Influence of long-term equine herpesvirus type 1 (EHV-1) infection on primary murine neurons-the possible effects of the multiple passages of EHV-1 on its neurovirulence.

Equine herpesvirus 1 (EHV-1), like other members of the Alphaherpesvirinae subfamily, is a neurotropic virus causing latent infections in the nervous system of the natural host. In the present study, we have investigated EHV-1 replication (wild-type Jan-E strain and Rac-H laboratory strain) during long-term infection and during the passages of the virus in cultured neurons. The studies were performed on primary murine neurons, which are an excellent in vitro model for studying neurotropism and neurovirulence of EHV-1. Using real-time cell growth analysis, we have demonstrated for the first time that primary murine neurons are able to survive long-term EHV-1 infection. Positive results of real-time PCR test indicated a high level of virus DNA in cultured neurons, and during long-term infection, these neurons were still able to transmit the virus to the other cells. We also compared the neurovirulence of Rac-H and Jan-E EHV-1 strains after multiple passages of these strains in neuron cell culture. The results showed that multiple passages of EHV-1 in neurons lead to the inhibition of viral replication as early as in the third passage. Interestingly, the inhibition of the EHV-1 replication occurred exclusively in neurons, because the equine dermal (ED) cells co-cultivated with neuroculture medium from the third passage showed the presence of large amount of viral DNA. In conclusion, our results showed that certain balance between EHV-1 and neurons has been established during in vitro infection allowing neurons to survive long-term infection.

Replication kinetics of neuropathogenic and non-neuropathogenic equine herpesvirus type 1 (EHV-1) strains in primary murine neurons and ED cell line.

Equine herpesvirus type 1 (EHV-1) causes respiratory infections, abortion and neurological disorders in horses. Molecular epidemiology studies have demonstrated that a single-point mutation in DNA polymerase gene, resulting in an amino acid variation (N752/D752), is significantly associated with the neuropathogenic potential of EHV-1 strains. The aim of the study was to elucidate if there are any differences between neuropathogenic (EHV-1 26) and non-neuropathogenic (Jan-E and Rac-H) EHV-1 strains in their ability to infect neuronal cells. For the tested EHV-1 strains, cytopathic effect (CPE) was manifested by changed morphology of cells, destruction of actin cytoskeleton and nuclei degeneration, which led to focal degeneration. Moreover, EHV-1 26 strain caused fusion of the infected cells to form syncytia in culture. Real-time PCR analysis demonstrated that both neuropathogenic and non-neuropathogenic EHV-1 strains replicated in neurons and ED cells (equine dermal cell line) at a similar level. We can assume that a point mutation in the EHV-1 polymerase does not affect viral replication in this cell type.

Equid herpesvirus type 1 (EHV-1) disrupts actin cytoskeleton during productive infection in equine leukocytes.

Equid herpesvirus type 1 (EHV-1) is a prevalent causative agent of equine diseases worldwide. After primary replication in the respiratory epithelium the virus disseminates systemically through a peripheral blood mononuclear cell (PBMC)-associated viraemia. EHV-1 is the only alphaherpes- virus known so far which is capable of establishing latent infection not only in neurons but also in immune system cells (mainly in lymphocytes and macrophages). Since leukocytes are not the target cells for viral replication but are used to transport EHV-1 to the internal organs, the questionremains how the virus avoids the immune response and whether it could potentially be associated with virus-induced cytoskeletal rearrangements. Therefore, the aim of this study was to investigate the progress of EHV-1 replication in leukocytes stimulated by phytohemagglutinin and the impact of EHV-1 infection on the actin cytoskeleton. Using the real-time PCR method we evaluated the quantity of viral DNA from samples collected at indicated time points post infection. In order to examine possible changes in actin cytoskeleton organization due to EHV-1 infection, we performed immunofluorescent staining using TRITC-phalloidin conjugate. The results showed that EHV-1 rep- licates in leukocytes at a restricted level but with the accompaniment of chromatin degradation. Simultaneously, infection with EHV-1 caused disruption of the actin cytoskeleton; this was particularly apparent in further stages of infection. Disruption of the actin cytoskeleton may lead to the limited release of the virus from the cells, but may be also beneficial for the virus, since at the same time it potentially impairs the immune function of leukocytes.

Systematic review with meta-analysis: early infant feeding and coeliac disease--update 2015.

BACKGROUND: New evidence emerged on early feeding practices and the risk of coeliac disease. AIM: To systematically update evidence on these practices to find out whether there is a need to revise current recommendations. METHODS: MEDLINE, EMBASE and the Cochrane Library were searched from July 2012 (end of last search) to February 2015 for studies of any design that assessed the effect of gluten consumption and breastfeeding on the development of coeliac disease and/or coeliac disease-related autoimmunity. RESULTS: We identified 21 publications, including two, new, large, randomised controlled trials performed in high-risk infants. Exclusive or any breastfeeding, as well as breastfeeding at the time of gluten introduction, did not reduce the risk of developing coeliac disease during childhood. For infants at high risk of developing coeliac disease, gluten introduction at 4 months of age in very small amounts, or at 6 or 12 months of age, resulted in similar rates of coeliac disease diagnosis in early childhood. Later gluten introduction was associated with later development of coeliac specific autoimmunity and coeliac disease during childhood, but not total risk reduction. Observational studies indicate that consumption of a higher amount of gluten at weaning may increase the risk for coeliac disease development. CONCLUSIONS: Infant feeding practices (breastfeeding, time of gluten introduction) have no effect on the risk of developing coeliac disease during childhood (at least at specific timeframes evaluated in the included studies), necessitating an update of current European recommendations.

Meta-analysis: Lactobacillus reuteri strain DSM 17938 (and the original strain ATCC 55730) for treating acute gastroenteritis in children.

Lactobacillus reuteri ATCC 55730 has been shown to provide a moderate clinical effect in the treatment of acute gastroenteritis (AGE) in children. However, as the L. reuteri ATCC 55730 strain was found to carry potentially transferable resistance traits for tetracycline and lincomycin, it was replaced by a new strain, L. reuteri DSM 17938, without unwanted plasmid-borne antibiotic resistance. Bioequivalence of the two strains has been suggested. We aimed to systematically evaluate data on the effectiveness of L. reuteri DSM 17938 and the original strain, L. reuteri ATCC 55730, in the treatment of AGE in children. The Cochrane Library, MEDLINE, and EMBASE databases, reference lists, and abstract books of major scientific meetings were searched in August 2013, with no language restrictions, for relevant randomised controlled trials (RCTs). Two RCTs (n=196) that evaluated L. reuteri DSM 17938 and three RCTs (n=156) that evaluated L. reuteri ATCC 55730, which involved hospitalised children aged 3 to 60 months, met the inclusion criteria. Compared with placebo or no treatment, DSM 17938 significantly reduced the duration of diarrhoea (mean difference -32 h, 95% confidence interval (CI): -41 to -24) and increased the chance of cure on day 3 (relative risk: 3.5, 95% CI: 1.2 to 10.8, random effects model). Similar results were obtained with the original strain, L. reuteri ATCC 55730. In conclusion, in hospitalised children, use of both strains of L. reuteri reduced the duration of diarrhoea, and more children were cured within 3 days. Data from outpatients and countryspecific cost-effectiveness analyses are needed. Given the limited data and the methodological limitations of the included trials, the evidence should be viewed with caution.

Equine herpesvirus type 1 (EHV-1)-induced rearrangements of actin filaments in productively infected primary murine neurons.

Equine herpesvirus type 1 (EHV-1) causes respiratory disease, abortion and neurological disorders in horses. In the present study, we investigated reorganization of the cytoskeleton in neurons infected with two EHV-1 strains: Jan-E (wild-type strain) and Rac-H (attenuated strain). The studies were performed on primary murine neurons, which are an excellent model for studying neurotropism and neurovirulence of EHV-1. We have demonstrated for the first time that EHV-1 infection causes rearrangements in the actin network of neurons that are dependent on the virus strain and its adaptation to cell culture in vitro. Immunofluorescent labeling and confocal microscopy revealed the formation of long, thin projections in neurons infected with the Jan-E strain, which was probably associated with enhanced intracellular spread of the virus. The EHV-1 Rac-H strain caused disruption of the microfilaments system and general depolymerization of actin, but treatment of neurons with cytochalasin D or latrunculin A resulted in limitation of viral replication. It can therefore be assumed that actin filaments are required only at the early stages of infection. Our results allow us to suggest that the actin cytoskeleton participates in EHV-1 infection of primary murine neurons but is not essential, and that other components of the cytoskeleton and/or cellular mechanisms may be also involved during EHV-1 infection.

Systematic review: early infant feeding and the prevention of coeliac disease.

BACKGROUND: PREVENTCD, Prevent Coeliac Disease, is an international project investigating the hypothesis of possible induction of tolerance to gluten in genetically predisposed children through introducing small quantities of gluten during the period of breastfeeding. AIM: To summarise current knowledge on the possible relationship between early feeding practices and the risk of coeliac disease (CD). METHODS: The Cochrane Library, MEDLINE, and EMBASE databases were searched in May 2011, and the search was updated in January 2012, and again in July 2012. RESULTS: Breastfeeding (BF) and CD: some studies show a protective effect of BF, while others show no effect. No studies have shown a long-term preventive effect. BF at the time of gluten introduction and CD: Results from a meta-analysis of five observational case-control studies suggest that BF at gluten introduction is associated with a lower risk of CD compared with formula feeding. It is unclear whether BF provides a permanent protection or only delays the onset of CD. Timing of gluten introduction: The data suggest that both early (≤4 months) and late (≥7 months) introduction of gluten may increase the risk of CD. Amount of gluten at weaning (and later) and CD: One incident case-referent study documented that the introduction of gluten in large amounts compared with small or medium amounts increased the risk of CD. CONCLUSIONS: In the absence of clear evidence, in order to decrease the risk of later coeliac disease, it is reasonable to avoid both early (<4 months) and late (≥7 months) introduction of gluten, and to introduce gluten while the infant is still being breastfed. Future studies may clarify the remaining uncertainties.

The xCELLigence system for real-time and label-free analysis of neuronal and dermal cell response to equine herpesvirus type 1 infection.

Real-time cell electronic sensing (RT-CES) based on impedance measurements is an emerging technology for analyzing the status of cells in vitro. It allows label-free, real time monitoring of the biological status of cells. The present study was designed to assess dynamic data on the cell processes during equine herpesvirus type 1 (EHV-1) infection of ED (equine dermal) cells and primary murine neuronal cell culture. We have demonstrated that the xCELLigence system with dynamic monitoring can be used as a rapid diagnostic tool both to analyze cellular behavior and to investigate the effect of viral infection.

Molecular dynamics studies on the water mixtures of pharmaceutically important ionic liquid lidocaine HCl.

In this paper the molecular dynamics of a common local-anesthetic drug, lidocaine hydrochloride (LD-HCl), and its water mixtures were investigated. By means of broadband dielectric spectroscopy and calorimetric measurements it was shown that even a small addition of water causes a significant effect on the relaxation dynamics of analyzed protic ionic liquid. Apart from the two well-resolved relaxations (σ- and γ-processes) and the ß-mode, identified as the JG-process, observed for anhydrous LD-HCl, a new relaxation peak (υ) is visible in the dielectric spectra of aqueous mixtures of this drug. Additionally, the significant effect of the water on the glass transition temperature of LD-HCl was found. The sample characterized with mole fraction of water X(w) = 0.44 reveals the glass transition temperature T(g), 42 K lower than that of anhydrous material (307 K). Finally, it was shown that by amorphization of the hydrochloride salt of lidocaine it is possible to obtain its room temperature ionic liquid form.

Apoptotic and necrotic changes in cultured murine neurons infected with equid herpesvirus 1.

Equid herpesvirus 1 (EHV-1), like other members of the Alphaherpesvirinae, is a neurotropic virus, that causes latent infections in the nervous system of the natural host. All alphaherpesviruses have developed sophisticated strategies to interfere with the host cell apoptotic mechanisms, but the ability of EHV-1 to induce apoptosis in neurons has not been determined yet. In this study, apoptotic and necrotic changes in cultured murine neurons were methods identifying key stages of apoptosis. These methods have demonstrated characteristic apoptosis features, like DNA fragmentation, chromatin condensation, membrane blebbing and cell shrinkage in the infected cells. It seems likely that apoptosis was the predominant way of cell death in EHV-1-infected murine neurons. However, we showed also that during acute EHV-1 infection the majority of infected neurons remained unchanged and survived for more than eight weeks in culture, suggesting some protective mechanisms induced by the virus. Furthermore, it was shown that infection of neurons with EHV-1 has no significant influence on the level of the caspase 3, 7, and 8. We speculate that the control of apoptosis may be the key mechanism regulating the balance between productive and latent infection at the site of virus persistence.

Opposite effects of two different strains of equine herpesvirus 1 infection on cytoskeleton composition in equine dermal ED and African green monkey kidney Vero cell lines: application of scanning cytometry and confocal-microscopy-based image analysis in a quantitative study.

Viruses can reorganize the cytoskeleton and restructure the host cell transport machinery. During infection viruses use different cellular cues and signals to enlist the cytoskeleton for their mission. However, each virus specifically affects the cytoskeleton structure. Thus, the aim of our study was to investigate the cytoskeletal changes in homologous equine dermal (ED) and heterologous Vero cell lines infected with either equine herpesvirus 1 (EHV-1) strain Rac-H or Jan-E. We found that Rac-H strain disrupted actin fibers and reduced F-actin level in ED cells, whereas the virus did not influence Vero cell cytoskeleton. Conversely, the Jan-E strain induced polymerization of both F-actin and MT in Vero cells, but not in ED cells. Confocal-microscopy analysis revealed that alpha-tubulin colocalized with viral antigen in ED cells infected with either Rac-H or Jan-E viruses. Alterations in F-actin and alpha-tubulin were evaluated by confocal microscopy, Microimage analysis and scanning cytometry. This unique combination allowed precise interpretation of confocal-based images showing the cellular events induced by EHV-1. We conclude that examination of viral-induced pathogenic effects in species specific cell lines is more symptomatic than in heterologous cell lines.

Equine herpesvirus type 1 (EHV-1) replication in primary murine neurons culture.

Equine herpesvirus-1 (EHV-1) infections cause significant economic losses for equine industries worldwide as a result of abortion, respiratory illness, and neurologic disease in all breeds of horses. The occurrence of abortions caused by EHV-1 has repeatedly been confirmed in Poland, but neurological manifestations of the infection have not been described yet. Also it is unknown how the infection of neurons with non-neuropathogenic strains is regulated. To further understand the virus-neuron interaction we studied two strains of EHV-1 in murine primary neuron cell cultures. Both strains were isolated from aborted fetuses: Rac-H, a reference strain isolated by Woyciechowska in 1959 (Woyciechowska 1960) and Jan-E isolated by Banbura et al. (Banbura et al. 2000). Upon infection of primary murine neuronal cell cultures with Jan-E or Rac-H strains, a cytopathic effect was observed, manifested by a changed morphology and disintegration of the cell monolayer. Positive results of immunofluorescence, nPCR and real-time PCR tests indicated high virus concentration in neurons, meaning that both EHV-1 strains were likely to replicate in mouse neurons in vitro without the need for adaptation. Moreover, we demonstrated that some neurons may survive (limited) virus replication during primary infection, and these neurons (eight weeks p.i.) harbour EHV-1 and were still able to transmit infection to other cells.

Equine herpesvirus type 1 quantification in different types of samples by a real-time PCR.

Equine herpesvirus type 1 (EHV-1) is one of the major viral agents causing diseases in horses common worldwide. A variety of techniques, including PCR, have been used to diagnose EHV-1 infections. In this paper, an attempt of real-time PCR has been described, which uses specific fluorochrome-labeled TaqMan probes for detection of viral DNA. This method does not require post-amplification manipulations, thereby reducing the risk of cross-contamination. The assay was sensitive enough to detect EHV-1 sequences in different clinical samples, as well in mice neuronal cell cultures. The technique was also very specific--here was no cross reaction with other human and equine herpesviruses. Compared to previously used nested PCR technique, the test was more sensitive and should be useful for the common diagnosis based on its specificity and rapidity.

Replication of equine herpesvirus type 1 in equine dermal cells transfected with Bam HI[G] restriction fragment of EHV-2 genome.

In previous experiments, we have demonstrated that the presence of equine herpesvirus 2 (EHV-2) enhanced plaque formation in cell cultures infected with equine herpesvirus type 1. To determine whether a specific region of the EHV-2 genome is responsible for this effect, we have constructed a library of Bam HI fragments of the EHV-2 genome ligated into pcDNA plasmid. Equine dermal (ED) cell cultures were subsequently transfected with the constructs, passaged 5 times, tested for the presence of the plasmids and infected with EHV-1 at MOI = 0.01. Only in cultures transfected with the pcDNA/Bam HI[G]construct, designated delta2/4, the mean number of plaques at 24 hrs p.i. was approximately 10 times higher than in non-transfected controls. Virus titers in culture supernatants as well as in freeze-thawed cells were 4- and 5-fold higher, respectively, than in non-transfected cultures. These differences were observed only at 24 hrs p.i. At 48 hrs p.i. cultures were completely destroyed and, surprisingly, the virus titer was slightly lower in the supernatant of transfected cells. However, the titer of EHV-1 in freeze-thawed culture was exactly the same as in the control. These results suggest that the presence of Bam HI[G] fragment of the EHV-2 genome stimulates (accelerates?) plaque formation only at earlier stages of infection but does not influence the total yield of EHV-1 at 48 hrs p.i. The exact mechanism of this stimulation remains unclear and further experiments are necessary to determine the role of putative EHV-2 proteins encoded by Bam HI [G] fragment of the EHV-genome.

Mass versus molar doses, similarities and differences.

Generally, they are two systems expressing the amounts of active substance in a given drug product, i.e. mass and molar dose. Currently, the dose system based on the mass is widely used in which doses are expressed in grams or milligrams. On the other hand, the molar dose system is in direct relation to the number of molecules. Hence, the objective of this work was to compare both systems in order to find their advantages and disadvantages. Active substances belonging to the groups of antibiotics, nootropic agents, beta-blockers, vitamins, GABA-analog, COX-2 inhibitors, calcium channel antagonists, benzodiazepine receptor agonists, lipid-modifying agents (fibrates), non-steroidal anti-inflammatory drugs (profens), estrogens, neuroleptics, analgesics and benzodiazepines were considered. Moreover, products containing two active substances were also taken into account. These are mixtures of hydrochlorothiazide with active substances influencing the renin-angiotensin system and combined oral contraceptives. For each active substance, belonging to the groups mentioned above molar doses were calculated from mass doses and molar mass. Hence, groups of drugs with a single active substance, drugs with similar pharmacological activities, pharmaceutical alternatives, and drugs with a single active ingredient manufactured in different doses were compared in order to find which dose system describes more adequately differences between and within the groups mentioned above. Comparisons were supported by a number of equations, which theoretically justify the data, and relationships derived from calculations.

Systematic review: racecadotril in the treatment of acute diarrhoea in children.

BACKGROUND: Racecadotril (acetorphan) is an antisecretory drug that exerts its antidiarrhoeal effects by inhibiting intestinal enkephalinase. AIM: To summarize studies testing the efficacy and safety of racecadotril for treating children with acute gastroenteritis. METHODS: Reports were gathered by searching electronic databases MEDLINE, EMBASE, the Cochrane Library (all up to April 2007), relevant journals, and bibliographies of reviewed articles. Only randomized-controlled trials were included. RESULTS: Three randomized-controlled trials (471 participants) met the inclusion criteria. Two trials reported stool output, and data suggested less stool output in the racecadotril group than in the control group. The duration of diarrhoea was significantly reduced in the three trials reporting this outcome. Achievement of a cure by day 5 was similar in both groups. Adverse effects were similar in both groups. CONCLUSIONS: The small number of included trials provided some evidence in favour of the use of racecadotril over placebo or no intervention, to reduce the stool output and duration of diarrhoea in children with acute gastroenteritis. However, more data in out-patients are needed. The safety as well as the cost-effectiveness of the therapy should be explored, before routine therapy with racecadotril is recommended.

RP-HPLC method with electrochemical detection for the determination of metoclopramide in serum and its use in pharmacokinetic studies.

A rapid and sensitive reversed-phase high performance liquid chromatographic method has been developed for the determination of metoclopramide in serum. The assay was performed after single extraction with ethyl ether using methyl parahydroxybenzoate as internal standard. Chromatographic separations were performed on C(18) stationary phase with a mobile phase composed of methanol-phosphate buffer pH 3 (30:70 v/v). Analytes were detected electrochemically. The quantification limit for metoclopramide in serum was 2 ng mL(-1). Linearity of the method was confirmed in the range of 5-120 ng mL(-1) (correlation coefficient 0.9998). Within-day relative standard deviations (RSDs) ranged from 0.3 to 5.5% and between-day RSDs from 0.8 to 6.0%. The analytical method was successfully applied for the determination of pharmacokinetic parameters after ingestion of 10 mg dose of metoclopramide. Studies were performed on 18 healthy volunteers of both sexes.

[The influence of premedication with diclofenac or pethidine on pain and postop treatment in lumbar discopathy]. / Wplyw premedykacji petydyna lub diklofenakiem na ból i leczenie pooperacyjne u chorych z dyskopatia ledzwiowa.

206 patients scheduled for spinal surgery (lumbar discopathy) were randomly premedicated with diclofenac, pethidine, diazepam or hydroxizine. The frequency of persisted postoperative pain was evaluated from the 3-ed. postoperative day to the end of hospitalisation--as the need for additional concomitant treatment with dexamethasone and intravenous analgesics. The frequency of persisted pain was significantly decreased in patients premedicated with diclofenac (together with diazepam) before spinal surgery (limited to fenestration) in comparison to patients premedicated with pethidine. The pre-emptive analgesic effect of diclofenac was even more evident in patients treated with non-steroidal anti-inflammatory drugs (NSAID) before surgery, but was not observed in patients after more traumatic surgery (laminectomy) premedicated with diazepam. The results are supporting the important role of NSAID given before surgery to decrease the frequency of persisted pain after spinal surgery (limited to fenestration), in patients treated with NSAID.

[Direct effects of the intrathecal administration of normal saline for the brain expansion after the removal of subdural hematoma]. / Bezposrednie skutki dokanalowego podawania fizjologicznego roztworu soli w celu rozprezenia mózgu po usunieciu krwiaka podtwardówkowego.

In a group of 360 patients the effects were studied of various management methods in case of lacking expansion of the brain after removal of chronic subdural haematoma on the therapeutic results. It was found that intrathecal infusion of normal saline was an effective procedure, with small frequency of transient complications, and was followed by a considerably lower number of re-operations than after external or internal drainage of haematoma cavity.

[Epidemiological analysis of the correlations between selected individual characteristics of patients with primary arterial hypertension]. / Epidemiologiczna analiza zaleznosci miedzy wybranymi cechami osobniczymi u chorych z nadcisnieniem tetniczym pierwotnym.

The purpose of the study was to find the correlations and to evaluate their strength between the progression of hypertension and age, sex, time between the diagnosis of hypertension and the beginning of treatment, body weight and other characteristics. The study was carried out in the population of patients treated in the Cardiology Department, Medical Academy in Lublin in the period 1981-1984. The computer-assisted analysis of the obtained material was carried out in the Department of Epidemiology, Medical Academy in Lublin. The studied population comprised 320 patients with essential hypertension. In the statistical analysis the chi square test was applied for finding of statistically significant correlations and Pearson test was used for estimation of correlation strength. Statistically significant correlations were obtained between the progression of hypertension and age, sex, time between hypertension diagnosis and present treatment and body weight.