Acute appendicitis: Factors associated with inconclusive ultrasound study and the need for additional computed tomography.

PURPOSE: To identify variables associated with inconclusive ultrasound examination and the need for further abdominopelvic computed tomography (CT) examination for the diagnosis of acute appendicitis. MATERIALS AND METHODS: A total of 105 adult patients with acute appendicitis were included. There were 55 patients (38 men, 17 women; mean age, 23±9 [SD] years; range: 15-58 years) with a diagnosis of acute appendicitis using ultrasound alone and 50 patients (30 men, 20 women; mean age, 31±14 [SD] years; range: 16-83 years) who required further CT. Demographic, clinical, and biological criteria and appendix location were compared between the two groups to search for variables associated with the need of further CT. RESULTS: Patients who required further CT were older (31.1±14 [SD] years) and had a greater body mass index (BMI) (26.7±4.3 [SD]kg/m2) than those who did not require CT (23±9 [SD] years and 22.9±3.4 [SD]kg/m2), respectively (P<0.01). A greater proportion of patients with complicated acute appendicitis was observed in patients who required further CT (9/50; 18%) than in those who had only ultrasound (1/55; 2%) (P=0.012). Atypical appendix location was more frequent in patients who required CT (19/50; 36%) than in those who had only ultrasound (6/55; 11%) (P<0.001). There were no significant differences regarding gender, inflammatory syndrome and hours of imaging (on call vs. working hours) between the two groups. CONCLUSION: Advanced age, high BMI, atypical appendix location, and complicated appendicitis are associated with inconclusive ultrasound and the need for further CT to diagnose acute appendicitis.

SERS of cells: What can we learn from cell lysates?

Surface-enhanced Raman spectroscopy (SERS) is a promising and emerging technique to analyze the cellular environment. We developed an alternative, rapid and label-free SERS-based method to get information about the cellular environment by analyzing cells lysates, thus avoiding the need to incorporate nanoparticles into cells. Upon sonicating and filtrating cells, we obtained lysates which, mixed with Au or Ag nanoparticles, yield stable and repeatable SERS spectra, whose overall profile depends on the metal used as substrate, but not on the buffer used for the lysis process. Bands appearing in these spectra were shown to arise mostly from the cytosol and were assigned to adenine, guanine, adenosine and reduced glutathione (GSH). Spectral differences among various cell types also demonstrated that this approach is suitable for cell type identification.

Toxic equivalency factors (TEFs) after acute oral exposure of azaspiracid 1, -2 and -3 in mice.

Azaspiracids (AZAs) are marine algal toxins that can be accumulated by edible shellfish to cause a foodborne gastrointestinal poisoning in humans. In the European Union, only AZA1, -2 and -3 are currently regulated and their concentration in shellfish is determined through their toxic equivalency factors (TEFs) derived from the intraperitoneal lethal potency in mice. Nevertheless, considering the potential human exposure by oral route, AZAs TEFs should be calculated by comparative oral toxicity data. Thus, the acute oral toxicity of AZA1, -2 and -3 was investigated in female CD-1 mice treated with different doses (AZA1: 135-1100µg/kg; AZA2 and AZA3: 300-1100µg/kg) and sacrificed after 24h or 14days. TEFs derived from the median lethal doses (LD50) were 1.0, 0.7 and 0.5, respectively for AZA1, -2 and -3. In fact, after 24h from gavage administration, LD50s were 443µg/kg (AZA1; 95% CL: 350-561µg/kg), 626µg/kg (AZA2; 95% CL: 430-911µg/kg) and 875µg/kg (AZA3; 95% CL: 757-1010µg/kg). Mice dead more than 5h after the treatment or those sacrificed after 24h (doses: ≥175µg AZA1/kg, ≥500µg AZA2/kg and ≥600µg AZA3/kg) showed enlarged pale liver, while increased serum markers of liver alteration were recorded even at the lowest doses. Blood chemistry revealed significantly increased serum levels of K+ ions (≥500mg/kg), whereas light microscopy showed tissue changes in the gastrointestinal tract, liver and spleen. No lethality, macroscopic, tissue or haematological changes were recorded two weeks post exposure, indicating reversible toxic effects. LC-MS/MS analysis of the main organs showed a dose-dependency in gastrointestinal absorption of these toxins: at 24h, the highest levels were detected in the stomach and, in descending order, in the intestinal content, liver, small intestine, kidneys, lungs, large intestine, heart as well as detectable traces in the brain. After 14days, AZA1 and AZA2 were still detectable in almost all the organs and intestinal content.

In vitro sensitivity to methyl-prednisolone is associated with clinical response in pediatric idiopathic nephrotic syndrome.

The aim of this study was to evaluate the in vitro steroid sensitivity as a predictor of clinical response to glucocorticoids in childhood idiopathic nephrotic syndrome (INS). Seventy-four patients (median age 4.33, interquartile range [IQR] 2.82-7.23; 63.5% male) were enrolled in a prospective multicenter study: in vitro steroid inhibition of patients' peripheral blood mononuclear cell proliferation was evaluated by [methyl-(3) H] thymidine incorporation assay at disease onset (T0) and after 4 weeks (T4) of treatment. Steroid dependence was associated with increased in vitro sensitivity at T4 assessed both as drug concentration inducing 50% of inhibition (IC50 ; odds ratio [OR] = 0.48, 95% confidence interval [CI] = 0.24-0.85; P = 0.0094) and maximum inhibition at the highest drug concentration (Imax ; OR = 1.13, 95% CI = 1.02-1.31; P = 0.017). IC50 > 4.4 nM and Imax < 92% at T4 were good predictors for optimal clinical response. These results suggest that this test may be useful for predicting the response to glucocorticoid therapy in pediatric INS.

Long noncoding RNA GAS5: a novel marker involved in glucocorticoid response.

Glucocorticoids (GCs) exert their effects through regulation of gene expression after activation in the cytoplasm of the glucocorticoid receptor (GR) encoded by NR3C1 gene. A negative feedback mechanism resulting in GR autoregulation has been demonstrated through the binding of the activated receptor to intragenic sequences called GRE-like elements, contained in GR gene. The long noncoding RNA growth arrest-specific transcript 5 (GAS5) interacts with the activated GR suppressing its transcriptional activity. The aim of this study was to evaluate the possible role of GAS5 and NR3C1 gene expression in the antiproliferative effect of methylprednisolone in peripheral blood mononuclear cells and to correlate the expression with individual sensitivity to GCs. Subjects being poor responders to GCs presented higher levels of GAS5 and NR3C1 in comparison with good responders. We suggest that abnormal levels of GAS5 may alter GC effectiveness, probably interfering with the mechanism of GR autoregulation.

Sandwich ELISA assay for the quantitation of palytoxin and its analogs in natural samples.

Palytoxins are potent marine biotoxins that have recently become endemic to the Mediterranean Sea, and are becoming more frequently associated with seafood. Due to their high toxicity, suitable methods to quantify palytoxins are needed. Thus, we developed an indirect sandwich ELISA for palytoxin and 42-hydroxy-palytoxin. An intralaboratory study demonstrated sensitivity (limit of detection, LOD = 1.1 ng/mL; limit of quantitation, LOQ = 2.2 ng/mL), accuracy (bias of 2.1%), repeatability (RSDr = 6% and 9% for intra- and interassay variability, respectively) and specificity: other common marine toxins (okadaic acid, domoic acid, saxitoxin, brevetoxin-3, and yessotoxin) do not cross-react in this assay. It performed well in three different matrices: observed LOQs were 11.0, 9.6, and 2.4 ng/mL for mussel extracts, algal net samples and seawater, respectively, with good accuracy and precision. The LOQ in seafood is 11 µg palytoxin/kg mussel meat, lower than that of the most common detection technique, LC-MS/MS.

The cytotoxic effect of palytoxin on Caco-2 cells hinders their use for in vitro absorption studies.

Palytoxin (PLTX), found in Palythoa zoanthids and Ostreopsis dinoflagellates, has also been detected in crabs and fish, through which it can enter into the food chain. Indeed, PLTX is considered the causative agent of several cases of human seafood poisoning resulting in systemic symptoms. Available epidemiological data on PLTX human toxicity suggest that the intestinal tract may be one of its in vivo targets and its potential site of access into the bloodstream. Hence, the purpose of this study was to investigate the suitability of the human intestinal Caco-2 cell line for evaluating PLTX oral absorption. A detailed analysis of PLTX cytotoxicity revealed a high sensitivity of Caco-2 cells: 4h toxin exposure reduced mitochondrial activity (MTT assay, EC(50) of 8.9±3.7×10(-12)M), cell density (SRB assay, EC(50) of 2.0±0.6×10(-11)M) and membrane integrity (LDH release, EC(50) of 4.5±1.4×10(-9)M and PI uptake, EC(50) of 1.0±0.8×10(-8)M). After low PLTX concentration (1.0×10(-11)M) exposure for 1-8h, followed by 24h recovery time in toxin-free medium, cell density reduction was only partially reversible. These results indicate that, due to the high susceptibility to PLTX cytotoxic effects, Caco-2 cells do not represent an appropriate and reliable model for investigating intestinal barrier permeation by this toxin.

Acute oral toxicity in mice of a new palytoxin analog: 42-hydroxy-palytoxin.

The acute oral toxicity of a new palytoxin congener, 42-hydroxy-palytoxin (42-OH-PLTX), was investigated in female CD-1 mice. The toxin (300-1697 µg/kg), administered by gavage, induced scratching, jumping, respiratory distress, cyanosis, paralysis and death of mice, with an LD50 of 651 µg/kg (95% confidence limits: 384-1018 µg/kg) within 24 h. Hematoclinical analyses showed increased plasma levels of lactate dehydrogenase and aspartate-aminotransferase at doses of 600 µg/kg and above, as well as of alanine-aminotransferase, creatine phosphokinase and potassium ions at ≥ 848 µg/kg. Histology revealed inflammatory lesions in the non-glandular area of the stomach of mice that survived up to 24 h after gavage (424-1200 µg/kg). Although no histological alterations were seen in skeletal and cardiac muscles, changes in some plasma biomarkers (creatine phosphokinase, lactate dehydrogenase) suggested involvement of these tissues in 42-OH-PLTX oral toxicity, in agreement with epidemiological data on seafood poisonings ascribed to palytoxins. Complete recovery of the tissue and hematological changes was observed two weeks post-exposure. Furthermore, 42-OH-PLTX induced in vitro delayed erythrocyte hemolysis at concentrations similar to those of PLTX (EC50 = 7.6 and 13.2 x 10⁻¹² M, respectively). This hemolysis could be completely neutralized by a monoclonal anti-PLTX antibody. The in vivo data, together with the in vitro data recorded for 42-OH-PLTX, seem to indicate Na+/K+-ATPase as one of the key cellular targets of this toxin.

Effects of the marine toxin palytoxin on human skin keratinocytes: role of ionic imbalance.

Palytoxin (PLTX), a marine toxin identified in Palythoa zoanthid corals and Ostreopsis dinoflagellates, represents an increasing hazard for human health. Recently, dermatological problems have been associated to cutaneous exposure to PLTX during Ostreopsis blooms arising the need for experimental data characterizing PLTX effects on the skin. This study highlights in vitro the cytotoxic effects of PLTX on human keratinocytes (HaCaT cell line). A short time exposure (4h) to PLTX reduced mitochondrial activity (MTT assay), cell mass (SRB assay) and plasma membrane integrity (LDH leakage) with different potencies: EC50 values of 6.1 ± 1.3×10⁻¹¹, 4.7 ± 0.9 × 10⁻¹° M and 1.8 ± 0.1 × 10⁻8 M, respectively. PLTX effect on mitochondrial activity was ouabain- and Na+-sensitive, but only partially sensitive to removal of Ca²+ ions. One hour exposure to the toxin also induced a Na+-dependent and Ca²+-independent superoxide anion production. These results indicate that among the chain of intracellular events following the interaction of PLTX with the Na+/K+-ATPase the first and crucial step is the increased intracellular Na+ concentration that triggers a sequence of cell dysfunction involving mitochondrial affection and oxidative stress, leading to an irreversible cell death. The PLTX concentrations inducing cytotoxicty seem to be lower than those of potential cutaneous human exposure during Ostreopsis ovata blooms, indicating the harmful potential of the toxin.