Hypertonic Saline Reduces Vascular Leakage in a Mouse Model of Severe Dengue

Dengue (DEN) is a mosquito-borne viral disease and represents a serious public health threat and an economical burden throughout the tropics. Dengue clinical manifestations range from mild acute febrile illness to severe DEN hemorrhagic fever/DEN shock syndrome (DHF/DSS). Currently, resuscitation with large volumes of isotonic fluid remains the gold standard of care for DEN patients who develop vascular leakage and shock. Here, we investigated the ability of small volume of hypertonic saline (HTS) suspensions to control vascular permeability in a mouse model of severe DEN associated with vascular leakage. Several HTS treatment regimens were considered and our results indicated that a single bolus of 7.5% NaCl at 4 mL per kg of body weight administered at the onset of detectable vascular leakage rapidly and significantly reduced vascular leak for several days after injection. This transient reduction of vascular leakage correlated with reduced intestine and liver damage with restoration of the hepatic functions, and resulted in delayed death of the infected animals. Mechanistically, we showed that HTS did not directly impact on the viral titers but resulted in lower immune cells counts and decreased systemic levels of soluble mediators involved in vascular permeability. In addition, we demonstrated that neutrophils do not play a critical role in DEN-associated vascular leakage and that the therapeutic effect of HTS is not mediated by its impact on the neutrophil counts. Together our data indicate that HTS treatment can transiently but rapidly reduce dengue-associated vascular leakage, and support the findings of a recent clinical trial which evaluated the efficacy of a hypertonic suspension to impact on vascular permeability in DSS children.     

Preprotachykinin-A gene products are key mediators of lung injury in polymicrobial sepsis

Preprotachykinin-A (PPT-A) gene products substance P and neurokinin-A have been shown to play an important role in neurogenic inflammation. To investigate the role of PPT-A gene products in lung injury in sepsis, polymicrobial sepsis was induced by cecal ligation and puncture in PPT-A gene-deficient mice (PPT-A(-/-)) and the wild-type control mice (PPT-A(+/+)). PPT-A gene deletion significantly protected against mortality, delayed the onset of lethality, and improved the long-term survival following cecal ligation and puncture-induced sepsis. PPT-A(-/-) mice also had significantly attenuated inflammation and damage in the lungs. The data suggest that deletion of the PPT-A gene may have contributed to the disruption in recruitment of inflammatory cells resulting in protection against tissue damage, as in these mice the sepsis-associated increase in chemokine levels is significantly attenuated.     

Activation of neurokinin-1 receptors up-regulates substance P and neurokinin-1 receptor expression in murine pancreatic acinar cells

Acute pancreatitis (AP) has been associated with an up-regulation of substance P (SP) and neurokinin-1 receptor (NK1R) in the pancreas. Increased SP-NK1R interaction was suggested to be pro-inflammatory during AP. Previously, we showed that caerulein treatment increased SP/NK1R expression in mouse pancreatic acinar cells, but the effect of SP treatment was not evaluated. Pancreatic acinar cells were obtained from pancreas of male swiss mice (25–30 g). We measured mRNA expression of preprotachykinin-A (PPTA) and NK1R following treatment of SP (10⁻⁶M). SP treatment increased PPTA and NK1R expression in isolated pancreatic acinar cells, which was abolished by pretreatment of a selective NK1R antagonist, CP96,345. SP also time dependently increased protein expression of NK1R. Treatment of cells with a specific NK1R agonist, GR73,632, up-regulated SP protein levels in the cells. Using previously established concentrations, pre-treatment of pancreatic acinar cells with Gö6976 (10 nM), rottlerin (5 μM), PD98059 (30 μM), SP600125 (30 μM) or Bay11-7082 (30 μM) significantly inhibited up-regulation of SP and NK1R. These observations suggested that the PKC-ERK/JNK-NF-κB pathway is necessary for the modulation of expression levels. In comparison, pre-treatment of CP96,345 reversed gene expression in SP-induced cells, but not in caerulein-treated cells. Overall, the findings in this study suggested a possible auto-regulatory mechanism of SP/NK1R expression in mouse pancreatic acinar cells, via activation of NK1R. Elevated SP levels during AP might increase the occurrence of a positive feedback loop that contributes to abnormally high expression of SP and NK1R.     

Nephrocystin-1 interacts directly with Ack1 and is expressed in human collecting duct

Nephronophthisis is characterised by renal fibrosis, tubular basement membrane disruption and corticomedullary cyst formation leading to end stage renal failure. Mutations in NPHP1 account for the underlying genetic defect in 25% of patients with nephronophthisis. Loss of urine concentration ability may be an early feature of nephronophthisis. Using yeast-2-library screening with the SH3 domain of nephrocystin-1 as bait, we identify Ack1 as a novel interaction partner. This interaction is confirmed using exogenous over-expression followed by co-immunoprecipitation. Ack1 is an activated Cdc42-associated kinase, and like nephrocystin-1, is a known interactor of p130Cas. Nephrocystin-1 partially colocalises with Ack1 at cell-cell contacts in IMCD3 cells. In human kidney, nephrocystin-1 expression is limited to cell-cell junctions in renal collecting duct cells. These data define Ack1 as a novel interaction partner of nephrocystin-1 and implicate cell-cell junctions and the renal collecting duct in the pathology of nephronophthisis.     

Identification of a novel family of proteins in snake venoms. Purification and structural characterization of nawaprin from Naja nigricollis snake venom

The three-dimensional structure of nawaprin has been determined by nuclear magnetic resonance spectroscopy. This 51-amino acid residue peptide was isolated from the venom of the spitting cobra, Naja nigricollis, and is the first member of a new family of snake venom proteins referred to as waprins. Nawaprin is relatively flat and disc-like in shape, characterized by a spiral backbone configuration that forms outer and inner circular segments. The two circular segments are held together by four disulfide bonds, three of which are clustered at the base of the molecule. The inner segment contains a short antiparallel beta-sheet, whereas the outer segment is devoid of secondary structures except for a small turn or 310 helix. The structure of nawaprin is very similar to elafin, a human leukocyte elastase-specific inhibitor. Although substantial parts of the nawaprin molecule are well defined, the tips of the outer and inner circular segments, which are hypothesized to be critical for binding interactions, are apparently disordered, similar to that found in elafin. The amino acid residues in these important regions in nawaprin are different from those in elafin, suggesting that nawaprin is not an elastase-specific inhibitor and therefore has a different function in the snake venom.     

Simple liquid chromatographic method for the determination of physostigmine and its metabolite eseroline in rat plasma: application to a pharmacokinetic study

Physostigmine, an anticholinergic drug, and its metabolite eseroline can be quantitated by high-performance liquid chromatography (HPLC) with photodiode-array detection. After addition of the structurally related internal standard (-)-N-methylphysostigmine, rat plasma samples were extracted and cleaned using a Varian Bond Elut C(18) column. The methanol-ammonia (98:2) eluate was evaporated to dryness and reconstituted with 0.01 M sodium dihydrogenphosphate (pH 3). Physostigmine and eseroline were separated on an Alltech Ultrasphere Silica column (250x4.6 mm I.D.; particle size 5 micrometer) at a flow-rate of 1 ml/min, with a mobile phase of 0.01 M sodium dihydrogenphosphate (pH 3)-acetonitrile (85:15). The limits of detection were 10 and 25 ng/ml for physostigmine and eseroline, respectively; the signal-to-noise ratio for this concentration was approximately 3:1. Spiked rat plasma containing 0.1-2.5 microgram/ml of physostigmine and eseroline gives good linearity. The average percentage recovery from five spiked plasma samples was 88.0+/-2.9 and 61.1+/-5.6% for physostigmine and eseroline, respectively. Within the concentration range 0.1-2.5 microgram/ml, the within-day precision was 1.9-8.3 and 3.0-7.7% for physostigmine and eseroline, respectively, and the between-day precision was 4.1-9.3 and 3.7-11% for physostigmine and eseroline, respectively. The method is rapid, simple and reliable, thus it is suitable for pharmacokinetic studies in the rat.     

Chemokines in acute respiratory distress syndrome

A characteristic feature of all inflammatory disorders is the excessive recruitment of leukocytes to the site of inflammation. The loss of control in trafficking these cells contributes to inflammatory diseases. Leukocyte recruitment is a well-orchestrated process that includes several protein families including the large cytokine subfamily of chemotactic cytokines, the chemokines. Chemokines and their receptors are involved in the pathogenesis of several diseases. Acute lung injury that clinically manifests as acute respiratory distress syndrome (ARDS) is caused by an uncontrolled systemic inflammatory response resulting from clinical events including major surgery, trauma, multiple transfusions, severe burns, pancreatitis, and sepsis. Systemic inflammatory response syndrome involves activation of alveolar macrophages and sequestered neutrophils in the lung. The clinical hallmarks of ARDS are severe hypoxemia, diffuse bilateral pulmonary infiltrates, and normal intracardiac filling pressures. The magnitude and duration of the inflammatory process may ultimately determine the outcome in patients with ARDS. Recent evidence shows that activated leukocytes and chemokines play a key role in the pathogenesis of ARDS. The expanding number of antagonists of chemokine receptors for inflammatory disorders may hold promise for new medicines to combat ARDS.     

A defect in lineage fate decision during fetal thymic invariant NKT cell development may regulate susceptibility to type 1 diabetes

A numerical and functional deficiency in invariant NKT (iNKT) cells detectable by 3 wk of age in the thymus and spleen mediates the pathogenesis of type 1 diabetes in NOD mice, but the stage of T cell development at which this deficiency first occurs is unknown. We report in this study that this deficiency develops after the CD4(+)CD8(+) double-positive stage of thymic T cell development and is due to a lineage-specific depletion of CD4(-)CD8(-) double-negative alphabeta T cells and iNKT cells from the thymus between embryonic day 18 and day 1 after birth. Thus, an inheritable defect in a lineage fate decision that elicits a deficiency in fetal thymic iNKT cell development may predispose to susceptibility to type 1 diabetes.     

The control of gene conversion properties and corresponding-site interference: the effects of conversion control factor 5 on conversion at locus w9 in Ascobolus immersus

The controls of various aspects and parameters of gene conversion at locus w-9 in the fungus Ascobolus immersus were investigated, along with positive and negative corresponding-site interference in meiotic chromatid pairing. When conversion control factor 5 alleles A and B were heterozygous, conversion frequencies at the closely linked target locus w9 were reduced to 3%, compared with 10.7% when A or B was homozygous, through effects on hybrid-DNA (h-DNA) formation parameters gamma1 and gamma2. In different ways, not related to whether ccf-5 alleles were homozygous or heterozygous, ccf-5 also affected parameters relating to the relative frequencies of asymmetric and symmetric h-DNA, the frequency with which the chromatid bearing the wild-type allele was the invading chromatid in asymmetric h-DNA, and h-DNA correction parameters for the frequency and direction of correction of mispairs. Corresponding-site interference is interference between the two pairs of non-sister chromatids of a bivalent in h-DNA formation at exactly corresponding sites. This interference was positive in the high conversion frequency crosses homozygous for ccf-5 alleles but was strongly negative in the low conversion frequency crosses heterozygous for ccf-5 alleles, through differential effects on parameters gamma1 and gamma2. Models of chromatid pairing are discussed.