Antibody Derived Peptides for Detection of Ebola Virus Glycoprotein

Background

Current Ebola virus (EBOV) detection methods are costly and impractical for epidemic scenarios. Different immune-based assays have been reported for the detection and quantification of Ebola virus (EBOV) proteins. In particular, several monoclonal antibodies (mAbs) have been described that bind the capsid glycoprotein (GP) of EBOV GP. However, the currently available platforms for the design and production of full-length mAbs are cumbersome and costly. The use of antibody fragments, rather than full-length antibodies, might represent a cost-effective alternative for the development of diagnostic and possibly even therapeutic alternatives for EBOV.

Methods/Principal Findings

We report the design and expression of three recombinant anti-GP mAb fragments in Escherichia coli cultures. These fragments contained the heavy and light variable portions of the three well-studied anti-GP full-length mAbs 13C6, 13F6, and KZ52, and are consequently named scFv-13C6, scFv-13F6, and Fab-KZ52, respectively. All three fragments exhibited specific anti-GP binding activity in ELISA experiments comparable to that of full-length anti-GP antibodies (i.e., the same order of magnitude) and they are easily and economically produced in bacterial cultures.

Conclusion/Significance

Antibody fragments might represent a useful, effective, and low cost alternative to full-length antibodies in Ebola related capture and diagnostics applications.     

Delineation of stage specific expression of Plasmodium falciparum EBA-175 by biologically functional region II monoclonal antibodies

Background

The malaria parasite Plasmodium falciparum EBA-175 binds its receptor sialic acids on glycophorin A when invading erythrocytes. The receptor-binding region (RII) contains two cysteine-rich domains with similar cysteine motifs (F1 and F2). Functional relationships between F1 and F2 domains and characterization of EBA-175 were studied using specific monoclonal antibodies (mAbs) against these domains.

Methods and Findings

Five mAbs specific for F1 or F2 were generated. Three mAbs specific for F2 potently blocked binding of EBA-175 to erythrocytes, and merozoite invasion of erythrocytes (IC₅₀ 10 to 100 µg/ml IgG in growth inhibition assays). A mAb specific for F1 blocked EBA-175 binding and merozoite invasion less effectively. The difference observed between the IC₅₀ of F1 and F2 mAbs was not due to differing association and disassociation rates as determined by surface plasmon resonance. Four of the mAbs recognized conformation-dependent epitopes within F1 or F2. Used in combination, F1 and F2 mAbs blocked the binding of native EBA-175 to erythrocytes and inhibited parasite invasion synergistically in vitro. MAb R217, the most potent, did not recognize sporozoites, 3-day hepatocyte stage parasites, nor rings, trophozoites, gametocytes, retorts, ookinetes, and oocysts but recognized 6-day hepatocyte stage parasites, and schizonts. Even though efficient at blocking binding to erythrocytes and inhibiting invasion into erythrocytes, MAb R217 did not inhibit sporozoite invasion and development in hepatocytes in vitro.

Conclusions

The role of the F1 and F2 domains in erythrocyte invasion and binding was elucidated with mAbs. These mAbs interfere with native EBA-175 binding to erythrocyte in a synergistic fashion. The stage specific expression of EBA-175 showed that the primary focus of activity was the merozoite stage. A recombinant RII protein vaccine consisting of both F1 and F2 domains that could induce synergistic activity should be optimal for induction of antibody responses that interfere with merozoite invasion of erythrocytes.     

Hypogalactosylation of serum N-glycans fails to predict clinical response to methotrexate and TNF inhibition in rheumatoid arthritis

Introduction

Rheumatoid arthritis (RA) is associated with hypogalactosylation of immunoglobulin G (IgG). We examined whether a proxy measure for galactosylation of IgG N-glycans could predict response to therapy or was differentially affected by methotrexate (MTX) or TNF blockade.

Methods

Using a previously defined normal phase high-performance liquid chromatography approach, we ascertained the galactosylation status of whole serum N-glycans in two well-defined RA clinical cohorts: the Autoimmune Biomarkers Collaborative Network (n = 98) and Nested I (n = 64). The ratio of agalactosylated to monogalactosylated N-glycans in serum (sG0/G1) was determined before and during therapy with MTX or TNF inhibition and correlated with anticitrullinated peptide antibody (ACPA) status and clinical response as assessed by 28-joint Disease Activity Score utilizing C-reactive peptide and European League Against Rheumatism response criteria.

Results

RA patients from both cohorts exhibited elevation of sG0/G1 at baseline. Improvement in clinical scores correlated with a reduction in sG0/G1 (Spearman''s ρ = 0.31 to 0.37; P < 0.05 for each cohort). However, pretreatment sG0/G1 was not predictive of clinical response. Changes in sG0/G1 were similar in the MTX and TNF inhibitor groups. Corrected for disease activity, ACPA positivity correlated with higher sG0/G1.

Conclusions

Baseline serum N-glycan hypogalactosylation, an index previously correlated with hypogalactosylation of IgG N-glycans, did not distinguish patients with rheumatoid arthritis who were likely to experience a favorable clinical response to MTX or TNF blockade. Clinical improvement was associated with partial glycan normalization. ACPA-positive patients demonstrated enhanced N-glycan aberrancy compared with ACPA-negative patients.     

High affinity human antibody fragments to dengue virus non-structural protein 3

Background

The enzyme activities catalysed by flavivirus non-structural protein 3 (NS3) are essential for virus replication. They are distributed between the N-terminal protease domain in the first one-third and the C-terminal ATPase/helicase and nucleoside 5′ triphosphatase domain which forms the remainder of the 618-aa long protein.

Methodology/Principal Findings

In this study, dengue full-length NS3 protein with residues 49 to 66 of NS2B covalently attached via a flexible linker, was used as bait in biopanning with a naïve human Fab phage-display library. Using a range of truncated constructs spanning the NS2B cofactor region and the full-length NS3, 10 unique Fab were identified and characterized. Of these, monoclonal Fab 3F8 was shown to bind α3″ (residues 526 through 531) within subdomain III of the helicase domain. The antibody inhibits the ATPase and helicase activites of NS3 in biochemical assays and reduces DENV replication in HEK293 cells that were previously transfected with Fab 3F8 compared with mock transfected cells.

Conclusions/Significance

Antibodies such as 3F8 are valuable tools for studying the molecular mechanisms of flaviviral replication and for the monospecific detection of replicating dengue virus in vivo.     

Loss and recovery of Mgat3 and GnT-III Mediated E-cadherin N-glycosylation is a mechanism involved in epithelial-mesenchymal-epithelial transitions

Background

N-acetylglucosaminyltransferase-III (GnT-III) is a glycosyltransferase encoded by Mgat3 that catalyzes the addition of β1,4-bisecting-N-acetylglucosamine on N-glycans. GnT-III has been pointed as a metastases suppressor having varying effects on cell adhesion and migration. We have previously described the existence of a functional feedback loop between E-cadherin expression and GnT-III-mediated glycosylation. The effects of GnT-III-mediated glycosylation on E-cadherin expression and cellular phenotype lead us to evaluate Mgat3 and GnT-III-glycosylation role during Epithelial-Mesenchymal-Transition (EMT) and the reverted process, Mesenchymal-Epithelial-Transition (MET).

Methodology/Principal Findings

We analyzed the expression profile and genetic mechanism controlling Mgat3 expression as well as GnT-III-mediated glycosylation, in general and specifically on E-cadherin, during EMT/MET. We found that during EMT, Mgat3 expression was dramatically decreased and later recovered when cells returned to an epithelial-like phenotype. We further identified that Mgat3 promoter methylation/demethylation is involved in this expression regulation. The impact of Mgat3 expression variation, along EMT/MET, leads to a variation in the expression levels of the enzymatic product of GnT-III (bisecting GlcNAc structures), and more importantly, to the specific modification of E-cadherin glycosylation with bisecting GlcNAc structures.

Conclusions/Significance

Altogether, this work identifies for the first time Mgat3 glycogene expression and GnT-III-mediated glycosylation, specifically on E-cadherin, as a novel and major component of the EMT/MET mechanism signature, supporting its role during EMT/MET.     

Potent neutralization of botulinum neurotoxin/b by synergistic action of antibodies recognizing protein and ganglioside receptor binding domain

Background

Botulinum neurotoxins (BoNTs), the causative agents for life-threatening human disease botulism, have been recognized as biological warfare agents. Monoclonal antibody (mAb) therapeutics hold considerable promise as BoNT therapeutics, but the potencies of mAbs against BoNTs are usually less than that of polyclonal antibodies (or oligoclonal antibodies). The confirmation of key epitopes with development of effective mAb is urgently needed.

Methods and Findings

We selected 3 neutralizing mAbs which recognize different non-overlapping epitopes of BoNT/B from a panel of neutralizing antibodies against BoNT/B. By comparing the neutralizing effects among different combination groups, we found that 8E10, response to ganglioside receptor binding site, could synergy with 5G10 and 2F4, recognizing non-overlapping epitopes within Syt II binding sites. However, the combination of 5G10 with 2F4 blocking protein receptor binding sites did not achieve synergistical effects. Moreover, we found that the binding epitope of 8E10 was conserved among BoNT A, B, E, and F, which might cross-protect the challenge of different serotypes of BoNTs in vivo.

Conclusions

The combination of two mAbs recognizing different receptors'' binding domain in BoNTs has a synergistic effect. 8E10 is a potential universal partner for the synergistical combination with other mAb against protein receptor binding domain in BoNTs of other serotypes.     

Improved Virus Neutralization by Plant-produced Anti-HIV Antibodies with a Homogeneous ��1,4-Galactosylated N-Glycan Profile

It is well established that proper N-glycosylation significantly influences the efficacy of monoclonal antibodies (mAbs). However, the specific immunological relevance of individual mAb-associated N-glycan structures is currently largely unknown, because of the heterogeneous N-glycan profiles of mAbs when produced in mammalian cells. Here we report on the generation of a plant-based expression platform allowing the efficient production of mAbs with a homogeneous β1,4-galactosylated N-glycosylation structure, the major N-glycan species present on serum IgG. This was achieved by the expression of a highly active modified version of the human β1,4-galactosyltransferase in glycoengineered plants lacking plant-specific glycosylation. Moreover, we demonstrate that two anti-human immunodeficiency virus mAbs with fully β1,4-galactosylated N-glycans display improved virus neutralization potency when compared with other glycoforms produced in plants and Chinese hamster ovary cells. These findings indicate that mAbs containing such homogeneous N-glycan structures should display improved in vivo activities. Our system, using expression of mAbs in tobacco plants engineered for post-translational protein processing, provides a new means of overcoming the two hurdles that limit the therapeutic use of anti-human immunodeficiency virus mAbs in global health initiatives, low biological potency and high production costs.About 40 million people are estimated to be infected with HIV-1,² and the HIV-1/AIDS epidemic continues to escalate, with the most devastating consequences seen in the most impoverished nations (1). Two strategies that have been pursued over the past 2 decades for stopping the AIDS pandemic/epidemic are the generation of vaccines to prevent HIV infection and the development of microbicides to prevent HIV transmission. Highly effective monoclonal antibodies (mAbs) are suitable to be used in both modalities. To date, only a handful of anti-HIV mAbs with neutralizing activities has been explored in more detail (2). In a recent clinical study, it has been demonstrated that a combination of three broadly neutralizing anti-HIV antibodies (2G12, 2F5, and 4E10) shows promise as AIDS treatment (3). However, despite effective in vitro neutralization activities, relatively modest in vivo effects were obtained, suggesting that the in vivo properties of these antibodies require further improvement (2). Noteworthy, these antibodies bind to HIV envelope proteins thus inhibiting viral entry into target cells (2, 4, 5). In addition to their potential use in therapeutic modalities, this renders them as promising candidates for microbicide development. However, high production costs using mammalian-cell technologies and insufficient efficacy of anti-HIV antibodies are remaining hurdles for their effective use. Among recent advances in generating antibodies with enhanced activities, glyco-engineering has been proven to be a powerful tool (6). It is well established that proper N-glycosylation significantly influences the efficacy of mAbs. Nevertheless, the specific immunological relevance of individual mAb-associated N-glycan structures is largely unknown, because of the heterogeneous N-glycan profiles of mAbs when produced in mammalian cells. A series of studies emphasize the critical role of IgG glycoforms lacking core α1,6-fucose for cell-mediated immunological activities (6). However, the immunological significance of N-glycans with terminal β1,4-galactose residues, the major N-glycan species present on serum IgG, has not yet been established.During the last 2 decades, plants have been under intensive investigation to provide an alternative system for cost-effective, highly scalable, and safe production of recombinant proteins. This resulted in a significant enhancement of expression levels (up to 100-fold) and a reduction of production time (7, 8), which makes the system economically interesting. Another important achievement was the generation of plant glycosylation mutants, which allows a controlled human-type glycosylation of recombinant glycoproteins (9, 10). Recently, we have generated different glycoforms of anti-HIV mAb 2G12 in the tobacco-related plant species Nicotiana benthamiana (9). All of them were functionally active, and HIV neutralization potency was comparable with CHO-derived 2G12. This process involved the generation of a plant glycosylation mutant (ΔXT/FT), which was found to produce mAbs carrying homogeneous N-glycans with terminal N-acetylglucosamine (Gn) residues (i.e. GnGn structures) lacking unwanted plant-specific β1,2-xylose and core α1,3-fucose residues. These glycans are devoid of any β1,4-linked galactose residues; thus in this study, we set out to glyco-engineer ΔXT/FT plants for quantitative β1,4-galactosylation. A highly active modified version of human β1,4-galactosyltransferase was used to transform ΔXT/FT and progeny screened for efficient protein β1,4-galactosylation. In total four glycoforms from the two anti-HIV mAbs 2G12 and 4E10 (plant- and CHO-derived) were generated and compared toward antigen binding and virus neutralization capacities.     

Increased sensitivity to broadly neutralizing antibodies of end-stage disease R5 HIV-1 correlates with evolution in Env glycosylation and charge

Background

Induction of broadly neutralizing antibodies, such as the monoclonal antibodies IgGb12, 2F5 and 2G12, is the objective of most antibody-based HIV-1 vaccine undertakings. However, despite the relative conserved nature of epitopes targeted by these antibodies, mechanisms underlying the sensitivity of circulating HIV-1 variants to broadly neutralizing antibodies are not fully understood. Here we have studied sensitivity to broadly neutralizing antibodies of HIV-1 variants that emerge during disease progression in relation to molecular alterations in the viral envelope glycoproteins (Env), using a panel of primary R5 HIV-1 isolates sequentially obtained before and after AIDS onset.

Principal Findings

HIV-1 R5 isolates obtained at end-stage disease, after AIDS onset, were found to be more sensitive to neutralization by TriMab, an equimolar mix of the IgGb12, 2F5 and 2G12 antibodies, than R5 isolates from the chronic phase. The increased sensitivity correlated with low CD4⁺ T cell count at time of virus isolation and augmented viral infectivity. Subsequent sequence analysis of multiple env clones derived from the R5 HIV-1 isolates revealed that, concomitant with increased TriMab neutralization sensitivity, end-stage R5 variants displayed envelope glycoproteins (Envs) with reduced numbers of potential N-linked glycosylation sites (PNGS), in addition to increased positive surface charge. These molecular changes in Env also correlated to sensitivity to neutralization by the individual 2G12 monoclonal antibody (mAb). Furthermore, results from molecular modeling suggested that the PNGS lost at end-stage disease locate in the proximity to the 2G12 epitope.

Conclusions

Our study suggests that R5 HIV-1 variants with increased sensitivity to broadly neutralizing antibodies, including the 2G12 mAb, may emerge in an opportunistic manner during severe immunodeficiency as a consequence of adaptive molecular Env changes, including loss of glycosylation and gain of positive charge.     

Intra-examiner and inter-examiner reproducibility of paraspinal thermography

Objective

The objective of this study was to evaluate the intra-examiner and inter-examiner reproducibility of paraspinal thermography using an infrared scanner.

Materials and Methods

The thermal functions of a commercially available infrared scanner (Insight Subluxation Station®) were evaluated for clinical reliability. Two practicing clinicians conducted the measures on 100 subjects. Intra class correlation coefficients (ICCs) and concordance correlation coefficients (CCCs) were calculated from the collected data.

Results

Mean bilateral paraspinal skin temperature was 89.78° F and ranged from 88.77° F to 91.43° F. Intra class correlation coefficients (ICCs) for agreement and consistency ranged from 0.959 to 0.976. Concordance correlation coefficients (CCCs) ranged from 0.783 to 0.859 with tight confidence intervals indicating robust estimates of these quantities.

Conclusion

This study revealed excellent intra-examiner and inter-examiner reproducibility of paraspinal thermography using a commercially available unit.     

Decreased Levels of Bisecting GlcNAc Glycoforms of IgG Are Associated with Human Longevity

Background

Markers for longevity that reflect the health condition and predict healthy aging are extremely scarce. Such markers are, however, valuable in aging research. It has been shown previously that the N-glycosylation pattern of human immunoglobulin G (IgG) is age-dependent. Here we investigate whether N-linked glycans reflect early features of human longevity.

Methodology/Principal Findings

The Leiden Longevity Study (LLS) consists of nonagenarian sibling pairs, their offspring, and partners of the offspring serving as control. IgG subclass specific glycosylation patterns were obtained from 1967 participants in the LLS by MALDI-TOF-MS analysis of tryptic IgG Fc glycopeptides. Several regression strategies were applied to evaluate the association of IgG glycosylation with age, sex, and longevity. The degree of galactosylation of IgG decreased with increasing age. For the galactosylated glycoforms the incidence of bisecting GlcNAc increased as a function of age. Sex-related differences were observed at ages below 60 years. Compared to males, younger females had higher galactosylation, which decreased stronger with increasing age, resulting in similar galactosylation for both sexes from 60 onwards. In younger participants (<60 years of age), but not in the older age group (>60 years), decreased levels of non-galactosylated glycoforms containing a bisecting GlcNAc reflected early features of longevity.

Conclusions/Significance

We here describe IgG glycoforms associated with calendar age at all ages and the propensity for longevity before middle age. As modulation of IgG effector functions has been described for various IgG glycosylation features, a modulatory effect may be expected for the longevity marker described in this study.     

Tissue Specificity of Human Angiotensin I-Converting Enzyme

Background

Angiotensin-converting enzyme (ACE), which metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling, as well as in reproductive functions, is expressed as a type-1 membrane glycoprotein on the surface of endothelial and epithelial cells. ACE also presents as a soluble form in biological fluids, among which seminal fluid being the richest in ACE content - 50-fold more than that in blood.

Methods/Principal Findings

We performed conformational fingerprinting of lung and seminal fluid ACEs using a set of monoclonal antibodies (mAbs) to 17 epitopes of human ACE and determined the effects of potential ACE-binding partners on mAbs binding to these two different ACEs. Patterns of mAbs binding to ACEs from lung and from seminal fluid dramatically differed, which reflects difference in the local conformations of these ACEs, likely due to different patterns of ACE glycosylation in the lung endothelial cells and epithelial cells of epididymis/prostate (source of seminal fluid ACE), confirmed by mass-spectrometry of ACEs tryptic digests.

Conclusions

Dramatic differences in the local conformations of seminal fluid and lung ACEs, as well as the effects of ACE-binding partners on mAbs binding to these ACEs, suggest different regulation of ACE functions and shedding from epithelial cells in epididymis and prostate and endothelial cells of lung capillaries. The differences in local conformation of ACE could be the base for the generation of mAbs distingushing tissue-specific ACEs.     

Effects of natalizumab treatment on Foxp3+ T regulatory cells

Background

Natalizumab, a monoclonal humanized antibody targeting the alpha-4 chain of very late activation antigen 4 (VLA-4) exerts impressive therapeutic effects in patients with relapsing-remitting multiple sclerosis. Our objective was to study impacts of Natalizumab therapy on Foxp3+ T regulatory cells (Tregs) in multiple sclerosis (MS) patients.

Methodology

A combined approach of in vitro and ex vivo experiments using T cells isolated from the peripheral blood of healthy donors and Natalizumab treated MS patients was chosen. We determined binding of Natalizumab and its effects on the frequency, transmigratory behaviour and suppressive function of Tregs.

Principal Findings

Binding of Natalizumab and expression of CD49d (alpha-4 chain of VLA-4) differed between non-regulatory and regulatory cells. Albeit Foxp3+ Tregs had lower levels of CD49d, Natalizumab blocked the transmigration of Foxp3+ Tregs similar to non-regulatory T cells. The frequency of peripheral blood Tregs was unaffected by Natalizumab treatment. Natalizumab does not alter the suppressive capacity of CD4+CD25^highCD127^lowFoxp3+ Tregs under in vitro conditions. Furthermore, the impaired function of Tregs in MS patients is not restored by Natalizumab treatment.

Conclusions

We provide a first detailed analysis of Natalizumab effects on the regulatory T cell population. Our prospective study shows that Foxp3+ Tregs express lower levels of VLA-4 and bind less Natalizumab. We further the understanding of the mechanisms of action of Natalizumab by demonstrating that unlike other immunomodulatory drugs the beneficial therapeutic effects of the monoclonal antibody are largely independent of alterations in Treg frequency or function.     

Liver stiffness measurement and biochemical markers in Senegalese chronic hepatitis B patients with normal ALT and high viral load

Background and Aims

Despite the high prevalence of chronic hepatitis B (CHB) in Africa, few studies have been performed among African patients. We sought to evaluate liver stiffness measurement by FibroScan® (LSM) and two biochemical scores (FibroTest®, Fibrometer®) to diagnose liver fibrosis in Senegalese CHB patients with HBV plasma DNA load ≥3.2 log₁₀ IU/mL and normal alanine aminotransferase (ALT) values.

Methods

LSM and liver fibrosis biochemical markers were performed on 225 consecutive HBV infected Senegalese patients with high viral load. Patients with an LSM range between 7 and 13 kPa underwent liver biopsy (LB). Two experienced liver pathologists performed histological grading using Metavir and Ishak scoring.

Results

225 patients were evaluated (84% male) and LB was performed in 69 patients, showing F2 and F3 fibrosis in 17% and 10% respectively. In these patients with a 7–13 kPa range of LSM, accuracy for diagnosis of significant fibrosis according to LB was unsatisfactory for all non-invasive markers with AUROCs below 0.70. For patients with LSM values below 7 kPa, FibroTest® (FT), and Fibrometer® (FM) using the cut-offs recommended by the test promoters suggested a fibrosis in 18% of cases for FT (8% severe fibrosis) and 8% for FM. For patients with LSM values greater than 13 kPa, FT, FM suggested a possible fibrosis in 73% and 70%, respectively.

Conclusion

In highly replicative HBV-infected African patients with normal ALT and LSM value below 13 kPa, FibroScan®, FibroTest® or Fibrometer® were unsuitable to predict the histological liver status of fibrosis.     

Strengths and weaknesses of immunotherapy for advanced non-small-cell lung cancer: a meta-analysis of 12 randomized controlled trials

Background

Lung cancer is one of the leading causes of cancer death worldwide. Non-small-cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers. Immunotherapy has yielded no consistent benefit to date for those patients. Assessing the objective efficacy and safety of immunotherapy for advanced NSCLC patients will help to instruct the future development of immunotherapeutic drugs.

Methodology and Principal Findings

We performed a meta-analysis of 12 randomized controlled trials including 3134 patients (1570 patients in the immunotherapy group and 1564 patients in the control group) with histologically confirmed stage IIIA, IIIB, or IV NSCLC. The analysis was executed with efficacy end points regarding overall survival (OS), progression-free survival (PFS), complete response (CR), partial response (PR), and total effective rate. Overall unstratified OS, PFS, PR, and total effective rate were significantly improved in advanced NSCLC patients in the immunotherapy group (P = 0.0007, 0.0004, 0.002, 0.003, respectively), whereas CR was not improved (P = 0.97). Subgroup analysis showed that monoclonal antibody (mAb) immunotherapy significantly improved the PFS, PR, and total effective rate and showed a trend of improving OS of advanced NSCLC patients compared with the control group, with one kind of adverse event being significantly dominant. Compared with the control group, the vaccine subgroup showed no significant difference with regard to serious adverse events, whereas cytokine immunotherapy significantly induced three kinds of serious adverse events.

Conclusions

Immunotherapy works efficiently on advanced NSCLC patients. Of several immunotherapies, mAb therapy may be a potential immunotherapy for advanced NSCLC patients, and become a standard complementary therapeutic approach in the future if the issues concerning toxicity and allergenicity of mAbs have been overcome.     

Glycomic profiling of carcinoembryonic antigen isolated from human tumor tissue

Background

Carcinoembryonic antigen (CEA) is a protein commonly found in human serum, with elevated CEA levels being linked to the progression of a wide range of tumors. It is currently used as a biomarker for malign tumors such as lung cancer and colorectal cancer [Urol Oncol: Semin Orig Invest 352: 644–648, 2013 and Lung Cancer 80: 45-49, 2013]. However, due to its low specificity in clinical applications, CEA can be used for monitoring only, rather than tumor diagnosis. The function of many glycoproteins is critically dependent on their glycosylation pattern, which in turn has the potential to serve in tumor detection. However, little is known about the detailed glycan patterns of CEA.

Methods

To determine these patterns, we isolated and purified CEA proteins from human tumor tissues using immunoaffinity chromatography. The glycan patterns of CEA were then analyzed using a Matrix-Assisted Laser Desorption/Ionization-Time of Flight-Mass Spectrometry³ (MALDI-TOF-MS³) approach.

Results

We identified 61 glycoforms in tumor tissue, where CEA is upregulated. These glycosylation entities were identified as bi-antennary, tri-antennary and tetra-antennary structures carrying sialic acid and fucose residues, and include a multitude of glycans previously not reported for CEA.

Conclusion

Our findings should facilitate a more precise tumor prediction than currently possible, ultimately resulting in improved tumor diagnosis and treatment.

Electronic supplementary material

The online version of this article (doi:10.1186/s12014-015-9088-3) contains supplementary material, which is available to authorized users.     

An Adenovirus Vector Incorporating Carbohydrate Binding Domains Utilizes Glycans for Gene Transfer

Background

Vectors based on human adenovirus serotype 5 (HAdV-5) continue to show promise as delivery vehicles for cancer gene therapy. Nevertheless, it has become clear that therapeutic benefit is directly linked to tumor-specific vector localization, highlighting the need for tumor-targeted gene delivery. Aberrant glycosylation of cell surface glycoproteins and glycolipids is a central feature of malignant transformation, and tumor-associated glycoforms are recognized as cancer biomarkers. On this basis, we hypothesized that cancer-specific cell-surface glycans could be the basis of a novel paradigm in HAdV-5-based vector targeting.

Methodology/Principal Findings

As a first step toward this goal, we constructed a novel HAdV-5 vector encoding a unique chimeric fiber protein that contains the tandem carbohydrate binding domains of the fiber protein of the NADC-1 strain of porcine adenovirus type 4 (PAdV-4). This glycan-targeted vector displays augmented CAR-independent gene transfer in cells with low CAR expression. Further, we show that gene transfer is markedly decreased in cells with genetic glycosylation defects and by inhibitors of glycosylation in normal cells.

Conclusions/Significance

These data provide the initial proof-of-concept for HAdV-5 vector-mediated gene delivery based on the presence of cell-surface carbohydrates. Further development of this new targeting paradigm could provide targeted gene delivery based on vector recognition of disease-specific glycan biomarkers.     

Spatial regulation of cell-wall structure in response to heavy metal stress: cadmium-induced alteration of the methyl-esterification pattern of homogalacturonans

Background and Aims

In flax hypocotyls, cadmium-induced reorientation of growth coincides with marked changes in homogalacturonan (HGA) epitopes that were recognized by JIM7 and JIM5 antibodies in the external tangential wall of the epidermis. In the present study, LM7 and 2F4 monoclonal antibodies were used, in addition to JIM5 and JIM7, to extend the investigation on the methyl-esterification pattern of HGA within various domains of the cortical tissues, including the cortical parenchyma where cell cohesion is crucial.

Methods

The PATAg (periodic acid thiocarbohydrazide–silver proteinate) test was applied to ultrathin sections so that the polysaccharides could be visualized and the ultrastructure studied. The monoclonal LM7, JIM5 and JIM7 antibodies that recognize differently methyl-esterified HGA were used. The monoclonal 2F4 antibody that is specific to a particular polygalacturonic acid conformation induced by a given calcium to sodium ratio was also applied. After immunogold labelling, the grids were stained with uranyl-acetate, the samples were observed using a transmission electron microscope and the gold particles were counted.

Key Results

In the presence of cadmium, the increase of LM7 labelling in external tangential wall of the epidermis, together with a decrease of JIM7 labelling, suggested a specific role for randomly partially de-esterified HGA to counteract the radial swelling stress. Enhanced JIM5 and 2F4 labelling in the junctions of the inner tissues indicated that the presence of blockwise de-esterified HGA might oppose cell separation.

Conclusions

The response of the hypocotyl to cadmium stress was to adapt the structure of the wall of cortical tissues by differently modulating the methyl-esterification pattern of HGA in various domains.     

Cartilage oligomeric matrix protein specific antibodies are pathogenic

Introduction

Cartilage oligomeric matrix protein (COMP) is a major non-collagenous component of cartilage. Earlier, we developed a new mouse model for rheumatoid arthritis using COMP. This study was undertaken to investigate the epitope specificity and immunopathogenicity of COMP-specific monoclonal antibodies (mAbs).

Methods

B cell immunodominant regions on the COMP molecule were measured with a novel enzyme-linked immunosorbent assay using mammalian expressed full-length mouse COMP as well as a panel of recombinant mouse COMP fragments. 18 mAbs specific to COMP were generated and the pathogenicity of mAbs was investigated by passive transfer experiments.

Results

B cell immunodominant epitopes were localized within 4 antigenic domains of the COMP but with preferential response to the epidermal growth factor (EGF)-like domain. Some of our anti-COMP mAbs showed interactions with the native form of COMP, which is present in cartilage and synovium. Passive transfer of COMP-specific mAbs enhanced arthritis when co-administrated with a sub-arthritogenic dose of a mAb specific to collagen type II. Interestingly, we found that a combination of 5 COMP mAbs was capable of inducing arthritis in naive mice.

Conclusions

We have identified the specificities of mAbs to COMP and their contribution to the development of arthritis. These findings will further improve our understanding of the autoantibody mediated immunopathologies occurring widely in rheumatoid arthritis (RA), as well as in other autoimmune disorders.     

Economic impact of cystic echinococcosis in peru

Background

Cystic echinococcosis (CE) constitutes an important public health problem in Peru. However, no studies have attempted to estimate the monetary and non-monetary impact of CE in Peruvian society.

Methods

We used official and published sources of epidemiological and economic information to estimate direct and indirect costs associated with livestock production losses and human disease in addition to surgical CE-associated disability adjusted life years (DALYs) lost.

Findings

The total estimated cost of human CE in Peru was U.S.$2,420,348 (95% CI:1,118,384–4,812,722) per year. Total estimated livestock-associated costs due to CE ranged from U.S.$196,681 (95% CI:141,641–251,629) if only direct losses (i.e., cattle and sheep liver destruction) were taken into consideration to U.S.$3,846,754 (95% CI:2,676,181–4,911,383) if additional production losses (liver condemnation, decreased carcass weight, wool losses, decreased milk production) were accounted for. An estimated 1,139 (95% CI: 861–1,489) DALYs were also lost due to surgical cases of CE.

Conclusions

This preliminary and conservative assessment of the socio-economic impact of CE on Peru, which is based largely on official sources of information, very likely underestimates the true extent of the problem. Nevertheless, these estimates illustrate the negative economic impact of CE in Peru.     

Effect of serotype on focus and mortality of invasive pneumococcal disease: coverage of different vaccines and insight into non-vaccine serotypes

Background

Differences in pathogenicity between pneumococcal serotypes are important when assessing the potential benefit of different valency vaccines. We investigated the effect of serotype on clinical presentation, outcome, and quality of life lost from invasive pneumococcal disease (IPD) in the context of the 7, 10, and 13 valent pneumococcal conjugate vaccines (PCV7, PCV10, PCV13).

Method

Serotyped IPD cases in England were linked to the national dataset of hospital admissions for April 2002 to March 2011. Based on patients’ diagnostic codes and vital status at the end of the admission, disease focus (meningitis, empyema, sepsis, or respiratory disease) and case fatality rates by serotype and age group (5, 5–64, and 65 years and over) were obtained. Using these data the quality adjusted life years (QALY) lost from the IPD remaining when use of PCV7 stopped in 2010 was estimated for the serotypes covered by higher valency vaccines.

Results

The linked dataset contained 23,688 cases with information on diagnosis, mortality, and serotype. There were significant differences between serotypes in the propensity to cause meningitis, death, and QALY loss in each of the investigated age groups. As a result, vaccines’ coverage of disease burden differed by endpoint. For example, in children under 5 years in 2009/10, PCV10 covered 39% of meningitis, 19% of deaths and 28% of the QALY loss of attributable to IPD, whereas the respective percentages for PCV13 were 65%, 67%, and 66%. The highest QALY loss per serotype in this age group was for 6A. Non-PCV serotypes causing the highest QALY loss were 22F and 33F in <5 year olds and 31 in older individuals.

Conclusion

Marked differences exist between serotypes in clinical presentation and outcome, and these should be considered when evaluating the potential impact of higher valency vaccines on overall disease burden and associated QALY loss.