Specificity and affinity of monoclonal antibodies against carcinoembryonic antigen.

The binding specificities of 52 well-characterized monoclonal antibodies (Mabs) against carcinoembryonic antigen (CEA) from 12 different research groups were studied by immunohistochemistry and immuno flow cytometry. In addition, the binding constant for the interaction between Mab and CEA was determined by a solution-phase assay. Cryostat sections of colon carcinoma and normal colon, stomach, liver, pancreas, and spleen were studied by immunohistochemistry. Peripheral blood granulocytes, monocytes, and lymphocytes were assayed by immuno flow cytometry. The Mabs used here have previously been classified into five essentially nonoverlapping epitope groups (GOLD 1-5) (Cancer Res., 49: 4852-4858, 1989). Most Mabs cross-reacted with different normal tissues, ranging from highly cross-reactive Mabs (positive reaction with 8 of 9 discriminating tissues) to relatively specific Mabs (positive reaction with 1 of 9 discriminating tissues). Five Mabs (10%) were specific, reacting only with colon carcinoma, normal colon mucosa, and normal gastric foveola. There was a correlation between epitope group and binding specificity. Mabs with a high degree of CEA specificity almost exclusively belonged to epitope groups 1, 2, and 3, while highly cross-reactive Mabs belonged to epitope groups 4 and 5. There was no correlation between antibody specificity and affinity for CEA. Specific Mabs with high as well as low affinity were found.

Differentiation of heroin and cocaine using dual-energy CT-an experimental study.

OBJECTIVE: To evaluate if heroin and cocaine can be distinguished using dual-energy CT. MATERIALS AND METHODS: Twenty samples of heroin and cocaine at different concentrations and standardized compression (SC) were scanned in dual-energy mode on a newest generation Dual Energy 64-row MDCT scanner. CT number, spectral graphs, and dual-energy index (DEI) were evaluated. Results were prospectively tested on six original samples from a body packer. Wilcoxon's test was used for statistical evaluation. RESULTS: Values are given as median and range. Under SC, the CT number of cocaine samples (-29.87 Hounsfield unit (HU) [-125.85; 16.16 HU]) was higher than the CT number of heroin samples (-184.37 HU [-199.81; -159.25 HU]; p < 0.01). Slope of spectral curves for cocaine was -2.36 HU/keV [-7.15; -0.67 HU/keV], and for heroin, 1.75 HU/keV [1.28; 2.5 HU/keV] (p < 0.01). DEI was 0.0352 [0.0081; 0.0528] for cocaine and significantly higher than for heroin samples (-0.0127 [-0.0097; -0.0159]; p < 0.001). While CT number was inconclusive, all six original packs were correctly classified after evaluation of the spectral curve and DEI. In contrast to the CT number, slope of the spectral curve and DEI were independent of concentration and compression. CONCLUSION: The slope of the spectral curve and the DEI from dual-energy CT data can be used to distinguish heroin and cocaine in vitro; these results are independent of compression and concentration in the measured range.

Swine monoclonal antibodies of high affinity and specificity to carcinoembryonic antigen.

To avoid the exclusive use of rodent monoclonal antibodies (MAbs) in patients for the detection of tumors by immunoscintigraphy and for radioimmunotherapy, swine MAbs were produced that are directed against carcinoembryonic antigen (CEA). Spleen cells from 2 pigs immunized with purified colon carcinoma CEA were fused with a nonsecreting mouse myeloma cell line by conventional methods, except that a particularly long immunization protocol and large amounts of spleen and myeloma cells were used. Of 1,200 growing hybrids tested, 20 were found initially to produce antibodies binding to radiolabeled CEA. Seven stable clones producing anti-CEA MAbs for more than 6 months were derived from these hybrids by repeated subcloning. The pig origin of the seven MAbs was demonstrated in a solid-phase CEA enzyme immunoassay where anti-pig immunoglobin (Ig) antibodies coupled to peroxidase gave a positive reaction while anti-mouse Ig antibodies were entirely negative. All swine MAbs were of the IgG isotype. Three anti-CEA MAbs showed no cross-reactivity with granulocytes, while four others gave various degrees of reactivity with different granulocyte glycoproteins. Competitive binding to CEA performed for two purified swine MAbs showed that they recognized two different epitopes. The affinity constants measured for these two MAbs by Scatchard plot on purified CEA were high (1.2 X 10(9) and 1.2 X 10(10) liter/mol). One of the MAbs was tested in vivo for tumor localization by injection, after radiolabeling, in nude mice bearing human colon carcinoma xenograft. High ratios of tumor to normal tissue were obtained with mean values of 10.5 for intact MAbs and of 26.8 for F(ab')2 fragments of the porcine MAb. The results showed that heterofusion with this particular protocol can be used to produce swine MAbs of high affinity and specificity for a well-defined tumor marker. These reagents may have an important clinical utility, particularly in patients who became sensitized to mouse immunoglobulins.

A dual functional origin of transfer in the ICEclc genomic island of Pseudomonas knackmussii B13.

Genomic islands (GEIs) are large DNA segments, present in most bacterial genomes, that are most likely acquired via horizontal gene transfer. Here, we study the self-transfer system of the integrative and conjugative element ICEclc of Pseudomonas knackmussii B13, which stands model for a larger group of ICE/GEI with syntenic core gene organization. Functional screening revealed that unlike conjugative plasmids and other ICEs ICEclc carries two separate origins of transfer, with different sequence context but containing a similar repeat motif. Conjugation experiments with GFP-labelled ICEclc variants showed that both oriTs are used for transfer and with indistinguishable efficiencies, but that having two oriTs results in an estimated fourfold increase of ICEclc transfer rates in a population compared with having a single oriT. A gene for a relaxase essential for ICEclc transfer was also identified, but in vivo strand exchange assays suggested that the relaxase processes both oriTs in a different manner. This unique dual origin of transfer system might have provided an evolutionary advantage for distribution of ICE, a hypothesis that is supported by the fact that both oriT regions are conserved in several GEIs related to ICEclc.

Biomechanical concept and clinical outcome of dual mobility cups.

Dual mobility cup systems in total hip arthroplasty consist of a metal back with a non-constrained liner, in which a constrained standard head articulates. While superior stability of such implants in comparison with standard total hip replacements is assumed, it is the purpose of this study to outline the biomechanical concept of dual mobility cups and to describe implant survival and dislocation rate based on the series published in the English-speaking and Francophone literature. A growing body of evidence indicates reduced dislocation rates in primary and revision total hip arthroplasty and in selected tumour cases. The limited availability of studies evaluating long-term implant survival and existing concerns with regard to increased wear rates and aseptic loosening, leads to the conclusion that such implants have to be used with prudence, particularly in standard primary hip arthroplasty and in young patients.

How can a dual oriT system contribute to efficient transfer of an integrative and conjugative element?

Integrative and conjugative elements (ICEs) are particularly interesting model systems for horizontal gene transfer, because they normally reside in an integrated state in the host chromosome but can excise and self-transfer under particular conditions, typically requiring exquisite regulatory cascades. Despite important advances in our understanding of the transfer mechanisms of a number of ICE, many essential details are lacking. Recently we reported that ICEclc, a 103 kb ICE of Pseudomonas knackmussii B13, has two active origins of transfer (oriTs), which is very much unlike conjugative plasmids that usually employ a single oriT. We discuss here how this dual oriT system could function and how it actually could have presented an evolutionary advantage for ICEclc distribution.

Phase-sensitive dual-inversion recovery for accelerated carotid vessel wall imaging.

OBJECTIVES: Dual-inversion recovery (DIR) is widely used for magnetic resonance vessel wall imaging. However, optimal contrast may be difficult to obtain and is subject to RR variability. Furthermore, DIR imaging is time-inefficient and multislice acquisitions may lead to prolonged scanning times. Therefore, an extension of phase-sensitive (PS) DIR is proposed for carotid vessel wall imaging. METHODS: The statistical distribution of the phase signal after DIR is probed to segment carotid lumens and suppress their residual blood signal. The proposed PS-DIR technique was characterized over a broad range of inversion times. Multislice imaging was then implemented by interleaving the acquisition of 3 slices after DIR. Quantitative evaluation was then performed in healthy adult subjects and compared with conventional DIR imaging. RESULTS: Single-slice PS-DIR provided effective blood-signal suppression over a wide range of inversion times, enhancing wall-lumen contrast and vessel wall conspicuity for carotid arteries. Multislice PS-DIR imaging with effective blood-signal suppression is enabled. CONCLUSIONS: A variant of the PS-DIR method has successfully been implemented and tested for carotid vessel wall imaging. This technique removes timing constraints related to inversion recovery, enhances wall-lumen contrast, and enables a 3-fold increase in volumetric coverage at no extra cost in scanning time.

Thrombin-sensitive dual fluorescence imaging and therapeutic agent for detection and treatment of synovial inflammation in murine rheumatoid arthritis.

We have developed a thrombin-sensitive polymeric photosensitizer prodrug (T-PS) to selectively image and eradicate inflammatory lesions in rheumatoid arthritis (RA). Thrombin is a serine protease up-regulated in synovial tissues of rheumatoid arthritis (RA) patients. T-PS consists of a polymeric backbone, to which multiple photosensitizer (PS) units are tethered via short thrombin-cleavable peptide linkers. Fluorescence emission and phototoxicity of the prodrug are efficiently quenched due to the interaction of neighboring photosensitizer units. The prodrug is passively delivered to the inflammation site via the enhanced permeability and retention (EPR) effect. Subsequent site-selective proteolytic cleavage of the peptide linkers restores its photoactivity by increasing the mutual distance between PS. Whole animal imaging in murine collagen-induced arthritis, an experimental model of RA revealed a dose-dependent fluorescence increase in arthritic paws after systemic prodrug injection. In addition, administration of T-PS resulted in much higher fluorescence selectivity for arthritic joints as compared to the free PS. Irradiation of the arthritic joints induced light dose dependent phototoxic effects such as apoptosis, vascular damage and local hemorrhage. Long-term observations showed complete regression of the latter. Irradiated non-arthritic tissues or non-irradiated arthritic tissues showed no histological effects after photodynamic therapy with T-PS. This illustrates that T-PS can localize inflammatory lesions with excellent selectivity and induce apoptosis and vascular shut down after irradiation.

Prediction of Recanalization Trumps Prediction of Tissue Fate: The Penumbra: A Dual-edged Sword.

BACKGROUND AND PURPOSE: To determine whether infarct core or penumbra is the more significant predictor of outcome in acute ischemic stroke, and whether the results are affected by the statistical method used. METHODS: Clinical and imaging data were collected in 165 patients with acute ischemic stroke. We reviewed the noncontrast head computed tomography (CT) to determine the Alberta Score Program Early CT score and assess for hyperdense middle cerebral artery. We reviewed CT-angiogram for site of occlusion and collateral flow score. From perfusion-CT, we calculated the volumes of infarct core and ischemic penumbra. Recanalization status was assessed on early follow-up imaging. Clinical data included age, several time points, National Institutes of Health Stroke Scale at admission, treatment type, and modified Rankin score at 90 days. Two multivariate regression analyses were conducted to determine which variables predicted outcome best. In the first analysis, we did not include recanalization status among the potential predicting variables. In the second, we included recanalization status and its interaction between perfusion-CT variables. RESULTS: Among the 165 study patients, 76 had a good outcome (modified Rankin score ≤2) and 89 had a poor outcome (modified Rankin score >2). In our first analysis, the most important predictors were age (P<0.001) and National Institutes of Health Stroke Scale at admission (P=0.001). The imaging variables were not important predictors of outcome (P>0.05). In the second analysis, when the recanalization status and its interaction with perfusion-CT variables were included, recanalization status and perfusion-CT penumbra volume became the significant predictors (P<0.001). CONCLUSIONS: Imaging prediction of tissue fate, more specifically imaging of the ischemic penumbra, matters only if recanalization can also be predicted.

On the dual contrast enhancement mechanism in frequency-selective inversion-recovery magnetic resonance angiography (IRON-MRA)

The susceptibility of blood changes after administration of a paramagnetic contrast agent that shortens T(1). Concomitantly, the resonance frequency of the blood vessels shifts in a geometry-dependent way. This frequency change may be exploited for incremental contrast generation by applying a frequency-selective saturation prepulse prior to the imaging sequence. The dual origin of vascular enhancement depending first on off-resonance and second on T(1) lowering was investigated in vitro, together with the geometry dependence of the signal at 3T. First results obtained in an in vivo rabbit model are presented.

Expression of dual angiogenic/neurogenic growth factors in human primary brain tumors.

Brain tumors, benign or malignant, are characterized by a very high degree of vascularization. Recent accumulating evidence suggests that during development the neuronal wiring follows the same routes as the vasculature and that these two systems may share some of the same factors for guidance. Thus, expression of dual angiogenic/neurogenic growth factors was evaluated by in situ hybridization in human primary brain tumors of three different types, i.e., astrocytomas, oligodendrogliomas, and ependymomas, of increasing grades, in relation with the grade and type of the tumor. For this evaluation we selected vascular endothelial growth factor (VEGF-A) and its receptors VEGF-R1 and VEGF-R2 and the neuropilins 1 and 2 (NRP-1 and NRP-2), which have proangiogenic properties, platelet-derived growth factor (PDGF) receptor-beta (PDGF-Rβ), which is required for the functional maturation of blood vessels, the ephrins and their Eph receptors, angiotensinogen (AGT) and thrombospondin-2 (TSP-2), which have potential antiangiogenic properties, and netrin-1 (Net-1), which regulates vascular architecture. We show that the expression of the VEGF-NRP system, PDGF-Rβ, TSP-2, AGT, and Net-1 are differentially regulated, either increased or decreased, in relation with the type and grade of the tumor, whereas regulation of the ephrinB system does not seem to be relevant in these human brain tumors.

Novel action of a wake-promoting neuropeptide in hippocampus : inhibition of nmda receptor function at excitatory synapses through orexin-a

One of the most intriguing functions of the brain is the ability to learn and memorize. The mechanism through which memory and learning are expressed requires the activation of NMDA receptors (NMDARs). These molecular entities are placed at the postsynaptic density of excitatory synapses and their function is tightly controlled by the actions of several modulators at the extracellular, intracellular and pore sites. A large part of the intracellular modulation comes from the action of G-protein coupled receptors (GPCRs). Through intracellular cascades typically involving kinases and phosphatases, GPCRs potentiate or inhibit NMDARs, controlling the conductive state but also the trafficking within the synapse. The GPCRs are involved in the modulation of a variety of brain functions. Many of them control cognition, memory and learning performance, therefore, their effects on NMDARs are extensively studied. The orexinergic system signals through GPCRs and it is well known for the regulation of waking, feeding, reward and autonomic functions. Moreover, it is involved in potentiating hippocampus-related cognitive tasks. Orexin receptors and fibers are present within the hippocampus, but whether these directly modulate hippocampal cells and synapses has not yet been determined. During my thesis, I studied orexinergic actions on excitatory synaptic transmission via whole-cell patch-clamp recordings in rat acute hippocampal slices. I observed that exogenously applied orexin-A (ox-A) exerted a strong inhibitory action on NMDAR-mediated synaptic potentials at mossy fiber (MF)-CA3 synapses, by postsynaptically activating orexin-2 receptors, a minor inhibition at Schaffer collateral-CAl synapses and did not affect other synapses with the CA3 area. Moreover, I demonstrated that the susceptibility of NMDARs to ox- A depends on the tone of endogenous orexin known to fluctuate during the day-night cycle. In fact, in slices prepared during the active period of the rats, when endogenous orexin levels are high, NMDAR-currents were not affected by exogenously applied ox-A. The inhibitory effect of ox-A was, however, reverted when interfering with the orexinergic system through intraperitoneal injections of almorexant, a dual orexin receptor antagonist, during the active phase prior to slice preparation. This thesis work suggests that the orexinergic system regulates NMDAR-dependent information flow through select hippocampal pathways depending on the time-of-day. The specific orexinergic modulation of NMDARs at MFs dampens the excitability of the hippocampal circuit and could impede the mechanisms related to memory formation, possibly also following extended periods of waking. -- La capacité d'apprentissage et de mémorisation est une des fonctions les plus intrigantes de notre cerveau. Il a été montré qu'elles requièrent l'activation des récepteurs NMDA (NMDARs). Ces entités moléculaires sont présentes au niveau de la densité post-synaptique des synapses excitatrices et leur fonction est étroitement contrôlée par l'action de nombreux modulateurs au niveau extracellulaire, intracellulaire et membranaire de ces récepteurs. Une grande partie de la modulation intracellulaire s'effectue via l'action de récepteurs couplés aux protéines G (GPCRs). Grace à leurs cascades intracellulaires typiquement impliquant des kinases et des phosphatases, les GPCRs favorisent l'activation ou l'inhibition des NMDARs, contrôlant ainsi leur perméabilité mais aussi leur mouvement à la synapse. Les GPCRs sont impliquées dans de nombreuses fonctions cérébrales telles que la cognition, la mémoire ainsi que la capacité d'apprentissage c'est pour cela que leurs effets sur les NMDARs sont très étudiés. Le système orexinergique fait intervenir ces GPCRs et est connu par son rôle dans la régulation de fonctions physiologiques telles que l'éveil, la prise alimentaire, la récompense ainsi que d'autres fonctions du système nerveux autonome. De plus, ce système est impliqué dans la régulation de tâches cognitives liées à l'hippocampe. Bien que les fibres et les récepteurs à l'orexine soient présents dans l'hippocampe, leur mécanisme d'action sur les cellules et les synapses de l'hippocampe n'a pas encore été élucidé. Durant ma thèse, je me suis intéressée aux effets de l'orexine sur la transmission synaptique excitatrice en utilisant la méthode d'enregistrement en patch-clamp en configuration cellule entière sur des tranches aiguës d'hippocampes de rats. J'ai observé que l'application exogène d'orexine A d'une part inhibe fortement les courants synaptiques dépendants de l'activation des NMDARs au niveau de la synapse entre les fibres moussues et CA3 via l'activation post-synaptique des orexine récepteurs 2 mais d'autre part n'inhibe que de façon mineure la synapse entre les collatérales de Schaffer et CAI et n'affecte pas les autres synapses impliquant CA3. J'ai également démontré que la sensibilité des NMDARs à l'orexine A dépend de sa concentration endogène qui fluctue durant le cycle éveil-sommeil. En effet, lorsque les coupes d'hippocampes sont préparées durant la période active de l'animal correspondant à un niveau endogène d'orexine élevé, l'application exogène d'orexine A n'a aucun effet sur les courants dépendants de l'activation des NMDARs. Cependant, l'injection dans le péritoine, durant la phase active de l'animal, d'un antagoniste des orexine récepteurs, l'almorexant, va supprimer l'effet inhibiteur de l'orexine A. Les résultats de ma thèse suggèrent donc que le système orexinergique module les informations véhiculées par les NMDARs via des voies de signalisation sélectives de l'hippocampe en fonction du moment de la journée. La modulation orexinergique des NMDARs au niveau des fibres moussues diminue ainsi l'excitabilité du circuit hippocampal et pourrait entraver les mécanismes liés à la formation de la mémoire, potentiellement après de longues périodes d'éveil.

The activity of the anti-apoptotic fragment generated by the caspase-3/p120 RasGAP stress-sensing module displays strict Akt isoform specificity.

The caspase-3/p120 RasGAP module acts as a stress sensor that promotes pro-survival or pro-death signaling depending on the intensity and the duration of the stressful stimuli. Partial cleavage of p120 RasGAP generates a fragment, called fragment N, which protects stressed cells by activating Akt signaling. Akt family members regulate many cellular processes including proliferation, inhibition of apoptosis and metabolism. These cellular processes are regulated by three distinct Akt isoforms: Akt1, Akt2 and Akt3. However, which of these isoforms are required for fragment N mediated protection have not been defined. In this study, we investigated the individual contribution of each isoform in fragment N-mediated cell protection against Fas ligand induced cell death. To this end, DLD1 and HCT116 isogenic cell lines lacking specific Akt isoforms were used. It was found that fragment N could activate Akt1 and Akt2 but that only the former could mediate the protective activity of the RasGAP-derived fragment. Even overexpression of Akt2 or Akt3 could not rescue the inability of fragment N to protect cells lacking Akt1. These results demonstrate a strict Akt isoform requirement for the anti-apoptotic activity of fragment N.