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This is the fourth contribution within an ongoing series dedicated to the phylogeny and taxonomy of powdery mildews. This particular installment undertakes a comprehensive evaluation of a group previously referred to as the "Uncinula lineage" within Erysiphe. The genus Erysiphe is too large to be assessed in a single paper; thus, the treatment of Erysiphe is split into three parts, according to phylogenetic lineages. The first paper, presented here, discusses the most basal lineage of Erysiphe and its relationship to allied basal genera within tribe Erysipheae (i.e., Brasiliomyces and Salmonomyces). ITS+28S analyses are insufficient to resolve the basal assemblage of taxa within the Erysipheae. Therefore, phylogenetic multilocus examinations have been carried out to better understand the evolution of these taxa. The results of our analyses favor maintaining Brasiliomyces, Bulbomicroidium, and Salmonomyces as separate genera, at least for the interim, until further phylogenetic multilocus data are available for additional basal taxa within the Erysipheae. The current analyses also confirmed previous results that showed that the "Uncinula lineage" is not exclusively composed of Erysiphe species of sect. Uncinula but also includes some species that morphologically align with sect. Erysiphe, as well as species that had previously been assigned to Californiomyces and Typhulochaeta. Numerous sequences of Erysiphe species from the "Uncinula lineage" have been included in the present phylogenetic analyses and were confirmed by their position in well-supported species clades. Several species have been sequenced for the first time, including Erysiphe clintonii, E. couchii, E. geniculata, E. macrospora, and E. parvula. Ex-type sequences are provided for 16 taxa including E. nothofagi, E. trinae, and E. variabilis. Epitypes are designated and ex-epitype sequences are added for 18 taxa including Erysiphe carpophila, E. densa, and U. geniculata var. carpinicola. The new species Erysiphe canariensis is described, and the new names E. hosagoudarii and E. pseudoprunastri and the new combination E. ampelopsidis are introduced. [ABSTRACT FROM AUTHOR]

Despite the enormous clinical importance for xenotransplantation, very little is known about the 3D structural basis for natural antibody recognition of the major carbohydrate xenoantigen, its derivatives and their peptide counterparts. Fundamentally, understanding the nature of peptide-carbohydrate cross-reactivity is necessary to allow a rational design of useful inhibitors. To satisfy this need, we have initiated a project to investigate the structural aspects of antibody recognition of carbohydrate xenoantigens and their peptide mimics by molecular docking. We aim to analyse critical ligand-protein interactions with a focus on identifying the degree of structural carbohydrate mimicry exhibited by peptide ligands. In this paper, we present docking simulations of complexes between a prototypical xenoreactive monoclonal antibody and two ligands: the major carbohydrate xenoantigen, terminal galactose-α(1,3)-galactose epitope [Galα(1,3)Gal] and a peptide inhibitor Galpep1 (DAHWESWL). [ABSTRACT FROM AUTHOR]

Systemic lupus erythematosus (SLE or lupus) is a complex disease with a multifactoral etiology, with genetic, hormonal, and environmental influences. Molecular mimicry as a result of viral infection may contribute to the development of lupus. The pattern of autoantibody development in lupus is consistent with initiation through molecular mimicry, as the initial autoantigenic epitopes that have been observed are limited and cross-reactive with viral proteins. Autoantibody specificity may then later diversify to other autoantigens through B-cell epitope spreading. Epstein-Barr virus (EBV) is an excellent candidate to be involved in molecular mimicry in lupus. EBV infection has been associated with lupus through serological and DNA studies. Infection with EBV results in the production of the viral protein Epstein-Barr virus nuclear antigen-1 (EBNA-1), antibodies against which cross-react with lupus-associated autoantigens, including Ro, Sm B/B', and Sm D1, in lupus patients. The immune response against EBV, and EBNA-1 in particular, differs among lupus patients and healthy controls, with controls maintaining a limited humoral response and failing to produce long-standing cross-reactive antibodies. We hypothesize that the humoral immune response to EBNA-1 in susceptible individuals leads to the generation of cross-reactive antibodies. Through the process of epitope spreading, these cross-reactive antibodies target additional, non-cross reactive autoepitopes, spread to additional autoantigens, and become pathogenic, leading eventually to clinical lupus. This paper reviews some of the current literature supporting roles for EBV exposure and epitope spreading in SLE. [ABSTRACT FROM AUTHOR]

H9N2 subtype of avian influenza virus (AIV) is primarily a bird virus, which is widespread in clinical avian disease, and reported in cases of human infection. As one of the surface proteins of AIV, the neuraminidase (NA) protein plays an important role mainly in viral budding. However, vaccine development and detection methods for NA of H9N2 AIVs are in urgent clinical need. In this study, a truncated NA gene (205–900 bp) was cloned from the NA sequence of H9N2 strain, and then expressed using pET-28a (+) vector. This purified recombinant NA protein was used to immunize BALB/c mice, and the monoclonal antibodies were screened through the indirect enzyme-linked immunosorbent assay (ELISA). Next, eight prokaryotic expression vectors were constructed for epitope identification. After cell fusion, three hybridoma cell lines producing the antibodies special to NA protein were screened by ELISA, western blotting, and indirect immunofluorescence; these were named 1B10, 2B6, and 5B2, respectively. Epitope scanning techniques were used to identify three B-cell epitopes recognized by these three monoclonal antibodies, 196KNATASIIYDGMLVD210, 210DSIGSWSKNIL220 and 221RTQESECVCI230. The subsequent homology analysis revealed the three epitopes were highly conserved in H9N2 AIV strains. The structural predictions of the antigenic epitopes indicated that all three epitopes were located in the catalytic region of NA. These results provide a basis for studying the function of the NA protein of H9N2 AIV and technical support for the development of a universal detection method based on anti-NA monoclonal antibodies. [ABSTRACT FROM AUTHOR]

Introduction: The availability of big data sets (‘OMICS’) has greatly impacted fundamental and translational science. High-throughput analysis of HLA class I and II associated peptidomes by mass spectrometry (MS) has generated large datasets, with the last decade witnessing tremendous growth in the breadth and number of studies. Areas covered: For this, we first analyzed naturally processed peptide (NP) data captured within the IEDB to survey and characterize the current state of NP data. We next asked to what extent the NP data overlap with existing T cell epitope and MHC binding data. Expert commentary: The current collection of NP data represents a large and diverse set of class I/II peptides mostly derived from self-antigens. These data overlap only marginally with existing immunogenicity and binding data and it is thus difficult to ascertain the correspondence between the different assay methodologies. This highlights a need for unbiased studies benchmarking in model antigen systems how well MHC binding and NP data predicts immunogenicity. Going forward, efforts at generating an integrated process for capturing all NP, curating associated metadata and accessing NP data from an immunological viewpoint will be important for development of novel methods for identifying optimal target antigens and for class I and II epitope prediction. [ABSTRACT FROM PUBLISHER]

Neuropilin-1 (NRP-1) is a non-tyrosine kinase receptor and when overexpressed, leads to angiogenesis. High expression of NRP-1 has been observed in various cancers. Unique characteristic of nanobodies (small size, high affinity and stability, and ease production) make them potential therapeutic tools. Oligoclonal nanobodies which detect multiple functional epitopes on the target antigen could be potential tools for inhibition of cancer resistance problems due to escape variant of tumor cells. In this study, oligoclonal anti-NRP-1 nanobodies were selected from camel immune library and their binding activities as well as in vitro functionality were evaluated. Anti-NRP-1 nanobodies were expressed in an Escherichia coli host, and purified using nickel affinity chromatography. The effect of each individual and oligoclonal nanobodies on human endothelial cells were evaluated by MTT, Tube formation, and migration assay as well. Results showed that oligoclonal anti-NRP-1 nanobodies detected different epitopes of NRP-1 antigen and inhibited in vitro angiogenesis of human endothelial cells better than each individual nanobody. Results indicate promising oligoclonal anti-NRP-1 nanobodies for inhibition of angiogenesis. [ABSTRACT FROM AUTHOR]

The second contribution to a new series devoted to the phylogeny and taxonomy of powdery mildews is presented. An overview of Neoerysiphe species is given, including references to ex-type sequences or, if unavailable, representative reference sequences for phylogenetic-taxonomic purposes are provided. The new species N. stachydis is described, and Striatoidium jaborosae is reduced to synonymy with Neoerysiphe macquii. Epitypes with ex-epitype sequences are designated for Alphitomorpha ballotae, A. labiatarum, Erysiphe galii, E. chelones, and E. galeopsidis. Based on phylogenetic analyses, it has been demonstrated that Neoerysiphe cumminsiana is confined to its type host, Roldana hartwegii (= Senecio seemannii), and other North and South American parasites on Asteraceae hosts, previously assigned to this species, pertain to N. macquii. The first record of N. macquii from Europe (Germany) on cultivated Bidens aurea was confirmed by sequencing. Sequence analysis of type material of N. rubiae reveals that this species should be excluded from Neoerysiphe; however, the true affinity of this taxon is not yet clear. [ABSTRACT FROM AUTHOR]

Aim: Lujo is a modern zoonotic virus that is potentially fatal and spreads by bodily fluids. In this research, immunoinformatic tools are used to build a vaccine. Methodology: The epitopes of cytotoxic T-lymphocytes, helper T-lymphocytes and linear B-lymphocytes were predicted from the most antigenic protein. The designed vaccine's physiochemical properties and 3D structure have been forecasted. Low free energy and strong binding affinity estimated in molecular docking against Toll-like receptor 4 (TLR4) and dynamic simulation. Furthermore, in silico cloning in the Escherichia coli K12 host system was performed for high level of expression. Conclusion: Finally, immune simulation was used to determine immune responses to the vaccine that was formulated confirming the developed vaccine as a good candidate against Lujo virus. [ABSTRACT FROM AUTHOR]

Gambierdiscus species produce toxins that cause ciguatera fish poisoning (CFP), the most common nonbacterial illness associated with fish consumption worldwide. Understanding the role that individual Gambierdiscus species play in causing CFP is hampered because the morphologically similar species in the genus are difficult to distinguish. Ambiguities in the description of the type species Gambierdiscus toxicus also exist. This paper presents detailed line drawings along with additional scanning electron microscopy (SEM) and light micrographs to more fully characterize the six known Gambierdiscus species in addition to describing four new species, Gambierdiscus caribaeus sp. nov., Gambierdiscus carolinianus sp. nov., Gambierdiscus carpenteri sp. nov., and Gambierdiscus ruetzleri sp. nov. All four of the new species are photosynthetic and epibenthic and have a Kofoidian plate formula of Po, 3′, 7″, 6C, 6 or 7S, 5′″, 1p, and 2‴′. Establishment of these species is supported by both morphological differences and three independent phylogenetic analyses utilizing small-subunit, as well as D1-D3 and D8-D10 large-subunit rDNA gene sequences. During the course of this study it became apparent that the description of the type species, G. toxicus, included more than one species. Since no type material exists we propose that Fig. 1 in Adachi and Fukuyo (1979) be designated as the lectotype for this species and that the epithecal view of isolate GTT-91 shown in Fig. 1 from Chinain et al. (1999) be designated as the epitype. The GTT-91 isolate has been well characterized genetically and the original SEM stub of this isolate archived at the Smithsonian Institution has been re-examined to provide a more detailed morphological analysis of the epitype. [ABSTRACT FROM AUTHOR]

Aim: In order to curb the transmission of SARS-CoV-2, nation-wide travel restrictions at different levels were implemented in different countries. Country-specific mutational profile may exist and have an impact on vaccine efficacy. Materials & methods: We identified nonsynonymous mutations in approximately 215,000 SARS-CoV-2 sequences during the first year of the pandemic in 35 countries. Mutational profiles on a bimonthly basis were traced over time. We also examined the mutations that overlapped with the spike protein vaccine epitopes. Results: Several new mutations emerged over time and were dominating in specific countries. Many nonsynonymous mutations were within multiple spike protein epitopes that might impact the vaccine efficacy. Conclusion: Our study advocates requirement of active monitoring of country-specific mutations and vaccine efficacies in respective countries. [ABSTRACT FROM AUTHOR]

Carbohydrate epitopes on the arabinogalactan protein (AGP) gum arabic recognised by a panel of seven monoclonal antibodies (MAbs), raised in a previous study, have been further characterised. These MAbs have now been divided into three groups based on their response to mild hydrolysis and elimination (neutral hydrolysis) when heated in buffers at different pH values. We have also investigated the binding of these antibodies to three fractions of gum arabic separated by hydrophobic interaction chromatography. MAb groups I and III produce consistent responses when tested against heat-treated whole and fractionated gum arabic, whilst group II display a varied response suggesting that these MAbs recognise complex epitopes, the structure of which is not immediately apparent. It appears that neutral hydrolysis is capable of increasing or decreasing the affinity of certain epitopes, whilst hydrolysis with a strong acid (TFA; tri-fluoro acetic acid) under harsh conditions reduces the affinity for all antigens. The results obtained have shown that the MAbs can be divided into three categories, two of which have distinctive and easily reproducible responses to treatment of the gum and would therefore be suitable for the investigation of Acacia gums which have been subjected to food preparation procedures. [ABSTRACT FROM AUTHOR]

New SARS-CoV-2 mutants have been continuously indentified with enhanced transmission ever since its outbreak in early 2020. As an RNA virus, SARS-CoV-2 has a high mutation rate due to the low fidelity of RNA polymerase. To study the single nucleotide polymorphisms (SNPs) dynamics of SARS-CoV-2, 158 SNPs with high confidence were identified by deep meta-transcriptomic sequencing, and the most common SNP type was C > T. Analyses of intra-host population diversity revealed that intra-host quasispecies' composition varies with time during the early onset of symptoms, which implicates viral evolution during infection. Network analysis of co-occurring SNPs revealed the most abundant non-synonymous SNP 22,638 in the S glycoprotein RBD region and 28,144 in the ORF8 region. Furthermore, SARS-CoV-2 variations differ in an individual's respiratory tissue (nose, throat, BALF, or sputum), suggesting independent compartmentalization of SARS-CoV-2 populations in patients. The positive selection analysis of the SARS-CoV-2 genome uncovered the positive selected amino acid G251V on ORF3a. Alternative allele frequency spectrum (AAFS) of all variants revealed that ORF8 could bear alternate alleles with high frequency. Overall, the results show the quasispecies' profile of SARS-CoV-2 in the respiratory tract in the first two months after the outbreak. [ABSTRACT FROM AUTHOR]

The ongoing life-threatening pandemic of coronavirus disease 2019 (COVID-19) has extensively affected the world. During this global health crisis, it is fundamentally crucial to find strategies to combat SARS-CoV-2. Despite several efforts in this direction and continuing clinical trials, no vaccine has been approved for it yet. To find a preventive measure, we have computationally designed a multi-epitopic subunit vaccine using immuno-informatic approaches. The structural proteins of SARS-CoV-2 involved in its survival and pathogenicity were used to predict antigenic epitopes. The antigenic epitopes were capable of eliciting a strong humoral as well as cell-mediated immune response, our predictions suggest. The final vaccine was constructed by joining the all epitopes with specific linkers and to enhance their stability and immunogenicity. The physicochemical property of the vaccine was assessed. The vaccine 3D structure prediction and validation were done and docked with the human TLR-3 receptor. Furthermore, molecular dynamics simulations of the vaccine-TLR-3 receptor complex are employed to assess its dynamic motions and binding stability in-silico. Based on this study, we strongly suggest synthesizing this vaccine, which further can be tested in-vitro and in-vivo to check its potency in a cure for COVID-19. [ABSTRACT FROM AUTHOR]

Podosphaera tridactyla (s. lat.) is a powdery mildew species occurring on a wide range of Prunus spp. almost worldwide. We have investigated the phylogeny of the Po. tridactyla complex, with special emphasis on potential aspects of cryptic speciation. The results suggested that Po. tridactyla represents a species complex consisting of at least 12 different species. Based on detailed morphological examinations and molecular sequence analyses, we propose dividing Po. tridactyla s. lat. into 10 species, encompassing 7 new species (Po. ampla, Po. pruni-avium, Po. pruni-cerasoidis, Po. prunigena, Po. pruni-lusitanicae, Po. prunina, and Po. pruni-japonicae) and 3 known species (Po. longiseta, Po. salatai, and Po. tridactyla s. str.). Oidium passerinii on Pr. laurocerasus is confirmed as a synonym of Po. tridactyla s. str. Epitypes are designated for Po. tridactyla and Oidium passerinii. [ABSTRACT FROM AUTHOR]

Keywords: Cross-protective immunity; human rhinovirus; immune epitopes; rational vaccine design; rhinovirus capsid proteins Although the antipeptide polyclonal antibody titers were much lower compared to a rabbit antiserum raised against purified whole-virion RV14 virus, these antibodies were broadly reactive and effectively neutralized about 60% of 48 rhinovirus serotypes tested, while only a few RV serotypes were neutralized by the anti-RV14 antiserum. J Immunol Baltim Md. 1950; 2016 (197): 3214 - 3224.. • Several conserved epitopes of rhinovirus capsid proteins were experimentally confirmed and their role in immune response to rhinovirus infection was shown. Cross-protective immunity, human rhinovirus, immune epitopes, rational vaccine design, rhinovirus capsid proteins. [Extracted from the article]

Introduction: Beta-lactams (BL) are the main cause of allergic drug reactions mediated by specific immunological mechanisms. Reactions can be IgE or effector T-cell mediated. The new antigenic determinants are recognized by the immunological system in the context of the common beta-lactam structure or the specific differences in the side chains of the antibiotics of this family plus the protein carrier. Areas covered: We have reviewed the recent clinical literature concerning new clinical entities, the progress in diagnosis including the difficulties for in in vivo and or in vitro testing as well as the new algorithms proposed for delabelling subjects classified as allergic to beta-lactams, and recommendations for desensitization procedures. Expert opinion: The knowledge gained over the last years on beta-lactam hypersensitivity has enabled a better understanding and management of cases with allergic reactions to beta-lactams. [ABSTRACT FROM AUTHOR]

The expression of protamines and the binding of these small arginine-rich proteins to DNA complete the process of spermatid chromatin reorganization and the global inactivation of the male’s haploid genome that occurs during the final stages of sperm development in mammals. While a number of anti-protamine antibodies have been created during the last 40 years, only a few have proven useful for detecting the presence of the protamines, determining the timing of their expression and deposition in chromatin, and investigating their structure and function in both maturing spermatids and sperm. The aim of this effort was to develop an additional set of monoclonal antibodies (MAbs) that not only recognize new P1 and P2 protamine epitopes but also work well as IHC reagents for detecting and identifying mammalian protamines in testicular tissue and ejaculated sperm. Using a combination of native and synthetic human protamines as antigens, 38 hybridoma clones recognizing human protamine P1 or P2 were generated. Antibodies produced by the 12 best clones were screened for selectivity by enzyme-linked immunosorbent assay, and two were found to recognize only human protamine P1 or P2, while a number of the others bound to both the human and mouse proteins. One MAb recognized every protamine tested. All the antibodies, including one recognizing stallion P1 and another recognizing stallion P2, bound to the native protamines in the chromatin of spermatids or sperm. While the majority labeled only elongating spermatids or sperm, several of the antibodies were found to also bind to the cytoplasm or nuclei of cells that lack protamine, which indicates these MAbs must recognize epitopes present in the protamines that are also found in other proteins. Thirteen overlapping human protamine P1 peptides were synthesized and subsequently used to identify the epitopes recognized by the six best antibodies. Abbreviations: BSA: bovine serum albumin; ELISA: enzyme-linked immunosorbent assay; HCl: hydrochloric acid; IHC: immunohistochemistry; i.p: intraperitoneal; LIS: lithium diiodosalicylate; MAb: monoclonal antibody; PBS: phosphate buffered saline [ABSTRACT FROM AUTHOR]

In this study, 49 species of Hydnum are recognized worldwide. Twenty-two of them are described here as new species. Epitypes are proposed for H. repandum and H. rufescens. The majority of the species are currently known only from a single continent. The barcodes produced in this study are deposited in the RefSeq database and used as a basis to name species hypotheses in UNITE. Eleven infrageneric clades recovered in a phylogenetic analysis are supported by morphological characteristics and formally recognized: subgenera Alba, Hydnum, Pallida, and Rufescentia; sections Hydnum, Olympica, Magnorufescentia, and Rufescentia; and subsections Mulsicoloria, Rufescentia, and Tenuiformia. [ABSTRACT FROM AUTHOR]

Salivarian trypanosomes evade the host immune system by continually swapping their protective variant surface glycoprotein (VSG) coat. Given that VSGs from various trypanosome stocks exhibited cross-reactivity (Camargo et al., Vet. Parasitol. 207, 17-33, 2015), we analyzed here which components are the antigenic determinants for this cross-reaction. Soluble forms of VSGs were purified from four Venezuelan animal trypanosome isolates: TeAp-N/D1, TeAp-ElFrio01, TeAp-Mantecal01, and TeGu-Terecay323. By using the VSG soluble form from TeAp-N/D1, we found that neither the inositol-1,2-cyclic phosphate moiety of the cross-reacting determinant nor the carbohydrate chains were exclusively responsible for its cross-reactivity. Then, all four purified glycoproteins were digested with papain and the resulting peptides were separated by high-performance liquid chromatography. Dot blot evaluation of the fractions using sera from trypanosome-infected animals yielded peptides that possessed cross-reaction activity, demonstrating for the first time that proteinaceous epitopes are also responsible for the cross-reactivity of trypanosome VSGs. [ABSTRACT FROM AUTHOR]

Purpose: To report a novel plasminogen gene mutation and detection of anti-plasminogen antibodies in a patient with ligneous conjunctivitis successfully treated with 60% fresh frozen plasma (FFP).Methods: Retrospective data collected on a 45-year-old Caucasian female presenting with unilateral chronic membranous lesions.Results: Laboratory investigation demonstrated decreased plasminogen antigen level, plasminogen activity, and rate of plasminogen activation by u-PA or t-PA, and elevated plasminogen activator inhibitor-1. Anti-plasminogen IgG and IgA antibodies were detected. DNA analysis revealed a novel Asp432Asn heterozygous missense mutation in the plasminogen gene (exon 11). The patient was treated with topical 60% FFP, achieved complete remission after four months, and remained membrane-free for over five years of follow-up.Conclusions: A novel plasminogen gene mutation, deficiency of plasminogen antigen and activity, and anti-plasminogen IgG and IgA antibodies were identified in a patient with adult-onset ligneous conjunctivitis. Sixty percent FFP maintained this patient disease-free for over five years. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is an autoimmune connective tissue disease, associated with the presence of anti-citrullinated protein antibodies (ACPA). These antibodies have been found in approximately 70% of patients suffering from RA and they are currently used for diagnosis of RA. Although they exhibit an absolute need for citrulline for antibody reactivity, no precise cognate antigen for these antibodies has been determined. In this study, we analyzed the reactivity of ACPA to various citrullinated peptides by modified enzyme-linked immunosorbent assays, in order to determine the dependency of specific amino acids for antibody reactivity. A non-human protein (ovalbumin) and antigens directly related to RA were used as templates for synthesis of non-modified and citrullinated peptides, becoming potential target epitopes. Mainly peptides containing a Cit-Gly motif were recognized by ACPAs, while no particular amino acids N-terminal of citrulline were found to be essential for antibody reactivity. Moreover, ACPA reactivity was not restricted to antigens known to be associated with ACPA-positive RA alone, but also to proteins without relation to RA, primarily illustrating that any protein in theory can be turned into an RA autoantigen, by introducing Cit-Gly motifs. Knowledge about the interaction between ACPAs and their citrullinated targets is important for understanding autoimmune ACPA responses in RA, which are known to contribute to the pathophysiology. [ABSTRACT FROM AUTHOR]

Little is known about the behavioral determinants that underlie cancer patients’ intention to express concerns during a consultation. This information can be relevant to developing effective interventions for cancer patients. In this study, the integrative model of behavioral prediction (IMBP) is used as a framework to unravel the determinants of patients’ intention to express concerns. The objectives of this study are to examine which of the IMBP determinants (attitude, perceived social norm, and/or self-efficacy) are significantly related to intention and what content of these determinants can be targeted to effect a change in patients’ intention. An online survey based on the IMBP determinants was distributed. A total of 236 cancer patients and cancer survivors participated. The results of the survey showed that patients’ attitudes and perceived social norm were the most important determinants of their intention to express concerns. The largest change in patients’ intention might be achieved by targeting the affective attitude, referring to the extent to which patients believe that expressing concerns is (un)pleasant, and the social norm, referring to the extent to which patients feel (un)supported by significant others in expressing concerns. [ABSTRACT FROM PUBLISHER]

Tetanus is a highly fatal disease caused by tetanus neurotoxin (TeNT) and remains a major threat to human and animal health, despite preventive strategies. TeNT is composed of heavy and light chain linked by a disulfide bond. The antibody response to TeNT is polyclonal and directed to multiple epitopes within both the light and heavy chains, leading to toxin neutralization. This study was undertaken to localize and compare neutralizing epitopes recognized by human and mouse TeNT-specific antibodies at a clonal level. In the present study, 22 murine hybridoma clones and 50 human lymphoblastoid cell lines secreting monoclonal antibodies (mAb) were generated against TeNT. The specificity of these mAb was determined using different recombinant fragments of tetanus toxin. Moreover, this study investigated thein vitrotoxin neutralizing activity of these mAb by a ganglioside GT1b assay. The results showed that tetanus toxoid immunization in humans and BALB/c mice induced a vigorous humoral immune response against different fragments of TeNT, particularly the carboxyl-terminal fragment of the heavy chain (known as fragment C). The fragment C-specific human and mouse mAb could largely neutralize TeNT. However, while all fragment C-specific human mAb reacted with the carboxyl-terminal part of this fragment (HCC), the majority of the mouse mAb failed to recognize this region. These results suggested that fragment C is the major target for the TeNT neutralizing antibodies, although different epitopes seem to be targeted by human and mouse antibodies. [ABSTRACT FROM AUTHOR]

Immunohistochemistry (IHC) is applied routinely in surgical and clinical pathology, because it is essential for diagnosis and sub-classification of many neoplastic lesions. Despite its extensive use for more than 40 years, lack of standardization is a major problem; many factors during the pre-analytical, analytical and post-analytical phases affect the final results. Nordic immunohistochemical Quality Control (NordiQC) was established in 2003 to evaluate the inter-laboratory consistency of IHC, focusing mainly on the analytical part. More than 26,000 IHC slides have been evaluated during the period 2003-2013; 15 − 300 laboratories have participated in each assessment. Overall, 71% of the staining results assessed have been evaluated as sufficient for diagnostic use, while 29% were judged insufficient. All IHC protocols used for the stained slides submitted to NordiQC have been evaluated by focusing on the technical calibration performed by the laboratories, and specific parameters that gave sufficient or insufficient results have been identified. The most common causes for insufficient results were: inadequate calibration of the primary antibody, use of an inadequate primary antibody, inappropriate choice of epitope retrieval method, insufficient heat induced epitope retrieval (HIER) and use of an inadequate detection kit. Approximately 90% of the insufficient results were characterized by either a signal that was too weak or false negative staining, whereas in the remaining 10%, a poor signal-to-noise ratio or false positive staining was seen. Identification of positive and negative tissue controls to ensure appropriate calibration of the IHC assay combined with individually tailored suggestions for protocol optimization have improved IHC staining for many markers and thus inter-laboratory consistency of the IHC results. The overall data generated by NordiQC during 11 years clearly indicates that external quality assessment is a valuable and necessary supplement to internal quality control. [ABSTRACT FROM AUTHOR]

The exposure ofAzospirillum brasilensecarbohydrate epitopes was investigated by electro-optical analysis of bacterial cell suspensions. To study changes in the electro-optical (EO) properties of the suspensions, we used antibodies generated to the complete lipopolysaccharide ofA. brasilensetype strain Sp7 and also antibodies to the smooth and rough O polysaccharides of Sp7. After 18 hr of culture growth, the EO signal of the suspension treated with antibodies to smooth O polysaccharide was approximately 20% lower than that of the suspension treated with antibodies to complete lipopolysaccharide (control). After 72 hr of culture growth, the strongest EO signal was observed for the cells treated with antibodies to rough O polysaccharide (approximately 46% greater than the control), whereas for the cells treated with antibodies to smooth O polysaccharide, it was much lower (approximately 23% of the control). These data were confirmed by electron microscopy. The results of the study may have importance for the rapid evaluation of changes in lipopolysaccharide form in microbial biotechnology, when the antigenic composition of the bacterial surface requires close control. [ABSTRACT FROM PUBLISHER]

Widely recognized as the gold standard for biological recognition, antibodies have been used to develop bioscavengers, enzyme-like catalytic therapies, and highly specific diagnostic clinical tests. The discovery of camelid antibodies in the 1990s has extended the useful range of antibodies into pH extremes, high temperature, and high salt conditions due to increased stability. However, determining the binding properties of these molecules prior to use under these conditions has not been widely employed because the most precise biophysical methods, surface plasmon resonance, and isothermal titration calorimetry, are not compatible with highly complex matrices. Herein, we demonstrate the use of microscale thermophoresis as a rapid, simple method to detect binding of a single-domain antibody in the presence of jet fuel/water mixes; a complex, harsh environment. This work provides key data to quantifying binding for the development of a sensor for detection of microbial growth in fuel. [ABSTRACT FROM AUTHOR]

Psoriasis is a chronic skin disorder that affects 1% to 3% of the general population worldwide. Streptococcal infection, especially streptococcal pharyngitis, has been shown to be a significant trigger of psoriasis in some patients, possibly by sensitizing T cells to keratin epitopes in the skin. Due to the role of the palatine tonsils as an immunological organ that may generate autoreactive T cells, tonsillectomy has been investigated as a treatment for psoriasis. Tonsillectomy originally gained acceptance in Japan as a treatment for palmoplantar pustulosis, a condition that shares features with pustular psoriasis. Subsequently, tonsillectomy has been used for the treatment of plaque psoriasis and guttate psoriasis. Recently, the first randomized, controlled clinical trial of tonsillectomy was performed. Here, we review the available evidence for the benefit of tonsillectomy as a treatment for palmoplantar pustulosis and psoriasis. We also discuss molecular studies aimed at understanding the role of tonsils in skin disease. [ABSTRACT FROM AUTHOR]

Neisseria meningitidis is a major cause of childhood morbidity and mortality worldwide. We describe an exceptional case of an immunocompetent 15-month-old child presenting with a unilateral anterior uveitis, hypopyon, and sepsis. Anterior chamber aspirate demonstrated gram-negative cocci before Neisseria meningitidis was identified in blood and cerebrospinal fluid. Meningococcal endophthalmitis presents variably with sepsis, meningitis, or isolated ocular symptoms. Diagnosis is a clinical challenge, requiring diagnostic sampling and treatment from both pediatricians and ophthalmologists. Delayed or incorrect treatment risks blindness, disability, or death. Simultaneous invasion of meningococcus across intact blood-brain and blood-ocular barriers in this child suggests antigenic correlates between meningeal and ocular endothelial interfaces. Meningococcus is an exclusively human pathogen; research is hampered by the lack of animal models. This clinical observation suggests the potential of a novel in vitro experimental approach of using ocular tissue from eye banks to further elucidate the meningococcal-endothelial interaction that underpins meningococcal disease. [ABSTRACT FROM AUTHOR]

Knowledge about antibody-antigen interactions is important for the understanding of the immune system mechanisms and for supporting development of drugs and biomarkers. A tool for identification of these antigenic epitopes of specific antibodies is epitope mapping. In this study, a modified enzyme-linked immunosorbent assay was applied for epitope mapping of a mouse monoclonal vimentin antibody using overlapping resin-bound peptides covering the entire vimentin protein. The minimal epitope required for binding was identified as the LDSLPLVD sequence using N- and C-terminally truncated peptides. The peptide sequence LDSLPLVDTH was identified as the complete epitope, corresponding to amino acids 428-437 in the C-terminal end of the human vimentin protein. Alanine scanning and functionality scanning applying substituted peptides were used to identify amino acids essential for antibody reactivity. In particular, the two aspartate residues were found to be essential for antibody reactivity since these amino acids could not be substituted without a reduction in antibody reactivity. The majority of the remaining amino acids could be substituted without reducing antibody reactivity notably. These results confirm that charged amino acids are essential for antibody reactivity and that the vimentin antibody is dependent on side-chain interactions in combination with backbone interactions. [ABSTRACT FROM AUTHOR]

Soybean meal in the post-weaning diet of pigs results in a growth lag correlated with Immunoglobulin G (IgG) production against soybean proteins, including the seed storage protein β-conglycinin. We screened sera from 30 pigs for IgG directed against β-conglycinin. Analysis of the same sera on protein blots identified sera that had IgG against the α-subunit of β-conglycinin. A tiled peptide array (16 amino acids with an 11 amino acid overlap) of the α-subunit of β-conglycinin was screened to identify the epitopes of the α-subunit that bind IgG. One major IgG binding epitope previously identified was confirmed. A second major IgG binding epitope was identified that bound IgG from half of the animals tested. Minor epitopes that bound IgG from one or two other sera were also identified. Binding of IgG to β-conglycinin in an enzyme-linked immunosorbent assay (ELISA) was inhibited by a synthetic peptide representing one of the minor epitopes. [ABSTRACT FROM PUBLISHER]

Plasmodial lactate dehydrogenase, terminal enzyme of the glycolytic pathway, has been shown to be biochemically, immunologically and structurally different from the mammalian enzyme. The substrate specific loop region of plasmodial lactate dehydrogenase (pLDH) has 5 amino acids insert (DKEWN) important for anti-malarial drug targeting. In the present study, we have produced six monoclonal antibodies, which are against three different epitopes of Plasmodium falciparum LDH (PfLDH). Two of these monoclonal antibodies (10C4D5 and 10D3G2) are against the substrate specific loop region of PfLDH (residues 98-109, AGFTKAPGKSDKEWNR). The 10C4D5 and 10D3G2 monoclonals bind to substrate specific loop region resulting in inhibition of PfLDH activity. A Microplate Sandwich ELISA was developed employing high affinity non-inhibitory (10A5H5, Kaff 1.272 ± 0.057 nM) and inhibitory (10C4D5, Kaff 0.306 ± 0.011 nM) monoclonal antibodies and evaluated using gossypol, a well known inhibitor of pLDH. The binding of gossypol to substrate specific loop region resulted in inhibition of binding of 10C4D5 monoclonal. This Microplate Sandwich ELISA can be utilized for identification of compounds inhibitory to PfLDH (binding to substrate specific loop region of parasite LDH) from combinatory chemical libraries or medicinal plants extracts. The Microplate Sandwich ELISA has also shown potential for specific diagnosis of malaria using finger prick blood samples. [ABSTRACT FROM AUTHOR]

This study was performed to produce a strong antibody against repeated peptides for a specific region of an antigen. Nine amino acids at C-terminal from human brainβ-tubulin III were selected as a target sequence since it is well known for a high conservancy of sequence and resistance to antibody production. The synthetic oligonucleotides had six bases (5′-CGAC—GT-3′, 5′-TC—CAGC-3′) appended at each end and were repeatedly linked and directly joined by ligase in the presence of restriction enzymes (AccI,AvaI) to allow for the construction of head-to-tail repeats. Ligated oligonucleotides containing more than 10-unit repeats were eluted from agarose gels and cloned into theAccI site of a modified pGEX expression vector. The cloned repeated target sequences were subsequently expressed in a bacterial system. A large amount of glutathione-S-transferase-fusion protein containing the unit repeats was purified by affinity chromatography on glutathione-agarose beads and injected into Balb/c mice to produce polyclonal and monoclonal antibodies (Abs). Total protein from several mammalian brains was extracted and utilised to test the quality of the generated Abs in immunoblot assays. The generated Abs specifically recognised the 55-kDa human brainβ-tubulin protein but not any other mammalian brainβ-tubulins. The potential uses of this system could be applied to an effective epitope generation for an antibody and to the development of a new type of subunit vaccine. [ABSTRACT FROM PUBLISHER]

In silico analysis was used to predict MHC class I and class II promiscuous epitopes and potential antigens, from 24 novel T cell antigens of Mycobacterium tuberculosis. Majority of the antigens (16/24) had high affinity peptides to both MHC class I and class II alleles and higher population coverage compared to well-proven T cell antigens ESAT-6, CFP-10 and Ag85B. Among these, highest population coverage were calculated for three novel T cell antigens Rv0733 (97.24%), Rv0462 (96.9%) and Rv2251 (96.3%). The prediction results were experimentally tested by in vitro stimulation of these novel T cell antigens with blood drawn from QuantiFERON-TB Gold In-Tube (QFT-IT) positive healthy household contacts of tuberculosis patients and pulmonary TB patients. Significantly higher level interferon-γ (IFN-γ) was observed, with these novel T cell antigens, in healthy household contacts compared to pulmonary TB subjects ( p = 0.0001). In silico analysis also resulted in prediction of 36 promiscuous epitopes from the novel 24 T cell antigens. Population coverage for 4 out of the 36 promiscuous epitopes was >90% [67 VVLLWSPRS (Rv1324), 42 VVGVTTNPS (Rv1448c), 178 MRFLLSAKS (Rv0242c) and 842 IRLMALVEY (Rv3800c)]. Our results shows that these novel antigens and promiscuous epitopes identified from our analysis can further be investigated for their usefulness for subunit vaccine development. [ABSTRACT FROM AUTHOR]

Clostridium tetani causes a life-threatening infectious disease by production of tetanus neurotoxin (TeNT), a 150 kDa molecule composed of light (LC) and heavy chain (HC) polypeptides. The TeNT HC contains an N-terminal domain critical for LC translocation and a C-terminal toxin receptor-binding domain known as fragment C. Despite extensive investigations on epitope specificity of anti-TeNT antibodies, the immunodominant neutralizing epitopes of the toxin are poorly defined. This study describes the generation and characterization of four monoclonal antibodies (MAb) specific for TeNT. The characteristics of each MAb were explored in terms of isotype, specificity, affinity, and immuno-globulin heavy chain variable region (IGHV) gene usage using ELISA, Western blotting, and sequencing techniques. The toxin neutralizing activity of the MAbs was also investigated using the in vitro GT1b neutralizing assay. The data demonstrated that all MAbs bind to tetanus toxin and toxoid. Sub-fragments binding analysis showed that two MAbs react with fragment C, one with both fragment C and LC, and one with LC. Only the two fragment C-specific MAbs were able to neutralize the toxin. Sequencing of the expressed VH and VL genes revealed rearrangements of various VH and VL gene segments in all hybridoma clones. Clonality of the hybridomas was also confirmed by a competition assay that showed recognition of distinct epitopes by these MAbs. The results suggest the importance of TeNT fragment C in terms of immunogenicity and toxin neutralization activity. [ABSTRACT FROM AUTHOR]

Ankylosing spondylitis (AS) is a chronic systemic arthritic disease that leads to significant disability and loss of quality of life in the ∼0.5% of the worldwide human population it affects. There is currently no cure for AS and mechanisms underlying its pathogenesis remain unclear. AS is highly genetic, with over 70% of the genetic risk being associated with the presence of HLA-B27 and endoplasmic reticulum aminopeptidase-1 (ERAP1) alleles. Furthermore, gene-gene interactions between HLA-B27 and ERAP1 AS risk alleles have recently been confirmed. Here, we demonstrate that various ERAP1 alleles can differentially mediate surface expression of antigens presented by HLA-B27 on human cells. Specifically, for all peptides tested, we found that an ERAP1 variant containing high AS risk SNPs reduced the amount of the peptide presented by HLA-B27, relative to low AS risk ERAP1 variants. These results were further validated using peptide catalysis assays in vitro, suggesting that high AS risk alleles have an enhanced catalytic activity that more rapidly destroys many HLA-B27-destined peptides, a result that correlated with decreased HLA-B27 presentation of the same peptides. These findings suggest that one mechanism underlying AS pathogenesis may involve an altered ability for AS patients harboring both HLA-B27 and high AS risk ERAP1 alleles to correctly display a variety of peptides to the adaptive arm of the immune system, potentially exposing such individuals to higher AS risk due to abnormal display of pathogen or self-derived peptides by the adaptive immune system. [ABSTRACT FROM AUTHOR]

This study was conducted to determine the broad-spectrum safety of a novel, water-soluble undenatured type II collagen (NEXT-II) derived from chicken sternum cartilage. The presence of epitope in NEXT-II was confirmed by using a commercial kit. The acute oral LD50 of NEXT-II was found to be greater than 5000 mg/kg bw in rats, while the single-dose acute dermal LD50 was greater than 2000 mg/kg bw. The primary dermal irritation index (PDII) of NEXT-II was found to be 1.8 and classified as slightly irritating to the skin. In primary eye irritation studies, the maximum mean total score (MMTS) of NEXT-II was observed to be 7.3 and classified as minimally irritating to the eye. Long-term safety studies were conducted in dogs over a period of 150 d, and no significant changes were observed in body weight, heart rate, respiration rate and blood chemistry. NEXT-II does not induce mutagenicity in the bacterial reverse mutation test in five Salmonella typhimurium strains either with or without metabolic activation. Furthermore, two experiments were conducted to assess the potential of NEXT-II to induce mutations with and without metabolic activation at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. No biologically relevant increase of mutants was observed. Also, no dose-dependent toxicity was observed. Furthermore, colony sizing showed no clastogenic effects induced by NEXT-II under the experimental conditions. These studies demonstrated the broad spectrum of safety of NEXT-II. [ABSTRACT FROM AUTHOR]

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Aurora kinases are serine/threonine kinases which play an important role in the process of mitosis and cell cycle regulation. Aurora kinase inhibitors are described to sensitize malignant cells to cytosine arabinoside and specific antibodies by mediating apoptosis. Aurora kinases are overexpressed in most acute leukemias but also in solid tumors. In this study we investigated whether epitopes derived from Aurora kinase A and B are able to elicit cellular immune responses in patients with acute myeloid leukemia (AML) to investigate their role as potential targets for specific immunotherapy. Samples of eight patients with AML were analyzed in enzyme-linked immunosorbent spot (ELISpot) assays and compared with immune responses of nine healthy volunteers (HVs). Specific CD8 + T cell responses were detected against the epitopes Aura A1, A2, B1, B2, B3, B4 and B5. Immune responses for epitopes derived from Aura B were induced more frequently compared to Aura A. The antigens with the most frequent cytotoxic T-lymphocyte (CTL) responses were Aura B3, B4 and B5, although the number of patients tested for these antigens was low. Aura B5 did not elicit specific CTL responses in HVs. For epitope Aura B6 no immune response was detected in HVs or patients. Taken together, with the combination of Aurora kinase inhibitors and an immunotherapeutic approach, an effective blast and minimal residual disease elimination might be achieved. [ABSTRACT FROM AUTHOR]

It is now well over a decade since the US Institute of Medicine of the National Academy of Sciences assigned the highest priority for a vaccine to prevent congenital human CMV infection, which was subsequently endorsed by the US National Vaccine Program Office. In spite of extensive efforts over many years, successful licensure of a CMV vaccine formulation remains elusive. While the understanding of immune regulation of CMV infection in healthy virus carriers and diseased patients has dramatically improved, traditional vaccine development programs have failed to exploit this knowledge. Until recently, most efforts have concentrated on designing vaccine formulations that block CMV infection through neutralizing antibodies. However, studies carried out in various disease settings, especially in transplant patients, have clearly emphasized the importance of cellular immunity and it is indeed encouraging to see that recent CMV vaccine development programs have started to incorporate this arm of the immune system. A number of new vaccine candidates have been found to be effective in preclinical studies, and are able to induce CMV-specific immune responses in clinical studies, although firm evidence for long-term efficacy is not yet available. For successful implementation of these vaccines in clinical settings, it will be important to demonstrate that the vaccine can induce effective levels of immunity for prevention of transmission of viral infection from mother to unborn baby and thus reduce CMV-related pathogenesis. For transplant recipients, vaccine strategies should be aimed at the induction of immunity that restricts viral reactivation and limits development of disease. [ABSTRACT FROM AUTHOR]

An all-in-one epitope retrieval buffer was developed to solubilize molten paraffin. This study illustrates that common all-in-one buffer additives such as surfactants and wetting agents do not solubilize molten paraffin but a water-soluble organic solvent (WSOS) does. This study also shows that the WSOS does not negatively impact immunohistochemical staining reactions. The addition of a WSOS into an all-in-one epitope retrieval buffer formulation maintains paraffin in solution when slides mounted with tissue sections are removed from the buffer. This action is an advantage for buffer tanks in which slides are in a vertical orientation; the slides are not recoated with molten paraffin. [ABSTRACT FROM AUTHOR]

Objectives: Progression of joint destruction is an important phenotypic feature in rheumatoid arthritis (RA). When factors have small effect sizes, both the avoidance of phenotypic misclassification and discerning true effects from noise are challenging. Assembling radiological measurements repeatedly in time harbours a smaller risk of misclassification than single measurements. Given serial measurements, different methods of analysis can be applied. This study evaluates different statistical methods of analysing longitudinal data. Methods: Three statistical methods were studied: linear regression (LR), generalized estimating equations (GEE), and multivariate normal regression analysis (MRA). All were applied longitudinally, testing for differences in radiological progression rates. As genetic variants are known to have small effect sizes, two genetic variants were studied as examples: rs675520 (located in the TNFAIP3-OLIG3 region) and the presence of the human leucocyte antigen (HLA) shared epitope (SE) alleles. Radiological data for 602 early RA patients with yearly radiographs and 7-years of follow-up were used. The powers obtained with the methods and the robustness against missingness were evaluated as outcome measures. Results: The presence of the rs675520 polymorphism and the HLA-SE risk genotype was associated with a 0.65-0.77 and 1.17-1.51 fold increased rate of joint destruction, respectively. The analyses performed with MRA resulted in smaller 95% confidence intervals (CIs) than the analyses using LR or GEE. In addition, the 95% CIs increased with the number of radiographs per patient. The power of MRA was higher than that of GEE. MRA was more robust against selective missingness than GEE or LR with a two-step approach (LRts). Conclusions: A multivariate normal regression model on subsequent radiographs is a powerful and robust method for analysing longitudinal joint destruction data. [ABSTRACT FROM AUTHOR]

The introduction of a One Fungus-One Name (1F1N) concept has led to intensive discussions among taxonomists. Based on the Xylariaceae, a hyperdiverse family of Ascomycota with over 1300 species, the advantages and pitfalls of these nomenclatural changes and their consequences for taxonomy and applied mycology are discussed. Historically, in the Xylariaceae, an 1F1N concept had already been realised: the types of all important genera are conserved. Most teleomorphs were discovered long before the anamorphic states and the latter did not receive separate names, hence no drastic taxonomic changes are expected. The new nomenclature calls for abandonment of some ill-defined anamorph genera, such as Muscodor. Other anamorph genera will be retained because their names refer to morphological symplesiomorphies that occur in several teleomorph genera. Various important taxa are only known from ancient specimens, and no DNA-based data are available. Much work still remains to be done to recollect these fungi, select epitypes, and settle their phylogenetic relationships. Until then, we recommend that taxonomic changes be applied at the suprageneric ranks whenever possible to maintain nomenclatural stability. [ABSTRACT FROM AUTHOR]

Vaccines encoding xenoantigens, "non-self" proteins that are highly homologous to their autologous counterparts, have been investigated as a means to increase immunogenicity and overcome tolerance to "self" antigens. We have previously shown that DNA vaccines encoding native prostatic acid phosphatase (PAP ) were able to elicit PAP -specific T cells in both rats and humans, but required multiple immunization courses. In this study, we investigated in a preclinical model whether immunizations with a DNA vaccine encoding a xenoantigen could elicit a cross-reactive immune response to the native protein, potentially requiring fewer immunizations. Lewis rats were immunized with a DNA vaccine encoding human PAP and splenocytes from immunized rats were screened with a human peptide library containing overlapping, 15-mer PAP -derived peptides using T-cell proliferation and interferon γ (IFNγ) release as measures of the immune response. One dominant PAP -specific, RT1.Al-restricted, epitope was identified. Direct immunization with the immunodominant peptide (HP 201-215) containing this epitope demonstrated that it included a naturally presented MHC Class I epitope recognized by CD8+ T cells in Lewis rats. However, no cross-reactive immune response was elicited to the corresponding rat peptide despite a difference of only three amino acids. Immunization with DNA vaccines encoding rat PAP (rPAP ) in which this foreign dominant epitope was included as well as with DNA vaccines coding for a variant of the xenoantigen from which this epitope was deleted, did not elicit responses to the native antigen. Overall, these results indicate that the immunization with a xenoantigen-coding DNA vaccine can lead to an immune response which potentially favors foreign epitopes and hence limits any cross-reactive response to the native antigen. [ABSTRACT FROM AUTHOR]

A safe and effective HIV vaccine has eluded the scientific community for over three decades. With the failure of vaccines based on neutralizing antibody and cytotoxic T cells, researchers are seeking novel approaches. The partially successful RV144 vaccine trial focused scientific interest on binding antibodies, such as those that mediate antibody-dependent cellular cytotoxicity (ADCC). The biological importance of HIV-specific ADCC is strongly suggested by the generation of ADCC-escape HIV variants and passive transfer experiments. Newer assays for HIV-specific ADCC have defined new epitopes other than in the envelope protein. Such ADCC epitopes could be useful in novel HIV vaccine design. Researchers have shown that recombinant viral vectors such as canarypox or adenovirus boosted with recombinant HIV proteins can induce ADCC and lead to partial protection. These significant developments pave the way for trialing ADCC-based technology in novel HIV vaccine studies. INSET: Key issues. [ABSTRACT FROM AUTHOR]

Functional T-cell epitope discovery is a key process for the development of novel immunotherapies, particularly for cancer immunology. In silico epitope prediction is a common strategy to try to achieve this objective. However, this approach suffers from a significant rate of false-negative results and epitope ranking lists that often are not validated by practical experience. A high-throughput platform for the identification and prioritization of potential T-cell epitopes is the iTopia™ Epitope Discovery System™, which allows measuring binding and stability of selected peptides to MHC Class I molecules. So far, the value of iTopia combined with in silico epitope prediction has not been investigated systematically. In this study, we have developed a novel in silico selection strategy based on three criteria: (1) predicted binding to one out of five common MHC Class I alleles; (2) uniqueness to the antigen of interest; and (3) increased likelihood of natural processing. We predicted in silico and characterized by iTopia 225 candidate T-cell epitopes and fixed-anchor analogs from three human tumor-associated antigens: CEA , HE R2 and TERT. HLA-A2-restricted fragments were further screened for their ability to induce cell-mediated responses in HLA-A2 transgenic mice. The iTopia binding assay was only marginally informative while the stability assay proved to be a valuable experimental screening method complementary to in silico prediction. Thirteen novel T-cell epitopes and analogs were characterized and additional potential epitopes identified, providing the basis for novel anticancer immunotherapies. In conclusion, we show that combination of in silico prediction and an iTopia-based assay may be an accurate and efficient method for MHC Class I epitope discovery among tumor-associated antigens. [ABSTRACT FROM AUTHOR]

Autoantibodies to thyroid peroxidase (TPO), the major target autoantigen in autoimmune thyroid diseases, recognize conformational epitopes limited to two immunodominant regions (IDRs) termed IDR-A and -B. The apparent restricted heterogeneity of TPO autoantibodies was discovered using TPO-specific mouse monoclonal antibodies (mAbs) and later confirmed by human recombinant Fabs. In earlier studies we identified key amino acids crucial for the interaction of human autoantibodies with TPO. Here we show the critical residues that participate in binding of five mAbs to the conformational epitopes on the TPO surface. Using ELISA we tested the reactivity of single and multiple TPO mutants expressed in CHO cells with a panel of mAbs specifically recognizing IDR-A (mAb 2 and 9) and IDR-B (mAb 15, 18, 64). We show that antibodies recognizing very similar regions on the TPO surface may interact with different sets of residues. We found that residues K713 and E716 contribute to the interaction between mAb 2 and TPO. The epitope for mAb 9 is critically dependent on residues R646 and E716. Moreover, we demonstrate that amino acids E604 and D630 are part of the functional epitope for mAb 15, and amino acids D624 and K627 for mAb 18. Finally, residues E604, D620, D624, K627, and D630 constitute the epitope for mAb 64. This is the first detailed study identifying the key resides for binding of mAbs 2, 9, 15, 18, and 64. Better understanding of those antibodies' specificity will be helpful in elucidating the properties of TPO as an antigen in autoimmune disorders. [ABSTRACT FROM AUTHOR]

A large number of food allergens, usually proteins capable of inducing allergic symptoms, including severe, even life-threatening reactions in predisposed individuals, have been identified and characterized. As most of these proteins are from our daily dietary intake, they are often difficult to avoid. However, the proteins that cause such immunoglobulin E (IgE)-mediated reactions can be assigned to only a limited number of protein families. Detailed knowledge about the characteristics of food allergens, their structures, biological activity, and stability, may be helpful in improving diagnosis of food allergy, avoiding unnecessary exclusion of diets, and assessing the risk of cross-reactive allergies to other food sources. The purpose of this review is to shed light on the sources and molecular properties of the allergenic proteins, their stability, the mechanisms of the allergenic responses, and recent findings related to prevention of this serious issue. [ABSTRACT FROM AUTHOR]

Antibody manufacturers often recommend antigen retrieval for formalin-fixed paraffin-embedded tissue sections before immunohistochemical staining. The most commonly used methods involve high temperatures (90-120°C) in buffers. Low-temperature retrieval methods allow the use of tissue flotation baths but their incubation times are often long. This study investigated retrieval of some antigens at low temperature (65°C) for 10 minutes in citrate buffer (pH 6·0) using a tissue flotation bath. Sections were obtained from two tissue specimens, breast and O. volvulus nodules, which were formalin-fixed and paraffin-embedded. The antigens of lactate dehydrogenase, pyruvate dehydrogenase kinase-2, malic enzyme-1, succinate dehydrogenase-B, glucose-6-phosphate dehydrogenase, alpha/beta-hydrolase-4, estrogen receptors (ER), and progesterone receptors (PR) were retrieved at low temperature and in the cases of ER and PR, were also retrieved at high temperature (120°C), using a pressure cooker, for comparison. All the retrievals were carried out in citrate buffer, pH 6·0. The results showed that all the enzymes scored 2+ (i.e. a strong reaction) in staining intensity and the staining for ER and PR were similar for both methods (2+). In immunohistochemical staining, the most important stage is antigen retrieval. The low-temperature antigen retrieval in citrate buffer at pH 6·0 provided staining intensities comparable to those by high-temperature methods. The results indicate that low-temperature antigen retrieval is cost-effective, rapid, and reliable. [ABSTRACT FROM AUTHOR]