Antigens Research Papers - Academia.edu (original) (raw)

- by and +1
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- Antibodies, Mice, The, Experimental Medicine

The development and validation of an HPLC method for the quantification of eight peptide antigens from the therapeutic cancer vaccine DPX-0907 is described. The antigens were formulated in DepoVax TM , a patented liposomal vaccine delivery platform used in a phase 1 study for breast, ovarian, and prostate cancers. A gradient reversed-phase method with UV detection was optimized for separating and quantifying the peptide mixture. Several extraction methods investigated to extract the peptides from the lipids led to poor recovery of one or more of the peptides. A simple, reproducible, and high-recovery extraction procedure for the simultaneous quantification of hydrophilic and hydrophobic peptides was discovered using a liquid-liquid extraction with water-saturated n-butanol and sodium bicarbonate (0.1 M). The method was found to be specific, linear, accurate, precise, and reliable within the range of 50-150% of the nominal concentration for DPX-0907. The validated method was successfully applied to the assay of peptide content in pre-clinical and clinical batches of DPX-0907.

- by Andrea Penwell
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- Chemistry, Analytical Chemistry, Medicine, Cancer Vaccines

Successful use of liposomes as immunological adjuvants in vaccines requires simple, easy to scale up technology capable of high-yield antigen entrapment. Recent work from this laboratory has led to the development of techniques that can generate liposomes of various sizes containing soluble antigens such as proteins or particulate antigens such as whole, live, or attenuated bacteria or viruses. Entrapment of proteins is carried out by the dehydration-rehydration procedure, which entails freeze-drying of a mixture of "empty" small unilamellar vesicles and free antigens. Upon rehydration, the large multilamellar vesicles that are formed incorporate up to 80% of the antigen used. When such liposomes are microfluidized in the presence of nonentrapped material, their size is reduced to about 100 nm in diameter, with much of the originally entrapped antigen still associated with the vesicles. A similar technique applied to the entrapment of particulate antigens (e.g., Bacillus s...

- by Gregory Gregoriadis
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- Vaccines, Liposomes, Peptides, Proteins

Impaired fibrinolysis as a result of increased plasminogen activator inhibitor-1 (PAI-1) levels in plasma is a common finding in patients with deep vein thrombosis (DVT). A 4G/5G polymorphism in the promoter region of the PAI-1 gene has been reported to influence the levels of PAI-1. The 4G allele was found to be associated with higher plasma PAI-1 activity (act), but contradictory results on the incidence of the 4G allele in DVT patients have been reported. The aim of this study was to analyse whether the PAI-1 promoter 4G/5G genotype increases the risk of venous thrombosis in subjects with thrombophilic defects, and to determine the distribution of the PAI-1 4G/5G genotype and its relation to plasma PAI-1 levels in 190 unrelated patients with DVT in comparison with a control group of 152 healthy subjects. No differences between the 4G/5G allele distribution in the DVT group (0´43/0´57) and in the control group (0´42/0´58) were observed. However, the presence of the 4G allele significantly increased the risk of thrombosis in patients with other thrombophilic defects.

- by Fernando Ferrando
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- Genetics, Nonparametric Statistics, Polymorphism, British

antigen expression and potentiate CD8+ T cell immune responses CD4+ T lymphocytes mediate in vivo clearance of plasmid DNA vaccine (5019 articles) Immunobiology Articles on similar topics can be found in the following Blood collections... more

- by Nico Van Rooijen
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- Immune response, Mice, Blood, DNA vaccines
- by Carmen Vigo
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- Biology, Medicine, Mice, Immunotherapy

Using matrices based upon Affi-Gel and Affi-Prep, we have examined conditions during the immobilization of antibodies (immunoglobulin G, IgG) that influence the performance of immunosorbents. Such conditions include: coupling pH, coupling kinetics, antibody density on the immunosorbent and the activation chemistries utilized for the immobilization process. These studies have shown that the capacity for antigen does not increase with increased antibody coupling efficiency. Presumably, increased coupling times or efficiencies lead to multi-site attachment of the antibody to the matrix, thereby causing inactivation. Immunosorbents containing low densities of IgG were found to have greater capacity for antigen on a per mole IgG basis. This suggests steric crowding of antigen at high antibody density. Finally, immunosorbents prepared through IgG carbohydrate linkages (oriented coupling) show dramatic increases in antigen capacity over those prepared by stochastic (random) coupling through IgG primary amino groups. A combination of low IgG density and oriented coupling of the IgG via the carbohydrate moiety may represent the best strategy for the preparation of immunosorbents.

- by Robert Matson
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- Engineering, Technology, Chromatography, Kinetics
- by Naomi Luban
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- Adolescent, Child, Blood Pressure, Exercise

Nanoemulsions are adjuvants that enhance antigen penetration in the nasal mucosa, increase cellular uptake of antigens by both epithelial dendritic cells, and promote migration of antigen-loaded dendritic cells to regional lymph nodes within a day of vaccine administration. The objective of this study was to determine whether the W 80 5EC nanoemulsion adjuvant enhances immune response not only by direct uptake of antigen by dendritic cells, but also indirectly, by phagocytosis of antigen-primed, apoptotic, epithelial cells. Consistent with this, we show that exposure of both epithelial cells (TC-1s) and dendritic cells (JAWS II or bone marrow derived dendritic cells (BMDCs)) to nanoemulsion exhibited augmented antigen uptake in cell culture. TC-1 cells subsequently underwent G 2 /M cell cycle arrest and apoptosis, and when co-cultured with JAWS II or BMDCs were rapidly engulfed by the dendritic cells, which responded by up-regulating dendritic cell maturation marker CD86. Altogether these results suggest that the effectiveness of nanoemulsions as adjuvants stems, at least in part, from the engulfment of antigen-loaded epithelial cells, leading to enhanced antigen processing and a strong and balanced mucosal and systemic immune response.

- by James Baker
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- Dendritic Cells, Biological Sciences, Antibodies, Mice

A general problem in immunocytochemistry is the development of a reliable multiple immunolabeling method with primary antibodies originating from the same host species. Here, we briefly outline different approaches intended to close this technological gap and focus on multiple immunolabeling with monoclonal primary antibodies. To this end, we generated a basic universal protocol for the use of secondary antibodies selectively recognizing different isotypes/subclasses of monoclonal primary antibodies. This approach is widely applicable and offers a simple procedure for simultaneously detecting two or more antigens.

- by Igor Buchwalow
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- Antibodies, Color, Fluorescent Antibody Technique, Antigens
- by A. Benhida and +1
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- Signal Transduction, T lymphocytes, Cell communication, Antigens

The aim of this study was to develop a new approach to testing the impact of nickel antigen on in vitro cell-proliferation assay, to identify adverse reactions to casting alloys among orthodontic patients. Cellproliferation assay in vitro was used as the basic methodology to assess the influence of such variables as source of nickel antigen, type of serum used to supplement the culture medium, and number of cells in the culture. We selected 35 orthodontic patients who were classified as nickel sensitive and non-nickel sensitive, based on their clinical records. Our results showed that hexahydrated nickel sulfate at 10 g/mL, 10% of autologous sera, and 2 ϫ 10 5 cells was the best condition for inducing the most marked nickel proliferation response in vitro. This optimized method was able to distinguish nickel-sensitive from non-nickel-sensitive dental patients and also to discriminate those with positive skin tests. Our data suggest that continuous exposure to nickel casting alloys might lead to oral tolerance mechanisms that modulate nickel sensitivity, as evidenced by the lower cell proliferation index in patients undergoing orthodontic treatment over 24 months. Finally, our findings demonstrated a known nickel-induced type 2 immune response and a marked lack of type 1 immunity (interferon ␥) as the hallmarks of nickel-sensitive patients. Further studies are needed to clarify the major cell phenotype associated with this type 2 immune response and the lack of type 1 immunity observed in nickel-sensitive people.

- by Alexandre Sylvio
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- Dentistry, Biomedical Engineering, Immune response, Adolescent

To identify specific markers of rectovaginal endometriotic nodule vasculature, highly enriched preparations of vascular endothelial cells and pericytes were obtained from endometriotic nodules and control endometrial and myometrial tissue by laser capture microdissection (LCM), and gene expression profiles were screened by microarray analysis. Of the 18 400 transcripts on the arrays, 734 were significantly overexpressed in vessels from fibromuscular tissue and 923 in vessels from stromal tissue of endometriotic nodules, compared with vessels dissected from control tissues. The most frequently expressed transcripts included known endothelial cell-associated genes, as well as transcripts with little or no previous association with vascular cells. The higher expression in blood vessels was further corroborated by immunohistochemical staining of six potential markers, five of which showed strong expression in pericytes. The most promising marker was matrix Gla protein, which was found to be present in both glandular epithelial cells and vascular endothelial cells of endometriotic lesions, although it was barely expressed at all in normal endometrium. LCM, combined with microarray analysis, constitutes a powerful tool for mapping the transcriptome of vascular cells. After immunohistochemical validation, markers of vascular endothelial and perivascular cells from endometriotic nodules could be identified, which may provide targets to improve early diagnosis or to selectively deliver therapeutic agents.

It has long been suggested that the overall shape of the antigen combining site (ACS) of antibodies is correlated with the nature of the antigen. For example, deep pockets are characteristic of antibodies that bind haptens, grooves indicate peptide binders, while antibodies that bind to proteins have relatively flat combining sites. In 1996, MacCallum, Martin and Thornton used a fractal shape descriptor and showed a strong correlation of the shape of the binding region with the general nature of the antigen.

- by Abigail Colli
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- Molecular Biology, Antibodies, Autoimmune Disease, Mice

Specific targeting and delivery as well as the display of antigens on the surface of professional antigen-presenting cells (APCs) are key issues in the design and development of new-generation vaccines aimed at the induction of both humoral and cellmediated immunity. Prophylactic vaccination against infectious diseases in general aims at the induction of humoral immune responses to prevent infection. This humoral immune response is mediated by antibody-producing B cells. On the other hand, therapeutic immunisation against virally infected cells and tumour cells requires the induction of cytotoxic T lymphocytes (CTLs) that can specifically recognise and lyse infected cells or transformed tumour cells. The induction of Major Histocompatibility Complex (MHC) class I restricted CTL activity is optimally achieved by synthesis of antigens within APCs, for example, after immunisation with live attenuated virus. However, immunisation with live vaccines bears the risk of causing disease. Therefore, alternative vaccine delivery systems, which enable introduction of nonreplicating antigen into the MHC class I presentation pathway, are sought. Furthermore, for the induction of effective humoral and cellular responses, MHC class II restricted activation of T helper cells (Th cells) is required. Among other delivery systems, as described in this theme issue of Advanced Drug Delivery Reviews, virosomes seem ideally suited for delivery of antigens into both MHC pathways. In this review, we will focus on the use of virosomes as carrier vehicles for the intracellular delivery of protein antigens and DNA, and the induction of a cellular immune response against encapsulated protein antigens and proteins expressed by virosome-associated plasmids. D

- by Toos Daemen
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- Drug delivery, Infectious Disease, Advanced Drug Delivery Research, Membrane Fusion
- by Patricia Nelson and +1
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- Immune response, Cytokines, Multidisciplinary, Mycobacterium tuberculosis

Dendrimeric platforms such as MAPs can be synthesized either entirely by solid-phase methods (SPPS, direct approach) or by conjugation in solution of preformed, SPPS-made building blocks (indirect approach). Although MAPs and MAP-like constructs have been extensively and successfully used for various biological (mainly immunological) applications, experimental reports are most often lacking in chemical detail about their preparation and characterization. Here, we provide complete accounts of the synthesis and analytical documentation of MAPs and similar dendrimers by either all-SPPS (direct) or chemoselective thioether ligation (indirect) methods. We have chosen as model epitopes a 24-residue sequence of the ectodomain of protein M2 from influenza virus (M2e), which is found to be a rather challenging peptide epitope, and a far more manageable, shortened (12-residue) version of the same peptide. The advantages and shortcomings of both direct and indirect methods are discussed.

- by David Andreu
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- Peptide Science, Peptides, Amino Acid Sequence, Epitopes

We studied the feasibility of using halloysite clay nanotubes (HNTs) and carboxyl-functionalised multi-walled carbon nanotubes (COOH-MWCNTs) as antigen carriers to improve immune responses against a recombinant LipL32 protein (rLipL32). Immunisation using the HNTs or COOH-MWCNTs significantly increased the rLipL32specific IgG antibody titres (p < 0.05) of Golden Syrian hamsters. None of the vaccines tested conferred protection against a challenge using a virulent Leptospira interrogans strain. These results demonstrated that nanotubes can be used as antigen carriers for delivery in hosts and the induction of a humoral immune response against purified leptospiral antigens used in subunit vaccine preparations.

- by Odir Dellagostin
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- Carbon Dioxide, Medical Microbiology, Feasibility Studies, Lipoproteins
- by Giuseppe Cannella
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- Chronic kidney disease, Calcium, Cardiovascular disease, Complication

recognized (with respect to effectiveness and availability) are automated perimetry in the central 10 degrees and Amsler grid testing. Multifocal ERG and fundus autofluorescence may play an increasing role in centres where testing is available. 10 This case demonstrates the importance of regular screening for chloroquine toxicity. With severe toxicity, diffuse ocular involvement and permanent severe vision loss are possible, especially in more susceptible individuals. We recommend investigation for other causes in advanced or atypical cases, to rule out underlying systemic or ocular disease.

- by Louis Miller
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- Immunology, Immune response, Clinical Trial, Membrane Proteins

Non-immune (naïve) phage antibody libraries have become an important source of antibodies for reagent, diagnostic, and therapeutic use. To date, reported naïve libraries have been constructed in phagemid vectors as fusions to pIII, yielding primarily single copy (monovalent) display of antibody fragments. For this work, we subcloned the single chain Fv (scFv) gene repertoire from a naïve phagemid antibody library into a true phage vector to create a multivalently displayed scFv phage library. Compared to monovalently displayed scFv, multivalent phage display resulted in improved efficiency of display as well as antibody selection. A greater number of antibodies were obtained and at earlier rounds of selection. Such increased efficiency allows the screening for binding antibodies after a single round of selection, greatly facilitating automation. Expression levels of antigen-binding scFv were also higher than from the phagemid library. In contrast, the affinities of scFv from the phage library were lower than from the phagemid library. This could be overcome by utilizing the scFv in a multivalent format, by affinity maturation, or by converting the library to monovalent display after the first round of selection.

- by Arup Roy-Burman
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- Molecular Biology, Kinetics, Bacteriophages, Western blotting
- by Joseph El-on
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- Microbiology, Leishmania, Experimental parasitology, Temperature

Although the field of cancer immunotherapy is intensively investigated, there is still a need for generic strategies that allow easy, mild and efficient formulation of vaccine antigens. Here we report on a generic polymer-protein ligation strategy to formulate protein antigens into reversible polymeric conjugates for enhanced uptake by dendritic cells and presentation to CD8 T-cells. A N-hydroxypropylmethacrylamide (HPMA) based co-polymer was synthesized via RAFT polymerization followed by introduction of pyridyldisulfide moieties. To enhance ligation efficiency to ovalbumin, that is used as model protein antigen, protected thiols were introduced onto lysine residues and deprotected in situ in presence of the polymer. The ligation efficiency was compared for both the thiol-modified versus unmodified ovalbumin and the reversibility was confirmed. Furthermore the obtained nano-conjugates were tested in vitro for their interaction and

- by Martijn Schuijs
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- Engineering, Dendritic Cells, Biological Sciences, Cell line

During a primary immune response generally two classes of antibody are produced, immunogtobulin M (IgM) and immunoglobulin G (IgG). It is currently thought that some lymphocytes which initially produce IgM switch to the production of IgG with the same specificity for antigen. During a secondary immune response IgG is the predominant antibody made throughout the response. In this paper we address the question of why such apparently complicated modes of response should have been adapted by evolution.

- by Byron Goldstein and +1
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- Immune response, Mathematical Biology, Kinetics, Biological Sciences
- by Scott Commins
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- Immunology, Adolescent, Cats, Child

This paper describes a sequential staining procedure for double immunoenzymatic staining of pairs of antigens in frozen tissue sections and cell smears using monoclonal antibodies. This technique involves performance of an indirect immunoperoxidase sandwich (including development of the enzyme reaction) followed by an unlabelled immuno-alkaline phosphatase sandwich (the APAAP method). The two enzyme labels are revealed using DAB/H202 for peroxidase and naphthol AS-MX plus fast blue or fast red for alkaline phosphatase. When compared with a hapten-sandwich/biotin-avidin system, the sequential staining procedure proved to be simpler and more sensitive and was also more suitable for double immunoenzymatic staining when monoclonal antibodies were only available in small amounts. The sequential staining procedure is particularly useful for the identification of antigens distributed in different cell populations or in different sites (e.g., nucleus and cytoplasm or cell surface) of the same cell. In contrast, this method does not appear to be very suitable for demonstrating two antigens located in the same site (e.g., surface membrane) of the same cell for which purpose double immunofluorescence remains the first choice.

- by Zidane Abdulaziz
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- Immunology, Monoclonal Antibodies, Avidin, Biotin

A new low frequency antigen, Rba, has been found in three blood donors. Studies on their families show that the antigen is inherited as a Mendelian autosomal dominant character. Rba segregates independently from ABO, MNSs, P,, Rh, Kell, Duffy, Kidd, ACP, and PGM,. Anti-Rbn is not uncommon in sera containing multiple antibodies to low frequency antigens.

- by Marcela Contreras
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- Antibodies, Pedigree, Clinical Sciences, Antigens
- by Mark Berryman
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- Electron Microscopy, Immunohistochemistry, Liver, Intestines

The intestinal epithelium is a critical interface between the organism and its environment. The cell polarity and structural properties of the enterocytes, limiting the amount of antigen reaching the epithelial surface, form the basis of the integrity of the epithelium. However, apart from their participation in digestive processes, the enterocytes perform more than just a passive barrier function. The resistance of the tight junctions regulates the paracellular transport of antigens. Furthermore, the enterocytes take up and process antigens, involving two functional pathways. In the major pathway, enzymes in the lysosomes degrade the antigens. In the minor direct transcytotic pathway, the antigens are not degraded and are released into the interstitial space. Moreover, the enterocytes can present processed antigens directly to T cells and are often directly involved in immune processes. In inflammatory conditions, the properties of the epithelial barrier and the outcome of the immune response to luminal antigens can be changed.

- by Bruno Goddeeris
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- Microbiology, Immunology, Medical Microbiology, Intestinal absorption
- by Patricia Calarco
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- Cell Adhesion, Biological Sciences, Spleen, Cerebellum

A method is described which permits the adaption of ELISA techniques for measurement of antibody against bacteria~ polysaccharides. First, the polysaccharide antigen is covalently bound to poly-L-lysine, using cyanuric chloride as the coupling agent. The poly-L-lysine then adsorbs to the walls of plastic tubes, thus immobilizing the polysaccharide coupled to the poly-L-lysine. The method is simple, rapid, and utilizes small amounts of polysaccharide antigen.

- by Brian Gray
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- Immunology, Streptococcus pneumoniae, Adsorption, Methods

We report 3 cases of lymphoid neoplasms with mixed lineage features of T-, NK-, or B-cell marker expression and clonal gene rearrangement for both T-cell receptor and immunoglobulin light chain IgK. A characteristic of our cases was the lack of expression of the specific B-cell transcription factor, Pax5, which is essential for maintaining the identity and function of mature B cells during late B lymphopoiesis. In the absence of Pax5, B cells in vitro can differentiate into macrophages, dendritic cells, granulocytes, and T/NK cells. Methylation analysis of the Pax5 gene in our cases suggests that its inactivation by this epigenetic event in a committed or mature B cell, before plasma cell differentiation, may well be a common pathogenetic mechanism in mature lymphoid neoplasms with expression of multilineage antigens. In particular, case 1 may represent a mixed NK-and B-cell lineage; and cases 2 and 3 may represent mixed T and B-cell lineage, respectively. Aberrations in the DNA methylation patterns are currently recognized as a hallmark of human cancer. Cases with aberrant phenotypes require molecular analysis for lineage assignment. Studies of such cases may be helpful to better elucidate whether they represent a distinct entity with clinical, immunophenotypic, and molecular characteristics or an incidental phenomenon during malignant transformation. Interestingly, these cases were all characterized by poor clinical outcome.

- by Stefano Pileri
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- Immunohistochemistry, Lymphoma, Natural Killer cells, DNA methylation

Red deer (Cervus elaphus) and white-tailed deer (Odocoileus virginianus) are hosts for different tick species and tick-borne pathogens and play a role in tick dispersal and maintenance in some regions. These factors stress the importance of controlling tick infestations in deer and several methods such as culling and acaricide treatment have been used. Tick vaccines are a cost-effective alternative for tick control that reduced cattle tick infestations and tick-borne pathogens prevalence while reducing the use of acaricides. Our hypothesis is that vaccination with vector protective antigens can be used for the control of tick infestations in deer. Herein, three experiments were conducted to characterize (1) the antibody response in red deer immunized with recombinant BM86, the antigen included in commercial tick vaccines, (2) the antibody response and control of cattle tick infestations in white-tailed deer immunized with recombinant BM86 or tick subolesin (SUB) and experimentally infested with Rhipicephalus (Boophilus) microplus, and (3) the antibody response and control of Hyalomma spp. and Rhipicephalus spp. field tick infestations in red deer immunized with mosquito akirin (AKR), the SUB ortholog and candidate protective antigen against different tick species and other ectoparasites. The results showed that deer produced an antibody response that correlated with the reduction in tick infestations and was similar to other hosts vaccinated previously with these antigens. The overall vaccine efficacy was similar between BM86 (E = 76%) and SUB (E = 83%) for the control of R. microplus infestations in white-tailed deer. The field trial in red deer showed a 25-33% (18-40% when only infested deer were considered) reduction in tick infestations, 14-20 weeks after the first immunization. These results demonstrated that vaccination with vector protective antigens could be used as an alternative method for the control of tick infestations in deer to reduce tick populations and dispersal in regions where deer are relevant hosts for these ectoparasites.

- by Francisco Ruiz-fons and +1
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- Vaccines, Biological Sciences, Red deer, Deer

B cells encounter both soluble Ag (sAg) and membrane-associated Ag (mAg) in the secondary lymphoid tissue, yet how the physical form of Ag modulates B cell activation remains unclear. This study compares actin reorganization and its role in BCR signalosome formation in mAg-and sAg-stimulated B cells. Both mAg and sAg induce F-actin accumulation and actin polymerization at BCR microclusters and at the outer rim of BCR central clusters, but the kinetics and magnitude of F-actin accumulation in mAgstimulated B cells are greater than those in sAg-stimulated B cells. Accordingly, the actin regulatory factors, cofilin and gelsolin, are recruited to BCR clusters in both mAg-and sAg-stimulated B cells but with different kinetics and patterns of cellular redistribution. Inhibition of actin reorganization by stabilizing F-actin inhibits BCR clustering and tyrosine phosphorylation induced by both forms of Ag. Depolymerization of F-actin leads to unpolarized microclustering of BCRs and tyrosine phosphorylation in BCR microclusters without mAg and sAg, but with much slower kinetics than those induced by Ag. Therefore, actin reorganization, mediated via both polymerization and depolymerization, is required for the formation of BCR signalosomes in response to both mAg and sAg.

- by Wenxia Song
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- Immunology, Biology, Immunology of the Gut, Medicine

When an unknown amount of antigen is allowed to diffuse radially from a well in a uniformly thin layer of antibody-containing agar for a sufficient time to allow all antigen to combine, the final area reached by the precipitate is directly proportional to the amount of antigen employed, and inversely proportional to the concentration of antibody. It is also shown that the temperature at which the plates are incubated has no perceptible influence upon the results. By standardizing the technical conditions of the experiment it is possible to use this principle for the immunochemical determination of antigens. In the experimental albumin-antialbumin system here described, the lower limit of the method was found to correspond to 0·0025 μg of antigen, and to an antigen concentrations of 1·25 μg per ml. The standard deviation of the antigen determinations was less than 2 per cent of the mean.

- by angelo angelo
- •
- Immunology, Molecular Immunology, Immunochemistry, Rabbits

Among experimental models of perinatal ischemic stroke, Renolleau's model mimics selected types of stroke at birth, including ischemia and reperfusion. However, its behavioural consequences on development have been poorly described. Here, ischemia-reperfusion was performed in 7-day-old Wistar rats. Between the ages of 9 and 40 days, sensorimotor and memory functions were assessed. The infarcted area was analysed by immunohistochemistry at 40 days of age. The remaining lesion was in the parietal cortex, in the form of a cone-shaped area. This area contained glial cells but neither neurons nor macrophages. Transient focal neonatal ischemia led to sensorimotor alterations in early adulthood, such as postural asymmetry, motor coordination and somatosensory deficits, and hyperactivity, as well as cognitive impairments, such as spatial reference memory deficits. Based on these results, we propose here a selection of behavioural tests that should constitute meaningful tools for assessing sensory and cognitive functions after experimental neonatal ischemic stroke.

- by Etienne Jacotot and +1
- •
- Immunohistochemistry, Memory, Brain, Pregnancy

Spinal cord injury presents a significant therapeutic challenge since the treatments available are mostly vain. The use of stem cells to treat this condition represents a promising new therapeutic strategy; therefore, a variety of stem cell treatments have been recently examined in animal models of CNS trauma. In this work, we analyzed the effects of third trimester amniotic fluid cells in a mouse model of spinal cord injury. Among the different cultures used for transplantation, some were able to induce a significant improvement in motor recovery (cultures #3.5, #3.6 and #7.30), evaluated by means of open field free locomotion. All effective cell cultures expressed the surface marker nerve/glial antigen 2, ortholog of the human chondroitin sulfate proteoglycan 4, which is present on several types of immature progenitor cells. The improved motor functional recovery was correlated with higher myelin preservation in the ventral horn white matter and an increased vascularization in the peri-lesion area. Real-Time PCR analysis showed higher expression levels of vascular endothelial growth factor and hypoxia-inducible factor-1α mRNA two days after cells transplantation compared to PBS-treated animals, indicating that an angiogenic pathway might have been activated by these cells, possibly through the production of hepatocyte growth factor. This cytokine appears to be produced mostly in filtering organs, such as the lung, of the transplanted animals and is likely released in the blood suggesting an endocrine role of hepatocyte growth factor in targeting the injury site.

- by Alfredo Gorio and +2
- •
- Psychology, Induced Pluripotent Stem Cells (I Psc), Treatment Outcome, Stem Cell Transplantation

Lipid peroxidation has been implicated in a variety of diseases. 4-Hydroxy-2-nonenal (HNE), a major oxidation by-product, is cytotoxic, mutagenic, and genotoxic, being involved in disease pathogenesis. Naturally occurring pharmacologically active small molecules are very attractive as natural nonsteroidal anti-inflammatory agents. Interest has greatly increased recently in the pharmacotherapeutic potential of curcumin, the yellow pigment found in the rhizomes of the perennial herb Curcuma longa (turmeric). Curcumin is efficacious against colon cancer, cystic fibrosis, and a variety of other disorders. Curcumin's full pharmacological potential is limited owing to its extremely limited water solubility. We report here that the water solubility of curcumin could be increased from 0.6 g/ml to 7.4 g/ml (12-fold increase) by the use of heat. Spectrophotometric (400-700 nm) and mass spectrometric profiling of the heat-extracted curcumin displays no significant heat-mediated disintegration of curcumin. Using an enzyme-linked immunosorbent assay that employed HNE modification of solid-phase antigen, we found that the heat-solubilized curcumin inhibited HNE-protein modification by 80%. Thus, inhibition of HNE modification may be a mechanism by which curcumin exerts its effect. We also report a simple assay to detect curcumin spectrophotometrically. Curcumin was solubilized in methanol and serially diluted in methanol to obtain a set of standards that were then read for optical density at 405 nm. Curcumin in the heatsolubilized samples was determined from this standard. Heat-solubilized curcumin should be considered in clinical trials involving curcumin, especially in the face of frustrating results obtained regarding curcumin-mediated correction of cystic fibrosis defects. 567 tion of cellular targets would be critical as pharmacotherapeutic agents.

- by Biji Kurien
- •
- Heat Treatment, Antioxidants, Curcumin, Surface plasmon resonance

This review highlights the recent developments in the area of nanocarrier-based mucosal delivery of therapeutic biomolecules and antigens. Macromolecular drugs have the unique power to tackle challenging diseases but their structure, physicochemical properties, stability, pharmacodynamics, and pharmacokinetics place stringent demands on the way they are delivered into the body (e.g., inability to cross mucosal surfaces and biological membranes). Carrier-based drug delivery systems can diminish the toxicity of therapeutic biomolecules, improve their bioavailability and make possible their administration via less-invasive routes (e.g., oral, nasal, pulmonary, etc.). Thus, the development of functionalized nanocarriers and nanoparticle-based microcarriers for the delivery of macromolecular drugs is considered an important scientific challenge and at the same time a business breakthrough for the biopharmaceutical industry. In order to be translated to the clinic the nanocarriers need to be biocompatible, biodegradable, stable in biological media, non-toxic and non-immunogenic, to exhibit mucoadhesive properties, to cross mucosal barriers and to protect their sensitive payload and deliver it to its target site in a controlled manner, thus increasing significantly its bioavailability and efficacy.

- by Costas Kiparissides
- •
- Biomedical Engineering, Vaccines, Nanoparticles, Controlled release

Rabbit antiserum raised against the crude extract of normal human prostatic tissue contained antibodies to a prostatic tissue-specific antigen as shown by immunoprecipitation techniques. Using this antiserum a prostate antigen was detected in normal, benign hypertrophic, and malignant prostatic tissues, but not in other human tissues. The prostate antigen was purified to homogeneity from prostatic tissues and showed a single protein band on analytical polyacrylamide gel electrophoresis and isoelectric focusing. This report thus presents the first demonstration of the purification of a prostate-specific antigen that does not represent prostatic acid phosphatase.

- by luis valenzuela
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- Urology, Technique, Prostate, Immunoprecipitation

The immune response of infected or immunized dromedaries contains a diverse repertoire of conventional and heavy chain-only antibodies, both functional in antigen binding. By de fi nition, a heavy chain antibody is devoid of a light chain and in the case of the heavy chain antibodies in camelids the CH1 domain is also missing. Consequently a camelid heavy chain antibody associates with its cognate antigen via a single domain, the variable heavy chain domain of a heavy chain antibody or VHH. An antigen-speci fi c VHH, also known as Nanobody, with excellent biochemical properties can be obtained in various ways. Their recombinant expression provides access to user-friendly tools for a wide variety of applications.

- by Serge Muyldermans
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- Molecular Biology, Antibodies, Protein Binding, Antigens

Objective: To compare the enzyme activity of different presentations of papain solution to validate in-house preparations. Methods: Two papain solutions were prepared, and the third presentation was a commercial solution. Tests were carried out with samples of red cells typed as weak RhD. Results: In-house prepared papain solutions showed similar enzyme reactivity, and statistically no differences compared to the enzyme activity of the commercial solution. Conclusion: Evaluating the cost-benefit ratio, the in-house prepared papain solutions present more economic advantages, and can be incorporated into immunohematological routines as a way to cope with periods of financial crisis and cost-containment policies.

- by Journal einstein (São Paulo)
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- Enzymes, Allergy and Immunology, Antigens, Papain
- by Roberto Furlan
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- Multiple sclerosis, Mice, Callithrix, Experimental Autoimmune Encephalomyelitis
- by Biji Kurien
- •
- Immunology, Western blotting, Diffusion, Proteins

Camel single-domain antibody fragments (VHHs) are promising tools in numerous biotechnological and medical applications. However, some conditions under which antibodies are used are so demanding that they can be met by only the most robust VHHs. A universal framework offering the required properties for use in various applications (e.g. as intrabody, as probe in biosensors or on micro-arrays) is highly valuable and might be further implemented when employment of VHHs in human therapy is envisaged. We identified the VHH framework of cAbBCII10 as a potential candidate, useful for the exchange of antigen specificities by complementarity determining region (CDR) grafting. Due to the large number of CDR-H loop structures present on VHHs, this grafting technique was expected to be rather unpredictable. Nonetheless, the plasticity of the cAbBCII10 framework allows successful transfer of antigen specificity from donor VHHs onto its scaffold. The cAbBCII10 was chosen essentially for its high level of stability (47 kJ mol K1 ), good expression level (5 mg l K1 in E. coli) and its ability to be functional in the absence of the conserved disulfide bond. All five chimeras generated by grafting CDR-Hs, from donor VHHs belonging to subfamily 2 that encompass 75% of all antigen-specific VHHs, on the framework of cAbBCII10 were functional and generally had an increased thermodynamic stability. The grafting of CDR-H loops from VHHs belonging to other subfamilies resulted in chimeras of reduced antigen-binding capacity.

- by A. Matagne
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- Molecular Biology, Biotechnology, Stability, Macromolecular X-Ray Crystallography
- by Yvonne Chen
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- Materials Science, Thermodynamics, International Business, Molecular Biology
- by Julia Pinkhasov
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- Medicinal Plants, Mice, High Pressure Liquid Chromatography, Plant extracts

biochemical activities of Vipera berus (European viper) venom. Toxicon 31, 743-753, 1993.-Vipera berus is widely distributed throughout the northern part of Europe and Asia. Characterization of several toxic effects of its venom in the mouse, as well as of in vitro enzymatic activities was performed. Vipera berus venom displayed in vitro proteolytic, fibrinolytic, anticoagulant, and phospholipase AZ activities . The i.p. LD s p Of the venom for Swiss mice was 0.86 aug/g (95% confidence limits 0.71-1.01 hg/g). Sïgnificant local tissue-damaging effects, including edema, hemorrhage and myonecrosis, were observed . The local edema was characterized by rapid onset, reaching a maximum after 0.~1 hr, and with dose-dependent persistence. The hemorrhagic potency was measured by a skin tent, giving a minimum hemorrhagic dose value of 3.2 hg. The venom also induced a moderate local myonecrosis, evidenced by histological evaluation of injected tissue (gastrocnemius), and by biochemical parameters (increase of plasma creatine kinase activity, and decrease of muscle residual MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide)-reducing activity). Characterization of the venom by SDS-polyacrylamide gel electrophoresis revealed 10 (reduced) or 11 (unreduced) main protein bands, which were further analyzed in relation to mol. wt and relative concentration by densitometry . A rabbit antiserum to V. berus venom recognized all main venom bands by immunoblotting. This antiserum cross-reacted to a variable extent with several crotaline venoms, as assessed by enzyme immunoassay.

- by Bruno Lomonte
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- Antibodies, Mice, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Edema