Immunogenicity and mechanisms of action of PnuBioVax, a multi-antigen serotype-independent prophylactic vaccine against infection with Streptococcus pneumoniae.

Streptococcus pneumoniae has multiple protein antigens on the surface in addition to the serotype specific polysaccharide capsule antigen. Whilst the capsule antigen is the target of the polysaccharide vaccines, bacterial proteins can also act as targets for the immune system. PnuBioVax (PBV) is being developed as a multi-antigen, serotype-independent prophylactic vaccine against S. pneumoniae disease. In this study we have sought to elucidate the immune response to PBV in immunised rabbits. Sera from PBV immunised rabbits contained high levels of IgG antibodies to the PBV vaccine, and pneumococcal antigens PspA, Ply, PsaA and PiuA which are components of PBV, when compared with control sera. The PBV sera supported killing of the vaccine strain TIGR4 in an opsonophagocytic killing assay and heterologous strains 6B, 19F and 15B. In addition, incubation in PBV sera led to agglutination of several strains of pneumococci, inhibition of Ply-mediated lysis of erythrocytes and reduced bacterial invasion of lung epithelial cells in vitro. These data suggest that PBV vaccination generates sera that has multiple mechanisms of action that may provide effective protection against pneumococcal infection and give broader strain coverage than the current polysaccharide based vaccines.

Comparison Between Cardiac Allograft Vasculopathy and Native Coronary Atherosclerosis by Optical Coherence Tomography.

We sought to explore differences in distribution and morphology of coronary lesions comparing cardiac allograft vasculopathy and native coronary atherosclerosis (NCA) using intravascular imaging with optical coherence tomography (OCT). At the time of routine surveillance angiography, 17 heart transplant (HT) recipients with a history of high-grade cellular rejection (HGR) and 43 HT recipients with none/mild (low)-grade rejection underwent OCT imaging of the left anterior descending and were compared to 60 patients with NCA without HT. Compared with patients with NCA, patients with HGR had similar intima areas but smaller external elastic lamina areas (7.9 mm(2) [6.3, 11.2] versus 6.6 mm(2) [4.8, 7.5], p = 0.02) resulting in smaller lumen areas (4.5 mm(2) [3.4, 6.6] versus 3.3 mm(2) [2.8, 4.7], p = 0.04) in distal segments and smaller lumen diameters in side branches (1.28 mm [1.19, 1.37] versus 1.09 mm [0.94, 1.24], p = 0.04). Compared with patients with NCA, lesions in patients with HT were more homogeneous, involving the entire coronary vascular tree. Patients with HGR had a higher prevalence of macrophages involving ≥1 quadrant in all 3 segments compared with patients with NCA. The number of microvessels was greater in patients with both HGR and LGR HT versus NCA. In conclusion, distinct findings in the distribution and morphology of coronary lesions between HT recipients and patients with NCA are evident by OCT imaging, suggesting that OCT might be useful to help differentiate cardiac allograft vasculopathy from NCA in vivo.

Increased coronary lipid accumulation in heart transplant recipients with prior high-grade cellular rejection: novel insights from near-infrared spectroscopy.

Cardiac allograft vasculopathy is a major cause of morbidity and mortality among patients after heart transplantation. We sought to assess the amount of lipid accumulation in the coronary arteries of transplant patients according to rejection grade. Overall, 39 consecutive heart transplant recipients undergoing annual routine surveillance coronary angiography underwent near-infrared spectroscopy and intravascular ultrasound imaging of 1 coronary artery. Rejection history was graded according to the International Society of Heart and Lung Transplantation (ISHLT) classification as none/mild/moderate-grade rejection (ISHLT 0, 1A/1B, or 2) compared to high-grade rejection (≥3A). Patients with prior history of high-grade rejection had larger plaque burden in the distal coronary segments [45.7 % (25.5-63.7) vs 25.1 % (19.9-37.8), p = 0.02] and a higher maximum lipid core burden index in any 4-mm long segment (maxLCBI(4mm)) [243 (91-400) vs 41 (1-170), p = 0.016] as compared with patients with prior history of none/mild/moderate-grade rejection. By multivariable linear regression analysis, prior history of high-grade rejection was an independent predictor for maxLCBI(4mm). A maxLCBI(4mm) >200 distinguished prior history of high-grade from none/mild/moderate rejection with a sensitivity of 61.5 % and specificity of 84.6 %. The current study demonstrates that the coronary arteries of post heart-transplant patients with a prior history of high-grade cellular rejection have increasing amounts of lipid-rich plaque. MaxLCBI(4mm) >200 might differentiate patients with previous high-grade cellular rejection from heart transplant recipients with none/mild/moderate-grade rejection.

In vivo comparison between cardiac allograft vasculopathy and native atherosclerosis using near-infrared spectroscopy and intravascular ultrasound.

AIMS: The aim was to compare cardiac allograft vasculopathy to native atherosclerosis by near-infrared spectroscopy-intravascular ultrasound (NIRS-IVUS). METHODS AND RESULTS: Twenty-seven atherosclerotic (non-transplant) patients and 28 heart transplant recipients undergoing routine surveillance coronary angiography underwent NIRS-IVUS imaging of the left anterior descending coronary artery. In each proximal, middle, and distal coronary artery segment, the maxLCBI4mm [4-mm long segment with maximum lipid core burden index (LCBI)] and corresponding IVUS parameters were compared. MaxLCBI4mm was significantly greater among atherosclerotic patients than the transplant patients in both proximal and middle coronary artery segments, but not in the distal segment. There was a positive linear correlation between maxLCBI4mm and maximum plaque burden in both groups, but atherosclerotic patients demonstrated a smaller maxLCBI4mm than transplant recipients among segments with plaque burden <40%. Among segments with a maximum plaque burden ≥40%, native-atherosclerosis patients had a greater maxLCBI4mm compared with transplant patients (P = 0.015). Calcification was present in 72.9% of native atherosclerosis and 14.7% of transplant segments (P< 0.001). Among the 165 analysed segments, prevalence of lipid-rich plaque (LRP) with superficial attenuation (30.9 vs. 1.2%, P < 0.001) or calcified LRP (13.6 vs. 2.4%, P = 0.03) was significantly greater in native atherosclerosis compared with transplant patients. Conversely, the proportion of segments with non-LRP (46.4 vs. 11.1%, P < 0.001) was higher in transplant patients. CONCLUSION: NIRS-IVUS imaging demonstrated early and accelerated lipid accumulation with smaller plaque burden and less calcium in patients after heart transplant when compared with patients with native atherosclerosis.

Robotic-Enhanced PCI Compared to the Traditional Manual Approach.

Remote-controlled robotic-enhanced percutaneous coronary intervention (PCI) was developed to improve procedural outcomes, reduce operator radiation exposure, and improve ergonomics. Critics questioned whether protection of the operator might result in increased radiation exposure to the patient and increase contrast media use. We studied this in a single-center comparison of robotic-enhanced versus traditional PCIs. A total of 40 patients who enrolled in the PRECISE study and had PCI with the CorPath 200 robotic system (Corindus Vascular Robotics) were compared to 80 consecutive patients who underwent conventional PCI. All patients had obstructive coronary artery disease, evidence of myocardial ischemia, and clinical indications for single-vessel PCI. Baseline demographics of the 40 robotic and 80 traditional PCIs were similar. Only 2 robotic-assisted cases required conversion to manual PCI. All patients had a final residual stenosis <30%. Robotic-enhanced PCI was associated with trends toward lower duration of fluoroscopy (10.1 ± 4.7 min vs 12.3 ± 7.6 min; P=.05), radiation dose (1389 ± 599 mGy vs 1665 ± 1026 mGy; P=.07), and contrast volume (121 ± 47 mL vs 137 ± 62 mL; P=.11). In conclusion, the initial experience with robotic-enhanced PCI was not associated with increased fluoroscopy duration, radiation, or contrast media exposure to patients, and compared favorably to the traditional approach.

Optical coherence tomographic evaluation of transplant coronary artery vasculopathy with correlation to cellular rejection.

BACKGROUND: Cardiac allograft vasculopathy is an accelerated fibroproliferative process that affects the coronary arteries of transplanted hearts. Intracoronary imaging with optical coherence tomography enables detection of subangiographic cardiac allograft vasculopathy. METHODS AND RESULTS: At the time of routine surveillance coronary angiography, 48 consecutive heart transplant recipients underwent optical coherence tomographic imaging of 1 coronary artery. Imaging findings were compared per rejection history that was graded according to the International Society of Heart and Lung Transplantation classification as none/mild (International Society of Heart and Lung Transplantation 0, 1A/1B, or 2) or high-grade rejection (≥3A). Compared with the none/mild rejection group (37 patients) using Mann-Whitney U test, patients in the high-grade rejection group (11 patients) had a thicker intima in all coronary segments (distal: 0.22 mm [0.09-0.41] versus 0.09 mm [0.06-0.17], P=0.02; middle: 0.35 mm [0.00-0.45] versus 0.14 mm [0.08-0.24], P=0.002; and proximal: 0.34 mm [0.21-0.44] versus 0.15 mm [0.11-0.21], P=0.005) and a higher prevalence of foamy macrophages (distal: 55% versus 9%, P=0.003; middle: 55% versus 22%, P=0.004; and proximal: 44% versus 13%, P=0.05) using χ(2) statistics. Side branches in the high-grade rejection group had smaller lumen diameters and a higher prevalence of intimal thickening (54% versus 36%; P=0.01). Intimal microvessels were also more prevalent in the high-grade rejection group versus the none/mild rejection group (46% versus 11%; P=0.02). CONCLUSIONS: Coronary optical coherence tomographic evaluation revealed that patients with a history of high-grade cellular rejection, compared with those with none/mild rejection, had more coronary artery intimal thickening with macrophage infiltration, involving all coronary segments and side branches. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT01403142.

A sporulation-specific, sigF-dependent protein, SspA, affects septum positioning in Streptomyces coelicolor.

The RNA polymerase sigma factor SigF controls late development during sporulation in the filamentous bacterium Streptomyces coelicolor. The only known SigF-dependent gene identified so far, SCO5321, is found in the biosynthetic cluster encoding spore pigment synthesis. Here we identify the first direct target for SigF, the gene sspA, encoding a sporulation-specific protein. Bioinformatic analysis suggests that SspA is a secreted lipoprotein with two PepSY signature domains. The sspA deletion mutant exhibits irregular sporulation septation and altered spore shape, suggesting that SspA plays a role in septum formation and spore maturation. The fluorescent translational fusion protein SspA-mCherry localized first to septum sites, then subsequently around the surface of the spores. Both SspA protein and sspA transcription are absent from the sigF null mutant. Moreover, in vitro transcription assay confirmed that RNA polymerase holoenzyme containing SigF is sufficient for initiation of transcription from a single sspA promoter. In addition, in vivo and in vitro experiments showed that sspA is a direct target of BldD, which functions to repress sporulation genes, including whiG, ftsZ and ssgB, during vegetative growth, co-ordinating their expression during sporulation septation.

An ultra scale-down characterization of low shear stress primary recovery stages to enhance selectivity of fusion protein recovery from its molecular variants.

Fusion proteins offer the prospect of new therapeutic products with multiple functions. The primary recovery is investigated of a fusion protein consisting of modified E2 protein from hepatitis C virus fused to human IgG1 Fc and expressed in a Chinese hamster ovary (CHO) cell line. Fusion protein products inevitably pose increased challenge in preparation and purification. Of particular concerns are: (i) the impact of shear stress on product integrity and (ii) the presence of product-related contaminants which could prove challenging to remove during the high resolution purification steps. This paper addresses the use of microwell-based ultra scale-down (USD) methods to develop a bioprocess strategy focused on the integration of cell culture and cell removal operations and where the focus is on the use of operations which impart low shear stress levels even when applied at eventual manufacturing scale. An USD shear device was used to demonstrate that cells exposed to high process stresses such as those that occur in the feed zone of a continuous non-hermetic centrifuge resulted in the reduction of the fusion protein and also the release of glycosylated intracellular variants. In addition, extended cell culture resulted in release of such variants. USD mimics of low shear stress, hydrohermetic feed zone centrifugation and of depth filtration were used to demonstrate little to no release during recovery of these variants with both results verified at pilot scale. Furthermore, the USD studies were used to predict removal of contaminants such as lipids, nucleic acids, and cell debris with, for example, depth filtration delivering greater removal than for centrifugation but a small (~10%) decrease in yield of the fusion protein. These USD observations of product recovery and carryover of contaminants were also confirmed at pilot scale as was also the capacity or throughput achievable for continuous centrifugation or for depth filtration. The advantages are discussed of operating a lower yield cell culture and a low shear stress recovery process in return for a considerably less challenging purification demand.

Coiled-coil protein Scy is a key component of a multiprotein assembly controlling polarized growth in Streptomyces.

Polarized growth in eukaryotes requires polar multiprotein complexes. Here, we establish that selection and maintenance of cell polarity for growth also requires a dedicated multiprotein assembly in the filamentous bacterium, Streptomyces coelicolor. We present evidence for a tip organizing center and confirm two of its main components: Scy (Streptomyces cytoskeletal element), a unique bacterial coiled-coil protein with an unusual repeat periodicity, and the known polarity determinant DivIVA. We also establish a link between the tip organizing center and the filament-forming protein FilP. Interestingly, both deletion and overproduction of Scy generated multiple polarity centers, suggesting a mechanism wherein Scy can both promote and limit the number of emerging polarity centers via the organization of the Scy-DivIVA assemblies. We propose that Scy is a molecular "assembler," which, by sequestering DivIVA, promotes the establishment of new polarity centers for de novo tip formation during branching, as well as supporting polarized growth at existing hyphal tips.

Survey of current enteral nutrition practices in treatment of amyotrophic lateral sclerosis.

BACKGROUND AND AIMS: Enteral nutrition (EN) is commonly prescribed for dysphagia and weight loss in amyotrophic lateral sclerosis (ALS), but there are currently no ALS-specific EN guidelines. We aimed to survey current practices prescribing EN to ALS patients. METHODS: An online survey was distributed using list servers administered by the Academy of Nutrition and Dietetics (AND), Muscular Dystrophy Association (MDA), and ALS Association (ALSA). RESULTS: A total of 148 dietitians, nurses, and physicians participated in the survey, of whom 50% were dietitians and 68% were associated with an ALS clinic. Only 47% of respondents reported their patients to be fully compliant with EN recommendations. Side effects (fullness, diarrhea, constipation, and bloating) were the most important reason for patient noncompliance, followed by dependence on caregivers. By contrast, only 3% of providers rated depression/hopelessness as the most important reason for noncompliance. Half of those surveyed reported that more than 25% of patients continued to lose weight after starting EN. CONCLUSIONS: Our survey results show a high frequency of gastrointestinal side effects and weight loss in ALS patients receiving EN. These findings may be limited by sampling error and non-response bias. Prospective studies are needed to help establish EN guidelines for ALS.

A prospective cohort study of neuropsychological test performance in ALS.

Our objective was to determine the prevalence and predictors of cognitive impairment in ALS, measure differences in survival among impaired and unimpaired patients, and assess changes in neuropsychological test performance over time. Fifty patients were enrolled in a prospective cohort study of neuropsychological performance. ANOVA and chi(2) tests assessed differences in clinical characteristics and neuropsychologic test results; general estimating equations assessed change in test performance; multiple regression determined which variables contributed to cognitive status; and Cox models compared survival. Thirty-six patients were categorized as cognitively normal, and 14 were impaired. Impaired patients were older at testing (p = 0.024), but no more likely to have bulbar signs. Predicators of impairment were symptom duration (p < 0.001), motor function (p < 0.001), and rate of ALS progression (p < 0.001). The Benton recognition (p < 0.001), Boston naming (p = 0.001), Wisconsin Card Sort (p = 0.001) and word generation (p = 0.001) tests contributed most strongly to cognitive status. Survival was worse in impaired patients (p = 0.027). Over time, only animal word generation declined (p = 0.016). In conclusion, 28% percent of patients were cognitively impaired. Older age and more severe ALS were associated with impairment. The strongest neuropsychological predictors of cognitive status were measures of executive, episodic memory and language function. Cognitively impaired patients had shorter survival time.

A service model of short-term case management for elderly people at risk of hospital admission.

This article presents the model of a short-term case management program focused on reducing emergency department presentations and unplanned hospital admissions for a targeted group of older people with complex care needs. As a semi-integrated health care program, Treatment Response and Assessment for Aged Care (TRAAC) is implemented by short-term case managers located in a variety of community agencies as well as acute and sub-acute hospital settings. The article discusses the features of the model including case finding, early intervention and risk screening, combined with the rapid mobilisation of specialised geriatric assessment services. The model has the potential to contribute to positive results in managing the complex health needs of this group. Evaluation outcomes including reductions in hospital use for the target group, and positive client and staff perceptions of the service model are discussed in relation to the unique features of the intervention program.

A novel compartment, the 'subapical stem' of the aerial hyphae, is the location of a sigN-dependent, developmentally distinct transcription in Streptomyces coelicolor.

Streptomyces coelicolor has nine SigB-like RNA polymerase sigma factors, several of them implicated in morphological differentiation and/or responses to different stresses. One of the nine, SigN, is the focus of this article. A constructed sigN null mutant was delayed in development and exhibited a bald phenotype when grown on minimal medium containing glucose as carbon source. One of two distinct sigN promoters, sigNP1, was active only during growth on solid medium, when its activation coincided with aerial hyphae formation. Transcription from sigNP1 was readily detected in several whi mutants (interrupted in morphogenesis of aerial mycelium into spores), but was absent from all bld mutants tested, suggesting that sigNP1 activity was restricted to the aerial hyphae. It also depended on sigN, thus sigN was autoregulated. Mutational and transcription studies revealed no functional significance to the location of sigN next to sigF, encoding another SigB-like sigma factor. We identified another potential SigN target, nepA, encoding a putative small secreted protein. Transcription of nepA originated from a single, aerial hyphae-specific and sigN-dependent promoter. While in vitro run-off transcription using purified SigN on the Bacillus subtilis ctc promoter confirmed that SigN is an RNA polymerase sigma factor, SigN failed to initiate transcription from sigNP1 and from the nepA promoter in vitro. Additional in vivo data indicated that further nepA upstream sequences, which are likely to bind a potential activator, are required for successful transcription. Using a nepA-egfp transcriptional fusion we located nepA transcription to a novel compartment, the 'subapical stem' of the aerial hyphae. We suggest that this newly recognized compartment defines an interface between the aerial and vegetative parts of the Streptomyces colony and might also be involved in communication between these two compartments.