The quest for a better understanding of chronic kidney disease complications: an update on uremic toxins

Chronic kidney disease is characterized by a progressive reduction of glomerular filtration rate and/or the appearance of proteinuria, and subsequently the progressive retention of organic waste compounds called uremic toxins (UT). Over the last decades, a large number of such compounds have been identified and their effects on organs and tissues, especially the cardiovascular system, has been demonstrated. In this review, we present the current classification of UT, as proposed by the EUTox Group, and the effects of some of the probably most important UTs, such as phosphate, FGF-23, PTH, AGEs, indoxyl sulfate and para-cresyl sulfate. We provide an overview on therapeutic approaches aimed to increase their extracorporeal removal via convective and/or adsorptive strategies and to lower their intestinal production/ absorption via dietetic and pharmacological interventions. The recognition that multiple toxins contribute to the uremia supports the need for new therapeutic targets, with a potentially positive impact on CKD progression and survival.

Synthesis of 4-hydroxycinnamic amides of di-, tri-, and tetraamines : potential insect toxins /

The monoconjugates of phenolic acids (i.e. coumaric acid) with polyamines such as spermidine and spermine are strikingly similar to some toxins from spiders and predatory wasps. Many plants contain phenolic acid polyamine conjugates and there is some reliable information supporting their roles as plant defense chemicals. Eleven monoacylated compounds of diamines, triamines, tetraamines and oxa-polyamine amines were prepared in three to seven steps: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 and 32. The synthesis proceeds through stepwise construction of the polyamine backbone (as in 62 and 72), followed by protection and deprotection steps of the amino functions. Desymmetrization of readily available and prepared symmetrical polyamines is a key step in the synthesis. The protecting groups employed were tert-butoxycarbonyl (BOC) and trifluoroacetyl (TFA) group which were removed under different conditions: acid and base respectively. Deprotection and refunctionalization of the polyamine reagent demonstrated the versatility of these systems for N-acylation.

Rat brain serotonin neurones that express neuronal nitric oxide synthase have increased sensitivity to the substituted amphetamine serotonin toxins 3,4-methylenedioxymethamphetamine and p-chloroamphetamine

Substituted amphetamines such as p-chloroamphetamine and the abused drug methylenedioxymethamphetamine cause selective destruction of serotonin axons in rats, by unknown mechanisms. Since some serotonin neurones also express neuronal nitric oxide synthase, which has been implicated in neurotoxicity, the present study was undertaken to determine whether nitric oxide synthase expressing serotonin neurones are selectively vulnerable to methylenedioxymethamphetamine or p-chloroamphetamine. Using double-labeling immunocytochemistry and double in situ hybridization for nitric oxide synthase and the serotonin transporter, it was confirmed that about two thirds of serotonergic cell bodies in the dorsal raphe nucleus expressed nitric oxide synthase, however few if any serotonin transporter immunoreactive axons in striatum expressed nitric oxide synthase at detectable levels. Methylenedioxymethamphetamine (30 mg/kg) or p-chloroamphetamine (2 x 10 mg/kg) was administered to Sprague-Dawley rats, and 7 days after drug administration there were modest decreases in the levels of serotonin transporter protein in frontal cortex, and striatum using Western blotting, even though axonal loss could be clearly seen by immunostaining. p-Chloroamphetamine or methylenedioxymethamphetamine administration did not alter the level of nitric oxide synthase in striatum or frontal cortex, determined by Western blotting. Analysis of serotonin neuronal cell bodies 7 days after p-chloroamphetamine treatment, revealed a net down-regulation of serotonin transporter mRNA levels, and a profound change in expression of nitric oxide synthase, with 33% of serotonin transporter mRNA positive cells containing nitric oxide synthase mRNA, compared with 65% in control animals. Altogether these results support the hypothesis that serotonin neurones which express nitric oxide synthase are most vulnerable to substituted amphetamine toxicity, supporting the concept that the selective vulnerability of serotonin neurones has a molecular basis.

Staphylococcal extracellular adherence protein induces platelet activation by stimulation of thiol isomerases

OBJECTIVE: Staphylococcus aureus can induce platelet aggregation. The rapidity and degree of this correlates with the severity of disseminated intravascular coagulation, and depends on platelet peptidoglycans. Surface-located thiol isomerases play an important role in platelet activation. The staphylococcal extracellular adherence protein (Eap) functions as an adhesin for host plasma proteins. Therefore we tested the effect of Eap on platelets. METHODS AND RESULTS: We found a strong stimulation of the platelet-surface thiol isomerases protein disulfide isomerase, endoplasmic reticulum stress proteins 57 and 72 by Eap. Eap induced thiol isomerase-dependent glycoprotein IIb/IIIa activation, granule secretion, and platelet aggregation. Treatment of platelets with thiol blockers, bacitracin, and anti-protein disulfide isomerase antibody inhibited Eap-induced platelet activation. The effect of Eap on platelets and protein disulfide isomerase activity was completely blocked by glycosaminoglycans. Inhibition by the hydrophobic probe bis(1-anilinonaphthalene 8-sulfonate) suggested the involvement of hydrophobic sites in protein disulfide isomerase and platelet activation by Eap. CONCLUSIONS: In the present study, we found an additional and yet unknown mechanism of platelet activation by a bacterial adhesin, involving stimulation of thiol isomerases. The thiol isomerase stimulatory and prothrombotic features of a microbial secreted protein are probably not restricted to S aureus and Eap. Because many microorganisms are coated with amyloidogenic proteins, it is likely that the observed mechanism is a more general one.

Aeromonas detection and their toxins from drinking water from reservoirs and drinking fountains

Aeromonads are inhabitants of aquatic ecosystems and are described as being involved in intestinal disturbances and other infections. A total of 200 drinking water samples from domestic and public reservoirs and drinking fountains located in Sao Paulo (Brazil), were analyzed for the presence of Aeromonas. Samples were concentrated by membrane filtration and enriched in APW. ADA medium was used for Aeromonas isolation and colonies were confirmed by biochemical characterization. Strains isolated were tested for hemolysin and toxin production. Aeromonas was detected in 12 samples (6.0%). Aeromonas strains (96) were isolated and identified as: A. caviae (41.7%), A. hydrophila (15.7%), A. allosacharophila (10.4%), A. schubertii (1.0%) and Aeromonas spp. (31.2%). The results revealed that 70% of A. caviare, 66.7% of A. hydrophila, 80% of A. allosacharophila and 46.6% of Aeromonas spp. were hemolytic. The assay for checking production of toxins showed that 17.5% of A. caviae, 73.3% of A. hydrophila, 60% of A. allosacharophila, 100% of A. schubertii, and 33.3% of Aeromonas spp. were able to produce toxins. The results demonstrated the pathogenic potential of Aeromonas, indicating that the presence of this emerging pathogen in water systems is a public health concern.

Proteomics of the neurotoxic fraction from the sea anemone Bunodosoma cangicum venom: Novel peptides belonging to new classes of toxins

In contrast to the many studies on the venoms of scorpions, spiders, snakes and cone snails, tip to now there has been no report of the proteomic analysis of sea anemones venoms. In this work we report for the first time the peptide mass fingerprint and some novel peptides in the neurotoxic fraction (Fr III) of the sea anemone Bunodosoma cangicum venom. Fr III is neurotoxic to crabs and was purified by rp-HPLC in a C-18 column, yielding 41 fractions. By checking their molecular masses by ESI-Q-Tof and MALDI-Tof MS we found 81 components ranging from near 250 amu to approximately 6000 amu. Some of the peptidic molecules were partially sequenced through the automated Edman technique. Three of them are peptides with near 4500 amu belonging to the class of the BcIV, BDS-I, BDS-II, APETx1, APETx2 and Am-II toxins. Another three peptides represent a novel group of toxins (similar to 3200 amu). A further three molecules (similar to similar to 4900 amu) belong to the group of type 1 sodium channel neurotoxins. When assayed over the crab leg nerve compound action potentials, one of the BcIV- and APETx-like peptides exhibits an action similar to the type 1 sodium channel toxins in this preparation, suggesting the same target in this assay. On the other hand one of the novel peptides, with 3176 amu, displayed an action similar to potassium channel blockage in this experiment. In summary, the proteomic analysis and mass fingerprint of fractions from sea anemone venoms through MS are valuable tools, allowing us to rapidly predict the occurrence of different groups of toxins and facilitating the search and characterization of novel molecules without the need of full characterization of individual components by broader assays and bioassay-guided purifications. It also shows that sea anemones employ dozens of components for prey capture and defense. (C) 2008 Elsevier Inc. All rights reserved.

Functional Diversity of Heat-labile Toxins (LT) Produced by Enterotoxigenic Escherichia coli

Heat-labile toxins (LTs) have ADP-ribosylation activity and induce the secretory diarrhea caused by enterotoxigenic Escherichia coli (ETEC) strains in different mammalian hosts. LTs also act as adjuvants following delivery via mucosal, parenteral, or transcutaneous routes. Previously we have shown that LT produced by human-derived ETEC strains encompass a group of 16 polymorphic variants, including the reference toxin (LT1 or hLT) produced by the H10407 strain and one variant that is found mainly among bacterial strains isolated from pigs (LT4 or pLT). Herein, we show that LT4 ( with six polymorphic sites in the A (K4R, K213E, and N238D) and B (S4T, A46E, and E102K) subunits) displays differential in vitro toxicity and in vivo adjuvant activities compared with LT1. One in vitro generated LT mutant (LTK4R), in which the lysine at position 4 of the A subunit was replaced by arginine, showed most of the LT4 features with an similar to 10-fold reduction of the cytotonic effects, ADP-ribosylation activity, and accumulation of intracellular cAMP in Y1 cells. Molecular dynamic studies of the A subunit showed that the K4R replacement reduces the N-terminal region flexibility and decreases the catalytic site crevice. Noticeably, LT4 showed a stronger Th1-biased adjuvant activity with regard to LT1, particularly concerning activation of cytotoxic CD8(+) T lymphocytes when delivered via the intranasal route. Our results further emphasize the relevance of LT polymorphism among human-derived ETEC strains that may impact both the pathogenicity of the bacterial strain and the use of these toxins as potential vaccine adjuvants.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA): molecular background, virulence, and relevance for public health

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Occurrence of coagulase-positive Staphylococcus in various food products commercialized in Botucatu, SP, Brazil and detection of toxins from food and isolated strains

The aim of this work was to assess the microbiological quality of commercialized desserts, sandwiches and finger food in Botucatu, SP, for human consumption. A total of 172 food samples were analyzed for fecal coliforms and coagulase-positive Staphylococcus and 69 (40.1%) were in disagreement with the standards established by Decree No. 12 (Brazilian Food Sanitation Standard, 2001). Coagulase-positive Staplylococcus was isolated from 26 (15.1%) samples. Toxins were not isolated directly from foods but 27 (54%) coagulase-positive Staphylococcus strains were enterotoxigenic, and toxin type C was the most frequently detected. These results suggest that these products may act as an important vehicle of transmission for well-established pathogens. (c) 2006 Elsevier Ltd. All rights reserved.

Predictive factors of outcome following staphylococcal peritonitis in continuous ambulatory peritoneal dialysis

Background and aims: Staphylococcus epidermidis and other coagulase-negative staphylococci (CoNS) are the most common agents of continuous ambulatory peritoneal dialysis (CAPD) peritonitis. Episodes caused by Staphylococcus aureus evolve with a high method failure rate while CoNS peritonitis is generally benign. The purpose of this study was to compare episodes of peritonitis caused by CoNS species and S. aureus to evaluate the microbiological and host factors that affect outcome. Material and methods: Microbiological and clinical data were retrospectively studied from 86 new episodes of peritonitis caused by staphylococci species between January 1996 and December 2000 in a university dialysis center. The influence of microbiological and host factors (age, sex, diabetes, use of vancomycin, exchange system and treatment time on CAPD) was analyzed by logistic regression model. The clinical outcome was classified into two results (resolution and non-resolution). Results: the odds of peritonitis resolution were not influenced by host factors. Oxacillin susceptibility was present in 30 of 35 S. aureus lineages and 22 of 51 CoNS (p = 0.001). There were 32 of 52 (61.5%) episodes caused by oxacillin-susceptible and 20 of 34 (58.8%) by oxacillin-resistant lineages resolved (p = 0.9713). of the 35 cases caused by S. aureus, 17 (48.6%) resolved and among 51 CoNS episodes 40 (78.4%) resolved. Resolution odds were 7.1 times higher for S. epidermidis than S. aureus (p = 0.0278), while other CoNS had 7.6 times higher odds resolution than S. epidermidis cases (p = 0.052). Episodes caused by S. haemolyticus had similar resolution odds to S. epidermidis (p = 0.859). Conclusions: S. aureus etiology is an independent factor associated with peritonitis non-resolution in CAPD, while S. epidermidis and S. haemolyticus have a lower resolution rate than other CoNS. Possibly the aggressive nature of these agents, particularly S. aureus, can be explained by their recognized pathogenic factors, more than antibiotic resistance.

Neutralization of snake venom phospholipase A(2) toxins by aqueous extract of Casearia sylvestris (Flacourtiaceae) in mouse neuromuscular preparation

Aqueous extract of Casearia sylvestris (Flacourtiaceae) has been shown to inhibit enzymatic and biological properties of some Bothrops and Crotalus venoms and their purified phospholipase A(2) (PLA(2)) toxins. In this work we evaluated the influence of C sylvestris aqueous extract upon neuromuscular blocking and muscle damaging activities of some PLA(2)S (crotoxin from C. durissus terrificus, bothropstoxin-I from B.jararacussu, piratoxin-I from B. pirajai and myotoxin-II from B. moojeni) in mouse phrenic-diaphragm preparations. Crotoxin (0.5 mu M) and all other PLA2 toxins (1.0 mu M) induced irreversible and time-dependent blockade of twitches. Except for crotoxin, all PLA2 toxins induced significant muscle damage indices, assessed by microscopic analysis. Preincubation of bothropstoxin-I, piratoxin-I or myotoxin-II with C. sylvestris extract (1:5 (w/w), 30 min, 37 degrees C significantly prevented the neuromuscular blockade of preparations exposed to the mixtures for 90 min; the extent of protection ranged from 93% to 97%. The vegetal extract also neutralized the muscle damage (protection of 80-95%). Higher concentration of the C. sylvestris extract (1: 10, w/w) was necessary to neutralize by 90% the neuromuscular blockade induced by crotoxin. These findings expanded the spectrum of C. sylvestris antivenom activities, evidencing that it may be a good source of potentially useful PLA2 inhibitors. (c) 2007 Elsevier B.V.. All rights reserved.

Molecular detection of enterotoxins E, G, H and I in Staphylococcus aureus and coagulase-negative staphylococci isolated from clinical samples of newborns in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

PCR detection of staphylococcal enterotoxin genes in Staphylococcus aureus strains isolated from raw and pasteurized milk

Milk is considered a nutritious food because it contains several important nutrients including proteins and vitamins. Conversely, it can be a vehicle for several pathogenic bacteria such as Staphylococcus aureus. This study aimed to analyze the frequency of genes encoding the staphylococcal enterotoxins (SEs) SEA, SEB, SEC, SED, SEE, SEG, SEH, SEI and SEJ in S. aureus strains isolated from raw or pasteurized bovine milk. S. aureus was found in 38 (70.4%) out of 54 raw milk samples at concentrations of up to 8.9 X 10(5) CFU/ml. This microorganism was present in eight samples of pasteurized milk before the expiration date and in 11 samples analyzed on the expiration date. of the 57 strains studied, 68.4% were positive for one or more genes encoding the enterotoxins, and 12 different genotypes were identified. The gene coding for enterotoxin A, sea, was the most frequent ( 16 strains, 41%), followed by sec (8 strains, 20.5%), sed (5 strains, 12.8%). seb (3 strains. 7.7%) and see (2 strains, 5.1%). Among the genes encoding the other enterotoxins, seg was the most frequently observed (11 strains. 28.2%), followed by sei (10 strains) and seh and sej (3 strains each). With the recent identification of new SEs, the perceived frequency of enterotoxigenic strains has increased. suggesting that the pathogenic potential of staphylococci may be higher than previously thoughts however, further studies are required to assess the expression of these new SEs by S. aureus. and their impact in foodborne disease. The quality of Brazilian milk is still low. and efforts from the government and the entire productive chain are required to attain consumer safety. (C) 2008 Elsevier B.V. All rights reserved.

Polymerase Chain Reaction Detection of Enterotoxins Genes in Coagulase-Negative Staphylococci Isolated from Brazilian Minas Cheese

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)