Histone variant H2A.J accumulates in senescent cells and promotes inflammatory gene expression.

The senescence of mammalian cells is characterized by a proliferative arrest in response to stress and the expression of an inflammatory phenotype. Here we show that histone H2A.J, a poorly studied H2A variant found only in mammals, accumulates in human fibroblasts in senescence with persistent DNA damage. H2A.J also accumulates in mice with aging in a tissue-specific manner and in human skin. Knock-down of H2A.J inhibits the expression of inflammatory genes that contribute to the senescent-associated secretory phenotype (SASP), and over expression of H2A.J increases the expression of some of these genes in proliferating cells. H2A.J accumulation may thus promote the signalling of senescent cells to the immune system, and it may contribute to chronic inflammation and the development of aging-associated diseases.

Recommended Immunological Strategies to Screen for Botulinum Neurotoxin-Containing Samples.

Botulinum neurotoxins (BoNTs) cause the life-threatening neurological illness botulism in humans and animals and are divided into seven serotypes (BoNT/A-G), of which serotypes A, B, E, and F cause the disease in humans. BoNTs are classified as "category A" bioterrorism threat agents and are relevant in the context of the Biological Weapons Convention. An international proficiency test (PT) was conducted to evaluate detection, quantification and discrimination capabilities of 23 expert laboratories from the health, food and security areas. Here we describe three immunological strategies that proved to be successful for the detection and quantification of BoNT/A, B, and E considering the restricted sample volume (1 mL) distributed. To analyze the samples qualitatively and quantitatively, the first strategy was based on sensitive immunoenzymatic and immunochromatographic assays for fast qualitative and quantitative analyses. In the second approach, a bead-based suspension array was used for screening followed by conventional ELISA for quantification. In the third approach, an ELISA plate format assay was used for serotype specific immunodetection of BoNT-cleaved substrates, detecting the activity of the light chain, rather than the toxin protein. The results provide guidance for further steps in quality assurance and highlight problems to address in the future.

Mediator independently orchestrates multiple steps of preinitiation complex assembly in vivo.

Mediator is a large multiprotein complex conserved in all eukaryotes, which has a crucial coregulator function in transcription by RNA polymerase II (Pol II). However, the molecular mechanisms of its action in vivo remain to be understood. Med17 is an essential and central component of the Mediator head module. In this work, we utilised our large collection of conditional temperature-sensitive med17 mutants to investigate Mediator's role in coordinating preinitiation complex (PIC) formation in vivo at the genome level after a transfer to a non-permissive temperature for 45 minutes. The effect of a yeast mutation proposed to be equivalent to the human Med17-L371P responsible for infantile cerebral atrophy was also analyzed. The ChIP-seq results demonstrate that med17 mutations differentially affected the global presence of several PIC components including Mediator, TBP, TFIIH modules and Pol II. Our data show that Mediator stabilizes TFIIK kinase and TFIIH core modules independently, suggesting that the recruitment or the stability of TFIIH modules is regulated independently on yeast genome. We demonstrate that Mediator selectively contributes to TBP recruitment or stabilization to chromatin. This study provides an extensive genome-wide view of Mediator's role in PIC formation, suggesting that Mediator coordinates multiple steps of a PIC assembly pathway.

Development of sensitive direct chemiluminescent enzyme immunoassay for the determination of dihydroartemisinin in plasma.

Despite significant progress in prevention and therapy, malaria is still one of the world's leading major diseases due to its high morbidity and mortality. Recommended treatments by the World Health Organization include the use of artemisinin and artemisinin derivative-based combination therapies. To allow efficient patient monitoring during antimalarial therapy without the use of expensive apparatus, we developed a sensitive direct chemiluminescent enzyme immunoassay for the determination of dihydroartemisinin in biological fluids. To produce specific antibodies against dihydroartemisinin (DHA), a synthetic DHA derivative was coupled to bovine serum albumin as the immunogen. In parallel, a new, rapid, and efficient procedure to covalently link glycoprotein to all amine-containing molecules has been established and the enzyme tracer was prepared by chemically coupling the DHA derivative in combination with SBP rather than the more commonly used HRP. It allowed us to develop, after optimization of the luminescent reagent, a sensitive and stable luminescent EIA, with a LLOQ of 90 pg mL(-1). This assay compares favorably with the most efficient HPLC methods previously reported with a LLOQ close to 1 ng mL(-1) and shows good precision and efficiency since recovery from human plasma spiked with DHA ranged between 91 and 103%, with coefficients of variation of <13%. To date, no immunoassay for DHA has been applied to plasma analysis and this EIA should be very useful in all clinical laboratories for rapid and cost-effective analysis.

Phage amplification and immunomagnetic separation combined with targeted mass spectrometry for sensitive detection of viable bacteria in complex food matrices.

We have developed and describe here for the first time a highly sensitive method for the fast and unambiguous detection of viable Escherichia coli in food matrices. The new approach is based on using label-free phages (T4), obligate parasites of bacteria, which are attractive for pathogen detection because of their inherent natural specificity and ease of use. A specific immunomagnetic separation was used to capture the progeny phages produced. Subsequently, T4 phage markers were detected by liquid chromatography coupled to targeted mass spectrometry. Combining the specificity of these three methodologies is of great interest in developing an alternative to conventional time-consuming culture-based technologies for the detection of viable bacteria for industrial applications. First, optimization experiments with phage T4 spiked in complex matrices (without a phage amplification event) were performed and demonstrated specific, sensitive, and reproducible phage capture and detection in complex matrices including Luria-Bertani broth, orange juice, and skimmed milk. The method developed was then applied to the detection of E. coli spiked in foodstuffs (with a phage amplification event). After having evaluated the impact of infection duration on assay sensitivity, we showed that our assay specifically detects viable E. coli in milk at an initial count of ≥1 colony-forming unit (cfu)/mL after an 8-h infection. This excellent detection limit makes our new approach an alternative to PCR-based assays for rapid bacterial detection.

Polyclonal antibodies: a cheap and efficient tool for screening of enantioselective catalysts.

Enantioselective polyclonal antibodies have been produced and characterized to develop a high-throughput screening method for lipase activity fingerprinting, with a view to the enantioselective hydrolysis of azlactones.

Immunological and metabolomic impacts of administration of Cry1Ab protein and MON 810 maize in mouse.

We have investigated the immunological and metabolomic impacts of Cry1Ab administration to mice, either as a purified protein or as the Cry1Ab-expressing genetically modified (GM) MON810 maize. Humoral and cellular specific immune responses induced in BALB/cJ mice after intra-gastric (i.g.) or intra-peritoneal (i.p.) administration of purified Cry1Ab were analyzed and compared with those induced by proteins of various immunogenic and allergic potencies. Possible unintended effects of the genetic modification on the pattern of expression of maize natural allergens were studied using IgE-immunoblot and sera from maize-allergic patients. Mice were experimentally sensitized (i.g. or i.p. route) with protein extracts from GM or non-GM maize, and then anti-maize proteins and anti-Cry1Ab-induced immune responses were analyzed. In parallel, longitudinal metabolomic studies were performed on the urine of mice treated via the i.g. route. Weak immune responses were observed after i.g. administration of the different proteins. Using the i.p. route, a clear Th2 response was observed with the known allergenic proteins, whereas a mixed Th1/Th2 immune response was observed with immunogenic protein not known to be allergenic and with Cry1Ab. This then reflects protein immunogenicity in the BALB/c Th2-biased mouse strain rather than allergenicity. No difference in natural maize allergen profiles was evidenced between MON810 and its non-GM comparator. Immune responses against maize proteins were quantitatively equivalent in mice treated with MON810 vs the non-GM counterpart and no anti-Cry1Ab-specific immune response was detected in mice that received MON810. Metabolomic studies showed a slight "cultivar" effect, which represented less than 1% of the initial metabolic information. Our results confirm the immunogenicity of purified Cry1Ab without evidence of allergenic potential. Immunological and metabolomic studies revealed slight differences in mouse metabolic profiles after i.g. administration of MON810 vs its non-GM counterpart, but no significant unintended effect of the genetic modification on immune responses was seen.

Toward the limits of sandwich immunoassay of very low molecular weight molecules.

A model study aiming at exploring the limits of sandwich immunoassays of very small molecules is described. Combinatorial association of antibody couples to detect small molecules constituted by two small epitopes connected via different linear spacers was used to investigate the minimum size of compounds susceptible to be simultaneously bound by two distinct antibodies. The results clearly indicated that despite the fact that below 10 carbon atoms unfavorable interactions between antibodies took place, molecules bearing two epitopes separated by only 5 carbon atoms might be directly detected by sandwich immunoassays.

In vitro digestion of Cry1Ab proteins and analysis of the impact on their immunoreactivity.

A pepsin resistance test performed at pH 1.2 and with high pepsin to protein ratio is one of the steps of the weight-of-evidence approach used for assessment of allergenicity of new proteins. However, the use of other in vitro digestibility tests, performed in more physiologically relevant conditions and in combination with immunological assays so as to increase the value of the information gained from the studies of stability of a novel protein to digestion for the overall allergenicity assessment, has been proposed. This study then aimed to investigate the stability to digestion of Cry1Ab protoxin and toxin, insecticidal proteins expressed in genetically modified crops, using simulated gastric fluid (SGF) at different pH values and pepsin-to-substrate ratios, in the presence or absence of physiological surfactant phosphatidylcholine (PC). Electrophoresis and immunoblot patterns and residual immunoreactivity of digesta were analyzed. Although Cry1Ab protoxin is extensively degraded at pH 1.2 with high pepsin-to-protein ratio, it is only slightly degraded at pH 2.0 and conserved its immunoreactivity. Furthermore, Cry1Ab proteins were demonstrated to be stable in a more physiologically relevant in vitro digestibility test (pH 2.5, pepsin-to-substrate ratio 1:20 (w/w) with PC). Factors such as pH, SGF composition, and pepsin-to-substrate ratio then greatly influence the digestion of Cry1Ab proteins, confirming that new and more physiologically relevant in vitro digestibility tests should be also considered to study the relationship between the resistance of a protein to digestion and its allergenicity.

Design, synthesis and studies of triphosphate analogues for the production of anti AZT-TP antibodies.

Triphosphates anabolites are the active chemical species of nucleosidic reverse transcriptase inhibitors in HIV-therapy. Herein, we describe (i) the design of stable triphosphate analogues of AZT using molecular modelling, (ii) their synthesis and (iii) their use for producing anti AZT-TP antibodies in the aim of developing an immunoassay for therapeutic drug monitoring.

Sensitive and specific enzyme immunoassays for antigenic trisaccharide from Bacillus anthracis spores.

A straightforward synthesis of an anthrose-containing trisaccharide derived from Bacillus anthracis was achieved. Antibodies raised against this hapten provide a highly sensitive enzyme immunoassay with a detection limit of 8.5 pmol mL(-1). By investigating the specificity of the antibodies obtained using different mono-, di- and trisaccharide synthetic analogues, we demonstrated that the epitope was mainly made up of the methyl group at C-5, the butamido group at C-4 and the hydroxyl at C-3 of the anthrose unit, the other parts of the trisaccharide appearing little involved in the recognition.

Comparison of ABC transporter modulation by atazanavir in lymphocytes and human brain endothelial cells: ABC transporters are involved in the atazanavir-limited passage across an in vitro human model of the blood-brain barrier.

Efflux pumps, P-glycoprotein (P-gp), multidrug resistance-associated proteins (MRPs), and breast cancer resistance protein (BCRP) have been shown to extrude HIV protease inhibitors from cells. These transporters are present on many barrier sites such as the blood-brain barrier (BBB) and on many circulating cells such as lymphocytes, and could reduce protease inhibitor concentration in sanctuary or HIV-1 target sites. This study compares the potential of the antiretroviral drug atazanavir to modulate P-gp and MRP expression and function in total lymphocytes and in human fetal brain endothelial cells (HBMECs). We address the question of atazanavir transport across the human BBB. Following incubation with atazanavir, P-gp and MRP1 expression was determined by direct immunofluorescence. Transporter function was assessed by measuring fluorescent dye efflux, either with or without specific inhibitors. Atazanavir substrate properties were determined by transport quantification through a validated in vitro human BBB model. Our results show that in contrast to HBMECs, in lymphocytes, atazanavir has no effect on MRP1 and P-gp expression. However, there were overall changes in P-gp function increasing its activity in lymphocytes and HBMECs. Using the in vitro human BBB model, we confirm the interaction of atazanavir with P-gp, MRPs, and BCRP in preventing its passage across this barrier and thus its entry into the central nervous system.

Synthesis of an anthrose derivative and production of polyclonal antibodies for the detection of anthrax spores.

A straightforward synthesis of a derivative of anthrose, the non-reducing terminal fragment of the antigenic tetrasaccharide from Bacillus anthracis, was achieved starting from d-galactose. This hapten is able to induce a highly specific and sensitive immune response in rabbit when attached to a carrier protein.

A sensitive sandwich enzyme immunoassay for free or complexed Clostridium botulinum neurotoxin type A.

Fourteen monoclonal antibodies (mAbs) against Clostridium botulinum type A neurotoxin were raised in mice using a carboxy terminal fragment of the toxin for immunization. A two-site immunometric assay was developed which allowed detection of 8 LD(50)/ml (40 pg/ml) botulinum neurotoxin A and an accurate quantification close to 25 LD(50)/ml (150 pg/ml). No cross-reactivity was observed with other toxinotypes. During the development of this assay, interference induced by associated protein was observed. By comparing the effect of different buffers, a buffer composed with Tris-HCl and NaCl salts was demonstrated to dissociate protein complexed with the neurotoxin A. Applied to the measurement of the toxin in different matrices, this dissociating buffer ensures the correct quantification.

Development of a competitive immunoassay for efavirenz: hapten design and validation studies.

The reverse transcriptase inhibitor efavirenz (EFV) is widely used in human immunodeficiency virus (HIV) therapy. Knowledge of the plasma and intracellular concentrations of the drug is of prime importance to get further insight into EFV action in vivo and would be useful for therapeutic drug monitoring (TDM). The aim of this study was to develop a sensitive and specific competitive enzyme immunoassay (EIA) for EFV in biological fluids. Two haptens that differed by the position of the linker were synthesized using two different ways and coupled to BSA. Anti-EFV polyclonal antibodies (pAb) were raised in rabbits using the corresponding immunogens. By comparing results obtained with EIA study with those observed with high-performance liquid chromatography (HPLC) we have shown that the position of the linker appears to be crucial for the specificity of the pAb. EIA was then developed in microtitration plates using the most specific pAb. The assay was performed on a minimum of 30 microL of plasma. It showed good precision and efficiency as well as good cross-validation with HPLC. The lowest limit of quantification (LLOQ) was 150 pg mL(-1), i.e., a value at least 10 times lower than those currently achieved using previously described techniques. This EIA should be useful in the clinical laboratory for monitoring patients during antiretroviral therapy especially young children as well as for measuring EFV in intracellular studies requiring lower amounts of biological material.

Quantitative immunoassay to measure plasma and intracellular atazanavir levels: analysis of drug accumulation in cultured T cells.

We have developed an enzyme immunoassay to measure atazanavir (ATV) levels in plasma and cells. Anti-ATV polyclonal antibodies were raised in rabbits by using a synthetic ATV derivative coupled to bovine serum albumin as the immunogen, and the enzyme tracer was prepared by chemically coupling the ATV derivative with acetylcholinesterase. These reagents were used to develop a sensitive competitive enzyme immunoassay performed in microtitration plates, and the lowest limit of quantification was 150 pg/ml, which is about 10 times more sensitive than previously published techniques. The plasma assay was performed, after a simple methanol extraction, with a minimum of 30 microl of plasma. This assay showed good precision and efficiency, since the rates of recovery from human plasma and cell extracts spiked with ATV ranged form 93 to 113%, with coefficients of variation of less than 10%. ATV concentrations were measured in peripheral blood mononuclear cells incubated with various ATV concentrations and in CEM cells in the absence or presence of antiretroviral drugs and drug transporter inhibitors. The results indicated a dose-dependent uptake (intracellular concentration/extracellular concentration ratio range, 0.04 to 19). A significant increase in the accumulation of ATV was noticed in the presence of P-glycoprotein and MRP1 inhibitors (dipyridamole, inter alia). Interestingly, efavirenz significantly increased the baseline accumulation of ATV, whereas nevirapine induced a marked reduction. This new enzyme immunoassay for measuring plasma and intracellular ATV levels was fully validated and provides an inexpensive and useful tool for routine therapeutic drug monitoring. Moreover, in vitro results suggested the implication of drug transporters and interactions with other antiviral drugs that should be further explored in human immunodeficiency virus-infected patients.

Sensitive enzyme immunoassay for measuring plasma and intracellular nevirapine levels in human immunodeficiency virus-infected patients.

We have developed an enzyme immunoassay to measure nevirapine (NVP) in plasma and peripheral blood mononuclear cells. Anti-NVP polyclonal antibodies were raised in rabbits by using a synthetic NVP derivative coupled to keyhole limpet hemocyanin as the immunogen, and the enzyme tracer was prepared by chemically coupling the NVP derivative with acetylcholinesterase. These reagents were used to develop a sensitive competitive enzyme immunoassay performed in microtitration plates with a 100-pg ml(-1) limit of detection and thus approximately 100 times more sensitive than previously published techniques. The plasma assay was performed directly without extraction (in this case, a 500-pg ml(-1) limit of detection was observed) on a minimum of 30 micro l of plasma. This assay shows good precision and efficiency, since recovery from human plasma and cell extracts spiked with NVP ranged between 87 and 104%, with coefficients of variation of <10%. A pharmacokinetic analysis of plasma NVP was performed for seven patients infected with human immunodeficiency virus (HIV), and it gave results similar to published findings. Intracellular concentrations of NVP were measured in cultured human T-lymphoblastoid cells and peripheral blood mononuclear cells from HIV-infected patients. The results indicated a very low intracellular/extracellular concentration ratio (0.134), thus demonstrating the absence of intracellular drug accumulation. This is the first intracellular assay of a nonnucleoside reverse-transcriptase inhibitor, and this method could be useful in monitoring plasma and intracellular NVP levels in HIV-infected patients.

An enzyme immunoassay for the quantification of plasma and intracellular lopinavir in HIV-infected patients.

The protease inhibitor lopinavir (LPV; [1S-[1R*(R*), 3R*,4R*]]-N-[4-[[(2,6-dimethylphenoxy)-acetyl]amino]-3-hydroxy-5-phenyl-1-(phenylmethyl) pentyl] tetrahydro-alpha-(1-methylethyl)-2-oxo-1(2H)-pyrimide acetamide) is widely used in anti-human immunodeficiency virus (HIV) therapy. Knowledge of the plasma and intracellular concentrations of the drug would be useful for a better understanding of LPV action and for therapeutic monitoring. The aim of this study was to develop a sensitive and specific immunoassay for LPV in plasma and cells. Anti-LPV polyclonal antibodies were raised in rabbits using a synthetic LPV derivative coupled to keyhole limpet hemocyanin (KLH) as immunogen. The enzyme tracer was prepared by chemically coupling the LPV derivative with acetylcholinesterase. These reagents were used to develop a competitive enzyme immunoassay (EIA) performed in microtitration plates. The assay was performed on a minimum of 50 microl of plasma or 2 x 10(6) cells. It showed good precision and efficiency in as much as recovery from human plasma and cell extracts spiked with LPV ranged between 87% and 104%, with coefficients of variation of less than 10%. The limit of detection (LOD) was 100 pg/ml, i.e., a value at least 10 times lower than those currently achieved using previously described techniques. Cross-validation with high-performance liquid chromatography (HPLC) revealed a good correlation between methods (r2=0.96). Intracellular concentrations of LPV were measured in cultured human T lymphoblastoid cells (CEM). A pharmacokinetic analysis of plasma and intracellular LPV was performed on a healthy volunteer, and measurements were done in patients infected with HIV. The results obtained indicated a high intracellular/extracellular concentration ratio in cultured cells (19.3) but not in cells from HIV patients (1.3). In contrast, in peripheral blood mononuclear cells (PBMC) the accumulation of ritonavir (RTV) was six times higher than LPV. To date, this is the first reported immunoassay for LPV, and this method is sensitive enough for monitoring plasma and intracellular LPV levels in HIV-infected patients and for intracellular studies.