Dimeric recombinant IgA directed against carcino-embryonic antigen, a novel tool for carcinoma localization.

Carcinoembryonic antigen (CEA) has been shown to be one of the best markers for in vivo tumor targeting of radiolabeled antibodies, despite the fact that it is localized predominantly at the apical side of human colon carcinoma cells within the fairly closed pseudolumen structures formed by these tumors. Due to this particular histological localization, a large proportion of the CEA molecules may remain inaccessible to the intravenously injected radiolabeled anti-CEA antibodies of IgG isotype, which are widely used in the clinic. In order to improve targeting, we made a recombinant dimeric IgA, which should have the capacity to translocate from the basolateral to the apical side of the pseudolumen formed by colon carcinoma cells after binding to the polyIg receptor (pIgR). A genomic chimeric mouse-human IgA2 construct was made using one of our most specific anti-CEA hybridomas, CE-25. The chimeric IgA (chIgA) was expressed in the Sp2/0 myeloma cell line. The secreted recombinant antibody was found to consist mostly of a dimeric form of IgA with a molecular weight of about 350 kDa. The dimeric chIgA was shown to translocate efficiently in vitro across a monolayer of epithelial cells expressing the pIgR and to retain full CEA binding activity.

Radiolabeled fragments of monoclonal antibodies against carcinoembryonic antigen for localization of human colon carcinoma grafted into nude mice.

Four monoclonal antibodies against carcinoembryonic antigen (CEA) have been selected from 32 hybrids that produce antibodies against this antigen, by the criteria of high affinity for CEA and low cross-reactivity with granulocyte glycoprotein(s). The specificity of tumor localization in vivo of the four MAb, and their F(ab')2 and Fab fragments was compared in nude mice bearing grafts of a serially transplanted, CEA-producing, human colon carcinoma. The distribution of radiolabeled MAb and their fragments after intravenous injection was analyzed by direct measurement of radioactivity in tumor and normal organs, as well as by whole-body scanning and by autoradiography of tumor sections. Paired labeling experiments, in which 131I-labeled antibody or fragments and 125I-labeled control IgG are injected simultaneously, were undertaken to determine the relative tumor uptakes of each labeled protein. The tumor antibody uptake divided by that of control IgG defines the specificity index of localization. Tumor antibody uptakes (as compared with the whole mouse), ranging between 7 and 15, and specificity indices ranging between 3.4 and 6.8, were obtained with the four intact MAb at day 4-5 after injection. With F(ab')2 fragments of the four MAb, at day 3, the tumor antibody uptakes ranged between 12 and 24 and the specificity indices between 5.3 and 8.2. With the Fab fragments prepared from the two most promising MAb, the antibody uptakes reached values of 34 and 82 at day 2-3 and the specificity indices were as high as 12 and 19. The scanning results paralleled those obtained by direct measurement of radioactivity. With intact MAb, tumor grafts of 0.5-1 g gave very contrasted positive scans 3 d after injection. Using MAb fragments, tumors of smaller size were detectable earlier. The best results were obtained with Fab fragments of MAb 35, which gave clear detections of tumors weighing only 0.1 g as early as 48 h after injection. Autoradiographs of tumor sections from mice injected with 125I-labeled MAb demonstrated that the radioactivity was localized in the tumor tissues and not in the stromal connective tissue of mouse origin. The highest radioactivity concentration was localized in areas known to contain CEA such as the pseudolumen of glands and the apical side of carcinoma cells. The penetration of radioactivity in the central part of tumor nodules and the pseudolumen appeared to be increased with the use of MAb fragments.

Lipid metabolism in myelinating glial cells: lessons from human inherited disorders and mouse models.

The integrity of central and peripheral nervous system myelin is affected in numerous lipid metabolism disorders. This vulnerability was so far mostly attributed to the extraordinarily high level of lipid synthesis that is required for the formation of myelin, and to the relative autonomy in lipid synthesis of myelinating glial cells because of blood barriers shielding the nervous system from circulating lipids. Recent insights from analysis of inherited lipid disorders, especially those with prevailing lipid depletion and from mouse models with glia-specific disruption of lipid metabolism, shed new light on this issue. The particular lipid composition of myelin, the transport of lipid-associated myelin proteins, and the necessity for timely assembly of the myelin sheath all contribute to the observed vulnerability of myelin to perturbed lipid metabolism. Furthermore, the uptake of external lipids may also play a role in the formation of myelin membranes. In addition to an improved understanding of basic myelin biology, these data provide a foundation for future therapeutic interventions aiming at preserving glial cell integrity in metabolic disorders.

The murine interleukin-2 receptor gamma chain gene: organization, chromosomal localization and expression in the adult thymus.

Defects in the interleukin-2 receptor gamma (IL-2R gamma) chain in the man result in an X-linked severe combined immunodeficiency, SCIDX1, characterized by an absence of T-cell differentiation. This phenotype may result from pertubations in IL-2, IL-4-, IL-7- or IL-15-mediated signaling, as the IL-2R gamma chain forms an integral component of these receptor systems. We have isolated and characterized cDNA and genomic clones for the murine IL-2R gamma. The gene (Il2rg) is well conserved between mouse and man with respect to overall structure and size, and contains regions of high conservation in the promoter region as well. Il2rg maps to mouse X chromosome region 40, in a region of synteny with human Xq12-13.1. We have also explored the expression of the IL-2R gamma during thymocyte development. IL-2R gamma transcripts are detected in the earliest thymocyte precursor cells and persist throughout intrathymic development into the mature peripheral compartment. Genomic clones for the murine IL-2R gamma will allow for further studies on the regulation and function of this gene in vivo.

Apport de l'angiographie au vert d'indocyanine (ICG) dans la localisation des néovascularisations chororoidiennes occultes [Value of indocyanine green angiography in localization of occult choroid neovascularization]

PURPOSE: To determine the role of Indocyanin Green (ICG) angiography in localizing occult new vessels associated with age-related macular degeneration (ARMD) and assess the possibilities of ICG guided laser photocoagulations. PATIENTS AND METHODS: Fluorescein and ICG angiographies (IMAGEnet system) of 62 patients with occult new vessels (ONV), serous (SPED) or vascular (VPED) pigment epithelium detachment have been studied. RESULTS: Based on fondoscopic examination and fluorescein angiography, 43 eyes (69%) disclosed ONV, 8 (13%) SPED and 11 (18%) VPED. Choroidal neovascularisation was confirmed by ICG angiography in 37 ONV cases (86%), in 8 (72%) VPED cases, but in no SPED. Conversion of ONV in classical neovascular membranes was possible in 19 ONV cases (44%) and in 6 (54%) VPED cases, making a laser photocoagulation possible in 9 eyes (36%). CONCLUSION: ICG angiography plays an important role in the evaluation, classification and laser treatment of patients with ONV secondary to ARMD.

1H-[13C] NMR spectroscopy of the rat brain during infusion of [2-13C] acetate at 14.1 T.

Full signal intensity (1)H-[(13)C] NMR spectroscopy, combining a preceding (13)C-editing block based on an inversion BISEP (B(1)-insensitive spectral editing pulse) with a spin-echo coherence-based localization, was developed and implemented at 14.1 T. (13)C editing of the proposed scheme was achieved by turning on and off the (13)C adiabatic full passage in the (13)C-editing block to prepare inverted and noninverted (13)C-coupled (1)H coherences along the longitudinal axis prior to localization. The novel (1)H-[(13)C] NMR approach was applied in vivo under infusion of the glia-specific substrate [2-(13)C] acetate. Besides a approximately 50% improvement in sensitivity, spectral dispersion was enhanced at 14.1 T, especially for J-coupled metabolites such as glutamate and glutamine. A more distinct spectral structure at 1.9-2.2 ppm(parts per million) was observed, e.g., glutamate C3 showed a doublet pattern in both simulated (1)H spectrum and in vivo (13)C-edited (1)H NMR spectra. Besides (13)C time courses of glutamate C4 and glutamine C4, the time courses of glutamate C3 and glutamine C3 obtained by (1)H-[(13)C] NMR spectroscopy were reported for the first time. Such capability should greatly improve the ability to study neuron-glial metabolism using (1)H-observed (13)C-edited NMR spectroscopy.

Localization and characterization of an active fault in an urbanized area in central Guatemala by means of geoelectrical imaging

The Polochic and Motagua faults define the active plate boundary between the North American and Caribbean plates in central Guatemala. A splay of the Polochic Fault traverses the rapidly growing city of San Miguel Uspantan that is periodically affected by destructive earthquakes. This fault splay was located using a 2D electrical resistivity tomography (ERT) survey that also characterized the fault damage zone and evaluated the thickness and nature of recent deposits upon which most of the city is built. ERT images show the fault as a similar to 50 m wide, near-vertical low-resistivity anomaly, bounded within a few meters by high resistivity anomalies. Forward modeling reproduces the key aspects of the observed electrical resistivity data with remarkable fidelity thus defining the overall location, geometry, and internal structure of the fault zone as well as the affected lithologies. Our results indicate that the city is constructed on a similar to 20 m thick surficial layer consisting of poorly consolidated, highly porous, water-logged pumice. This soft layer is likely to amplify seismic waves and to liquefy upon moderate to strong ground shaking. The electrical conductivity as well as the major element chemistry of the groundwater provides evidence to suggest that the local aquifer might, at least in part, be fed by water rising along the fault. Therefore, the potential threat posed by this fault splay may not be limited to its seismic activity per se, but could be compounded its potential propensity to enhance seismic site effects by injecting water into the soft surficial sediments. The results of this study provide the basis for a rigorous analysis of seismic hazard and sustainable development of San Miguel Uspantan and illustrate the potential of ERT surveying for paleoseismic studies.

VGLUT1 is Present in Cortical, Hippocampal and Striatal Astrocytes

The last decade has presented studies providing evidence for astrocytic exocytosis of glutamate potentiating nerve signals. To make further investigations into this astrocytic attribute we investigated the localization of the vesicular glutamate transporter 1 (VGLUT1) in small processes of astrocytes close to glutamatergic terminals in frontal cortex, striatum, molecular layer of hippocampus and stratum radiatum of hippocampus. According to the importance of VGLUT1 in glutamate exocytosis the presence of VGLUT1 in astrocytic processes indicates the ability to exocytose glutamate. METHODS: For qualitative analysis we used immunoflourescence histochemistry. Sections from rat frontal cortex, striatum, molecular layer of hippocampus and stratum radiatum of hippocampus were labeled with antibodies against glutamine synthetase (an astrocytic marker) and VGLUT1. Z-stacks of 4.5-5 lm obtained by confocal microscopy from each section were deconvolved and 3D reconstructed in Amira. Small astrocytic processes were analysed for the presence of VGLUT1 inside the processes. The quantitative analysis was done by immunogold labeling. Ultrathin sections from each brain region were labeled for GLT (an astrocytic marker) and VGLUT1. Pictures obtained by electron microscopy were analysed and the point density (gold particles/nm2) for VGLUT1 in astrocytic processes was measured. RESULTS: Using confocal 3D reconstructions we were qualitatively able to identify VGLUT1 within small processes of astrocytes in all four brain regions. Reflecting our qualitative findings the electron microscopical immunogold quantifications showed a significant density of gold particles signaling VGLUT1 in astrocytic processes in all four brain regions. CONCLUSION: We extend the results of previous studies on glutamate release from astrocytes, which have focused on the hippocampus, proposing that astrocytic exocytosis of glutamate is a global phenomenon in the brain.

Spatial, temporal and subcellular localization of islet-brain 1 (IB1), a homologue of JIP-1, in mouse brain.

Islet-brain 1 (IB1) was recently identified as a DNA-binding protein of the GLUT2 gene promoter. The mouse IB1 is the rat and human homologue of the Jun-interacting protein 1 (JIP-1) which has been recognized as a key player in the regulation of c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways. JIP-1 is involved in the control of apoptosis and may play a role in brain development and aging. Here, IB1 was studied in adult and developing mouse brain tissue by in situ hybridization, Northern and Western blot analysis at cellular and subcellular levels, as well as by immunocytochemistry in brain sections and cell cultures. IB1 expression was localized in the synaptic regions of the olfactory bulb, retina, cerebral and cerebellar cortex and hippocampus in the adult mouse brain. IB1 was also detected in a restricted number of axons, as in the mossy fibres from dentate gyrus in the hippocampus, and was found in soma, dendrites and axons of cerebellar Purkinje cells. After birth, IB1 expression peaks at postnatal day 15. IB1 was located in axonal and dendritic growth cones in primary telencephalon cells. By biochemical and subcellular fractionation of neuronal cells, IB1 was detected both in the cytosolic and membrane fractions. Taken together with previous data, the restricted neuronal expression of IB1 in developing and adult brain and its prominent localization in synapses suggest that the protein may be critical for cell signalling in developing and mature nerve terminals.

Cross validation of experts versus registration methods for target localization in deep brain stimulation.

In the last five years, Deep Brain Stimulation (DBS) has become the most popular and effective surgical technique for the treatent of Parkinson's disease (PD). The Subthalamic Nucleus (STN) is the usual target involved when applying DBS. Unfortunately, the STN is in general not visible in common medical imaging modalities. Therefore, atlas-based segmentation is commonly considered to locate it in the images. In this paper, we propose a scheme that allows both, to perform a comparison between different registration algorithms and to evaluate their ability to locate the STN automatically. Using this scheme we can evaluate the expert variability against the error of the algorithms and we demonstrate that automatic STN location is possible and as accurate as the methods currently used.

Analysis of glial acidic fibrillary protein in the human entorhinal cortex during aging and in Alzheimer's disease.

Glial fibrillary acidic protein, GFAP, is a major intermediate filament protein of glial cells and major cytoskeletal structure in astrocytes. The entorhinal cortex has a key role in memory function and is one of the first brain areas to reveal hallmark structures of Alzheimer's disease and therefore provides an ideal tissue to investigate incipient neurodegenerative changes. Here we have analyzed age- and disease-related occurrence and composition of GFAP in the human entorhinal cortex by using one- and two-dimensional electrophoresis, Western blots and immunocytochemistry combined with confocal microscopy. A novel monoclonal antibody, GF-02, was characterized that mainly reacted with intact GFAP molecules and indicated that more acidic and soluble GFAP forms were also more susceptible to degradation. GFAP and vimentin increased with aging and in Alzheimer's disease (AD). Two-dimensional electrophoresis and Western blots revealed a complex GFAP pattern, both in aging and AD with different modification and degradation forms. Immunohistochemistry indicated that reactive astrocytes mainly accumulated in relation to neurofibrillary tangles and senile plaques in deeper entorhinal cortex layers. GFAP may be used as an additional but not exclusive diagnostic tool in the evaluation of neurodegenerative diseases because its levels change with age and respond to senile plaque and tangle formation.

Substance P and calbindin D-28k-immunoreactivity in primary sensory neurons of chick embryos: differential neuronal birthdates and transient co-localization.

During the ontogenesis of dorsal root ganglia (DRG), the immunoreactivity to substance P (SP) and calbindin D-28k (CaBP) appears in chickens at embryonic day 5 (E5) and E10 respectively. To establish the birthdates of primary sensory neurons expressing SP or CaBP, chick embryos were given repetitive intra-amniotic injections of [3H]-thymidine. The neuroblasts giving rise to SP-expressing neurons were labeled up to E6 while those generating CaBP-immunoreactive neurons stopped to incorporate [3H]-thymidine before E5.5. This finding indicates that neurons exhibiting distinct phenotypes may originate from neuroblasts which arrest to proliferate at close but distinct stages of development. To determine whether SP and CaBP are co-expressed or not in DRG neurons, chick embryos at E12, E18, and chickens two weeks after hatching were perfused and fixed to detect simultaneously SP- and CaBP-immunoreactivity in DRG sections. The results showed that SP and CaBP were transiently co-expressed by a subset of neurons at E12. Later, however, the SP-immunoreactivity was gradually lost by these ganglion cells, so that the SP- and CaBP-immunoreaction defined two distinct neuronal subpopulations after hatching. In conclusion, most CaBP-immunoreactive DRG cells derive from a subset of neurons in which SP and CaBP are transiently co-localized.

Video-assisted thoracoscopic resection of a small pulmonary nodule after computed tomography-guided localization with a hook-wire system. Experience in 45 consecutive patients.

BACKGROUND: This study is a single-institution validation of video-assisted thoracoscopic (VATS) resection of a small solitary pulmonary nodule (SPN) previously localized by a CT-guided hook-wire system in a consecutive series of 45 patients. METHODS: The records of all patients undergoing VATS resection for SPN preoperatively localized by CT-guided a hook-wire system from January 2002 to December 2004 were assessed with respect to failure to localize the lesion by the hook-wire system, conversion thoracotomy rate, duration of operation, postoperative complications, and histology of SPN. RESULTS: Forty-five patients underwent 49 VATS resections, with simultaneous bilateral SPN resection performed in 4. Preoperative CT-guided hook-wire localization failed in two patients (4%). Conversion thoracotomy was necessary in two patients (4%) because it was not possible to resect the lesion by a VATS approach. The average operative time was 50 min. Postoperative complications occurred in 3 patients (6%), one hemothorax and two pneumonia. The mean hospital stay was 5 days (range: 2-18 days). Histological assessment revealed inflammatory disease in 17 patients (38%), metastasis in 17 (38%), non-small-cell lung cancer (NSCLC) in 4 (9%), lymphoma in 3 (6%), interstitial fibrosis in 2 (4%), histiocytoma in one (2%), and hamartoma in one (2%). CONCLUSIONS: Histological analysis of resected SPN revealed unexpected malignant disease in more than 50% of the patients indicating that histological clarification of SPN seems warranted. Video-assisted thoracoscopic resection of SPN previously localized by a CT-guided hook-wire system is related to a low conversion thoracotomy rate, a short operation time, and few postoperative complications, and it is well suited for the clarification of SPN.