Identification of Immunogenic Salmonella enterica Serotype Typhi Antigens Expressed in Chronic Biliary Carriers of S. Typhi in Kathmandu,Nepal

Background

Salmonella enterica serotype Typhi can colonize and persist in the biliary tract of infected individuals, resulting in a state of asymptomatic chronic carriage. Chronic carriers may act as persistent reservoirs of infection within a community and may introduce infection to susceptible individuals and new communities. Little is known about the interaction between the host and pathogen in the biliary tract of chronic carriers, and there is currently no reliable diagnostic assay to identify asymptomatic S. Typhi carriage.

Methodology/Principal Findings

To study host-pathogen interactions in the biliary tract during S. Typhi carriage, we applied an immunoscreening technique called in vivo-induced antigen technology (IVIAT), to identify potential biomarkers unique to carriers. IVIAT identifies humorally immunogenic bacterial antigens expressed uniquely in the in vivo environment, and we hypothesized that S. Typhi surviving in the biliary tract of humans may express a distinct antigenic profile. Thirteen S. Typhi antigens that were immunoreactive in carriers, but not in healthy individuals from a typhoid endemic area, were identified. The identified antigens included a number of putative membrane proteins, lipoproteins, and hemolysin-related proteins. YncE (STY1479), an uncharacterized protein with an ATP-binding motif, gave prominent responses in our screen. The response to YncE in patients whose biliary tract contained S. Typhi was compared to responses in patients whose biliary tract did not contain S. Typhi, patients with acute typhoid fever, and healthy controls residing in a typhoid endemic area. Seven of 10 (70%) chronic carriers, 0 of 8 bile culture-negative controls (0%), 0 of 8 healthy Bangladeshis (0%), and 1 of 8 (12.5%) Bangladeshis with acute typhoid fever had detectable anti-YncE IgG in blood. IgA responses were also present.

Conclusions/Significance

Further evaluation of YncE and other antigens identified by IVIAT could lead to the development of improved diagnostic assays to identify asymptomatic S. Typhi carriers.     

Genetic monogamy despite variable ecological conditions and social environment in the cooperatively breeding apostlebird

Mating strategies may be context‐dependent and may vary across ecological and social contexts, demonstrating the role of these factors in driving the variation in genetic polyandry within and among species. Here, we took a longitudinal approach across 5 years (2006–2010), to study the apostlebird (Struthidea cinerea), an Australian cooperatively breeding bird, whose reproduction is affected by ecological “boom and bust” cycles. Climatic variation drives variation in the social (i.e., group sizes, proportion of males and females) and ecological (i.e., plant and insect abundance) context in which mating occurs. By quantifying variation in both social and ecological factors and characterizing the genetic mating system across multiple years using a molecular parentage analysis, we found that the genetic mating strategy did not vary among years despite significant variation in rainfall, driving primary production, and insect abundance, and corresponding variation in social parameters such as breeding group size. Group sizes in 2010, an ecologically good year, were significantly smaller (mean = 5.8 ± 0.9, n = 16) than in the drought affected years, between 2006 and 2008, (mean = 9.1 ± 0.5, n = 63). Overall, apostlebirds were consistently monogamous with few cases of multiple maternity or paternity (8 of 78 nests) across all years.     

Contrasting mitochondrial diversity of European starlings (Sturnus vulgaris) across three invasive continental distributions

European starlings (Sturnus vulgaris) represent one of the most widespread and problematic avian invasive species in the world. Understanding their unique population history and current population dynamics can contribute to conservation efforts and clarify evolutionary processes over short timescales. European starlings were introduced to Central Park, New York in 1890, and from a founding group of about 100 birds, they have expanded across North America with a current population of approximately 200 million. There were also multiple introductions in Australia in the mid‐19th century and at least one introduction in South Africa in the late 19th century. Independent introductions on these three continents provide a robust system to investigate invasion genetics. In this study, we compare mitochondrial diversity in European starlings from North America, Australia, and South Africa, and a portion of the native range in the United Kingdom. Of the three invasive ranges, the North American population shows the highest haplotype diversity and evidence of both sudden demographic and spatial expansion. Comparatively, the Australian population shows the lowest haplotype diversity, but also shows evidence for sudden demographic and spatial expansion. South Africa is intermediate to the other invasive populations in genetic diversity but does not show evidence of demographic expansion. In previous studies, population genetic structure was found in Australia, but not in South Africa. Here we find no evidence of population structure in North America. Although all invasive populations share haplotypes with the native range, only one haplotype is shared between invasive populations. This suggests these three invasive populations represent independent subsamples of the native range. The structure of the haplotype network implies that the native‐range sampling does not comprehensively characterize the genetic diversity there. This study represents the most geographically widespread analysis of European starling population genetics to date.     

Integrative Advances for OCT-Guided Ophthalmic Surgery and Intraoperative OCT: Microscope Integration,Surgical Instrumentation,and Heads-Up Display Surgeon Feedback

Purpose

To demonstrate key integrative advances in microscope-integrated intraoperative optical coherence tomography (iOCT) technology that will facilitate adoption and utilization during ophthalmic surgery.

Methods

We developed a second-generation prototype microscope-integrated iOCT system that interfaces directly with a standard ophthalmic surgical microscope. Novel features for improved design and functionality included improved profile and ergonomics, as well as a tunable lens system for optimized image quality and heads-up display (HUD) system for surgeon feedback. Novel material testing was performed for potential suitability for OCT-compatible instrumentation based on light scattering and transmission characteristics. Prototype surgical instruments were developed based on material testing and tested using the microscope-integrated iOCT system. Several surgical maneuvers were performed and imaged, and surgical motion visualization was evaluated with a unique scanning and image processing protocol.

Results

High-resolution images were successfully obtained with the microscope-integrated iOCT system with HUD feedback. Six semi-transparent materials were characterized to determine their attenuation coefficients and scatter density with an 830 nm OCT light source. Based on these optical properties, polycarbonate was selected as a material substrate for prototype instrument construction. A surgical pick, retinal forceps, and corneal needle were constructed with semi-transparent materials. Excellent visualization of both the underlying tissues and surgical instrument were achieved on OCT cross-section. Using model eyes, various surgical maneuvers were visualized, including membrane peeling, vessel manipulation, cannulation of the subretinal space, subretinal intraocular foreign body removal, and corneal penetration.

Conclusions

Significant iterative improvements in integrative technology related to iOCT and ophthalmic surgery are demonstrated.     

Large-scale ex vivo expansion and characterization of natural killer cells for clinical applications

Abstract Background aims. Interest in natural killer (NK) cell-based immunotherapy has resurged since new protocols for the purification and expansion of large numbers of clinical-grade cells have become available. Methods. We have successfully adapted a previously described NK expansion method that uses K562 cells expressing interleukin (IL)-15 and 4-1 BB Ligand (BBL) (K562-mb15-41BBL) to grow NK cells in novel gas-permeable static cell culture flasks (G-Rex). Results. Using this system we produced up to 19 × 10(9) functional NK cells from unseparated apheresis products, starting with 15 × 10(7) CD3(-) CD56 (+) NK cells, within 8-10 days of culture. The G-Rex yielded a higher fold expansion of NK cells than conventional gas-permeable bags and required no cell manipulation or feeding during the culture period. We also showed that K562-mb15-41BBL cells up-regulated surface HLA class I antigen expression upon stimulation with the supernatants from NK cultures and stimulated alloreactive CD8 (+) T cells within the NK cultures. However, these CD3 (+) T cells could be removed successfully using the CliniMACS system. We describe our optimized NK cell cryopreservation method and show that the NK cells are viable and functional even after 12 months of cryopreservation. Conclusions. We have successfully developed a static culture protocol for large-scale expansion of NK cells in the gas permeable G-Rex system under good manufacturing practice (GMP) conditions. This strategy is currently being used to produce NK cells for cancer immunotherapy.     

Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina)

Trichoderma reesei is the main industrial source of cellulases and hemicellulases used to depolymerize biomass to simple sugars that are converted to chemical intermediates and biofuels, such as ethanol. We assembled 89 scaffolds (sets of ordered and oriented contigs) to generate 34 Mbp of nearly contiguous T. reesei genome sequence comprising 9,129 predicted gene models. Unexpectedly, considering the industrial utility and effectiveness of the carbohydrate-active enzymes of T. reesei, its genome encodes fewer cellulases and hemicellulases than any other sequenced fungus able to hydrolyze plant cell wall polysaccharides. Many T. reesei genes encoding carbohydrate-active enzymes are distributed nonrandomly in clusters that lie between regions of synteny with other Sordariomycetes. Numerous genes encoding biosynthetic pathways for secondary metabolites may promote survival of T. reesei in its competitive soil habitat, but genome analysis provided little mechanistic insight into its extraordinary capacity for protein secretion. Our analysis, coupled with the genome sequence data, provides a roadmap for constructing enhanced T. reesei strains for industrial applications such as biofuel production.     

Application of in vivo induced antigen technology (IVIAT) to Bacillus anthracis

In vivo induced antigen technology (IVIAT) is an immuno-screening technique that identifies bacterial antigens expressed during infection and not during standard in vitro culturing conditions. We applied IVIAT to Bacillus anthracis and identified PagA, seven members of a N-acetylmuramoyl-L-alanine amidase autolysin family, three P60 family lipoproteins, two transporters, spore cortex lytic protein SleB, a penicillin binding protein, a putative prophage holin, respiratory nitrate reductase NarG, and three proteins of unknown function. Using quantitative real-time PCR comparing RNA isolated from in vitro cultured B. anthracis to RNA isolated from BALB/c mice infected with virulent Ames strain B. anthracis, we confirmed induced expression in vivo for a subset of B. anthracis genes identified by IVIAT, including L-alanine amidases BA3767, BA4073, and amiA (pXO2-42); the bacteriophage holin gene BA4074; and pagA (pXO1-110). The exogenous addition of two purified putative autolysins identified by IVIAT, N-acetylmuramoyl-L-alanine amidases BA0485 and BA2446, to vegetative B. anthracis cell suspensions induced a species-specific change in bacterial morphology and reduction in viable bacterial cells. Many of the proteins identified in our screen are predicted to affect peptidoglycan re-modeling, and our results support significant cell wall structural remodeling activity during B. anthracis infection. Identification of L-alanine amidases with B. anthracis specificity may suggest new potential therapeutic targets.     

Overestimates of survival after HAART: implications for global scale-up efforts

Background

Monitoring the effectiveness of global antiretroviral therapy scale-up efforts in resource-limited settings is a global health priority, but is complicated by high rates of losses to follow-up after treatment initiation. Determining definitive outcomes of these lost patients, and the effects of losses to follow-up on estimates of survival and risk factors for death after HAART, are key to monitoring the effectiveness of global HAART scale-up efforts.

Methodology/Principal Findings

A cohort study comparing clinical outcomes and risk factors for death after HAART initiation as reported before and after tracing of patients lost to follow-up was conducted in Botswana''s National Antiretroviral Therapy Program. 410 HIV-infected adults consecutively presenting for HAART were evaluated. The main outcome measures were death or loss to follow-up within the first year after HAART initiation. Of 68 patients initially categorized as lost, over half (58.8%) were confirmed dead after tracing. Patient tracing resulted in reporting of significantly lower survival rates when death was used as the outcome and losses to follow-up were censored [1-year Kaplan Meier survival estimate 0.92 (95% confidence interval, 0.88–0.94 before tracing and 0.83 (95% confidence interval, 0.79–0.86) after tracing, log rank P<0.001]. In addition, a significantly increased risk of death after HAART among men [adjusted hazard ratio 1.74 (95% confidence interval, 1.05–2.87)] would have been missed had patients not been traced [adjusted hazard ratio 1.41 (95% confidence interval, 0.65–3.05)].

Conclusions/Significance

Due to high rates of death among patients lost to follow-up after HAART, survival rates may be inaccurate and important risk factors for death may be missed if patients are not actively traced. Patient tracing and uniform reporting of outcomes after HAART are needed to enable accurate monitoring of global HAART scale-up efforts.