Determination of crucial epitopes in the sperm protein calsperin employing synthetic peptides and monoclonal antibodies.

The chaperone protein calsperin is exclusively expressed in the testes and is essential for sperm migration from the uterus into the oviduct. During spermatogenesis, calsperin interacts with ADAM3, a spermatozoon membrane protein required for fertilization. In this study, we characterized a calsperin epitope by using two monoclonal antibodies and resin-bound calsperin peptides, which were tested for reactivity using a modified enzyme-linked immunosorbent assay. An epitope located at the C-terminal end of calsperin corresponding to amino acids 228 WEKHFLDAS237 was identified. Three hot spot amino acids were essential for antibody binding whereas the remaining amino acids in the identified epitope appeared to be essential for bringing the critical contact residues into an α-helix structure. No notable sequence similarity was determined between the identified calsperin epitope and calreticulin, a chaperone homologue with sequence similarity, indicating that the identified epitope was specific for calsperin. Characterization of the calsperin epitope and of the two antibodies tested may be used in assays for further characterization of calsperin, where knowledge about the binding sites is necessary, for example, in sandwich assays. Moreover, studies like these may be used to study the function of calsperin during spermatogenesis and fertilization in detail and to develop new male contraception methods by targeting calsperin and mediating neutralization of its function.

Advances in Antibody Design and Antigenic Peptide Targeting 2.0.

Antibodies possess numerous important functions in diagnostics, both as therapeutics and as research tools [...].

Antibody Cross-Reactivity in Auto-Immune Diseases.

Autoimmunity is defined by the presence of antibodies and/or T cells directed against self-components. Although of unknown etiology, autoimmunity commonly is associated with environmental factors such as infections, which have been reported to increase the risk of developing autoimmune diseases. Occasionally, similarities between infectious non-self and self-tissue antigens may contribute to immunological cross-reactivity in autoimmune diseases. These reactions may be interpreted as molecular mimicry, which describes cross-reactivity between foreign pathogens and self-antigens that have been reported to cause tissue damage and to contribute to the development of autoimmunity. By focusing on the nature of antibodies, cross-reactivity in general, and antibody-antigen interactions, this review aims to characterize the nature of potential cross-reactive immune reactions between infectious non-self and self-tissue antigens which may be associated with autoimmunity but may not actually be the cause of disease onset.

Yeast Secretes High Amounts of Human Calreticulin without Cellular Stress.

The ER chaperone calreticulin (CALR) also has extracellular functions and can exit the mammalian cell in response to various factors, although the mechanism by which this takes place is unknown. The yeast Saccharomyces cerevisiae efficiently secretes human CALR, and the analysis of this process in yeast could help to clarify how it gets out of eukaryotic cells. We have achieved a secretion titer of about 140 mg/L CALR in our S. cerevisiae system. Here, we present a comparative quantitative whole proteome study in CALR-secreting yeast using non-equilibrium pH gradient electrophoresis (NEPHGE)-based two-dimensional gel electrophoresis (2DE) as well as liquid chromatography mass spectrometry in data-independent analysis mode (LC-MSE). A reconstructed carrier ampholyte (CA) composition of NEPHGE-based first-dimension separation for 2DE could be used instead of formerly commercially available gels. Using LC-MSE, we identified 1574 proteins, 20 of which exhibited differential expression. The largest group of differentially expressed proteins were structural ribosomal proteins involved in translation. Interestingly, we did not find any signs of cellular stress which is usually observed in recombinant protein-producing yeast, and we did not identify any secretory pathway proteins that exhibited changes in expression. Taken together, high-level secretion of human recombinant CALR protein in S. cerevisiae does not induce cellular stress and does not burden the cellular secretory machinery. There are only small changes in the cellular proteome of yeast secreting CALR at a high level.

Human herpesvirus infections and circulating microvesicles expressing galectin-3 binding protein in patients with systemic lupus erythematosus.

OBJECTIVES: Circulating microvesicles (MVs) expressing the type 1 interferon-inducible protein galectin-3 binding protein (G3BP) are potentially major sources of autoantigens in systemic lupus erythematosus (SLE). In this study, we explore if plasma concentrations of G3BP-expressing MVs correlate with signs of various active human herpesvirus (HHV) infections in SLE patients, suggesting a virus-induced mechanism for the generation of these vesicles. METHODS: In 49 SLE patients, the plasma levels of immunoglobulin G (IgG) against cytomegalovirus (CMV) pp52, Epstein-Barr virus (EBV) early antigen diffuse (EA/D), and HHV6 p41 were measured by ELISAs and used as humoral markers of ongoing/recently active viral infection. MVs in platelet-poor plasma were quantified and characterised by flow cytometry, with regard to the binding of Annexin V (AnxV) and the expression of G3BP. Spearman's rho and the Wilcoxon rank-sum test were applied for associative evaluation of virus serology with MV subsets, and clinical and demographic data. RESULTS: The CMV pp52-directed antibodies correlated positively with the high G3BP-expressing MVs; either low (rho=0.4, p-value=0.005) or high (rho=0.37, p-value=0.01) in AnxV-expression. Furthermore, these MV subsets were higher in individuals with high and low IgG levels against CMV pp52 and EBV EA/D, respectively, relative to subjects with low and high IgG levels against these HHV antigens. Importantly, none of the associations were explained by immunosuppressants or antimalarials. CONCLUSIONS: Ongoing/recently active CMV infection is associated with circulating MVs expressing G3BP in SLE patients, supporting a link between specific viral infections and potentially pathogenic MVs in SLE.

In Vitro Angiogenesis Inhibition and Endothelial Cell Growth and Morphology.

A co-culture assay with human umbilical vein endothelial cells (HUVECs) and normal human dermal fibroblasts (NHDFs) was used to study whether selected angiogenesis inhibitors were able to inhibit differentiation and network formation of HUVECs in vitro. The effect of the inhibitors was determined by the morphology and the calculated percentage area covered by HUVECs. Neutralizing VEGF with avastin and polyclonal goat anti-VEGF antibody and inhibiting VEGFR2 with sorafenib and vatalanib resulted in the formation of HUVEC clusters of variable sizes as a result of inhibited EC differentiation. Furthermore, numerous inhibitors of the VEGF signaling pathways were tested for their effect on the growth and differentiation of HUVECs. The effects of these inhibitors did not reveal a cluster morphology, either individually or when combined to block VEGFR2 downstream pathways. Only the addition of N-methyl-p-bromolevamisole revealed a similar morphology as when targeting VEGF and VEGFR2, meaning it may have an inhibitory influence directly on VEGFR signaling. Additionally, several nuclear receptor ligands and miscellaneous compounds that might affect EC growth and differentiation were tested, but only dexamethasone gave rise to cluster formation similarly to VEGF-neutralizing compounds. These results point to a link between angiogenesis, HUVEC differentiation and glucocorticoid receptor activation.

Peptide Antibody Reactivity to Homologous Regions in Glutamate Decarboxylase Isoforms and Coxsackievirus B4 P2C.

Two isoforms of the glutamate decarboxylase (GAD) enzyme exist, GAD65 and GAD67, which are associated with type 1 diabetes (T1D) and stiff-person syndrome (SPS), respectively. Interestingly, it has been reported that T1D patients seldom develop SPS, whereas patients with SPS occasionally develop T1D. In addition, coxsackievirus B4 (CVB4) has previously been proposed to be involved in the onset of T1D through molecular mimicry. On this basis, we aimed to examine antibody cross-reactivity between a specific region of GAD65 and GAD67, which has high sequence homology to the nonstructural P2C protein of CVB4 to determine potential correlations at antibody level. Monoclonal peptide antibodies generated in mice specific for a region with high similarity in all three proteins were screened for reactivity along with human sera in immunoassays. In total, six antibodies were generated. Two of the antibodies reacted to both GAD isoforms. However, none of the antibodies were cross-reactive to CVB, suggesting that antibody cross-reactivity between GAD65 and CVB, and GAD67 and CVB may not contribute to the onset of T1D and SPS, respectively.

Production and Characterization of Peptide Antibodies to the C-Terminal of Frameshifted Calreticulin Associated with Myeloproliferative Diseases.

Myeloproliferative Neoplasms (MPNs) constitute a group of rare blood cancers that are characterized by mutations in bone marrow stem cells leading to the overproduction of erythrocytes, leukocytes, and thrombocytes. Mutations in calreticulin (CRT) genes may initiate MPNs, causing a novel variable polybasic stretch terminating in a common C-terminal sequence in the frameshifted CRT (CRTfs) proteins. Peptide antibodies to the mutated C-terminal are important reagents for research in the molecular mechanisms of MPNs and for the development of new diagnostic assays and therapies. In this study, eight peptide antibodies targeting the C-terminal of CRTfs were produced and characterised by modified enzyme-linked immunosorbent assays using resin-bound peptides. The antibodies reacted to two epitopes: CREACLQGWTE for SSI-HYB 385-01, 385-02, 385-03, 385-04, 385-07, 385-08, and 385-09 and CLQGWT for SSI-HYB 385-06. For the majority of antibodies, the residues Cys1, Trp9, and Glu11 were essential for reactivity. SSI-HYB 385-06, with the highest affinity, recognised recombinant CRTfs produced in yeast and the MARIMO cell line expressing CRTfs when examined in Western immunoblotting. Moreover, SSI-HYB 385-06 occasionally reacted to CRTfs from MPN patients when analysed by flow cytometry. The characterized antibodies may be used to understand the role of CRTfs in the pathogenesis of MPNs and to design and develop new diagnostic assays and therapeutic targets.

Structural Analysis of Calreticulin, an Endoplasmic Reticulum-Resident Molecular Chaperone.

Calreticulin (Calr) is an endoplasmic reticulum (ER) chaperone involved in protein quality control, Ca2+ regulation and other cellular processes. The structure of Calr is unusual, reflecting different functions of the protein: a proline-rich ß-hairpin arm and an acidic C-terminal tail protrude from a globular core, composed of a ß-sheet sandwich and an α-helix. The arm and tail interact in the presence of Ca2+ and cover the upper ß-sheet, where a carbohydrate-binding site gives the chaperone glycoprotein affinity. At the edge of the carbohydrate-binding site is a conserved, strained disulphide bridge, formed between C106 and C137 of human Calr, which lies in a polypeptide-binding site. The lower ß-sheet has several conserved residues, comprised of a characteristic triad, D166-H170-D187, Tyr172 and the free C163. In addition to its role in the ER, Calr translocates to the cell surface upon stress and functions as an immune surveillance marker. In some myeloproliferative neoplasms, the acidic Ca2+-binding C-terminal tail is transformed into a polybasic sequence.

Molecular Recognition and Advances in Antibody Design and Antigenic Peptide Targeting.

Molecular recognition, the specific interaction between molecules by a combination of physical forces, has been a subject of scientific investigation for decades [...].

The microbiome reflects diagnosis and predicts disease severity in paediatric onset inflammatory bowel disease.

Objectives: A microbiotic profile characterized by decreased abundance and richness has been described in inflammatory bowel disease (IBD). Recently, sequencing the microbiome to the species level has become possible, which can improve our understanding of the gut to host interaction in IBD. We aimed to describe the microbiotic profile in paediatric IBD and compare it to disease phenotype and disease course. Methods: Faecal samples were collected from a cross-sectional cohort. The microbiome analysis was performed using 16S and 18S rRNA sequencing with the miSeq instrument. Inflammatory activity was assessed by faecal calprotectin. Data regarding medical treatment and surgery in the year after faecal sampling were collected from patient charts. Results: One hundred and forty-three (143) paediatric IBD patients and 34 healthy controls (HC) were included. We found a reduced richness in IBD patients compared to HCs (controls vs. ulcerative colitis (UC), p < .001 and controls vs. Crohn's disease (CD), p = .04)). Moreover, a high degree of intestinal inflammation and extensive disease extent was associated with reduced richness in UC (p = .02 and p = .04, respectively). Nine species were significantly associated with a healthy microbiome and three species were associated with IBD. Lastly, we found that the composition of the microbiome could distinguish between CD, UC and HCs. Conclusions: In this study, we found that the microbiome could discriminate between IBD phenotypes and predict which patients were at risk of surgery. In the future, this could be included as part of the diagnostic work-up in IBD patients.

Serum Calprotectin in Adolescents With Inflammatory Bowel Disease-A Pilot Investigation.

OBJECTIVES: Fecal calprotectin (FC) is a well-integrated parameter in the monitoring of adolescent patients with inflammatory bowel disease (IBD). However, measurement of FC is limited by day-to-day-variation and by the feces consistency. Furthermore, adolescents are often noncompliant to deliver fecal sampling leading to suboptimal monitoring. Consequently, we see the need of a substitute biomarker whenever measurement of FC fails and aimed to investigate serum calprotectin (SC) in adolescents with IBD. METHODS: In cross sectional data from 19 ulcerative colitis (UC) patients <18 years old, a Spearman correlation was used to analyze the correlation between SC, FC, C-reactive protein (CRP) and endoscopic and symptom scores. In longitudinal data collected from 20 UC and Crohn disease (CD) patients (10-17 years old), Mixed Effect Models (MEM) were used to analyze the association between SC, FC, CRP, and symptom scores. RESULTS: We found positive correlations between SC (19 samples) and the endoscopic score, symptom score, and CRP (r = 0.56, P = 0.01; r = 0.64, P = 0.003; r = 0.97, P < 0.0001). We found no significant correlation between SC and FC. In 27 samples from UC patients, the association of SC with FC and CRP were positive and significant (P = 0.004, estimate = 0.32; P = 0.0001, estimate = 0.002). The association between SC and symptom score was insignificant. In 49 samples from CD patients, the association between SC and CRP was significant (P = 0.02, estimate = 0.002) whereas associations between SC and FC and symptom score were insignificant. CONCLUSIONS: In the current pilot study, we found a correlation between SC and the endoscopically assessed inflammation in UC. SC may have the potential to improve disease monitoring of adolescent patients.

Increased antibody levels to stage-specific Epstein-Barr virus antigens in systemic autoimmune diseases reveal a common pathology.

The immune responses to antigens from different stages of the Epstein-Barr virus (EBV) life cycle were investigated in systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Sjögren's syndrome (SS), and systemic sclerosis (SSc) to gain knowledge of EBV's involvement in the etiology of systemic autoimmune diseases (SADs) and for an overview of the humoral immune responses against EBV. Investigations were performed by the use of ELISA. IgM, IgA, and IgG antibody binding to 11 EBV antigens: EBNA1, EBNA2, BALF5, EAD, BALF2, EA/R, VCA p18, VCA p23, gB, gp350, and gp42 were examined in serum pools from SAD patients and healthy controls (HCs). Increased antibody levels against the 11 EBV antigens in the SAD pools were seen compared to the HC pool. Specifically, SLE was characterized by strongly increased IgA to EAD both compared to HCs and other SADs, and RA was characterized by increased IgM levels to several EBV antigens. The SADs may be partly distinguished by their differential immune responses to various antigens in the EBV life cycle. All together, these findings support an association between EBV infection and SADs.

Peptides, Antibodies, Peptide Antibodies and More.

The applications of peptides and antibodies to multiple targets have emerged as powerful tools in research, diagnostics, vaccine development, and therapeutics. Antibodies are unique since they, in theory, can be directed to any desired target, which illustrates their versatile nature and broad spectrum of use as illustrated by numerous applications of peptide antibodies. In recent years, due to the inherent limitations such as size and physical properties of antibodies, it has been attempted to generate new molecular compounds with equally high specificity and affinity, albeit with relatively low success. Based on this, peptides, antibodies, and peptide antibodies have established their importance and remain crucial reagents in molecular biology.

Fine Mapping of Glutamate Decarboxylase 65 Epitopes Reveals Dependency on Hydrophobic Amino Acids for Specific Interactions.

Characterization of multiple antibody epitopes has revealed the necessity of specific groups of amino acid residues for reactivity. This applies to the majority of antibody-antigen interactions, where especially charged and hydrophilic amino acids have been reported to be essential for antibody reactivity. This study describes thorough characterization of glutamic acid decarboxylase (GAD) 65 antigenic epitopes, an immunodominant autoantigen in type 1 diabetes (T1D). As linear epitopes are sparsely described for GAD65 in T1D, we aimed to identify and thoroughly characterize two GAD65 antibodies using immunoassays. A monoclonal antibody recognized an epitope in the N-terminal domain of GAD65, 8FWSFGSE14, whereas a polyclonal antibody recognized two continuous epitopes in the C-terminal domain, corresponding to amino acids 514RTLED518 and 549PLGDKVNF556. Hydrophobic amino acids were essential for antibody reactivity, which was verified by competitive inhibition assays. Moreover, the epitopes were located in flexible linker regions and turn structures. These findings confirm the versatile nature of antibody-antigen interactions and describe potential continuous epitopes related to T1D, which predominantly have been proposed to be of discontinuous nature.

Mapping of the binding sites of human diamine oxidase (DAO) monoclonal antibodies.

OBJECTIVE: Recently we characterized five mouse monoclonal antibodies that allow the specific and sensitive detection of human diamine oxidase (DAO). To understand differences in binding characteristics and recognition of enzyme variants, we mapped the antibody binding sites. METHODS: Fragments of human DAO were expressed as glutathione-S-transferase fusion proteins that were used for testing antibody binding on immunoblots. Combined information from species cross-reactivity, sequence comparison and binding site-prediction software were used to localize the epitope recognized by each antibody. RESULTS: All five monoclonal DAO antibodies bound to linear epitopes between the N3 and enzymatic domains of the 732 amino acid protein. The binding sites could be mapped onto amino acid regions V262-E278 and P279-R288, respectively, which exhibit considerable sequence variation in mammals explaining the fact that the human DAO antibodies do not cross-react with DAO from other species. The antibodies efficiently bind only denatured human DAO but not the native protein. CONCLUSIONS: Characterization of the binding sites of the DAO antibodies revealed that the antibodies bind two adjacent epitopes and exhibit similar binding characteristics and species cross-reactivity. As the epitopes do not overlap any of the amino acid substitutions described for clinically significant DAO gene polymorphisms, our antibodies will also be useful for analyses of the mutant DAO proteins.

Smoking, disease characteristics and serum cytokine levels in patients with primary Sjögren's syndrome.

Smoking affects several disease processes. Epidemiological studies have previously found a negative association between primary Sjögren's syndrome (pSS) and smoking. The aim of this study was to examine whether markers of disease activity and cytokine expression in pSS patients differ between ever and never smokers. Fifty-one consecutive pSS patients and 33 population controls were included in the study. Clinical and standard laboratory parameters were registered. Serum cytokines (IL-1ß, IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17, IL-18, IL-33, IFN-α, IFN-γ, TNF-α, EGF, BAFF, Fas-ligand, RANTES, TGF-ß1) were assessed. A positive lip biopsy was less prevalent among ever smoking patients compared to never smokers (81 vs 100%; p = 0.03). However, except for TNF-α, which was higher in ever smokers, no differences in cytokine levels were found when comparing ever and never smoking pSS patients. Furthermore, no significant differences were found between ever and never smoking patients in the ESSDAI total score, IgG levels, or complement levels. However, IL-6, IL-12, IL-17 and IL-18 were significantly increased in pSS patients compared to controls. In this study, a negative association between ever smoking and positive lip biopsy was found, confirming previous reports. Expected differences in cytokine levels compared to controls were noted, but no major differences were found between ever and never smoking pSS patients. Taking into account the negative association between pSS diagnosis and smoking in epidemiological studies, possible explanations include a local effect of smoking on salivary glands rather than systemic effects by cigarette smoke.

Human MHC-II with Shared Epitope Motifs Are Optimal Epstein-Barr Virus Glycoprotein 42 Ligands-Relation to Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disorder of unknown etiology, which is characterized by inflammation in the synovium and joint damage. Although the pathogenesis of RA remains to be determined, a combination of environmental (e.g., viral infections) and genetic factors influence disease onset. Especially genetic factors play a vital role in the onset of disease, as the heritability of RA is 50-60%, with the human leukocyte antigen (HLA) alleles accounting for at least 30% of the overall genetic risk. Some HLA-DR alleles encode a conserved sequence of amino acids, referred to as the shared epitope (SE) structure. By analyzing the structure of a HLA-DR molecule in complex with Epstein-Barr virus (EBV), the SE motif is suggested to play a vital role in the interaction of MHC II with the viral glycoprotein (gp) 42, an essential entry factor for EBV. EBV has been repeatedly linked to RA by several lines of evidence and, based on several findings, we suggest that EBV is able to induce the onset of RA in predisposed SE-positive individuals, by promoting entry of B-cells through direct contact between SE and gp42 in the entry complex.

Mapping of the binding sites of human histamine N-methyltransferase (HNMT) monoclonal antibodies.

OBJECTIVE: Recently, we characterized mouse monoclonal antibodies that allow the specific and sensitive detection of human histamine N-methyltransferase (HNMT). To understand differences in binding characteristics and recognition of enzyme variants we mapped the antibody binding sites. METHODS: Fragments of human HNMT were expressed as glutathione S-transferase fusion proteins that were used for testing antibody binding on immunoblots. Combined information from species cross-reactivity, sequence comparison, protein structure, and binding site prediction software were used to localize the epitope recognized by each antibody. RESULTS: All eight monoclonal HNMT antibodies bound to linear epitopes in the C-terminal domain of the 292 amino acid protein. Of the five antibodies cross-reacting with HNMT from other species, one bound region L182-T223, three region M224-E261, and one region L262-A292. All three antibodies recognising only human HNMT bound the C-terminal region L262-A292 that contains residues present only in the human protein. CONCLUSIONS: Our HNMT monoclonal antibodies bind in three different regions of the protein and those binding the same putative epitope exhibit similar binding characteristics and species cross-reactivity. Antibodies binding non-overlapping epitopes will facilitate analyses of all clinically relevant variants described for HNMT.

Monoclonal antibodies for human and porcine histamine N-methyltransferase (HMT) facilitate protein expression and localization studies.

OBJECTIVE: The lack of suitable antibodies for the histamine inactivating enzyme histamine N-methyltransferase (HMT) has so far prevented the direct analysis of HMT proteins in man and other mammals. METHODS: A series of monoclonal antibodies was produced by immunizing mice with human and porcine HMT expressed in vitro. Antibodies were characterized by immunoblotting and immunohistochemical staining. RESULTS: Six different monoclonal antibodies specific for human HMT and four different monoclonal antibodies specific for porcine HMT were obtained that can detect HMT with up to tenfold greater sensitivity than the most sensitive enzymatic assays currently available. Using these antibodies allowed us to confirm the expression and cellular localization of HMT in various human and porcine tissues, where the presence of the enzyme had previously been deduced from activity measurement and HMT mRNA analysis. Immunohistochemical staining of human and porcine tissue sections clearly showed that HMT is a cytosolic protein, which is localized in specific cells of most mammalian tissues. CONCLUSIONS: The new monoclonal antibodies not only allow a comprehensive quantitative evaluation of the expression of HMT at the cellular level in man and other mammals but will also facilitate sensitive analyses of disease-associated alterations of this protein.