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-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (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. We show how protein microarray can be used to determine 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), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation 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, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.

Characterization of Behavioral, Signaling and Cytokine Alterations in a Rat Neurodevelopmental Model for Schizophrenia, and Their Reversal by the 5-HT6 Receptor Antagonist SB-399885.

Post-weaning social isolation of rats produces neuroanatomical, neurochemical and behavioral alterations resembling some core features of schizophrenia. This study examined the ability of the 5-HT6 receptor antagonist SB-399885 to reverse isolation-induced cognitive deficits, then investigated alterations in hippocampal cell proliferation and hippocampal and frontal cortical expression of selected intracellular signaling molecules and cytokines. Male Lister hooded rats (weaned on post-natal days 21-24 and housed individually or in groups of 3-4) received six i.p. injections of vehicle (1% Tween 80, 1 mL/kg) or SB-399885 (5 or 10 mg/kg) over a 2-week period starting 40 days post-weaning, on the days that locomotor activity, novel object discrimination (NOD), pre-pulse inhibition of acoustic startle and acquisition, retention and extinction of a conditioned freezing response (CFR) were assessed. Tissue was collected 24 h after the final injection for immunohistochemistry, reverse-phase protein microarray and western blotting. Isolation rearing impaired NOD and cue-mediated CFR, decreased cell proliferation within the dentate gyrus, and elevated hippocampal TNFα levels and Cdc42 expression. SB-399885 reversed the NOD deficit and partially normalized CFR and cell proliferation. These effects were accompanied by altered expression of several members of the c-Jun N-terminal Kinase (JNK) and p38 MAPK signaling pathways (including TAK1, MKK4 and STAT3). Although JNK and p38 themselves were unaltered at this time point hippocampal TAK1 expression and phosphorylation correlated with visual recognition memory in the NOD task. Continued use of this neurodevelopmental model could further elucidate the neurobiology of schizophrenia and aid assessment of novel therapies for drug-resistant cognitive symptoms.

Autoantibodies of IgM and IgG classes show differences in recognition of multiple autoantigens in chronic obstructive pulmonary disease.

Autoimmunity occurs in chronic obstructive pulmonary disease (COPD). We describe an antigen microarray for detecting serum autoantibodies (AAbs) to determine how IgM, as well as IgG, AAbs distinguish patients with COPD from controls with a history of smoking without COPD. All COPD patients' sera contained elevated levels of AAbs to some of 30 autoantigens. There were significant differences in the autoantigenic specificities of IgM AAbs compared to IgG AAbs in the COPD sera: for example, AAbs to histone and scl-70 were mainly IgG, whereas AAbs to CENP-B and La/ssB were mainly IgM; by contrast, IgM and IgG AAbs to collagen-V were equally prevalent. Thus, a combination of IgM and IgG AAbs specific for multiple autoantigens are detected in all cases of COPD at a level at which all non-COPD controls are negative for AAbs. This highlights the importance of different classes of AAbs to a range of autoantigens in COPD.

A signalome screening approach in the autoinflammatory disease TNF receptor associated periodic syndrome (TRAPS) highlights the anti-inflammatory properties of drugs for repurposing.

TNF receptor associated periodic syndrome (TRAPS) is an autoinflammatory disease caused by mutations in TNF Receptor 1 (TNFR1). Current therapies for TRAPS are limited and do not target the pro-inflammatory signalling pathways that are central to the disease mechanism. Our aim was to identify drugs for repurposing as anti-inflammatories based on their ability to down-regulate molecules associated with inflammatory signalling pathways that are activated in TRAPS. This was achieved using rigorously optimized, high through-put cell culture and reverse phase protein microarray systems to screen compounds for their effects on the TRAPS-associated inflammatory signalome. 1360 approved, publically available, pharmacologically active substances were investigated for their effects on 40 signalling molecules associated with pro-inflammatory signalling pathways that are constitutively upregulated in TRAPS. The drugs were screened at four 10-fold concentrations on cell lines expressing both wild-type (WT) TNFR1 and TRAPS-associated C33Y mutant TNFR1, or WT TNFR1 alone; signalling molecule levels were then determined in cell lysates by the reverse-phase protein microarray. A novel mathematical methodology was developed to rank the compounds for their ability to reduce the expression of signalling molecules in the C33Y-TNFR1 transfectants towards the level seen in the WT-TNFR1 transfectants. Seven high-ranking drugs were selected and tested by RPPA for effects on the same 40 signalling molecules in lysates of peripheral blood mononuclear cells (PBMCs) from C33Y-TRAPS patients compared to PBMCs from normal controls. The fluoroquinolone antibiotic lomefloxacin, as well as others from this class of compounds, showed the most significant effects on multiple pro-inflammatory signalling pathways that are constitutively activated in TRAPS; lomefloxacin dose-dependently significantly reduced expression of 7/40 signalling molecules across the Jak/Stat, MAPK, NF-κB and PI3K/AKT pathways. This study demonstrates the power of signalome screening for identifying candidates for drug repurposing.

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.

The role of indoleamine 2,3-dioxygenase-aryl hydrocarbon receptor pathway in the TLR4-induced tolerogenic phenotype in human DCs.

A controlled inflammatory response is required for protection against infection, but persistent inflammation causes tissue damage. Dendritic cells (DCs) have a unique capacity to promote both inflammatory and anti-inflammatory processes. One key mechanism involved in DC-mediated immunosuppression is the expression of tryptophan-metabolizing enzyme indoleamine 2,3-dioxygenase (IDO). IDO has been implicated in diverse processes in health and disease but its role in endotoxin tolerance in human DCs is still controversial. Here we investigated the role of IDO in shaping DCs phenotype and function under endotoxin tolerance conditions. Our data show that TLR4 ligation in LPS-primed DCs, induced higher levels of both IDO isoforms together with the transcription factor aryl-hydrocarbon receptor (AhR), compared to unprimed controls. Additionally, LPS conditioning induced an anti-inflammatory phenotype in DCs - with an increase in IL-10 and higher expression of programmed death ligand (PD-L)1 and PD-L2 - which were partially dependent on IDO. Furthermore, we demonstrated that the AhR-IDO pathway was responsible for the preferential activation of non-canonical NF-κB pathway in LPS-conditioned DCs. These data provide new insight into the mechanisms of the TLR4-induced tolerogenic phenotype in human DCs, which can help the better understanding of processes involved in induction and resolution of chronic inflammation and tolerance.

Peripheral killer cells do not differentiate between asthma patients with or without fixed airway obstruction.

OBJECTIVE: The three main types of killer cells - CD8+ T cells, NK cells and NKT cells - have been linked to asthma and chronic obstructive pulmonary disease (COPD). However, their role in a small subset of asthma patients displaying fixed airway obstruction (FAO), similar to that seen in COPD, has not been explored. The objective of the present study was to investigate killer cell numbers, phenotype and function in peripheral blood from asthma patients with FAO, asthma patients without FAO, and healthy individuals. METHODS: Peripheral CD8+ T cells (CD8+CD3+CD56-), NK cells (CD56+CD3-) and NKT-like cells (CD56+CD3+) of 14 asthma patients with FAO (post-bronchodilator FEV/FVC <0.7, despite clinician-optimised treatment), 7 asthma patients without FAO (post-bronchodilator FEV/FVC ≥ 0.7), and 9 healthy individuals were studied. RESULTS: No significant differences were seen between the number, receptor expression, MAPK signalling molecule expression, cytotoxic mediator expression, and functional cytotoxicity of peripheral killer cells from asthma patients with FAO, asthma patients without FAO and healthy individuals. CONCLUSIONS: Peripheral killer cell numbers or functions do not differentiate between asthma patients with or without fixed airway obstruction.

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 pathway.

SMAD4 loss enables EGF, TGFß1 and S100A8/A9 induced activation of critical pathways to invasion in human pancreatic adenocarcinoma cells.

Epidermal Growth Factor (EGF) receptor overexpression, KRAS, TP53, CDKN2A and SMAD4 mutations characterize pancreatic ductal adenocarcinoma. This mutational landscape might influence cancer cells response to EGF, Transforming Growth Factor ß1 (TGFß1) and stromal inflammatory calcium binding proteins S100A8/A9. We investigated whether chronic exposure to EGF modifies in a SMAD4-dependent manner pancreatic cancer cell signalling, proliferation and invasion in response to EGF, TGFß1 and S100A8/A9. BxPC3, homozigously deleted (HD) for SMAD4, and BxPC3-SMAD4+ cells were or not stimulated with EGF (100 ng/mL) for three days. EGF pre-treated and non pretreated cells were stimulated with a single dose of EGF (100 ng/mL), TGFß1 (0,02 ng/mL), S100A8/A9 (10 nM). Signalling pathways (Reverse Phase Protein Array and western blot), cell migration (Matrigel) and cell proliferation (XTT) were evaluated. SMAD4 HD constitutively activated ERK and Wnt/ß-catenin, while inhibiting PI3K/AKT pathways. These effects were antagonized by chronic EGF, which increased p-BAD (anti-apoptotic) in response to combined TGFß1 and S100A8/A9 stimulation. SMAD4 HD underlied the inhibition of NF-κB and PI3K/AKT in response to TGFß1 and S100A8/A9, which also induced cell migration. Chronic EGF exposure enhanced cell migration of both BxPC3 and BxPC3-SMAD4+, rendering the cells less sensitive to the other inflammatory stimuli. In conclusion, SMAD4 HD is associated with the constitutive activation of the ERK and Wnt/ß-catenin signalling pathways, and favors the EGF-induced activation of multiple signalling pathways critical to cancer proliferation and invasion. TGFß1 and S100A8/A9 mainly inhibit NF-κB and PI3K/AKT pathways and, when combined, sinergize with EGF in enhancing anti-apoptotic p-BAD in a SMAD4-dependent manner.

Human Blood Autoantibodies in the Detection of Colorectal Cancer.

Colorectal cancer (CRC) is the second most common malignancy in the western world. Early detection and diagnosis of all cancer types is vital to improved prognosis by enabling early treatment when tumours should be both resectable and curable. Sera from 3 different cohorts; 42 sera (21 CRC and 21 matched controls) from New York, USA, 200 sera from Pittsburgh, USA (100 CRC and 100 controls) and 20 sera from Dundee, UK (10 CRC and 10 controls) were tested against a panel of multiple tumour-associated antigens (TAAs) using an optimised multiplex microarray system. TAA specific IgG responses were interpolated against the internal IgG standard curve for each sample. Individual TAA specific responses were examined in each cohort to determine cutoffs for a robust initial scoring method to establish sensitivity and specificity. Sensitivity and specificity of combinations of TAAs provided good discrimination between cancer-positive and normal serum. The overall sensitivity and specificity of the sample sets tested against a panel of 32 TAAs were 61.1% and 80.9% respectively for 6 antigens; p53, AFP, K RAS, Annexin, RAF1 and NY-CO16. Furthermore, the observed sensitivity in Pittsburgh sample set in different clinical stages of CRC; stage I (n = 19), stage II (n = 40), stage III (n = 34) and stage IV (n = 6) was similar (73.6%, 75.0%, 73.5% and 83.3%, respectively), with similar levels of sensitivity for right and left sided CRC. We identified an antigen panel of sufficient sensitivity and specificity for early detection of CRC, based upon serum profiling of autoantibody response using a robust multiplex antigen microarray technology. This opens the possibility of a blood test for screening and detection of early colorectal cancer. However this panel will require further validation studies before they can be proposed for clinical practice.

Clinical and biological significance of RAD51 expression in breast cancer: a key DNA damage response protein.

Impaired DNA damage response (DDR) may play a fundamental role in the pathogenesis of breast cancer (BC). RAD51 is a key player in DNA double-strand break repair. In this study, we aimed to assess the biological and clinical significance of RAD51 expression with relevance to different molecular classes of BC and patients' outcome. The expression of RAD51 was assessed immunohistochemically in a well-characterised annotated series (n = 1184) of early-stage invasive BC with long-term follow-up. A subset of cases of BC from patients with known BRCA1 germline mutations was included as a control group. The results were correlated with clinicopathological and molecular parameters and patients' outcome. RAD51 protein expression level was also assayed in a panel of cell lines using reverse phase protein array (RPPA). RAD51 was expressed in the nuclei (N) and cytoplasm (C) of malignant cells. Subcellular co-localisation phenotypes of RAD51 were significantly associated with clinicopathological features and patient outcome. Cytoplasmic expression (RAD51C(+)) and lack of nuclear expression (RAD51 N(-)) were associated with features of aggressive behaviour, including larger tumour size, high grade, lymph nodal metastasis, basal-like, and triple-negative phenotypes, together with aberrant expression of key DDR biomarkers including BRCA1. All BRCA1-mutated tumours had RAD51C(+)/N(-) phenotype. RPPA confirmed IHC results and showed differential expression of RAD51 in cell lines based on ER expression and BRCA1 status. RAD51 N(+) and RAD51C(+) tumours were associated with longer and shorter breast cancer-specific survival (BCSS), respectively. The RAD51 N(+) was an independent predictor of longer BCSS (P < 0.0001). Lack of RAD51 nuclear expression is associated with poor prognostic parameters and shorter survival in invasive BC patients. The significant associations between RAD51 subcellular localisation and clinicopathological features, molecular subtype and patients' outcome suggest that the trafficking of DDR proteins between the nucleus and cytoplasm might play a role in the development and progression of BC.

The prognostic significance of STAT3 in invasive breast cancer: analysis of protein and mRNA expressions in large cohorts.

Signal transducer and activator of transcription (STAT) transcription factors family are involved in diverse cellular biological functions. Reports regarding the prognostic impact of STAT3 expression in breast cancer (BC) are variable whether being a factor of poor or good prognosis. Immunohistochemical expression of phospho-STAT3 (pSTAT3) was studied in large series of invasive BC (n = 1270). pSTAT3 and STAT3 were quantified using reverse phase protein array (RPPA) on proteins extracted from macro-dissected FFPE tissues (n = 49 cases). STAT3 gene expression in the METABRIC cohort was also investigated. STAT3 gene expression prognostic impact was externally validated using the online BC gene expression data (n = 26 datasets, 4.177 patients). pSTAT3 was expressed in the nuclei and cytoplasm of invasive BC cells. Nuclear pSTAT3 overexpression was positively associated with smaller tumour size, lower grade, good NPI, negative lymphovascular invasion (LVI), ER+, PgR+, p53-, HER2-, and low Ki67LI and an improved breast cancer-specific survival (BCSS), independently of other factors. On RPPA, the mean pSTAT3 and STAT3 expressions were higher in ER+, PgR+, and smaller size tumours. Higher STAT3 transcripts in the METABRIC cohort were observed in cases with favourable prognostic criteria and as well as improved BCSS within the whole cohort, ER+ cohort with and without hormonal therapy, and ER- cohort including those who did not receive adjuvant chemotherapy. Pooled STAT3 gene expression data in the external validation cohort showed an association with improved patients' outcome (P < 0.001, HR = 0.84, 95 % CI 0.79-0.90). Results of this study suggest nuclear localisation of pSTAT3 as favourable prognostic marker in invasive BC, results re-enforced by analysis of STAT3 gene expression data. This good prognostic advantage was maintained in patients who received and who did not receive adjuvant therapy. Therefore, STAT3 could have context-dependent molecular roles of in BC, results which warrant further prospective verification in clinical trials.

The mannose receptor negatively modulates the Toll-like receptor 4-aryl hydrocarbon receptor-indoleamine 2,3-dioxygenase axis in dendritic cells affecting T helper cell polarization.

BACKGROUND: Dendritic cells (DCs) are key players in the induction and re-elicitation of TH2 responses to allergens. We have previously shown that different C-type lectin receptors on DCs play a major role in allergen recognition and uptake. In particular, mannose receptor (MR), through modulation of Toll-like receptor (TLR) 4 signaling, can regulate indoleamine 2,3-dioxygenase (IDO) activity, favoring TH2 responses. Interestingly, the aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor with an emerging role in immune modulation, has been implicated in IDO activation in response to TLR stimulation. OBJECTIVE: Here we investigated how allergens and lectins modulate the TLR4-AhR-IDO axis in human monocyte-derived DCs. METHODS: Using a combination of genomics, proteomics, and immunologic studies, we investigated the role of MR and AhR in IDO regulation and its effect on T helper cell differentiation. RESULTS: We have demonstrated that LPS induces both IDO isoforms (IDO1 and IDO2) in DCs, with partial involvement of AhR. Additionally, we found that, like mannan, different airborne allergens can effectively downregulate TLR4-induced IDO1 and IDO2 expression, most likely through binding to the MR. Mannose-based ligands were also able to downregulate IL-12p70 production by DCs, affecting T helper cell polarization. Interestingly, AhR and some components of the noncanonical nuclear factor κB pathway were shown to be downregulated after MR engagement, which could explain the regulatory effects of MR on IDO expression. CONCLUSION: Our work demonstrates a key role for MR in the modulation of the TLR4-AhR-IDO axis, which has a significant effect on DC behavior and the development of immune responses against allergens.

Clinical utility of reverse phase protein array for molecular classification of breast cancer.

Reverse Phase Protein Array (RPPA) represents a sensitive and high-throughput technique allowing simultaneous quantitation of protein expression levels in biological samples. This study aimed to confirm the ability of RPPA to classify archival formalin-fixed paraffin-embedded (FFPE) breast cancer tissues into molecular classes used in the Nottingham prognostic index plus (NPI+) determined by immunohistochemistry (IHC). Proteins were extracted from FFPE breast cancer tissues using three extraction protocols: the Q-proteome FFPE Tissue Kit (Qiagen, Hilden, Germany) and two in-house methods using Laemmli buffer with either incubation for 20 min or 2 h at 105 °C. Two preparation methods, full-face sections and macrodissection, were used to assess the yield and quality of protein extracts. Ten biomarkers used for the NPI+ (ER, PgR, HER2, Cytokeratins 5/6 and 7/8, EGFR, HER3, HER4, p53 and Mucin 1) were quantified using RPPA and compared to results determined by IHC. The Q-proteome FFPE Tissue Kit produced significantly higher protein concentration and signal intensities. The intra- and inter-array reproducibility assessment indicated that RPPA using FFPE lysates was a highly reproducible and robust technique. Expression of the biomarkers individually and in combination using RPPA was highly consistent with IHC results. Macrodissection of the invasive tumour component gave more reliable results with the majority of biomarkers determined by IHC, (80 % concordance) compared with full-face sections (60 % concordance). Our results provide evidence for the technical feasibility of RPPA for high-throughput protein expression profiling of FFPE breast cancer tissues. The sensitivity of the technique is related to the quality of extracted protein and purity of tumour tissue. RPPA could provide a quantitative technique alternative to IHC for the biomarkers used in the NPI+.

Tumour necrosis factor receptor I blockade shows that TNF-dependent and TNF-independent mechanisms synergise in TNF receptor associated periodic syndrome.

TNF receptor associated periodic syndrome (TRAPS) is an autoinflammatory disease involving recurrent episodes of fever and inflammation. It is associated with autosomal dominant mutations in TNF receptor superfamily 1A gene localised to exons encoding the ectodomain of the p55 TNF receptor, TNF receptor-1 (TNFR1). The aim of this study was to investigate the role of cell surface TNFR1 in TRAPS, and the contribution of TNF-dependent and TNF-independent mechanisms to the production of cytokines. HEK-293 and SK-HEP-1 cell lines were stably transfected with WT or TRAPS-associated variants of human TNF receptor superfamily 1A gene. An anti-TNFR1 single domain antibody (dAb), and an anti-TNFR1 mAb, bound to cell surface WT and variant TNFR1s. In HEK-293 cells transfected with death domain-inactivated (R347A) TNFR1, and in SK-HEP-1 cells transfected with normal (full-length) TNFR1, cytokine production stimulated in the absence of exogenous TNF by the presence of certain TNFR1 variants was not inhibited by the anti-TNFR1 dAb. In SK-Hep-1 cells, specific TRAPS mutations increased the level of cytokine response to TNF, compared to WT, and this augmented cytokine production was suppressed by the anti-TNFR1 dAb. Thus, TRAPS-associated variants of TNFR1 enhance cytokine production by a TNF-independent mechanism and by sensitising cells to a TNF-dependent stimulation. The TNF-dependent mechanism requires cell surface expression of TNFR1, as this is blocked by TNFR1-specific dAb.

Profiling Humoral Immune Responses to Clostridium difficile-Specific Antigens by Protein Microarray Analysis.

Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotype-specific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated <10% coefficient of variation (CV). Significant correlation was observed between microarray and ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses to C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost.

The novel S59P mutation in the TNFRSF1A gene identified in an adult onset TNF receptor associated periodic syndrome (TRAPS) constitutively activates NF-κB pathway.

INTRODUCTION: Mutations in the TNFRSF1A gene, encoding tumor necrosis factor receptor 1 (TNF-R1), are associated with the autosomal dominant autoinflammatory disorder, called TNF receptor associated periodic syndrome (TRAPS). TRAPS is clinically characterized by recurrent episodes of long-lasting fever and systemic inflammation. A novel mutation (c.262 T > C; S59P) in the TNFRSF1A gene at residue 88 of the mature protein was recently identified in our laboratory in an adult TRAPS patient. The aim of this study was to functionally characterize this novel TNFRSF1A mutation evaluating its effects on the TNF-R1-associated signaling pathways, firstly NF-κB, under particular conditions and comparing the results with suitable control mutations. METHODS: HEK-293 cell line was transfected with pCMV6-AC construct expressing wild-type (WT) or c.262 T > C (S59P), c.362G > A (R92Q), c.236C > T (T50M) TNFRSF1A mutants. Peripheral blood mononuclear cells (PBMCs) were instead isolated from two TRAPS patients carrying S59P and R92Q mutations and from five healthy subjects. Both transfected HEK-293 and PBMCs were stimulated with tumor necrosis factor (TNF) or interleukin 1ß (IL-1ß) to evaluate the expression of TNF-R1, the activation of TNF-R1-associated downstream pathways and the pro-inflammatory cytokines by means of immunofluorescent assay, array-based technique, immunoblotting and immunometric assay, respectively. RESULTS: TNF induced cytoplasmic accumulation of TNF-R1 in all mutant cells. Furthermore, all mutants presented a particular set of active TNF-R1 downstream pathways. S59P constitutively activated IL-1ß, MAPK and SRC/JAK/STAT3 pathways and inhibited apoptosis. Also, NF-κB pathway involvement was demonstrated in vitro by the enhancement of p-IκB-α and p65 nuclear subunit of NF-κB expression in all mutants in the presence of TNF or IL-1ß stimulation. These in vitro results correlated with patients' data from PBMCs. Concerning the pro-inflammatory cytokines secretion, mainly IL-1ß induced a significant and persistent enhancement of IL-6 and IL-8 in PBMCs carrying the S59P mutation. CONCLUSIONS: The novel S59P mutation leads to defective cellular trafficking and to constitutive activation of TNF-R1. This mutation also determines constitutive activation of the IL-1R pathway, inhibition of apoptosis and enhanced and persistent NF-κB activation and cytokine secretion in response to IL-1ß stimulation.

Clinical and biological significance of glucocorticoid receptor (GR) expression in breast cancer.

The glucocorticoid receptor (GR) is a member of the nuclear receptor superfamily of transcription factors, which exerts anti-proliferative and anti-apoptotic activities. The GR is expressed in a large proportion of breast cancer (BC) although levels generally decrease during cancer progression. This study aimed to determine the clinical and biological significance of GR expression using a large series of early-stage BC with long-term follow-up and BC cell lines. Immunohistochemistry was used to assess the expression of GR in 999 cases of primary invasive BC prepared as tissue microarrays. Reverse phase protein microarray was used to assess the expression of GR in MCF7 and MDA-MB-231 cell lines. Nuclear expression of GR was observed in 61.6 % of breast tumours and was associated with features of good prognosis including smaller tumour size and lower grade with less pleomorphism and low mitotic count. GR expression was positively correlated with expression of oestrogen (ER) and progesterone receptors. In ER-positive tumours, GR was associated with other features of favourable outcome including FOXA1, GATA3 and BEX1 expression, while low GR expression was associated with high Ki67, p53 and CD71 expression. GR expression is associated with features of good outcome but does not provide prognostic information independent of size, stage and grade. Understanding the receptor and its effects on BC behaviour is essential for avoiding any unwanted effects from the use of glucocorticoids in routine oncology practice.

Prognostic and biological significance of peroxisome proliferator-activated receptor-gamma in luminal breast cancer.

Peroxisome proliferator-activated receptor-gamma (PPARγ) is an adopted orphan receptor that belongs to the nuclear receptor superfamily of transcription factors. PPARγ is regarded as a differentiation factor and it plays an important role in regulating adipogenesis, cell growth, proliferation and tumour progression. In breast cancer (BC), PPARγ agonists were reported to inhibit proliferation and growth invasion and promote phenotypic changes associated with a less malignant and more differentiated status. This study aims to assess the prognostic and biological roles of PPARγ protein expression in a large cohort of BC patients (n = 1100) with emphasis on the luminal oestrogen receptor (ER) positive class. Immunohistochemistry was used to assess the levels of PPARγ expression in BC series prepared as tissue microarrays (TMAs). PPARγ antibody specificity was confirmed using Western blotting. PPARγ nuclear expression was detected in 79 % of the cases and its expression was positively correlated with the hormonal receptors (ER, progesterone receptor and androgen receptor). PPARγ levels were significantly higher in tumours with lobular subtype, smaller size and lower grade, while HER2-positive, ductal or medullary tumours were associated with lower PPARγ levels. Survival analysis showed that PPARγ is associated with better outcome in the whole series as well as in luminal ER-positive class. Cox regression model showed that PPARγ is an independent predictor of outcome. Higher PPARγ was associated with longer survival in patients with ER-positive tumours who did not receive hormone therapy. PPARγ is a good prognostic marker associated with hormone receptors. In patients with luminal BCs, PPARγ is a marker of better prognosis and is associated with longer survival.

The mammalian target of rapamycin complex 1 (mTORC1) in breast cancer: the impact of oestrogen receptor and HER2 pathways.

The mammalian target of rapamycin complex 1 (mTORC1) is a downstream of the PI3K/Akt pathway which affects cancer development. mTORC1 has many downstream signalling effectors that can enhance different cellular responses. This study aims to investigate the expression of mTORC1 in breast cancer (BC) and correlate it with key clinicopathological and molecular features of BC especially to proteins related to oestrogen receptor (ER) and HER2 pathways in different BC classes. Moreover, mTORC1 expression was assessed in 6 BC cell lines including ER+ and ER- cell lines with and without HER2 transfection. Immunohistochemistry was used to assess the expression of phospho (p) mTORC1 in a large (n = 1300) annotated BC series prepared as tissue microarray. Reverse phase protein array (RPPA) was used to assess its expression in the different BC cell lines. The expression of p-mTORC1 was cytoplasmic with moderate/high expression noted in 44 % of BC. p-mTORC1 expression was associated with clinicopathological variables characteristic of good prognosis. Positive correlation with ER, ER-related proteins AKT, PI3K and luminal differentiation markers were observed in the whole series and in the ER+HER2- subgroup. Association with HER2 was mainly observed in the ER-negative class. RPPA indicated that p-mTORC1 expression was mainly related to ER expression and with better outcome in the Akt positive tumours. p-mTORC1 is associated with good prognostic features. Its expression is related to ER and ER related proteins in addition to AKT and PI3K. Its relation with HER2 expression is mainly seen in the absence of ER expression.