Early diagnosis of bladder cancer through the detection of urinary tyrosine-phosphorylated proteins.

BACKGROUND: A noninvasive, highly sensitive and specific urine test is needed for bladder cancer (BC) diagnosis and surveillance in addition to the invasive cystoscopy. We previously described the diagnostic effectiveness of urinary tyrosine-phosphorylated proteins (UPY) and a new assay (UPY-A) for their measurement in a pilot study. The aim of this work was to evaluate the performances of the UPY-A using an independent cohort of 262 subjects. METHODS: Urinary tyrosine-phosphorylated proteins were measured by UPY-A test. The area under ROC curve, cutoff, sensitivity, specificity and predictive values of UPY-A were determined. The association of UPY levels with tumour staging, grading, recurrence and progression risk was analysed by Kruskal-Wallis and Wilcoxon's test. To test the probability to be a case if positive at the UPY-A, a logistic test adjusted for possible confounding factor was used. RESULTS: Results showed a significant difference of UPY levels between patients with BC vs healthy controls. For the best cutoff value, 261.26 Standard Units (SU), the sensitivity of the assay was 80.43% and the specificity was 78.82%. A statistically significant difference was found in the levels of UPY at different BC stages and grades between Ta and T1 and with different risk of recurrence and progression. A statistically significant increased risk for BC at UPY-A ⩾261.26 SU was observed. CONCLUSIONS: The present study supplies important information on the diagnostic characteristics of UPY-A revealing remarkable performances for early stages and allowing its potential use for different applications encompassing the screening of high-risk subjects, primary diagnosis and posttreatment surveillance.

A high-throughput assay for the detection of Tyr-phosphorylated proteins in urine of bladder cancer patients.

BACKGROUND: Bladder cancer has the peculiarity of shedding neoplastic cells and their components in urine representing a valuable opportunity to detect diagnostic markers. Using a semi-quantitative method we previously demonstrated that the levels of Tyr-phosphorylated proteins (TPPs) are highly increased in bladder cancer tissues and that soluble TPPs can also be detected in patient's urine samples. Although the preliminary evaluation showed very promising specificity and sensitivity, insufficient accuracy and very low throughput of the method halted the diagnostic evaluation of the new marker. To overcome this problem we developed a quantitative methodology with high sensitivity and accuracy to measure TPPs in urine. METHODS: The Immobilized Metal Affinity Chromatography (IMAC) was miniaturized in a 96 well format. Luminescence, visible and infrared fluorescence antibody-based detection methods were comparatively evaluated. RESULTS: Due to their low abundance we evidenced that both phosphoprotein enrichment step and very sensitive detection methods are required to detect TPPs in urine samples. To pursue high throughput, reproducibility and cost containment, which are required for bladder cancer screening programs, we coupled the pre-analytical IMAC procedure with high sensitive detection phases (infrared fluorescence or chemiluminescence) in an automated platform. CONCLUSIONS: A high throughput method for measuring with high sensitivity TPP levels in urine samples is now available for large clinical trial for the establishment of the diagnostic and predictive power of TPPs as bladder cancer marker. GENERAL SIGNIFICANCE: The new assay represents the first quantitative and high throughput method for the measurement of TPPs in urine.

A common variant upstream of the PAX6 gene influences islet function in man.

AIMS/HYPOTHESIS: Impaired glucose tolerance and impaired insulin secretion have been reported in families with PAX6 mutations and it is suggested that they result from defective proinsulin processing due to lack of prohormone convertase 1/3, encoded by PCSK1. We investigated whether a common PAX6 variant would mimic these findings and explored in detail its effect on islet function in man. METHODS: A PAX6 candidate single nucleotide polymorphism (rs685428) was associated with fasting insulin levels in the Diabetes Genetics Initiative genome-wide association study. We explored its potential association with glucose tolerance and insulin processing and secretion in three Scandinavian cohorts (N = 8,897 individuals). In addition, insulin secretion and the expression of PAX6 and transcriptional target genes were studied in human pancreatic islets. RESULTS: rs685428 G allele carriers had lower islet mRNA expression of PAX6 (p = 0.01) and PCSK1 (p = 0.001) than AA homozygotes. The G allele was associated with increased fasting insulin (p (replication) = 0.02, p (all) = 0.0008) and HOMA-insulin resistance (p (replication) = 0.02, p (all) = 0.001) as well as a lower fasting proinsulin/insulin ratio (p (all) = 0.008) and lower fasting glucagon (p = 0.04) and gastric inhibitory peptide (GIP) (p = 0.05) concentrations. Arginine-stimulated (p = 0.02) insulin secretion was reduced in vivo, which was further reflected by a reduction of glucose- and potassium-stimulated insulin secretion (p = 0.002 and p = 0.04, respectively) in human islets in vitro. CONCLUSIONS/INTERPRETATION: A common variant in PAX6 is associated with reduced PAX6 and PCSK1 expression in human islets and reduced insulin response, as well as decreased glucagon and GIP concentrations and decreased insulin sensitivity. These findings emphasise the central role of PAX6 in the regulation of islet function and glucose metabolism in man.

Impairment of macrophage functions after ingestion of Plasmodium falciparum-infected erythrocytes or isolated malarial pigment.

Human monocyte-derived macrophages ingest diamide-treated red blood cells (RBC), anti-D immunoglobulin (Ig)G-opsonized RBC, or Plasmodium falciparum ring-stage parasitized RBC (RPRBC), degrade ingested hemoglobin rapidly, and can repeat the phagocytic cycle. Monocytes fed with trophozoite-parasitized RBC (TPRBC), which contain malarial pigment, or fed with isolated pigment are virtually unable to degrade the ingested material and to repeat the phagocytic cycle. Monocytes fed with pigment display a long-lasting oxidative burst that does not occur when they phagocytose diamide-treated RBC or RPRBC. The phorbol myristate acetate-elicited oxidative burst is irreversibly suppressed in monocytes fed with TPRBC or pigment, but not in monocytes fed with diamide-treated or IgG-opsonized RBC. This pattern of inhibition of phagocytosis and oxidative burst suggests that malarial pigment is responsible for the toxic effects. Pigment iron released in the monocyte phagolysosome may be the responsible element. 3% of total pigment iron is labile and easily detached under conditions simulating the internal environment of the phagolysosome, i.e., pH 5.5 and 10 microM H2O2. Iron liberated from pigment could account for the lipid peroxidation and increased production of malondialdehyde observed in monocytes fed with pigment or in RBC ghosts and liposomes incubated at pH 6.5 in presence of pigment and low amounts of H2O2. Removal of the labile iron fraction from pigment by repeated treatments with 0.1 mM H2O2 at pH 5.5 reduces pigment toxicity. It is suggested that iron released from ingested pigment is responsible for the intoxication of monocytes. In acute and chronic falciparum infections, circulating and tissue-resident phagocytes are seen filled with TPRBC and pigment particles over long periods of time. Moreover, human monocytes previously fed with TPRBC are unable to neutralize pathogenic bacteria, fungi, and tumor cells, and macrophage responses decline during the course of human and animal malaria. The present results may offer a mechanistic explanation for depression of cellular immunity in malaria.

Solubility study and intensification of extraction of phenolic and anthocyanin compounds from Oryza sativa L. 'Violet Nori'.

Oryza sativa L. 'Violet Nori' is an Italian cultivar of spontaneous growing aromatic purple rice which is particularly rich in polyphenolic compounds, especially anthocyanins, conferring it an excellent antioxidant capacity. The present study aimed at increasing the extraction yields of its antioxidant compounds with green strategies and it is divided into two steps. The first step concerned a solubility study of the targeted polyphenols in different ethanol:water mixtures by means of a theoretical prediction method, using the simulation program COSMO-RS, and the subsequently confirmation of the computational results by practical experiments. Once the best extraction mixture was identified, the second step of the work was performed, with the purpose of intensifying the extraction yield. Therefore, various innovative green extraction techniques, including ultrasound, using both the probe system and the ultrasonic bath, bead milling, microwave and accelerated solvent extractions were tested and compared to conventional maceration. Results, expressed in terms of total phenolic and total monomeric anthocyanin contents, showed that the best extracting solvent for 'Violet Nori' rice was the mixture ethanol:water (60:40 v/v), being COSMO-RS computational predictions in good correlation with the experimental results. Moreover, the most efficient techniques to extract the antioxidant compounds resulted to be both ultrasound-assisted extraction probe and bead milling, that in only 5 min got the same extractive efficiency obtained after 3 h of conventional maceration.

Phagocytosis of P. falciparum malarial pigment hemozoin by human monocytes inactivates monocyte protein kinase C.

Hemozoin (malarial pigment) is a ferriprotoporphyrin IX-rich hemoglobin degradation product present in parasitized RBC. Avidly phagocytosed hemozoin abolishes phagocyte TPA-induced oxidative burst. Membrane-associated PKC increased transiently in hemozoin-fed monocytes by 50% after 30 min and decreased irreversibly to 20% of initial value within 5 h after phagocytosis. Control RBC-fed monocytes showed transient decay of membrane-associated PKC followed by complete recovery 12 h after phagocytosis. Cytosolic PKC was not impaired within 12 h and diminished drastically 24 h after phagocytosis of hemozoin. Results are compatible with increased degradation of membrane-translocated PKC, possibly by iron/H2O2-mediated damage of cysteine-rich regulatory domains of PKC.

Role of Na+/H+ exchange in thrombin- and arachidonic acid-induced Ca2+ influx in platelets.

Platelet activation is accompanied by an increase of cytosolic free Ca2+ concentration, [Ca2+]i, (due to both extracellular Ca2+ influx and Ca2+ movements from the dense tubular system) and an Na+ influx associated with H+ extrusion. The latter event is attributable to the activation of Na+/H+ exchange, which requires Na+ in the extracellular medium and is inhibited by amiloride and its analogs. The present study was carried out to determine whether a link exists between Ca2+ transients (measured by the quin2 method and the 45CaCl2 technique) and Na+/H+ exchange activation (studied with the pH-sensitive intracellular probe, 6-carboxyfluorescein) during platelet stimulation. Washed human platelets, stimulated with thrombin and arachidonic acid, showed: (1) a large and rapid [Ca2+]i rise, mostly due to a Ca2+ influx through the plasma membrane; (2) a marked intracellular alkalinization. Both phenomena were markedly inhibited in the absence of extracellular Na+ or in the presence of an amiloride analog (EIPA). Monensin, a cation exchanger which elicits Na+ influx and alkalinization, and NH4Cl, which induces alkalinization only, were able to evoke an increase in [Ca2+]i, mostly as an influx from the extracellular medium. Our results suggest that Ca2+ influx induced by thrombin and arachidonic acid in human platelets is strictly dependent on Na+/H+-exchange activation.

Binding of naturally occurring antibodies to oxidatively and nonoxidatively modified erythrocyte band 3.

Both oxidative clustering (elicited by diamide treatment) and nonoxidative clustering (elicited by zinc/BS3 (bis[sulfosuccinimidyl]suberate) treatment) of erythrocyte integral membrane proteins induce binding of autologous antibodies with anti-band 3 specificity, followed by complement deposition and phagocytosis. Autologous antibodies eluted from nonoxidatively clustered erythrocytes bind to and stimulate phagocytosis of oxidatively damaged erythrocytes. Those eluted antibodies bind specifically to disulfide-crosslinked band 3 dimers generated by diamide treatment. Band 3 dimerization and antibody binding are abrogated by cleavage of band 3 cytoplasmic domain. Thus, disulfide-crosslinked band 3 dimers are the minimal band 3 aggregate with enhanced affinity for anti-band 3 antibodies. The eluted antibodies do not bind to band 3 dimers generated nonoxidatively by BS3 treatment but bind avidly to larger band 3 clusters generated nonoxidatively by zinc/BS3 treatment. Possibly, disulfide crosslinking of cytoplasmic domain cysteines induces reorientation of intramembrane domains as to expose putative anti-band 3 epitopes and allow bivalent binding of anti-band 3 antibodies. Extensive nonoxidative band 3 clustering appears to disrupt the native band 3 conformation and generate reoriented dimers which expose putative anti-band 3 epitopes in the proper distance and orientation as to allow bivalent antibody binding.

Video laparoscopic surgery: is out-of-hospital thromboprophylaxis necessary?

Despite widespread use of laparoscopic procedures, no adequate data are available to support specific recommendations for venous thromboprophylaxis in patients undergoing laparoscopic surgery. This prospective, randomized trial is the first to be designed to evaluate a regimen of out-of-hospital thromboprophylaxis after laparoscopic surgery. Consecutive patients admitted for laparoscopic surgery were considered for the study. The thromboprophylaxis regimen used for each patient was based on a risk score. Possible thromboprophylactic measures included elastic stockings and pre- and postoperative Dalteparin or early ambulation. At discharge, patients were randomly allocated either to continue Dalteparin for 1 week, or to receive no further prophylaxis. Patients judged to be at low risk were not randomized. Compression ultrasound of the leg veins was performed in all patients 4 weeks after hospital discharge. Fifty-three patients, all with acute appendicitis, were judged to be at low risk of deep vein thrombosis and were not included in the randomized study. The remaining 209 patients fell into two groups: 104 patients received postdischarge Dalteparin and 105 patients did not. The incidence of deep vein thrombosis was 0% (0 of 104) vs. 0.95% (one of 105), respectively (P = 1.00). The risk of postdischarge venous thromboembolism is low in patients undergoing laparoscopic surgery who receive in-hospital thromboprophylaxis. Given this low risk, a clinical trial powered to determine if extending prophylaxis in such patients reduces the risk of clinically apparent deep vein thrombosis would be unfeasibly large.

Decreased band 3 anion transport activity and band 3 clusterization in congenital dyserythropoietic anemia type II.

Congenital dyserythropoietic anemia type II (CDA-II) is the most common form of inherited dyserythropoiesis. Erythroid precursor and red blood cells (RBCs) show characteristic morphological abnormalities. Biochemical studies have shown that this disease is associated with reduced glycosylation activity, which endows band 3 (anion transporter) with peculiar characteristics. The life span of RBCs may be shortened in patients with CDA-II, a phenomenon that has been ascribed to this membrane defect. We analyzed seven unrelated patients with CDA-II and five control subjects. In all of the CDA-II patients, erythrocytes presented a band 3 that was thinner than usual and also migrated slightly faster on SDS-PAGE. Analysis of anion transport function in CDA-II RBC samples demonstrated decreased anion exchange activity per band 3 molecule. Furthermore, we observed that the CDA-II RBCs contained larger amounts of aggregate band 3 than control erythrocytes. Aggregate band 3 has been reported to bind naturally occurring antibodies that mediate the phagocytic removal of RBCs. We provide evidence that both the phagocytic index (RBCs/macrophage) and the amount of membrane-bound immunoglobulin (IgG) are elevated in CDA-II erythrocytes. Our results suggest that the mild hemolysis observed in patients with CDA-II may be ascribed to clusterization of band 3, which leads to IgG binding and phagocytosis, and not to a secondary modification of the cytoskeletal structure of RBCs.

Reduction of nitroxide free radical by normal and G6PD deficient red blood cells.

The electron spin resonance signal of Tempol decays in the presence of red cells. The decay is due to reduction of oxidant, paramagnetic nitroxide group by the metabolic activity of the red cell. In normal red cells, GSH level was stable and Tempol reduction rate followed a first-order kinetics. In G6PD-deficient red cells, GSH dropped and Tempol reduction rate was slower and followed a second-order kinetics. In normal red cells, diamide reversibly oxidized GSH. First-order kinetics of Tempol reduction rate was attained after a delay time proportional to the diamide concentration and corresponding to the full regeneration of GSH. In diamide-treated G6PD deficient, and in NEM-treated, normal red cells, irreversible disappearance of GSH was followed by irreversible dose-dependent decrease in Tempol reduction rate. A correlation between GSH levels and Tempol reduction rate was observed. A correlation was also established between Tempol reduction rate and stimulation of pentosephosphate shunt activity.

Enhanced IgG- and complement-independent phagocytosis of sulfatide-enriched human erythrocytes by human monocytes.

Phagocytosis by adherent human monocytes of human erythrocytes (RBC), sulfatide-enriched by incubation with 10(-12) to 10(-9) M cerebroside sulfate, was enhanced approx. 6-fold. Increased phagocytosis was observed only in RBC opsonized with fresh plasma, and not in non-opsonized or serum-opsonized RBC. Increased phagocytosis was immunoglobulin- and complement independent. Thrombospondin and von Willebrand factor, present in plasma but not in serum, and binding selectively to sulfatides, are likely mediators of the enhanced phagocytosis.

Plasmodium falciparum glutathione metabolism and growth are independent of glutathione system of host erythrocyte.

Plasmodium falciparum parasites grew normally in glutathione (GSH)-depleted normal and G6PD-deficient (Mediterranean variant) erythrocytes (RBC). Growth inhibition was observed only at less than approximately 6-12% residual GSH. Parasites studied separately with the Sendai virus technique synthesized GSH de novo and regenerated reduced GSH 10-20 times faster than non-parasitized RBC. Electron spin resonance measurement of Tempol reduction indicated that the ability to reduce free radicals was restricted to the parasite. The marked efflux of oxidized GSH was mainly derived from the parasite. In conclusion, parasites are endowed with powerful and host-independent mechanisms which de novo synthesize or regenerate GSH and allow undisturbed parasite development in GSH-depleted RBC.

Simulated microgravity inhibits the genetic expression of interleukin-2 and its receptor in mitogen-activated T lymphocytes.

Experiments conducted in space in the last two decades have shown that T lymphocyte activation in vitro is remarkably reduced in microgravity. The data indicate that a failure of the expression of the interleukin-2 receptor (measured as protein secreted in the supernatant) is responsible of the loss of activity. To test such hypothesis we have studied the genetic expression of interleukin-2 and of its receptor in concanavalin A-activated lymphocytes with the RT-PCR technology. Microgravity conditions were simulated in the fast rotating clinostat and in the random positioning machine. The latter is an instrument introduced recently to study gravitational effects on single cells. Our data clearly show that the expression of both IL-2 and IL-2Ralpha genes is significantly inhibited in simulated O X g. Thus full activation is prevented.

Characterization of membrane surface proteins of Mycoplasma agalactiae during natural infection.

We have analyzed antigenic variation of seven M. agalactiae wild strains using different sera from naturally infected sheep. Only 30 day sera recognized all surface proteins and inhibited the growth of mycoplasmas. Furthermore, we have observed that two strongly immunogenic proteins: 55 and 35 kDa were digested using 500 micrograms/ml of trypsin. These two bands are immunoprecipitated together with four other proteins but only the 35 kDa protein is recognized by eluted antibodies.

Antimalarial drugs inhibit the phagocytosis of erythrocytes infected with Plasmodium falciparum.

Phagocytic cells constitute the first line of defence against malarial parasites. They perform their role by delivering oxidative radicals and by phagocytosing infected red blood cells (IRBC). Phagocytosis is mediated by antibody binding to clustered band 3 antigen in the IRBC membrane and activation of the alternative complement pathway. In this study we showed that treatment of IRBC containing Plasmodium falciparum with therapeutically-relevant concentrations of antimalarial drugs considerably reduced the binding of immunoglobulin G (IgG) to, and the phagocytosis of, IRBC. Opsonization of IRBC by fresh serum before drug treatment prevented this inhibitory action of drugs. Removal of the drug restored IgG binding and the phagocytic susceptibility of IRBC in a time-dependent fashion. Direct measurement of the effect of chloroquine on the clustering of band 3 in IRBC, however, failed to reveal any disruption of the aggregation. We conclude that antimalarial drugs are able to alter, by an as yet unresolved mechanism, the affinity of IgG to clustered band 3. This affinity of IRBC seems to be determined by a dynamic process that depends on the metabolic activity of the parasite.

Acute haemolysis in childhood falciparum malaria.

Acute haemolysis associated with clinical episodes of high-level Plasmodium falciparum parasitaemia was studied in 20 children from an holoendemic area (coastal Tanzania). The change in blood haemoglobin (Hb) concentration ranged from -46 to g/L during the 72-h observation period and was linearly related to maximum parasitaemia. Balance studies between loss of blood Hb, increase in plasma Hb and appearance of Hb in the urine indicated that extravascular clearance of red cells was the predominant mode of erythrocyte clearance. Most subjects, however, showed minor signs of intravascular haemolysis. The plasma Hb was << 1% of blood Hb and haemoglobinuria was detected in 14/20 children but the excretion of Hb in urine was < 0.5% of total Hb loss. Haemoglobinuria was, however, a marker of severe haemolysis, since the maximum blood Hb loss in children without haemoglobinuria was 10 g/L. Erythrocyte-bound opsonins known to induce erythrophagocytosis, i.e., complement C3c fragments and autologous IgG, were increased in all patients. In the patients with major haemolysis, the changes correlated to the haemolysis over time. Hence, a similar mechanism for predominantly extravascular erythrocyte clearance may be operative in acute malarial anaemia, normal erythrocyte senescence and other forms of acute haemolysis.

Neutrophils and monocytes from subjects with the Mediterranean G6PD variant: effect of Plasmodium falciparum hemozoin on G6PD activity, oxidative burst and cytokine production.

Glucose 6-phosphate dehydrogenase (G6PD) activity and oxidative burst were measured in neutrophils and monocytes from five, hemizygous, G6PD-deficient (Mediterranean variant) individuals and five normal controls. Additionally, tumor necrosis factor (TNF), interleukin-10 (IL-10), interleukin-12 (IL-12) release and phagocytosis of the malarial pigment hemozoin or opsonized erythrocytes (RBC) were measured in monocytes recovered from G6PD-deficient and normal individuals. G6PD activity was significantly lower in "deficient monocytes" (38% residual activity, p = 0.01) and not significantly different in "deficient neutrophils" (79% residual activity, p = 0.83) compared to homologous leukocytes recovered from normal controls. Oxidative burst was not significantly different in "deficient" versus "normal" neutrophils and monocytes. Previous phagocytosis of hemozoin decreased the phorbol ester induced oxidative burst in "deficient" and "normal" monocytes but not in neutrophils. Phagocytosis of hemozoin and RBC strongly stimulated cytokine production. With the exception of IL-10, the cytokine production pattern was comparable in "deficient" versus "normal" cells. Incubation with high concentrations of hemozoin (equivalent to 300 RBC per monocyte) strongly stimulated TNF production. Lipopolysaccharide (LPS) had an additive effect on TNF production induced by hemozoin or opsonized RBC. IL-12 production was induced only by the presence of large amounts of hemozoin. IL-10 production was increased in normal monocytes incubated with RBC or hemozoin. LPS increased IL-10 production significantly in monocytes incubated with RBC or low amounts of hemozoin (equivalent to 30 RBC per monocyte), but had no effect when given alone or in conjunction with high concentrations of hemozoin. Interestingly, deficient monocytes produced less IL-10 than normal cells under these conditions. In conclusion, except for IL-10 production, we did not find major functional differences between neutrophils and monocytes from individuals with or without the Mediterranean G6PD mutation.

Effects of propranolol compared with clonidine on portal haemodynamics: a double-blind cross-over study using duplex-Doppler ultrasonography.

BACKGROUND: Patients with liver cirrhosis and large oesophageal varices run a high risk of digestive haemorrhage due to the rupture of oesophageal varices, an event associated with a high mortality. At present, the only treatment for the prevention of first bleeding from oesophageal varices on which there is general agreement is drug-based. In order to tailor drug treatment to the requirements of individual patients more precisely, an ever-increasing number of drugs is being investigated. DESIGN: Double-blind cross-over study. METHODS: Sixteen cirrhotic patients with large oesophageal varices were studied by means of duplex-Doppler ultrasonography to determine variations in portal haemodynamics after oral administration of 0.150 mg clonidine and to compare these with the variations observed after oral administration of 40 mg propranolol. RESULTS: Propranolol caused a significant reduction in maximum portal flow velocity (P < 0.001), whereas clonidine failed to cause any such variation (P = 0.194). Considering as responders those patients who exhibited at least a 10% decrease in maximum portal flow velocity, 11 patients responded to propranolol; of these, three also responded to clonidine. No patient responded only to clonidine. CONCLUSION: The absence of any effects on the parameters of portal haemodynamics would appear to deny clonidine any significant role in preventing first bleeding resulting from the rupture of oesophageal varices.

Human immune cells as space travelers.

Experiments in space have shown that T lymphocyte function is altered in more than 50% of space crew members. There is strong evidence that such effect is due to stress rather than to weightlessness per se. However the health of astronauts was never threatened so far. Experiments in-vitro with cultures of human peripheral blood lymphocytes (not from astronauts) have shown that T cell function is dramatically reduced. Recent work with the random positioning machine, a new instrument to simulate conditions similar to microgravity, indicate that there are direct gravitational effects on the genetic expression of interleukin-2 and of its receptor in T lymphocytes.