Influence of the dietary fibre levels on faecal hair excretion after 14 days in short and long-haired domestic cats.

Hairballs are a common problem in cats and may result in intestinal obstruction. Dietary fibre has been recommended to stimulate hair faecal excretion. The objective of this study was to assess the influence of psyllium and different levels of total dietary fibre (6% vs. 11% vs. 15% TdF) on hair faecal excretion in short and long-haired (LH) domestic cats. Twenty-one adult cats were divided into three panels: shedding panel, short-haired (SH) panel and LH panel. Shedding panel was used to assess shedding throughout the study with a normalized brushing. In parallel, SH panel and LH panel were used to evaluate the impact of the diets on faecal hair excretion. They were fed a low-fibre diet during phase 1 (diet 6, 6.0% TdF). In phase 2, panels were fed either diet 11 (11% TdF) or diet 15 (15% TdF). In phase 3, cats returned to diet 6 before a crossover on the other diet in phase 4. Those diets were fed to cats for 14 days. Faecal hair excretion was quantified daily. Data were analysed using a generalized linear model procedure. The assessment of shedding showed that the study did not occur during moulting season. In the LH panel, diet 11 and diet 15 increased (P < 0.001) faecal hair excretion by 81% and 113% respectively, compared to the control diet. In the SH panel, no influence of the diets was observed. This study supports that fibre affects faecal hair excretion in LH cats, suggesting that a diet with psyllium and 11% or 15% TdF might minimize hairball formation in 14 days and between shedding periods. In SH cats, 11% and 15% fibre levels had no effect on faecal hair excretion. This could be explained by the low quantity of hair ingested outside the shedding season or by the short duration of the study.

Development of monoclonal antibodies to human kallikrein-related peptidase 6 (KLK6) and their use in an immunofluorometric assay for free KLK6.

We have raised monoclonal antibodies against KLK6 and constructed a 'sandwich' type immunoassay using 8A8G3 as capture and 3H3E9 as tracer antibodies. 8A8G3 bound to one side of KLK6, whereas 3H3E9 probably bound near its catalytic site. The assay did not detect complexed forms of KLK6, indicating that it quantifies only free KLK6 (fKLK6). fKLK6 was higher in serum of patients with ovarian cancer (11.34 µg/l±1.37) than in controls (3.39 µg/l±0.42). The cerebrospinal fluid contained high concentrations of fKLK6 (257-965 µg/l). This assay could facilitate the evaluation of fKLK6 in human diseases.

Prostate cancer biomarker annexin A3 detected in urines obtained following digital rectal examination presents antigenic variability.

OBJECTIVES: Annexin A3 (ANXA3) is a potential marker for prostate cancer (PCa). We aimed to develop robust immunoassays suitable for quantifying ANXA3 in urine samples obtained following digital rectal examination (DRE) in order to facilitate the diagnostic performance evaluation of this marker. DESIGN AND METHODS: Anti-ANXA3 monoclonal antibodies were generated and their epitopes mapped. Two different ANXA3 assay prototypes were established on the VIDAS® automated immunoanalyser and analytical validation was carried out using post-DRE urine samples obtained from patients with PCa (n=23) or benign prostate hyperplasia (n=31). RESULTS: The assays had the same capture antibody (TGC44) but different detection antibodies (13A12 or 5C5), recognizing novel distinct epitopes. Both had a lower limit of quantification <1ng/mL and were highly specific for ANXA3, not cross-reacting with other annexins. Interassay imprecision was ≤11% and ≤15% for 13A12 and 5C5 assays, respectively. Surprisingly, a total lack of correlation was observed between ANXA3 levels measured by these two assays in post-DRE urines, indicating detection of distinct antigenic variants. Two freeze-thaw cycles did not affect analyte stability in either assay, whereas a lack of stability of antigenic variants was observed when samples were stored at -80°C for 1month. CONCLUSIONS: Two different antigenic variants of ANXA3 are present in post-DRE urines and their clinical significance for diagnosis of prostate cancer should be further investigated. These variants are not stable over time in samples preserved at -80°C. Until this issue is resolved, ANXA3 should only be measured in freshly collected samples.

Risk of hormone escape in a human prostate cancer model depends on therapy modalities and can be reduced by tyrosine kinase inhibitors.

Almost all prostate cancers respond to androgen deprivation treatment but many recur. We postulated that risk of hormone escape--frequency and delay--are influenced by hormone therapy modalities. More, hormone therapies induce crucial biological changes involving androgen receptors; some might be targets for escape prevention. We investigated the relationship between the androgen deprivation treatment and the risk of recurrence using nude mice bearing the high grade, hormone-dependent human prostate cancer xenograft PAC120. Tumor-bearing mice were treated by Luteinizing-Hormone Releasing Hormone (LHRH) antagonist alone, continuous or intermittent regimen, or combined with androgen receptor (AR) antagonists (bicalutamide or flutamide). Tumor growth was monitored. Biological changes were studied as for genomic alterations, AR mutations and protein expression in a large series of recurrent tumors according to hormone therapy modalities. Therapies targeting Her-2 or AKT were tested in combination with castration. All statistical tests were two-sided. Tumor growth was inhibited by continuous administration of the LH-RH antagonist degarelix (castration), but 40% of tumors recurred. Intermittent castration or complete blockade induced by degarelix and antiandrogens combination, inhibited tumor growth but increased the risk of recurrence (RR) as compared to continuous castration (RR(intermittent): 14.5, RR(complete blockade): 6.5 and 1.35). All recurrent tumors displayed new quantitative genetic alterations and AR mutations, whatever the treatment modalities. AR amplification was found after complete blockade. Increased expression of Her-2/neu with frequent ERK/AKT activation was detected in all variants. Combination of castration with a Her-2/neu inhibitor decreased recurrence risk (0.17) and combination with an mTOR inhibitor prevented it. Anti-hormone treatments influence risk of recurrence although tumor growth inhibition was initially similar. Recurrent tumors displayed genetic instability, AR mutations, and alterations of phosphorylation pathways. We postulated that Her-2/AKT pathways allowed salvage of tumor cells under castration and we demonstrated that their inhibition prevented tumor recurrence in our model.

Nonstructural protein NS1 immunodominant epitope detected specifically in dengue virus infected material by a SELDI-TOF/MS based assay.

Dengue virus (DV) infection is the most common mosquito-born viral disease of public health significance. Though most patients only suffer from flu-like symptoms, a small group of patients experiences more severe forms of the disease. The viral nonstructural protein 1 (NS1), a secreted protein correlating with viremia, is a key element used for dengue diagnosis with potential implications in severe dengue prognosis. Capture-ELISAs for the early detection of the NS1 protein in the sera during the acute febrile stage are commonly used in routine by diagnostic laboratories. In this study, the detection of NS1 protein in DV-infected material was assessed by an alternative method combining a single NS1-directed monoclonal antibody and the SELDI-TOF/MS technology. According to the epitope mapping, the antibodies used are mainly directed against an immuno-dominant peptide located on the C-terminal part of the protein. The NS1 SELDI-TOF assay is specific, has a sensitivity level close to capture-ELISAs and is potentially useful for a coupled serotyping/detection assay or for the detection of subtle post-translational modifications on the protein.

Crystal structure of human prostate-specific antigen in a sandwich antibody complex.

Human prostate-specific antigen (PSA or human kallikrein-related peptidase 3) present in small quantities in the sera of healthy men becomes elevated in prostate cancer (PCa) and other prostate disorders. The ability to identify the free PSA fraction associated with PCa could increase the reliability of the PSA diagnostic test. Here we present the crystal structure of human PSA from seminal fluid in a sandwich complex with two monoclonal antibodies (mAbs). MAb 5D5A5 captures total PSA with exceptionally high affinity, and mAb 5D3D11 selectively discriminates between free PSA subforms that are more abundant in sera from patients with PCa. Although the antigen is not of seric origin, several insights into cancer diagnosis can be discerned from this complex. MAb 5D3D11 recognizes a PSA conformation different from that previously reported. Interacting with the kallikrein loop, the PSA N-linked glycan attached to asparagine 61 is an uncommonly complex sialated triantennary chain. O-linked glycosylation is observed at threonine 125. The description of how PSA subforms in prostatic fluid can be discriminated using pairs of antibodies is a first step in the design of new strategies that are capable of real discrimination among PSA subforms, which will lead to the formulation of more reliable diagnostic tests. In a companion article [Muller, B. H., Savatier, A., L'Hostis, G., Costa, N., Bossus, M., Michel, S., et al. (2011). In vitro affinity maturation of an anti-PSA antibody for prostate cancer diagnostic assay. J. Mol. Biol.], we describe engineering efforts to improve the affinity of mAb 5D3D11, a first step towards such goal.

In vitro affinity maturation of an anti-PSA antibody for prostate cancer diagnostic assay.

Prostate-specific antigen (PSA) is a serum marker that is widely used for the diagnosis of prostatic diseases. Various subforms of free PSA, which are associated with prostate cancer differently, have been identified in sera. Thus, specific detection of certain subforms could permit discrimination between benign and malignant cases. Although the monoclonal antibody 5D3D11 displays the desired selectivity, its relative weak binding affinity prevents its development into an effective diagnostic tool. The directed-evolution strategy presented here succeeds in enhancing affinity and immunoassay sensitivity while maintaining selectivity. Starting without structural data, we constructed four independent phage-display single-chain variable fragment (scFv) libraries targeting hot spots from CDR-L1, H1, H2, and H3. Mutations derived from each library were combined, yielding further affinity gains. This constitutes the first demonstration of additivity for independently selected complementarity-determining region (CDR) hot-spot mutations. The X-ray structure of the Fab' 5D3D11-PSA complex (after it became available) inspired the design of two new libraries targeting CDR-L3 that resulted in other higher-affinity variants. Attempts at combining the new variants with previous ones did not result in further gains, suggesting that mutations from the two strategies provide alternative but noncomplementary solutions for affinity enhancement of 5D3D11. The results can be interpreted to provide a plausible explanation for the observed lack of additivity. Finally, with respect to the wild-type scFv, the best binders show an enhancement of sensitivity in sandwich immunoassay. Its ability to discriminate between prostate cancer sera and benign prostatic hyperplasia sera has now been confirmed through the dosage of 63 patients.

Crystal structure of a ternary complex between human prostate-specific antigen, its substrate acyl intermediate and an activating antibody.

Human prostate-specific antigen (PSA or KLK3) is an important marker for the diagnosis and management of prostate cancer. This is an androgen-regulated glycoprotein of the kallikrein-related protease family secreted by prostatic epithelial cells. Its physiological function is to cleave semenogelins in the seminal coagulum and its enzymatic activity is strongly modulated by zinc ions. Here we present the first crystal structure of human PSA in complex with monoclonal antibody (mAb) 8G8F5 that enhances its enzymatic activity. The mAb recognizes an epitope composed of five discontinuous segments including residues from the kallikrein loop and stabilizes PSA in an "open and active conformation" that accelerates catalysis. We also present the crystal structure of PSA in complex with both the mAb 8G8F5 and a fluorogenic substrate Mu-KGISSQY-AFC, derived from semenogelin I. By exploiting the inhibition of PSA by zinc ions, we were able to obtain a substrate acyl intermediate covalently linked to the catalytic serine, at pH 7.3 but not at pH 5.5. Moreover, the inhibition of PSA activity by zinc was found to be modulated by pH variations but not by the antibody binding. The correlation of the different data with the physiological conditions under which PSA can cleave semenogelins is discussed.

Characterization of prostate-specific antigen binding peptides selected by phage display technology.

Prostate-specific antigen (PSA) is an important marker for the diagnosis and management of prostate cancer. Free PSA has been shown to be more extensively cleaved in sera from benign prostatic hyperplasia patients than in sera from prostate cancer patients. Moreover, the presence of enzymatically activatable PSA was characterized previously in sera from patients with prostate cancer by the use of the specific anti-free PSA monoclonal antibody (mAb) 5D3D11. As an attempt to obtain ligands for the specific recognition of different PSA forms including active PSA, phage-displayed linear and cyclic peptide libraries were screened with PSA coated directly into microplate wells or presented by two different anti-total PSA mAbs. Four different phage clones were selected for their ability to recognize PSA and the inserted peptides were produced as synthetic peptides. These peptides were found to capture and to detect specifically free PSA, even in complex biological media such as sera or tumour cell culture supernatants. Alanine scanning of peptide sequences showed the involvement of aromatic and hydrophobic residues in the interaction of the peptides with PSA whereas Spotscan analysis of overlapping peptides covering the PSA sequence identified a peptide binding to the kallikrein loop at residues 82-87, suggesting that the peptides could recognize a non-clipped form of PSA. Moreover, the PSA-specific peptides enhance the enzymatic activity of PSA immobilized into microplate wells whereas the capture of PSA by the peptides inhibited totally its enzymatic activity while the peptide binding to PSA had no effect in solution. These PSA-specific peptides could be potential tools for the recognition of PSA forms more specifically associated to prostate cancer.

Usefulness of the phage display technology for the identification of a hepatitis C virus NS4A epitope recognized early in the course of the disease.

A dodecapeptide phage-displayed library was screened with the mouse monoclonal antibody (mAb) 2E3C2 which competed with human antibodies for the binding to the HCV c100 recombinant protein. Four mimotopes shared a consensus motif with the HCV 1701-1707 sequence corresponding to the carboxyl-terminal domain of the non-structural protein NS4A. However, these mimotopes reacted with 2E3C2 only, whereas the corresponding NS4 epitope defined at the sequence 1698-1709 and displayed on phage was recognized by both 2E3C2 and sera from HCV infected patients. Using the Spot method of multiple peptide synthesis and alanine replacement analysis, the respective reactivities of mAb 2E3C2 and anti-NS4A human antibodies against NS4 were shown to be directed against two slightly different overlapping minimal linear sequences and to involve different critical residues. The phage clone displaying the NS4 epitope was used to study the specific recognition of this epitope by different individual HCV positive sera as well as by two seroconversion panels of sera from HCV infected patients. Compared with the detection by RIBA of the different HCV antigens and c100 particularly, these results indicated that the antibodies directed against the NS4 (1698-1709) epitope were produced early during the course of the disease and decreased later.

Selective recognition of enzymatically active prostate-specific antigen (PSA) by anti-PSA monoclonal antibodies.

Prostate-specific antigen (PSA) is widely used as a serum marker for the diagnosis of prostate cancer. To evaluate two anti-free PSA monoclonal antibodies (mAbs) as potential tools in new generations of more relevant PSA assays, we report here their properties towards the recognition of specific forms of free PSA in seminal fluids, LNCaP supernatants, 'non-binding' PSA and sera from cancer patients. PSA from these different origins was immunopurified by the two anti-free PSA mAbs (5D3D11 and 6C8D8) as well as by an anti-total PSA mAb. The composition of the different immunopurified PSA fractions was analysed and their respective enzymatic activities were determined. In seminal fluid, enzymatically active PSA was equally purified with the three mAbs. In LNCaP supernatants and human sera, 5D3D11 immunopurified active PSA mainly, whereas 6C8D8 immunopurified PSA with residual activity. In sera of prostate cancer patients, we identified the presence of a mature inactive PSA form which can be activated into active PSA by use of high saline concentration or capture by an anti-total PSA mAb capable of enhancing PSA activity. According to PSA models built by comparative modelling with the crystal structure of horse prostate kallikrein described previously, we assume that active and activable PSA could correspond to mature intact PSA with open and closed conformations of the kallikrein loop. The specificity of 5D3D11 was restricted to both active and activable PSA, whereas 6C8D8 recognized all free PSA including intact PSA, proforms and internally cleaved PSA.

Characterization of mimotopes mimicking an immunodominant conformational epitope on the hepatitis C virus NS3 helicase.

The hepatitis C virus (HCV) nonstructural 3 (NS3) protein is composed of an amino terminal protease and a carboxyl terminal RNA helicase. NS3 contains major antigenic epitopes. The antibody response to NS3 appears early in the course of infection and is focused on the helicase region. However, this response cannot be defined by short synthetic peptides indicating the recognition of conformation-dependent epitopes. In this study, we have screened a dodecapeptide library displayed on phage with anti-NS3 mouse monoclonal antibodies (mAbs) that compete with each other and human anti-HCV NS3 positive sera. Two peptides (mimotopes) were selected that appeared to mimic an immunodominant epitope since they were recognized specifically by the different anti-NS3 mAbs of the study and by human sera from HCV infected patients. Homology search between the two mimotopes and the NS3 sequence showed that one of the two peptides shared amino acid similarities with NS3 at residues 1396-1398 on a very accessible loop as visualized on the three-dimensional structure of the helicase domain whereas the other one had two amino acids similar to nearby residues 1376 and 1378. Reproduced as synthetic dodecapeptides, the two mimotopes were recognized specifically by 19 and 22, respectively, out of 49 sera from HCV infected patients. These mimotopes allowed also the detection of anti-NS3 antibodies in sera of HCV patients at the seroconversion stage. These results suggest that the two NS3 mimotopes are potential tools for the diagnosis of HCV infection.

[Structural variations of the eye linked to daily vertical migrations of Daphnia longispina]. / Variations structurales de l'oeil, liées aux migrations verticales journalières de Daphnia longispina.

The role of the eyes, and more precisely that of the ommatidian pigments, in the control of daily rhythms of movement of Daphnia longispina is investigated. In the laboratory, under permanent light (LL), the pigmentary modifications observed are globally similar to those observed in situ, except in their timing, around dusk, LL cycles do not coincide with the ascent of daphnids but precede it. This is the expression of an endogenous free-running rhythm. Therefore, in D. longispina, an internal oscillator controls DVMs, which are circadian and not nycthemeral, and, in situ, illumination at dawn suffices to synchronize migrations on dark/light alternation.