In vivo localisation of radiolabelled antibodies to carcinoembryonic antigen in human colon carcinoma grafted into nude mice.

SEVERAL attempts have been made to show the specific localisation in vivo of anti-tumour antibodies. Most of these studies, however, either in experimental animals1,2 or in humans3 were performed with antibodies obtained by adsorption and elution from poorly characterised crude tumour fractions.

CpG are efficient adjuvants for specific CTL induction against tumor antigen-derived peptide.

The identification of CTL-defined tumor-associated Ags has allowed the development of new strategies for cancer immunotherapy. To potentiate the CTL responses, peptide-based vaccines require the coadministration of adjuvants. Because oligodeoxynucleotides (ODN) containing CpG motifs are strong immunostimulators, we analyzed the ability of CpG ODN to act as adjuvant of the CTL response against tumor-derived synthetic peptide in the absence or presence of IFA. Mice transgenic for a chimeric MHC class I molecule were immunized with a peptide analog of MART-1/Melan-A(26-35) in the presence of CpG ODN alone or CpG ODN emulsified in IFA. The CTL response was monitored ex vivo by tetramer staining of lymphocytes. In blood, spleen, and lymph nodes, peptide mixed with CpG ODN alone was able to elicit a stronger systemic CTL response as compared with peptide emulsified in IFA. Moreover, CpG ODN in combination with IFA further enhanced the CTL response in terms of the frequency of tetramer+CD8+ T cells ex vivo. The CTL induced in vivo against peptide analog in the presence of CpG ODN are functional, as they were able to recognize and kill melanoma cells in vitro. Overall, these results indicate that CpG ODN by itself is a good candidate adjuvant of CTL response and can also enhance the effect of classical adjuvant.

Intelligence-led crime scene processing. Part II : Intelligence and crime scene examination.

A better integration of the information conveyed by traces within intelligence-led framework would allow forensic science to participate more intensively to security assessments through forensic intelligence (part I). In this view, the collection of data by examining crime scenes is an entire part of intelligence processes. This conception frames our proposal for a model that promotes to better use knowledge available in the organisation for driving and supporting crime scene examination. The suggested model also clarifies the uncomfortable situation of crime scene examiners who must simultaneously comply with justice needs and expectations, and serve organisations that are mostly driven by broader security objectives. It also opens new perspective for forensic science and crime scene investigation, by the proposal to follow other directions than the traditional path suggested by dominant movements in these fields.

Non-Destructive Evaluation of Iowa Pavements Phase 2: Development of a Fully Automated Software System for Rapid Analysis/Processing of the Falling Weight Deflectometer Data, 2009

The Office of Special Investigations at Iowa Department of Transportation (DOT) collects FWD data on regular basis to evaluate pavement structural conditions. The primary objective of this study was to develop a fully-automated software system for rapid processing of the FWD data along with a user manual. The software system automatically reads the FWD raw data collected by the JILS-20 type FWD machine that Iowa DOT owns, processes and analyzes the collected data with the rapid prediction algorithms developed during the phase I study. This system smoothly integrates the FWD data analysis algorithms and the computer program being used to collect the pavement deflection data. This system can be used to assess pavement condition, estimate remaining pavement life, and eventually help assess pavement rehabilitation strategies by the Iowa DOT pavement management team. This report describes the developed software in detail and can also be used as a user-manual for conducting simulation studies and detailed analyses. *********************** Large File ***********************

Human leukocyte antigen class II variants and adult-onset asthma: does occupational allergen exposure play a role?

Recently, a locus centred on rs9273349 in the HLA-DQ region emerged from genome-wide association studies of adult-onset asthma. We aimed to further investigate the role of human leukocyte antigen (HLA) class II in adult-onset asthma and a possible interaction with occupational exposures. We imputed classical HLA-II alleles from 7579 single-nucleotide polymorphisms in 6025 subjects (1202 with adult-onset asthma) from European cohorts: ECRHS, SAPALDIA, EGEA and B58C, and from surveys of bakers and agricultural workers. Based on an asthma-specific job-exposure matrix, 2629 subjects had ever been exposed to high molecular weight (HMW) allergens. We explored associations between 23 common HLA-II alleles and adult-onset asthma, and tested for gene-environment interaction with occupational exposure to HMW allergens. Interaction was also tested for rs9273349. Marginal associations of classical HLA-II alleles and adult-onset asthma were not statistically significant. Interaction was detected between the DPB1*03:01 allele and exposure to HMW allergens (p = 0.009), in particular to latex (p = 0.01). In the unexposed group, the DPB1*03:01 allele was associated with adult-onset asthma (OR 0.67, 95%CI 0.53-0.86). HMW allergen exposures did not modify the association of rs9273349 with adult-onset asthma. Common classical HLA-II alleles were not marginally associated with adult-onset asthma. The association of latex exposure and adult-onset asthma may be modified by DPB1*03:01.

Sandwich enzyme immunoassay using three monoclonal antibodies against different epitopes of carcinoembryonic antigen (CEA).

Purified monoclonal antibodies (Mab) produced by 3 hybridomas and reacting with 3 different epitopes of carcinoembryonic antigen (CEA) were used in a solid phase enzyme immunoassay. Two Mabs were physically adsorbed to polystyrene balls and the third Mab was coupled to alkaline phosphatase using the bifunctional reagent N-succinimidyl-3-(2-pyridyldithio)-propionate. During a first incubation, CEA from heat-extracted serum samples was immunoadsorbed to the antibody coated balls. After washing of the balls, bound CEA was detected by a second incubation with the enzyme coupled Mab. The sensitivity of the assay was 0.6 ng per ml of serum. A total of 196 serum samples from patients with various types of carcinoma, with liver cirrhosis, or from healthy blood donors with or without smoking habits, were tested. The results obtained with the monoclonal enzyme immunoassay (M-EIA) were compared with those obtained with perchloric acid extracts of the same serum samples tested by an inhibition radioimmunoassay using conventional goat anti-CEA antiserum. There was an excellent correlation between the two assays. In particular, the new M-EIA gave good results for the detection of tumor recurrences in the follow-up of colon carcinoma patients. However, despite the use of exclusively monoclonal antibodies the new assay detected a similar percentage of slightly elevated CEA values as the conventional assay in patients with non-malignant disease, suggesting that the CEA associated with non-malignant diseases is immunologically identical to the CEA released by colon carcinoma.

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.

Molecular insights into T cell activation : study of the TRAF, Bcl10 and Carma 1 proteins

Abstract : Activation of naïve T lymphocytes is essential for the onset of an adaptive immune response against a pathogenic threat. T lymphocytes are activated through the engagement of their highly specific cell surface antigen-receptor (TCR), together with co-stimulatory receptors, by activated antigen-presenting cells that display antigenic peptide fragments from the pathogen that they have detected. Dissection of the mechanisms that modulate TCR- and co-stimulation- induced signals is therefore crucial for the understanding of the molelcular basis of adaptive immune responses. Following antigen-receptor triggering, the Carma1, Bcl10 and Malt1 (CBM) proteins assemble into an oligomeric complex, which is essential for activation of the NF-κB and JNK signaling pathways in lymphocytes. In this work, by using human epithelial and lymphocytic cell lines, we identified the TNF-receptor-associated factor (TRAF) proteins TRAF3 and TRAF7 as new binding partners of Bcl10 and Carma1, respectively. We could show that TRAF3 is required for the proper transcriptional upregulation of IL-2 in activated T cells, and that endogenous TRAF3 is recruited to Bcl10 following TCR engagement. Although the mechanisms used by TRAF3 to modulate the transcriptional activation of the IL-2 promoter are not elucidated, the stimulus-dependent association ofTRAF3 with its direct binding partner Bcl10 suggests that TRAF3 is regulating Bcl10 function in TCR-activated lymphocytes. We also demonstrated that TRAF7 acts as a negative regulator of Carma1-induced NFκB-and AP1-dependent transcription by overexpression in 293T cells. These data suggest that TRAF7 could contribute to the negative regulation of TCR-dependent Carma1 functions. Finally, we showed that Carma1 is processed upon antigen-receptor triggering in B and T cell lines, as well as in primary human CTLs, and that this processing is dependent on the proteolytic activity of Malt1. Collectively, this work contributes to describe new proteins and regulatory mechanisms that modulate CBM-dependent functions in activated lymphocytes. Furthermore, it uncovers new tracks that could lead to a better molecular understanding of the complex interplay between the activatory and inhibitory regulators associated with the CBM complex. Résumé : L'activation des lymphocytes T naifs est une étape essentielle à la mise en place d'une réponse immunitaire adaptative pour combattre une infection. Après la détection d'un pathogène, les cellules présentatrices d'antigènes exposent à leur surface des fragments peptidiques provenant du pathogène, qui activent le récepteur à antigène (TCR) spécifique des lymphocytes T, ainsi que des molécules co-stimulatrices qui contribuent à l'activation complète des lymphocytes T. La caractérisation des mécanismes qui modulent les cascades de signaux émanant du TCR et des récepteurs de co-stimulation est essentielle à la compréhension du fonctionnement moléculaire de la réponse immunitaire adaptative. La ligation du TCR induit la formation d'un complexe oligomérique comprenant les protéines Carma1, Bcl10 et Malt1, qui est essentiel à l'activation des voies de signalisation cellulaires NF-κB et JNK induisant l'activation complète des lymphorctes T. Dans cette étude, à l'aide de lignées de cellules humaines épithéliales et lymphocytaires, nous avons identifié que deux protéines de la famille des TRAF (Tumor Necrosis Factor Receptor-Associated Factor), TRAF3 et TRAF7, s'associent à Bc110 et à Carma1, respectivement. Les TRAFs sont d'importants régulateurs des voies de signalisation dans les cellules du système immunitaire inné et adaptatif. Nous avons démontré que TRAF3 était important pour permettre la transcription de l'interleukine-2 (IL-2) dans les lymphocytes T activés, et que TRAF3 s'associait à Bc110 à la suite de la stimulation du TCR Les mécanismes que TRAF3 utilise pour moduler l'activation du promoteur de l'IL-2 ne sont pas connus, mais l'association de TRAF3 à Bc110 suite à la stimulation du TCR suggère que TRAF3 régule la fonction de Bc110. Nous avons également identifié TRAF7 comme un nouveau régulateur négatif des voies NF-κB et JNK induites par surexpression de la protéine Carma1. Nos données suggèrent que TRAF7 pourrait également contribuer à la régulation négative de la fonction de Carma1 dans les lymphocytes activés. Enfin, nous avons découvert que Carma1 était clivé suite à la stimulation du TCR, et que ce clivage dépendait de l'activité protéolytique de Malt1. Cette étude contribue ainsi à la description de nouvelles protéines et de nouveaux mécanismes qui modulent l'activité du complexe CBM dans les lymphocytes activés, et ouvre la voie à la caractérisation moléculaire de ces nouveaux mécanismes importants pour la régulation de la réponse immunitaire adaptative.

gp100(209-2M) peptide immunization of human lymphocyte antigen-A2+ stage I-III melanoma patients induces significant increase in antigen-specific effector and long-term memory CD8+ T cells.

Thirty-five HLA-A2(+) patients with completely resected stage I-III melanoma were vaccinated multiple times over 6 months with a modified melanoma peptide, gp100(209-2M), emulsified in Montanide adjuvant. Direct ex vivo gp100(209-2M) tetramer analysis of pre- and postvaccine peripheral blood mononuclear cells (PBMCs) demonstrated significant increases in the frequency of tetramer(+) CD8(+) T cells after immunization for 33 of 35 evaluable patients (median, 0.36%; range, 0.05-8.9%). Ex vivo IFN-gamma cytokine flow cytometry analysis of postvaccine PBMCs after brief gp100(209-2M) in vitro activation showed that for all of the patients studied tetramer(+) CD8(+) T cells produced IFN-gamma; however, some patients had significant numbers of tetramer(+) IFN-gamma(-) CD8(+)T cells suggesting functional anergy. Additionally, 8 day gp100(209-2M) in vitro stimulation (IVS) of pre- and postvaccine PBMCs resulted in significant expansion of tetramer(+) CD8(+) T cells from postvaccine cells for 34 patients, and these IVS tetramer(+) CD8(+) T cells were functionally responsive by IFN-gamma cytokine flow cytometry analysis after restimulation with either native or modified gp100 peptide. However, correlated functional and phenotype analysis of IVS-expanded postvaccine CD8(+) T cells demonstrated the proliferation of functionally anergic gp100(209-2M)- tetramer(+) CD8(+) T cells in several patients and also indicated interpatient variability of gp100(209-2M) stimulated T-cell proliferation. Flow cytometry analysis of cryopreserved postvaccine PBMCs from representative patients showed that the majority of tetramer(+) CD8+ T cells (78.1 +/- 4.2%) had either an "effector" (CD45 RA(+)/CCR7(-)) or an "effector-memory" phenotype (CD45RA(-)/CCR7(-)). Notably, analysis of PBMCs collected 12-24 months after vaccine therapy demonstrated the durable presence of gp100(209-2M)-specific memory CD8(+) T cells with high proliferation potential. Overall, this report demonstrates that after vaccination with a MHC class I-restricted melanoma peptide, resected nonmetastatic melanoma patients can mount a significant antigen-specific CD8(+) T-cell immune response with a functionally intact memory component. The data further support the combined use of tetramer binding and functional assays in correlated ex vivo and IVS settings as a standard for immunomonitoring of cancer vaccine patients.

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.