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.

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.

Development of a set of multiplex PCR assays for the simultaneous identification of enterotoxigenic, enteropathogenic, enterohemorrhagic and enteroinvasive Escherichia coli.

Diarrheagenic E. coli comprise a diverse group of microorganisms responsible for gastrointestinal diseases in humans. On the basis of their virulence traits they are distinguished from the non-pathogenic E. coli and classified in several categories. Molecular methods represent the most reliable techniques for distinguishing pathogenic from non-pathogenic E. coli and characterising their pathogenic features. In this paper we report the development of a set of three multiplex PCR assays for the simultaneous and rapid identification of diarrheagenic E. coli belonging to ETEC, EPEC, EHEC and EIEC groups. Assay 1 utilizes primer pairs specific for genes coding for ST and LT toxins of ETEC, and for the E. coli beta-glucuronidase (uidA); assay 2 detects the presence of the eae and bfpA genes of EPEC, and assay 3 recognizes stx1 and stx2 of EHEC, and ial of EIEC. This technique has been validated on 190 E. coli isolated in Angola, Italy and Mozambique from feces of children with diarrhea. Results obtained with the set of multiplex PCR demonstrated 100% accordance with those obtained for the same isolates by PCR on single target genes. The proposed set of multiplex PCRs is the first reported assay that allows the simultaneous characterization of the four categories of diarrheagenic E. coli.

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.

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.

Identification of HIV patients with active pulmonary tuberculosis using urine based polymerase chain reaction assay.

BACKGROUND: Despite the increased dissemination of tuberculosis among HIV infected patients, the diagnosis is difficult to establish. Traditional microbiological methods lack satisfactory sensitivity. We have developed a highly sensitive and specific nested polymerase chain reaction (PCR) capable of detecting Mycobacterium tuberculosis DNA in urine specimens and have used this test to examine urine specimens from HIV patients with active pulmonary tuberculosis. METHODS: Urine specimens from 13 HIV infected patients with microbiologically proven active pulmonary tuberculosis, 10 AIDS patients with non-tuberculous mycobacterial infection (documented by blood culture), 53 AIDS patients with no evidence of mycobacterial disease, and 80 healthy subjects (25 with positive skin test to purified protein derivative) were tested for M tuberculosis using PCR, acid fast staining (AFS), and culture. RESULTS: Of the urine specimens from patients with active tuberculosis, all tested positive by PCR, two by culture, and none by AFS. No reactivity was observed in urine specimens from patients with non-tuberculous mycobacterial infection. Of the 53 AIDS patients without mycobacterial infection, one had a positive urine PCR. Normal subjects were all negative. CONCLUSIONS: Urine based nested PCR for M tuberculosis may be a useful test for identifying HIV patients with pulmonary tuberculosis.

Metabolic indicators of oxidative stress correlate with haemichrome attachment to membrane, band 3 aggregation and erythrophagocytosis in beta-thalassaemia intermedia.

Haematological data, genotype, transfusion requirements, metabolic indicators of oxidative stress (flux via hexose-monophosphate shunt (HMPS); steady state level of GSH and GSSG, NADPH and NADP; activity of anti-oxidant enzymes), parameters of membrane damage (aggregated band 3; membrane-bound haemichromes, autologous immunoglobulins (Igs) and C3 complement fragments) and erythrophagocytosis were measured in erythrocytes (RBC) of 15 beta-thalassaemia intermedia patients (nine splenectomized) with low, if any, transfusion requirements. Patients presented increased aggregated band 3, bound haemichromes, Igs and C3 complement fragments, and increased erythrophagocytosis. Bound haemichromes strongly correlated with aggregated band 3. Anti-band 3 Igs were predominantly associated with aggregated band 3. Erythrophagocytosis positively correlated with aggregated band 3, haemichromes and Igs, suggesting the involvement of haemichrome-induced band 3 aggregation in phagocytic removal of beta-thalassaemic RBC. Splenectomized patients showed higher degrees of membrane damage and phagocytosis, significantly higher numbers of circulating RBC precursors, and tendentially higher numbers of reticulocytes. Basal flux via HMPS was increased twofold, but HMPS stimulation by methylene blue was decreased, as was the glucose flux via HMPS. GSH was remarkably decreased, whereas NADPH was increased. Except for unchanged catalase and glutathione reductase, anti-oxidant enzymes had increased activity. Negative correlation between HMPS stimulation by methylene blue and bound haemichromes indicated that the ability to enhance HMPS may counteract haemichrome precipitation and limit consequent membrane damage leading to erythrophagocytosis.

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.

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.

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.

Contact-dependent disruption of the host cell membrane skeleton induced by Trichomonas vaginalis.

This report presents evidence showing that the pathogenetic process of the protozoan parasite Trichomonas vaginalis involves degradation of the target cell membrane skeleton; spectrin, the most representative protein within this structure, has been identified as the main molecular target. Degradation of the target cell spectrin is accomplished only upon contact with the parasite, and immunochemical and immunofluorescence studies performed with the erythrocyte as a model demonstrate that degradation of the protein takes place before target cell lysis. A preliminary characterization of the effectors involved has led to the identification of a nonsecreted 30-kDa proteinase which is characterized by a high specificity for spectrin. This molecule is suggested as the main effector responsible for cytoskeletal disruption.

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.

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.

Characterization of the autologous antibodies that opsonize erythrocytes with clustered integral membrane proteins.

In earlier studies we presented evidence that the clustering of the integral membrane protein, band 3, can serve as a signal for immune recognition and clearance of senescent or abnormal erythrocytes from circulation. In this study, we have exploited the capacity of 1 mmol/L Zn+2 to mildly and reversibly cluster band 3 in situ to characterize the nature of the autologous antibodies specific for the clustered state. We report that the autologous IgG elute almost exclusively in a high molecular weight complex with other proteins when C12E8 detergent extracts of Zn clustered membranes are chromatographed on Sepharose CL-6B. The complex was also seen to contain complement component C3, hemoglobin, and a cross-linked oligomer of band 3. Autologous IgG and complement were virtually absent from all other fractions. When the band 3 clusters were disaggregated by removal of the Zn+2, the autologous IgG eluted from the erythrocyte surface. Collection of this IgG and use of the antibody in immunoblots of erythrocyte membranes showed that the band 3 monomer, dimer, and oligomers were the major antigenic species. Except for a minor unidentified band at approximately 78,000 d, no other proteins were significantly stained. Curiously, band 3 showed an uneven staining pattern, with oligomers and the leading edge of the monomers appearing more intensely than expected from their abundances in the Coomassie blue-stained gels. Typing of the same autologous IgG with monoclonal antibodies specific for the different subclasses of IgG showed the presence of only subtypes 2 and 3. Taken together, these data suggest that a specific population of autologous IgG recognizes sites of integral membrane protein clustering (a common lesion in senescent and abnormal red blood cells) and that the antigen within these clusters involves an aggregated state of band 3.

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.