Novel relationship between Vegf expression and retinal pigmented epithelium permeability following light damage in the mouse retina

Purpose:In the retina, the balance between pro- and anti-angiogenic factors is critical for angiogenesis control but is also involved in cell survival and maintenance. For instance, the anti-angiogenic factor PEDF is neuroprotective for photoreceptors (PRs) in models of retinal degeneration. We previously reported upregulation of VEGF (24h to 48h post lesion) in the light-damage (LD) model. Furthermore, systemic delivery of PEDF, as well as lentiviral gene transfer of an anti-VEGF antibody rescue PRs from cell death. Studies in vitro show that VEGF induces retinal endothelial cells apoptosis via the alteration of the Akt1/p38 MAPK signalling pathway under hypoxic conditions. Thus, in this study, we investigate the effect of high levels of VEGF on retinal pigmented epithelium (RPE) permeability and molecular targets expression after light-induced PR degeneration. Methods:To characterize the action of VEGF in the retina during the course of LD, we exposed adult Balb/c mice to 5'000 lux for 1h, and we collected neural retinas and eye-cups (containing RPE) at different time points after the LD. We analysed protein expression by Elisa and Western blotting. In order to study RPE cell permeability after the LD we stained β-catenin on flat mounted RPE. Results:In the neural retina, preliminary results indicate that high levels of VEGF induce a significant upregulation of VEGF receptor 2, whereas VEGF receptor 1 expression is decreased. Concomitantly with VEGF upregulation, LD increases the Src phosphorylation between 24h to 48h. Furthermore, we observe that β-catenin translocates to the cytoplasm of RPE cells between 24h to 36h after the lesion, indicating an increase on the RPE permeability, which could contribute indirectly to the deleterious effect of VEGF observed during light-induced PR apoptosis. Conclusions:This study further involves VEGF in LD and highlights the prime importance of angiogenic factor balance for PR survival. Our results suggest that PR apoptosis is augmented by RPE cell permeability, which may induce high level of VEGF and could be deleterious. The specific action of RPE permeability on PR survival and the role of Src in the retina are under investigation.

An alternative technique to reveal polysaccharides in Toxoplasma gondii tissue cysts

Ultrathin sections of tissue cysts isolated from the brain of Toxoplasma gondii infected mice were submitted to two different methodologies derived from the periodic acid - Schiff's reagent (PAS) technique. The use of osmium tetroxide vapor as a developing agent of the aldehyde oxidation to reveal polysaccharides with periodic acid resulted in positive reaction in amylopectin granules in bradyzoites, as well as in the wall and matrix of the cysts, with excellent increment of the ultrastructural morphology. This technique can be used for study of T. gondii-host cell intracellular cycle, the differentiation tachyzoite-bradyzoite, and also for the formation of cysts into the host cells.

A case of megacolon in Rio Grande Valley as a possible case of Chagas disease

We have been searching for evidence of Chagas disease in mummified human remains. Specifically, we have looked for evidence of alteration of intestinal or fecal morphology consistent with megacolon, a condition associated with Chagas disease. One prehistoric individual recovered from the Chihuahuan Desert near the Rio Grande exhibits such pathology. We present documentation of this case. We are certain that this individual presents a profoundly altered large intestinal tract and we suggest that further research should focus on confirmation of a diagnosis of Chagas disease. We propose that the prehistoric activity and dietary patterns in Chihuahua Desert hunter/gatherers promoted the pathoecology of Chagas disease.

The in vitro cytopathology of a porcine and the simian (SA-11) strains of rotavirus

Rotaviruses have been implicated as the major causal agents of acute diarrhoea in mammals and fowls. Experimental rotavirus infection have been associated to a series of sub-cellular pathologic alterations leading to cell lysis which may represent key functions in the pathogenesis of the diarrhoeic disease. The current work describes the cytopathic changes in cultured MA-104 cells infected by a simian (SA-11) and a porcine (1154) rotavirus strains. Trypan blue exclusion staining showed increased cell permeability after infection by both strains, as demonstrated by cell viability. This effect was confirmed by the leakage of infected cells evaluated by chromium release. Nuclear fragmentation was observed by acridine orange and Wright staining but specific DNA cleavage was not detected. Ultrastructural changes, such as chromatin condensation, cytoplasm vacuolisation, and loss of intercellular contact were shown in infected cells for both strains. In situ terminal deoxynucleotidyl transferase (Tunel) assay did not show positive result. In conclusion, we demonstrated that both strains of rotavirus induced necrosis as the major degenerative effect.

Cytotoxicity of three new triazolo-pyrimidine derivatives against the plant trypanosomatid: Phytomonas sp. isolated from Euphorbia characias

There is no effective chemotherapy against diseases caused by Phytomonas sp., a plant trypanosomatid responsible for economic losses in major crops. We tested three triazolo-pyrimidine complexes [two with Pt(II), and another with Ru(III)] against promastigotes of Phytomonas sp. isolated from Euphorbia characias. The incorporation of radiolabelled precursors, ultrastructural alterations and changes in the pattern of metabolite excretion were examined. Different degrees of toxicity were found for each complex: the platinun compound showed an inhibition effect on nucleic acid synthesis, provoking alterations on the levels of mitochondria, nucleus and glycosomes. These results, together with others reported previously in our laboratory about the activity of pyrimidine derivatives, reflect the potential of these compounds as agents in the treatment of Phytomonas sp.

Differences in the stability of the plasmids of Yersinia pestis cultures in vitro: impact on virulence

Plasmid and chromosomal genes encode determinants of virulence for Yersinia pestis, the causative agent of plague. However, in vitro, Y. pestis genome is very plastic and several changes have been described. To evaluate the alterations in the plasmid content of the cultures in vitro and the impact of the alterations to their pathogenicity, three Y. pestis isolates were submitted to serial subculture, analysis of the plasmid content, and testing for the presence of characteristic genes in each plasmid of colonies selected after subculture. Different results were obtained with each strain. The plasmid content of one of them was shown to be stable; no apparent alteration was produced through 32 subcultures. In the other two strains, several alterations were observed. LD50 in mice of the parental strains and the derived cultures with different plasmid content were compared. No changes in the virulence plasmid content could be specifically correlated with changes in the LD50.

Early white matter alterations in men predisposed to FXTAS revealed by MT imaging.

Introduction: The Fragile X - associated Tremor Ataxia Syndrome (FXTAS) is a recently described, and under-diagnosed, late onset (≈ 60y) neurodegenerative disorder affecting male carriers of a premutation in the Fragile X Mental Retardation 1 (FMR1) gene. The premutation is an CGG (Cytosine-Guanine-Guanine) expansion (55 to 200 CGG repeats) in the proximal region of the FMR1 gene. Patients with FXTAS primarily present with cerebellar ataxia and intention tremor. Neuroradiological features of FXTAS include prominent white matter disease in the periventricular, subcortical, middle cerebellar peduncles and deep white matter of the cerebellum on T2-weighted or FLAIR MR imaging (Jacquemmont 2007, Loesch 2007, Brunberg 2002, Cohen 2006). We hypothesize that a significant white matter alteration is present in younger individuals many years prior to clinical symptoms and/or the presence of visible lesions on conventional MR sequences and might be detectable by magnetization transfer (MT) imaging. Methods: Eleven asymptomatic premutation carriers (mean age = 55 years) and seven intra-familial controls participated to the study. A standardized neurological examination was performed on all participants and a neuropsychological evaluation was carried out before MR scanning performed on a 3T Siemens Trio. The protocol included a sagittal T1-weighted 3D gradient-echo sequence (MPRAGE, 160 slices, 1 mm^3 isotropic voxels) and a gradient-echo MTI (FA 30, TE 15, matrix size 256*256, pixel size 1*1 mm, 36 slices (thickness 2mm), MT pulse duration 7.68 ms, FA 500, frequency offset 1.5 kHz). MTI was performed by acquiring consecutively two set of images; first with and then without the MT saturation pulse. MT images were coregistered to the T1 acquisition. The MTR for every intracranial voxel was calculated as follows: MTR = (M0 - MS)/M0*100%, creating a MTR map for each subject. As first analysis, the whole white matter (WM) was used to mask the MTR image in order to create an histogram of the MTR distribution in the whole tissue class over the two groups examined. Then, for each subject, we performed a segmentation and parcellation of the brain by means of Freesurfer software, starting from the high resolution T1-weighted anatomical acquisition. Cortical parcellations was used to assign a label to the underlying white matter by the construction of a Voronoi diagram in the WM voxels of the MR volume based on distance to the nearest cortical parcellation label. This procedure allowed us to subdivide the cerebral WM in 78 ROIs according to the cortical parcellation (see example in Fig 1). The cerebellum, by the same procedure, was subdivided in 5 ROIs (2 per each hemisphere and one corresponding to the brainstem). For each subject, we calculated the mean value of MTR within each ROI and averaged over controls and patients. Significant differences between the two groups were tested using a two sample T-test (p<0.01). Results: Neurological examination showed that no patient met the clinical criteria of Fragile X Tremor and Ataxia Syndrome yet. Nonetheless, premutation carriers showed some subtle neurological signs of the disorder. In fact, premutation carriers showed a significant increase of tremor (CRST, T-test p=0.007) and increase of ataxia (ICARS, p=0.004) when compared to controls. The neuropsychological evaluation was normal in both groups. To obtain general characterizations of myelination for each subject and premutation carriers, we first computed the distribution of MTR values across the total white matter volume and averaged for each group. We tested the equality of the two distributions with the non parametric Kolmogorov-Smirnov test and we rejected the null-hypothesis at a p=0.03 (fig. 2). As expected, when comparing the asymptomatic permutation carriers with control subjects, the peak value and peak position of the MTR values within the whole WM were decreased and the width of the distribution curve was increased (p<0.01). These three changes point to an alteration of the global myelin status of the premutation carriers. Subsequently, to analyze the regional myelination and white matter integrity of the same group, we performed a ROI analysis of MTR data. The ROI-based analysis showed a decrease of mean MTR value in premutation carriers compared to controls in bilateral orbito-frontal and inferior frontal WM, entorhinal and cingulum regions and cerebellum (Fig 3). The detection of these differences in these regions failed with other conventional MR techniques. Conclusions: These preliminary data confirm that in premutation carriers, there are indeed alterations in "normal appearing white matter" (NAWM) and these alterations are visible with the MT technique. These results indicate that MT imaging may be a relevant approach to detect both global and local alterations within NAWM in "asymptomatic" carriers of premutations in the Fragile X Mental Retardation 1 (FMR1) gene. The sensitivity of MT in the detection of these alterations might point towards a specific physiopathological mechanism linked to an underlying myelin disorder. ROI-based analyses show that the frontal, parahippocampal and cerebellar regions are already significantly affected before the onset of symptoms. A larger sample will allow us to determine the minimum CGG expansion and age associated with these subclinical white matter alterations.

Programmed cell death in Trypanosoma cruzi induced by Bothrops jararaca venom

Cells die through a programmed process or accidental death, know as apoptosis or necrosis, respectively. Bothrops jararaca is a snake whose venom inhibits the growth of Trypanosoma cruzi epimastigote forms causing mitochondrion swelling and cell death. The aim of the present work was to determine the type of death induced in epimastigotes of T. cruzi by this venom. Parasite growth was inhibited after venom treatment, and 50% growth inhibition was obtained with 10 µg/ml. Ultrastructural observations confirmed mitochondrion swelling and kinetoplast disorganization. Furthermore, cytoplasmic condensation, loss of mitochondrion membrane potential, time-dependent increase in phosphatidylserine exposure at the outer leaflet plasma membrane followed by permeabilization, activation of caspase like protein and DNA fragmentation were observed in epimastigotes throughout a 24 h period of venom treatment. Taken together, these results indicate that the stress induced in epimastigote by this venom, triggers a programmed cell death process, similar to metazoan apoptosis, which leads to parasite death.

Histophatology and ultrastructure of Henneguya caudalongula sp. n. infecting Prochilodus lineatus (Pisces: Prochilodontidae) cultivated in the state of São Paulo, Brazil

The histological and ultrastructural characteristics of a new species of Henneguya and the host reactions to infection by this species are reported. Henneguya caudalongula sp. n. was found in the inter and intralamellar regions of the gills of Prochilodus lineatus (Valenciennes, 1836) cultivated at Center for the Research and Management of Continental Fishing Resources located in the municipality of Pirassununga, state of São Paulo, Brazil. The plasmodia were white and round or ellipsoidal and measured 0.2 to 1 mm in length. The development of the parasite was asynchronous and the mature spores were fusiform, with a total length 71 ± 1.4 µm, body length of 16.6 ± 0.54 µm and width 4.6 ± 0.2 µm. The caudal process was 52.6 ± 1.5 µm long. The polar capsules were elongate (length 6.1 ± 0.19 µm, width 1.6 ± 0.15 µm) and of equal size. The polar filament was coiled in 10-11 turns. The prevalence of the parasite was 48.3% and did not vary significantly with the season or host size.

Monitoring of resistance to the pyrethroid cypermethrin in Brazilian Aedes aegypti (Diptera: Culicidae) populations collected between 2001 and 2003

Resistance to cypermethrin of different Aedes aegypti Brazilian populations, collected at two successive periods (2001 and 2002/2003), was monitored using the insecticide-coated bottles bioassay. Slight modifications were included in the method to discriminate between mortality and the knock down effect. Although this pyrethroid was recently started to be used in the country to control the dengue vector, a decrease in susceptibility was noted between both periods analyzed, particularly in the city of Rio de Janeiro. The results indicate that resistance is due at least in part to a target site alteration.

Experimental infection of Leishmania (L.) chagasi in a cell line derived from Lutzomyia longipalpis (Diptera:Psychodidae)

The present work describes the in vitro infection of a cell line Lulo, derived from Lutzomyia longipalpis embryonic tissue, by Leishmania chagasi promastigotes. This infection process is compared with a parallel one developed using the J774 cell line. The L. chagasi MH/CO/84/CI-044B strain was used for experimental infection in two cell lines. The cells were seeded on glass coverslips in 24-well plates to reach a final number of 2 x 10(5) cells/well. Parasites were added to the adhered Lulo and J774 cells in a 10:1 ratio and were incubated at 28 and 37ºC respectively. After 2, 4, 6, 8, and 10 days post-infection, the cells were extensively washed with PBS, fixed with methanol, and stained with Giemsa. The number of internalized parasites was determined by counting at least 400 cultured cells on each coverslip. The results showed continuous interaction between L. chagasi promastigotes with the cell lines. Some ultrastructural characteristics of the amastigote forms were observed using transmission electron microscopy. The highest percentage of infection in Lulo cells was registered on day 6 post-infection (29.6%) and on day 4 in the J774 cells (51%). This work shows similarities and differences in the L. chagasi experimental infection process in the two cell lines. However, Lulo cells emerge as a new model to study the life-cycle of this parasite.

Spinal modulations accompany peripheral fatigue during prolonged tennis playing.

To examine the time course of alteration in neural process (spinal loop properties) during prolonged tennis playing, 12 competitive players performed a series of neuromuscular tests every 30 min during a 3-h match protocol. Muscle activation (twitch interpolation) and normalized EMG activity were assessed during maximal voluntary contraction (MVC) of plantar flexors. Spinal reflexes and M-waves were evoked at rest (i.e., H(max) and M(max) , respectively) and during MVC (i.e., H(sup) , V-wave, M(sup) , respectively). MVC torque declined significantly (P<0.001) across the match protocol, due to decrease (P<0.001) in muscle activation and in normalized EMG activity. The impairment in MVC was significantly correlated (r=0.77; P<0.05) with the decline in muscle activation. H(max) /M(max) (P<0.001), H(sup) /M(sup) (P<0.01) and V/M(sup) (P<0.05) ratios were depressed with fatigue and decreased by ∼80%, 46% and 61% at the end of exercise, respectively. Simultaneously, peak twitch torque and M-wave amplitude were significantly (P<0.01) altered with exercise, suggesting peripheral alterations. During prolonged tennis playing, the compromised voluntary strength capacity is linked to a reduced neural input to the working muscles. This central activation deficit partly results from a modulation in spinal loop properties.

Efficacy of Citrus reticulata and Mirazid in treatment of Schistosoma mansoni

This work has been carried out to investigate the effect of Schistosoma mansoni infection on mice livers after treatment with the ethanolic extract of Citrus reticulata root or the oleo-resin extract from Myrrh of Commiphora molmol tree (Mirazid), as a new antishistosomal drug. Marker enzymes for different cell organelles were measured; succinate dehydrogenase (SDH); lactate dehydrogenase (LDH) and its isoenzymes; glucose-6-phosphatase (G-6-Pase); acid phosphatase (AP) and 5'- nucleotidase. Liver function enzymes; aspartate aminotransferase (AST); alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were also estimated. Parasitological studies through ova count and worm burden will also be taken into consideration. The results showed a marked reduction in SDH, LDH, AST, and ALT enzyme activities and a significant increase in G-6-Pase, AP, 5'- nucleotidase, and ALP after S. mansoni infection. A noticeable alteration in LDH subunits were also noticed. Treatment with C. reticulata or Mirazid improved all the previous enzyme activities with a noticeable reduction in ova count and worm burden.

Lymphatic vessel contractile activity and intestinal inflammation

Edema is a consistent observation in inflamatory bowel disease (IBD), and immune responses are inevitable in inflammation. Because the lymphatic system is an integral part of both tissue fluid homeostasis and immune reactions, it is likely that lymphatics play a role in the complex etiology of IBD. Despite the consistent findings that the lymphatic system is altered during gastrointestinal inflammation, the majority of studies conducted on the disease only mention the lymphatic system in passing. The effects of inflammatory mediators on lymphatic vessel function also remain poorly defined, despite its essential role in immunity and prevention of tissue edema. Processes allowing effective lymph transport are altered during inflammation, however, the mode of alteration and reason why lymphatics are ineffective in inflammatory reactions need to be further investigated. In addition, these processes have not yet been examined in an appropriate animal model and little has been done using in vivo methods of investigation in any model of gastrointestinal inflammation. This paper reviews the role of the lymphatic system in intestinal inflammation, as well as the role of the inflammatory products in mediating lymphatic contractile function.

The effect of Bulgarian propolis against Trypanosoma cruzi and during its interaction with host cells

Propolis has shown activity against pathogenic microorganisms that cause diseases in humans and animals. The ethanol (Et-Blg) and acetone (Ket-Blg) extracts from a Bulgarian propolis, with known chemical compositions, presented similar activity against tissue culture-derived amastigotes. The treatment of Trypanosoma cruzi-infected skeletal muscle cells with Et-Blg led to a decrease of infection and of the intracellular proliferation of amastigotes, while damage to the host cell was observed only at concentration 12.5 times higher than those affecting the parasite. Ultrastructural analysis of the effect of both extracts in epimastigotes revealed that the main targets were the mitochondrion and reservosomes. Et-Blg also affected the mitochondrion-kinetoplast complex in trypomastigotes, offering a potential target for chemotherapeutic agents.