A protein microarray assay for serological determination of antigen-specific antibody responses following Clostridium difficile infection

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-C. difficile antibody levels in human sera and in separate preparations of polyclonal IVIg. The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. Herein, a combination of isotype (IgG, IgA IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), precursor form of B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027) and control proteins (Tetanus toxoid and Candida albicans) were determined by protein microarray. Microarrays were probed with sera from individuals with C. difficile infection (CDI), cystic fibrosis (CF) without diarrhea, healthy controls and individuals pre- and post-IVIg therapy for treatment of CDI, combined immunodeficiency disorder and chronic inflammatory demyelinating polyradiculopathy. Significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels were seen between patient groups, commercial preparations of IVIg and sera before and following IVIg administration. A significant correlation was observed between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C. difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, it is anticipated that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner

Immune responses to hepatitis C virus (HCV): investigation of the role of L-ficolin and anti-E1E2

Hepatitis C virus (HCV) causes acute and chronic liver diseases in humans. Its two envelope proteins, E1 and E2, are heavily glycosylated. They interact with host cell receptors and provide a target for host immune recognition. The host virus interactions determine the pathogenesis and outcome of HCV infection. L-ficolin is a soluble pattern recognition molecule of importance in innate immune defence against microorganisms. It activates the lectin complement pathway upon binding to carbohydrate recognition patterns on microorganisms. It was hypothesised that L-ficolin could interact with HCV glycoproteins. Both recombinant and serum derived L-ficolin were investigated for binding to the envelope glycoprotein E1E2 of HCV. Specific, dose-dependent binding of L-ficolin to HCV glycoprotein E1E2 was observed. The interaction between L-ficolin and HCV particles in infected sera was also demonstrated. Binding of L-ficolin to HCV pseudoparticles expressing E1E2 glycoproteins resulted in neutralisation of virus infectivity. The serum L-ficolin level was significantly higher in patients with mild HCV liver fibrosis compared to patients with severe HCV liver fibrosis. These results suggest a potential protective effect of L-ficolin, as an innate immune defence, against HCV infection. To study the role of anti-HCV E1 and E2 (anti-E1E2) in HCV disease, the levels of anti-E1E2 antibodies were evaluated in 230 sera of patients with chronic hepatitis C by enzyme-linked immunosorbent assay. The antigens used were recombinant HCV glycoproteins derived from genotype 1 (H77c) and genotype 3 (UKN3A1.28). Seroreactivity was greater when sera were tested against antigen derived from their homologous genotype than against heterologous antigen. The seroreactivity was inversely proportional to the viral load and to the degree of liver fibrosis. These results demonstrate that seroreactivity against E1E2 depends upon the genotypic origin of the E1E2 antigens and the infecting genotype, and suggest a possible protective effect of anti-E1E2 against disease progression

Impact of body mass index on the incidence and severity of post-endoscopic retrograde cholangiopancreatography pancreatitis

Background: Pancreatitis is a potential major complication after endoscopic retrograde cholangiopancreatography (post-ERCP pancreatitis; PEP). Obesity has been associated with increased severity of acute pancreatitis. However, the correlation between obesity and PEP is controversial. Therefore, our study aimed to clarify the relationship between body mass index (BMI) and the incidence and severity of PEP. Methods: A retrospective cohort study was conducted to elucidate the relationship between BMI and PEP in all patients who underwent ERCP in a tertiary referral center between January 2009 and October 2016. Patient characteristics and procedure details were collected. PEP was defined by consensus criteria. Multivariate logistic regression was used to determine the association between BMI and PEP. Results: The analysis included 2236 patients whose BMI was recorded and had adequate follow up (921 with BMI≥30 kg/m2, 1315 with BMI<30 kg/m2). PEP was diagnosed in 107 (4.8%) patients. PEP was seen in 49 obese patients (5.3%) and 58 non-obese patients (4.4%). In the univariate and multivariate analysis BMI≥30 kg/m2 was not associated with PEP (odds ratio 1.2, 95%CI 0.8-1.8; P=0.32). A subgroup analysis of different BMI subcategories found that BMI was not associated with the incidence or severity of PEP. Conclusion: In the largest study to date, neither obesity nor low body weight increased the incidence or severity of PEP

Development and validation of protein microarray technology for simultaneous inflammatory mediator detection in human sera

Biomarkers, including cytokines, can help in the diagnosis, prognosis, and prediction of treatment response across a wide range of disease settings. Consequently, the recent emergence of protein microarray technology, which is able to quantify a range of inflammatory mediators in a large number of samples simultaneously, has become highly desirable. However, the cost of commercial systems remains somewhat prohibitive. Here we show the development, validation, and implementation of an in-house microarray platform which enables the simultaneous quantitative analysis of multiple protein biomarkers. The accuracy and precision of the in-house microarray system were investigated according to the Food and Drug Administration (FDA) guidelines for pharmacokinetic assay validation. The assay fell within these limits for all but the very low-abundant cytokines, such as interleukin- (IL-) 10. Additionally, there were no significant differences between cytokine detection using our microarray system and the “gold standard” ELISA format. Crucially, future biomarker detection need not be limited to the 16 cytokines shown here but could be expanded as required. In conclusion, we detail a bespoke protein microarray system, utilizing well-validated ELISA reagents, that allows accurate, precise, and reproducible multiplexed biomarker quantification, comparable with commercial ELISA, and allowing customization beyond that of similar commercial microarrays

Homoclinicity in the dynamics of forced fluidized bed catalytic reactors

The dynamic behaviour of the non-isothermal oscillating fluidized bed catalytic reactor subjected to high and low frequency forcing is investigated. Resonance horns and period doubling loci inside the horns are constructed, using efficient algorithms, for different positions of the centre of forcing relative to the homoclinical orbit (infinite period orbit) of the autonomous (unforced) system. The analysis shows that the reactor behaviour is very sensitive to the position of the centre of forcing relative to the homoclinical orbit. For high frequency forcing (ω/ω0 = 5/1) with centre of forcing close to the homoclinical orbit, the system shows period doubling to chaos at very small amplitudes. For low frequency forcing (ω/ω0 = 2/1), incomplete period doubling not leading to chaos is observed in some cases and the new period adding bifurcation is also uncovered for other cases. The interactions between different resonance horns are investigated and the resulting bistabilities are elucidated. Quantitative methods proved that the strange attractors encountered are chaotic with one positive Lyapunov exponent and a Lyapunov fractal dimension between 1 and 2

Immunization with a synthetic consensus hepatitis C virus E2 glycoprotein ectodomain elicits virus-neutralizing antibodies

Global eradication of hepatitis C virus (HCV) infection will require an efficacious vaccine capable of eliciting protective immunity against genetically diverse HCV strains. Natural spontaneous resolution of HCV infection is associated with production of broadly neutralizing antibodies targeting the HCV glycoproteins E1 and E2. As such, production of cross-neutralizing antibodies is an important endpoint for experimental vaccine trials. Varying success generating cross-neutralizing antibodies has been achieved with immunogens derived from naturally-occurring HCV strains. In this study the challenge of minimising the genetic diversity between the vaccine strain and circulating HCV isolates was addressed. Two novel synthetic E2 glycoprotein immunogens (NotC1 and NotC2) were derived from consensus nucleotide sequences deduced from samples of circulating genotype 1 HCV strains. These two synthetic sequences differed in their relative positions in the overall genotype 1a/1b phylogeny. Expression of these constructs in Drosophila melanogaster S2 cells resulted in high yields of correctly-folded, monomeric E2 protein, which were recognised by broadly neutralizing monoclonal antibodies. Immunization of guinea pigs with either of these consensus immunogens, or a comparable protein representing a circulating genotype 1a strain resulted in high titres of cross-reactive anti-E2 antibodies. All immunogens generated antibodies capable of neutralizing the H77 strain, but NotC1 elicited antibodies that more potently neutralized virus entry. These vaccine-induced antibodies neutralized some viruses representing genotype 1, but not strains representing genotype 2 or genotype 3. Thus, while this approach to vaccine design resulted in correctly folded, immunogenic protein, cross-neutralizing epitopes were not preferentially targeted by the host immune response generated by this immunogen. Greater immunofocussing by vaccines to common epitopes is necessary to successfully elicit broadly neutralizing antibodies

Clinicopathological and prognostic significance of mitogen-activated protein kinases (MAPK) in breast cancers

Background Mitogen-activated protein kinases (MAPKs) are signalling transduction molecules that have different functions and diverse behaviour in cancer. In breast cancer, MAPK is related to oestrogen receptor (ER) and HER2. Methods Protein expression of a large panel of MAPKs (JNK1/2, ERK, p38, C-JUN and ATF2 including phosphorylated forms) were assessed immunohistochemically in a large (n = 1400) and well-characterised breast cancer series prepared as tissue microarray. Moreover, reverse phase protein array was applied to quantify protein expression of MAPKs in six breast cancer cell lines with different phenotypes including HER2-transfected cells. Results MAPKs expression was associated with clinicopathological variables characteristic of good prognosis. These associations were most significant in the whole series and in the ER? subgroup compared to other BC classes. Most of MAPKs showed a positive association with ER, BCL2 and better outcome and were negatively associated with the proliferation marker Ki67 and p53. Association of MAPK with HER2 was mainly seen in the ER- subgroup. Reverse phase protein array confirmed immunohistochemistry results and revealed differential expression of MAPK proteins in ER? and ER- cell lines. Conclusions MAPKs are associated with good prognosis and their expression is mainly related to ER. Studying a large panel rather than individual biomarkers may provide improved understanding of the pathwa

High prevalence of subclass-specific binding and neutralizing antibodies against Clostridium difficile toxins in adult cystic fibrosis sera: possible mode of immunoprotection against symptomatic C. difficile infection

Objectives: Despite multiple risk factors and a high rate of colonization for Clostridium difficile, the occurrence of C. difficile infection in patients with cystic fibrosis is rare. The aim of this study was to compare the prevalence of binding C. difficile toxin-specific immunoglobulin (Ig)A, IgG and anti-toxin neutralizing antibodies in the sera of adults with cystic fibrosis, symptomatic C. difficile infection (without cystic fibrosis) and healthy controls. Methods: Subclass-specific IgA and IgG responses to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B only expressing strain (CCUG 20309) and precursor form of B fragment of binary toxin, pCDTb, were determined by protein microarray. Neutralizing antibodies to C. difficile toxins A and B were evaluated using a Caco-2 cell-based neutralization assay. Results: Serum IgA anti-toxin A and B levels and neutralizing antibodies against toxin A were significantly higher in adult cystic fibrosis patients (n=16) compared with healthy controls (n=17) and patients with symptomatic C. difficile infection (n=16); p≤0.05. The same pattern of response prevailed for IgG, except that there was no difference in anti-toxin A IgG levels between the groups. Compared with healthy controls (toxins A and B) and patients with C. difficile infection (toxin A), sera from cystic fibrosis patients exhibited significantly stronger protective anti-toxin neutralizing antibody responses. Conclusion: A superior ability to generate robust humoral immunity to C. difficile toxins in the cystic fibrosis population is likely to confer protection against symptomatic C. difficile infection. This protection may be lost in the post-transplantation setting, where sera-monitoring of anti-C. difficile toxin antibody titers may be of clinical value

Patients with TNF Receptor Associated Periodic Syndrome (TRAPS) are hypersensitive to Toll‐like receptor 9 stimulation

Tumour necrosis factor receptor‐associated periodic syndrome (TRAPS) is an hereditary autoinflammatory disorder characterised by recurrent episodes of fever and inflammation. It is associated with autosomal dominant mutations in TNFRSF1A, which encodes tumour necrosis factor receptor‐1 (TNFR1). Our aim was to understand the influence of TRAPS mutations on the response to stimulation of the pattern recognition receptor TLR9. Peripheral blood mononuclear cells (PBMCs) and serum were isolated from TRAPS patients and healthy controls: Serum levels of fifteen pro‐inflammatory cytokines were measured to assess the initial inflammatory status. IL‐1β, IL‐6, IL‐8, IL17, IL22, TNF‐α, VEGF, IFN‐γ, MCP‐1 and TGF‐β were significantly elevated in TRAPS patients sera, consistent with constitutive inflammation. Stimulation of PBMCs with TLR9 ligand (ODN2006) triggered significantly greater upregulation of pro‐inflammatory signalling intermediates (TRAF3, IRAK2, TOLLIP, TRAF6, pTAK, TAB2, pTAB2, IRF7, RIP, NF‐kB p65, pNF‐κB p65, and MEK1/2) in TRAPS patients’ PBMCs. This upregulation of proinflammatory signalling intermediates and raised serum cytokines occurred despite concurrent anakinra treatment and no overt clinical symptoms at time of sampling. These novel findings further demonstrate the wide‐ranging nature of the dysregulation of innate immune responses underlying the pathology of TRAPS and highlights the need for novel pathway‐specific therapeutic treatments for this disease