Differential Effects of Fingolimod and Natalizumab on B Cell Repertoires in Multiple Sclerosis Patients.

Natalizumab and fingolimod are effective multiple sclerosis (MS) therapies that disrupt lymphocyte migration but have differential effects on B cell maturation and trafficking. We investigated their effects on peripheral blood (PB) and cerebrospinal fluid (CSF) B cell repertoires using next-generation deep sequencing. Paired CSF and PB B cell subsets (naïve, CD27+ memory, and CD27-IgD- double-negative B cells and plasmablasts) were collected by applying flow cytometry at baseline and after 6 months of treatment and their respective heavy-chain variable region repertoires assessed by Illumina MiSeq. Treatment with fingolimod contracted, whereas natalizumab expanded circulating PB B cells. CSF B cell numbers remained stable following fingolimod treatment but decreased with natalizumab therapy. Clonal overlap between CSF and PB B cells was reduced with natalizumab treatment but remained stable with fingolimod therapy. Lineage analyses of pre- and posttreatment CSF B cell repertoires revealed large, clonally expanded B cell clusters in natalizumab-treated MS patients but no intrathecal clonal expansion following fingolimod therapy. Our findings suggest that natalizumab diminishes the exchange of peripheral and intrathecal B cells without impacting intrathecal clonal expansion. In contrast, fingolimod treatment fails to alter blood-brain barrier B cell exchange but diminishes intrathecal clonal expansion. Sphingosine-1 phosphate receptor inhibition may alter intrathecal B cell biology in MS.

Report of a Delphi exercise to inform the design of a research programme on screening for thoracic aortic disease.

OBJECTIVES: To inform the design of a clinical trial of a targeted screening programme for relatives of individuals affected by thoracic aortic disease, we performed a consensus exercise as to the acceptability of screening, the optimal sequence and choice of tests, long-term patient management, and choice of trial design. METHODS: Working with the Aortic Dissection Awareness UK & Ireland patient association, we performed a Delphi exercise with clinical experts, patients, and carers, consisting of three rounds of consultation followed by a final multi-stakeholder face-to-face workshop. RESULTS: Thirty-five experts and 84 members of the public took part in the surveys, with 164 patients and clinicians attending the final workshop. There was substantial agreement on the need for a targeted screening pathway that would employ a combined approach (imaging + genetic testing). The target population would include the first- and second-degree adult (> 15 years) relatives, with no upper age limit of affected patients. Disagreement persisted about the screening process, sequence, personnel, the imaging method to adopt, computed tomography (CT) scan vs magnetic resonance imaging (MRI), and the specifics of a potential trial, including willingness to undergo randomisation, and measures of effectiveness and acceptability. CONCLUSION: A Delphi process, initiated by patients, identified areas of uncertainty with respect to behaviour, process, and the design of a targeted screening programme for thoracic aortic disease that requires further research prior to any future trial.

Coronary Artery Disease Associated Transcription Factor TCF21 Regulates Smooth Muscle Precursor Cells that Contribute to the Fibrous Cap.

TCF21 is a basic helix-loop-helix transcription factor that has recently been implicated as contributing to susceptibility to coronary heart disease based on genome wide association studies. In order to identify transcriptionally regulated target genes in a major disease relevant cell type, we performed siRNA knockdown of TCF21 in in vitro cultured human coronary artery smooth muscle cells and compared the transcriptome of siTCF21 versus siCONTROL treated cells. The raw (FASTQ) as well as processed (BED) data from 3 technical replicates per treatment has been deposited with Gene Expression Omnibus (GSE44461).

Metal uptake via phosphate fertilizer and city sewage in cereal and legume crops in Pakistan.

Crop irrigation with heavy metal-contaminated effluents is increasingly common worldwide and necessitates management strategies for safe crop production on contaminated soils. This field study examined the phytoavailability of three metals (Cd, Cu, and Zn) in two cereal (wheat, maize) and legume (chickpea, mungbean) crops in response to the application of either phosphatic fertilizer or sewage-derived water irrigation over two successive years. Five fertilizer treatments, i.e. control, recommended nitrogen (N) applied alone and in combination of three levels of phosphorus (P), half, full and 1.5 times of recommended P designated as N0P0, N1P0, N1P0.5, N1P1.0, and N1P1.5, respectively. Tissue concentrations of Cd, Cu, Zn, and P were determined in various plant parts, i.e., root, straw, and grains. On the calcareous soils studied while maximum biomass production was obtained with application of P at half the recommended dose, the concentrations of metals in the crops generally decreased with increasing P levels. Tissue metal concentrations increased with the application of N alone. Translocation and accumulation of Zn and Cu were consistently higher than Cd. And the pattern of Cd accumulation differed among plant species; more Cd being accumulated by dicots than monocots, especially in their grains. The order of Cd accumulation in grains was maize > chickpea > mungbean > wheat. Mungbean and chickpea straws also had higher tissue Cd concentration above permissible limits. The two legume species behaved similarly, while cereal species differed from each other in their Cd accumulation. Metal ion concentrations were markedly higher in roots followed by straw and grains. Increasing soil-applied P also increased the extractable metal and P concentrations in the post-harvest soil. Despite a considerable addition of metals by P fertilizer, all levels of applied P effectively decreased metal phytoavailability in sewage-irrigated soils, and applying half of the recommended dose of P fertilizer was the most feasible solution for curtailing plant metal uptake from soils. These findings may have wide applications for safer crop production of monocot species when irrigating crops with sewage effluent-derived waters.

Urocortin--from Parkinson's disease to the skeleton.

Urocortin (Ucn 1), a 40 amino acid long peptide related to corticotropin releasing factor (CRF) was discovered 19 years ago, based on its sequence homology to the parent molecule. Its existence was inferred in the CNS because of anatomical and pharmacological discrepancies between CRF and its two receptor subtypes. Although originally found in the brain, where it has opposing actions to CRF and therefore confers stress-coping mechanisms, Ucn 1 has subsequently been found throughout the periphery including heart, lung, skin, and immune cells. It is now well established that this small peptide is involved in a multitude of physiological and pathophysiological processes, due to its receptor subtype distribution and promiscuity in second messenger signalling pathways. As a result of extensive studies in this field, there are now well over one thousand peer reviewed publications involving Ucn 1. In this review, we intend to highlight some of the less well known actions of Ucn 1 and in particular its role in neuronal cell protection and maintenance of the skeletal system, both by conventional methods of reviewing the literature and using bioinformatics, to highlight further associations between Ucn 1 and disease conditions. Understanding how Ucn 1 works in these tissues, will help to unravel its role in normal and pathophysiological processes. This would ultimately allow the generation of putative medical interventions for the alleviation of important diseases such as Parkinson's disease, arthritis, and osteoporosis.

In vivo transcostal histotripsy therapy without aberration correction.

This study investigates the in vivo therapeutic capabilities of transcostal histotripsy without using aberration correction mechanisms and its thermal impact on overlying tissues. Non-invasive liver treatments were conducted in eight pigs, with four lesions generated through transcostal windows with full ribcage obstruction and four lesions created through transabdominal windows without rib coverage. Treatments were performed by a 750 kHz focused transducer using 5 cycle pulses at 200 Hz PRF, with estimated in situ peak negative pressures of 13-17 MPa. Temperatures on overlying tissues including the ribs were measured with needle thermocouples inserted superficially beneath the skin. Treatments of approximately 40 min were applied, allowing overlying tissue temperatures to reach saturation. Lesions yielded statistically comparable ablation volumes of 3.6 ± 1.7 cm(3) and 4.5 ± 2.0 cm(3) in transcostal and transabdominal treatments, respectively. The average temperature increase observed in transcostal treatments was 3.9 ± 2.1 °C, while transabdominal treatments showed an increase of 1.7 ± 1.3 °C. No damage was seen on the ribcage or other overlying tissues. These results indicate that histotripsy can achieve effective treatment through the ribcage in vivo without requiring correction mechanisms, while inducing no substantial thermal effects or damage to overlying tissues. Such capabilities could benefit several non-invasive therapy applications involving transcostal treatment windows.

Effect of gallium on growth of Streptococcus mutans NCTC 10449 and dental tissues.

Gallium-doped phosphate-based glasses (Ga-PBG) were assessed for their impact on Streptococcus mutans and dental mineralisation, firstly by disc diffusion assays followed by biofilms grown on nitrocellulose filter membrane (NFM) and constant-depth film fermentor (CDFF). Short-time exposure (10 min) effects of Ga-PBG on S. mutans biofilm were compared with that of 0.2% chlorhexidine. The effects of Ga-PBG on bovine enamel (which was investigated under pH-cycling condition) and dentine were analysed using transverse microradiography (TMR), profilometry and inductively coupled plasma optical-emission spectrometry (ICP-OES). The disc diffusion assays showed inhibition zones of 24.5 ± 0.5 mm for Ga-PBG compared with controls (C-PBG). Ga-PBG showed statistically significant growth inhibition of S. mutans biofilms on NFM (p = 0.001) and CDFF (p < 0.046) compared with hydroxyapatite (HA) and C-PBG. The CDFF assay revealed a maximum of 2.11 log colony-forming unit (CFU) reduction at 48 h, but short-time exposure effects were comparable with that of 0.2% chlorhexidine only on older biofilms (maximum of 0.59 vs. 0.69 log CFU reduction at 120 h). TMR analyses of the enamel revealed non-significant mineral loss (p = 0.37) only in the case of Ga-PBG samples compared with controls including sodium fluoride. ICP-OES analyses indicated transient gallium adsorption into dentine by calcium displacement. The results confirmed that gallium inhibited S. mutans growth and appears to have the potential to protect the enamel surface under conditions representative of the oral environment. Further work is needed to establish whether it has an application in daily oral hygiene procedures to prevent or reduce caries.

Decline in extractable antibiotics in manure-based composts during composting.

A wide variety of antibiotics have been detected in natural water samples and this is of potential concern because of the adverse environmental effects of such antibiotic residues. One of the main sources of antibiotics effluence to the surrounding environment is livestock manures which often contain elevated concentrations of veterinary antibiotics (VAs) which survive digestion in the animal stomach following application in animal husbandry practices. In Korea, livestock manures are normally used for compost production indicating that there is potential for antibiotic release to the environment through compost application to agricultural lands. Therefore, reduction of the amount of VAs in composts is crucial. The purpose of this study was to understand the influence of the composting process and the components of the compost on the levels of three common classes of antibiotics (tetracyclines, sulfonamides, and macrolides). Composted materials at different stages of composting were collected from compost manufacturing plants and the variation in antibiotic concentrations was determined. Three different antibiotics, chlortetracycline (CTC), sulfamethazine (SMZ), and tylosin (TYL) at three different concentrations (2, 10, and 20mgkg(-1)) were also applied to a mixture of pig manure and sawdust and the mixtures incubated using a laboratory scale composting apparatus to monitor the changes in antibiotic concentrations during composting together with the physicochemical properties of the composts. During composting, in both field and lab-scale investigations, the concentrations of all three different antibiotics declined below the relevant Korean guideline values (0.8mgkg(-1) for tetracyclines, 0.2mgkg(-1) for sulfonamides and 1.0mgkg(-1) for macrolides). The decline of tetracycline and sulfonamide concentrations was highly dependent on the presence of sawdust while there was no influence of sawdust on TYL decline.

Non-invasive pulsed cavitational ultrasound for fetal tissue ablation: feasibility study in a fetal sheep model.

OBJECTIVES: Currently available fetal intervention techniques rely on invasive procedures that carry inherent risks. A non-invasive technique for fetal intervention could potentially reduce the risk of fetal and obstetric complications. Pulsed cavitational ultrasound therapy (histotripsy) is an ablation technique that mechanically fractionates tissue at the focal region using extracorporeal ultrasound. In this study, we investigated the feasibility of using histotripsy as a non-invasive approach to fetal intervention in a sheep model. METHODS: The experiments involved 11 gravid sheep at 102-129 days of gestation. Fetal kidney, liver, lung and heart were exposed to ultrasound pulses (< 10 µs) delivered by an external 1-MHz focused ultrasound transducer at a 0.2-1-kHz pulse-repetition rate and 10-16 MPa peak negative pressure. Procedures were monitored and guided by real-time ultrasound imaging. Treated organs were examined by gross and histological inspection for location and degree of tissue injury. RESULTS: Hyperechoic, cavitating bubble clouds were successfully generated in 19/31 (61%) treatment attempts in 27 fetal organs beneath up to 8 cm of overlying tissue and fetal bones. Histological assessment confirmed lesion locations and sizes corresponding to regions where cavitation was monitored, with no lesions found when cavitation was absent. Inability to generate cavitation was primarily associated with increased depth to target and obstructing structures such as fetal limbs. CONCLUSION: Extracorporeal histotripsy therapy successfully created targeted lesions in fetal sheep organs without significant damage to overlying structures. With further improvements, histotripsy may evolve into a viable technique for non-invasive fetal intervention procedures.

Applicability of the Charm II system for monitoring antibiotic residues in manure-based composts.

The effluence of veterinary antibiotics (VAs) to aquatic and terrestrial environments is of concern due to the potential adverse effects on human health, such as the production of antibiotic resistant bacteria. One of the main pathways for antibiotics to enter the environment is via the application of manure and/or manure-based composts as an alternative organic fertilizer to agricultural lands. While a wide diversity of manure-based composts are produced in Korea, there is currently no regulatory guideline for VA residues. Hence, monitoring and limiting the concentration of VA residues in manure and/or manure-based composts prior to application to the lands is important to mitigate any environmental burden. The current study was conducted to examine the applicability of the Charm II antibiotic test system for monitoring tetracyclines, sulfonamides and macrolides in manure-based composts. The Charm II system was a highly reproducible method for determining whether VA residue concentrations in manure-based compost exceeded specific guideline values. A wide range of manure-based composts and liquid fertilizers commercially available in Korea were examined using the Charm II system to monitor the residues of the target VAs. For this, the guideline concentrations of VA residues (0.8 mg kg(-1) for tetracyclines, 0.2 mg kg(-1) for sulfonamides, and 0.1 mg kg(-1) for macrolides) stated in 'Official Standard of Feeds' under the 'Control of Livestock and Fish Feed Act' in Korea were adopted to establish control points. Of the 70 compost samples examined 12 exceeded 0.8 mg kg(-1) for tetracyclines and 21 exceeded 0.2 mg kg(-1) for sulfonamides. Of the 25 liquid fertilizer samples examined most samples exceeded these prospective guidelines.

Absence of Epstein-Barr virus in the brain and CSF of patients with multiple sclerosis.

OBJECTIVE: Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that becomes latent in B-lymphocytes and has been implicated in the pathogenesis of multiple sclerosis (MS). We searched for latent and active EBV infection in MS brain and CSF. METHODS: Nested and non-nested real-time PCR were used to detect cell-specific and EBV-specific transcripts in 15 fresh-frozen and 5 formalin-fixed paraffin-embedded MS plaques and in single MS CSF B-lymphocytes and plasma cells. Intrathecal anti-EBV antibody synthesis was measured by ELISA. Immunocytochemistry was used to detect binding of MS CSF and recombinant antibodies (rAbs) generated from clonally expanded plasma cells in MS CSF to EBV-infected cells. RESULTS: No EBV RNA was found in MS CSF B-lymphocytes or plasma cells. In active MS plaques, EBV-encoded RNA (EBER)-1 was the only and rarely detected transcript. The frequency of detected intrathecal anti-EBV antibody synthesis in patients with MS did not differ from that in non-MS inflammatory CNS disease control patients. Anti-EBV antibodies were detected in the CSF of patients with MS, but MS rAbs did not react with EBV. CONCLUSIONS: Application of real-time PCR to multiple sclerosis brain and single B-lymphocytes in CSF did not reveal any evidence of active Epstein-Barr virus infection.

Development and characterization of promoterless helper RNAs for the production of alphavirus replicon particle.

Alphavirus-based replicon systems are frequently used as preclinical vectors and as antigen discovery tools, and they have recently been assessed in clinical vaccine trials. Typically, alphavirus replicon RNAs are delivered within virus-like replicon particles (VRP) that are produced following transfection of replicon RNA and two helper RNAs into permissive cells in vitro. The non-structural proteins expressed from the replicon RNA amplify the replicon RNA in cis and the helper RNAs in trans, the latter providing the viral structural proteins necessary to package the replicon RNA into VRP. Current helper RNA designs incorporate the alphavirus 26S promoter to direct the transcription of high levels of structural gene mRNAs. We demonstrate here that the 26S promoter is not required on helper RNAs to produce VRP and propose that such promoterless helper RNAs, by design, reduce the probability of generating replication-competent virus that may otherwise result from RNA recombination.

Measles virus-specific plasma cells are prominent in subacute sclerosing panencephalitis CSF.

OBJECTIVE: To demonstrate the specificity of expanded CD138(+) plasma cell clones recovered from the CSF of a patient with subacute sclerosing panencephalitis (SSPE) for measles virus (MV). METHODS: IgG variable region sequences of single-antibody-secreting CD138(+) cells sorted from SSPE CSF were amplified by single-cell PCR and analyzed. Human IgG1 recombinant antibodies (rAbs) were produced from four expanded CD138(+) clones and assayed for immunoreactivity against MV proteins. RESULTS: Clonal expansion was a prominent feature of the SSPE plasma cell repertoire, and each of the four rAbs assayed was specific for either the MV fusion or the MV nucleocapsid protein. CONCLUSIONS: Expanded plasma cell clones in the CSF of patients with subacute sclerosing panencephalitis produce disease-relevant antibodies. Recombinant antibodies derived from CSF B cells could provide a tool to identify target antigens in idiopathic inflammatory disorders.

Conditional mouse models to study developmental and pathophysiological gene function in muscle.

This chapter will review conditional mouse model systems that have been developed to study gene function in skeletal, cardiac, and vascular smooth muscle cells in vivo with an emphasis on the utility of these models for investigating developmental and pathophysiological gene function in muscle. In general, these systems have utilized muscle-specific/selective promoter-enhancers in conjunction with site-specific DNA recombinases, e.g., Cre-loxP, and fusion proteins with these recombinases that confer temporal control, such as tamoxifen-inducible CreER systems. A major focus of this chapter will be to discuss unique challenges of studying Cre-mediated mutagenesis/gene targeting in these muscle types during development and in the adult animal, some of which are inherent of the muscle cell type being studied. For example, unlike cardiac and skeletal muscle cells, the vascular SMC is extremely plastic and able to undergo rapid phenotypic modulation to various environmental cues in vivo. Thus, employing SMC marker gene promoter enhancers for conditional gene targeting in SMCs must take into account the possibility and/or certainty that the particular SMC promoter enhancers used may or may not be transcriptionally active in SMCs of a vessel wall under normal and some pathophysiological conditions. Moreover, individual floxed loci within the same muscle cell type and tissue have different degrees of sensitivity to Cre, most likely dependent on the chromatin state of that particular gene, i.e., closed/condensed state or open/active state. Thus, Cre recombination may be ineffective for specific floxed gene DNA. Lastly, rigorous efforts must be made to confirm the degree of recombination in a tissue, taking into full account the multicellularity of the tissue, to understand the extent of the physiological effect in that organ.

Alphavirus replicon approach to promoterless analysis of IRES elements.

Here we describe a system for promoterless analysis of putative internal ribosome entry site (IRES) elements using an alphavirus (family Togaviridae) replicon vector. The system uses the alphavirus subgenomic promoter to produce transcripts that, when modified to contain a spacer region upstream of an IRES element, allow analysis of cap-independent translation of genes of interest (GOI). If the IRES element is removed, translation of the subgenomic transcript can be reduced >95% compared to the same transcript containing a functional IRES element. Alphavirus replicons, used in this manner, offer an alternative to standard dicistronic DNA vectors or in vitro translation systems currently used to analyze putative IRES elements. In addition, protein expression levels varied depending on the spacer element located upstream of each IRES. The ability to modulate the level of expression from alphavirus vectors should extend the utility of these vectors in vaccine development.

A randomized trial of local anesthesia with intravenous sedation vs general anesthesia for the vaginal correction of pelvic organ prolapse.

The purpose of this study is to compare the feasibility of local anesthesia with IV sedation versus general anesthesia for vaginal correction of pelvic organ prolapse. Patients with pelvic organ prolapse who were scheduled for an anterior or posterior colporrhaphy, or an obliterative procedure, and who did not have a contraindication or preference to type of anesthesia were randomized to one of the two anesthesia groups. Nineteen patients were randomized to the general group and 21 patients were randomized to the local group. Mean operating room, anesthesia, and surgical time were similar in each group, and 10 patients in the local group bypassed the recovery room. Requests and doses of antiemetics, postoperative verbal numerical pain scores and length of hospital stay were similar between the two groups. Mean recovery room and total hospital costs were significantly lower in the local group. Local anesthesia with IV sedation is a feasible alternative for vaginal surgery to correct pelvic organ prolapse.

Smooth muscle of telokin-deficient mice exhibits increased sensitivity to Ca2+ and decreased cGMP-induced relaxation.

Cyclic nucleotides can relax smooth muscle without a change in [Ca2+]i, a phenomenon termed Ca2+ desensitization, contributing to vasodilation, gastrointestinal motility, and airway resistance. The physiological importance of telokin, a 17-kDa smooth muscle-specific protein and target for cyclic nucleotide-induced Ca2+ desensitization, was determined in telokin null mice bred to a congenic background. Telokin null ileal smooth muscle homogenates compared to wild type exhibited an approximately 30% decrease in myosin light-chain phosphatase (MLCP) activity, which was reflected in a significant leftward shift (up to 2-fold at pCa 6.3) of the Ca2+ force relationship accompanied by an increase in myosin light-chain phosphorylation. No difference in the Ca2+ force relationship occurred in telokin WT and knockout (KO) aortas, presumably reflecting the normally approximately 5-fold lower telokin content in aorta vs. ileum smooth muscle. Ca2+ desensitization of contractile force by 8-Br-cGMP was attenuated by 50% in telokin KO intestinal smooth muscle. The rate of force relaxation reflecting MLCP activity, in the presence of 50 microM 8-Br-cGMP, was also significantly slowed in telokin KO vs. WT ileum and was rescued by recombinant telokin. Normal thick filaments in telokin KO smooth muscles indicate that telokin is not required for filament formation or stability. Results indicate that a primary role of telokin is to modulate force through increasing MLCP activity and that this effect is further potentiated through phosphorylation by cGMP in telokin-rich smooth tissues.

LPP expression during in vitro smooth muscle differentiation and stent-induced vascular injury.

Lipoma preferred partner (LPP) has been identified as a protein highly expressed in smooth muscle (SM) tissues. The aim of the present study was to determine mechanisms that regulate LPP expression in an in vitro model of SM cell (SMC) differentiation and in stent-induced pig coronary vessel injury. All trans-retinoic acid treatment of A404 cells induced a strong increase in LPP, as well as SM alpha-actin, SM myosin heavy chain, and smoothelin mRNA levels, in a Rho kinase (ROK)-dependent manner. Adenovirus mediated overexpression of myocardin in A404 cells significantly increased LPP mRNA expression. Interestingly, inactivation of RhoA with C3-exoenzyme or treatment with ROK inhibitors strongly inhibited myocardin mRNA expression in retinoic acid-treated A404 cells or human iliac vein SMCs. LPP silencing with short interfering RNA significantly decreased SMC migration. LPP expression was also markedly decreased in focal adhesion kinase (FAK)-null cells known to have impaired migration but rescued with inducible expression of FAK. LPP expression in FAK-null fibroblasts enhanced cell spreading. In stented pig coronary vessels, LPP was expressed in the neointima of cells lacking smoothelin and showed expression patterns identical to those of SM alpha-actin. In conclusion, LPP appears to be a myocardin-, RhoA/ROK-dependent SMC differentiation marker that plays a role in regulating SMC migration.