A New ELISA Using the ANANAS Technology Showing High Sensitivity to diagnose the Bovine Rhinotracheitis from Individual Sera to Pooled Milk

Diagnostic tests for veterinary surveillance programs should be efficient, easy to use and, possibly, economical. In this context, classic Enzyme linked ImmunoSorbent Assay (ELISA) remains the most common analytical platform employed for serological analyses. The analysis of pooled samples instead of individual ones is a common procedure that permits to certify, with one single test, entire herds as “disease-free”. However, diagnostic tests for pooled samples need to be particularly sensitive, especially when the levels of disease markers are low, as in the case of anti-BoHV1 antibodies in milk as markers of Infectious Bovine Rhinotracheitis (IBR) disease. The avidin-nucleic-acid-nanoassembly (ANANAS) is a novel kind of signal amplification platform for immunodiagnostics based on colloidal poly-avidin nanoparticles that, using model analytes, was shown to strongly increase ELISA test performance as compared to monomeric avidin. Here, for the first time, we applied the ANANAS reagent integration in a real diagnostic context. The monoclonal 1G10 anti-bovine IgG1 antibody was biotinylated and integrated with the ANANAS reagents for indirect IBR diagnosis from pooled milk mimicking tank samples from herds with IBR prevalence between 1 to 8%. The sensitivity and specificity of the ANANAS integrated method was compared to that of a classic test based on the same 1G10 antibody directly linked to horseradish peroxidase, and a commercial IDEXX kit recently introduced in the market. ANANAS integration increased by 5-fold the sensitivity of the 1G10 mAb-based conventional ELISA without loosing specificity. When compared to the commercial kit, the 1G10-ANANAS integrated method was capable to detect the presence of anti-BHV1 antibodies from bulk milk of gE antibody positive animals with 2-fold higher sensitivity and similar specificity. The results demonstrate the potentials of this new amplification technology, which permits improving current classic ELISA sensitivity limits without the need for new hardware investments.     

Nitric oxide impairs mitochondrial movement in cortical neurons during hypoxia

Cortical nitric oxide (NO) production increases during hypoxia/ischemia in the immature brain and is associated with both neurotoxicity and mitochondrial dysfunction. Mitochondrial redistribution within the cell is critical to normal neuronal function, however, the effects of hypoxia on mitochondrial dynamics are not known. This study tested the hypothesis that hypoxia impairs mitochondrial movement via NO-mediated pathways. Fluorescently labeled mitochondria were studied using time-lapse digital video microscopy in cultured cortical neurons exposed either to hypoxia/re-oxygenation or to diethyleneamine/nitric oxide adduct, DETA-NO (100-500 microm). Two NO synthase inhibitors, were used to determine NO specificity. Mitochondrial mean velocity, the percentage of movement (i.e. the time spent moving) and mitochondrial morphology were analyzed. Exposure to hypoxia reduced mitochondrial movement to 10.4 +/- 1.3% at 0 h and 7.4 +/- 1.7% at 1 h of re-oxygenation, versus 25.6 +/- 1.4% in controls (p < 0.05). Mean mitochondrial velocity (microm s(-1)) decreased from 0.374 +/- 0.01 in controls to 0.146 +/- 0.01 at 0 h and 0.177 +/- 0.02 at 1 h of re-oxygenation (p < 0.001). Exposure to DETA-NO resulted in a significant decrease in mean mitochondrial velocity at all tested time points. Treatment with NG-nitro-L-arginine methyl ester (L-NAME) prevented the hypoxia-induced decrease in mitochondrial movement at 0 h (30.1 +/- 1.6%) and at 1 h (26.1 +/- 9%) of re-oxygenation. Exposure to either hypoxia/re-oxygenation or NO also resulted in the rapid decrease in mitochondrial size. Both hypoxia and NO exposure result in impaired mitochondrial movement and morphology in cultured cortical neurons. As the effect of hypoxia on mitochondrial movement and morphology can be partially prevented by a nitric oxide synthase (NOS) inhibitor, these data suggest that an NO-mediated pathway is at least partially involved.     

Cyclic ADP-ribose is a second messenger in the lipopolysaccharide-stimulated activation of murine N9 microglial cell line

Lipopolysaccharide, the main component of the cell wall of Gram-negative bacteria, is known to activate microglial cells following its interaction with the CD14/Toll-like receptor complex (TLR-4). The activation pathway triggered by lipopolysaccharide in microglia involves enhanced basal levels of intracellular calcium ([Ca2+]i) and terminates with increased generation of cytokines/chemokines and nitric oxide. Here we demonstrate that in lipopolysaccharide-stimulated murine N9 microglial cells, cyclic ADP-ribose, a universal and potent Ca2+ mobiliser generated from NAD+ by ADP-ribosyl cyclases (ADPRC), behaves as a second messenger in the cell activation pathway. Lipopolysaccharide induced phosphorylation, mediated by multiple protein kinases, of the mammalian ADPRC CD38, which resulted in significantly enhanced ADPRC activity and in a 1.7-fold increase in the concentration of intracellular cyclic ADP-ribose. This event was paralleled by doubling of the basal [Ca2+]i levels, which was largely prevented by the cyclic ADP-ribose antagonists 8-Br-cyclic ADP-ribose and ryanodine (by 75% and 88%, respectively). Both antagonists inhibited, although incompletely, functional events downstream of the lipopolysaccharide-induced microglia-activating pathway, i.e. expression of inducible nitric oxide synthase, overproduction and release of nitric oxide and of tumor necrosis factor alpha. The identification of cyclic ADP-ribose as a key signal metabolite in the complex cascade of events triggered by lipopolysaccharide and eventually leading to enhanced generation of pro-inflammatory molecules may suggest a new therapeutic target for treatment of neurodegenerative diseases related to microglia activation.     

Specific and selective probes for Pseudomonas aeruginosa from phage-displayed random peptide libraries

The design of novel biosensors for the detection of biological threats, such as Pseudomonas aeruginosa, requires probes that specifically bind biological agents and insure their immediate and efficient recognition. Advanced bio-selective sensors may meet the requests for isolation, concentration of the agents and their real-time detection. There is a need for robust and inexpensive affinity probes alternative to antibodies. These probes may be recruited from random peptide libraries displayed on filamentous phage. In this study, we identified from two phage-displayed random peptide libraries phage clones displaying peptides capable of specific and strong binding to P. aeruginosa cell surface. The ability of the phage clones to interact specifically with P. aeruginosa was demonstrated by using enzyme-linked immunosorbent assay (ELISA). We assessed selectivity of phage-bacteria-binding by comparing the binding ability of the selected clones to the selector bacterium and a panel of other bacterial species; we also demonstrated by dot spot and immunoblotting that the most reactive and selective phage peptide bound with high avidity the bacterial cell surface. In addition, as proof-of-concept, we tested the possibility to immobilize the affinity-selected phage to a putative biosensor surface. The quality of phage deposition was monitored by ELISA, and phage-bacterial-binding was confirmed by high-power optical phase contrast microscopy. Overall, the results of this work validate the concept of affinity-selected recombinant filamentous phages as probes for detecting and monitoring bacterial agents under any conditions that warrant their recognition, including clinical-based diagnostics and possibly biological warfare applications.     

Adenylic dinucleotides produced by CD38 are negative endogenous modulators of platelet aggregation

Diadenosine 5',5'-P1,P2-diphosphate (Ap2A) is one of the adenylic dinucleotides stored in platelet granules. Along with proaggregant ADP, it is released upon platelet activation and is known to stimulate myocyte proliferation. We have previously demonstrated synthesis of Ap2A and of two isomers thereof, called P18 and P24, from their high pressure liquid chromatography retention time, by the ADP-ribosyl cyclase CD38 in mammalian cells. Here we show that Ap2A and its isomers are present in resting human platelets and are released during thrombin-induced platelet activation. The three adenylic dinucleotides were identified by high pressure liquid chromatography through a comparison with the retention times and the absorption spectra of purified standards. Ap2A, P18, and P24 had no direct effect on platelet aggregation, but they inhibited platelet aggregation induced by physiological agonists (thrombin, ADP, and collagen), with mean IC(50) values ranging between 5 and 15 mum. Moreover, the three dinucleotides did not modify the intracellular calcium concentration in resting platelets, whereas they significantly reduced the thrombin-induced intracellular calcium increase. Through binding to the purinergic receptor P2Y(11), exogenously applied Ap2A, P18, and P24 increased the intracellular cAMP concentration and stimulated platelet production of nitric oxide, the most important endogenous antiaggregant. The presence of Ap2A, P18, and P24 in resting platelets and their release during thrombin-induced platelet activation at concentrations equal to or higher than the respective IC(50) value on platelet aggregation suggest a role of these dinucleotides as endogenous negative modulators of aggregation.     

Osservazioni ultrastrutturali su particolari organelli polimembranosi in Euglena gracilis

Abstract

Ultrastructural observations on polymembranous organelles in Euglena gracilis. Polymembranous organelles, similar to hypertrophied mitochondria, seen in the cytoplasm of bleached or photosynthetic Euglena gracilis cells are presented.     

Regulation of PCDH15 function in mechanosensory hair cells by alternative splicing of the cytoplasmic domain

Protocadherin 15 (PCDH15) is expressed in hair cells of the inner ear and in photoreceptors of the retina. Mutations in PCDH15 cause Usher Syndrome (deaf-blindness) and recessive deafness. In developing hair cells, PCDH15 localizes to extracellular linkages that connect the stereocilia and kinocilium into a bundle and regulate its morphogenesis. In mature hair cells, PCDH15 is a component of tip links, which gate mechanotransduction channels. PCDH15 is expressed in several isoforms differing in their cytoplasmic domains, suggesting that alternative splicing regulates PCDH15 function in hair cells. To test this model, we generated three mouse lines, each of which lacks one out of three prominent PCDH15 isoforms (CD1, CD2 and CD3). Surprisingly, mice lacking PCDH15-CD1 and PCDH15-CD3 form normal hair bundles and tip links and maintain hearing function. Tip links are also present in mice lacking PCDH15-CD2. However, PCDH15-CD2-deficient mice are deaf, lack kinociliary links and have abnormally polarized hair bundles. Planar cell polarity (PCP) proteins are distributed normally in the sensory epithelia of the mutants, suggesting that PCDH15-CD2 acts downstream of PCP components to control polarity. Despite the absence of kinociliary links, vestibular function is surprisingly intact in the PCDH15-CD2 mutants. Our findings reveal an essential role for PCDH15-CD2 in the formation of kinociliary links and hair bundle polarization, and show that several PCDH15 isoforms can function redundantly at tip links.     

Synergistic interactions between HDAC and sirtuin inhibitors in human leukemia cells

Aberrant histone deacetylase (HDAC) activity is frequent in human leukemias. However, while classical, NAD(+)-independent HDACs are an established therapeutic target, the relevance of NAD(+)-dependent HDACs (sirtuins) in leukemia treatment remains unclear. Here, we assessed the antileukemic activity of sirtuin inhibitors and of the NAD(+)-lowering drug FK866, alone and in combination with traditional HDAC inhibitors. Primary leukemia cells, leukemia cell lines, healthy leukocytes and hematopoietic progenitors were treated with sirtuin inhibitors (sirtinol, cambinol, EX527) and with FK866, with or without addition of the HDAC inhibitors valproic acid, sodium butyrate, and vorinostat. Cell death was quantified by propidium iodide cell staining and subsequent flow-cytometry. Apoptosis induction was monitored by cell staining with FITC-Annexin-V/propidium iodide or with TMRE followed by flow-cytometric analysis, and by measuring caspase3/7 activity. Intracellular Bax was detected by flow-cytometry and western blotting. Cellular NAD(+) levels were measured by enzymatic cycling assays. Bax was overexpressed by retroviral transduction. Bax and SIRT1 were silenced by RNA-interference. Sirtuin inhibitors and FK866 synergistically enhanced HDAC inhibitor activity in leukemia cells, but not in healthy leukocytes and hematopoietic progenitors. In leukemia cells, HDAC inhibitors were found to induce upregulation of Bax, a pro-apoptotic Bcl2 family-member whose translocation to mitochondria is normally prevented by SIRT1. As a result, leukemia cells become sensitized to sirtuin inhibitor-induced apoptosis. In conclusion, NAD(+)-independent HDACs and sirtuins cooperate in leukemia cells to avoid apoptosis. Combining sirtuin with HDAC inhibitors results in synergistic antileukemic activity that could be therapeutically exploited.     

Abscisic Acid Released by Human Monocytes Activates Monocytes and Vascular Smooth Muscle Cell Responses Involved in Atherogenesis

Abscisic acid (ABA) is a phytohormone recently identified as a new endogenous pro-inflammatory hormone in human granulocytes. Here we report the functional activation of human monocytes and vascular smooth muscle cells by ABA. Incubation of monocytes with ABA evokes an intracellular Ca²⁺ rise through the second messenger cyclic ADP-ribose, leading to NF-κB activation and consequent increase of cyclooxygenase-2 expression and prostaglandin E₂ production and enhanced release of MCP-1 (monocyte chemoattractant protein-1) and of metalloprotease-9, all events reportedly involved in atherogenesis. Moreover, monocytes release ABA when exposed to thrombin-activated platelets, a condition occurring at the injured vascular endothelium; monocyte-derived ABA behaves as an autocrine and paracrine pro-inflammatory hormone-stimulating monocyte migration and MCP-1 release, as well as vascular smooth muscle cells migration and proliferation. These results, and the presence of ABA in human arterial plaques at a 10-fold higher concentration compared with normal arterial tissue, identify ABA as a new signal molecule involved in the development of atherosclerosis and suggest a possible new target for anti-atherosclerotic therapy.     

P2X7-mediated Increased Intracellular Calcium Causes Functional Derangement in Schwann Cells from Rats with CMT1A Neuropathy

Charcot-Marie-Tooth (CMT) is the most frequent inherited neuromuscular disorder, affecting 1 person in 2500. CMT1A, the most common form of CMT, is usually caused by a duplication of chromosome 17p11.2, containing the PMP22 (peripheral myelin protein-22) gene; overexpression of PMP22 in Schwann cells (SC) is believed to cause demyelination, although the underlying pathogenetic mechanisms remain unclear. Here we report an abnormally high basal concentration of intracellular calcium ([Ca²⁺]_i) in SC from CMT1A rats. By the use of specific pharmacological inhibitors and through down-regulation of expression by small interfering RNA, we demonstrate that the high [Ca²⁺]_i is caused by a PMP22-related overexpression of the P2X7 purinoceptor/channel leading to influx of extracellular Ca²⁺ into CMT1A SC. Correction of the altered [Ca²⁺]_i in CMT1A SC by small interfering RNA or with pharmacological inhibitors of P2X7 restores functional parameters of SC (migration and release of ciliary neurotrophic factor), which are typically defective in CMT1A SC. More significantly, stable down-regulation of the expression of P2X7 restores myelination in co-cultures of CMT1A SC with dorsal root ganglion sensory neurons. These results establish a pathogenetic link between high [Ca²⁺]_i and impaired SC function in CMT1A and identify overexpression of P2X7 as the molecular mechanism underlying both abnormalities. The development of P2X7 inhibitors is expected to provide a new therapeutic strategy for treatment of CMT1A neuropathy.Charcot-Marie-Tooth disease type 1 (CMT1)³ is a progressive hereditary motor and sensory neuropathy, characterized by distal muscle wasting and weakness, foot deformities, and severe slowing of nerve conduction, because of progressive demyelination (1). With a prevalence of 1 case in 2500, CMT1 is the most common hereditary neurologic disorder, and in the majority of cases (CMT1A) the disease is associated with a duplication on chromosome 17p11.2 of the gene for PMP22 (peripheral myelin protein 22) (2). PMP22 is a 22-kDa glycoprotein mainly expressed by myelinating Schwann cells (SC) and localized in compact myelin (3). The transgenic rat model of CMT1A, obtained by overexpression of PMP22 (4), confirms a role of PMP22 in the pathogenesis of CMT1A. Both PMP22 overexpression because of gene duplication and point mutations of PMP22 are associated with a CMT1A phenotype.The biochemical mechanisms correlating PMP22 dysfunction with demyelination are still unclear. Some reports indicate that a perturbed homeostasis of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) might be causally involved in the demyelination process. Conditions inducing an increased [Ca²⁺]_i in SC impair cell differentiation and myelination (5, 6), similarly to what occurs in CMT1A. Incubation of intact rat nerves with Ca²⁺ and ionophores causes a progressive demyelination, spreading from the paranodes and invading regions of formerly compact myelin, which is dependent upon a rise in the [Ca²⁺]_i of SC (5).Additional evidence for the detrimental effect of a [Ca²⁺]_i elevation on myelin production by SC comes from application of ATP to murine SC monocultures, inducing an immediate and large increase in the [Ca²⁺]_i. As a result of ATP treatment, maturation and differentiation of SC, as well as expression of the myelin basic protein and production of compact myelin, are completely prevented (6). Taken together, the above observations indicate that abnormally elevated Ca²⁺ levels are causally related to impairment of myelin production by SC.In this study, we addressed the possible correlation between PMP22 overexpression and alteration of the [Ca²⁺]_i homeostasis in SC from a rat model of CMT1A. We recorded higher levels of basal [Ca²⁺]_i in affected than in control cells, and we identified the mechanisms responsible for the perturbation of the [Ca²⁺]_i levels in CMT1A SC. Experiments with pharmacological inhibitors and with small interfering RNA (siRNA) unequivocally demonstrated a correlation in CMT1A SC between overexpression of the purinergic receptor P2X7 and influx of extracellular [Ca²⁺]_i across this plasma membrane receptor/channel. In addition, correction of the abnormally elevated [Ca²⁺]_i levels by the use of a P2X7 antagonist or through down-regulation of the expression of P2X7 by transfection with siRNA or with short hairpin RNA-expressing plasmid (shRNA) restored the normal phenotype in CMT1A SC. These findings suggest that CMT1A should be considered as a “calcium disease.” Identification of P2X7 activation as the pathogenetic mechanism underlying demyelination may provide the rationale for a new therapeutic strategy for CMT1A, a disease with no currently available treatment.