The specific response of gypsy moth A1 neurosecretory neurons to different environmental stressors

Changes in the number and morphometric parameters of A1 neurosecretory neurons (nsn) were analyzed in Lymantria dispar 4^th instar caterpillars, exposed for 3 days to different stressors: cadmium, high temperature and tannic acid. The relative cytoplasm density of A1 nsn was also estimated. Caterpillars reared on a diet supplemented with cadmium exhibited increased size of A1 nuclei (10 and 250 μg Cd per g of dry food weight), increased number of nucleolii in nuclei and raised relative cytoplasm density in all experimental groups. Cadmium obviously induces intensive synthetic activity in A1 nsn. The second stressor was high environmental temperature of 35°C. Decrease of all analyzed morphometric parameters suggests that acute exposure of 4^th instar caterpillars to 35°C, as well as 12 h recovery at optimal temperature of 23°C, reduced the activity of A1 nsn. Tannic acid was added to the artificial diet in the following concentrations: 1%, 2.5% and 5%. All estimated morphological parameters did not change after addition 1 and 2.5% of tannic acid. After addition of 5% of tannic acid, the activity of A1 nsn declined.     

Clearance of Measles Virus from Persistently Infected Cells by Short Hairpin RNA

Subacute sclerosing panencephalitis (SSPE) is a demyelinating central nervous system disease caused by a persistent measles virus (MV) infection of neurons and glial cells. There is still no specific therapy available, and in spite of an intact innate and adaptive immune response, SSPE leads inevitably to death. In order to select effective antiviral short interfering RNAs (siRNAs), we established a plasmid-based test system expressing the mRNA of DsRed2 fused with mRNA sequences of single viral genes, to which certain siRNAs were directed. siRNA sequences were expressed as short hairpin RNA (shRNA) from a lentiviral vector additionally expressing enhanced green fluorescent protein (EGFP) as an indicator. Evaluation by flow cytometry of the dual-color system (DsRed and EGFP) allowed us to find optimal shRNA sequences. Using the most active shRNA constructs, we transduced persistently infected human NT2 cells expressing virus-encoded HcRed (piNT2-HcRed) as an indicator of infection. shRNA against N, P, and L mRNAs of MV led to a reduction of the infection below detectable levels in a high percentage of transduced piNT2-HcRed cells within 1 week. The fraction of virus-negative cells in these cultures was constant over at least 3 weeks posttransduction in the presence of a fusion-inhibiting peptide (Z-Phe-Phe-Gly), preventing the cell fusion of potentially cured cells with persistently infected cells. Transduced piNT2 cells that lost HcRed did not fuse with underlying Vero/hSLAM cells, indicating that these cells do not express viral proteins any more and are “cured.” This demonstrates in tissue culture that NT2 cells persistently infected with MV can be cured by the transduction of lentiviral vectors mediating the long-lasting expression of anti-MV shRNA.The neurodegenerative human disease subacute sclerosing panencephalitis (SSPE) occurs with an incidence rate of approximately 1:10,000 after infection with wild-type measles virus (MV) (4, 38). The course of the illness is quite variable, usually lasting from 1 to 3 years. Much more rapid forms that lead to death within a few months as well as prolonged courses with a duration of more than 20 years have been described (40). Neuropathological findings include diffuse encephalitis, affecting both the gray and white matters, characterized by perivascular cuffing and diffuse lymphocytic infiltrations. Neurons, oligodendrocytes, fibrous astrocytes, and some brain microvascular endothelial cells contain large aggregates of intranuclear inclusion bodies consisting of MV nucleocapsid structures (1, 16). In these persistently infected cells, viral ribonucleoprotein particles (RNPs) replicate intracellularly, whereas the budding of complete viruses and cell-cell fusion are not observed. A characteristic feature of this central nervous system disease is that the expression of viral envelope proteins (matrix [M], fusion [F], and hemagglutinin [H] proteins) is restricted by various means. In particular, the M protein and the cytoplasmic part of the F protein harbor single or hypermutations or deletions, which prevent their proper expression (2, 3, 9, 10). The lack of M reduces budding, supports cell fusion, and enhances the intracellular replication of RNPs (7, 8, 32, 37). As far as is known, the cell-to-cell spread of infectivity in the human brain occurs in the presence of normal cellular and strong humoral antiviral immune responses with very high anti-MV antibody titers in the cerebrospinal fluid. This, however, cannot prevent virus spread.A variety of approaches to the treatment of SSPE have been attempted, but an evaluation of their efficiency has been extremely difficult, since clinical trials are based on small numbers of patients, the course of SSPE is highly variable, and spontaneous remissions may also occur. Intrathecal or intraventricular administration of alpha interferon, inosiplex, and/or ribavirin is a common regimen, but despite many efforts, the establishment of an effective therapy has not been possible. Since the immune systems of the patients appear normal, and given the fact that virus spreads in the form of intracellular RNPs, a promising specific therapy must target this intracellular replication of MV.RNA interference (RNAi) may provide such a means and has already been used successfully to inhibit the expression of a number of viral infections, including the Ebola, influenza A, hepatitis B and C, human immunodeficiency, respiratory syncytial, and West Nile viruses, and several RNAi-based therapeutics are already in preclinical test phases (for reviews, see references 6 and 24). Small interfering RNAs (siRNAs) have also been described to be active against MV (20, 29, 32), including an MV isolate from an SSPE patient (SSPE-Kobe-1) (28). In the latter approach, the authors generated recombinant adenoviruses (rAdV) expressing siRNA against MV L mRNA and assessed them in freshly infected Vero/SLAM cells. In contrast to this work, we constructed lentiviral vectors expressing short hairpin RNAs (shRNAs) and transduced persistently infected human NT2 cells with these vectors. This lentiviral approach provided the proof of principle that a preexisting persistent MV infection can be cured by shRNA.     

A method for detecting the effect of magnetic field on activity changes of neuronal populations of Morimus funereus (Coleoptera, Cerambycidae)

Modification of a new method for detecting changes in the activities of neuronal population and the nearest neuron is described. Preliminary measurements of the influence of a static magnetic field (2 mT) on neuronal population activity on eight individuals of an endangered insect species Morimus funereus are included. Five minutes exposure produced both excitatory (5/8) and inhibitory (3/8) effect on the activity of neuronal population of M. funereus antennal lobe. However, when the reversibility of induced effects was quantitatively analyzed, our results showed that they were prevailingly irreversible: (7/8) for the population, (6/8) for the nearest neuron.     

Foxp3+ regulatory T cells control persistence of viral CNS infection

We earlier established a model of a persistent viral CNS infection using two week old immunologically normal (genetically unmodified) mice and recombinant measles virus (MV). Using this model infection we investigated the role of regulatory T cells (Tregs) as regulators of the immune response in the brain, and assessed whether the persistent CNS infection can be modulated by manipulation of Tregs in the periphery. CD4(+) CD25(+) Foxp3(+) Tregs were expanded or depleted during the persistent phase of the CNS infection, and the consequences for the virus-specific immune response and the extent of persistent infection were analyzed. Virus-specific CD8(+) T cells predominantly recognising the H-2D(b)-presented viral hemagglutinin epitope MV-H(22-30) (RIVINREHL) were quantified in the brain by pentamer staining. Expansion of Tregs after intraperitoneal (i.p.) application of the superagonistic anti-CD28 antibody D665 inducing transient immunosuppression caused increased virus replication and spread in the CNS. In contrast, depletion of Tregs using diphtheria toxin (DT) in DEREG (depletion of regulatory T cells)-mice induced an increase of virus-specific CD8(+) effector T cells in the brain and caused a reduction of the persistent infection. These data indicate that manipulation of Tregs in the periphery can be utilized to regulate virus persistence in the CNS.     

Effect of magnetic fields on antioxidative defense and fitness-related traits of Baculum extradentatum (insecta, phasmatodea)

This study aimed to determine the effect of magnetic fields on the antioxidative defense and fitness-related traits of Baculum extradentatum. Following exposure to magnetic fields, antioxidative defense (superoxide dismutase (SOD), catalase (CAT) activities, and total glutathione (GSH) content) and fitness-related traits (egg mortality, development dynamics, and mass of nymphs) were monitored in nymphs. The experimental groups were: control (kept out of influence of the magnets), a group exposed to a constant magnetic field (CMF) of 50 mT, and a group exposed to an alternating magnetic field (AMF) of 50 Hz, 6 mT. We found increased SOD and CAT activities in animals exposed to constant and AMFs, whereas GSH activity was not influenced by experimental magnetic fields. No differences were found in egg mortality between control and experimental groups. Significant differences in the time of development between the control and the CMF group were observed, as well as between the CMF and the AMF group. No differences were found in the mass of the nymphs between the three experimental groups. In conclusion, CMF and AMF have the possibility to modulate the antioxidative defense and some of the fitness-related traits in B. extradentatum.     

Spontaneously beating neonatal rat heart myocyte culture-a model to characterize angiotensin II at(1) receptor autoantibodies in patients with preeclampsia

This report focuses on angiotensin II AT(1) receptor autoantibodies (anti-AT(1)-AABs) in preeclamptic women. An enzyme-linked immunosorbent assay was described. Biotinylated peptide was incubated with anti-AT(1)-AABs. Streptavidin-coated magnetic particles bind the protein-autoantibody complex. Detection of anti-AT(1)-AABs was performed using anti-human IgG3 peroxidase-coupled antibody. The color reaction of tetramethylbenzidine solution was stopped by adding 0.5 M H(2)SO(4). Optical density was measured at 450 nm (620 nm reference filter). Seventy-nine percent of anti-AT(1)-AAB-positive patients (measured by bioassay) showed an increase in optical density (>145%). The same biotinylated peptide was successfully used for purification of 6/6 anti-AT(1)-AABs. Chronotropic effects of purified antibodies were registered on primary cultured neonatal rat cardiomyocytes with the computer imaging system IMAGOQUANT. Western blot of coimmunoprecipitation of angiotensin II AT(1) receptor shows one band (molecular weight >40.0 kDa) in potassium thiocyanate eluate.     

The embryonic and post-embryonic development in two Drosophila species exposed to the static magnetic field of 60 mT

In this study, a static magnetic field influence on development and viability in two different species, Drosophila melanogaster and Drosophila hydei, was investigated. Both species completed development (egg-adult), in and out of the static magnetic field induced by double horseshoe magnet. Treated vials with eggs were placed in the gap between magnetic poles (47 mm) and exposed to the average magnetic induction of 60 mT, while control ones were kept far enough from magnetic field source. We found that exposure to the static magnetic field reduced development time in both species, but statistical significance was found only for D. hydei. Furthermore, we found that the average viability of both Drosophila species exposed to the magnetic field was significantly weaker compared to control ones. These results indicate that 60 mT static magnetic field could be considered as a potential stressor, influencing on different levels the embryonic and post-embryonic development of individuals.