Role of a FMRFamide-like family of neuropeptides in the pharyngeal nervous system of Caenorhabditis elegans

The nervous system of C. elegans has a remarkable abundance of flp genes encoding FMRFamide-like (FLP) neuropeptides. To provide insight into the physiological relevance of this neuropeptide diversity, we have tested more than 30 FLPs (encoded by 23 flps) for bioactivity on C. elegans pharynx. Eleven flp genes encode peptides that inhibit pharyngeal activity, while eight flp genes encode peptides that are excitatory. Three potent peptides (inhibitory, FLP-13A, APEASPFIRFamide; excitatory, FLP-17A, KSAFVRFamide; excitatory, FLP-17B, KSQYIRFamide) are encoded by flp genes, which, according to reporter gene constructs, are expressed in pharyngeal motoneurons. Thus, they may act through receptors localized on the pharyngeal muscle. The two other potent peptides, FLP-8 (excitatory AF1, KNEFIRFamide,) and FLP-11A (inhibitory, AMRNALVRFamide), appear to be expressed in extrapharyngeal neurons and are therefore likely to act either indirectly or as neurohormones. Intriguingly, a single neuron can express peptides that have potent but opposing biological activity in the pharynx. Only five flp genes encode neuropeptides that have no observable effect on the pharynx, but none of these have shown reporter gene expression in the pharyngeal nervous system. To examine the roles of multiple peptides produced from single precursors, a comparison was made between the bioactivity of different neuropeptides for five flp genes (flp-3, flp-13, flp-14, flp-17, and flp-18). For all but one gene (flp-14), the effects of peptides encoded by the same gene were similar. Overall, this study demonstrates the impressive neurochemical complexity of the simple circuit that regulates feeding in the nematode, C. elegans.     

Costimulation via CD55 on human CD4+ T cells mediated by CD97

Decay-accelerating factor (CD55) is a complement regulatory protein, which is expressed by most cells to protect them from complement-mediated attack. CD55 also binds CD97, an EGF-TM7 receptor constitutively expressed on granulocytes and monocytes and rapidly up-regulated on T and B cells upon activation. Early results suggested that CD55 could further enhance T cell proliferation induced by phorbol ester treatment. The present study demonstrates that coengagement of CD55, using either cross-linking mAbs or its natural ligand CD97, and CD3 results in enhanced proliferation of human peripheral blood CD4(+) T cells, expression of the activation markers CD69 and CD25, and secretion of IL-10 and GM-CSF. Recently, an increase in T cell responsiveness in CD55(-/-) mice was shown to be mediated by a lack of complement regulation. In this study, we show that direct stimulation of CD55 on CD4(+) T cells with CD97 can modulate T cell activation but does not interfere with CD55-mediated complement regulation. Our results support a multifaceted role for CD55 in human T cell activation, constituting a further link between innate and adaptive immunity.     

A generic, homogenous method for measuring kinase and inhibitor activity via adenosine 5'-diphosphate accumulation

The authors describe an assay to measure the generation of adenosine 5'-diphosphate (ADP) resulting from phosphorylation of a substrate by a kinase. ADP accumulation is detected by conversion to a fluorescent signal via a coupled enzyme system. The technology has potential applications for the assessment of inhibitor potency and mode of action as well as kinetic analysis of enzyme activity. The assay has a wide dynamic range (0.25-75 microM) and has been validated with several kinases including the highly active cyclic adenosine monophosphate-dependent protein kinase (PKAalpha), casein kinase 1 (CK1), and the weakly active kinase Jun N-terminal kinase 2 (Jnk2alpha2). Kinase activity can be measured either in an end point or continuous mode. Assay performance in end point mode was compared with an adenosine 5'-triphosphate (ATP) depletion assay and in continuous mode with a pyruvate kinase/lactate dehydrogenase coupled assay. The ability to characterize kinase kinetics was demonstrated by deriving ATP/substrate affinity (Michaelis-Menten constant; K(m)) values for PKAalpha, CK1, and Jnk2alpha2. The assay readily measured activity with kinase reactions using protein substrates, indicating the suitability for use with large macromolecules. A wide range of inhibitor activities could be determined even in the presence of high ATP concentrations, making the assay highly suitable to characterize the mode of action of the inhibitor in question. Collectively, this assay provides a homogenous, generic method for a number of applications in kinase drug discovery.     

Anthelmintic cyclcooctadepsipeptides: complex in structure and mode of action

The broad-spectrum anthelmintic cyclooctadepsipeptide PF1022A is a fungal metabolite from Rosellinia sp. PF1022, which is a Mycelia sterilia found on the leaves of Camellia japonica. A broad range of structurally related cyclooctadepsipeptides has been characterized and tested for anthelmintic activities. These metabolites have been used as starting points to generate semisynthetic derivatives with varying nematocidal capacity. Predominant among these compounds is emodepside, which exhibits a broad nematocidal potential against gastrointestinal and extraintestinal parasites. Here we review the chemical biology and mode of action of cyclooctadepsides with particular attention to PF1022A and emodepside. We illustrate how they target nematode neuromuscular function, opening up new avenues for antiparasitic treatments with potential capability for important selective toxicity.     

Characterization of X-ray Dose in Murine Animals Using microCT,a New Low-Dose Detector and nanoDot Dosimeters

Background

Dose continues to be an area of concern in preclinical imaging studies, especially for those imaging disease progression in longitudinal studies. To our knowledge, this work is the first to characterize and assess dose from the Inveon CT imaging platform using nanoDot dosimeters. This work is also the first to characterize a new low-dose configuration available for this platform.

Methodology/Principal Findings

nanoDot dosimeters from Landauer, Inc. were surgically implanted into 15 wild type mice. Two nanoDots were placed in each animal: one just under the skin behind the spine and the other located centrally within the abdomen. A manufacturer-recommended CT protocol was created with 1 projection per degree of rotation acquired over 360 degrees. For best comparison of the low dose and standard configurations, noise characteristics of the reconstructed images were used to match the acquisition protocol parameters. Results for all dose measurements showed the average dose delivered to the abdomen to be 13.8 cGy±0.74 and 0.97 cGy±0.05 for standard and low dose configurations respectively. Skin measurements of dose averaged 15.99 cGy±0.72 and 1.18 cGy±0.06. For both groups, the standard deviation to mean was less than 5.6%. The maximum dose received for the abdomen was 15.12 cGy and 0.97 cGy while the maximum dose for the skin was 17.3 cGy and 1.32 cGy. Control dosimeters were used for each group to validate that no unwanted additional radiation was present to bias the results.

Conclusions/Significance

This study shows that the Inveon CT platform is suitable for imaging mice both for single and longitudinal studies. Use of low-dose detector hardware results in significant reductions in dose to subjects with a >12x (90%) reduction in delivered dose. Installation of this hardware on another in vivo microCT platform resulted in dose reductions of over 9x (89%).     

High avidity cytotoxic T lymphocytes can be selected into the memory pool but they are exquisitely sensitive to functional impairment

High avidity cytotoxic T lymphocytes (CTL) are important in viral clearance and anti-tumor immunity, however, mechanisms for their optimal generation and maintenance in vivo remain unclear. Immunizing mice with an antibody-DNA vaccine encoding a single CTL epitope, induces a 100 fold higher avidity response than peptide vaccination with the identical epitope. The high avidity response is retained into memory and can be efficiently reactivated with an antibody-DNA boost. In contrast, reactivation of high avidity CTL with peptide, stimulated responses with a significant drop in avidity, suggesting loss or conversion of the high avidity CTL to lower avidity. Similarly, high avidity T cells maintained ex vivo were exquisitely sensitive to signaling with low doses of peptide (1 ng/ml) giving optimal TCR stimulation and resulting in retained avidity, proliferation and ability to kill specific targets. In contrast, high avidity T cells maintained ex vivo with supraoptimal TCR stimulation (10 μg/ml peptide) resulted in reduced avidity and failure to kill tumor cells. They also failed to proliferate, showed a significant increase in apoptosis and expressed high levels of the exhaustion marker programmed death-1 (PD-1) and low levels of the lymphocyte-activation gene 3 (LAG-3). This suggests high avidity T cells are recruited to the memory pool but can be lost by supraoptimal stimulation in vitro and in vivo. This is characterized by loss of function and an increase in cell death. The remaining CTL, exhibit low functional avidity that is reflected in reduced anti-tumor activity. This could contribute to failure of the immune system to control the growth of tumors and has implications for vaccination strategies and adoptive transfer of T cells.     

Lipopolysaccharide-Induced Lung Injury Is Independent of Serum Vitamin D Concentration

Vitamin D deficiency is increasing in incidence around the world. Vitamin D, a fat-soluble vitamin, has documented effects on the innate and adaptive immune system, including macrophage and T regulatory (Treg) cell function. Since Treg cells are important in acute lung injury resolution, we hypothesized that vitamin D deficiency increases the severity of injury and delays injury resolution in lipopolysaccharide (LPS) induced acute lung injury. Vitamin D deficient mice were generated, using C57BL/6 mice, through diet modification and limited exposure to ultraviolet light. At 8 weeks of age, vitamin D deficient and sufficient mice received 2.5 g/kg of LPS or saline intratracheal. At 1 day, 3 days and 10 days, mice were anesthetized and lung elastance measured. Mice were euthanized and bronchoalveolar lavage fluid, lungs and serum were collected. Ex vivo neutrophil chemotaxis was evaluated, using neutrophils from vitamin D sufficient and deficient mice exposed to the chemoattractants, KC/CXCL1 and C5a, and to bronchoalveolar lavage fluid from LPS-exposed mice. We found no difference in the degree of lung injury. Leukocytes were mildly decreased in the bronchoalveolar fluid of vitamin D deficient mice at 1 day. Ex-vivo, neutrophils from vitamin D deficient mice showed impaired chemotaxis to KC but not to C5a. Vitamin D deficiency modestly impairs neutrophil chemotaxis; however, it does not affect lung injury or its resolution in an LPS model of acute lung injury.     

A simplified 4-site economical intradermal post-exposure rabies vaccine regimen: a randomised controlled comparison with standard methods

Background

The need for economical rabies post-exposure prophylaxis (PEP) is increasing in developing countries. Implementation of the two currently approved economical intradermal (ID) vaccine regimens is restricted due to confusion over different vaccines, regimens and dosages, lack of confidence in intradermal technique, and pharmaceutical regulations. We therefore compared a simplified 4-site economical PEP regimen with standard methods.

Methods

Two hundred and fifty-four volunteers were randomly allocated to a single blind controlled trial. Each received purified vero cell rabies vaccine by one of four PEP regimens: the currently accepted 2-site ID; the 8-site regimen using 0.05 ml per ID site; a new 4-site ID regimen (on day 0, approximately 0.1 ml at 4 ID sites, using the whole 0.5 ml ampoule of vaccine; on day 7, 0.1 ml ID at 2 sites and at one site on days 28 and 90); or the standard 5-dose intramuscular regimen. All ID regimens required the same total amount of vaccine, 60% less than the intramuscular method. Neutralising antibody responses were measured five times over a year in 229 people, for whom complete data were available.

Findings

All ID regimens showed similar immunogenicity. The intramuscular regimen gave the lowest geometric mean antibody titres. Using the rapid fluorescent focus inhibition test, some sera had unexpectedly high antibody levels that were not attributable to previous vaccination. The results were confirmed using the fluorescent antibody virus neutralisation method.

Conclusions

This 4-site PEP regimen proved as immunogenic as current regimens, and has the advantages of requiring fewer clinic visits, being more practicable, and having a wider margin of safety, especially in inexperienced hands, than the 2-site regimen. It is more convenient than the 8-site method, and can be used economically with vaccines formulated in 1.0 or 0.5 ml ampoules. The 4-site regimen now meets all requirements of immunogenicity for PEP and can be introduced without further studies.

Trial Registration

Controlled-Trials.com ISRCTN 30087513