In vitro basal cytotoxicity assay applied to estimate acute oral systemic toxicity of grandisin and its major metabolite

Preclinical investigations can start with preliminary in vitro studies before using animal models. Following this approach, the number of animals used in preclinical acute toxicity testing can be reduced. In this study, we employed an in-house validated in vitro cytotoxicity test based on the Spielmann approach for toxicity evaluation of the lignan grandisin, a candidate anticancer agent, and its major metabolite. the 4-O-demethylgrandisin, by neutral red uptake (NRU) assay, on mouse fibroblasts Balb/c 3T3 cell line. Using different concentrations of grandisin and its major metabolite (2.31; 1.16; 0.58; 0.29; 0.14; 0.07; 0.04; 0.002 mu M) in Balb/c 3T3-A31 NRU cytotoxicity assay, after incubation for 48 h, we obtained IC(50) values for grandisin and its metabolite of 0.078 and 0.043 mu M, respectively. The computed LD(50) of grandisin and 4-O-demethylgrandisin were 617.72 and 429.95 mg/kg, respectively. Both were classified under the Globally Harmonized System as category 4. Since pharmacological and toxicological data are crucial in the developmental stages of drug discovery, using an in vitro assay we demonstrated that grandisin and its metabolite exhibit distinct toxicity profiles. Furthermore, results presented in this work can contribute to reduce the number of animals required in subsequent pharmacological/toxicological studies. (C) 2010 Elsevier GmbH. All rights reserved.

Conformational preferences for some 2-substituted N-methoxy-N-methylacetamides through spectroscopic and theoretical studies

The analysis of the IR carbonyl band of the 2-substituted N-methoxy-N-methylacetamides Y-CH(2)C(O)-N(OMe)Me (Y = F1, OMe 2, OPh 3, Cl 4), supported by B3LYP/6-311++G(3df, 3pd) calculations along with the NBO analysis for 1-4, indicated the existence of cis-gauche conformers i.e. (c) and (g) for 1 and 3, (c(1), c(2)) and (g(1), g(2)) for 2, and (c) and (g(1), g(2)) for 4. In the gas phase, the g conformer population prevails over the c one, for 1 and 3, the (c(1) + c(2)) population prevails over the (g(1) + g(2)) one for 2, and the (g(1) + g(2)) conformer population is more abundant than (c) one for 4. In n-hexane solution, the cis conformer is more abundant for 1-3. The occurrence of Fermi resonance in the nu(CO) region, in n-hexane, precludes the estimative of relative populations of the (c, g(1), g(2)) conformers for 4. The SCI-PCM calculations agree with the solvent effect on the nu(CO) band component relative intensities for 1-3. NBO analysis showed that the n(N) -> pi.(CO), orbital interaction is the main factor which stabilizes the gauche (g, g(1), g(2)) conformers for 1-4 into a larger extent relative to the cis (c, c(1), c(2)) ones. The n(y) -> pi(.)(Co,) sigma(C-Y) -> pi.(CO,) pi(CO) -> sigma(C-Y) and 7co orbital interactions still contribute, but into a minor extent for the stabilization of the gauche conformers relative to the cis ones. The existence of some pyramidalization at the nitrogen atom of the Weinreb amides 1-4 is responsible for the occurrence of Y(delta)-(4)center dot center dot center dot O(delta)-(9) and Y(delta)-(4)center dot center dot center dot N(delta)-(7) short contacts in the gauche (g, g(1), g(2)) conformers, which originates strong repulsive Coulombic interactions, acting in opposition to the large orbital stabilization of the gauche conformer with respect to the cis one. Therefore, a delicate balance of the Coulombic and orbital interactions seems to be responsible for the observed stabilization of the gauche (g, g(1), g(2)) and cis (c, c(1), c(2)) conformers, both in the gas phase and in the solution for 1-4. However, the cis conformer predominance, in non polar solvents, for the 2-substituted N-methoxy-N-methyl acetamides 1-3, bearing in a first raw (fluorine and oxygen) atoms, is in the opposite direction to the gauche conformer preference for the corresponding 2-substituted N,N-dialkyl-acetamides. (C) 2010 Elsevier B.V. All rights reserved.

Detection of Paraquat in Oral Fluid, Plasma, and Urine by Capillary Electrophoresis for Diagnosis of Acute Poisoning

FAPESP

Impact of socioeconomic and clinical factors on child oral health-related quality of life (COHRQoL)

Child oral health-related quality of life (COHRQoL) has been increasingly assessed; however, few studies appraised the influence of socioeconomic status on COHRQoL in developing countries. This study assessed the relationship of COHRQoL with socioeconomic backgrounds and clinical factors. This study followed a cross-sectional design, with a multistage random sample of 792 schoolchildren aged 12 years, representative of Santa Maria, a southern city in Brazil. Participants completed the Brazilian version of the Child Perceptions Questionnaire (CPQ(11-14)), their parents or guardians answered questions on socioeconomic status, and a dental examination provided information on the prevalence of caries, dental trauma and occlusion. The assessment of association used hierarchically adjusted Poisson regression models. Higher impacts on COHRQoL were observed for children presenting with untreated dental caries (RR 1.20; 95% CI 1.07-1.35) and maxillary overjet (RR 1.19; 95% CI 1.02-1.40). Socioeconomic factors also associated with COHRQoL; poorer scores were reported by children whose mothers have not completed primary education (RR 1.30; 95% CI 1.17-1.44) and those with lower household income (RR 1.13; 95% CI 1.02-1.26). Poor socioeconomic standings and poor dental status have a negative impact on COHRQoL; reducing health inequalities may demand dental programmes and policies targeting deprived population.

Effects of physiotherapy on hemodynamic variables in newborns with Acute Respiratory Distress Syndrome

Background: Acute respiratory distress syndrome (ARDS) is a frequent respiratory disturbance in preterm newborns. Preceding investigations evaluated chronic physiotherapy effects on newborns with different lung diseases; however, no study analyzed acute physiotherapy treatment on premature newborns with ARDS. In this study we aimed to evaluate the acute effects of chest and motor physiotherapy treatment on hemodynamic variables in preterm newborns with ARDS. Methods: We evaluated heart rate (HR), respiratory rate (RR), systolic (SAP), mean (MAP) and diastolic arterial pressure (DAP), temperature and oxygen saturation (SO(2)%) in 44 newborns with ARDS. We compared all variables between six periods in one day: before first physiotherapy treatment vs. after first physiotherapy treatment vs. before second physiotherapy treatment vs. after second physiotherapy treatment vs. before third physiotherapy treatment vs. after third physiotherapy treatment. Variables were measured 2 minutes before and 5 minutes after each physiotherapy session. We applied Anova one way followed by post hoc Bonferroni test. Results: HR (147.5 +/- 9.5 bpm vs. 137.7 +/- 9.3 bpm; p<0.001), RR (45.5 +/- 8.7cpm vs. 41.5 +/- 6.7 cpm; p=0.001), SAP (70.3 +/- 10.4 mmHg vs. 60.1 +/- 7.1 mmHg; p=0.001) and MAP (55.7 +/- 10 mmHg vs. 46 +/- 6.6 mmHg; p=0.001) were significantly reduced after the third physiotherapy treatment compared to before the first session. There were no significant changes regarding temperature, DAP and SO(2) %. Conclusion: Chest and motor physiotherapy acutely improves HR, RR, SAP, MAP and SO(2) % in newborns with ARDS.

Characteristics of Acute Kidney Injury in Patients Infected with the 2009 Influenza A (H1N1) Virus

Background and objectives: There have been few studies investigating acute kidney injury (AKI) in patients infected with the 2009 pandemic influenza A (H1N1) virus. Therefore, the objective of this study was to identify the factors associated with AKI in H1N1-infected patients. Design, setting, participants, & measurements: This was a study of 47 consecutive critically ill adult patients with reverse transcriptase-PCR-confirmed H1N1 infection in Brazil. Outcome measures were AKI (as defined by the Risk, Injury, Failure, Loss, and End-stage renal failure [RIFLE] criteria) and in-hospital death. Results: AKI was identified in 25 (53%) of the 47 H1N1-infected patients. AKI was associated with vasopressor use, mechanical ventilation, high Acute Physiology and Chronic Health Evaluation II (APACHE II) scores, and severe acidosis as well as with higher levels of C-reactive protein and lactic dehydrogenase upon intensive care unit (ICU) admission. A nephrology consultation was requested for 16 patients (64%), and 8 (50%) required dialysis. At ICU admission, 7 (15%) of the 25 AKI patients had not yet progressed to AKI. However, by 72 hours after ICU admission, no difference in RIFLE score was found between AKI survivors and nonsurvivors. Of the 47 patients, 9 (19%) died, all with AKI. Mortality was associated with mechanical ventilation, vasopressor use, dialysis, high APACHE II score, high bilirubin levels, and a low RIFLE score at ICU admission. Conclusions: Among critically ill H1N1-infected patients, the incidence of AKI is high. In such patients, AKI is mainly attributable to shock. Clin J Am Soc Nephrol 5: 1916-1921, 2010. doi: 10.2215/CJN.00840110

Reduced venous endothelial responsiveness after oral lipid overload in healthy volunteers

The aim of this study was to investigate endothelial venous function, mflammatory markers, and systemic oxidative stress after an oral lipid overload (OLO). We studied 18 healthy adults (9 men; age, 29.2 +/- 0.9 years; body mass index, 22.3 +/- 0.4 kg/m(2)). Blood samples were collected in the fasting state and 3, 4, and 5 hour after the OLO (1000 kcal, 58% fat) for metabolic variables, oxidative stress, inflammatory markers, adiponectin, and resistin. Changes in vein diameter to phenylephrine, acetylcholine, and sodium nitroprusside (dorsal hand vein technique) were measured before and after the OLO. Oral lipid overload increased triglycerides (61 +/- 6 vs 134 +/- 17 mg/dL, P <.001), insulin (7.2 +/- 0.8 vs 10.7 +/- 1.3 mu U/mL, P <.05), and resistin (5.38 +/- 0.5 vs 6.81 +/- 0.7 ng/mL, P <.05) and reduced antioxidant capacity (plasma total antioxidant capacity: 186.7 +/- 56 vs 161.8 +/- 50 U Trolox per microliter plasma, P <.01), vascular reactivity (171.3 +/- 85 vs 894.4 +/- 301 ng/mL, P <.001), and maximum acetylcholine venodilation (105.9% +/- 9% vs 61.0% +/- 7%, P <.05). No changes were observed for sodium nitroprusside. Post-OLO triglycerides were positively correlated with phenylephrine dose (rho = 0.38, P <.05) and resistin (rho = 0.43, P <.01) and negatively correlated with the maximum acetylcholine venodilation (rho = -0.36, P <.05). In conclusion, an OLO impaired venoconstriction responsiveness in healthy subjects, probably because of a reduction in the antioxidant capacity. (C) 2008 Elsevier Inc. All rights reserved.

Structural changes in the epithelium of the small intestine and immune cell infiltration of enteric ganglia following acute mucosal damage and local inflammation

An acute enteritis is commonly followed by intestinal neuromuscular dysfunction, including prolonged hyperexcitability of enteric neurons. Such motility disorders are associated with maintained increases in immune cells adjacent to enteric ganglia and in the mucosa. However, whether the commonly used animal model, trinitrobenzene sulphonate (TNBS)-induced enteritis, causes histological and immune cell changes similar to human enteric neuropathies is not clear. We have made a detailed study of the mucosal damage and repair and immune cell invasion following intralumenal administration of TNBS. Intestines from untreated, sham-operated and TNBS-treated animals were examined at 3 h to 56 days. At 3 h, the mucosal surface was completely ablated, by 6 h an epithelial covering was substantially restored and by 1 day there was full re-epithelialisation. The lumenal epithelium developed from a squamous cell covering to a fully differentiated columnar epithelium with mature villi at about 7 days. Prominent phagocytic activity of enterocytes occurred at 1-7 days. A surge of eosinophils and T lymphocytes associated with the enteric nerve ganglia occurred at 3 h to 3 days. However, elevated immune cell numbers occurred in the lamina propria of the mucosa until 56 days, when eosinophils were still three times normal. We conclude that the disruption of the mucosal surface that causes TNBS-induced ileitis is brief, a little more than 6 h, and causes a transient immune cell surge adjacent to enteric ganglia. This is much briefer than the enteric neuropathy that ensues. Ongoing mucosal inflammatory reaction may contribute to the persistence of enteric neuropathy.

Laminin-derived peptide AG73 regulates migration, invasion, and protease activity of human oral squamous cell carcinoma cells through syndecan-1 and beta 1 integrin

Squamous cell carcinoma is a prevalent head and neck tumor with high mortality. We studied the role played by laminin alpha 1 chain peptide AG73 on migration, invasion, and protease activity of cells (OSCC) from human oral squamous cell carcinoma. Immunohistochemistry and immunofluorescence analyzed expression of laminin alpha 1 chain and MMP9 in oral squamous cells carcinoma in vivo and in vitro. Migratory activity of AG73-treated OSCC cells was investigated by monolayer wound assays and in chemotaxis chambers. AG73-induced invasion was assessed in Boyden chambers. Invasion depends on MMPs. Conditioned media from cells grown on AG73 was subjected to zymography. We searched for AG73 receptors related to these activities in OSCC cells. Immunofluorescence analyzed AG73induced colocalization of syndecan-1 and beta 1 integrin. Cells had these receptors silenced by siRNA, followed by treatment with AG73 and analysis of migration, invasion, and protease activity. Oral squamous cell carcinoma expresses laminin alpha 1 chain and MMP9. OSCC cells treated with AG73 showed increased migration, invasion, and protease activity. AG73 induced colocalization of syndecan-1 and beta 1 integrin. Knockdown of these receptors decreased AG73-dependent migration, invasion, and protease activity. Syndecan-1 and beta 1 integrin signaling downstream of AG73 regulate migration, invasion, and MMP production by OSCC cells.

Bone marrow mononuclear cells attenuate fibrosis development after severe acute kidney injury

One of the early phases that lead to fibrosis progression is inflammation. Once this stage is resolved, fibrosis might be prevented. Bone marrow mononuclear cells (BMMCs) are emerging as a new therapy for several pathologies, including autoimmune diseases, because they enact immunosuppression. In this study we aimed to evaluate the role of BMMC administration in a model of kidney fibrosis induced by an acute injury. C57Bl6 mice were subjected to unilateral severe ischemia by clamping the left renal pedicle for 1 h. BMMCs were isolated from femurs and tibia, and after 6 h of reperfusion, 1 x 10(6) cells were administrated intraperitoneally. At 24 h after surgery, treated animals showed a significant decrease in creatinine and urea levels when compared with untreated animals. Different administration routes were tested. Moreover, interferon (IFN) receptor knockout BMMCs were used, as this receptor is necessary for BMMC activation. Labeled BMMCs were found in ischemic kidney on FACS analysis. This improved outcome was associated with modulation of inflammation in the kidney and systemic modulation, as determined by cytokine expression profiling. Despite non-amelioration of functional parameters, kidney mRNA expression of interleukin (IL)-6 at 6 weeks was lower in BMMC-treated animals, as were levels of collagen 1, connective tissue growth factor (CTGF), transforming growth factor-beta (TGF-beta) and vimentin. Protective molecules, such as IL-10, heme oxygenase 1 (HO-1) and bone morphogenetic 7 (BMP-7), were increased in treated animals after 6 weeks. Moreover, Masson and Picrosirius red staining analyses showed less fibrotic areas in the kidneys of treated animals. Thus, early modulation of inflammation by BMMCs after an ischemic injury leads to reduced fibrosis through modulation of early inflammation.

It takes guts for tolerance: The phenomenon of oral tolerance and the regulation of autoimmune response

The intestinal tract is a peculiar environment due to its constant contact with the microbiota agents, food antigens and other molecules. Such exposure requires the establishment of important regulatory mechanisms in order to avoid inflammatory response and self aggression. In this context, the GALT plays a very relevant role due to the presence of several different cellular populations which are the main players in this phenomenon. Moreover, it was described a while ago that the oral ingestion of a given molecule is able to induce systemic tolerance to the same molecule when it is used as an immunogen by parenteral route, known as oral tolerance. This observation led researches to use these mechanisms to induce tolerance against cognate antigens of different autoimmune diseases. In this context, in this review we focused on several tolerance inducing mechanisms which are relevant not only for the maintenance of intestinal tract but also for the suppression of T effector cells, such as Th1, Th2 and the newly described Th17 cells. To name a few, CD103(+) dendritic cells, Tr1 cells derived IL-10 secretion, Foxp3 conversion and CD4(+)LAP(+) regulatory cells induction are among the recently described features of the tolerogenic environment of the intestinal tract. (C) 2009 Elsevier B.V. All rights reserved.

Detection of the Tim-3 ligand, galectin-9, inside the allograft during a rejection episode

Introduction: Tim-3 is a Th1 lymphocytes membrane protein with inhibitory function. Its ligand, galectin-9, was recently identified and it is expressed in some lymphocyte subpopulation. In addition, endothelial cells and fibroblasts can also express galectin-9 according to the local cytokine milieu. Both molecules can act as important regulatory tools in the immune system. Aim: Evaluate the expression of these immunoregulatory molecules inside kidney allografts during acute rejection episodes. Methods: By using a quantitative polymerase chain reaction assay, we measured the levels of messenger RNA (mRNA) for galectin-9 and Tim-3 in 21 samples obtained at allograft nephrectomy. Five samples received the histological diagnosis of acute non-vascular rejection (ANVR), twelve of acute vascular rejection (AVR), and five of loss of non-immune cause (LNIC; as control). As cytolytic response markers we measured mRNA levels of granzyme B, interferon-gamma and perforin. The statistic analysis was performed using one way analysis of variance (ANOVA) and Pearson correlation. Results: The mean levels of Tim-3 mRNA expression were 13.99 +/- 6.99 for LNIC, 48.13 +/- 54.47 for RACNV and 238.63 +/- 333.14 for RAV (p = 0.004). For galectin-9, the mean values were 0.57 +/- 0.49 for LNIC, 0.66 +/- 0.36 for RACNV and 2.34 +/- 1.62 for RAV (p = 0.006). Furthermore, there was a positive correlation between both molecules (r = 0.526, p = 0.016). Also. granzyme B, perforin and interferon-gamma mRNA expression were different among the three groups. Conclusion: Messenger RNA level expressions of all the studied molecules were higher inside allografts with more severe rejection. Moreover, there was a positive correlation between galectin-9 and Tim-3 mRNA levels. The simultaneous expression of galectin-9 and Tim-3 may indicate an immunoregulatory function, during the ongoing cytotoxic response. (C) 2008 Elsevier B.V. All rights reserved.

Toll-like receptors-related genes in kidney transplant patients with chronic allograft nephropathy and acute rejection

Introduction: Toll-like receptors (TLR) comprehend an emerging family of receptors that recognize pathogen-associated molecular patterns and promote the activation of leukocytes. Surgical trauma and ischemia-reperfusion injury are likely to provide exposure to endogenous ligands for TLR in virtually all kidney transplant recipients. Methods: Macroarray (GEArray OHS-018.2 Series-Superarray) analyses of 128 genes involved in TLR signaling pathway were performed in nephrectomy samples of patients with chronic allograft nephropathy (CAN) and acute rejection (AR, vascular and non vascular). The analysis of each membrane was performed by GEArray Expression Analysis Suite 2.0. Results: Macroarray profile identified a gene expression signature that could discriminate CAN and AR. Three genes were significantly expressed between CAN and vascular AR: Pellino 2; IL 8 and UBE2V1. In relation to vascular and non-vascular AR, there were only two genes with statistical significance: IL-6 and IRAK-3. Conclusion: Vascular and non-vascular AR and CAN showed different expression of a few genes in TLR pathway. The analysis of nephrectomy showed that activation of TLR pathway is present in AR and CAN. (C) 2008 Elsevier B.V. All rights reserved.

Early modulation of inflammation by mesenchymal stem cell after acute kidney injury

Therapy with stem cells has showed to be promising for acute kidney injury (AKI), although how it works is still controversial. Modulation of the inflammatory response is one possible mechanism. Most of published data relies on early time and whether the protection is still maintained after that is not known. Here, we analyzed whether immune modulation continues after 24 h of reperfusion. MSC were obtained from male Wistar rats. After 3-5 passages, cells were screened for CD73, CD90, CD44, CD45, CD29 and CD 31. In addition, MSC were submitted to differentiation in adipocyte and in osteocyte. AKI was induced by bilaterally clamping of renal pedicles for 60 min. Six hours after injury, MSC (2 x 105 cells) were administered intravenously. MSC-treated animals presented the lowest serum creatinine compared to non-treated animals (24 h: 1.3 +/- 0.21 vs. 3.23 +/- 0.89 mg/dl, p<0.05). The improvement in renal function was followed by a lower expression of IL-1b, IL-6 and TNF-alpha and higher expression of IL-4 and IL-10. However, 48 h after reperfusion, this cytokine profile has changed. The decrease in Th1 cytokines was less evident and IL-6 was markedly up regulated. PCNA analysis showed that regeneration occurs faster in kidney tissues of MSC-treated animals than in controls at 24 h. And also ratio of Bcl-2/Bad was higher at treated animals after 24 and 48 h. Our data demonstrated that the immunomodulatory effects of MSC occur at very early time point, changing the inflammation profile toward a Th2 profile. (C) 2009 Elsevier B.V. All rights reserved.

Acute toxicity of a single gavage dose of fumonisin B(1) in rabbits

The aim of this study was to determine the clinical, pathological and mycotoxicological effects of oral administration of fumonisin B, (FBI) in rabbits. Eighteen rabbits were randomly assigned to two experimental groups: control group, 0 mg FB(1): fumonisin group. 31.5 mg FB(1)/kg body weight, corresponding to about 630 mg FB(1)/kg diet. Fumonisin administered as a single oral dose to rabbits resulted in acute toxicity, significantly interfering with body and liver weight. Serum biochemical analysis revealed a significant increase of total protein, alkaline phosphatase (AP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyltransferase (GGT), urea and creatinine in the group receiving FBI compared to control animals, a finding characterizing hepatic and renal injury in this group. Urinary protein concentrations were markedly elevated at 12,24,48 and 72 h after dosing, although visible pathological abnormalities were not observed, probably because of rapid repair of the damage. FBI was detected in feces, with a maximum concentration at 24h after administration, indicating that the enterohepatic circulation is important in rabbits. FBI concentrations found in urine were low, with peak elimination at 12 h after intoxication. The highest FBI concentrations were observed in feces compared to urine and liver, demonstrating that feces are the main routes of excretion. (C) 2009 Elsevier Ireland Ltd. All rights reserved.