Genotypic Diversity and Pathogenic Potential of Clinical and Environmental Vibrio parahaemolyticus Isolates From Brazil.

Vibrio parahaemolyticus strains recovered from human diarrheal stools (one in 1975 and two in 2001) and environmental sources (four, between 2008 and 2010) were investigated for the presence of virulence genes (trh, tdh, and vpadF), pandemic markers (orf8, toxRS new), and with respect to their pathogenic potential in two systemic infection models. Based only on the presence or absence of these genetic markers, they were classified as follows: the environmental strains were non-pathogenic, whereas among the clinical strains, the one isolated in 1975 was pathogenic (non-pandemic), and the other two were pathogenic (pandemic). The pathogenic potential of the strains was evaluated in mice and Galleria mellonella larvae infection models, and except for the clinical (pathogenic, non-pandemic) isolate, the others produced lethal infection in both organisms, regardless of their source, serotype, and genotype (tdh, orf8, toxRS new, and vpadF). Based on mice and larval mortality rates, the strains were then grouped according to virulence (high, intermediate, and avirulent), and remarkably similar results were obtained by using these models: The clinical strain (pathogenic and non-pandemic) was classified as avirulent, and other strains (four non-pathogenic and two pandemic) were considered of high or intermediate virulence. In summary, these findings demonstrate that G. mellonella larvae can indeed be used as an alternative model to study the pathogenicity of V. parahaemolyticus. Moreover, they raise doubts about the use of traditional virulence markers to predict pathogenesis of the species and show that reliable models are indispensable to determine the pathogenic potential of environmental isolates considered non-pathogenic, based on the absence of the long-standing virulence indicators.

N­glycosylation and receptor tyrosine kinase signaling affect claudin­3 levels in colorectal cancer cells.

Changes in protein levels in different components of the apical junctional complex occur in colorectal cancer (CRC). Claudin­3 is one of the main constituents of tight junctions, and its overexpression can increase the paracellular flux of macromolecules, as well as the malignant potential of CRC cells. The aim of this study was to investigate the molecular mechanisms involved in the regulation of claudin­3 and its prognostic value in CRC. In silico evaluation in each of the CRC consensus molecular subtypes (CMSs) revealed that high expression levels of CLDN3 (gene encoding claudin­3) in CMS2 and CMS3 worsened the patients' long­term survival, whereas a decrease in claudin­3 levels concomitant with a reduction in phosphorylation levels of epidermal growth factor receptor (EGFR) and insulin­like growth factor 1 receptor (IGF1R) could be achieved by inhibiting N­glycan biosynthesis in CRC cells. We also observed that specific inactivation of these receptor tyrosine kinases (RTKs) led to a decrease in claudin­3 levels, and this regulation seems to be mediated by phospholipase C (PLC) and signal transducer and activator of transcription 3 (STAT3) in CRC cells. RTKs are modulated by their N­linked glycans, and inhibition of N­glycan biosynthesis decreased the claudin­3 levels; therefore, we evaluated the correlation between N­glycogenes and CLDN3 expression levels in each of the CRC molecular subtypes. The CMS1 (MSI immune) subtype concomitantly exhibited low expression levels of CLDN3 and N­glycogenes (MGAT5, ST6GAL1, and B3GNT8), whereas CMS2 (canonical) exhibited high gene expression levels of CLDN3 and N­glycogenes (ST6GAL1 and B3GNT8). A robust positive correlation was also observed between CLDN3 and B3GNT8 expression levels in all CMSs. These results support the hypothesis of a mechanism integrating RTK signaling and N­glycosylation for the regulation of claudin­3 levels in CRC, and they suggest that CLDN3 expression can be used to predict the prognosis of patients identified as CMS2 or CMS3.

Dual role of histamine on microglia-induced neurodegeneration.

Several hypotheses have been raised about the dual role of histamine in neurological disorders, and evidences have shown its crucial involvement in the modulation of microglia-mediated neuroinflammation. Previously, we reported that the administration of histamine induces a deleterious effect by promoting a pro-inflammatory phenotype on microglia that in turn compromises dopaminergic neuronal survival. Contrary, under lipopolysaccharide challenge, histamine inhibits the injurious effect of microglia-mediated inflammation, protecting dopaminergic neurons, suggesting that the modulation of microglial activity is dependent on the environmental context. Thus, histamine and/or histamine receptor agonists may serve to develop new therapeutic approaches to overcome neurodegenerative disorders.

GDNF contributes to oestrogen-mediated protection of midbrain dopaminergic neurones.

Parkinson's disease (PD) is characterised by the preferential loss of dopaminergic neurones from the substantia nigra (SN) that leads to the hallmark motor disturbances. Animal and human studies suggest a beneficial effect of oestrogen to the nigrostriatal system, and the regulation of neurotrophic factor expression by oestrogens has been suggested as a possible mechanism contributing to that neuroprotective effect. The present study was designed to investigate whether the neuroprotection exerted by 17ß-oestradiol on nigrostriatal dopaminergic neurones is mediated through the regulation of glial cell line-derived neurotrophic factor (GDNF) expression. Using an in vivo rat model of PD, we were able to confirm the relevance of 17ß-oestradiol in defending dopaminergic neurones against 6-hydroxydopamine (6-OHDA) toxicity. 17ß-oestradiol, released by micro-osmotic pumps, implanted 10 days before intrastriatal 6-OHDA injection, prevented the loss of dopaminergic neurones induced by 6-OHDA. 17ß-oestradiol treatment also promoted an increase in GDNF protein levels both in the SN and striatum. To explore the relevance of GDNF increases to 17ß-oestradiol neuroprotection, we analysed, in SN neurone-glia cultures, the effect of GDNF antibody neutralisation and RNA interference-mediated GDNF knockdown. The results showed that both GDNF neutralisation and GDNF silencing abolished the dopaminergic protection provided by 17ß-oestradiol against 6-OHDA toxicity. Taken together, these results strongly identify GDNF as an important player in 17ß-oestradiol-mediated dopaminergic neuroprotection.

Evaluation of the impact of diabetes on retinal metabolites by NMR spectroscopy.

PURPOSE/AIM OF THE STUDY: Diabetic retinopathy (DR) is a leading cause of blindness in working age adults in developed countries. Changes in metabolites and in metabolic pathways of the retina caused by hyperglycemia may compromise the physiology of the retina. Using nuclear magnetic resonance (NMR) spectroscopy, we aimed to investigate the effect of diabetes on the levels of intermediate metabolites in rat retinas and the metabolic pathways that could be affected. MATERIALS AND METHODS: Diabetes was induced in male Wistar rats with a single injection of streptozotocin (65 mg/Kg, i.p.). Metabolic alterations were analyzed in streptozotocin-induced diabetic rat retinas by (1)H NMR spectroscopy. Glucose uptake was measured with 2-deoxy-D-[1-(3)H]glucose. Lactate production was evaluated by (1)H NMR spectroscopy using [U-(13)C]glucose. RESULTS: Tissue levels of several metabolic intermediates were quantified, but no significant changes in the levels of most metabolites were detected, with the exceptions of glucose, significantly increased, and lactate, significantly reduced in diabetic rat retinas, as compared to age-matched controls. The cytosolic redox ratio, indirectly evaluated by lactate-to-pyruvate ratio, was significantly reduced in diabetic rat retinas, as well as glucose uptake. Parallel studies demonstrated that lactate production rates were significantly diminished, suggesting a reduction in the glycolytic flux. CONCLUSIONS: These results suggest that diabetes may significantly decrease glycolysis in the retina since higher intracellular glucose levels do not translate into higher intracellular lactate levels or into higher rates of lactate production. These changes may alter the normal functioning of the retina during diabetes and may contribute for vision loss in DR.

High glucose changes extracellular adenosine triphosphate levels in rat retinal cultures.

Diabetic retinopathy (DR) is the leading cause of blindness in adults. In diabetes, there is activation of microglial cells and a concomitant release of inflammatory mediators. However, it remains unclear how diabetes triggers an inflammatory response in the retina. Activation of P2 purinergic receptors by adenosine triphosphate (ATP) may contribute to the inflammatory response in the retina, insofar as it has been shown to be associated with microglial activation and cytokine release. In this work, we evaluated how high glucose, used as a model of hyperglycemia, considered the main factor in the development of DR, affects the extracellular levels of ATP in retinal cell cultures. We found that basal extracellular ATP levels were not affected by high glucose or mannitol, but the extracellular elevation of ATP, after a depolarizing stimulus, was significantly higher in retinal cells cultured in high glucose compared with control or mannitol-treated cells. The increase in the extracellular ATP was prevented by application of botulinum neurotoxin A or by removal of extracellular calcium. In addition, degradation of exogenously added ATP was significantly lower in high-glucose-treated cells. It was also observed that, in retinal cells cultured under high-glucose conditions, the changes in the intracellular calcium concentrations were greater than those in control or mannitol-treated cells. In conclusion, in this work we have shown that high glucose alters the purinergic signaling system in the retina, by increasing the exocytotic release of ATP and decreasing its extracellular degradation. The resulting high levels of extracellular ATP may lead to inflammation involved in the pathogenesis of DR.

High glucose induces caspase-independent cell death in retinal neural cells.

Diabetic retinopathy is a leading cause of blindness among adults in the western countries. It has been reported that neurodegeneration may occur in diabetic retinas, but the mechanisms underlying retinal cell death are poorly understood. We found that high glucose increased the number of cells with condensed nuclei and the number of TUNEL-positive cells, and caused an increase in the translocation of phosphatidylserine to the outer leaflet of the plasma membrane, indicating that high glucose induces apoptosis in cultured retinal neural cells. The activity of caspases did not increase in high glucose-treated cells, but apoptosis-inducing factor (AIF) levels decreased in the mitochondria and increased in the nucleus, indicating a translocation to the nucleus where it may cause DNA fragmentation. These results demonstrate that elevated glucose induces apoptosis in cultured retinal neural cells. The increase in apoptosis is not dependent on caspase activation, but is mediated through AIF release from the mitochondria.

Inflammation and neurogenesis in temporal lobe epilepsy.

The aim of the present review is to discuss the evidence supporting the hypothesis that inflammation and neurogenesis play an important role in temporal lobe epilepsy (TLE) and to examine whether possible strategies that involve the pharmacological manipulation of inflammation/neurogenesis can lead to the development of novel approaches for the treatment of epilepsy. Since it is not yet clear whether the neuron-glia response obtained in this pathology is a secondary effect of an aggressive inflammation or if it is somehow related to the cause of the epileptic condition, with the present review we guide the readers through the complex and ambiguous crosstalk between neuroimmunology and epilepsy.

Evaluation of lesion clustering in irradiated plasmid DNA.

PURPOSE: To measure the yield of DNA strand breaks and clustered lesions in plasmid DNA irradiated with protons, helium nuclei, and y-rays. MATERIALS AND METHODS: Plasmid DNA was irradiated with 1.03, 19.3 and 249 MeV protons (linear energy transfer = 25.5, 2.7, and 0.39 keV microm(-1) respectively), 26 MeV helium nuclei (25.5 keV microm) and gamma-rays (137Cs or 60Co) in phosphate buffer containing 2 mM or 200 mM glycerol. Single-and double-strand breaks (SSB and DSB) were measured by gel electrophoresis, and clustered lesions containing base lesions were quantified by converting them into irreparable DSB in transformed bacteria. RESULTS: For protons, SSB yield decreased with increasing LET (linear energy transfer). The yield of DSB and all clustered lesions seemed to reach a minimum around 3 keV microm(-1). There was a higher yield of SSB, DSB and total clustered lesions for protons compared to helium nuclei at 25.5 keV microm(-1). A difference in the yields between 137Cs and 60Co gamma-rays was also observed, especially for SSB. CONCLUSION: In this work we have demonstrated the complex LET dependence of clustered-lesion yields, governed by interplay of the radical recombination and change in track structure. As expected, there was also a significant difference in clustered lesion yields between various radiation fields, having the same or similar LET values, but differing in nanometric track structure.

Ca2+ stores in the chick embryo retina cells.

The Ca2+ stores of digitonin permeabilized chick embryo retina cells in culture were characterized, by using the fluorescence of Fluo-3 potassium salt to follow continuously the free [Ca2+] in the medium. After ATP dependent Ca2+ accumulation, the Ca2+ release was induced by several agents; 10 microM cyclic-ADP-ribose (cADPR), 40 microM Ins (1,4,5)P3 10 microM thapsigargin (Th), 25 microM ionomycin (Ion), 15 microM CCCP together with 4.5 micrograms/ml oligomycin (CCCP/Olig), 50 microM arachidonic acid (AA). Neither Ins(1,4,5)P3 nor cADPR were able to mobilize Ca2+ from internal stores in these cells, but Th and AA were effective in releasing Ca2+. Four major Ca2+ stores in chick embryo retina cells were distinguished: i) the thapsigargin sensitive Ca2+ store, most likely the ER; ii) the Ca2+ store sensitive to oligomycin and CCCP, most likely the mitochondrial Ca2+ store, iii) an AA sensitive Ca2+ store, which is distinct from the previous two; and, iv) the Ca2+ store only sensitive to ionomycin. The capacities of these different Ca2+ stores of the chick embryo retina cells, relative to the total intracellular stores, are: 63.3%, 14.1%, 8.2%, for the ER, the mitochondrial and for the AA sensitive Ca2+ stores, respectively.

Ontogeny of the L-type voltage sensitive calcium channels in chick embryo retinospheroids.

The L-type voltage sensitive calcium channels (VSCC) of chick embryo retinospheroids were characterized during the development in vitro. Functionally, the activity of VSCC was characterized by continuously monitoring the changes in the intracellular free Ca2+ concentration (delta[Ca2+]i) with indo-1, in response to 30 mM KCl. The contribution of the L-type VSCC was evaluated using the L-type VSCC antagonist, nitrendipine. We also characterized the binding of [3H]nitrendipine to retinospheroid membranes during development, and determined the Kd and Bmax values. We observed that the changes in [Ca2+]i in response to 30 mM KCl increased from 159.46 +/- 6.62 nM at 0 days in vitro (DIV) retinospheroids to 704.4 +/- 59.9 nM at 14 DIV retinospheroids. Nitrendipine (2 microM) blocked the delta[Ca2+]i response by approximately 67% in all ages tested. No significant difference in the Kd values for the nitrendipine binding was observed during in vitro development of the retinospheroids. However, the Bmax increased from 27.99 +/- 1.95 fmol/mg protein in 0 DIV retinospheroids to 131.09 +/- 14.24 fmol/mg protein in 14 DIV retinospheroids, supporting the delta[Ca2+]i results. The results presented suggest that the increase in [Ca2+]i during development was due to an increase in the number of L-type channels. Therefore, the expression of L-type VSCC is developmentally regulated during retinogenesis in vitro and accompanies neuronal maturation, probably regulating the Ca2+ input crucial to the onset of important intracellular Ca2+-dependent functions.

Voltage-sensitive Ca2+ channels in rat striatal synaptosomes: role on the [Ca2+]i responses to membrane depolarization.

The fluorescent Ca2+ indicator Indo-1 was used to study the effect of depolarization evoked by KCl or 4-aminopyridine (4-AP) on the intracellular free calcium concentration responses (delta[Ca2+]i) in rat striatal synaptosomes. Depolarization of the synaptosomes with [KCl] > 7.5 mM induced a rapid increase of the [Ca2+]i followed by a decay towards a plateau. The size of the [Ca2+]i response varied sigmoidally with the synaptosomal membrane potential, with a transition potential of -27.3 mV. Depolarization with 4-AP evoked a dose-dependent sustained increase of the [Ca2+]i. Nitrendipine, omega-Conotoxin GVIA (omega-CgTx) and omega-Agatoxin IVA (omega-Aga IVA) were used to evaluate the relative role of L-, N-, P- and possibly Q-type voltage-sensitive Ca2+ channels (VSCCs) on the [Ca2+]i changes evoked by each of the two depolarizing agents. Nitrendipine caused only about 10% inhibition of the effect of either agent on the [Ca2+]i, suggesting that the L-type VSCCs have a modest contribution. The omega-CgTx decreased the response to KCl and 4-AP by 15 and 30%, respectively, but the latter effect may be partially due to a non-specific effect on Na+ channels. The omega-Aga IVA reduced the response to 4-AP by 26.5%, and this effect was additive to that of omega-CgTx, further suggesting that the striatal nerve terminals possess P- and/or Q-type, in addition to N-type Ca2+ channels. Neomycin (0.35 mM), tentatively used as an antagonist of the P-type channels, had a potent effect, decreasing the response to K(+)-depolarization and to 4-AP by, respectively, 32.5 and 48.5%. It is suggested that at the concentration used the antibiotic also partially blocks VSCCs which do not belong to the L-, N-, P- or Q-type VSCCs. We conclude that striatal nerve endings are equipped with at least four to five pharmacologically distinct classes of VSCCs, which are sensitive to well known antagonists of the L-, N-, P-, and Q-type VSCCs.

Ins(1,4,5)P3 induces Ca2+ release from brain microsomes loaded either by the Ca2+ ATPase or by the Na+/Ca2+ exchanger.

In this study we investigated the release of Ca2+ in brain microsomes after Ca2+ loading by the Ca(2+)-ATPase or by the Na+/Ca2+ exchanger. The results show that in microsomes loaded with Ca2+ by the Ca(2+)-ATPase, Ins(1,4,5)P3 (5 microM) released 21 +/- 2% of the total Ca2+ accumulated, and that in the microsomes loaded with Ca2+ by the Na+/Ca2+ exchanger, Ins(1,4,5)P3 released 28 +/- 3% of the total Ca2+ accumulated. These results suggest that receptors of Ins(1,4,5)P3 may be co-localized with the Na+/Ca2+ exchanger in the endoplasmic reticulum membrane or that there are Ins(1,4,5)P3 receptors in the plasma membrane where the Na+/Ca2+ exchanger is normally present, or both. We also found that Ins(1,4,5)P3 inhibited the Ca(2+)-ATPase by 33.7%, but that it had no significant effect on the Na+/Ca2+ exchanger.

Eficácia da vacina Sabin em crianças subnutridas da amazônia / The efficacy of oral polio vaccine in malnourished Amazonial children

A eficácia da vacina Sabin foi determinada em 106 crianças normais e subnutridas da Amazônia, após a administraçäo de uma e duas doses de vacina oral (trivalente). Após a aplicaçäo de uma dose de vacina, verificou-se que apenas 9% das crianças com subnutriçäo pregressa (crônica) e 43% das crianças normais formaram anticorpos neutralizantes (protetores) contra dois ou três tipos de poliovírus (p=0,04). Após duas doses de vacina, os níveis de imunidade dos dois grupos de crianças estudadas acusaram, respectivamente, 32% e 75% (p=0,001). Estes resultados mostram que a resposta imunitária à vacina Sabin foi sensivelmente inferior no grupo das crianças subnutridas, do que no das crianças normais. Em decorrência disto, será necessário administrar um número maior de doses de vacina oral àquelas crianças, a fim de que níveis satisfatórios de imunidade contra a poliomielite sejam atingidos em toda a populaçäo infantil