Dispersal capacity of Haematopota spp. and Stomoxys calcitrans using a mark-release-recapture approach in Belgium.

The dispersion potential of mechanical vectors is an important factor in the dissemination of pathogens. A mark-release-recapture experiment was implemented using two groups (unfed and partially fed) of the Tabanidae (Diptera) (Haematopota spp.) and biting Muscidae (Diptera) (Stomoxys calcitrans) most frequently collected in Belgium in order to evaluate their dispersion potential. In total, 2104 specimens of Haematopota spp. were collected directly from horses and 5396 S. calcitrans were collected in a cattle farm using hand-nets. Some of these insects were partially fed in vitro and all were subsequently coloured. Overall, 67 specimens of S. calcitrans (1.2%) and 17 of Haematopota spp. (0.8%) were recaptured directly on horses. Stomoxys calcitrans flew maximum distances of 150 m and 300 m when partially fed and unfed, respectively. Haematopota spp. travelled maximum distances of 100 m and 200 m when partially fed and unfed, respectively. Segregation measures seem essential in order to reduce the risk for pathogen transmission. A distance of 150 m appears to be the minimum required for segregation to avoid the risk for mechanical transmission, but in areas of higher vector density, this should probably be increased.

Foci report on indigenous Dermacentor reticulatus populations in Belgium and a preliminary study of associated babesiosis pathogens.

The occurrence of autochthonous clinical cases of canine and equine babesiosis in Belgium during the last two decades suggests that the vector of the pathogens responsible for these diseases, Dermacentor reticulatus (Ixodida: Ixodidae), may be present in this country. Consequently, evidence for the presence of this tick species in different locations within Belgium was investigated. Four different locations were monitored by flagging in 2010; these included the locations at which D. reticulatus was previously found on a dog in 2009 and on two red deer in 2007. Two different species of tick were identified, Ixodes ricinus (Ixodida: Ixodidae) and D. reticulatus. A total of 282 D. reticulatus adult ticks (98 males, 184 females) were collected from the four sites. Ticks were found mainly from early March until the end of May and a peak in activity was apparent in March. A Babesia spp. (Piroplasmida: Babesiidae) genus-specific polymerase chain reaction test based on the amplification of a fragment of the 18S rRNA gene was used to investigate the potential presence of Babesia spp. All DNA extracts isolated from the total tick samples yielded negative results. Additional studies to accurately determine the distribution and vectorial capacity of this important tick species in Belgium are warranted.

Coprological survey of endoparasite infections in owned dogs and owners' perceptions of endoparasite control in Belgium and the Netherlands.

Infections with endoparasites are common in dogs. Some of these parasites are potentially zoonotic and therefore a public health concern. A survey was conducted in twenty-six small animal practices to evaluate the prevalence of endoparasites in Belgian and Dutch owned dogs older than 6 months as well as risk factors associated with infection. Out of 239 faecal samples screened (168 in Belgium and 71 in the Netherlands), 18 dogs were tested positive for at least one type of endoparasite with three dogs co-infected with two parasitic species. Toxocara sp. was the most frequently found endoparasite (4.6%). Three other dogs were positive for Angiostrongylus vasorum (1.4%) using the Baermann method and confirmed in one dog by the Angiodetect® test. Age and predation behaviour were identified as two risk factors associated with endoparasite infection. Although the majority (77%) of the owners in this study reported to administer at least one anthelmintic treatment per year, only a minority of them (24.3%) were aware of the risk to human health, indicating that owner awareness is sub-optimal. For dog owners, human toxocarosis and other potential zoonoses remain an underestimated health concern. The implementation of sustainable parasite control strategies should be promoted taking also into account the public health risk.

A comparative test of ixodid tick identification by a network of European researchers.

This study reports the results of a comparative test of identification of ticks occurring in Western Europe and Northern Africa. A total of 14 laboratories were voluntarily enrolled in the test. Each participant received between 22 and 25 specimens of adult and nymphal ticks of 11 species: Dermacentor marginatus, D. reticulatus, Haemaphysalis punctata, Hyalomma lusitanicum, Hy. marginatum, Ixodes ricinus, I. hexagonus, Rhipicephalus annulatus, R. bursa, R. rossicus, and/or R. sanguineus s.l. Ticks were morphologically identified by three of the co-authors and the identification confirmed by a fourth co-author who used molecular methods based on several genes. Then ticks were randomly selected and blindly distributed among participants, together with a questionnaire. Only specimens collected while questing and, if possible, in the same survey, were circulated. Because of the random nature of the test, a participant could receive several specimens of the same species. Species in the different genera had variable misidentification rates (MR) of 7% (Dermacentor), 14% (Ixodes), 19% (Haemaphysalis), 36% (Hyalomma), and 54% (Rhipicephalus). Within genera, the MR was also variable ranging from 5.4% for I. ricinus or 7.4% for D. marginatus or D. reticulatus to 100% for R. rossicus. The test provided a total misidentification rate of 29.6% of the species of ticks. There are no significant differences in MR according to the sex of the tick. Participants were requested to perform a second round of identifications on the same set of ticks, using only purposely prepared keys (without illustrations), circulated to the enrolled participants, including 2 species of the genus Dermacentor, 8 of Haemaphysalis, 10 of Hyalomma, 23 of Ixodes, and 6 of Rhipicephalus. The average MR in the second round was 28%: 0% (Dermacentor), 33% (Haemaphysalis), 30% (Hyalomma) 18% (Ixodes), and 50% (Rhipicephalus). Species which are not reported in the countries of a participating laboratory had always highest MR, i.e. purely Mediterranean species had highest MR by laboratories in Central and Northern Europe. Participants expressed their concerns about a correct identification for almost 50% of the ticks of the genera Hyalomma and Rhipicephalus. The results revealed less than total confidence in identifying the most prominent species of ticks in the Western Palearctic, and underpin the need for reference libraries for specialists involved in this task. Results also showed that a combination of certain genes may adequately identify the target species of ticks.

Tamoxifen inhibits nerve growth factor-induced proliferation of the human breast cancerous cell line MCF-7.

An array of polypeptide growth factors contribute to the development of breast cancer, the most common tumor-related cause of death in women of Western countries. Therefore, breast cancer therapy should be aimed at inhibition of growth factor-dependent breast cancerous cell proliferation. However, the relative contribution of each individual factor in the development and maintenance of the transformed phenotype is largely unknown. Here we report for the first time that the proliferative effects of nerve growth factor, (NGF) a typical neurotrophin, are similar to those of epidermal growth factor (EGF) and insulin-like growth factor II, and are enhanced by 17beta-estradiol in the human breast cancer cell line MCF-7. The effect of NGF appeared to be mediated by its trkA receptors (trkA(NGFR)), as suggested by the potent inhibition of both MCF-7 cell proliferation and trkA(NGFR) phosphorylation occurring upon treatment of cultures with the selective trkA(NGFR) inhibitor K252a. Surprisingly, the antiestrogen drug tamoxifen (TAM) inhibited NGF-induced MCF-7 cell proliferation and trkA(NGFR) phosphorylation in a concentration-related fashion. The effect of TAM seemed to be estrogen receptor-independent, because the pure estrogen receptor antagonist ICI 182.780 was unable to block NGF-induced trkA(NGFR) phosphorylation. Our data underline the new emerging role of trkA(NGFR) in breast tumor growth, and suggest a related novel therapeutic use of TAM in breast cancer.

Protective effects of estradiol on TRAIL-induced apoptosis in a human oligodendrocytic cell line: evidence for multiple sites of interactions.

Demyelinating diseases are high impact neurological disorders. Steroids are regarded as protective molecules in the susceptibility to these diseases. Here, we studied the interactions between tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), a potent proapoptotic molecule toxic to oligodendrocytes, and 17-beta-estradiol (E-17-beta), in human oligodendrocytic MO3.13 cells. Exposure of cells to TRAIL resulted in the upregulation of both death receptors DR4 and DR5 and apoptosis, as well as the activation of caspase-8 and -3, increased phosphorylation of Jun-N-terminal kinase and p38 kinase, and the reduction of bcl-2 and bcl-xL proteins. TRAIL-mediated MO3.13 cell apoptosis was abrogated by the dominant-negative form of the adaptor protein FADD and by caspase inhibitors. Preincubation with E-17-beta completely prevented both TRAIL-induced DR4 and DR5 upregulation and apoptosis. Estrogen-induced cytoprotection was time and concentration dependent and reverted by antiestrogens. Estrogen treatment per se reduced kinase phosphorylation, and upregulated bcl-2 and bcl-xL proteins. In conclusion, our data show that the detrimental role of TRAIL on oligodendrocytes can be effectively counteracted by estrogens, thus suggesting that the underlying molecular interactions can be of potential relevance in characterizing novel targets for therapy of demyelinating disorders.

A retrospective serological survey on human babesiosis in Belgium.

In Europe, most clinical babesiosis cases in humans have been attributed to Babesia divergens and Babesia sp. EU1. Babesia microti infection of humans occurs mainly in the United States; although a case of autochthonous B. microti infection and serological evidence of infection have been reported in Europe. The Indirect Fluorescent Antibody Test was used to screen sera from 199 anonymous Belgian patients with history of tick bite and clinical symptoms compatible with a tick-borne disease. The serological screen detected positive reactivity in 9% (n = 18), 33.2% (n = 66), and 39.7% (n = 79) of the samples against B. microti, B. divergens, and Babesia sp. EU1, respectively. Thus, evidence of contact among three potentially zoonotic species of Babesia and humans has been confirmed in Belgium. Preventive action and development of better diagnostic tools should help in prevention of clinical cases and to clarify the true burden of such infection for individuals and public health.

Higher-order structure of domain III in Escherichia coli 16S ribosomal RNA, 30S subunit and 70S ribosome.

We have investigated in detail the conformation of domain III of 16S rRNA (nucleotides 913-1408), using a variety of chemical and enzymatic structure probes. The sites of reaction were identified by primer extension with reverse transcriptase using appropriate oligodeoxyribonucleotide primers. This study has been done on 16S rRNA in its naked form, in the 30S subunit and in the 70S ribosome. Data obtained with naked RNA broadly confirm the secondary structure model proposed essentially by comparative sequence analysis, and allow identification of nucleotides involved in tertiary interactions. Our results are in reasonably good agreement with structure probing experiments of Moazed et al. [1]. However, several discrepancies have been observed. Within the 30S subunit, a high number of nucleotides become unreactive whereas other nucleotides show an enhanced reactivity. This probably reflects local conformational changes. Interestingly, they are located in strategic regions of the RNA, e.g. around C1400 (involved in tRNA binding) and C1192 (involved in spectinomycin recognition). Results are also discussed together with the topographical localization of the ribosomal proteins in this area. The study on the 70S particle allows identification of regions at the interface of subunits or exposed at the surface of the ribosome.

Divergent effects of corticotropin releasing hormone on endothelial cell nitric oxide synthase are associated with different expression of CRH type 1 and 2 receptors.

1. Endothelium is a target for an array of factors involved in inflammation. Endothelial cells express receptors for CRH, a neuropeptide produced during inflammation. We report both the concentration-dependent inhibitory effect of CRH upon cytokine-stimulated nitrite release by H5V murine endothelioma cells, and its stimulatory one in HUVEC cells. 2. Western blot analysis showed that CRH inhibits cytokine-stimulated iNOS protein in H5V cells, and, instead, potentiated it in HUVEC cells. 3. H5V cells expressed both CRH receptors (CRH-R1 and R2) mRNAs, whereas HUVEC cells expressed the CRH-R2 mRNA solely. 4. CRH increased medium nitrites and iNOS protein expression in H5V cells pretreated with the selective CRH-R1 antagonist CP 154,526. However, the selective CRH-R2 antagonist anti-Svg-30 failed to produce similar effects. In fact, anti-Svg-30 inhibited CRH-induced increase of nitrite release and iNOS expression in HUVEC cells. 5. Our results confirm the activating role of CRH on endothelial cells, although it suggests its possible inhibitory role in the late phase of the inflammatory response. NO-mediated effects of CRH on endothelial cells could be exploited in therapeutic strategies related to inflammatory and/or degenerative diseases.

Ellipticine derivatives interacting with model membranes. Influence of quaternarization of nitrogen-2.

Four compounds of the ellipticine family were examined in their interaction with liposomes and with an isolated bacterial membrane. The physicochemical methods used detected only minor differences between the properties of the amphiphilic drugs (ellipticine and 2-methyl-ellipticinium) and the two dipolar drugs (9-hydroxy-ellipticine and 2-methyl 9-hydroxy-ellipticinium). The amphiphilic drugs were able to become associated to anionic liposomes in a 20-30% excess of charge neutralization, and seem to penetrate deeper into the lipid layer than the two dipolar drugs. It was also shown that ellipticine penetrates deeper into liposomes membrane than into natural membrane used. In contrast with what can be postulated from the literature dealing with the behaviour of quaternarized drugs, it seems that ellipticine and its quaternarized analogues present fast diffusion through multilayered vesicles. On the whole, the membrane effects of the ellipticines studied here are not different for quaternarized drugs and for drugs not permanently charged, but are influenced by the existence on the molecules of a second polar function.

Apomorphine, dopamine and phenylethylamine reduce the proportion of phosphorylated insulin receptor substrate 1.

We tested the ability of dopamine, apomorphine, phenylethylamine and pergolide to inhibit the proliferation of fetal calf serum-stimulated human breast cancer (MCF)-7 cells. While the first three compounds were able to block the proliferation of MCF-7 cells, pergolide failed to do so (up to 100 microM). The inhibitory effect of dopamine, apomorphine and phenylethylamine was also evident in serum-starved insulin-stimulated MCF-7 cells. Apomorphine also inhibited the proliferation of the human oestrogen receptor-negative breast cancer (MDA-MB231) and prostate carcinoma (LNCaP) cell lines. In a second set of experiments, we measured the ability of dopamine, apomorphine, phenylethylamine and pergolide to inhibit the phosphorylation (or increase the dephosphorylation) of the insulin receptor substrate (IRS)-1, a major intracellular substrate of the insulin-like growth factor (IGF)-1 receptor. Dopamine, apomorphine and phenylethylamine all reduced to zero the level of phosphorylated IRS-1 with potencies ranging between 0.01 and 1 microM. Finally, we found that fibroblasts from IRS-1 null (-/-) mice were less sensitive to the anti-proliferative effect of apomorphine compared to fibroblasts from wild type-mice, suggesting that the inhibition of IRS-1 phosphorylation by apomorphine is an important aspect of the activity of this compound.

Behavioral and neurochemical effects of dopaminergic drugs in models of brain injury.

The dopaminergic drugs, ropinirole and dihydroergocryptine (DHECP) were injected subcutaneously (s.c.) at doses of 0.5 and 1 mg/kg/day for 7 days into male rats of the Sprague-Dawley strain. The drug pretreatment reverted amnesia induced in rats by hypobaric hypopxia and tested in active and passive avoidance tasks. Furthermore, a partial restoration of memory retention was found in animals with a 2-month brain occlusive ischemia induced by manipulation of the four major arteries of the brain. No major changes were found in spontaneous motor activity, but drug treatment increased ambulation of animals subjected to acute or chronic experimental manipulation. In a model of kainate-induced epilepsy, ropinirole or DHECP did not affect seizure parameters, but reduced mortality rate. At the end of behavioral procedures, in all animals subjected to hypobaric hypoxia or to brain occlusive ischemia glutathione redox index (glutathione reduced/glutathione oxidized ratio) was measured in the frontal cortex, striatum and hippocampus. It was found that experimental models of brain injury were followed by a decrease of reduced glutathione content in all brain areas. The glutathione redox index was augmented by ropinirole or DHECP treatment in all brain areas. These behavioral and neurochemical findings suggest that ropinirole and DHECP may exert either protective activity (as found in animals pretreated with these drugs and exposed to hypobaric hypoxia) or reversal of brain injury (as found in animals treated after two-month occlusive brain ischemia). Thus, both drugs may be studied as therapeutic agents in brain injuries of various origin.

Experimental study on the toxicity and the local and systemic tolerability of gemcitabine after topical treatment of the rabbit bladder.

This study investigated gemcitabine administered intravesically to establish the local and systemic tolerability necessary for clinical trials. Gemcitabine was directly administered via catheter into the bladders of 24 male New Zealand rabbits weighing an average of 1.9+/-0.08 kg. Three groups received weekly gemcitabine for 5 (50 mg/kg) or 8 (25 mg/kg or controls) weeks. Animals were inspected daily for signs of toxicity and distress, body weight changes, and water and food consumption; electrocardiogram, blood pressure, and urinalysis were recorded before dosing and after 4 and 8 weeks of treatment. The rabbits were euthanized, and a full necropsy was performed on day 1 after the last instillation. Principal organs (spleen, thymus, testis, and muscle) and plasma samples were analyzed for the systemic absorption of gemcitabine. The 25-mg/kg dose was well tolerated with no clinical side effects. At 50 mg/kg, signs of mild myelosuppression and severe symptomatic toxicity (leg weakness, and hair and body weight loss) was evident after 3 weeks of treatment and three of the seven animals in this group died after four doses. Necropsies revealed normal bone marrow cellularity and organ histology at both doses. No significant systemic drug absorption was seen. These findings suggest that intravesical administration of gemcitabine does not produce organ-specific toxicity, but the higher dose (50 mg/kg) may represent the threshold above which increasing morbidity may occur.

Red wine micronutrients as protective agents in Alzheimer-like induced insult.

Plant extract micronutrients are commonly added to diets for health and prevention of degenerative disease. However, there are barriers to the introduction of these products as antioxidant therapies in counteracting chronic human diseases, probably because the molecular bases of their therapeutic potential are poorly clarified. The present study was designed to evaluate the possible protective effect of combined micronutrients present in black grape skin on toxicity induced by 25-35 beta-amyloid peptid or by serum of Alzheimer's disease patients, in human umbilical vein endothelial cells (HUVECs). The hypothesis was tested by examining the results of lactic dehydrogenase (LDH) release to estimate cytoplasmic membrane breakdown; activity of mitochondrial complexes, reactive oxygen species (ROS) production and malonyl dialdehyde (MDA) levels as markers of oxidative stress induction and COMET assay to evaluate DNA fragmentation. The results demonstrate that black grape skin extract reduces the ROS production, protects the cellular membrane from oxidative damage, and consequently prevents DNA fragmentation. The experimental results suggest that this natural compound may be used to ameliorate the progression of pathology in AD disease therapy.

Increased ACTH and cortisol secretion after interleukin-alpha injection in the common marmoset (Callithrix jacchus jacchus).

We have studied the effect of intravenous injection of interleukin-1 (dose range: from 0.25 to 4.5 microg/kg of body weight) on plasma ACTH and cortisol levels in the marmoset, a primate paradygm of peripheral glucocorticoid resistance. Blood sampling were collected and body temperature recorded 0, 15, 30, 60, 120, 180, 240 and 300 min after injection. Interleukin-1 stimulated secretion of ACTH in a dose-dependent fashion. Maximal secretion occurred 120 min after injection, and lasted up to 240 min. Plasma ACTH levels returned to baseline 300 min after interleukin-1 injection. Plasma cortisol levels were related to ACTH levels. Body temperature elevation, which occurred 10-15 min after injection was dose-dependent, and lasted 3 h. Results suggest that the pyrogenic effect of interleukin is associated, in the marmoset, with integrated activation of the hypothalamic-pituitary-adrenal axis. In light of the proneness of marmosets to hyperimmune disorders, our data are consistent with the hypothesized central biological role of IL-1, as well as the pathophysiological relevance of the neuro-endocrine-immune cross-talk during the acute phase response.

Thymic factors influence on behavior in rodents.

We studied the effect of thymopentin, a synthetic thymic peptide, on spontaneous behavior and stress models in BALB/c mice in which a WEHI 164 clone 13 murine fibrosarcoma had been implanted, as well as in the intact Sprague-Dawley rat. In untreated animals with tumors, spontaneous behavior was significantly inhibited. Resistance to swimming in cold water was also decreased in untreated animals. Thymopentin (10, 100, 1,000, and 5,000 micrograms/kg body weight, IP, 20 min before the test) enhanced spontaneous behavior in tumor-implanted mice. In addition, thymopentin partially restored floating capability of tumor bearers in either freely moving or animals on which an additional weight had been applied. In the latter test, plasma corticotropin and corticosterone levels were relatedly modified according to treatment. Rats treated with thymopentin showed a decreased sensitivity to painful stimuli. The effect of thymopentin was comparable to acetylsalicylate. Finally, thymic factors appeared capable of restoring the diminished behavioral activity of animals bearing tumors, as well as of increasing resistance to stressful stimuli and pain.

An optimized DNA extraction and multiplex PCR for the detection of Fasciola sp. in lymnaeid snails.

This study deals with the development and validation of an original PCR protocol to assess the presence of Fasciola hepatica in Galba truncatula its main intermediate host in Western Europe. In the present study two DNA extraction techniques are compared and a new multiplex PCR is described. The Chelex(®) DNA extraction technique showed to be more appropriate than the classical Phenol/Chloroform/Proteinase K based method because of the absence of toxic organic solvent, shorter duration and lower cost, and a higher reproducibility regarding DNA concentrations and wavelength ratios. The multiplex PCR was set up to amplify the lymnaeid internal transcribed spacer 2 sequence (500-600 bp) that act as an internal control and a 124 bp Fasciola sp. sequence that is repeated more than 300,000 times in fluke whole genome. Ninety six snails were pooled and 6 snails (6.25%) found positive for Fasciola sp. The limit of detection is lower than the minimal biological infestation unit (one miracidium). DNA extracts from Paramphistomum daubneyi, Dicrocoelium lanceolatum, and Fascioloides magna did not cross react.