Management of patients with advanced prostate cancer: recommendations of the St Gallen Advanced Prostate Cancer Consensus Conference (APCCC) 2015.

The first St Gallen Advanced Prostate Cancer Consensus Conference (APCCC) Expert Panel identified and reviewed the available evidence for the ten most important areas of controversy in advanced prostate cancer (APC) management. The successful registration of several drugs for castration-resistant prostate cancer and the recent studies of chemo-hormonal therapy in men with castration-naïve prostate cancer have led to considerable uncertainty as to the best treatment choices, sequence of treatment options and appropriate patient selection. Management recommendations based on expert opinion, and not based on a critical review of the available evidence, are presented. The various recommendations carried differing degrees of support, as reflected in the wording of the article text and in the detailed voting results recorded in supplementary Material, available at Annals of Oncology online. Detailed decisions on treatment as always will involve consideration of disease extent and location, prior treatments, host factors, patient preferences as well as logistical and economic constraints. Inclusion of men with APC in clinical trials should be encouraged.

Vaccination with neutrophil inhibitory factor reduces the fecundity of the hookworm Ancylostoma ceylanicum.

Neutrophil inhibitory factor (NIF), a protein isolated from hookworms of the genus Ancylostoma, inhibits CD11b/18-dependent leucocyte function, binding to the I domain of CD11b. Historically, NIF was serendipitously isolated from whole worm extracts during a search for novel antihaemostatic agents, and little is known of its source or biological significance to the parasite. NIF has also been identified as a possible hookworm vaccine candidate. Ancylostoma ceylanicum recombinant NIF, expressed in its active form in Pichia pastoris, was purified and its functional activity confirmed using neutrophil adhesion assays and confirmatory immunoassay. Recombinant NIF was subsequently used in vaccination trials in the A. ceylanicum-hamster model system for human hookworm infection. Vaccinated and challenged animals were not protected in terms of worm burden or haematocrit values, despite the presence of high levels of specific antibody against NIF. However, adult worms resident in vaccinated animals showed a significant reduction in fecundity (85.8% by day 21 postinfection), indicating a degree of protection against subsequent transmission by vaccination. These data indicate that targeted vaccination with recombinant subunit material, derived from a known and effective immune suppressant secreted by the parasite, may offer partial protection against the transmission of hookworm infection. Furthermore, we can also report that a biological activity characteristic of NIF is detectable in the secretions of A. ceylanicum using two complementary bioassays. Complete neutralization of this secreted activity by vaccination in combination with other vaccine candidates may result in improved protection against A. ceylanicum infection.

Mechanisms underlying neutrophil adhesion to apical epithelial membranes.

Crypt abscesses allow prolonged apposition of activated neutrophils to the epithelial surface of the colon. Adhesion of neutrophils to both the vascular endothelium and basolateral epithelial membrane share common effector molecules but are distinct processes. This study aimed to define the mechanisms that effect adhesion, independent of transmigration, to the apical epithelium. HT29 (cl 19A) cells were grown to confluency and incubated with neutrophils under conditions of: (i) neutrophil stimulation with phorbol-myristate-acetate; (ii) monolayer stimulation with interferon gamma, tumour necrosis factor alpha (IFN gamma, TNF alpha); and (iii) recent epithelial cell trypsinisation. These experiments were carried out in the presence of neutralising antibodies to CD18, CD11b, LFA-1, E-selectin, P-selectin, intracellular adhesion molecule 1 (ICAM-1), and ICAM-2; a novel CD11b/CD18 antagonist, neutrophil inhibitory factor (rNIF); adenosine receptor agonists (5'N-ethycarboxamido adenosine/N6-cylopentyladenosine (NECA/CPA)) and a platelet activating factor (PAF) receptor antagonist lexipafant. Adhesion of stimulated neutrophils to resting monolayers was Mac-1, CD18 dependent and ICAM-1, ICAM-2, E-selectin, P-selectin, PAF independent. Cytokine activated monolayers exhibited higher binding of neutrophils which was inhibited by rNIF and aCD18. Recently trypsinised monolayers bound neutrophils in a CD11b/CD18 and CD18 independent manner. Adenosine agonists failed to influence neutrophil adhesion under any condition. This study shows neutrophil adhesion to apical epithelial membranes is similar to that at the epithelial basolateral membrane, though different to that seen at the vascular endothelium. These results highlight regional differences in neutrophil adhesion molecule usage.

Neutrophil inhibitory factor is neuroprotective after focal ischemia in rats.

We tested the neuroprotective potential of neutrophil inhibitory factor (rNIF), a novel 41-kd recombinant glycoprotein derived from a hookworm, in a model of focal cerebral ischemia in the rat. Male Wistar rats were assigned to treatment with rNIF and vehicle. Middle cerebral artery occlusion (MCAO) for 2 hours was induced by insertion of an intraluminal suture. Infusion of the drug was initiated at the onset of reperfusion. Infarct volume was determined 48 hours after reperfusion. Neutrophils were measured within the ischemic tissue by myeloperoxidase (MPO) staining. Treatment with rNIF resulted in a 48% reduction in cerebral infarction compared with control animals (p < 0.01). Neutrophil accumulation in the ischemic brains of rNIF-treated rats was reduced significantly (p < 0.01) compared with control animals. The number of neutrophils within the infarcted tissue correlated positively with the size of the area of infarction (p < 0.001, r = 0.6) within representative cerebral coronal sections. We demonstrated a significant neuroprotective effect of rNIF with continuous treatment for 48 hours following 2 hours of MCAO. The neuroprotective effect was correlated with a reduced number of neutrophils within the ischemic tissue. These results demonstrate potential therapeutic properties of rNIF in the management of stroke.

Neutrophil inhibitory factor prevents neutrophil-dependent lung injury.

Neutrophil inhibitory factor (NIF) is a recently cloned 41-kDa protein from the canine hookworm that binds CD11b/CD18 and inhibits CD11b/CD18-dependent neutrophil adhesion. We evaluated NIF's effects on neutrophil-dependent lung injury in guinea pigs. Pulmonary vascular endothelial CD54 (ICAM-1) was induced in buffer-perfused lungs by 90-min exposure to 1000 U/ml TNF-alpha. Human neutrophils (2 x 10(7)) were added to the perfusate and activated by 5 x 10(-9) PMA; in some lungs, the neutrophils were pretreated with NIF (100 nM) before their addition to the perfusate. Lung injury was assessed by wet:dry weight ratio, and neutrophil uptake by lung myeloperoxidase (MPO) activity. HUVEC exposed to TNF-alpha for 90 min were assayed for neutrophil adhesion, and we compared PMA-stimulated neutrophil adhesion to endothelial cells and fibrinogen-coated plates. PMA-induced pulmonary edema (lung wet:dry ratio increased from 8.8 +/- 0.7 to 18.8 +/- 4.4) was inhibited by NIF (10.0 +/- 1.0). Lung MPO activity concomitantly decreased from 17.1 +/- 6.1 to 8.7 +/- 1.8 U/mg dry lung tissue in the NIF-treated group, similar to controls (6.9 +/- 2.0). Endothelial monolayer experiments confirmed that NIF reduced neutrophil adherence (basal adhesion of 11 +/- 3% increased to 30 +/- 5% with TNF-alpha pretreatment of endothelial cells, an increase that was reduced to 10 +/- 4% with NIF). Moreover, NIF prevented PMA-induced neutrophil adhesion to fibrinogen, a CD11b/CD18-dependent event, but produced a smaller decrease in adherence to endothelial cells, which also involves CD11a/CD18 integrins. These studies indicate that NIF prevents neutrophil-dependent lung vascular injury by inhibiting neutrophil adhesion to the TNF-alpha-activated endothelium.

A natural glycoprotein inhibitor (NIF) of CD11b/CD18 reduces leukocyte adhesion in the liver after hemorrhagic shock.

This study was designed to assess the effect of neutrophil inhibitory factor (NIF), a novel specific inhibitor of CD11b/CD18 on hepatic leukocyte trafficking by intravital microscopy 5 h after hemorrhagic shock. Anesthetized rats were instrumented for invasive hemodynamical monitoring. Hemorrhagic shock was induced for 60 min by withdrawal of arterial blood (mean arterial blood pressure = 40 mmHg). Rats were adequately resuscitated for 5 h to achieve a mean arterial blood pressure > 100 mmHg and were randomly assigned to blinded treatment with NIF or placebo control protein administered as a single intravenous bolus (10 mg/kg) at the time of resuscitation. Intrahepatic leukocyte adhesion was evaluated by in vivo fluorescence microscopy. There were no significant differences observed in hemodynamic parameters between the shock groups throughout the study, however, NIF significantly reduced firm leukocyte adhesion in liver sinusoids. The results suggest that NIF may be beneficial in the attenuation of the pathological shock-induced leukocyte adhesion.

Complete inhibition of endotoxin-induced coagulation activation in chimpanzees with a monoclonal Fab fragment against factor VII/VIIa.

Gram-negative sepsis is oftentimes complicated by activation of coagulation with disseminated intravascular coagulation and microthrombosis. This may contribute to the associated morbidity, multiple organ failure and death. Recent studies have established that the tissue factor-dependent pathway of blood coagulation has a significant participatory role in the initial endotoxin-induced activation of coagulation. Tissue factor (TF), expressed on the surface of activated monocytes and endothelial cells forms cell surface complexes with free circulating factors VII and VIIa. The latter complex proteolytically activates factors X and IX. Recent in vivo experiments have shown that a rapidly neutralizing TF monoclonal antibody prevents and arrests the endotoxin-induced activation of coagulation and similar studies have shown to reduce mortality in baboons. In this study we describe the preparation of a factor VII/VIIa neutralizing monoclonal Fab fragment and characterize its effect on in vivo activation of coagulation during experimental endotoxemia in chimpanzees. Four chimpanzees received a bolus intravenous injection of 4 ng/kg endotoxin in combination with Fab fragments of a factor VII/VIIa neutralizing murine monoclonal antibody (12D10) at a dose of either 50 micrograms/kg (n = 2) or 100 micrograms/kg (n = 2). Four control animals received a bolus injection of endotoxin alone. Administration of the 12D10 Fab fragments, immediately preceding the endotoxin bolus injection, effectively blocked the endotoxin-induced activation of coagulation. Plasma levels of products of in vivo activation, namely F1 + 2, TAT complexes and FpA remained at baseline values. The administration of 12D10 resulted in a rapid decline in factor VII/VIIa antigen levels which remained below 5 ng/ml for 180-240 min, followed by a rapid return to baseline levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Ligand and cation binding are dual functions of a discrete segment of the integrin beta 3 subunit: cation displacement is involved in ligand binding.

The alpha IIb beta 3 integrin binds Arg-Gly-Asp-containing (RGD-containing) ligands in a cation-dependent interaction. A fourteen amino acid sequence, beta 3 (118-131), and an antibody to it, inhibited ligand binding functions of alpha IIb beta 3, and a 1:1 stoichiometric beta 3 (118-131)-RGD complex was detected by mass spectroscopy. Cation binding to beta 3 (118-131) was demonstrated by terbium luminescence and mass spectroscopy. Notably, ligand displaced cation from the beta 3(118-131) peptide and also from purified alpha IIb beta 3. Thus, beta 3 (118-131), a highly conserved region in integrin beta subunits, binds both ligand and cation. Formation of a ternary complex between cation, ligand, and receptor, with subsequent displacement of cation from beta 3 (118-131) and a second site within the receptor, may be central to the mechanism of ligand recognition by integrins.

Functional interaction between the integrin antagonist neutrophil inhibitory factor and the I domain of CD11b/CD18.

Neutrophil inhibitory factor (NIF) is a hookworm-derived glycoprotein ligand of the integrin CD11b/CD18 that inhibits human neutrophil function (Moyle, M., Foster, D. L., McGrath, D. E., Brown, S. M., Laroche, Y., De Meutter, J., Stanssens, P., Bogowitz, C. A., Fried, V. A., Ely, J. A., Soule, H. R., and Vlasuk, G. P. (1994) J. Biol. Chem. 269, 1008-10015). Here, we present evidence that recombinant NIF (rNIF) associates with the approximately 200-amino acid residue I domain of CD11b/CD18 and that this interaction is essential for inhibition of neutrophil function by NIF. First, radiolabeled rNIF binds to a recombinant glutathione S-transferase fusion protein that contains the CD11b I domain. This high affinity interaction has a partial dependence on divalent cations. The association of rNIF with the CD11b I domain is specific because 125I-rNIF does not bind either a glutathione S-transferase fusion protein that contains the I domain of the integrin CD11a/CD18 or recombinant glutathione S-transferase without the I domain. Second, the CD11b I domain fusion protein effectively competes with CD11b/CD18 on human neutrophils for 125I-rNIF binding. Third, the CD11b I domain fusion protein blocks the inhibition of certain neutrophil functions by rNIF, including adhesion of neutrophils to human endothelial cell monolayers and adhesion-dependent release of hydrogen peroxide from neutrophils. Specificity is demonstrated by the inability of the CD11a I domain fusion protein to block either rNIF binding to neutrophils or rNIF activity. Fourth, rNIF blocks the interaction between neutrophils and fibrinogen, a CD11b/CD18 ligand that is also thought to bind the I domain of CD11b. In contrast, rNIF does not appear to block the binding of factor X to CD11b/CD18 on neutrophils. These results suggest that CD11b/CD18 has multiple distinct binding sites for its cognate ligands, including, but not limited to, the I domain. NIF interferes with the binding of a subset of these CD11b/CD18 ligands in a highly selective manner.

Sequential imaging of indium-111-labeled monoclonal antibody in human mammary tumors hosted in nude mice.

Using a bifunctional chelating agent, indium-111 was attached to a monoclonal antibody 10- 3D2 , specific for a 126-kilodalton phosphoglycoprotein antigen associated with human mammary carcinoma, and was then used to localize and visualize human mammary tumors hosted in nude mice. Simultaneous tumor concentration of In-111-10- 3D2 was eight times greater than that of control I-125-MOPC-21. Uptake of F(ab')2 and Fab of 10- 3D2 was also compared. the scintigrams demonstrated that intact antibody provided the best images. Control In-111-labeled MOPC-21 and plasma did not show specific localization in the tumor. Uptake of In-111-labeled 10- 3D2 was also compared in two lines of human mammary tumors, BT-20 and HS- 578T . Imaging with 10- 3D2 was better for BT-20 than for HS- 578T . These studies demonstrated that (a) In-111-10- 3D2 can be utilized to image human mammary tumors hosted in nude mice; (b) intact antibody provided the best tumor images, although F(ab')2 had optimal target-to-background ratios for earlier imaging; and (c) different mammary tumor lines with possibly different concentrations of tumor-associated antigen showed different rates of uptake and apparent saturation with 10- 3D2 .

Membrane 126-kilodalton phosphoglycoprotein associated with human carcinomas identified by a hybridoma antibody to mammary carcinoma cells.

A large number of hybridomas producing antibodies to the membrane fraction of a human mammary carcinoma cell line have been produced. Among those hybridoma antibodies that bound to exposed surface membrane epitopes on the human mammary carcinoma cell line BT-20, one has been found to bind to all tested breast carcinoma cell lines as well as to a number of other human carcinomas by solid-phase radioimmunoassay. This hybridoma antibody, designated 10-3D2, does not bind to normal mammary epithelium, a variety of control cell lines, or selected normal human tissue homogenates. The 10-3D2 hybridoma antibody identifies by immunoprecipitation a membrane 126-kilodalton protein which is indistinguishable in different tumor cells. It is glycosylated by reference to incorporation of [3H]glucosamine and also is phosphorylated by reference to incorporation of 32P. It is present in a finely granular pattern on the cell surface of mammary carcinoma cells with concentration at the sites of apparent cell-cell contact. This 126-kilodalton phosphoglycoprotein, which we have consistently been unable to identify by immunoprecipitation of intrinsically labeled normal cells, may represent a heretofore unrecognized tumor-associated marker for human mammary carcinomas as well as certain other types of human neoplasms.

Detection of simian virus 40 surface-associated large tumor antigen by enzyme-catalyzed radioiodination.

To facilitate detection of SV40 surface-associated tumor antigen (T-ag), conditions were established to surface label T-ag on intact cells by lactoperoxidase-catalyzed radioiodination (125I/LPO). SDS-PAGE analysis of anti-T immunoprecipitates of SV40-transformed and -infected cells labelled with 125I/LPO revealed the presence of iodinated T-ag. Several types of control experiments were employed to guarantee the surface specificity of the 125I/LPO labelling technique. When SV40-transformed mouse cells were surface labelled with lactoperoxidase and glucose oxidase immobilized on insoluble beads, a preparation less readily internalized than soluble enzymes, T-ag was iodinated. Selective immunoprecipitation of surface antigens demonstrated that lactoperoxidase did not iodinate internally localized T-ag. A reconstruction experiment in which an extract of SV40-infected cells was added to uninfected cells prior to surface labelling suggested that T-ag released from lysed cells did not adhere significantly to monolayer surfaces and become iodinated. Finally, systematic omission of reactants from the iodination reaction revealed that exogenous addition of lactoperoxidase and H2O2 was necessary to generate an iodinated T-ag, indicating that endogenous host cell reactants do not contribute significantly to the iodination of T-ag. 125I-labelled T-ag was detectable on the surface of SV40 tsA-infected cells at the nonpermissive temperature 24 h post infection, indicating that the tsA lesion does not prevent the interaction of T-ag with the cell surface. When 125I/LPO-labelled transformed or infected cells were chased for 2.5 h after labelling, iodinated T-ag was no longer associated with the cell monolayer but was immunoprecipitable from culture supernatants. Cultures from which labelled T-ag had been shed could then be relabelled with 125I/LPO and surface-associated T-ag was again detectable. These data suggest that surface-associated T-ag is continuously shed from the cell surface and is rapidly replaced in the membrane by intracellular T-ag.

Antigenic and immunogenic characteristics of nuclear and membrane-associated simian virus 40 tumor antigen.

Antisera were prepared in syngeneic hosts against subcellular fractions of simian virus 40 (SV40)-transformed cells (MoalphaPM, MoalphaNuc), glutaraldehydefixed SV40-transformed cells (HaalphaH-50-G, MoalphaVLM-G), and electrophoretically purified denatured SV40 tumor antigen (T-ag) (RaalphaT). Immune sera were also collected from animals bearing tumors induced by SV40-transformed cells (HaalphaT, MoalphaT, HAF) and from SV40-immunized animals that had rejected a transplant of SV40-transformed cells (HaalphaS, MoalphaS). Immunological reagents prepared against cell surface (MoalphaPM, HaalphaS, MoalphaS, HaalphaH-50-G, MoalphaVLM-G) reacted exclusively with the surface of SV40-transformed cells by indirect immunofluorescence or protein A surface antigen radioimmunoassay. Immunological reagents prepared against the nuclear fraction (MoalphaNuc) or whole-cell determinants (HaalphaT, MoalphaT, HAF, RaalphaT) reacted with both the nuclei and surface of SV40-transformed or -infected cells. All reagents were capable of immunoprecipitating 96,000-molecular weight large T-ag from solubilized whole cell extracts of SV40-transformed cells. The exclusive surface reactivity of HaalphaS exhibited in immunofluorescence tests was abolished by solubilization of subcellular fractions, which then allowed immunoprecipitation of T-ag by HaalphaS from both nuclear and plasma membrane preparations. Specificity was established by the fact that all T-reactive reagents failed to react in serological tests against chemically transformed mouse cells, and sera from mice bearing transplants chemically transformed mouse cells (MoalphaDMBA-2) failed to react with SV40-transformed mouse or hamster cells. Reagents demonstrating positive surface immunofluorescence and protein A radioimmunoassay reactions against SV40-transformed cells were capable of blocking the surface binding of RaalphaT to SV40-transformed cells in a double-antibody surface antigen radioimmunoassay. This blocking ability demonstrated directly that a component specificity of each surface-reactive reagent is directed against SV40 T-ag. A model is presented which postulates that the differential detection of T-ag by the various serological reagents is a reflection of immunogenic and antigenic differences between T-ag polypeptides localized in nuclei and plasma membranes.

Subcellular Localization of simian virus 40 large tumor antigen.

The distribution of simian virus 40 large tumor antigen in subcellular fractions from simian virus 40-transformed hamster (H-50) and mouse (VLM) cells and from simian virus 40-infected monkey cells was determined. Solubilized [(35)S]-methionine- or (32)P(i)-labeled surface membrane and nuclear fractions were prepared, immunoprecipitated with hamster anti-T serum, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tumor antigen with an apparent molecular weight of approximately 96,000 was detected in both subcellular fractions. Minor components of approximately 68,000 and approximately 56,000 with anti-T reactivity which labeled with [(35)S]methionine were also detected in both fractions from H-50 cells, as were components of approximately 140,000 and approximately 56,000 from VLM cells. The 56,000 component appeared to be greatly reduced in (32)P(i)-labeled surface membrane fractions. Normal cells or cells transformed with a heterologous agent, such as polyoma virus or a chemical carcinogen, lacked immunoprecipitable tumor antigen. Cell fractionation was monitored by [(3)H]thymidine labeling, NADH-diaphorase activity, and Na(+)-K(+)-dependent ATPase activity. These analyses revealed only trace contamination of surface membranes by nuclei, extremely low levels of nuclear rupture during homogenization, and an approximate 10-fold enrichment of surface membrane. Reconstruction experiments demonstrated that soluble tumor antigen failed to associate or copurify with surface membranes during fractionation procedures. These results indicate the presence of a protein in the plasma membrane of cells transformed or infected by simian virus 40 that is immunologically indistinguishable from nuclear tumor antigen.

Role of the simian virus 40 gene A product in regulation of DNA synthesis in transformed cells.

Cells transformed by tsA mutants of simian virus 40 (SV40) are temperature sensitive for the maintenance of the transformed phenotype. The kinetics of induction of DNA synthesis were determined for hamster cell transformants shifted to the permissive temperature after a 48-h serum arrest at the nonpermissive temperature. DNAsynthesis was initiated in the tsA transformants by 8 h after shiftdown was maximal by 12 h. The presence or absence of fetal bovine serum at the time of temperature shift had no effect on the kinetics of initiation of DNA synthesis. Analysis of TTP in tsA transformants revealed similar levels of incorporation of [3H]thymidine into TTP at both permissive and nonpermissive temperatures. Autoradiography revealed that by 12 h after a shift to the permissive temperature, approximately 50% of the cells exhibited labeled nuclei after a 60-min pulse with [3H]thymidine, indicating that a majority of the cells were actively synthesizing DNA. By 8 to 12 h after a shiftup of confluent tsA transformants to the nonpermissive temperature, the number of labeled nuclei was reduced to approximately 16%, regardless of serum concentration. These data indicate that the SV40 gene A product, either directly or indirectly, regulates cellular DNA synthesis in transformed cells.