Luminex xMAP Technology Research Articles

The association between selected mid-trimester amniotic fluid candidate proteins and spontaneous preterm delivery

Objective: The aim of this study was to explore inflammatory response and identify early potential biomarkers in mid-trimester amniotic fluid associated with subsequent spontaneous preterm delivery (PTD).Methods: A cohort study was performed at Sahlgrenska University Hospital/Östra, Gothenburg, Sweden, between 2008 and 2010. Amniotic fluid was collected from consecutive women undergoing mid-trimester transabdominal genetic amniocentesis at 14–19 gestational weeks. Clinical data and delivery outcome variables were obtained from medical records. The analysis included 19 women with spontaneous PTD and 118 women who delivered at term. A panel of 26 candidate proteins was analyzed using Luminex xMAP technology. Candidate protein concentrations were analyzed with ANCOVA and adjusted for plate effects.Results: The median gestational age at delivery was 35 + 3 weeks in women with spontaneous PTD and 40 + 0 weeks in women who delivered at term. Nominally significantly lower amniotic fluid levels of adiponectin (PTD: median 130,695 pg/mL (IQR 71,852–199,414) vs term: median 185,329 pg/mL (IQR (135,815–290,532)), granulocyte-macrophage colony stimulating factor (PTD: median 137 pg/mL (IQR 74–156) vs term: median 176 pg/mL (IQR 111–262)), and macrophage migration inhibitory factor (PTD: median 3025 pg/mL (IQR 1885–3891) vs term: median 3400 pg/mL (IQR 2181–5231)) were observed in the spontaneous PTD group, compared with the term delivery group, after adjusting for plate effects. No significant differences remained after Bonferroni correction for multiple comparisons.Conclusions: Our results are important in the process of determining the etiology behind spontaneous PTD but due to the non-significance after Bonferroni correction, the results should be interpreted with caution. Further analyses of larger sample size will be required to determine whether these results are cogent and to examine whether microbial invasion of the amniotic cavity or intra-amniotic inflammation occurs in asymptomatic women in the mid-trimester with subsequent spontaneous PTD.

Establishment and optimization of a liquid bead array for the simultaneous detection of ten insect-borne pathogens

BackgroundInsect-borne diseases could induce severe symptoms in human and clinical signs in animals, such as febrility, erythra, arthralgia and hemorrhagic fever, and cause significant economic losses and pose public health threat all over the world. The significant advantages of Luminex xMAP technology are high-throughput, high parallel and automation. This study aimed to establish a liquid bead array based on Luminex xMAP technology that was able to simultaneously detect multiple insect-borne pathogens.MethodsSpecific probes and primers to detect the nucleic acid of 10 insect-borne pathogens were designed. Probes were coupled with fluorescent carboxylated microspheres. The parameters of the system were optimized, including ratio of forward/reverse primers (1:2), hybridization temperature (50 °C) and duration (30 min) and quantity of PCR product (2 μl). The sensitivity and specificity of the system were also evaluated. Moreover mixed nucleic acid of 10 insect-borne pathogens, including Bluetongue virus, Epizootic hemorrhagic disease virus of deer, Coxiella burnetii, African swine fever virus, West Nile fever virus, Borrelia burgdorferi, vesicular stomatitis virus, Rift Valley fever virus, Ebola virus and Schmalenberg’s disease virus, and 3000 clinical samples were tested for practicability.ResultsThe optimized detection system showed high sensitivity, specificity and reproducibility. Each probe showed specific fluorescence signal intensity without any cross-hybridization for the other insect-borne pathogens tested, which included dengue virus, tick-borne encephalitis virus, Japanese encephalitis virus, Xinjiang hemorrhagic fever virus, spotted fever group rickettsiae, ehrlichiae and chikungunya virus. The limit of detection was 10 copies of target gene. Insect-borne pathogens were successfully detected among the 3000 clinical samples, and the results were consistent with those obtained using gold-standard assays or commercial nucleic acid detection kits.ConclusionsThis optimized liquid array detection system was high-throughput and highly specific and sensitive in screening of the insect-borne pathogens. It was promising in detection of these pathogens for molecular epidemiological studies.

Blood Inflammatory Mediators and Cognitive Decline in Alzheimer's Disease: A Two Years Longitudinal Study.

Peripheral inflammatory processes are involved in Alzheimer's disease (AD). We aimed to determine whether plasma inflammatory mediator levels at diagnosis are associated with cognitive decline through a 2-year follow-up in AD patients. Patients (n = 109, mean age 79.44 (6.82) years) were included at diagnosis with MMSE scores between 16 and 25, with C-reactive protein <10 mg/L, and without any acute or chronic inflammation status. Plasma IL-1β, IL-6, TNF-α, and CCL5 were measured using Luminex X-MAP technology at baseline, and after one year and two years of follow-up. The mean values of IL-1β, IL-6, TNF-α, and CCL5 at diagnosis were 0.3, 1.94, 6.57, and 69,615.81 pg/mL, respectively. Mean cognitive decline in MMSE was 3.35 points. No correlation between plasmatic value of IL-1β, IL-6, TNF-α, or CCL5 at diagnosis and cognitive decline during the two years of follow-up was found. Cognitive decline in AD does not appear to be predictable by the tested inflammatory mediators.

Specific Profiles Of Circulating Mediators Characterize Older Persons With Physical Frailty And Sarcopenia

Chronic inflammation, changes in body composition, muscle loss (sarcopenia) and decreasing homeostatic reserve (frailty) are hallmarks of aging. Several circulating inflammatory markers have been associated with these age‐related conditions. However, a gold standard biomarker able to predict functional impairment in older adults is currently unavailable. Muscle is crucial for several metabolic processes, including protein/aminoacid metabolism. Perturbations in protein/aminoacid metabolism may play also a role in the development of physical frailty and sarcopenia (PF&amp;S). The simultaneous analysis of an array of circulating aminoacid/metabolites and their relationship with inflammatory mediators may help gain insights in the pathophysiology of PF&amp;S.To this aim, we analyzed the profile of circulating inflammatory mediators and amino acids in older people with and without PF&amp;S.More than five hundred persons aged 70+ years were screened. Of these, sixty (20 men and 40 women; mean age 76.9±4.8 years) were diagnosed with PF&amp;S. Thirty (14 men and 16 women) non‐sarcopenic, non‐frail persons were enrolled in the control group. A panel of 27 cytokines, chemokines and growth factors was analyzed via a multiplex, magnetic bead‐based immunoassay on a Bio‐Plex® System with Luminex xMap Technology. A panel of 37 serum amino acids and derivatives was assessed by UPLC‐MS. Multi‐block partial least squares discriminant analysis (PLS‐DA) was employed to explore the relationship among inflammatory and amino acid profiles of people with PF&amp;S. Double cross‐validation procedures were used to validate the predictive ability of the PLS‐DA model.The optimal complexity of the PLS‐DA model was found to be three latent variables. The proportion of correct classification was 85.5 ± 4.4 for persons with PF&amp;S and 88.3 ± 3.6 for controls. As for the amino acid profile, people with PF&amp;S showed higher levels of aspartic acid, asparagine, taurine, citrulline, alpha‐aminobutyric acid, methionine, and glutamic acid. Increased levels of interleukin‐8, myeloperoxidase, and platelet derived growth factor‐BB were also found in people with PF&amp;S.The dissection of these patterns may provide novel insights into the role played by inflammatory mediators and protein/amino acid perturbations in the disabling cascade associated with PF&amp;S and possible new targets for interventions.Support or Funding InformationThis study was supported by the “Centro Studi Achille e Linda Lorenzon”, intramural research grants from the Catholic University of the Sacred Heart (D3.2 2013 and D3.2 2015), Innovative Medicines Initiative Joint Undertaking (IMI‐JU 115621), and Fondazione Roma (NCDs Call for Proposal 2013)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

F23. LOW-GRADE INFLAMMATORY PROFILE IN FIRST-EPISODE PSYCHOSIS: RESULTS FROM THE STREAM STUDY IN BRAZIL

BackgroundThe inflammatory hypothesis of schizophrenia suggests that abnormalities in inflammatory factors may contribute to the onset of this disease early in adulthood. Nevertheless, no study has investigated possible effects of shared environment by looking at cytokine levels in healthy siblings of first-episode psychosis patients (FEP). The aim of this study was to investigate inflammatory cytokine (IL-6, IL-10, TNF-a) abnormalities in a sizeable epidemiological-based sample of FEP patients, their healthy siblings and population-based controls.MethodsThis study is part of the epidemiological investigation “Schizophrenia and Other Psychoses Translational Research: Environment and Molecular Biology” (STREAM), conducted in Ribeirão Preto (São Paulo, Brazil) and surrounding area, which is part of the international consortium “European Network of National Schizophrenia Networks Studying Gene-Environment Interactions” (EU-GEI). We recruited a total sample of 507 participants, composed by 166 FEP patients (64% males; mean age: 30.3 ± 12.2), 76 siblings (30.3% males; mean age: 31.5 ± 11.0) and 265 population-based controls (50.2% males; mean age: 31.7 ± 11.2). Plasma cytokines IL-6, TNF-a and IL-10 were analysed by Multiplex Bead Array (Luminex xMap technology). We performed the multivariate general linear model (GLM) analysis with age as covariate, cytokines as dependent variables and diagnostic group and sex as explanatory variables.ResultsWe found significant differences between the three groups [F (6,994) = 10.864; p < 0.001] in the plasma concentration of all the three cytokines (p < 0.001), independent of the sex of the participants (p = 0.115; interaction diagnosis & sex p = 0.115). FEP patients had significantly higher levels of IL-6, TNF-a, and IL-10 when compared to controls (p < 0.001 for all). When compared to their siblings, patients had higher levels of both TNF-a and IL-10 (p < 0.05 for both), and a trend to IL-6 (p = 0.058), whereas siblings did not differ from controls for any of the three cytokines analysed (p > 0.05 for all).DiscussionThis is the first study conducted in the South hemisphere to demonstrate the low-grade inflammatory profile in FEP patients, compared to their siblings and community-based controls. The fact that IL-6, TNF-a and IL-10 are all higher in the FEP group than their healthy siblings and community-based controls suggests the synergism of individual vulnerability factors in the development of this inflammatory profile.

Luminex® xMAP® technology is an effective strategy for high-definition human leukocyte antigen typing of cord blood units prior to listing.

Allele-level donor-recipient match at HLA-A, HLA-B, HLA-C and HLA-DRB1 loci impacts the outcome after cord blood transplantation for hematologic malignancies and modifies the strategy of donor selection. High definition of both class I and II HLA loci at time of listing is a way to improve the attractiveness of cord blood bank inventories, reducing the time for donor search and procurement and simplifying donor choice, in particular, for patients of non-European heritage. In 2014, Luminex® xMAP® technology was introduced in our laboratory practice and was applied to cord blood units typing. In this study, we evaluated the impact of this strategy in comparison with the platform in use until 2013, relying on LiPA reverse polymerase chain reaction-sequence-specific oligonucleotide (revPCR-SSO) plus polymerase chain reaction-sequence-specific primer (PCR-SSP). In 2014, the time for testing was shorter (141 vs 181 days on average), the number of test repetitions was lower (in particular for HLA-A locus, p = 0.026), and the cost reduced (240.7 vs 395.6 euros per unit on average) compared to 2013, demonstrating that Luminex xMAP technology is superior to the previous approach. Luminex xMAP platform has useful application in cord blood banking programs, to achieve high-definition HLA typing of cord blood units at the time of banking in a quick, accurate, and cost-effective manner.

Effects of Quercetin, Kaempferol, and Exogenous Glutathione on Phospho- and Total-AKT in 3T3-L1 Preadipocytes

ABSTRACTObesity has been reported to be a risk factor for some types of cancer, such as prostate and lung. The AKT or PI3K-AKT is a signal transduction pathway that promotes survival and growth in response to extracellular signals. The aim of this study was to investigate the effects of two flavonoids, quercetin and kaempferol, and exogenous glutathione (GSH) on the expressions of phospho- and total-AKT levels in 3T3-L1 preadipocytes. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels were measured in the treated samples and used as the internal standard. 3T3-L1 preadipocytes were exposed to each flavonoid and GSH at concentrations of 0, 5, 10, 15, 20, and 25 µM, and the levels of phospho- and total-Akt were measured by the MILLIPLEX MAP mates protocol, based on the Luminex xMAP technology (Millipore Corp., St. Charles, MI, USA). GAPDH levels in the preadipocytes were not significantly different at the doses tested for the flavonoids and exogenous GSH. However, significant (p <.05) decreases in phospho-AKT levels in cells treated with quercetin, kaempferol, and GSH at certain doses were observed compared to their respective controls. Total-AKT levels showed the same profile for all the tested compounds. Significant (p <.01) differences were observed for kaempferol (15–25 µM), quercetin at 10 and 20 µM, and GSH at 10 µM compared to their respective controls. Findings suggest that exposure of 3T3-L1 preadipocytes to quercetin, kaempferol, and GSH may block the activation of AKT, suggesting the role such compounds play in cell differentiation in 3T3-L1 cells.

Human aqueous humor levels of transforming growth factor-β2: Association with matrix metalloproteinases/tissue inhibitors of matrix metalloproteinases

The present study aims to investigate the association of transforming growth factor-β2 (TGF-β2) and matrix metalloproteinases (MMPs), MMP-2 and MMP-3, and tissue inhibitors of matrix metalloproteinases (TIMPs), TIMP-1, TIMP-2 and TIMP-3 in the aqueous humor of patients with high myopia or cataracts. The levels of TGF-β2 and MMPs/TIMPs were measured with the Luminex xMAP Technology using commercially available Milliplex xMAP kits. The association between TGF-β2 and MMPs/TIMPs levels was analyzed using the Spearmans correlation test. The levels of TGF-β2 were identified to be positively correlated with the levels of TIMP-1 and TIMP-3 (TIMP-1: r=0.334; P=0.007; TIMP-3: r=0.309; P=0.012). The levels of MMP-2, MMP-3 and TIMP-2 did not significantly correlate with TGF-β2 levels (P>0.05). A positive correlation was identified between TGF-β2 and TIMPs in the aqueous humor of human eyes with elongated axial length. It appears that TGF-β2 stimulates the expression of TIMPs as a compensatory reaction to the development of high myopia.

Effect of Single Sensitization Event on Human Leukocyte Antigen Alloimmunization in Kidney Transplant Candidates: A Single-Center Experience.

Human leukocyte antigen alloimmunization is caused by exposure to HLA antigens through transfusion, pregnancy, or transplant. Our study objective was to present the rate of positivity of anti-HLA antibody considering the effects of a single sensitization event in kidney transplant candidates at our center. Our study reviewed 606 kidney transplant candidates. Patient sera were analyzed using Luminex xMAP technology. Panel reactive antibody positivity rates and antibody strengths in patients were analyzed according to a single sensitization event. Our findings showed 246 patients (40.6%) with a panel reactive antibody > 0, of which 97 (39.4%) were sensitized from a single event, 119 (48.4%) were sensitized by multiple events, and 30 (12.2%) had no known sensitizing event. Considering patients sensitized by a single event with a panel reactive antibody > 0, we found that 25.8% had received transplant only, 49.5% had previous pregnancy only, and 24.7% had received transfusion only. The strength of antibodies was significantly higher in patients with previous transplant procedures than in those with transfusion for HLA-A (P < .01), HLA-B (P < .05), HLA-C (P < .05), HLA-DR (P < .001), HLA-DQ (P < .05), and HLA-DP (P < .05). Similarly, we observed significantly higher median fluorescence intensity values for HLA-A, -DR, -DQ, and -DP loci in patients with a previous transplant procedure versus pregnancy. The strength of antibodies against HLA-DR was significantly higher in patients with a previous pregnancy compared with those with transfusion (P < .01). This study documents the profile of HLA alloimmunization in kidney transplant candidates. In particular, transplant procedures appear to have a greater immunologic impact, followed by pregnancy and transfusion. Our data confirm and are in accordance with those of several studies in which the sensitization events were associated with higher prevalence of anti-HLA antibodies.

A multiplex liquid-chip assay based on Luminex xMAP technology for simultaneous detection of six common respiratory viruses.

We utilized one-step multiplex reverse transcription-PCR (RT-PCR) and Luminex xMAP technology to develop a respiratory multiplex liquid-chip assay (rMLA) for simultaneous detection of 6 common respiratory viruses, including influenza virus type A (FluA) and type B (FluB), para-influenza virus type 3 (PIV-3), respiratory syncytial virus (RSV), human metapneumovirus (MPV) and a threatening virus to China, Middle East Respiratory Syndrome coronavirus (MERS-CoV). Performance of rMLA was evaluated by comparing with real-time RT-PCR. Detection data from clinical specimens showed that the rMLA had diagnostic sensitivities of 97.10% for FluA, 94.59% for FluB, 98.68% for PIV-3, 94.87% for RSV and 95.92% for MPV (No Data for MERS-CoV due to the lack of positive specimens). Data of analytical sensitivities showed that the detection limits of the rMLA assay were 5–25 viral RNA copies per μl for FluA, FluB, PIV-3 and MERS-CoV, approximate to the real-time RT-PCR assay; while the values were 8 and 22copies/μl for MPV and RSV, lower than the real-time RT-PCR(78 and 114 copies/μl respectively). The results indicated that the rMLA is a sensitive, specific detection tool and comparable to real-time RT-PCR, especially suitable for high-throughput detection of respiratory specimens.

Comparison of the cervista HPV HR test and luminex XMAP technology for the diagnosis of cervical intraepithelial neoplasia

ObjectivesThis study was designed to compare the Cervista high risk (HR) human papillomavirus (HPV) test with Luminex XMAP technology for the detection of the relationship between HPV infection and cervical intraepithelial neoplasia. MethodsIn total, 3280 patients in a cervical specialty clinic were divided into two groups for either Cervista (1855 patients) or Luminex (1425 patients). Subsequent colposcopy examinations were performed in 1270 women with cytologic results showing ASCUS or higher level lesions. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were evaluated. ResultsThe positive rates of the Cervista and Luminex groups were 61.48% and 67.43%, respectively, and occurrence in those 30–40 years old was most common. The typing of HPV showed that A9 positive cases were the most prevalent genotype (33.53%), followed by A5/A6 (16.44%) and A7 (11.37%) in the Cervista group, and HPV-16 was the most prevalent genotype (25.81%), followed by HPV-18 (18.6%) and HPV-31 (8.6%) in the Luminex group. Moreover, the overall concordance rate was 96.26% (95% confidence interval, 93.51–99.00%) in the 187 women with cytologic results of ASCUS or higher. There were no significant differences in the positive rates of HPV between the Cervista and Luminex groups, and both had a high sensitivity and NPV. ConclusionsThe results showed a high good concordance between the two methods in diagnostic accuracy. Among the patients in the cervical specialty clinic, both the A9 group of HPV and HPV-16 showed the highest positive rate. Cervista and Luminex shared similar a clinical value in the detection of CIN2 or higher.

Analysis of aqueous humor concentrations of cytokines in retinoblastoma.

To investigate the components of the aqueous humor (AH) in patients with retinoblastoma (RB). We collected 0.1 ml AH of 35 children with RB and 20 patients with congenital cataracts as controls. Multiplex enzyme-linked immunosorbent assays (ELISAs) and Luminex xMAP technology were used to assess 45 cytokines/chemokines, matrix metalloproteinases (MMPs), and acute-phase proteins in the identification cohort. The concentrations of IL-6, IL-7, IL-8, IFN-γ, PIGF-1, VEGF-A, β-NGF, HGF, EGF and FGF-2 were significantly higher in the AH of patients with RB than those in the control group (P<0.05). The study showed that the higher levels of IP-10, IL-6, IL-7, IL-8, IFN-γ, PIGF-1, VEGF-A, β-NGF, HGF, EGF and FGF-2 in AH may be associated with RB. Our findings may facilitate a better understanding of the molecular pathways of tumors and solid molecular targets for new strategies for therapy and the earlier diagnosis of RB.

Plasma concentrations of free amyloid β cannot predict the development of Alzheimer's disease

IntroductionBiomarkers that identify individuals at risk of Alzheimer's disease (AD) development would be highly valuable. Plasma concentration of amyloid β (Aβ)—central in the pathogenesis of AD—is a logical candidate, but studies to date have produced conflicting results on its utility. MethodsPlasma samples from 339 preclinical AD cases (76.4% women, mean age 61.3 years) and 339 age- and sex-matched dementia-free controls, taken an average of 9.4 years before AD diagnosis, were analyzed using Luminex xMAP technology and INNO-BIA plasma Aβ form assays to determine concentrations of free plasma Aβ40 and Aβ42. ResultsPlasma concentrations of free Aβ40 and Aβ42 did not differ between preclinical AD cases and dementia-free controls, in the full sample or in subgroups defined according to sex and age group (<60 and ≥ 60 years). The interval between sampling and AD diagnosis did not affect the results. Aβ concentrations did not change in the years preceding AD diagnosis among individuals for whom longitudinal samples were available. DiscussionPlasma concentrations of free Aβ could not predict the development of clinical AD, and Aβ concentrations did not change in the years preceding AD diagnosis in this sample. These results indicate that free plasma Aβ is not a useful biomarker for the identification of individuals at risk of developing clinical AD.

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.

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.

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.