Plasticity in the timing of detachment of an Eurasian-African songbird tick, Ixodes frontalis.

In non-permanent parasites, host detachment should take place in an environment that ensures the continuation of their life cycle. Timing of detachment - in combination with the host's space use - affects dispersal and transmission success of the parasites and of the pathogens they vector. Before reaching the adult reproductive stage, ticks need to go through multiple immature developmental stages (larva and nymph), each feeding on host blood. In between the feeding bouts, they often remain in the off-host environment for considerable periods of time. With this study, we aimed to obtain more insight in Ixodes frontalis' off-host habitat use by comparing its detachment pattern in different life stages with that of two habitat-specialized ticks also found on birds: the endophilic tree-hole tick (Ixodes arboricola) and the exophilic sheep tick (Ixodes ricinus), the latter living in humid understory vegetation of forests. For this, we artificially infested hole-roosting (great tits, Parus major) and open-roosting (blackbirds, Turdus merula) birds with ticks under laboratory conditions, and recorded whether detachment occurred during the day or the night. We hypothesize that nocturnal detachment improves off-host mating opportunities and host localization, whereas diurnal detachment optimizes tick dispersal. Ixodes frontalis nymphs detached during the night, especially when feeding on blackbirds. This behaviour was very similar to that of I. arboricola (larva and nymph) feeding on great tits. In contrast, I. frontalis larvae detached during the day, especially when feeding on great tits, which resembles that of I. ricinus' feeding behaviour (larva and nymph). Ixodes frontalis left the host within seven days, immediately after completion of the blood meal. This is similar to both developmental stages of I. ricinus but contrasts with the very long (up to 20 days) feeding duration in I. arboricola. Thus I. frontalis shows strong plasticity, switching from dispersal-centered (larvae) to host-centered (nymphs) detachment behaviour. Findings are discussed with regard to the ticks' habitat use, dispersal, life history and host specificity.

Safe core-satellite magneto-plasmonic nanostructures for efficient targeting and photothermal treatment of tumor cells.

Magneto-plasmonic nanostructures functionalized with cell targeting units are of great interest for nanobiotechnology applications. Photothermal treatment of cells targeted with antibody functionalized nanostructures and followed by magnetic isolation, allows killing selected cells and hence is one of the applications of great interest. The magneto-plasmonic nanostructures reported herein were synthesized using naked gold and magnetite nanoparticles obtained through a green approach based on laser ablation of bulk materials in water. These particles do not need purifications steps for biocompatibility and are functionalized with a SERRS (surface enhanced resonance Raman scattering) active molecule for detection and with an antibody for targeting prostate tumor cells. Quantitative results for the cell targeting and selection efficiency show an overall accuracy of 94% at picomolar concentrations. The photothermal treatment efficiently kills targeted and magneto-selected cells producing a viability below 5% after 3 min of irradiation, compared with almost 100% viability of incubated and irradiated, but non targeted cells.

Highly sensitive electrochemiluminescence detection of a prostate cancer biomarker.

Prostate-specific membrane antigen (PSMA), a glycoprotein expressed in the prostatic epithelium endowed with enzymatic activity, is a very promising diagnostic marker for the early detection of prostate cancer. In this study, we report a novel electrochemiluminescence ELISA-like immunosensor based on carbon nanotubes and a highly specific sandwich immunoassay for the PSMA detection. To fabricate the device, an optically transparent electrode was modified with doubly functionalized multi-walled carbon nanotubes carrying amine groups and a monoclonal anti-PSMA antibody. Subsequently, to complete the sandwich immunosensing device, a second specific monoclonal anti-PSMA antibody was labelled with a electrochemiluminescent probe. Under optimized experimental conditions, the proposed sensing device exhibits a performance exceeding that of the state of-the-art in terms of the limit of detection (LOD) and limit of quantification (LOQ) as good as 0.88 ng mL-1 and 2.60 ng mL-1, respectively, in real complex samples such as cell lysates. In addition, the unique role of carbon nanotubes is also discussed by comparison with an analogue sensor assembled without the nanocarbon-based material.

Comparative analysis of monoclonal antibodies against prostate-specific membrane antigen (PSMA).

BACKGROUND: Prostate-specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II (GCPII), is generally recognized as a diagnostic and therapeutic cancer antigen and a molecular address for targeted imaging and drug delivery studies. Due to its significance in cancer research, numerous monoclonal antibodies (mAbs) against GCPII have been described and marketed in the past decades. Unfortunately, some of these mAbs are poorly characterized, which might lead to their inappropriate use and misinterpretation of the acquired results. METHODS: We collected the 13 most frequently used mAbs against GCPII and quantitatively characterized their binding to GCPII by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). Using a peptide library, we mapped epitopes recognized by a given mAb. Finally, we assessed the applicability of these mAbs to routine experimental setups, including Western blotting, immunohistochemistry, and flow cytometry. RESULTS: ELISA and SPR analyses revealed that mAbs J591, J415, D2B, 107-1A4, GCP-05, and 2G7 bind preferentially to GCPII in native form, while mAbs YPSMA-1, YPSMA-2, GCP-02, GCP-04, and 3E6 bind solely to denatured GCPII. mAbs 24.4E6 and 7E11-C5.3 recognize both forms of GCPII. Additionally, we determined that GCP-02 and 3E6 cross-react with mouse GCPII, while GCP-04 recognizes GCPII and GCPIII proteins from both human and mouse. CONCLUSION: This comparative analysis provides the first detailed quantitative characterization of the most commonly used mAbs against GCPII and can serve as a guideline for the scientific community to use them in a proper and efficient way.

A single-chain fragment against prostate specific membrane antigen as a tool to build theranostic reagents for prostate cancer.

Prostate carcinoma is the most common non-cutaneous cancer in developed countries and represents the second leading cause of death. Early stage androgen dependent prostate carcinoma responds well to conventional therapies, but relatively few treatment options exist for patients with hormone-refractory prostate cancer. One of the most suitable targets for antibody-mediated approaches is prostate specific membrane antigen (PSMA) which is a well known tumour associated antigen. PSMA is a type II integral cell-surface membrane protein that is not secreted, and its expression density and enzymatic activity are increased progressively in prostate cancer compared to normal prostate epithelium, thereby making PSMA an ideal target for monoclonal antibody imaging and therapy. To obtain a small protein that can better penetrate tissue, we have engineered a single-chain variable fragment (scFv) starting from the variable heavy and light domains of the murine anti-PSMA monoclonal antibody D2B. scFvD2B was analysed in vitro for activity, stability, internalisation ability and in vivo for targeting specificity. Maintenance of function and immunoreactivity as well as extremely high radiolabelling efficiency and radiochemical purity were demonstrated by in vitro assays and under different experimental conditions. Despite its monovalent binding, scFvD2B retained a good strength of binding and was able to internalise around 40% of bound antigen. In vivo we showed its ability to specifically target only PSMA expressing prostate cancer xenografts. Due to these advantageous properties, scFvD2B has the potential to become a good theranostic reagent for early detection and therapy of prostate cancers.

A dominant linear B-cell epitope of ricin A-chain is the target of a neutralizing antibody response in Hodgkin's lymphoma patients treated with an anti-CD25 immunotoxin.

Hodgkin's lymphoma patients treated with an anti-CD25 Ricin toxin A-chain (RTA)-based Immunotoxin (RFT5.dgA) develop an immune response against the toxic moiety of the immunoconjugate. The anti-RTA antibody response of 15 patients showing different clinical features and receiving different total amounts of RFT5.dgA was therefore studied in detail, considering antibody titre, IgG and IgM content, average binding efficacy and ability to inhibit in vitro the cytotoxicity of a RTA-based Immunotoxin. No correlations were found between these parameters and the clinical features of the patients or the total amount of Immunotoxin administered. However, using a peptide scan approach we have identified a continuous epitope recognized by all patients studied, located within the stretch L161-I175 of the RTA primary sequence, close to a previously identified T-cell epitope. The ability of anti-L161-I175 antibodies to recognize folded RTA and to affect the biological activity of RTA by inhibiting RTA-IT cytotoxicity in vitro revealed that they may exert an important role in IT neutralization in vivo. Discovery of RTA immunodominant epitopes which are the target of anti-RTA immune response may lead to the development of immunomodulating strategies and to more successful treatment schedules.

Identification of ricin A-chain HLA class II-restricted epitopes by human T-cell clones.

The identification of ricin toxin A-chain (RTA) epitopes and the molecular context in which they are recognized will allow strategies to be devised that prevent/suppress an anti-RTA immune response in patients treated with RTA-based immunotoxins. RTA-specific human T-cell lines and T-cell clones were produced by in vitro priming of PBMC. The T-cell clones used a limited set of Vbeta chains (Vbeta1, Vbeta2 and Vbeta8) to recognize RTA epitopes. The use of RTA deletion mutants demonstrated that T-cell lines and T-cell clones from three out of four donors responded to RTA epitopes within the domain D124-Q223, whereas one donor recognized the region I1-D124. The response to RTA peptides of T-cell lines and T-cell clones from two donors allowed the identification of immunogenic segments (D124-G140 and L161-T190) recognized in the context of different HLA-DRB1 alleles (HLA-DRB1*0801, and HLA-DRB1*11011 and B1*03011, respectively). The response to L161-T190 was investigated in greater detail. We found that the HLA-DRB1*03011 allele presents a minimal epitope represented by the sequence I175-Y183 of RTA, whereas the HLA-DRB1*11011 allele presents the minimal epitope M174-I184. RTA peptides and an I175A RTA point mutant allowed us to identify I175 as a crucial residue for the epitope(s) recognized by the two HLA-DRB1 alleles. Failure of T-cell clones to recognize ribosome inactivating proteins (RIPs) showing sequences similar but not identical to RTA further confirmed the role of I175 as a key residue for the epitope recognized in the context of HLA-DRB1*11011/03011 alleles.

Pharmacokinetics of intrathecal transferrin-ricin a chain immunotoxin.

We have studied the pharmacokinetics of an anti-transferrin receptor immunotoxin following intrathecal (i.t.) and intravenous (i.v.) bolus inoculation in healthy rats. After i.t. inoculation of 4.9 microg transferrin-ricin A-chain (Tfn-RTA) we have measured the immunotoxin concentration in the cerebrospinal fluid (CSF), in the brain tissue and in the peripheral blood. After i.v. administration of 4.9 microg Tfn-RTA the concentration of Tfn-RTA immunotoxin was evaluated in the peripheral blood. We found that the clearance of Tfn-RTA from the CSF is rapid (9.1 microLmin(-1)), the immunotoxin then diffuses into the brain tissue and in the peripheral blood where it reaches concentrations below the MTC50 (Minimum Toxin Concentration 50%). The rate of immunotoxin elimination from the peripheral blood following either i.v. or i.t. administration are similar (kel = 0.0021 min(-1) vs. 0.0025 min(-1)). Thus, in the healthy rat the immunotoxin does not accumulate following i.t. inoculation, reaching non toxic concentrations in the brain tissue and in the peripheral blood, whereas in the CSF as well as at the interface CSF/brain tissue the immunotoxin may reach potentially therapeutic concentrations. In conclusion we believe that the i.t. inoculation of an immunotoxin could be considered a potentially useful route of administration in the treatment of leptomeningeal carcinomatosis.

Expression of myelin basic protein (MBP) epitopes in human non-neural cells revealed by two anti-MBP IgM monoclonal antibodies.

Two monoclonal antibodies (1H6.2 and 45.30) were raised against MBP purified from human brain under experimental conditions that allowed MBP to retain binding to surrounding myelin lipids (human lipid-bound MBP (hLB-MBP)). 1H6.2 and 45.30 MoAbs were selected on the basis of their different binding properties to: hLB-MBP, human lipid-free-MBP (hLF-MBP) and bovine lipid-free-MBP (bLF-MBP). Although the isotype of both MoAbs was IgM, their specificity, as tested in ELISA assays against chemical haptens and unrelated protein antigens, was restricted to MBP. 1H6.2 and 45.30 MoAbs stained MBP from human brain white matter tissue extracts, as well as bLF-MBP, in Western blot assays. Both MoAbs stained oligodendrocytes and myelin in immunohistochemical analysis of white matter from human brain. Tissue sections from human peripheral nerves were labelled by 1H6.2 only, however, demonstrating that the MoAbs recognize two different epitopes. Epitopes recognized by 1H6.2 and 45.30 MoAbs were also expressed by a wide array of human non-neural cells of either normal or pathological origin, as evidenced by cytofluorimetric assays. In particular, MBP epitopes (MEs) were expressed by lymphoid cells as well as by cells which play a pivotal role in immune homeostasis and in the immune response, such as thymic epithelial cells and professional antigen-presenting cells. Both MoAbs were efficiently internalized by cells from a human B cell line, suggesting trafficking of MEs along the endocytic pathways. These findings support hypotheses regarding the role of MEs expressed by non-neural cells in establishing self-tolerance and/or in triggering the immune response against MBP antigen.

Effect of therapeutic macromolecules in spheroids.

Recent advances in biotechnology have allowed the production of new types of macromolecular therapeutic agents (antibodies, immunotoxins, cytokines, extracellular matrix molecule (ECM) proteins, vectors) that may eventually find broad clinical applications in the treatment of human tumors and other diseases. The model of the Multicellular Tumor Spheroids (MTS) represents a valuable tool to test the therapeutic potential of these new pharmacologic agents in a 3-D context. Specific questions pertaining to the behaviour in a 3-D setting of some of the macromolecules under evaluation for in vivo applications can also be addressed in the MTS model (e.g. 'binding site barrier', role of cell-cell and cell-ECM interactions). This paper reviews the most significant contributions regarding the delivery of macromolecules to MTS, the penetration and therapeutic effects of antibodies, radiolabelled antibodies, immunotoxins and other macromolecular compounds.

Genetic grafting of membrane-acting peptides to the cytotoxin dianthin augments its ability to de-stabilize lipid bilayers and enhances its cytotoxic potential as the component of transferrin-toxin conjugates.

Three chimeric proteins were obtained by fusing together the dianthin gene and DNA fragments encoding for the following membrane-acting peptides: the N-terminus of protein G of the vesicular stomatitis virus (KFT25), the N terminus of the HA2 hemagglutinin of influenza virus (pHA2), and a membrane-acting peptide (pJVE). Chimeric dianthins (KFT25DIA, pHA2DIA and pJVEDIA) retained full enzymatic activity in cell-free assays and showed increased ability to induce pH-dependent calcein release from large unilamellar vesicles (LUVs). pHA2DIA and pJVEDIA also showed faster kinetics of interaction with LUVs, while KFT25DIA and pHA2DIA displayed a reduced cytotoxicity as compared to wild-type dianthin. Conjugates made by chemically cross-linking KFT25DIA or pJVEDIA and human transferrin (Tfn) showed greater cell-killing efficiency than conjugates of Tfn and wild-type dianthin. As a consequence, by fusion of membrane-acting peptides to the dianthin sequence the specificity factor (i.e., the ratio between non-specific and specific toxicity) of Tfn-KFT25DIA, Tfn-pHA2DIA and Tfn-pJVEDIA was increased with respect to that of Tfn-based conjugates made with wild-type dianthin. Taken together, our results suggest that genetic fusion of membrane-acting peptides to enzymatic cytotoxins results in the acquisition of new physico-chemical properties exploitable for designing new recombinant cytotoxins and to tackle cell-intoxication mechanisms.

Improved method for carbohydrate-deficient transferrin determination in human serum by capillary zone electrophoresis.

Carbohydrate-deficient transferrin (CDT) is a reliable marker of chronic or repeated alcohol abuse. It indicates a group of isoforms of human transferrin (Tf), the main iron transport serum protein, deficient in sialic acid residues (asialo-, monosialo- and disialo-Tf) in comparison to the main isotransferrin which contains four sialic acid groups (tetrasialo-Tf). The aim of the present work was to develop a capillary electrophoretic method suitable for rapid determination of CDT components in serum. Serum samples (0.1 ml) were saturated with iron by incubation with 10 mM FeCl3 (2 microl) and 500 mM NaHCO3 (3 microl) for 30 min, then diluted 1:10 in water and injected by positive pressure (0.5 p.s.i. for 10 s). Separation was performed with a capillary zone electrophoretic method using bare fused-silica capillaries (57 cm x 20 microm I.D.) and a buffer composed of 100 mM sodium tetraborate adjusted with 6 M HCl to pH 8.3 added with 1.5 mM diaminobutane. Applied voltage was 20 kV and temperature 25 degrees C. Detection was by UV absorption at 200 nm wavelength. Under the described conditions, asialo-, monosialo-, disialo-, trisialo- and tetrasialo-transferrin were baseline separated. The limit of detection (signal-to-noise ratio of 2) was about 0.3% for disialo-Tf, and 0.5% of trisialo-Tf, expressed as percentages of the terasialo-Tf peak area. Day-to-day RSDs of relative migration times were < or = 0.2%. Quantitation showed day-to-day RDSs < or = 6.9% and < or = 10.9% for disialo- and trisialo-Tf, respectively. The results from 79 control subjects, including social drinkers, and 23 alcoholics showed disialo- and trisialo-Tf significantly increased in patients (P<0.0001 and <0.01, respectively). A clear interference from trisialo-Tf in an immunoassay for CDT was demonstrated. The present method is suitable for confirmation of CDT immunoassays by independent technique.

Self-potentiation of ligand-toxin conjugates containing ricin A chain fused with viral structures.

A chimeric protein was obtained by fusing together the ricin toxin A chain (RTA) gene and a DNA fragment encoding the N terminus of protein G of the vesicular stomatitis virus. Chimeric RTA (cRTA) retained full enzymic activity in a cell-free assay, but was 10-fold less toxic against human leukemic cells than either native RTA (nRTA) or unmodified recombinant RTA (rRTA). However, conjugates made with cRTA and human transferrin (Tfn) showed 10-20-fold greater cell killing efficacy than Tfn-nRTA or Tfn-rRTA conjugates despite equivalent binding of the three conjugates to target tumor cells. As a consequence, by fusion of the KFT25 peptide to the RTA sequence, the specificity factor (i.e. the ratio between nonspecific and specific cytotoxicity) of Tfn-cRTA was increased 90-240 times with respect to those of Tfn-nRTA and Tfn-rRTA. cRTA interacted with phospholipid vesicles with 15-fold faster kinetics than nRTA at acidic pH. Taken together, our results suggest that the ability of vesicular stomatitis virus protein G to interact with cell membranes can be transferred to RTA to facilitate its translocation to the cell cytosol. Our strategy may serve as a general approach for potentiating the cytotoxic efficacy of antitumor immunotoxins.

Heterogeneous response of individual multicellular tumour spheroids to immunotoxins and ricin toxin.

The cytoreductive effects of anti-transferrin receptor (anti-TfnR) immunotoxins (ITs) and of ricin toxin against tumour micromasses have been evaluated in a multicellular tumour spheroid (MTS) model. More than 600 (656) MTSs obtained with human breast carcinoma (MCF7) or rat glioblastoma (9L) cell lines were treated individually with ITs or toxin and the effects induced by the treatment were measured for each MTS as volume variation vs time by applying the Gompertz growth model. Two dose-dependent patterns of MTS growth were observed in MTSs of both cell lines in response to IT or toxin treatment: (1) complete inhibition of MTS growth ('sterilisation'); and (2) partial/complete inhibition ('heterogeneous response'). Within the range of IT or toxin concentrations resulting in partial inhibition of MTS growth, the sensitivity of treated MTSs was extremely heterogeneous (the cytoreductive effects varying between 0.1 and 4 logs of cells killed for a given IT or toxin concentration). Analysis of the post-treatment regrowth kinetics indicated that treated non-sterilised and control MTSs reached the same final limiting volumes. However, the doubling time estimated for the surviving cells of treated MCF7 and 9L MTSs ranged between 15 and 50 h, indicating that each MTS had individual growing potential. In conclusion, our results indicate that at substerilising IT concentrations individual heterogenicity of MTSs may greatly influence the cytoreductive potential of ITs. An implication of our study is that the efficacy of an IT treatment in eradicating disseminated micrometastases may not be predictable a priori. The MTS model that we describe in this paper may help in dissecting out factors limiting the effect of ITs in three-dimensional tumours.

Cardiovascular effects of befunolol, betaxolol and timolol eye drops.

A double-blind cross-over study was carried out on 15 glaucoma patients so as to verify the effects of three the beta blockers, beta-1 selective betaxolol, non-selective timolol and befunolol an agent with intrinsic sympathomimetic activity on the heart rate, monitored by a Holter apparatus, and with the blood pressure measured in standing and supine positions before and after eight days of topical therapy. The results showed that timolol induced a significant decrease in minimum heart rate (-4.2 +/- 2.9) (p less than 0.001) and in diastolic blood pressure in standing position (-8.0 +/- 12.5 mmHg) (p less than 0.05), betaxolol decreased systolic pressure in orthostatism (-7.5 +/- 12.3 mmHg) and in clinostatism (-12.1 +/- 16.2 mmHg) and diastolic pressure in orthostatism (-6.25 +/- 9.4 mmHg) (p less than 0.05). Befunolol alone did not alter the heart rate or blood pressure.