-based subunit vaccine induces cross-protective chlamydial immunity that is enhanced by CTA2B, the nontoxic derivative of cholera toxin

The Vibrio cholerae ghost (rVCG) platform is an effective carrier and delivery system for designing efficacious Chlamydia vaccines. We investigated whether CTA2B, the nontoxic derivative of cholera toxin, can augment protective immunity conferred by an rVCG-based chlamydial vaccine and enhance cross-protection against heterologous chlamydial strains. An rVCG vaccine coexpressing chlamydial major outer membrane protein and CTA2B was genetically constructed and antigens were targeted to the inner membrane of V. cholerae before ghost production by gene E -mediated lysis. Effective immunomodulation by CTA2B was demonstrated by the ability of the vaccine construct to enhance the activation and maturation of dendritic cells in vitro . Also, C57BL/6 mice immunized via mucosal and systemic routes showed increased specific mucosal and systemic antibody and T-helper type-1 (Th1) responses, irrespective of the route. The enhanced production of IFN-γ, but not IL-4 by genital mucosal and splenic T cells, indicated a predominantly Th1 response. Clearance of the Chlamydia muridarum vaginal infection was significantly enhanced by codelivery of the vaccine with CTA2B, with the intravaginal route showing a moderate advantage. These results indicate that the rVCG-based vaccine is capable of inducing cross-protection against heterologous chlamydial serovars and that incorporation of mucosal adjuvants, such as CTA2B in the rVCG delivery platform, may enhance protective immunity.     

Biomarkers: in combination they may do better

The field of biomarkers is a growing one, particularly in osteoarthritis (OA). OA is the most common disabling condition in older persons and a major cause of morbidity. While the debate continues about which of the involved tissues - cartilage, bone or synovium - is the most important in OA aetiology, there is no doubt that the three develop abnormalities in concert; perhaps a truly useful biomarker will reflect just that. While efforts continue to identify reliable biomarkers useful for characterising the status, prognosis and measurement of treatment response in OA, combining existing biomarkers to improve their accuracy looks promising.     

Standardizing clinical trials workflow representation in UML for international site comparison

Background

With the globalization of clinical trials, a growing emphasis has been placed on the standardization of the workflow in order to ensure the reproducibility and reliability of the overall trial. Despite the importance of workflow evaluation, to our knowledge no previous studies have attempted to adapt existing modeling languages to standardize the representation of clinical trials. Unified Modeling Language (UML) is a computational language that can be used to model operational workflow, and a UML profile can be developed to standardize UML models within a given domain. This paper''s objective is to develop a UML profile to extend the UML Activity Diagram schema into the clinical trials domain, defining a standard representation for clinical trial workflow diagrams in UML.

Methods

Two Brazilian clinical trial sites in rheumatology and oncology were examined to model their workflow and collect time-motion data. UML modeling was conducted in Eclipse, and a UML profile was developed to incorporate information used in discrete event simulation software.

Results

Ethnographic observation revealed bottlenecks in workflow: these included tasks requiring full commitment of CRCs, transferring notes from paper to computers, deviations from standard operating procedures, and conflicts between different IT systems. Time-motion analysis revealed that nurses'' activities took up the most time in the workflow and contained a high frequency of shorter duration activities. Administrative assistants performed more activities near the beginning and end of the workflow. Overall, clinical trial tasks had a greater frequency than clinic routines or other general activities.

Conclusions

This paper describes a method for modeling clinical trial workflow in UML and standardizing these workflow diagrams through a UML profile. In the increasingly global environment of clinical trials, the standardization of workflow modeling is a necessary precursor to conducting a comparative analysis of international clinical trials workflows.     

Micropropagation of Withania somnifera from germinating seeds and shoot tips

Shoot multiplication was achieved in vitro from shoot tips of aseptically germinated seedlings of Withania somnifera L. using low concentrations of 6-benzyladenine (BA), viz. 2.2, 4.4 and 8.9 M. Maximum number of shoots were obtained when 2.3 M 2,4-dichlorophenoxyacetic acid (2,4-D) or 2.5 M indolebutyric acid (IBA) was added to medium containing 4.4 M BA during initiation of shoot multiplication, but not when added later. Direct multiple shoot initiation was also obtained from germinating seeds in the presence of BA alone. Rooting was successful in excised shoots grown on growth regulator-free MS medium. Rooted shoots were successfully established in soil in a greenhouse.     

Vitamin D status and determinants of deficiency in non-supplemented athletes during the winter months in Tunisia

Recent reports suggest that hypovitaminosis D in athletes is as common as in the general population. This study was devised to examine vitamin D status and determinants of deficiency in athletes living in a sunny country (Tunisia). One hundred and fifty national elite athletes, training outdoors (n = 83) or indoors (n = 67), were enrolled from January to February 2012. Plasma 25-hydroxyvitamin D was measured by radioimmunoassay. Concentrations were between 50 and 75 nmol · l^-1 in 21.3% of participants, between 25 and 50 nmol · l^-1 in 55.3% of participants and <25 nmol · l^-1 in 14.7% of participants. The concentrations were significantly lower in indoor athletes than outdoor athletes (36.2±19.0 nmol · l^-1 vs. 49.1±19.2 nmol · l^-1; p < 0.001). In multivariate analysis, vitamin D deficiency (25-hydroxyvitamin D <50 nmol · l^-1) was associated with indoor sports [multi-adjusted odds ratio (95% confidence interval), 5.03 (1.64-15.4); p = 0.005], female gender [3.72 (1.44-9.65); p = 0.007] and age < 18 years [2.40 (1.01-5.85); p = 0.05]. Athletes living in sun-rich environments are exposed to a high risk of vitamin D inadequacy. Given the importance of vitamin D in health and athletic ability, targeting sufficient levels of plasma 25-hydroxyvitamin D in athletes is well justified.     

Methionine-oxidized horse heart cytochromes c. II. Conformation and heme configuration

The two products from the reaction of horse heart ferricytochrome c with Chloramine-T, the F^III and F^II CT-cytochromes, contain modification of the methionines to methionine sulfoxides, but they are distinct in their physiological functions. Conformational and heme-configurational characterization of the two CT-cytochromes has been carried out by using absorption, circular dichroism, fluorescence, proton magnetic resonance, and resonance Raman spectroscopy. The pH-absorption spectroscopic behavior, thermal stability, and ionization of the phenolic hydroxyls have also been reported. Spectroscopic studies of the heme c fragment, H8, in the presence of dimethylsulfoxide, as a model for CT-cytochrome heme configuration, were also conducted. The ferric and the ferrous CT-cytochromes above pH 7.5 have similar, yet distinct, spectroscopic properties, absorption, CD, resonance Raman, and PMR spectra, typical of low-spin hexacoordinated hemes, but distinct from those of the unmodified protein. The ferric spectrum lacks the 695-nm band, and the reduced spectrum contains an additional inflection at about 400 nm, a feature also observed in the spectra of ferrous H8-DMSO systems. The CD, resonance Raman, and PMR spectra are typical of a cytochrome with a loosened heme crevice and altered coordination configuration. The Methionine-80 proton resonances are absent in the uupfield PMR spectra of both the CT-ferricytochromes. The ferrous spectra, on the other hand, contain all the Met-80 resonances, but with smaller upfield shifts than those of the native protein. Both CT-ferric cytochromes are less stable in the acid region and convert to high-spin forms with a two-step transition and with a distinct set of pK _a values. The overall conformation is nearly identical to that of the native protein, but it is less stable to thermal unfolding. All the factors differentiating the modified preparations from the unmodified protein are more pronunced in the case of F^II, with F^III being the closest to the unmodified form. The two functionally distinct CT-cytochromes are two conformational isomers; conformationally and heme configurationally, they are spectroscopically very similar, yet distinct. Both contain an altered heme iron coordination configuration. The sulfur of Met-80 is repalced by the oxygen of Met-80 sulfoxide of a different configuration, R or S. Both contain a loosened heme crevice and are conformationally less stable than the native protein, F^II CT-cytochrome c being the most deranged.     

Multiplex quantitative PCR for detection of lower respiratory tract infection and meningitis caused by <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis>, <Emphasis Type="Italic">Haemophilus influenzae</Emphasis> and <Emphasis Type="Italic">Neisseria meningitidis</Emphasis>

Background  

Streptococcus pneumoniae and Haemophilus influenzae cause pneumonia and as Neisseria meningitidis they are important agents of meningitis. Although several PCR methods have been described for these bacteria the specificity is an underestimated problem. Here we present a quantitative multiplex real-time PCR (qmPCR) for detection of S. pneumoniae (9802 gene fragment), H. influenzae (omp P6 gene) and N. meningitidis (ctrA gene). The method was evaluated on bronchoalveolar lavage (BAL) samples from 156 adults with lower respiratory tract infection (LRTI) and 31 controls, and on 87 cerebrospinal fluid (CSF) samples from meningitis patients.