Active Acetylcholinesterase Immobilization on a Functionalized Silicon Surface.

In this work, we studied the attachment of active acetylcholinesterase (AChE) enzyme on a silicon substrate as a potential biomarker for the detection of organophosphorous (OP) pesticides. A multistep functionalization strategy was developed on a crystalline silicon surface: a carboxylic acid-terminated monolayer was grafted onto a hydrogen-terminated silicon surface by photochemical hydrosilylation, and then AChE was covalently attached through amide bonds using an activation EDC/NHS process. Each step of the modification was quantitatively characterized by ex-situ Fourier transform infrared spectroscopy in attenuated-total-reflection geometry (ATR-FTIR) and atomic force microscopy (AFM). The kinetics of enzyme immobilization was investigated using in situ real-time infrared spectroscopy. The enzymatic activity of immobilized acetylcholinesterase enzymes was determined with a colorimetric test. The surface concentration of active AChE was estimated to be Γ = 1.72 × 10(10) cm(-2).

Molecular monolayers on silicon as substrates for biosensors.

(111) silicon surfaces can be controlled down to atomic level and offer a remarkable starting point for elaborating nanostructures. Hydrogenated surfaces are obtained by oxide dissolution in hydrofluoric acid or ammonium fluoride solution. Organic species are grafted onto the hydrogenated surface by a hydrosilylation reaction, providing a robust covalent Si-C bonding. Finally, probe molecules can be anchored to the organic end group, paving the way to the elaboration of sensors. Fluorescence detection is hampered by the high refractive index of silicon. However, improved sensitivity is obtained by replacing the bulk silicon substrate by a thin layer of amorphous silicon deposited on a reflector. The development of a novel hybrid SPR interface by the deposition of an amorphous silicon-carbon alloy is also presented. Such an interface allows the subsequent linking of stable organic monolayers through Si-C bonds for a plasmonic detection. On the other hand, the semiconducting properties of silicon can be used to implement field-effect label-free detection. However, the electrostatic interaction between adsorbed species may lead to a spreading of the adsorption isotherms, which should not be overlooked in practical operating conditions of the sensor. Atomically flat silicon surfaces may allow for measuring recognition interactions with local-probe microscopy.

Semiquantitative study of the EDC/NHS activation of acid terminal groups at modified porous silicon surfaces.

Infrared spectroscopy is used to investigate the transformation of carboxyl-terminated alkyl chains immobilized on a surface into succinimidyl ester-terminated chains by reaction with an aqueous solution of N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The acid chains are covalently grafted at the surface of hydrogenated porous silicon whose large specific surface area allows for assessing the activation yield in a semiquantitative way by infrared (IR) spectroscopy and detecting trace amounts of surface products and/or reaction products of small IR cross section. In this way, we rationalize the different reaction paths and optimize the reaction conditions to obtain as pure as possible succinimidyl ester-terminated surfaces. A diagram mapping the surface composition after activation was constructed by systematically varying the solution composition. Results are accounted for by NHS surface adsorption and a kinetic competition between the various EDC-induced surface reactions.

Highly sensitive and reusable fluorescence microarrays based on hydrogenated amorphous silicon-carbon alloys.

We have designed a new architecture of fluorescent microarrays based on a thin layer of hydrogenated amorphous silicon-carbon alloy (a-Si(0.85)C(0.15):H) deposited on an aluminium-on-glass back reflector. These substrates are modified with an organic monolayer anchored through Si-C bonds and terminated with carboxyl groups, allowing for the covalent immobilization of biological probes. The fluorescence yield is maximized by optimization of the a-Si(0.85)C(0.15):H layer thickness. This approach is assessed for DNA recognition, demonstrating an increase in sensitivity by over one order of magnitude as compared to commercial slides, and the possibility of following in situ the molecular recognition event (hybridization). The immobilization chemistry provides these substrates with a superior chemical stability toward ageing or long-term exposure to physiological buffers, which allows for many successive hybridization/dehybridization cycles without measurable changes in performance.

Assessment of tumour response to chemotherapy for metastatic colorectal cancer: accuracy of the RECIST criteria.

Evaluation of tumour size modifications in response to treatment is a critical issue in the management of advanced malignancies. In 1981, the World Health Organization (WHO) established guidelines for tumour response assessment. These WHO1981 criteria were recently simplified in a revised version, named RECIST (Response Evaluation Criteria in Solid Tumours), which uses unidimensional instead of bidimensional measurements, a reduced number of measured lesions, withdrawal of the progression criteria based on isolated increase of a single lesion, and different shrinkage threshold for definitions of tumour response and progression. In order to validate these new guidelines, we have compared results obtained with both classifications in a prospective series of 91 patients receiving chemotherapy for metastatic colorectal cancer. Data from iterative tomographic measurements were fully recorded and reviewed by an expert panel. The overall response and progression rates according to the WHO1981 criteria were 19% and 58%, respectively. Using RECIST criteria, 16 patients were reclassified in a more favourable subgroup, the overall response rate being 28% and the progression rate 45% (non-weighted kappa concordance test 0.72). When isolated increase of a single measurable lesion is not taken into account for progression with the WHO1981 criteria, only 7 patients were reclassified and the kappa test was satisfying, i.e. > or =0.75, for the whole population as well as for each of the responding and progressive subgroups. Since it provides concordant results with a simplified method, the use of RECIST criteria is recommended for evaluation of treatment efficacy in clinical trials and routine practice.

Shared usage of the chemokine receptor CXCR4 by primary and laboratory-adapted strains of feline immunodeficiency virus.

Strains of the feline immunodeficiency virus (FIV) presently under investigation exhibit distinct patterns of in vitro tropism. In particular, the adaptation of FIV for propagation in Crandell feline kidney (CrFK) cells results in the selection of strains capable of forming syncytia with cell lines of diverse species origin. The infection of CrFK cells by CrFK-adapted strains appears to require the chemokine receptor CXCR4 and is inhibited by its natural ligand, stromal cell-derived factor 1alpha (SDF-1alpha). Here we found that inhibitors of CXCR4-mediated infection by human immunodeficiency virus type I (HIV-1), such as the bicyclam AMD3100 and short peptides derived from the amino-terminal region of SDF-1alpha, also blocked infection of CrFK by FIV. Nevertheless, we observed differences in the ranking order of the peptides as inhibitors of FIV and HIV-1 and showed that such differences are related to the species origin of CXCR4 and not that of the viral envelope. These results suggest that, although the envelope glycoproteins of FIV and HIV-1 are substantially divergent, FIV and HIV-1 interact with CXCR4 in a highly similar manner. We have also addressed the role of CXCR4 in the life cycle of primary isolates of FIV. Various CXCR4 ligands inhibited infection of feline peripheral blood mononuclear cells (PBMC) by primary FIV isolates in a concentration-dependent manner. These ligands also blocked the viral transduction of feline PBMC by pseudotyped viral particles when infection was mediated by the envelope glycoprotein of a primary FIV isolate but not by the G protein of vesicular stomatitis virus, indicating that they act at an envelope-mediated step and presumably at viral entry. These findings strongly suggest that primary and CrFK-adapted strains of FIV, despite disparate in vitro tropisms, share usage of CXCR4.

Delayed infection after immunization with a peptide from the transmembrane glycoprotein of the feline immunodeficiency virus.

Recent advances in the quantitative assessment of viral burden, by permitting the extension of criteria applied to assess the efficacy of vaccines from all-or-none protection to diminution of the viral burden, may allow the identification of original immunogens of value in combined vaccines. Peptides corresponding to three domains of the envelope glycoproteins of feline immunodeficiency virus that are recognized during natural infection were used to immunize cats. After challenge with a primary isolate of feline immunodeficiency virus, the development of acute infection was monitored by quantitative assessment of the viral burden in plasma and tissues by competitive reverse transcription-PCR, by measurement of the humoral response developed to viral components, and by lymphocyte subset analysis. Whereas immunization with two peptides derived from the surface glycoprotein had no effect on the early course of infection, immunization with a peptide derived from the transmembrane glycoprotein delayed infection, as reflected by a diminished viral burden in the early phase of primary infection and delayed seroconversion. This peptide, located in the membrane-proximal region of the extracellular domain, has homology to an epitope of human immunodeficiency virus type 1 recognized by a broadly neutralizing monoclonal antibody. These results suggest that lentivirus transmembrane glycoproteins share a determinant in the juxtamembrane ectodomain which could be of importance in the design of vaccines against AIDS.

Enhancement of feline immunodeficiency virus (FIV) infection after DNA vaccination with the FIV envelope.

Despite intensive experimentation to develop effective and safe vaccines against the human immunodeficiency viruses and other pathogenic lentiviruses, it remains unclear whether an immune response that does not afford protection may, on the contrary, produce adverse effects. In the present study, the effect of genetic immunization with the env gene was examined in a natural animal model of lentivirus pathogenesis, infection of cats by the feline immunodeficiency virus (FIV). Three groups of seven cats were immunized by intramuscular transfer of plasmid DNAs expressing either the wild-type envelope or two envelopes bearing mutations in the principal immunodominant domain of the transmembrane glycoprotein. Upon homologous challenge, determination of plasma virus load showed that the acute phase of viral infection occurred earlier in the three groups of cats immunized with FIV envelopes than in the control cats. Genetic immunization, however, elicited low or undetectable levels of antibodies directed against envelope glycoproteins. These results suggest that immunization with the FIV env gene may result in enhancement of infection and that mechanisms unrelated to enhancing antibodies underlay the observed acceleration.

Neutralization sensitivity and accessibility of continuous B cell epitopes of the feline immunodeficiency virus envelope.

Antibodies elicited during natural infection of domestic cats by the feline immunodeficiency virus (FIV) recognize continuous epitopes in nine domains of the virus envelope glycoproteins. Whereas antibodies directed against the V3 envelope region can neutralize laboratory-adapted virus, neutralization of FIV has been shown to depend upon cellular substrate, and virus adaptation to laboratory cell lines may alter sensitivity to neutralizing antibodies. We therefore undertook a systematic analysis of the continuous B cell epitopes of the envelope of a primary FIV isolate, Wo. The capacity of feline antisera elicited against nine envelope domains to neutralize primary and laboratory-adapted virus was evaluated in feline peripheral blood mononuclear cells (PBMC). The laboratory-adapted strain Petaluma was used to compare neutralization in PBMC and Crandell feline kidney cells (CrFK). Antibodies specific for the V3 region neutralized both primary and laboratory-adapted virus whether residual infectivity was measured in CrFK or in feline PBMC. However, a large discrepancy in the efficiency of neutralization was observed in these ex vivo models of infection, perhaps reflecting diversity in the interaction between virus and different cellular targets. We also examined the accessibility of epitopes on the functional oligomeric envelope complex of FIV. Most of the epitopes were poorly exposed on native envelope glycoproteins at the surface of live infected cells. The most accessible domain was the only domain sensitive to neutralizing antibodies. These results suggest that inaccessibility on oligomeric envelope glycoproteins may frequently underlie the insensitivity of diverse lentivirus B cell epitopes to neutralization.

Immunization trial of cats with a replication-defective adenovirus type 5 expressing the ENV gene of feline immunodeficiency virus.

Our aim was to develop a recombinant replication-defective adenovirus suitable for the vaccination of cats against feline immunodeficiency virus. We first demonstrated that this vector was able to transfer a marker gene (E. coli beta-galactosidase) in feline cells in vitro. We then constructed an adenovirus type 5 expressing the Feline Immunodeficiency Virus (FIV) envelope (ENV) gene of the Wo isolate in the absence of the rev gene (Ad-ENV-Wo). Ad-ENV-Wo was then tested in four cats in a 3 injections scheme (at day 0, day 30 and day 210). Four other control cats received Ad-gp50, a similar recombinant adenovirus expressing gp50 (Ad-gp50) of pseudorabies virus (PRV). Viruses were formulated in two different kind of oil adjuvants (water/oil and water/oil/water), a protocol previously shown to enhance the immune response against the virus-induced protein. The control cats developed neutralizing antibodies against PRV, demonstrating the potency of recombinant human adenovirus 5 (Ad5) as a vector in cats. Antibody responses appeared after the first injection and were higher with the water/oil/water formulation than with the water/oil controls. However, none of the four cats vaccinated with Ad-ENV-Wo developed antibodies against two peptides of the envelope protein. Animals were challenged with 20 infectious doses 50% of the strain Wo. All of them developed antibodies against FIV within 4 to 5 weeks, and FIV virus could be isolated from all.

Comparison of serological tests for the diagnosis of feline immunodeficiency virus infection of cats.

A new enzyme-linked-immunosorbent assay (ELISA) for the detection of antibodies to feline immunodeficiency virus was compared with previously described ELISAs. Serum samples from 184 infected or uninfected cats were tested using a whole virus lysate kit and ELISAs based on recognition of one of two synthetic peptides (P237 and P253) localized in the transmembrane domain of the viral envelope. The whole virus lysate commercial kit led to the detection of 6% false positive and 4.3% false negative sera. The ELISA based on peptide P253 gave no false positive result and failed to detect only one serum that was subsequently shown to be positive by radio-immunoprecipitation assay. A sandwich-ELISA test using Galanthus nivalis agglutinin, a lectin that specifically binds terminal mannose groups of the envelope proteins was used as a confirmatory test for equivocal results with peptide ELISA and gave similar results. This study indicates that recognition of P253 could serve as a sensitive and specific test for the diagnosis of seropositivity to feline immunodeficiency virus, and moreover that the Galanthus nivalis ELISA could be useful in equivocal cases as a confirmatory test.

Histopathological changes in lymph nodes of cats experimentally infected with the feline immunodeficiency virus (FIV).

Twelve specific-pathogen-free (SPF) kittens aged 8-12 weeks were serially infected in pairs every 6 weeks, by the intraperitoneal route, with the feline immunodeficiency virus (FIV). Three additional SPF kittens were kept as controls. The infected animals were killed 10 weeks after inoculation, during the primary phase of the FIV infection. Generalized lymphadenopathy (GL) was observed in the first three pairs of cats. All lymph nodes examined from the 12 infected cats showed histological changes. These included severe follicular hyperplasia with hyperactive follicular centres (FCs) which were either (1) naked, (2) infiltrated by lymphocytes, (3) seen to contain islets of lymphocytic mantle cells, or (4) disrupted by lymphocytes. The presence of both CD4+ and CD8+ T lymphocytes was demonstrated in the FCs immunocytochemically. The distribution of CD4 lymphocytes resembled that in control lymph nodes, but the CD8 cells were increased in number and either scattered or clustered in the follicles. In addition, varying degrees of interfollicular proliferation and medullary plasmacytosis were observed in the lymph nodes. These findings, which were common to all infected animals, represented distinct prodromal manifestations of FIV infection. The changes in lymphocyte subpopulation distribution observed in early FIV infection were reminiscent of findings encountered in human immunodeficiency virus (HIV) infection and reinforce the suggestion that FIV infection is an appropriate model for the study of HIV pathogenesis.

Serological diagnosis of feline immunodeficiency virus infection based on synthetic peptides from Env glycoproteins.

Feline immunodeficiency virus (FIV) is a lentivirus which infects domestic cats, causing an acquired immunodeficiency syndrome (AIDS). The aim of the present work was the development of an immunoassay for the diagnosis of FIV infection, using synthetic peptides from FIV envelope (Env) glycoproteins. Four peptides (8 to 11 amino acids long) corresponding to group-specific epitopes of FIV Env extracellular (SU) or transmembrane (TM) glycoproteins were synthesized. They were evaluated by enzyme-linked immunosorbent assay (ELISA) for immunoreactivity with sera from naturally or experimentally FIV-infected cats. One of these, P237, corresponds to a conserved nonapeptide of FIV TM, folded as a loop between two cysteines. ELISA performed with P237 on 171 sera from FIV-infected cats and 46 sera from specific-pathogen-free cats showed no false positive cases and 100% detection of infected cat sera. Moreover, 47 pet cat sera which were negative with a whole virus-based-ELISA were tested with the P237 ELISA: 2 out of 47 showed reactivity. FIV infection of these two cats was confirmed by radio-immunoprecipitation assay. Temporal studies performed on serial serum samples from experimentally infected cats detected antibodies to P237 three to five weeks after inoculation of virus. Thus, the P237 ELISA is a sensitive and specific immunoassay for early detection of antibodies to FIV. In addition, this synthetic nonapeptide is easier to produce and purify than virus preparations or recombinant proteins.

Structure and variations of feline immunodeficiency virus envelope glycoproteins.

We report the characterization of the env gene of a feline immunodeficiency virus isolate from France (FIV Wo). FIV Wo gag and env genes were cloned directly from cat peripheral blood mononuclear cells, using polymerase chain reaction. The env molecular clone was shown to be functional and to express antigenically relevant envelope glycoproteins in vitro. Alignment of FIV Wo sequences with available FIV sequences and application of a regionalization algorithm resulted in delineation of variable and conserved domains of FIV Env. These data were used to build a schematic folding model of FIV envelope glycoproteins. The Env molecular clone, variability map, and structural model constitute helpful tools for future studies of FIV envelope aimed at the determination of structure-function relationships or design of diagnostics or vaccine reagents.

Localisation of three epitopes of the env protein of feline immunodeficiency virus.

The envelope protein of the feline immunodeficiency virus (FIV) was analyzed using several epitope prediction programs based on profiles of hydrophilicity, antigenicity, and probability of residues to lie on the protein surface. Tentative homologies with the immunodominant epitope sites in simian virus (SIV) or human immunodeficiency virus (HIV) such as the V3 loop, the site of cleavage between surface envelope protein (SU) and transmembrane envelope protein (TM), and sites of N-glycosylation were thus identified. Five peptides corresponding to potential epitopes were synthesized. Four out of five peptides (P99, P100, P101, P103) were from the FIV surface envelope protein (SU). The last one (P102) was from the FIV transmembrane envelope protein TM. Three of these peptides (P99, P100, and P102) were recognized in ELISA by almost all the sera from infected cats. The peptide from TM (102) was recognized by sera from both naturally infected and inoculated cats, whereas peptides P99 and P100 (from SU) were recognized mainly by sera from naturally infected cats. On the basis of these results we propose that peptides P99, and P100 from SU and P102 from TM constitute epitopes on the FIV env protein.

In vitro properties and experimental pathogenic effect of three strains of feline immunodeficiency viruses (FIV) isolated from cats with terminal disease.

Three strains of virus isolated from peripheral blood mononuclear cells (PBMC) of sick cats were identified as feline immunodeficiency virus (FIV) on the basis of in vitro cytopathic effect, T-lymphotropism, ultrastructural morphology and magnesium-dependent reverse-transcriptase activity. The pathogenic properties of two isolates were studied in 13 experimentally infected cats. The primary phase of infection was characterised by a range of haematological (neutropenia, lymphopenia, presence of atypical lymphocytes) and clinical alterations (fever, various signs lasting several weeks, generalised lymphadenopathy persisting for several months) and specific seroconversion. A correlation between the inoculated dose of virus and the intensity and duration of clinical signs was observed. The primary phase was followed in the 10 surviving cats by a stage of asymptomatic seropositivity of undetermined duration but which has persisted for over 35 months for the earliest infections. Viruses reisolated several weeks or months after experimental infection retained the same in vitro properties as the initial isolates.