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.

Phagocytosis of malarial pigment haemozoin by human monocytes: a confocal microscopy study.

Haem from host erythrocyte (RBC) haemoglobin is polymerized in the digestive organelle of Plasmodium falciparum to haemozoin (HZ), a crystaLline, insoluble substance. Human monocytes avidly ingest HZ that persists undigested for long periods of time, and generates potent bioactive lipid peroxide derivatives. Protein kinase C, an effector of signal transduction, phagolysosome formation and acidification, is inhibited in HZ-fed monocytes. Inability to digest HZ might derive from impairment in phagolysosome formation or acidification. Time-course and extent of HZ phagocytosis and acidification of phagolysosomes were studied by quantitative confocal microscopy. From 180 min until 72 h after the start of phagocytosis approximately 75-79% of the monocytes contained massive amounts of HZ. Coincidence between red (HZ) and green (acidic organelles) fluorescent compartments was very high. Confocal images showed that at 30-60 min after the start of phagocytosis, HZ was preferentially present as separated particles, with full co-localization of red and green fluorescence. Later on HZ-laden phagolysosomes tended to fuse together. In conclusion, phagolysosome formation and acidification were normal in HZ-fed monocytes during the 72-h observation time. The presence of HZ in the phagolysosome, the site of antigen processing, may offer a physical link with immunodepression in malaria.

Growth of Plasmodium falciparum induces stage-dependent haemichrome formation, oxidative aggregation of band 3, membrane deposition of complement and antibodies, and phagocytosis of parasitized erythrocytes.

Plasmodium falciparum-parasitized erythrocytes (RBCs) are progressively transformed into non-self cells, phagocytosed by human monocytes. Haemichromes, aggregated band 3 (Bd3) and membrane-bound complement fragment C3c and IgG were assayed in serum-opsonized stage-separated parasitized RBCs. All parameters progressed from control to rings to trophozoites to schizonts: haemichromes, nil; 0.64 +/- 0.12; 5.6 +/- 1.91; 8.4 +/- 2.8 (nmol/ml membrane); Bd3, 1 +/- 0.1; 4.3 +/- 1.5; 23 +/- 5; 25 +/- 6 (percentage aggregated); C3c, 31 +/- 11; 223 +/- 86; 446 +/- 157; 620 +/- 120 (mOD405/min/ml membrane); IgG, 35 +/- 12; 65 +/- 23; 436 +/- 127; 590 +/- 196 (mOD405/min/ml membrane). All increments in rings versus controls and in trophozoites versus rings were highly significant. Parasite development in the presence of 100 micromol/l beta-mercaptoethanol largely reverted haemichrome formation, Bd3 aggregation, C3c and IgG deposition and phagocytosis. Membrane proteins extracted by detergent C12E8 were separated on Sepharose CL-6B. Haemichromes, C3c and IgG were present exclusively in the high-molecular-weight fractions together with approximately 30% of Bd3, indicating the oxidative formation of immunogenic Bd3 aggregates. Immunoblots of separated membrane proteins with anti-Bd3 antibodies confirmed Bd3 aggregates that, in part, did not enter the gel. Immunoprecipitated antibodies eluted from trophozoites reacted preferentially with aggregated Bd3. Changes in parasitized RBC membranes and induction of phagocytosis were similar to oxidatively damaged, senescent or thalassaemic RBC, indicating that parasite-induced oxidative modifications of Bd3 were per se sufficient to induce and enhance phagocytosis of malaria-parasitized RBC.

Hemozoin stability and dormant induction of heme oxygenase in hemozoin-fed human monocytes.

Human monocytes avidly ingest malarial pigment, hemozoin. Phagocytosed hemozoin persists in the monocyte for a long time and modifies important monocyte functions. Stability of phagocytosed hemozoin may depend on modifications of the hemozoin heme moiety or reduced ability to express heme-inducible heme oxygenase. We show here that the spectral characteristics of alkali-solubilized hemozoin were identical to those of authentic heme, although hemozoin was solubilized by alkali much more slowly than authentic heme. Alkali-solubilized hemozoin was a substrate of microsomal rat heme oxygenase and bilirubin reductase, with bilirubin as the main final product. Hemozoin feeding to human monocytes did not induce heme oxygenase, but authentic heme and alkali-solubilized hemozoin supplemented to hemozoin-fed monocytes induced heme oxygenase and were degraded normally. Lysosomes isolated from hemozoin-fed monocytes released only traces of heme while lysosomes from erythrocyte-fed monocytes liberated considerable quantities of heme. Lack of heme release from hemozoin did not depend on proteolysis-resistant, heme-binding proteins, since lysosomal proteases fully degraded hemozoin-associated proteins but did not solubilize hemozoin. In conclusion, our data indicate that lack of induction of HO1 is due to the intrinsic structural characteristics of hemozoin and not to hemozoin-mediated impairment of the mechanism of HO1 induction.

Early phagocytosis of glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes parasitized by Plasmodium falciparum may explain malaria protection in G6PD deficiency.

In population-based studies it has been established that inherited deficiency of erythrocyte (E) glucose-6-phosphate dehydrogenase (G6PD) confers protection against severe Plasmodium falciparum (P falciparum) malaria. Impaired growth of parasites in G6PD-deficient E in vitro has been reported in some studies, but not in others. In a systematic analysis, we have found that with five different strains of P falciparum (FCR-3, KI, C10, HB3B, and T9/96), there was no significant difference in either invasion or maturation when the parasites were grown in either normal or G6PD-deficient (Mediterranean variant) E. With all of these strains and at different maturation stages, we were unable to detect any difference in the amount of P falciparum-specific G6PD mRNA in normal versus deficient parasitized E. The rate of 14C-CO2 production from D-[1-14C] glucose (which closely reflects intracellular activity of G6PD) contributed by the parasite was very similar in intact normal and deficient E. By contrast, in studies of phagocytosis of parasitized E by human adherent monocytes, we found that when the parasites were at the ring stage (ring-stage parasitized E [RPE]), deficient RPE were phagocytosed 2.3 times more intensely than normal RPE (P = .001), whereas there was no difference when the parasites were at the more mature trophozoite stage (trophozoite-stage parasitized E [TPE]). Phagocytic removal markers (autologous IgG and complement C3 fragments) were significantly higher in deficient RPE than in normal RPE, while they were very similar in normal and deficient TPE. The level of reduced glutathione was remarkably lower in deficient RPE compared with normal RPE. We conclude that impaired antioxidant defense in deficient RPE may be responsible for membrane damage followed by phagocytosis. Because RPE, unlike TPE, are nontoxic to phagocytes, the increased removal by phagocytosis of RPE would reduce maturation to TPE and to schizonts and may be a highly efficient mechanism of malaria resistance in deficient subjects.

Spermidine acetyltransferase in rat hepatocytes cultured at different oxygen tensions.

To understand the mechanism involved in the liver zonation of polyamines, we have studied the possible role of oxygen tension. When hepatocytes were cultured at 21% and at 5% oxygen in atmosphere to mimic periportal and perivenous conditions, polyamine content was modified. The observed modifications suggested an effect on the interconversion pathway. Spermidine acetyltransferase (SAT) activity and N1-acetylspermidine were therefore measured in the same conditions. SAT activity was markedly increased after 6 hours and N1-acetylspermidine was accumulated in the cells. This was caused by new enzyme synthesis. The higher expression of SAT was accompanied by an increase in the content of the specific messenger RNA (mRNA). When liver cells were depleted of polyamines, SAT activity and the specific mRNA content were not enhanced by oxygen deprivation, but they increased when polyamines were added again. Polyamines therefore appear to be necessary to promote the increase in SAT mRNA.

Erythrocyte stages of Plasmodium falciparum exhibit a high nitric oxide synthase (NOS) activity and release an NOS-inducing soluble factor.

Nitric oxide (NO), a highly diffusible cellular mediator involved in a wide range of biological effects, has been indicated as one of the cytotoxic agents released by leukocytes to counteract malaria infection. On the other hand, NO has been implicated as a mediator of the neuropathological symptoms of cerebral malaria. In such circumstances NO production has been thought to be induced in host tissues by host-derived cytokines. Here we provide evidence for the first time that human red blood cells infected by Plasmodium falciparum (IRBC) synthesize NO. The synthesis of NO (measured as citrulline and nitrate production) appeared to be very high in comparison with human endothelial cells; no citrulline and nitrate production was detectable in noninfected red blood cells. The NO synthase (NOS) activity was very high in the lysate of IRBC (while not measurable in that of normal red blood cells) and was inhibited in a dose-dependent way by three different NOS inhibitors (L-canavanine, NG-amino-L-arginine, and NG-nitro-L-arginine). NOS activity in P. falciparum IRBC is Ca++ independent, and the enzyme shows an apparent molecular mass < 100 kD, suggesting that the parasite expresses an isoform different from those found in mammalian cells. IRBC release a soluble factor able to induce NOS in human endothelial cells. Such NOS-inducing activity is not tissue specific, is time and dose dependent, requires de novo protein synthesis, and is probably associated with a thermolabile protein having a molecular mass > 100 kD. Our data suggest that an increased NO synthesis in P. falciparum malaria can be directly elicited by soluble factor(s) by the blood stages of the parasite, without necessarily requiring the intervention of host cytokines.

Role of hemichrome binding to erythrocyte membrane in the generation of band-3 alterations in beta-thalassemia intermedia erythrocytes.

Nine splenectomized, hematologically well-compensated beta-thalassemia intermedia patients randomly chosen from a pool of 60 similar patients were studied. Membrane proteins solubilized with nondenaturing detergent C12E8 were gel filtered on Sepharose CL-6B (Pharmacia Fine Chemicals, Uppsala, Sweden). Fractions containing higher than 4,000-kD molecular-weight aggregates were isolated and analyzed. Four patients had remarkably increased amounts of membrane-bound hemichromes and Igs. In those patients, band 3 underwent oxidative modifications such as aggregation and a decrease in sulfhydryl groups. The other five patients had low amounts of membrane-bound hemichromes and less modifications of band 3. The same band-3 modifications could be reproduced by challenging normal membranes with artificially generated hemichromes or with hemolysates prepared from thalassemic erythrocytes of the high-hemichrome group. Addition of reduced glutathione to the challenged membranes did not hinder hemichrome binding, but prevented oxidative modifications of band 3 and Ig binding to high-molecular-weight band-3 aggregates. Hemichrome binding to band 3, hemichrome-mediated oxidation of band-3 cytoplasmic domains, generation of high-molecular-weight band-3 aggregates, and enhanced opsonization by anti-band-3 antibodies is a possible sequence of events leading to phagocytic removal of erythrocytes in thalassemia.

Modulation of ornithine aminotransferase activity by oxygen in rat hepatocyte cultures.

In hepatocytes in culture, ornithine aminotransferase activity remained higher when the cells were cultured at low oxygen tension (5%) than at high tension (21%), that is, it was higher in hepatovenous conditions. Northern blot analysis showed that the amount of the specific mRNA for the enzyme was also higher. Results of experiments performed in the presence of CoCl2, to replace the central Fe2+ in heme, or succinylacetone, to inhibit heme synthesis, support the view that a heme protein participates in the regulation of ornithine aminotransferase activity by oxygen. The oxygen sensor does not appear to act through phosphorylation by kinase C, as TPA has no significant effect on the process, but a phosphorylation dependent on cAMP might be involved.

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.

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.

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.

In vivo priming of human normal neutrophils by granulocyte-macrophage colony stimulating factor: effect on the production of platelet activating factor.

The effect of granulocyte-macrophage colony stimulating factor (GM-CSF) (recombinant, mammalian, glycosylated, Sandoz, Schering Plough; 4 micrograms/kg every 12 h for 3 d, s.c.) on platelet activating factor (PAF, 1-O-alkyl-2-acetyl-sn glycero-3 phosphorylcholine) production from neutrophils was studied in five cancer patients with normal haemopoiesis. Peripheral blood counts, PAF production and lyso-PAF: acetyl transferase (EC 2.3.1.67) (AT) activity in neutrophils were evaluated before treatment, during treatment and 3 d after treatment had been discontinued. GM-CSF induced a three-fold increase in the number of circulating neutrophils. Neutrophils obtained during treatment produced about twice as much PAF than before treatment in response to a variety of stimuli (N-formyl-methionyl-leucyl-phenylalanine, tumour necrosis factor-alpha, phagocytosis of baker's yeast spores opsonized with C3b). This increased PAF synthesis and release is concomitant with a 2-3-fold increase in AT activity. Moreover, lower concentrations of stimuli are sufficient to induce PAF synthesis from neutrophils obtained during GM-CSF treatment. Three days after treatment had been discontinued, stimulus induced PAF production had returned to baseline levels. Since GM-CSF induces a marked shift to the left in the Arneth score, the increased PAF release might have been due to the presence of younger granulocytes. This was, however, ruled out by experiments showing that normal neutrophils primed in vitro with GM-CSF produce more PAF when challenged with the same stimuli. The potential relevance of this effect of GM-CSF treatment lies on the crucial role of PAF in inflammatory reactions and its intervention in some immune reactions, including delayed hypersensitivity, and in endotoxic shock. Lastly, increased PAF production from neutrophils may explain some toxicities observed during treatment with high doses of GM-CSF.

Activation of a ribosomal S6 protein kinase in rapidly emerging diethylnitrosamine-induced gamma-glutamyltranspeptidase-positive hyperplastic liver lesions of the rat.

The gamma-glutamyl transpeptidase (GGT)-positive hyperplastic liver lesions which developed in the Fisher 344 rat 7 and 60 days following a single carcinogenic dose of diethylnitrosamine (DENA, 200 mg/kg body weight), short-term dietary exposure to 0.02% 2-acetylaminofluorene (AAF) to suppress the growth of normal hepatocytes, and partial hepatectomy to actuate rapid growth of DENA altered hepatocytes not suppressed by AFF, showed an increased activity of a kinase which specifically phosphorylates the ribosomal S6 protein in vitro. Sham-operated animals showed, on the contrary, no GGT-positive cells and low S6 kinase activity, under the same conditions. After partial hepatectomy, activation of S6 kinase and elevated levels of phosphorylated S6 protein in vitro were detected in the early phases of "normal" hepatocyte proliferation, during liver regeneration, in DENA-treated, GGT-negative preparations, when the "selection" agent AAF was omitted from the diet. The observed activation of S6 kinase in GGT-positive hepatocytes and/or liver nodules could represent an early manifestation of the enhanced proliferation of altered hepatocytes during tumor induction and/or promotion under these conditions.