xMAP Technology Research Articles

Inflammatory Cytokine Pattern Is Sex-Dependent in Mouse Cutaneous Melanoma Experimental Model.

We present the evaluation of inflammatory cytokines in mouse cutaneous melanoma experimental model, as markers of disease evolution. Moreover, to test our experimental model, we have used low doses of dacarbazine (DTIC). C57 BL/6J mouse of both sexes were subjected to experimental cutaneous melanoma and treated with low doses of DTIC. Clinical parameters and serum cytokines were followed during tumor evolution and during DTIC therapy. Cytokine/chemokine pattern was assessed using xMAP technology and the following molecules were quantified: interleukins (IL)-1-beta, IL-6, IL-10, IL-12 (p70), interferon (IFN)-gamma, granulocyte macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF)-alpha, macrophage inflammatory protein (MIP)-1alpha, monocyte chemoattractant protein (MCP-1), and keratinocyte-derived chemokine (KC). Significant differences were found between normal females and males mice, female mice having a statistically higher serum concentration of IL-1-beta compared to male mice, while males have a significantly higher concentration of MIP-1-alpha. During melanoma evolution in the female group, IL-1-beta, MIP-1-alpha, and KC circulatory levels were found 10-fold increased, while other cytokines doubled their values. In the male mice group, only circulatory KC increased 4 times, while IL-1-beta and TNF-alpha doubled their circulatory values. Various serum cytokines correlated with the disease evolution in cutaneous melanoma mouse model.

Levels of cytokines in drug hypersensitivity.

IntroductionMultiple drug intolerance is a serious complication of drug therapy and is an issue of allergology. The aim of the study was the investigation of cytokine status in patients with drug hypersensitivity and multiple drug hypersensitivity.Material and methods19 patients with multiple drug hypersensitivity, 34 patients with hypersensitivity to one drug, and 35 non-allergic subjects were involved. Only women were included in the study. A multiplex assay of 27 cytokines and chemokines was performed using xMap technology (Human Cytokine Panel I by Bio-Rad).ResultsWomen with drug allergy revealed increased IL-2 levels (p < 0.05). In the case of the study of cytokine status in patients with multiple drug hypersensitivity, the new data revealed the prevalence of pro-inflammatory cytokine status with the participation of cytokines IL-17, IL-9, TNF-α, IP-10, and MIP-1.ConclusionsVarious immune response arms Th2, Th17, as well as macrophages were the determining factors in the cytokine balance that was found in patients with multiple drug hypersensitivity.

Levels of cerebrospinal fluid biomarkers total tau and phosphorylated tau do not predict survival time after diagnosis of Alzheimer’s disease – An 18-year follow-up

Background: The pathological process in Alzheimer’s disease (AD) probably starts decades before the onset of symptoms and the clinical AD diagnosis. In patients with AD, the level of cerebrospinal fluid (CSF) amyloid-β1-42 (Aβ42) is usually lower, and the levels of total tau (T-tau) and phosphorylated tau (P-tau) higher than in healthy elderly people. However, the cutoffs differ between studies and the predictive values are too low to diagnose AD using only CSF biomarkers. Several previous reports have shown that the levels of T-tau and P-tau become pathological later in the course of AD compared with Aβ42, yet it is unclear if higher levels of tau shorten the individuals’ life expectancy after diagnosis. The current study aims to investigate whether pathological levels of T-tau and/or P-tau can predict survival in AD. Methods: The Swedish Alzheimer Treatment Study (SATS) is a prospective, observational, multicenter study for the longitudinal assessment of cholinesterase inhibitor treatment in a routine clinical setting. This presentation includes all 151 participants clinically diagnosed with AD, who underwent a lumbar puncture. Patients were evaluated regarding cognitive and functional abilities at baseline (time of diagnosis) and semi-annually over 3 years. Socio-demographic characteristics, concomitant medications and the date of death were recorded. CSF was collected in polypropylene tubes, stored at −80 °C and analyzed after the clinical follow-up of the study was completed. The levels of T-tau, P-tau phosphorylated at Thr181 and Aβ42 were determined using xMAP technology. Pathological levels of CSF biomarkers were defined as: T-tau >100 ng/ml, P-tau >51 ng/ml and Aβ42 =126 and >=129 ng/ml) and P-tau (>=65 and >=70 ng/ml), respectively, were also examined; their lifespan did not differ from the other individuals. The actual continuous values of the CSF biomarkers or dichotomously coded normal/pathological, respectively, were not significant in Cox regression models adjusted for the above-mentioned predictors. Conclusion: Mortality in AD is complex and depends on many factors e.g., demographic and clinical. In this clinical-practice-based long-term study, almost half of the participants with AD had normal levels of tau. We found no clear results that the levels of T-tau and/or P-tau affect survival after diagnosis in AD. This observation does not support the theory that these patients have a more advanced disease. However, the individuals with pathological levels of tau had fewer years of education and worse cognitive status indicating a lower cognitive reserve capacity, which might influence life expectancy. These findings might be useful when considering new diagnostic criteria and when interpreting outcomes from future clinical trials of potentially disease-modifying AD therapies. (Less)

Incorporating Aptamers in the Multiple Analyte Profiling Assays (xMAP): Detection of C-Reactive Protein.

Aptamers are short oligonucleotide sequences used in detection systems because of their high affinity binding to a variety of macromolecules. With the introduction of aptamers over 25 years ago came the exploration of their use in many different applications as a substitute for antibodies. Aptamers have several advantages; they are easy to synthesize, can bind to analytes for which it is difficult to obtain antibodies, and in some cases bind better than antibodies. As such, aptamer applications have significantly expanded as an adjunct to a variety of different immunoassay designs. The Multiple-Analyte Profiling (xMAP) technology developed by Luminex Corporation commonly uses antibodies for the detection of analytes in small sample volumes through the use of fluorescently coded microbeads. This technology permits the simultaneous detection of multiple analytes in each sample tested and hence could be applied in many research fields. Although little work has been performed adapting this technology for use with apatmers, optimizing aptamer-based xMAP assays would dramatically increase the versatility of analyte detection. We report herein on the development of an xMAP bead-based aptamer/antibody sandwich assay for a biomarker of inflammation (C-reactive protein or CRP). Protocols for the coupling of aptamers to xMAP beads, validation of coupling, and for an aptamer/antibody sandwich-type assay for CRP are detailed. The optimized conditions, protocols and findings described in this research could serve as a starting point for the development of new aptamer-based xMAP assays.

Characterisation of B killer cell immunoglobulin-like receptor genes and telomeric and centromeric motifs in hematopoietic stem cell transplantation donors in Vojvodina, Serbia

The outcome of HSCT is strongly in?uenced by the genetic similarity or identity in the HLA genes that affects the incidence of graft-versus-host disease (GvHD). Successful allogeneic HSCT, however, depends also on T-cell mediated graft-versus-leukemia (GvL) effect, in which donor-derived T cells and natural killer (NK) cells kill these malignant cells in the patient, therefore playing a crucial role in relapse prevention. The aim of this study was to make the predictive analysis of the structure and distribution of B KIR alleles and centromeric and telomeric KIR genotypes in HSCT donors in Vojvodina with regard to their contribution to protection from relapse. A total of 124 first-degree relatives of patients with hematological malignancies were examined for the presence or absence of 15 KIR genes by using of PCR-SSO technique with Luminex xMap technology. The percentage of individuals carrying each KIR gene, centromeric and telomeric KIR haplotypes and genotypes was determined by direct counting. Sixty two percent of the HSCT donors in Vojvodina carry A KIR haplotype, while nearly 38% carry B KIR haplotype. The distribution of B KIR genes showed that among 124 studied HSCT donors, 31(25%) do not carry none of the KIR genes belonging to B group, 71.77% of donors have two or more B KIR genes, 61.29% of them carry KIR 2DL2 and 2DS2 or more B KIR genes. The analysis of centromeric and telomeric KIR genotypes, showed that Cen-A1/Tel-A1 genotype had a highest frequency of 51.47% and Cen-B2/Tel-B1 the lowest frequency of 1.30%. The usage of donor KIR B gene content and centromeric and telomeric KIR gene structure could be used in development of a simple algorithm to identify donors who will provide the most protection against the relapse in related HSC transplants.

Inflammation as a contributing factor among postmenopausal Saudi women with osteoporosis.

Postmenopausal osteoporosis is an important metabolic bone disease characterized by rapid bone loss occurring in the postmenopausal period. Recently, the most prevalent form of clinically significant osteopenia and osteoporosis involves various inflammatory conditions. The aim of the study is to evaluate the association between proinflammatory markers (interleukin [IL]-1β, IL-6, TNF-α) with bone turnover markers (BTMs) in postmenopausal Saudi women with and without osteoporosis. A total of 200 postmenopausal Saudi women ≥50 years old, 100 with osteoporosis and 100 without osteoporosis (control) were recruited under the supervision of qualified physicians in King Salman Hospital and King Fahd Medical City, Riyadh, Saudi Arabia. Serum tumor necrosis factor alpha (TNF-α), IL-1, IL-4, IL-6, and parathyroid hormone (PTH) were determined using Luminex xMAP technology. N-telopeptides of collagen type I (NTx) was assessed using ELISA, 25(OH) vitamin D and osteocalcin were determined using electrochemiluminescence, serum calcium and inorganic phosphate (Pi) were measured by a chemical analyzer. Serum IL-1β, IL-6, NTx, and PTH levels in women with osteoporosis were significantly higher than controls. Although IL-4 and osteocalcin were significantly lower than controls. IL-1β and TNF-α were positively associated with NTx in osteoporosis women. TNF-α, IL-6, and TNF-α were positively correlated with IL-lβ in both groups. A significant negative correlation between osteocalcin and IL-1β in healthy women and women with osteoporosis were observed. Findings of the present study implicate a role for cytokine pattern-mediated inflammation in patients with osteoporosis.

Blood Serum Affects Polysaccharide Production and Surface Protein Expression in S. Aureus.

BackgroundS. aureus biofilm serves a major role in pathogenesis. Two of the major components of bacterial biofilm are Polysaccharides intercellular adhesions (PIA) and surface proteins. It is not known how PIA and surface proteins expressions are affected in presence of blood serum. Analyses of surface proteins expressions will provide more effective biomarker discovery that might lead to development of antimicrobial therapeutics to meet the challenges of biofilm-related infections.MethodSecondary cultures of S. aureus Philips, a biofilm-forming bacterium, were generated by inoculating 1 ml of overnight culture into 50 ml of TSB. Bacteria were cultured at several concentrations of blood serum and found that 12.5% supplemented blood serum provide s similar growth curve as normal TSB (100%). One and 2 D SASPAGE were used to separate proteins and the differentially expressed proteins were identified by nano-LC/MS.ResultsPolysaccharide intercellular adhesions production was significantly increased due to the addition of blood serum in the media. We also identified two serum proteins, apolipoprotein and globulin (Fc and Fab), that remained attached with the membrane fraction of bacterial proteins.ConclusionThese results have strongly demonstrated that blood serum influences the exopolysaccharide expression in S. aureus.

Abstract A07: Identification of endocrine/metabolic biomarkers associated to early PDC in a transgenic mouse model

Abstract Background: Pancreatic ductal adenocarcinoma (PDC) is a deadly disease, even surgery contributes little to survival if PDC is not diagnosed at an initial stage. Glucose homeostasis alteration associated to PDC was described to identify an high risk population for screening. Moreover the weight loss often manifests several months before cachexia. The crosstalk between metabolism and cancer could be a key to early diagnosis. Our aim is to find endocrine/metabolic biomarkers of early PDC development in a gold standard murine model. Materials and Methods: Candidate markers were investigated using LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre (KPC) mouse, a genetically-engineered model that develops PDC with 100% penetrance. KPC has been fed with standard diet (SD) or high fat diet (HFD) introducing a further risk factor for diabetes and metabolic alterations. To overcome the problem of asynchronous development of PDC in KPC, 7T-MRI was used to classify mice in actual pathological stage: normal (S1); cystic degeneration (S2); small tumor (S3) and advanced pancreatic cancer (S4). We performed metabolic test (OGTT) and serum endocrine/metabolic profile (xMAP technology and clinical biochemistry) in n=27 KPC (and control mice PDX-1 Cre) fed with HFD vs n=26 KPC (and control mice PDX-1 Cre) fed with SD. To prioritize markers we computed the ROC curve analysis. Results: i) Glucose metabolism: KPC were normoglycemic regardless of the diet; the AUC in OGTT increases in KPC fed with HFD (HFD vs SD: S1 p&amp;lt;0,001, S2 p&amp;lt;0,001, S3 p=0,002, Cre p&amp;lt;0,001) ii) Serum levels of 29 endocrine/metabolic biomarkers were assessed during disease progression. The more relevant results are: increase of PYY (p&amp;lt;0,003) and decrease of leptin (p&amp;lt;0,02), GIP (p&amp;lt;0.005) albumin (p&amp;lt;0.001) both in HFD and SD fed KPC (Cre vs S2); increase of amylase in stage 1 HFD-fed KPC (p=0,037). Moreover PYY was able to discriminate control from stage 2 KPC with an AUC=0,929 in the ROC curve analysis (p&amp;lt;0,001). Conclusions: Unlike human, in the mouse model the development of PDC is not associated with an hyperglycemic status, regardless of the diet. However KPC fed with HFD experience a mild impairment of glucose metabolism (as well as in control mice) and a mild pancreatic damage. Our results identify Leptin, PYY, GIP and albumin as markers of early stage PDC in a preclinical model with a diagnostic potential to be validated in human patients. Citation Format: Valentina Pasquale, Erica Dugnani, Daniela Liberati, Paolo Marra, Tamara Canu, Antonio Esposito, Lorenzo Piemonti1.{Authors}. Identification of endocrine/metabolic biomarkers associated to early PDC in a transgenic mouse model. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A07.

Cross-sectional and longitudinal associations between serum inflammatory cytokines and knee bone marrow lesions in patients with knee osteoarthritis.

To describe cross-sectional and longitudinal associations between serum levels of interleukin (IL) - 6, IL-17A, IL-17F, IL-23 and knee bone marrow lesions (BMLs) in patients with knee osteoarthritis (OA). Patients (n=192) with symptomatic knee OA (mean 63 years, range 50-79, female 53%) were assessed at baseline and after 24 months. At each time point, serum IL-6, IL-17A, IL-17F and IL-23 were measured using Bio-Plex® Multiplex Immunoassays with Luminex xMAP technology. Knee BMLs were scored using the modified whole organ MRI score (WORMS) from T2 weighted fat-suppressed fast spin echo magnetic resonance imaging (MRI). Multivariable linear regression and log binominal regression were used to determine the associations between cytokines and BMLs. Baseline IL-6 (quarters) were significantly associated with total knee BMLs (P<0.01 for the trend) as well as associated with an increase in BML score (P=0.05 for the trend), after adjustment for confounders. Baseline IL-17F and IL-23 (highest quarters vs others) was associated with an increase in BML score in females (P=0.04 for IL-17F; P=0.01 for IL-23), but not in males, in multivariable analyses. In contrast, IL-17A was not significantly associated with BMLs in either females or males. IL-6 is associated with increased knee BMLs in both females and males with OA. Serum IL-17F and IL-23 predicted increased knee BML scores in females only, suggesting that inflammation is involved in BML pathogenesis in knee OA, especially in women. ClinicalTrials.gov identifier: NCT01176344; Australian New Zealand Clinical Trials Registry: ACTRN12610000495022.

Correlations Between MMPs and TIMPs Levels in Aqueous Humor from High Myopia and Cataract Patients

ABSTRACTPurpose: To study the relationships between matrix metalloproteinases (MMP)-2, MMP-3, and tissue inhibitors of matrix metalloproteinases (TIMP)-1, TIMP-2, and TIMP-3 aqueous humor levels in patients with high myopia or cataract.Materials and Methods: MMPs and TIMPs protein levels in 65 aqueous humor samples collected from patients with high myopia or cataract during cataract or clear lens extraction surgery were measured with the Luminex xMAP Technology. The relationship between MMPs and TIMPs levels was analyzed with Spearman’s correlation test.Results: MMP-2 levels, but not MMP-3 levels, were increased in the aqueous humor from high-myopia patients. Levels of TIMP-1, -2, and -3 were positively and very significantly correlated with the MMP-2 levels (TIMP-1: r=0.626, p < 0.001; TIMP-2: r = 0.545, p < 0.001; TIMP-3: r = 0.439, p < 0.001). TIMP-2 and-3 levels did not significantly correlate with MMP-3 levels (TIMP-2: r = 0.175, p > 0.05; TIMP-3: r = 0.127, p > 0.05) and TIMP-1 levels only marginally correlated with MMP-3 levels (r = 0.278, 0.01< P < 0.05).Conclusions: Compared to the present findings with the relationship of MMPs and TIMPs in other fields of medicine, our results are consistent with the homeostasis hypothesis that the increase of TIMPs serves as a compensation reaction to inhibit the excessive degradation caused by the increase of MMPs and limits the development of myopia.

Increased Proliferation of the Ileal Epithelium as a Remote Effect of Ulcerative Colitis.

Aside from cases of backwash ileitis, the ileal mucosa of patients with ulcerative colitis (UC), an idiotypic inflammatory bowel disease, has received little attention despite the fact that colitis is known to trigger alterations in morphology and/or functions of the small intestine remotely. The ileal mucosa was studied in patients with UC and in a spontaneous model of colitis (Il10/Nox1 mice) mimicking the histological and clinical features of UC and was also studied in acute and chronic murine models of chemically induced colitis. Proliferation and apoptosis were assessed using morphological and immunohistological methods and Western blot analysis. Peyer's patch immune cell subsets were analyzed. Cytokines levels were quantified using quantitative PCR and Luminex xMAP technology. Total RNA from isolated ileal crypts was used for whole genome transcriptome analysis. The most striking features were an increased ileal crypt length associated with an enhanced cell proliferation of the transit-amplifying cells along with activation of the Wnt/β-catenin and MAPkinase pathways. These changes did not result from intestinal inflammation as assessed by histology and/or pro-inflammatory cytokine expression levels. The increased proliferation rate was dependent on the duration but not on the severity of colitis and was observed in different mouse models of colitis, including the Il10/Nox1 model and 2,4,6-trinitrobenzenesulfonic acid-treated mice. Interestingly, the ileal mucosa of patients with UC also displayed longer crypts and enhanced cell proliferation compared with control patients. These data show that despite the absence of inflammation in the small intestine, alterations in the ileal mucosa homeostasis are present in UC.

Physiological changes of adipokines during the menstrual cycle

Background. The cyclical effects of hormones during the menstrual cycle (MC) are responsible for driving ovulation. The information about roles of adipokines within the scope of MC are not definite. Leptin plays a role in sexual function and regulating the onset of puberty. Thin girls often fail to ovulate or release an egg from an ovary during menstruation cycles. Leptin also acts on specific receptors in the hypothalamus to inhibit appetite. Levels of leptin are increased in women suffering from premenstrual syndrome.Aim. The aim of our study was to describe physiological changes of selected steroids and adipokines at healthy women during the MC.Methods. Twenty-seven women with regular menstrual cycles were included in the study. Each sample was collected in cooled EDTA tubes, centrifuged at 2000 rpm in a refrigerated centrifuge, and stored at –80 °C. For all samples we measured luteinizing hormone (LH), follicularstimulating hormone (FSH), sex hormone-binding globulin (SHBG), testosterone, dehydroepiandrosterone (DHEA), estradiol, 7α-DHEA, 7β-DHEA, 7-oxoDHEA, 17-hydroxyprogesterone (17-OH P), progesterone, cortisol, adrenocorticotropic hormone (ACTH) by RIA and IRMA. Levels in plasma of hormones associated with food intake (c-peptide, ghreline, GIP, GLP, GLP-1, glucagon, insulin, leptin, PAI-1, resistin and visfatin) were measured using magnetic bead-based multiple assays (x-MAP technology, Luminex Corporation). Two kits were used: the 10-plex Bio-Plex Pro Human Diabetes assay and the 2-plex Bio-Plex Pro Human Adiponectin and Adipsin assay (both Bio-Rad Laboratories).Patient. Twenty-seven women with regular menstrual cycles (cycle length 28±2 days) were included in the study. The average age of the women was 31.8±3.56, and average BMI 22.9±2.8. The women used no hormonal contraceptives or other medicines influencing the production of steroid hormones, and were non-smokers. Before enrollment in the study, all signed informed consent that was approved by the local ethical committee of the Institute of Endocrinology.Intervention. Fasting blood samples were taken in the morning between 7 and 8 am. The first sampling was done at the start of the menstrual cycle (1st or 2nd day). Subsequent samples were taken at regular intervals every three days, for a total of 10 samples taken during the study.Main outcome measures. During the MC we found increased levels of testosterone, estradiol, progesterone, and 17-hydroxyprogesterone during ovulation. SHBG gradually increased after ovulation. There was a significant decrease in resistin levels during ovulation, followed by an increase in the latter part of the cycle. Adipsin showed a notable increase during ovulation, but this increase was not statistically significant.Results. Classical changes in gonadotropins, estrogens and progesterone during the menstrual cycle are accompanied by less striking but significant changes in 17-hydroxyprogesterone and testosterone. No significant changes show dehydroepiandrosterone and its 7-oxygenated metabolites. Adipokines show a tendency to increase during ovulation, while ghrelin and resistin decrease. There is also a remarkable association of sex hormone binging globuline (SHBG) on the day of the cycle.Conclusions. Our results demonstrate that changes to adipokines during the menstrual cycle are not substantial. Differing leptin levels are characteristic for premenstrual syndrome. Precise descriptions of physiological changes in healthy women are important in helping us understand the significance of the changes accompanying various pathological states.

Abstract 3378: Using multiplex immunoassays for profiling the circulating and tissue angiogenic biomarkers in mouse and human cancer

Abstract Monitoring the expression levels of multiple pro- and anti-angiogenic factors in circulation and tumor microenvironment is crucial in examining the role of angiogenesis in cancer pathological processes or in identifying potential biomarkers for oncology drug development. The Luminex xMAP technology allows simultaneous detection of multiple proteins in a single sample and has been used widely in cancer biomarker discovery. Using the Luminex xMAP technology, we developed two Human Angiogenesis Panels (17-plex and 20-plex) and a Mouse Angiogenesis Panel (27-plex) for the simultaneous detection of multiple angiogenic biomarkers in less than 50 μL of human or mouse samples. We report here the protein biomarker profiles of these 37 human angiogenic factors in 1) serum samples, collected from breast cancer and colorectal cancer patients and normal donors, 2) tumor extracts, collected from breast tumor and colorectal tumor extracts and their matched adjacent normal tissue, 3) conditioned media in cultured tumor cells and stromal cells, and in addition, the protein biomarker profiles of 27 mouse angiogenic factors in the conditioned media of various mouse tumor cells and stromal cells. For example, of the 37 human angiogenesis serum protein markers tested, 16 biomarkers [angiopoietin-2 (ANGPT-2), EGF, endoglin, follistatin, G-CSF, HGF, PLGF, sAXL, PDGF-AB/BB, osteopontin (OPN), sHGFR/c-Met, suPAR, sTie-2, sHer2, soluble neuropilin-1 (sNRP-1), and tenascin-C] showed significant difference in expression levels between normal and breast cancer serum samples (p&amp;lt;0.05) and 13 biomarkers [ANGPT-2, EGF, PLGF, sAXL, sHer3, OPN, thrombospondin-2 (TSP-2), sHGFR/c-Met, suPAR, sTie-2, sNRP-1, sIL-6R, and tenascin-C] showed differential expression levels between normal and colorectal cancer serum samples (p&amp;lt;0.05). Altogether, the results of this study demonstrate the utility of these human and mouse angiogenesis multiplex panels as a useful tool in studying circulating and tissue protein biomarker profiles in cancer research. Citation Format: Wen-Rong Lie, Jehangir Mistry. Using multiplex immunoassays for profiling the circulating and tissue angiogenic biomarkers in mouse and human cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3378.

Abstract 3265: Homeobox B9 (HOXB9) sustains anti-VEGF treatment resistance in gastrointestinal tumors

Abstract Resistance to anti-angiogenic therapies poses one of the greatest challenges in gastrointestinal tumors research. We recently identified several proinflammatory secreted factors, including interleukin-1 (IL1), CXC ligand (CXCL) 1 and 8, Transforming Growth Factor β (TGFβ)1, and Angiopoietin-like Protein 2 (ANGPTL2), that were overexpressed in murine models of tumors resistant to the anti-VEGF antibody bevacizumab (BEV). These factors induced epithelial-to-mesenchymal transition (EMT) and increased aggressiveness. HOXB9 has been identified as a key transcription factor in common for these factors. Here, we hypothesized that HOXB9 might be responsible for BEV resistance in gastrointestinal tumors. HOXB9 expression and activation were measured in BEV-sensitive COLO357FG and in their BEV-resistant counterpart FGBR pancreatic cancer cell lines, and in LOVO, MDST8, LIM2099, CCK81, GP5D, and SNUC4 colon cancer cell lines by EMSA and DAPA. Serum levels of IL1, CXCL1 and 8, TGFβ1 and ANGPTL2 in nude mice bearing tumors were measured by multiplex xMAP technology. Immunohistochemical analyses were performed in pre- vs. post-progression biopsies from colorectal cancer patients receiving BEV. HOXB9 protein was more expressed and activated in FGBR than did in COLO357FG cells. shRNA to knock down HOXB9 significantly (P&amp;lt;0.01) reduced proinflammatory factors and EMT markers expression, and migration in FGBR cells. Mice bearing HOXB9-silenced tumors had significantly (P&amp;lt;0.01) lower serum levels of IL1, CXCL1 and 8, TGFβ1 and ANGPTL2 than did their respective controls. BEV significantly reduced tumor burden (P≤0.0001) and prolonged survival duration (P = 0.0006) in mice bearing HOXB9-silenced FGBR tumors, whereas it was ineffective on FGBR scramble tumors. HOXB9-positive LOVO, MDST8, and LIM2099 cells had significantly higher expression levels of proinflammatory factors and EMT markers, and migration rates than did HOXB9-negative CCK81, GP5D, and SNUC4 colon cancer cell lines. In vivo, BEV significantly reduced tumor burden and prolonged survival duration in mice bearing CCK81 or GP5D tumors (P&amp;lt;0.05), whereas it was ineffective on MDST8 and LIM2099 tumors. HOXB9 shRNA significantly reduced proinflammatory factors and EMT markers expression, and migration in MDST8 cells. BEV significantly prolonged survival duration in mice bearing HOXB9-silenced MDST8 tumors (P = 0.0025), but did not on MDST8 scramble tumors. Validation of the differential expression of HOXB9 in biopsies from colorectal patients progressing under BEV therapy will be available at the time of the meeting. In conclusion, we identified HOXB9 as crucial transcription factor to sustain BEV resistance in gastrointestinal tumors. Silencing of HOXB9 is a promising approach to modulate this resistance. Our results candidate HOXB9 as potential biomarker for selecting patients with colorectal and pancreatic cancer for antiangiogenic therapy. Citation Format: Carmine Carbone, Geny Piro, Francesca Simionato, Fotios Loupakis, Chiara Cremolini, Gabriella Fontanini, Federica Di Nicolantonio, Elisa Moratti, Francesca Ligorio, Marco Zanotto, Raffaela Santoro, Maria Mihaela Mina, Aldo Scarpa, Alberto Bardelli, Giampaolo Tortora, Davide Melisi. Homeobox B9 (HOXB9) sustains anti-VEGF treatment resistance in gastrointestinal tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3265.

Isotypic analysis of antibodies against activated Factor VII in patients with Factor VII deficiency using the x-MAP technology

While the immune response to hemophilic factors in hemophilia has been widely studied, little is known about the development of anti-Factor VII (FVII) antibodies in FVII deficiency. We developed a robust technique based on the x-MAP technology to detect the presence of antibodies against FVII and characterize their isotype and validated this method using blood samples from 100 patients with FVII deficiency (median FVII clotting activity [FVII:C]: 6%) and 95 healthy controls. Anti-FVII antibodies were detected in patients but also in some controls, although the concentration of total immunoglobulin G (IgGt) and IgG1 and IgG4 subclasses was significantly different between groups. The IgG1 subclass concentrations remained significantly different also when only untreated patients were compared with controls. This difference could partially be related to the F7 genotype, particularly in patients harboring the p.Arg139Gln mutation. This x-MAP-based method might be useful for assessing the immunogenicity of novel FVII compounds and of activated FVII (FVIIa) concentrates. Further prospective studies are needed to better understand the clinical relevance of these antibodies in the management of patients with FVII deficiency.

Prevalence of HPV infection among 28,457 Chinese women in Yunnan Province, southwest China

Human papillomavirus (HPV) infection plays a key role in the development of cervical cancer. The aim of the current study was to investigate the HPV type distribution in Chinese women from Yunnan Province, southwest China. A total of 28,457 individuals ranging in age from 17–84 years were recruited from 13 clinical hospitals located in 10 different regions of Yunnan Province. Cervicovaginal swabs were collected from each participant, and HPV screening was performed using Luminex xMAP technology. Our results showed that the HPV prevalence was 12.9% in Yunnan Province. Overall, 10.6% of the individuals were positive for a single HPV type, and 2.3% were positive for multiple types. Among the individuals who tested positive for a single HPV type and multiple HPV types, the three most prevalent high-risk types were 52, 16, and 58. Age subgroup analysis showed two peaks for the frequencies of single and multiple HPV infections, one for the group of women under 25 years old, and the other for the group over 56 years old. Here, we present data regarding the prevalence and type distribution of HPV infection, which will aid in the estimation of the potential clinical benefit and cost-effectiveness of HPV screening and vaccination in China.

Assessment of cardiovascular risk from the use of an interleukin-1 inhibitor in patients with severe tophaceous gout

It is suggested that interleukin (IL)-1β inhibition may promote a reduction in the risk of cardiovascular diseases; however, the mechanism of this impact has not been adequately investigated. Objective: to evaluate the impact of canakinumab therapy on the structural and functional properties of the vascular bed and the stiffness of the vessel wall. Patients and methods. An open-labeled prospective study was conducted in 20 patients with chronic tophaceous gout at the V.A. Nasonova Research Institute of Rheumatology. Among the patients, there were 17 (85%) men and 3 (15%) women. All the patents received a single subcutaneous injection of canakinumab 150 mg. Estimation of high-sensitivity C-reactive protein (hs-CRP) and IL-6 levels, 24-hour blood pressure (BP) monitoring, duplex scanning of the carotid arteries (intimamedia thickness (IMT)), and determination of central artery stiffness (aortic pulse wave velocity (APWV), m/sec) were done before and 14 and 120 days after drug administration; the level of hs-CRP was quantitatively measured using a high-sensitivity immunoturbidimetric assay and the concentration of IL-6 was determined by xMAP technology (Human Grp I Cytokine 27-plex panel on a BioPlex-200 analyzer (Bio-Rad, USA). Antihypertensive lipidlowering therapy was not changed during the investigation. Statistical analysis was carried out using a package of applied Statistica 10.0 programs (StatSoft/Inc., USA). Results. Examination of the common carotid artery indicated that the median IMT that was 0.83 [0.71; 0.94] mm at baseline decreased to 0.74 [0.69; 0.84] mm by the end of the investigation (p = 0.022). The median APWV that was 14.12 [10; 17] m/sec at baseline dropped to 13.1 [11; 14] by the end of the investigation, but statistically insignificantly (p = 0.79). By the end of the investigation, there was a reduction in APWV as related to the baseline level in 13 (65%) patients: 7 of them had not one swollen joint and only 5 patients had a CRP level of > 5 mg/l, which was significantly less frequently than that in patients without positive changes in this index: in 6 (86%) of the 7 patients (p = 0.043). There was a direct correlation of changes in APW with those in serum CRP levels (p = 0.012 and p = 0.047, respectively) after 14 and 120 days and with those in serum IL-6 concentrations following 14 days (p = 0.003). 24-hour BP monitoring showed no significant BP change in the patients after 14 and 120 days after injection. The mean level of total cholesterol and triglycerides remained unchanged. Conclusion. The IL-6 inhibitor canakinumab can have a positive effect on IMT and arterial stiffness. The effect of the drug on the structural and functional properties of the vascular bed is due to the magnitude of its anti-inflammatory activity

Diagnostic value of determination of urine cytokines in children with urinary tract diseases

Objective: to determine the urinary concentrations of cytokines for the diagnosis of the localization of inflammation in the urinary system and to establish the contribution of mediators of the immune system to the pathogenesis of the pathology in question. Twenty-nine children aged 2.7 to 11.5 years with pyelonephritis (n=16) and the unspecified localization of urinary infection or cystitis (n=13) were examined. A control group consisted of 15 healthy children. Cytokine levels were determined in the morning urine portion using the multiplex xMAP technology and then recalculated to the concentration of creatinine. Urinary tract infection was accompanied by an avalanche-like increase in the total urinary cytokine concentration, which was 20-fold greater than the reference values in the children with pyelonephritis. Interleukin 8 (IL-8) constituted the highest proportion of cytokines. IL-10 and interferon-γ (IFN-γ) made up the smallest percentage in the total mass of cytokines. Higher IL-6 and IFN-γ values and a simultaneous increase in the level of 2 or more cytokines are of the greatest diagnostic value in determining the renal site of urinary tract infection.

Serological diagnosis of Leptospirosis in bovine serum samples using a microsphere immunoassay

Leptospirosis causes significant economic loss within the cattle industry worldwide. Current diagnostic methods are generally inadequate for dealing with large numbers of samples, are outdated, and provide little useful diagnostic...

Abstract B01: Profiling of serum and plasma angiogenic biomarkers in breast cancer and colon cancer using Luminex xMAP technology-based human angiogenesis multiplex immunoassay panels

Abstract Tumors require neovascularization for growth. Consequently, the angiogenesis process is key to understanding tumor development and progression, as well as diagnosis and treatment. Measuring the levels of pro- and anti-angiogenic factors in plasma or serum in cancer patients may provide prognostic information concerning disease processes and mechanisms and the elucidation of potential biomarkers for epidemiological studies or clinical trials. The Luminex xMAP technology, a bead-based multiplex immunoassay for the simultaneous measurement of multiple analytes in a small sample size, has been widely used in cancer biomarker discoveries. We report here the use of two newly developed multiplex human angiogenesis panels to profile the circulating biomarkers commonly used in the angiogenesis pathway analysis. The two panels are 1) MILLIPLEX MAP Human Angiogenesis Panel 1, a 17-plex multiplex immunoassay for the simultaneous quantification of the following 17 angiogenesis biomarkers: Angiopoietin-2, BMP-9, EGF, Endoglin, Endothelin-1, FGF-1, FGF-2, Follistatin, G-CSF, HB-EGF, HGF, IL-8, Leptin, PLGF, VEGF-A, VEGF-C, and VEGF-D, and 2) MILLIPLX MAP Human Angiogenesis Panel 2, a 20-plex multiplex immunoassay for simultaneous quantitation of the following 20 human angiogenesis biomarkers: Angiostatin, soluble E-Selectin, Osteopontin, PDGF-AB/BB, PECAM-1/sCD31, Tenascin C, Thrombospondin-2 (TSP-2), soluble AXL, soluble c-Kit, soluble EGFR, soluble Her2/ErbB2, soluble Her3/ErbB3, soluble HGFR/c-Met, soluble IL-6Rα, soluble Neuropilin-1, soluble Tie-2, soluble urokinase receptor (suPAR), soluble VEGFR1, soluble VEGFR2, and soluble VEGFR3. In this study, we report the use of these 37 angiogenic, growth factor and soluble receptor biomarkers to profile serum samples and plasma samples from a panel of breast cancer and colon cancer patients, with a similar number of normal serum and plasma samples, as a corresponding set, to generate a tumor profile of angiogenic factors using the Luminex xMAP platform. The results of this study demonstrated the utility of these human angiogenesis panels in studying cancer development and cancer biomarker discovery, as well as its potential application in translational research. Citation Format: Wen-Rong Lie, Deborah Droll, Jehangir Mistry. Profiling of serum and plasma angiogenic biomarkers in breast cancer and colon cancer using Luminex xMAP technology-based human angiogenesis multiplex immunoassay panels. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr B01.