Clinical value of circulating endothelial cells and of soluble CD146 levels in patients undergoing surgery for non-small cell lung cancer.

BACKGROUND: Previous studies indicate that endothelial injury, as demonstrated by the presence of circulating endothelial cells (CECs), may predict clinical outcome in cancer patients. In addition, soluble CD146 (sCD146) may reflect activation of angiogenesis. However, no study has investigated their combined clinical value in patients undergoing resection for non-small cell lung cancer (NSCLC). METHODS: Data were collected from preoperative blood samples from 74 patients who underwent resection for NSCLC. Circulating endothelial cells were defined, using the CellSearch Assay, as CD146+CD105+CD45-DAPI+. In parallel, sCD146 was quantified using an ELISA immunoassay. These experiments were also performed on a group of 20 patients with small-cell lung cancer, 60 healthy individuals and 23 patients with chronic obstructive pulmonary disease. RESULTS: The CEC count and the plasma level of sCD146 were significantly higher in NSCLC patients than in the sub-groups of controls (P<0.001). Moreover, an increased CEC count was associated with higher levels of sCD146 (P=0.010). Both high CEC count and high sCD146 plasma level at baseline significantly correlated with shorter progression-free survival (P<0.001, respectively) and overall survival (P=0.005; P=0.009) of NSCLC patients. CONCLUSIONS: The present study provides supportive evidence to show that both a high CEC count and a high sCD146 level at baseline correlate with poor prognosis and may be useful for the prediction of clinical outcome in patients undergoing surgery for NSCLC.

Activation of human T cells is associated with tyrosine phosphorylation of several cellular proteins.

Human T lymphocytes are activated to proliferate after triggering the T Cell Antigen Receptor Complex. CD3-Ti, with either antigen, mitogenic lectins or monoclonal antibodies against its different subunits. Stimulation of Jurkat leukemic human T cells with anti-CD3 or anti-Ti monoclonal antibodies was found to induce, within 1 min, an increase in the phosphorylation of a set of cellular proteins that can be precipitated with anti-phosphotyrosine antibodies. Seven phosphotyrosine-containing proteins were separated with respective mol. wt of 21, 25, 38, 55, 70, 80 and 110 kDa, among which the 38 kDa species is predominant. Moreover, incubation of Jurkat T cells with sodium orthovanadate, a potent inhibitor of phosphotyrosine protein-phosphatases, was found to potentiate the effects of anti-CD3 mAb on tyrosine phosphorylation. In addition vanadate also induced IL-2 secretion in Jurkat cells when associated with the phorbol ester TPA, further demonstrating the importance of these phosphorylation reactions in the process of T cell activation. Our results therefore allow us to identify several protein substrates of a tyrosine kinase activity, whose stimulation appears to be an early event in human T cell activation through the antigen receptor pathway.

Effect of pH on the recovery of human alpha-fetoprotein from immunosorbent columns. An improved elution procedure.

A slow decrease in pH was shown to be more efficient in eluting human alpha-fetoprotein bound to Sepharose-immobilized antibodies than a pH shock. With this method, nearly 50% of the antigen absorbed onto the column was recovered in a single peak whereas the yield was 3 times lower in the case of a single elution at pH 2.6. Our result suggests that dissociation of the antigen-antibody complex occurred only within a narrow pH range.

Reverse ELISPOT assay for clonal analysis of cytokine production. II. Enumeration of interleukin-1-secreting cells by amplified (avidin-biotin anti-peroxidase) assay.

Detection of cytokine-producing cells can be accomplished by reverse modifications of the ELISPOT assay using cytokine-specific unconjugated and enzyme-labelled antibodies as solid phase capture system and detecting reagents, respectively. However, in certain situations where the secreted cytokine is produced in minute amounts such as in the case of interleukin-1 (IL-1), the sensitivity of the indicator immunoenzyme system employed may be insufficient to permit detection of the corresponding secreting cells. We have developed a novel immunoenzyme amplification procedure that involves the use of a biotinylated secondary anti-enzyme antibody reagent to enhance the signal provided by the primary enzyme-labelled antibody conjugate. Following addition of enzyme-conjugated avidin, ELISPOT assay wells are developed with a suitable chromogen substrate yielding spots located at the former position of cells secreting the analyte under study. As a model system, the detection of IL-1 beta-secreting cells by human peripheral blood monocytes is described.

Coupling of gamma-globulin to microcrytstalline cellulose by periodate oxidation.

Anti-rabbit IgG sheep gamma-globulins were covalently coupled to periodate oxidized microcrystalline cellulose using the Schiff reaction. Optimal conditions (sodium m-periodate concentration and gamma-globulin amount) were studied measuring the ability of this solid-phase antibody to bind glucose oxidase-labeled rabbit IgG.

Enzymoimmunoassay of human alpha-fetoprotein.

A non-competitive method for the determination of human alpha-fetoprotein (AFP) in serum, using a pure specific antibody linked to glucose oxidase is described. When applied to human AFP, this method gives reproducible results in the range 0.7 to 15 ng/ml, in a relatively short time (6 hr). AFP sera levels of healthy human adults, pregnant women and adults with liver diseases, were tested both by enzymoimmunoassay and radioimmunoassay. In all cases, good agreement was noted between the two methods.

Human interleukin 2. Detection at the picomolar level by sandwich enzyme immunoassay.

Human interleukin 2 was detected at the pM level by a simple sequential sandwich enzyme immunoassay. The lymphokine to be assayed was first extracted from supernatants of mitogen-activated Jurkat leukemic T cells or peripheral blood lymphocytes using anti-recombinant interleukin 2 rabbit IgG insolubilized onto polystyrene microtiter plates and was revealed by an anti-interleukin 2 Fab' fragment conjugated to peroxidase. The whole method could be performed within 8 h and allowed the measurement of interleukin 2 irrespective of its degree of glycosylation. Among the currently used mitogens, only ConA at a concentration above 10 micrograms/ml interfered with the assay. The method was carefully compared to the reference bioassay and was found to be only 3-5 times less sensitive.

Production and characterization of monoclonal and polyclonal antibodies to human alpha 2-HS: development of a two-site ELISA test.

A convenient and sensitive indirect sandwich ELISA test was developed for measuring both 63 kDa human alpha 2-HS secreted by human hepatoma cell lines and the 59 kDa alpha 2-HS species present in serum/plasma. Monoclonal and rabbit antibodies to plasma alpha 2-HS were produced and selected by immunoprecipitation techniques using iodinated alpha 2-HS or 35S-labeled alpha 2-HS. Various monoclonal antibodies recognizing both forms of the protein were coated onto microtiter plates and after binding of alpha 2-HS, biotinylated monoclonal antibodies with compatible binding or biotinylated immunopurified F(ab')2 fragments from the rabbit antiserum were added and subsequently revealed with avidin-biotin peroxidase complex. Formats using a rabbit detector antibody were the most sensitive and one was selected for the whole study. The test developed was capable of detecting plasma alpha 2-HS devoid of connecting peptide and HepG2 hepatoma cell line derived alpha 2-HS at the ng/ml level. The test has been used to measure levels of alpha 2-HS in both serum and supernatants from HepG2 cell lines.

An enzyme immunoassay design using labelled antibodies for the determination of haptens. Application to methotrexate assay.

A competitive enzyme immunoassay with labelled antibodies has been developed for methotrexate (MTX). Methotrexate in the sample and a constant quantity of this hapten physically absorbed to polystyrene spheres through a methylated bovine albumin carrier were allowed to compete for a limiting amount of peroxidase labelled antibody. After washing, the residual enzyme activity bound to the solid phase was measured. This test was able to detect 10 fm of MTX per sample. A comparative study of this test with a commercial radioimmunoassay kit using the same antiserum and a high pressure liquid chromatography method showed that the sensitivity, specificity and precision of this test were as good as of the radioimmunoassay. The high pressure liquid chromatography method was 500 times less sensitive. Good agreement was found among the 3 methods on 83 serum samples from patients receiving methotrexate therapy.

Detection of human IL-1 alpha and IL-1 beta at the subpicomolar level by colorimetric sandwich enzyme immunoassay.

Two separate convenient sandwich enzyme immunoassay methods were developed for measuring the production of the monokines interleukin-1 alpha (IL-1 alpha) and interleukin-1 beta (IL-1 beta) from peripheral blood mononuclear cells. Polyclonal antisera raised against the recombinant proteins and selected on the basis of their ability to neutralize IL-1-induced IL-2 secretion were used for coating microtiter plates or preparing peroxidase-Fab' conjugates. Both techniques were able to accurately and specifically detect monokines from various sources in the sub-picomolar range and were not influenced by compounds currently used for cell activation. A high molecular weight form of IL-1 beta was demonstrated under certain conditions and the two enzyme immunoassays were successfully applied to the detection of IL-1 alpha and IL-1 beta present in cell supernatants following stimulation with mitogenic or chemical agents.

A sandwich method of enzyme immunoassay. III. Assay for human beta-2 microglobulin compared with radioimmunoassay.

A sandwich enzyme immunoassay has been developed to measure human beta-2 microglobulin (beta 2m) in bilogical fluids. beta 2m is first bound by specific antibody covalently coupled to microcrystalline cellulose. The solid phase is washed and reincubated with glucose oxidase-labeled anti-beta 2m antibody. After washing, the enzymic activity of the solid phase is measured by incubation with the appropriate chromogenic substrate. The OD is directly related to the quantity of beta 2m to be measured. A second sandwich assay has also been developed which uses plastic microplates coated with the IgG fraction of rabbit anti-beta 2m serum. Reproducible results are obtained in the range 2-150 and 0.75-48 microgram/l respectively. These tests detect 17 fmoles of beta 2m. Assays of 27 sera showed good agreement between these two enzyme immunoassay methods and two radioimmunoassays.

A novel enzyme immunoassay for total thyroxine using immobilized antibodies and hydrophobic chromatography purified thyroxine-peroxidase conjugate.

A new and simple competitive enzyme immunoassay method for the measurement of total thyroxine in serum is described. Anti-thyroxine antibodies, raised in sheep with a bovine serum albumin-thyroxine conjugate prepared with carbodiimide as coupling initiator, were physically adsorbed onto a large surface area polypropylene support. Competition occurred between thyroxine in the sample and a thyroxine-peroxidase conjugate prepared with a glutaraldehyde spacer and further purified by octyl Sepharose hydrophobic chromatography. The entire assay was performed in 2 h with a useful range of 10-300 micrograms/l. The coefficients of variation ranged from 6.9 to 12% and good agreement (r = 0.93-0.97) was found between this method and 2 radioimmunoassays on 71 serum samples having well distributed thyroxine values.

A sandwich method of enzyme-immunoassay. II. Quantification of rheumatoid factor.

A sandwich enzyme-immunoassay (EIA) has been applied to the determination of the rheumatoid factor (RF). This non-competeitive assay comprises 3 steps: 1) the RF to be assayed is extracted for the biological medium by an immunosorbent of aggregated IgG linked to cellulose; 2) the solid phase is then incubated with the enzyme-labeled aggregated IgG; 3) the enzymatic activity of the immunosorbent is then measured with a suitable chromogenic reagent. This activity is a direct function of the amount of RF to be assayed. This assay gave reproducible results in the range 0.5-50.0 IU/ml. A good agreement was obtained between the EIA and the Waaler-Rose test but no correlation was obtained with the latex slide-test. This assay permits a quantitation of RF with a good reproducibility (coefficient of variation in the range of 10% for moderately elevated values) and thus allows a closer follow-up of patients. The results do not depend on the interpretation of the technician performing the test, which can be easily automated. Finally, it may detect some RF devoid of agglutinating activity.

Non-competitive enzyme immunoassay of human trypsin 1.

A sandwich enzyme immunoassay was developed for human pancreatic trypsin 1 using polystyrene balls coated with specific IgG as the first antibody and peroxidase-labeled IgG as the second antibody. The entire assay takes 6 h and the detection limit is 0.5 microgram/l. The assay can be performed on sera samples or on discs carrying dried blood spots. Good agreement was found with a radioimmunoassay kit. This simple assay could be widely applied to confirm the elevated immunoreactive serum trypsin described in newborn children with cystic fibrosis.

Measurement of the anti-HIV agent 2',3'-didehydro-2',3'-dideoxythymidine (D4T) by competitive ELISA.

2',3'-didehydro-2',3'-dideoxythymidine (D4T) is a thymidine analogue with potent anti-HIV activity in vitro and is currently being investigated therapeutically in patients with advanced HIV infection. We describe a first one-step competitive ELISA method developed for D4T measurement. Anti-D4T rabbit antibodies were raised against a D4T hemisuccinate-bovine serum albumin immunogen. A D4T-hemisuccinate-horseradish peroxidase conjugate and a monoclonal anti-rabbit IgG antibody insolubilized onto a microtiter plate were used as a tracer and capture system, respectively. The method was capable of detecting 2 ng/ml of D4T in cell cultures and 20 ng/ml of D4T in plasma samples previously separated in microconcentrator devices. Cross-reactivity analysis showed that thymidine, D4T monophosphate, or azidothymidine, were weakly recognized by the ELISA and that thymine or other nucleosides were unreactive. The test was successfully used for the quantification of D4T in cell extracts from CEM or Molt 4 cell lines cultured with D4T and in the plasma of patients with advanced HIV infection, receiving D4T therapy. Moreover this ELISA could be used for the indirect quantification of D4T phosphorylated intracellular metabolites previously separated by reverse phase HPLC and hydrolyzed with alkaline phosphatase.

In vivo involvement of polymorphonuclear neutrophils in Leishmania infantum infection.

BACKGROUND: The role of lymphocytes in the specific defence against L. infantum has been well established, but the part played by polynuclear neutrophil (PN) cells in controlling visceral leishmaniasis was much less studied. In this report we examine in vivo the participation of PN in early and late phases of infection by L. infantum. RESULTS: Promastigote phagocytosis and killing occurs very early after infection, as demonstrated by electron microscopy analyses which show in BALB/c mouse spleen, but not in liver, numerous PN harbouring ultrastructurally degraded parasites. It is shown, using mAb RB6-8C5 directed against mature mouse granulocytes, that in chronically infected mice, long-term PN depletion did not enhance parasite counts neither in liver nor in spleen, indicating that these cells are not involved in the late phase of L. infantum infection. In acute stage of infection, in mouse liver, where L. infantum load is initially larger than that in spleen but resolves spontaneously, there was no significant effect of neutrophils depletion. By contrast, early in infection the neutrophil cells crucially contributed to parasite killing in spleen, since PN depletion, performed before and up to 7 days after the parasite inoculation, resulted in a ten-fold increase of parasite burden. CONCLUSIONS: Taken together these data show that neutrophil cells contribute to the early control of the parasite growth in spleen but not in liver and that these cells have no significant effect late in infection in either of these target organs.

Efficacy of long-term therapy with low doses of omeprazole in the control of gastric acid secretion in Zollinger-Ellison syndrome patients.

Thirteen patients with Zollinger-Ellison syndrome were investigated: 8 without, and 5 with, previous gastric surgery. After 7-34 months of treatment with famotidine, 8 out of 13 patients were resistant to this drug. Omeprazole 60 mg/day was administered to these 8 patients; after one month, the dose was reduced to 40 mg/day, and after another month to 20 mg/day. Basal acid secretion was inhibited by every dose of omeprazole. The patients were then treated with a low dose (20 mg/day) of omeprazole for a longer period. Periodic clinical and endoscopic assessments, and measurement of basal acid secretion showed the efficacy of this low dose of omeprazole in our Zollinger-Ellison syndrome patients. The drug was discontinued after 12-32 months of omeprazole treatment, and gastric acid recovery was evaluated. Four patients recovered 50% of their 'initial basal acid secretion' after 5 days, while two patients who had been treated with omeprazole for a longer time (30-32 months) recovered only 38 and 40%, respectively, of their 'initial basal acid secretion' at the tenth day. Our results indicate that the omeprazole dosage to be used in the treatment of Zollinger-Ellison syndrome must be chosen principally on the basis of basal acid secretion determination. A low daily dose of omeprazole is able to control acid secretion in Zollinger-Ellison syndrome for a long period (10-30 months). The slow recovery of gastric secretory function demonstrates the prolonged inhibitory effects of omeprazole.

Human interleukin-6: detection of 10 attomoles by colorimetric sandwich ELISA using immunopurified polyclonal anti-IL-6 antibodies.

A convenient and sensitive sequential sandwich colorimetric ELISA test was established for quantitating IL-6 in culture supernatants or in serum. Immunopurified HRP-labelled rabbit Fab' fragment was used as the tracer and IgG-coated microtiter plate as the capture antibody. The limit of detection was as low as 10 attomoles of analyte (2.5 pg/ml). Unglycosylated recombinant IL-6 and the natural glycosylated cytokine were recognized equally. In addition, IL-6 measurements were unaffected by the presence of various cytokines and assay sensitivity was only slightly reduced in the presence of undiluted serum samples. The technique was applied to the study of in vitro IL-6 production from activated monocytes and to the in vivo determination of IL-6 in various pathological states.

Paraformaldehyde fixation of LPS-stimulated human monocytes: technical parameters permitting the study of membrane IL-1 activity.

The existence of IL-1 activity on the cell surface of stimulated mononuclear phagocytes is a matter of controversy. In particular, fixation of IL-1-expressing cells for 15 min in 1% paraformaldehyde (PFA) is commonly used to evidence such "membrane-associated" IL-1 activity but other authors have attributed this to passive leakage of IL-1 alpha from the cells and report no activity with longer fixation times. Using specific IL-1 alpha and IL-1 beta assays, we found that after the mild standard PFA fixation procedure, not only IL-1 alpha but also IL-1 beta were released into the supernatants for up to 96 h following fixation; membrane IL-1 activity cannot thus be measured in these conditions. However, using conditions in which neither immunoreactive IL-1 molecules nor IL-1 activity are found in the supernatants (i.e. assay at 144 h, increased fixation time), we were still able to detect IL-1 activity on LPS-stimulated, PFA-fixed monocytes. This activity was independent of the duration of PFA fixation and was inhibited by anti-IL-1 alpha but not anti-IL-1 beta antibodies. Our data thus underline the importance of technical conditions in the study of membrane-associated IL-1 activity.

Prolonged administration of dexamethasone induces limited reactivation of visceral leishmaniasis in chronically infected BALB/c mice.

Leishmania parasites persist in their vertebrate host after the treatment-induced clinical cure and in the asymptomatic infection. They confer resistance to reinfection but represent a risk of occurrence of acute leishmaniosis in immunosuppressed conditions. We examined the effects of prolonged dexamethasone administration on a chronic Leishmania infantum infection. Splenic T cell populations from the long-term-infected BALB/c mice were reduced by 55%, whereas those from uninfected controls were depleted by 85%. The ability of the remaining spleen cells to produce IL-2, IFN-gamma, IL-4 and TNF-alpha after in vitro specific stimulation decreased twofold, and the specific anti-leishmanial antibodies declined 3- to 5-fold. Liver, spleen and bone marrow are the main L. infantum targets in natural and experimental infections. Three-fold increase of amastigote burden was evidenced in the spleen, after dexamethasone administration was prolonged for over 2 months. No reactivation of Leishmania proliferation was disclosed in the liver and bone marrow. These results show a decreased sensitivity of splenic T cells to dexamethasone in a chronic Leishmania infection and a distinct response of the Leishmania-infected target organs to the dexamethasone-induced immunosuppression.