Immediate increase of plasma protein complement C3 in response to an acute stressor.

Whereas chronic stress has immunosuppressive effects, the more immediate immunologic consequences of acute stressors are less known. We postulated that, as part of their 'fight or flight' response, rainbow trout would rapidly increase the efficacy of their natural immune system by means of increased concentrations of crucial plasma proteins. Plasma samples were taken from resting fish and from fish 5, 10 or 20 min after initiation of a stressful regime. Using crossed immunoelectrophoresis, we documented increases in concentrations of complement C3 and 3 other proteins within 5 min of initiation of stress. The concentration of C3 nearly doubled within 10 min of initiation of stress and had returned to near resting level by 20 min. This rapid kinetics preclude dependence on gene activation, the basis of the acute phase response. Potentiation of natural immunity, which can reasonably be expected to be selectively advantageous during or immediately after acute stressors may be one result of this increase.

Immunoelectrophoresis for the Characterization of Allergen Extracts.

Immunoelectrophoresis can be used for analysis of individual proteins in complex mixtures. The conditions involved in immunoelectrophoresis are mild, avoiding the risk of denaturation, and it is possible to perform relative quantification of individual components. The fundamental disadvantage is the dependence on rabbit antisera as reagents. The usefulness of immunoelectrophoresis in allergy research is greatly enhanced by the possibility of identification of allergens to which the individual in question has IgE.The common principle is characterized by two independent electrophoreses having direction of current perpendicular to each other, i.e., crossed immunoelectrophoresis (CIE). This ultimately results in the formation of characteristic bell-shaped precipitates, each precipitate representing one antigen. There is a linear relationship between the amount of antigen and size of precipitate for a given antibody concentration for each precipitate and so relative quantification can be performed. The sensitivity and resolution power of CIE are very high and there are multiple variations of the technique, some of which will be illustrated in this chapter.

Identification of common immunodominant antigens of Eimeria tenella, Eimeria acervulina and Eimeria maxima by immunoproteomic analysis.

Clinical chicken coccidiosis is mostly caused by simultaneous infection of several Eimeria species, and host immunity against Eimeria is species-specific. It is urgent to identify common immunodominant antigen of Eimeria for developing multivalent anticoccidial vaccines. In this study, sporozoite proteins of Eimeria tenella, Eimeria acervulina and Eimeria maxima were analyzed by two-dimensional electrophoresis (2DE). Western bot analysis was performed on the yielded 2DE gel using antisera of E. tenella E. acervulina and E. maxima respectively. Next, the detected immunodominant spots were identified by comparing the data from MALDI-TOF-MS/MS with available databases. Finally, Eimeria common antigens were identified by comparing amino acid sequence between the three Eimeria species. The results showed that analysis by 2DE of sporozoite proteins detected 629, 626 and 632 protein spots from E. tenella, E. acervulina and E. maxima respectively. Western bot analysis revealed 50 (E. tenella), 64 (E. acervulina) and 57 (E. maxima) immunodominant spots from the sporozoite 2DE gels of the three Eimeria species. The immunodominant spots were identified as 33, 27 and 25 immunodominant antigens of E. tenella, E. acervulina and E. maxima respectively. Fifty-four immunodominant proteins were identified as 18 ortholog proteins among the three Eimeria species. Finally, 5 of the 18 ortholog proteins were identified as common immunodominant antigens including elongation factor 2 (EF-2), 14-3-3 protein, ubiquitin-conjugating enzyme domain-containing protein (UCE) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In conclusion, our results not only provide Eimeria sporozoite immunodominant antigen map and additional immunodominant antigens, but also common immunodominant antigens for developing multivalent anticoccidial vaccines.

Scedosporium prolificans immunomes against human salivary immunoglobulin A.

The filamentous fungus Scedosporium prolificans is an emerging multidrug resistant pathogen related to serious infections mainly affecting immunocompromised individuals. Considering that it is frequently isolated from anthropic environments and penetrates mainly through the airways, the human mucosal immune system may play an important protective role against S. prolificans. To advance in the search for biomarkers and targets both for diagnosis and treatment, we analysed the S. prolificans immunomes recognized by human salivary Immunoglobulin A. Using indirect immunofluorescence, it was observed that conidia were strongly recognized, while hyphae were not. By 2-D immunoblotting and peptide mass fingerprinting, 25 immunodominant antigens in conidia and 30 in hyphae were identified. These included catalase, putative glyceronetransferase, translation elongation factor-1α, serine/threonine protein kinase, putative superoxide dismutase, putative mitochondrial cyclophilin 1 and peptidyl-prolyl cis-trans isomerase in conidiospores, and putative Hsp60, ATP synthase ß chain, 40S ribosomal protein S0, citrate synthase and putative ATP synthase in hyphae. The functional study showed that metabolism - and protein fate - related enzymes were the most abundant antigens in conidia, whereas metabolism - , translation - , or energy production - related enzymes were in hyphae. The immunogenic proteins identified are proposed as candidates for the development of novel diagnostic tools or therapeutic strategies.

Neurobiological signatures of alcohol dependence revealed by protein profiling.

Alcohol abuse causes dramatic neuroadaptations in the brain, which contribute to tolerance, dependence, and behavioral modifications. Previous proteomic studies in human alcoholics and animal models have identified candidate alcoholism-related proteins. However, recent evidences suggest that alcohol dependence is caused by changes in co-regulation that are invisible to single protein-based analysis. Here, we analyze global proteomics data to integrate differential expression, co-expression networks, and gene annotations to unveil key neurobiological rearrangements associated with the transition to alcohol dependence modeled by a Chronic Intermittent Ethanol (CIE), two-bottle choice (2BC) paradigm. We analyzed cerebral cortices (CTX) and midbrains (MB) from male C57BL/6J mice subjected to a CIE, 2BC paradigm, which induces heavy drinking and represents one of the best available animal models for alcohol dependence and relapse drinking. CIE induced significant changes in protein levels in dependent mice compared with their non-dependent controls. Multiple protein isoforms showed region-specific differential regulation as a result of post-translational modifications. Our integrative analysis identified modules of co-expressed proteins that were highly correlated with CIE treatment. We found that modules most related to the effects of CIE treatment coordinate molecular imbalances in endocytic- and energy-related pathways, with specific proteins involved, such as dynamin-1. The qRT-PCR experiments validated both differential and co-expression analyses, and the correspondence among our data and previous genomic and proteomic studies in humans and rodents substantiates our findings. The changes identified above may play a key role in the escalation of ethanol consumption associated with dependence. Our approach to alcohol addiction will advance knowledge of brain remodeling mechanisms and adaptive changes in response to drug abuse, contribute to understanding of organizational principles of CTX and MB proteomes, and define potential new molecular targets for treating alcohol addiction. The integrative analysis employed here highlight the advantages of systems approaches in studying the neurobiology of alcohol addiction.

Characterization of fluorescent poly(isobutylcyanoacrylate) nanoparticles obtained by copolymerization of a fluorescent probe during Redox Radical Emulsion Polymerization (RREP).

PURPOSE: The purpose of the work was to demonstrate that a polymerizable fluorescent labeled was incorporated in the core of chitosan/pluronic® F68-coated Poly(IsobutylCyanoAcrylate) (PIBCA) nanoparticles thanks to a covalent linkage. It was also aimed to show that the labeling did not modify the complement activation capacity of the nanoparticles which are designed as drug carriers for the in vivo delivery of siRNA. METHOD: Fluorescent nanoparticles were prepared by adding a fluorescent monomer dye, methacryloxyethyl thiocarbamoyl rhodamine B during the preparation of nanoparticles by redox radical emulsion polymerization. The structure and composition of the fluorescent nanoparticles was investigated. The capacity of the fluorescent nanoparticles to activate the complement system was evaluated by 2D immunoelectrophoresis. RESULTS: Results from the analysis of the composition and structure of polymers forming the nanoparticles showed that the fluorescent dye was incorporated in the core of the nanoparticles by formation of a stable covalent linkage with PIBCA. The labeled nanoparticles showed the same surface properties as the corresponding non-labeled nanoparticles based on analysis of the polymer structure, physicochemical properties and evaluation of their capacity to activate the complement system. CONCLUSION: This work showed that the fluorescent PIBCA nanoparticles were labeled by incorporation of the fluorescent probe in the nanoparticle core and that the fluorescent probe did not modify the nanoparticle surface properties. These fluorescent nanoparticles can be proposed as relevant models to investigate how they deliver siRNA to their biological target in cell cultures and during in vivo experiments.

Causes of alternative pathway dysregulation in dense deposit disease.

BACKGROUND AND OBJECTIVES: This study was designed to investigate the causes of alternative pathway dysregulation in a cohort of patients with dense deposit disease (DDD). DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Thirty-two patients with biopsy-proven DDD underwent screening for C3 nephritic factors (C3Nefs), factor H autoantibodies (FHAAs), factor B autoantibodies (FBAAs), and genetic variants in CFH. C3Nefs were detected by: ELISA, C3 convertase surface assay (C3CSA), C3CSA with properdin (C3CSAP), two-dimensional immunoelectrophoresis (2DIEP), and immunofixation electrophoresis (IFE). FHAAs and FBAAs were detected by ELISA, and CFH variants were identified by Sanger sequencing. RESULTS: Twenty-five patients (78%) were positive for C3Nefs. Three C3Nef-positive patients were also positive for FBAAs and one of these patients additionally carried two novel missense variants in CFH. Of the seven C3Nef-negative patients, one patient was positive for FHAAs and two patients carried CFH variants that may be causally related to their DDD phenotype. C3CASP was the most sensitive C3Nef-detection assay. C3CASP and IFE are complementary because C3CSAP measures the stabilizing properties of C3Nefs, whereas IFE measures their expected consequence-breakdown of C3b. CONCLUSIONS: A test panel that includes C3CSAP, IFE, FHAAs, FBAAs, and genetic testing for CFH variants will identify a probable cause for alternative pathway dysregulation in approximately 90% of DDD patients. Dysregulation is most frequently due to C3Nefs, although some patients test positive for FHAAs, FBAAs, and CFH mutations. Defining the pathophysiology of DDD should facilitate the development of mechanism-directed therapies.

Valoración de la translucencia nucal y anatomía fetal en la ecografía de 11-13+6 semanas: técnica bidimensional frente a tridimensional / Assessment of nuchal translucency and foetal anatomy in the 11-13.6 weeks ultrasound: two-dimensional versus three-dimensional technique

Introducción. se valora la posibilidad de evaluar la anatomía fetal y la medición de la translucencia nucal (TN) a partir del estudio diferido de un volumen capturado mediante ecografía tridimensional (3D). Objetivo. comparar los resultados obtenidos mediante la exploración ecográfica bidimensional (2D) y 3D. Método. estudio prospectivo realizado en 100 gestaciones únicas, que acuden para cribado de aneuploidías entre la 11 y 13,6 semanas. Se practica ecografía 2D vía abdominal por un primer explorador (E1), con estudio anatómico (definido en base a un score anatómico), valoración de TN (según criterios de la Fetal Medicine Foundation) y mapa Doppler color (ductus venoso y vasos umbilicales). El mismo explorador (E1) captura un volumen fetal total 3D vía abdominal, con y sin Doppler color. Los volúmenes 3D se valoran en diferido mediante navegación multiplanar por dos exploradores (E1, E2). Resultados. la medición del CRL pudo hacerse por ambos exploradores en el 100% de los casos en 2D y 3D, sin diferencias significativas entre ambos. La TN pudo valorarse en el 100% de los casos mediante la ecografía 2D, y en el 63 y el 48% mediante ecografía 3D en E1 y E2, respectivamente. Los porcentajes de valoración de la anatomía son inferiores mediante la exploración 3D, aunque alcanza el 90-100% en estructuras como cabeza, tórax, abdomen, estómago y extremidades. No se encuentran diferencias en el tiempo de exploración entre ambas técnicas. Se demuestra que a mayor experiencia del explorador, menor es el tiempo de análisis en diferido, aunque este tiempo se estabiliza a partir de 20 volúmenes analizados (curva de aprendizaje). Conclusión. la obtención de un solo volumen fetal total 3D vía transabdominal entre las 11 y las 13,6 semanas permite una valoración en diferido de la anatomía básica y de la TN, aunque en cifras inferiores al 2D(AU)

Introduction. An evaluation is made of the possibility of assessing foetal anatomy and measuring nuchal translucency (NT) from the deferred study of the volume captured using three-dimensional (3D) ultrasound. Objective. To compare the results obtained by the two-dimensional (2D) and 3D ultrasound examination. Method. A prospective study performed on 100 single pregnancies, who came for aneuploidy screening between 11-13.6 weeks gestation. A 2D abdominal ultrasound was performed by a first examiner (E1), with an anatomical study (defined based on an anatomy score), an NT evaluation (based on criteria of the Foetal Medicine Foundation) and a colour Doppler map (ductus venosus and umbilical vessels. The same examiner (E1) captured a total foetal volume by abdominal 3D, with and without colour Doppler. The 3D volumes were assessed by two examiners (E1 and E2) in deferred mode using multiplanar navigation. Results. Measurement of the crown-rump length (CRL) could be made in 2D and 3D by both examiners in 100% of cases, with no significant differences between them. The NT could be assessed in 100% of cases using 2D ultrasound, and in 63% and 48% of cases using 3D ultrasound by E1 and E2, respectively. The anatomy assessment percentages were lower with 3D, although they reached 90-100% in structures such as the head, thorax, abdomen, stomach and limbs. There were no differences in the examination times between the techniques. It showed that the more experienced the examiner, the lower the time of deferred analysis; although this time was established from 20 volumes analysed (learning curve). Conclusion. The obtaining a single total foetal volume by trans-abdominal 3D ultrasound between 11-13.6 weeks allows an assessment to be made of the basic anatomy and the NT, although in percentages lower than in 2D(AU)

Immunomodulation of serum complement (C3) and macrophages by synthetic pyrethroid fenvalerate: in vitro study.

Fenvalerate, a type II synthetic pyrethroid, has emerged as one of the most potent indoor toxicants. Despite its widespread usage, the adverse effect of this insecticide on immune defense mechanism has not been comprehensively investigated. In this in vitro study we report the effect of fenvalerate on two pivotal components of the immune network, namely the complement system and macrophages. Fenvalerate treated human sera showed serum complement activation as evident by significant (p<0.05) increase in C3b, C3d and C3a levels and a significant (p<0.05) decline in CH50 levels. Further detailed study demonstrates that the activation of complement system is through alternative pathway. This is possibly responsible for various allergic manifestations often reported in subjects exposed to fenvalerate. In addition, fenvalerate induce cellular apoptosis and cytotoxicity, as demonstrated by cytoplasmic vacuolization, heterochromatin condensation, hypodiploid nuclei and DNA fragmentation in macrophages. Considerable deleterious effects on macrophages in conjunction with uncontrolled serum complement activation are probably one of the major mechanisms contributing for the immunosuppressive effects of fenvalerate.

The memory enhancing effect of the APP-derived tripeptide Ac-rER is mediated through CRMP2.

The diasteromeric (D/L) form of the acetylated tripeptide rER (NH2-D-arg-L-glu-D-arg-COOH), derived from the external domain of amyloid precursor protein, protects against amyloid-ß induced memory loss for a passive avoidance task in young chicks and enhances retention for a weak version of the task when injected peripherally up to 12 h prior to training. The tripeptide readily crosses the blood-brain barrier, binds to receptor sites in the brain and is without adverse effects on general behaviour. The mechanisms of its action are unknown, as are its target molecules/pathways. Here, we report the binding partners for Ac-rER are collapsin response mediator protein 2 (CRMP2), syntaxin binding protein 1 and heat shock protein 70. Behavioural studies of the effects of Ac-rER on memory retention confirmed that the effect of Ac-rER is mediated via CRMP2, as anti-CRMP2 antibodies if injected intracranially 30 min pre-training, induced amnesia for the passive avoidance task. However, Ac-rER, if injected prior to the anti-CRMP2, rescues the memory deficits induced by anti-CRMP2 antibodies. As CRMP2 is placed at the junction of many different cellular processes during brain development and in adult neuronal plasticity as well as being implicated in Alzheimer's disease, this strengthens the claim that Ac-rER may be a potential therapeutic agent in Alzheimer's disease, although its precise mode of action remains to be elucidated.

Immunoproteomics of extracellular proteins of the Aeromonas hydrophila China vaccine strain J-1 reveal a highly immunoreactive outer membrane protein.

Aeromonas hydrophila is a gram-negative bacterium that can infect a variety of aquatic and terrestrial animals. It is essential to develop a vaccine to reduce the economic losses caused by this bacterium in aquaculture worldwide. Here, an immunoproteomic assay was used to identify the immunogenic extracellular proteins of the Chinese vaccine strain J-1. Ten unique immunogenic proteins were identified from the two-dimensional electrophoresis immunoblot profiles by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) or MALDI-TOF-TOF-MS. One protein of interest, Omp38, was detected by antisera on two-dimensional immunoblots, suggesting that it might be located both extracellularly and in the membrane. In exploring the potential of Omp38 as a vaccine candidate in fish, we found the omp38 gene to be prevalent by PCR among different (36/48) A. hydrophila isolates. The recombinant Omp38 induced a strong antibody response in rabbits, and the polyclonal antibody could recognize a band of approximately 38 kDa in the immunoblots of outer membrane protein extracts from most (24/40) of the A. hydrophila strains, including different predominant serotypes in China. These results indicated that the outer membrane antigen identified in this study could be developed as a vaccine candidate to induce protective immunity against A. hydrophila infection.

Use of (32)P to study dynamics of the mitochondrial phosphoproteome.

Protein phosphorylation is a well-characterized regulatory mechanism in the cytosol, but remains poorly defined in the mitochondrion. In this study, we characterized the use of (32)P-labeling to monitor the turnover of protein phosphorylation in the heart and liver mitochondria matrix. The (32)P labeling technique was compared and contrasted to Phos-tag protein phosphorylation fluorescent stain and 2D isoelectric focusing. Of the 64 proteins identified by MS spectroscopy in the Phos-Tag gels, over 20 proteins were correlated with (32)P labeling. The high sensitivity of (32)P incorporation detected proteins well below the mass spectrometry and even 2D gel protein detection limits. Phosphate-chase experiments revealed both turnover and phosphate associated protein pool size alterations dependent on initial incubation conditions. Extensive weak phosphate/phosphate metabolite interactions were observed using nondisruptive native gels, providing a novel approach to screen for potential allosteric interactions of phosphate metabolites with matrix proteins. We confirmed the phosphate associations in Complexes V and I due to their critical role in oxidative phosphorylation and to validate the 2D methods. These complexes were isolated by immunocapture, after (32)P labeling in the intact mitochondria, and revealed (32)P-incorporation for the alpha, beta, gamma, OSCP, and d subunits in Complex V and the 75, 51, 42, 23, and 13a kDa subunits in Complex I. These results demonstrate that a dynamic and extensive mitochondrial matrix phosphoproteome exists in heart and liver.

Cross-reactivity between storage and dust mites and between mites and shrimp.

Many patients have sensitivities to multiple species of storage and house dust mites. It is not clear if this is because patients have multiple sensitivities to species-specific mite allergens or if these mites share many cross-reacting allergens. Our objective was to further define the cross-allergenicity between several species of storage and house dust mites using crossed-immunoelectrophoresis (CIE), crossed-radioimmunoelectrophoresis (CRIE), immunoblotting, and ELISA. CIE and CRIE reactions revealed that storage mites shared two cross-antigenic molecules and one of these bound IgE in a serum pool from mite allergic patients. Antibody in anti-sera built to each species of mite recognized many SDS-PAGE resolved proteins of other mite species and this suggested the potential for other cross-reactive allergens. Among patient sera, IgE bound to many different proteins but few had IgE that bound to a protein with common molecular weights across the mite species and this suggested mostly species-specific allergens. Antiserum built to each mite species precipitated one protein in shrimp extracts that bound anti-Der p 10 (tropomyosin) and IgE in the serum pool. Anti-Der p 10 showed strong binding to shrimp tropomyosin but very little to any of the mite proteins. ELISA showed the mite extracts contained very little tropomyosin. The storage and dust mites investigated contain mostly species-specific allergens and very small amounts of the pan-allergen tropomyosin compared to shrimp and snail.

Immunomodulatory effect of DDT (bis[4-chlorophenyl]-1,1,1-trichloroethane) on complement system and macrophages.

DDT (bis[4-chlorophenyl]-1,1,1-trichloroethane) is responsible for many immuno-dysregulatory functions in exposed animals, but data particularly on complement system and macrophages are limited. In this study we have shown that DDT activates the complement system through the alternative pathway in the absence of any pathogen. A significant (p<0.05) increase in C3b, C3d and C3a generation, and decline in complement hemolytic activity was observed in insecticide exposed sera. The uncontrolled complement consumption reduces the lytic activity of the complement, which enhances the susceptibility to pyogenic infection if the exposure to DDT remains unabated. Further, DDT induced the significant (p<0.05) production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) in macrophages and thus contributes inflammatory reactions, cytokine imbalance and immune-dysregulation. These molecular changes in macrophages lead to structural aberrations like heterochromatin condensation, loss of pseudopodia, cytoplasmic vacuolization, DNA fragmentation and hypodiploid nuclei as seen in our study, suggesting apoptosis. However, in presence lipopolysaccharide, DDT induced significant (p<0.05) suppression of TNF-alpha and NO generation, suggestive of impairment of macrophage microbiocidal effects. This study concludes that the functional and structural derangements of macrophages in association with uncontrolled and excessive complement consumption by DDT are perhaps one of the major mechanisms contributing to the immunosuppressive effects of insecticide.

Immunoelectrophoresis for the characterization of allergen extracts.

Immunoelectrophoresis can be used for analysis of individual proteins in complex mixtures. The conditions involved in immunoelectrophoresis are mild, avoiding the risk of denaturation, and it is possible to perform relative quantification of individual components. The principle disadvantage is the dependence on rabbit antisera as reagents. The usefulness of immunoelectrophoresis in allergy research is greatly enhanced by the possibility of identification of allergens to which the individual in question has IgE. The common principle is characterized by two independent electrophoreses having direction of current perpendicular to each other, i.e., crossed immunoelectrophoresis (CIE). This ultimately results in the formation of characteristic bell-shaped precipitates, each precipitate representing one antigen. There is a linear relationship between the amount of antigen and size of precipitate for a given antibody concentration for each precipitate and so relative quantification can be performed. The sensitivity and resolution power of CIE is very high and there are multiple variations of the technique, some of which will be illustrated in this chapter.

Comparison of rocket and crossed immuno-electrophoresis assays for determination of the level of actin complexing of Gc globulin.

OBJECTIVE: Gc globulin (vitamin D-binding protein) is a component of the extracellular actin scavenger system. The level of Gc globulin is reduced in patients with fulminant hepatic failure, septic shock and trauma. Furthermore, low levels of Gc globulin in patients with fulminant hepatic failure and multiple trauma have been found to correlate with the morbidity and mortality of patients. Owing to a large increase in the turnover of Gc globulin upon complex formation with actin, it may be important to determine both the total Gc globulin concentration and the degree of complexing with actin for estimating the clinical prognosis of a patient. For this reason, we have compared a crossed immuno-electrophoresis method (CIE), suitable for visualizing the degree of complexing with actin, with a rocket immuno-electrophoresis method (RIE), previously used for determination of the complex degree. MATERIAL AND METHODS: Sera from healthy donors and from patients with acetaminophen-induced liver disease or trauma were investigated using CIE, RIE and enzyme-linked immunosorbent assay (ELISA). RESULTS: Using the CIE, no Gc globulin-actin complexes were detected among healthy donors. Complexes were present in 21 of 39 patients with liver disease and 3 of 37 trauma patients. High complex ratios (> 20 %) were found in 6 of 7 patients with hepatic encephalopathy. Using the RIE, complexes were detected in most samples. CONCLUSION: The results show that the CIE method may be used for determining the degree of actin complexing in conjunction with ELISA or RIE in determining the levels of total Gc globulin.

Identification of immuno-reactive proteins from a sheep gastrointestinal nematode, Trichostrongylus colubriformis, using two-dimensional electrophoresis and mass spectrometry.

Gastrointestinal nematode infections of livestock animals are prevalent and costly problems worldwide. Currently, infections are controlled by anthelmintic chemicals but increasing drug resistance has prompted research interest to shift towards alternative methods of control such as vaccine development and selection of worm-resistant animals. The present study analyses proteins from Trichostrongylus colubriformis infective L3s that are recognised by IgG of immune sheep. Following protein separation via two-dimensional electrophoresis and Western blot probing with plasma from sheep resistant to T. colubriformis, mass spectrometry-based proteomic analyses were used to identify immuno-reactive protein spots. We were able to identify 28 immune targets, including aspartyl protease inhibitor, enolase, chaperone proteins, galectin, glycolytic enzymes, kinase, phosphatase and structural muscle proteins such as myosin, paramyosin, calponin and DIM-1. The data suggest that immune responses to T. colubriformis are dispersed over a relatively large number of parasite antigens, including several cytoplasmically expressed proteins. The results have new implications for understanding the molecular mechanisms that underpin host-parasite interaction during gastrointestinal nematode infections.

[Immuno-proteomic screening of human pancreatic cancer associated membrane antigens for early diagnosis].

OBJECTIVE: To screen and identify the immunogenic membrane antigens in human pancreatic cancer for early diagnosis. METHODS: Membrane protein was extracted from pancreatic cancer cell lines and separated by using 2-DE. One of the two parallel 2-DE gels went for staining while the other underwent immunoblot. Serum IgG, which was purified from clinically collected sera of pancreatic cancer patients, was used as the primary antibodies for the immunoblot. Positive dots of immunoblot were identified by MALDI-TOF mass spectrometry and PMF matching, and then evaluated by bio-informatics methods. The candidate membrane antigens were further validated respectively in cell lines and tissues by RT-PCR and immunohistochemistry. RESULTS: The immunoblot of mixed membrane protein with serum IgG from cancer patients showed eight positive dots. These dots were identified with MALDI and PMF as: VDAC-1, VDAC-2, CHCHD3, SLP-2 and TOM40. RT-PCR showed that these membrane antigens were expressed in several pancreatic cancer cell lines. Immunohistochemistry showed prominent SLP-2 over expression in cancer tissue. CONCLUSIONS: VDAC-1, VDAC-2, CHCHD3, SLP-2, and TOM40 are the new candidate immunogenic membrane antigens of pancreatic cancer. These membrane antigens can be subsequently tested in high dangerous population for early diagnosis of pancreatic cancer.

Immuno-proteomic screening of human pancreatic cancer associated membrane antigens for early diagnosis / 中华外科杂志

<p><b>OBJECTIVE</b>To screen and identify the immunogenic membrane antigens in human pancreatic cancer for early diagnosis.</p><p><b>METHODS</b>Membrane protein was extracted from pancreatic cancer cell lines and separated by using 2-DE. One of the two parallel 2-DE gels went for staining while the other underwent immunoblot. Serum IgG, which was purified from clinically collected sera of pancreatic cancer patients, was used as the primary antibodies for the immunoblot. Positive dots of immunoblot were identified by MALDI-TOF mass spectrometry and PMF matching, and then evaluated by bio-informatics methods. The candidate membrane antigens were further validated respectively in cell lines and tissues by RT-PCR and immunohistochemistry.</p><p><b>RESULTS</b>The immunoblot of mixed membrane protein with serum IgG from cancer patients showed eight positive dots. These dots were identified with MALDI and PMF as: VDAC-1, VDAC-2, CHCHD3, SLP-2 and TOM40. RT-PCR showed that these membrane antigens were expressed in several pancreatic cancer cell lines. Immunohistochemistry showed prominent SLP-2 over expression in cancer tissue.</p><p><b>CONCLUSIONS</b>VDAC-1, VDAC-2, CHCHD3, SLP-2, and TOM40 are the new candidate immunogenic membrane antigens of pancreatic cancer. These membrane antigens can be subsequently tested in high dangerous population for early diagnosis of pancreatic cancer.</p>