Mapping the common antigenic determinants in avidin and streptavidin

An epitope scan analysis of the whole sequence of avidin and core streptavidin using polyclonal antibodies to these two antigens reveal the presence of multiple common epitopes in both the proteins. These antigenic determinants consist mostly of either identical or similar residues. The antibody recognition sites in both antigens are shown to be localized to homologous regions.

Antigenic determinants on chicken riboflavin carrier protein. A study with monoclonal antibodies

Monoclonal antibodies raised against chicken egg white riboflavin carrier protein were classified into seven categories each recognizing a distinct epitope. Of these, six were directed against conformation dependent epitopes and one to a sequential epitope. The roles of lysine residues and the post-translationally attached phosphate and oligosaccharide moieties in the antigenicity of riboflavin carrier protein recognized by the monoclonal antibodies were investigated. The binding region of three monoclonal antibodies could be located within the 87–219 amino acid sequence of the protein and one antibody among these recognized a sequence of 182–204 amino acid residues. All the monoclonal antibodies were able to recognize riboflavin carrier proteins present in the sera of pregnant rats, cows and humans indicating that the epitopes to which they are directed are conserved through evolution from chicken to the human.

Antigenic determinants of human serum retinol binding protein as probed with monoclonal antibodies

Monoclonal antibodies raised against human serum retinol-binding protein (hRBP) were used as probes for the study of the antigenic determinants of hRBP and those shared with the same protein from other species. The antibodies could be classified into four distinct groups and react with the homologous proteins from the rat as well as the rabbit sera. Three of these antibodies recognize sequential or continuous epitopes while the remaining antibody is directed against a discontinuous or conformational epitope. By chemical cleavage with cyanogen bromide, the domains recognized by the monoclonal antibodies could be delineated. By solid-phase synthetic approach, the core sequences recognized by two of these monoclonal antibodies were identified to amino acid sequences 45–51 and 128–131 of the primary amino acid sequence of hRBP.

Antigenic determinants at the carboxy terminus of chicken egg white riboflavin carrier protein (RCP): Epitope mapping and antibody-mediated pregnancy curtailment in rodents

The epitopic core sequences recognized by three monoclonal antibodies raised to chicken riboflavin carrier protein (RCP) were mapped to the C-terminal tail-end of the protein using the pepscan method A 21-residue synthetic peptide corresponding to residues 200-219 of the protein and comprising the regions corresponding to the antibodies was synthesized. Administration of polyclonal antibodies specific to this peptide led to termination of early pregnancy in mice. Also, active immunization of rats with the peptide-purified protein derivative conjugate inhibited establishment of pregnancy. These results demonstrate the functional importance of the C-terminal 200-219 region of chicken RCP. Copyright (C) 1996 Elsevier Science Ltd.

Chemical camouflage of antigenic determinants: Stealth erythrocytes

In a number of clinical circumstances it would be desirable to artificially conceal cellular antigenic determinants to permit survival of heterologous donor cells. A case in point is the problem encountered in transfusions of patients with rare blood types or chronically transfused patients who become allosensitized to minor blood group determinants. We have tested the possibility that chemical modification of the red blood cell (RBC) membrane might serve to occlude antigenic determinants, thereby minimizing transfusion reactions. To this end, we have covalently bound methoxy(polyethylene glycol) (mPEG) to the surface of mammalian RBC via cyanuric chloride coupling. Human RBC treated with this technique lose ABO blood group reactivity as assessed by solution–phase antisera agglutination. In accord with this, we also find a profound decrease in anti-blood group antibody binding. Furthermore, whereas human monocytes avidly phagocytose untreated sheep RBC, mPEG-derivatized sheep RBC are ineffectively phagocytosed. Surprisingly, human and mouse RBC appear unaffected by this covalent modification of the cell membrane. Thus, mPEG-treated RBC are morphologically normal, have normal osmotic fragility, and mPEG-derivatized murine RBC have normal in vivo survival, even following repeated infusions. Finally, in preliminary experiments, mPEG-modified sheep RBC intraperitoneally transfused into mice show significantly improved (up to 360-fold) survival when compared with untreated sheep RBC. We speculate that similar chemical camouflage of intact cells may have significant clinical applications in both transfusion (e.g., allosensitization and autoimmune hemolytic disease) and transplantation (e.g., endothelial cells and pancreatic β cells) medicine.

Is the generation of neo-antigenic determinants by free radicals central to the development of autoimmune rheumatoid disease?

Biomolecules are susceptible to many different post-translational modifications that have important effects on their function and stability, including glycosylation, glycation, phosphorylation and oxidation chemistries. Specific conversion of aspartic acid to its isoaspartyl derivative or arginine to citrulline leads to autoantibody production in models of rheumatoid disease, and ensuing autoantibodies cross-react with native antigens. Autoimmune conditions associate with increased activation of immune effector cells and production of free radical species via NADPH oxidases and nitric oxide synthases. Generation of neo-antigenic determinants by reactive oxygen and nitrogen species ROS and RNS) may contribute to epitope spreading in autoimmunity. The oxidation of amino acids by peroxynitrite, hypochlorous acid and other reactive oxygen species (ROS) increases the antigenicity of DNA, LDL and IgG, generating ligands for which autoantibodies show higher avidity. This review focuses on the evidence for ROS and RNS in promoting the autoimmune responses observed in diseases rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). It considers the evidence for ROS/RNS-induced antigenicity arising as a consequence of failure to remove or repair ROS/RNS damaged biomolecules and suggests that an associated defect, probably in T cell signal processing or/or antigen presentation, is required for the development of disease.

Major histocompatibility complex binding affinity of an antigenic determinant is crucial for the differential secretion of interleukin 4/5 or interferon gamma by T cells.

Differential activation of CD4+ T-cell precursors in vivo leads to the development of effectors with unique patterns of lymphokine secretion. To investigate whether the differential pattern of lymphokine secretion is influenced by factors associated with either the display and/or recognition of the ligand, we have used a set of ligands with various class II binding affinities but unchanged T-cell specificity. The ligand that exhibited approximately 10,000-fold higher binding to I-Au considerably increased the frequency of interferon gamma-producing but not interleukin (IL) 4- or IL-5-secreting cells in vivo. Using an established ligand-specific, CD4+ T-cell clone secreting only IL-4, we also demonstrated that stimulation with the highest affinity ligand resulted in interferon gamma production in vitro. In contrast, ligands that demonstrated relatively lower class II binding induced only IL-4 secretion. These data suggest that the major histocompatibility complex binding affinity of antigenic determinants, leading to differential interactions at the T cell-antigen-presenting cell interface, can be crucial for the differential development of cytokine patterns in T cells.

Influence of culture conditions on the molecular signature of mesenchymal stem cells

Cell based therapies require cells capable of self renewal and differentiation, and a prerequisite is the ability to prepare an effective dose of ex vivo expanded cells for autologous transplants. The in vivo identification of a source of physiologically relevant cell types suitable for cell therapies is therefore an integral part of tissue engineering. Bone marrow is the most easily accessible source of mesenchymal stem cells (MSCs), and harbours two distinct populations of adult stem cells; namely hematopoietic stem cells (HSCs) and bone mesenchymal stem cells (BMSCs). Unlike HSCs, there are yet no rigorous criteria for characterizing BMSCs. Changing understanding about the pluripotency of BMSCs in recent studies has expanded their potential application; however, the underlying molecular pathways which impart the features distinctive to BMSCs remain elusive. Furthermore, the sparse in vivo distribution of these cells imposes a clear limitation to their in vitro study. Also, when BMSCs are cultured in vitro there is a loss of the in vivo microenvironment which results in a progressive decline in proliferation potential and multipotentiality. This is further exacerbated with increased passage number, characterized by the onset of senescence related changes. Accordingly, establishing protocols for generating large numbers of BMSCs without affecting their differentiation potential is necessary. The principal aims of this thesis were to identify potential molecular factors for characterizing BMSCs from osteoarthritic patients, and also to attempt to establish culture protocols favourable for generating large number of BMSCs, while at the same time retaining their proliferation and differentiation potential. Previously published studies concerning clonal cells have demonstrated that BMSCs are heterogeneous populations of cells at various stages of growth. Some cells are higher in the hierarchy and represent the progenitors, while other cells occupy a lower position in the hierarchy and are therefore more committed to a particular lineage. This feature of BMSCs was made evident by the work of Mareddy et al., which involved generating clonal populations of BMSCs from bone marrow of osteoarthritic patients, by a single cell clonal culture method. Proliferation potential and differentiation capabilities were used to group cells into fast growing and slow growing clones. The study presented here is a continuation of the work of Mareddy et al. and employed immunological and array based techniques to identify the primary molecular factors involved in regulating phenotypic characteristics exhibited by contrasting clonal populations. The subtractive immunization (SI) was used to generate novel antibodies against favourably expressed proteins in the fast growing clonal cell population. The difference between the clonal populations at the transcriptional level was determined using a Stem Cell RT2 Profiler TM PCR Array which focuses on stem cell pathway gene expression. Monoclonal antibodies (mAb) generated by SI were able to effectively highlight differentially expressed antigenic determinants, as was evident by Western blot analysis and confocal microscopy. Co-immunoprecipitation, followed by mass spectroscopy analysis, identified a favourably expressed protein as the cytoskeletal protein vimentin. The stem cell gene array highlighted genes that were highly upregulated in the fast growing clonal cell population. Based on their functions these genes were grouped into growth factors, cell fate determination and maintenance of embryonic and neural stem cell renewal. Furthermore, on a closer analysis it was established that the cytoskeletal protein vimentin and nine out of ten genes identified by gene array were associated with chondrogenesis or cartilage repair, consistent with the potential role played by BMSCs in defect repair and maintaining tissue homeostasis, by modulating the gene expression pattern to compensate for degenerated cartilage in osteoarthritic tissues. The gene array also presented transcripts for embryonic lineage markers such as FOXA2 and Sox2, both of which were significantly over expressed in fast growing clonal populations. A recent groundbreaking study by Yamanaka et al imparted embryonic stem cell (ESCs) -like characteristic to somatic cells in a process termed nuclear reprogramming, by the ectopic expression of the genes Sox2, cMyc and Oct4. The expression of embryonic lineage markers in adult stem cells may be a mechanism by which the favourable behaviour of fast growing clonal cells is determined and suggests a possible active phenomenon of spontaneous reprogramming in fast growing clonal cells. The expression pattern of these critical molecular markers could be indicative of the competence of BMSCs. For this reason, the expression pattern of Sox2, Oct4 and cMyc, at various passages in heterogeneous BMSCs population and tissue derived cells (osteoblasts and chondrocytes), was investigated by a real-time PCR and immunoflourescence staining. A strong nuclear staining was observed for Sox2, Oct4 and cMyc, which gradually weakened accompanied with cytoplasmic translocation after several passage. The mRNA and protein expression of Sox2, Oct4 and cMyc peaked at the third passage for osteoblasts, chondrocytes and third passage for BMSCs, and declined with each subsequent passage, indicating towards a possible mechanism of spontaneous reprogramming. This study proposes that the progressive decline in proliferation potential and multipotentiality associated with increased passaging of BMSCs in vitro might be a consequence of loss of these propluripotency factors. We therefore hypothesise that the expression of these master genes is not an intrinsic cell function, but rather an outcome of interaction of the cells with their microenvironment; this was evident by the fact that when removed from their in vivo microenvironment, BMSCs undergo a rapid loss of stemness after only a few passages. One of the most interesting aspects of this study was the integration of factors in the culture conditions, which to some extent, mimicked the in vivo microenvironmental niche of the BMSCs. A number of studies have successfully established that the cellular niche is not an inert tissue component but is of prime importance. The total sum of stimuli from the microenvironment underpins the complex interplay of regulatory mechanisms which control multiple functions in stem cells most importantly stem cell renewal. Therefore, well characterised factors which affect BMSCs characteristics, such as fibronectin (FN) coating, and morphogens such as FGF2 and BMP4, were incorporated into the cell culture conditions. The experimental set up was designed to provide insight into the expression pattern of the stem cell related transcription factors Sox2, cMyc and Oct4, in BMSCs with respect to passaging and changes in culture conditions. Induction of these pluripotency markers in somatic cells by retroviral transfection has been shown to confer pluripotency and an ESCs like state. Our study demonstrated that all treatments could transiently induce the expression of Sox2, cMyc and Oct4, and favourably affect the proliferation potential of BMSCs. The combined effect of these treatments was able to induce and retain the endogenous nuclear expression of stem cell transcription factors in BMSCs over an extended number of in vitro passages. Our results therefore suggest that the transient induction and manipulation of endogenous expression of transcription factors critical for stemness can be achieved by modulating the culture conditions; the benefit of which is to circumvent the need for genetic manipulations. In summary, this study has explored the role of BMSCs in the diseased state of osteoarthritis, by employing transcriptional profiling along with SI. In particular this study pioneered the use of primary cells for generating novel antibodies by SI. We established that somatic cells and BMSCs have a basal level of expression of pluripotency markers. Furthermore, our study indicates that intrinsic signalling mechanisms of BMSCs are intimately linked with extrinsic cues from the microenvironment and that these signals appear to be critical for retaining the expression of genes to maintain cell stemness in long term in vitro culture. This project provides a basis for developing an “artificial niche” required for reversion of commitment and maintenance of BMSC in their uncommitted homeostatic state.

Grass pollen allergens globally: The contribution of subtropical grasses to burden of allergic respiratory diseases

Grass pollens of the temperate (Pooideae) subfamily and subtropical subfamilies of grasses are major aeroallergen sources worldwide. The subtropical Chloridoideae (e.g. Cynodon dactylon; Bermuda grass) and Panicoideae (e.g. Paspalum notatum; Bahia grass) species are abundant in parts of Africa, India, Asia, Australia and the Americas, where a large and increasing proportion of the world's population abide. These grasses are phylogenetically and ecologically distinct from temperate grasses. With the advent of global warming, it is conceivable that the geographic distribution of subtropical grasses and the contribution of their pollen to the burden of allergic rhinitis and asthma will increase. This review aims to provide a comprehensive synthesis of the current global knowledge of (i) regional variation in allergic sensitivity to subtropical grass pollens, (ii) molecular allergenic components of subtropical grass pollens and (iii) allergic responses to subtropical grass pollen allergens in relevant populations. Patients from subtropical regions of the world show higher allergic sensitivity to grass pollens of Chloridoideae and Panicoideae grasses, than to temperate grass pollens. The group 1 allergens are amongst the allergen components of subtropical grass pollens, but the group 5 allergens, by which temperate grass pollen extracts are standardized for allergen content, appear to be absent from both subfamilies of subtropical grasses. Whilst there are shared allergenic components and antigenic determinants, there are additional clinically relevant subfamily-specific differences, at T- and B-cell levels, between pollen allergens of subtropical and temperate grasses. Differential immune recognition of subtropical grass pollens is likely to impact upon the efficacy of allergen immunotherapy of patients who are primarily sensitized to subtropical grass pollens. The literature reviewed herein highlights the clinical need to standardize allergen preparations for both types of subtropical grass pollens to achieve optimal diagnosis and treatment of patients with allergic respiratory disease in subtropical regions of the world. © 2014 John Wiley & Sons Ltd.

IgE cross-reactivity between Pas n 1 of Bahia grass pollen and other group 1 grass pollen allergens

Background The subtropical Bahia grass (Paspalum notatum) is an important source of pollen allergens with an extended season of pollination and wide distribution in warmer climates. The immunological relationship between pollen allergens of Bahia grass and temperate grasses is unresolved. Methods Serum IgE reactivity of grass pollen-allergic patients with Bahia, Ryegrass and Bermuda grass pollen extracts and their purified group 1 allergens, Pas n 1, Lol p 1 and Cyn d 1, were compared by immunoblotting, ELISA, inhibition ELISA, basophil activation by flow cytometry and molecular modeling. Results Differences in antibody recognition of allergenic components between Bahia grass and Ryegrass pollen were observed by immunoblotting. Eight grass pollen-allergic patients from a temperate region showed greater serum IgE reactivity with Ryegrass pollen than Bahia grass by ELISA. For seven of these sera, Ryegrass pollen inhibited IgE reactivity with Bahia grass pollen but not the converse. For 51 sera from grass pollen-allergic patients in this temperate region, IgE reactivity with Lol p 1 was greater than Pas n 1 or Cyn d 1. IgE reactivity with Lol p 1 was not inhibited by Pas n 1 or Cyn d 1, but Pas n 1 IgE reactivity was inhibited by Lol p 1. Two group 1 grass pollen allergen-specific mAb distinguished between temperate and subtropical grass pollens. Basophil activation for three patients tested was greater by Ryegrass pollen than Bahia or Bermuda grass, and by Lol p 1 than Pas n 1 or Cyn d 1. In contrast, two patients from a subtropical region had higher serum IgE reactivity with Bahia grass pollen than Ryegrass and Bahia grass pollen inhibited IgE reactivity with Ryegrass. A structural model of Pas n 1 showed amino acids implicated in IgE epitopes of other group 1 allergens were juxtaposed on the surface. Conclusion Allergens from subtropical Bahia grass pollen, including Pas n 1, share antigenic determinants with temperate grass pollen allergens, but patients exhibit higher serum IgE reactivity to their locally predominant grass pollen. Basophil activation by Bahia grass pollen and Pas n 1 in patients from a temperate climate indicates clinically relevant cross-sensitization between temperate and subtropical grass pollens.

Characterization of epitopes for virus-neutralizing monoclonal antibodies to Ross River virus E2 using phage-displayed random peptide libraries

Ross River virus (RRV) is the predominant cause of epidemic polyarthritis in Australia, yet the antigenic determinants are not well defined. We aimed to characterize epitope(s) on RRV-E2 for a panel of monoclonal antibodies (MAbs) that recognize overlapping conformational epitopes on the E2 envelope protein of RRV and that neutralize virus infection of cells in vitro. Phage-displayed random peptide libraries were probed with the MAbs T1E7, NB3C4, and T10C9 using solution-phase and solid-phase biopanning methods. The peptides VSIFPPA and KTAISPT were selected 15 and 6 times, respectively, by all three of the MAbs using solution-phase biopanning. The peptide LRLPPAP was selected 8 times by NB3C4 using solid-phase biopanning; this peptide shares a trio of amino acids with the peptide VSIFPPA. Phage that expressed the peptides VSIFPPA and LRLPPAP were reactive with T1E7 and/or NB3C4, and phage that expressed the peptides VSIFPPA, LRLPPAP, and KTAISPT partially inhibited the reactivity of T1E7 with RRV. The selected peptides resemble regions of RRV-E2 adjacent to sites mutated in neutralization escape variants of RRV derived by culture in the presence of these MAbs (E2 210-219 and 238-245) and an additional region of E2 172-182. Together these sites represent a conformational epitope of E2 that is informative of cellular contact sites on RRV.

Use of α- and β-subunit specific antibodies in studying interaction of hCG with Leydig cell receptors

Antisera (a/s) raised to individual α- and β-subunits of human chorionic gonadotropin (hCG) have been characterized for specificity using immunoaffinity procedures and used to study the disposition of the two subunits when intact hCG is complexed with luteinizing hormone (LH) receptor of the Leydig cells. Three kinds of experiments were done. (a) The ability of the preformed hormone-antibody (H-Ab) complex to bind to receptor and stimulate a response; (b) the ability of the a/s to dissociate hCG from its complex with the receptor and thereby terminate response; and (c) the ability of the premixed antibody and receptor to compete for binding of labeled hCG. Although the subunit specific a/s used here were equipotent in binding hCG (capacity to bind and Ka being very similar), their behavior once the receptor preparation or Leydig cell is introduced into the system was drastically different. The β-subunit antibody relative to the α-subunit antibody, appeared to be poorly effective in preventing hCG from either binding to the receptor or inhibiting the continuation of response. The results suggest that hCG upon interaction with the receptor loses the determinants specific to the β-region more rapidly compared to those specific to the α-region suggesting thereby that the initial interaction of hCG with the receptor should be occurring through sites in the β-subunit. Although the α-subunit portion of the hCG molecule is available for binding to the antibody for a relatively longer time, the biological response of the cell seems very sensitive to such binding with the antibody as it invariably results in loss of response. In the Leydig cell system, the ability of the a/s to bind hCG that is already complexed to the receptor appears to be dependent upon the time of addition of the antibody to the incubation medium. The antisera were totally ineffective in inhibiting steroidogenic response to hCG if added 60 min after addition of hCG. This would suggest that the hormone-receptor complex once formed perhaps continues to change its orientation with the result that with time relatively less and less of antigenic determinants become available for antibody binding.