Multiplex Electrochemiluminescence Assay Research Articles

Abstract 15748: Association of Clonal Hematopoiesis in the Setting of Human Immunodeficiency Virus Infection With Subclinical Atherosclerosis and Vascular Inflammation

Background: Clonal hematopoiesis of indeterminate potential (CHIP) is associated with cardiovascular disease (CVD) and common in HIV, but whether CHIP contributes to atherosclerosis in HIV is unknown. We hypothesized CHIP is associated with atherosclerosis and arterial inflammation among people with HIV (PWH). Methods: We studied treated, suppressed PWH ages 35-70 years old with ≥1 CVD risk factor. CHIP mutations were detected with a validated targeted sequencing assay. We measured carotid intima-media thickness (IMT) longitudinally with ultrasound and aortic inflammation and lymph node activity using cross-sectional 18 F-FDG-PET. Inflammatory biomarkers were measured with a multiplex electrochemiluminescence assay. We used linear regression with adjustment for age, sex, nadir CD4 count, smoking, hypertension, diabetes, and hyperlipidemia. Results: We included 231 PWH (52±9 years, 7% female). 32 (14%) had CHIP with median variant allele fraction of 3.1%. Common mutations were in DNM3TA (n=21) and TET2 (n=6). Only age was associated with CHIP (OR 2.3 per decade older, 95%CI 1.2-3.9; p=0.003). Among N=165 (CHIP=22), mean IMT was 1.0 mm with and without CHIP (p=0.63), unchanged after adjustment (Figure). CHIP was not associated with prevalent or incident plaque. Over 3.2 years, IMT progression was faster among those with CHIP (0.033 mm/year; p=0.10), attenuated after adjustment (0.022 mm/year; p=0.27). Among 80 with FDG-PET, CHIP (n=12) was associated with higher lymph node activity (SUV p=0.04) that was attenuated in reference to background activity and adjusted for risk factors. CHIP was not associated with arterial inflammation (p=0.83), inflammatory markers, or viral persistence markers. Conclusions: Among PWH, CHIP mutations were not associated with subclinical atherosclerosis or arterial inflammation, proposed mechanisms of how CHIP could cause CVD. Clinical outcomes studies are needed to ascertain the impact of CHIP on CVD in HIV.

Identifying immune biomarkers associated with human trauma as potential therapeutic targets

Abstract Severe trauma can induce systemic immunosuppression, which makes understanding the biomarker profiles of trauma patients critical for therapeutic development and for predicting potential immune responses to treatments such as biomedical implants. Our goal was to establish a data set containing levels of a large array of biomarkers measured in blood from human trauma patients. Multiplex electrochemiluminescence assays were conducted to measure the levels of 59 biomarkers in 1000 blood samples collected from patients at the time of admission to trauma centers. Injury types were categorized as motor vehicle crashes (n=448), falls (n=221), gun wounds (n=78), stabbings (n=117), or other traumatic injuries. Patients ranged in age from 18 to 101, with a mean age of 42.7 for male patients (n= 725) and 49.8 for female patients (n= 272). There were no significant differences in biomarker expression levels between the different injury types. Differences in age distributions of male and female patients for each injury type were not found to be statistically significant, except in the case of falls, which had a higher average age for female patients compared to male patients. MIF, MIP-5, and YKL-40 had the highest expression, with concentrations of 554148 ± 62938 pg/mL, 17409 ± 919 pg/mL, and 98793 ± 10015 pg/mL (mean ± 95 % CI) respectively, at 1:100 dilution. Further experiments are currently being conducted to establish biomarker profiles for control uninjured human samples, and data comparing the profiles of trauma patients and uninjured controls will be presented. Overall, this work provides a resource for understanding immune responses to injury and potentially developing new therapeutics with these biomarker profiles in mind. This work was supported by the National Institute of Biomedical Imaging and Bioengineering

Preventing rheumatoid arthritis: Lessons from that of type 1 diabetes

Preventing rheumatoid arthritis: Lessons from that of type 1 diabetes

Topical review- salivary biomarkers in chronic muscle pain.

Muscle related temporomandibular disorders (myogenous TMD), one of the most common orofacial pain conditions, is characterized by facial pain and often accompanied by jaw movement limitations. Although the underlying biological mechanisms are still unclear, a cluster of proteins and peptides is assumed tobe involved in the pathophysiology. These proteins andpeptides may be measured in a simple non-invasive saliva sample. This work investigated whether saliva can be used to sample algogenic substances that can serve asmolecular biomarkers for TMD myalgia. Saliva and blood samples were collected from healthy individuals (n=69) and patients diagnosed with TMD myalgia (n=39) according to the Diagnostic Criteria for TMD. Unstimulated and stimulated whole, parotid, and sublingual saliva were analysed. The protein profiles were investigated using two-dimensional gel electrophoresis followed by identification with liquid chromatography tandem mass spectrometry. Levels of nerve growth factor (NGF), calcitonin gene-related peptide (CGRP), and brain derived neuro-tropic factor (BDNF) were determined using western blotting based technology and multiplex electro-chemiluminescence assay panel. Glutamate, serotonin, and substance p (SP) were determined using commercially available methods. Different saliva collection approaches resulted insignificant differences in the protein profile as well as inthe expression of NGF, BDNF, CGRP, SP, and glutamate. Stimulated whole saliva showed least variability in protein concentration (35%) and was correlated to plasma levels ofglutamate. Unlike SP and glutamate, NGF and BDNF expressed a rhythmic variation in salivary expression withhigher levels in the morning (p<0.05). Patients with a diagnosis of TMD myalgia had significantly higher levels of salivary glutamate but lower salivary NGF and BDNF compared to controls; in addition, the lower NGF and BDNF levels correlated to psychological dysfunction. Thequantitative proteomics data revealed 20 proteins that were significantly altered in patients compared to controls. The identified proteins are involved in metabolic processes, immune response, and stress response. Dissimilarities in protein profile and clinical variables were observed between TMD myalgia and myofascial pain. The work highlights the importance of consistency in saliva collection approaches, including thetiming of the collection. It displayed significant changes in pain specific mediators and protein profile in TMD myalgia and furthermore dissimilarities between subclasses indicating different pathophysiology. After extensive validation, potential salivary biomarkers can becombined with clinical features to better understand and diagnose TMD myalgia.

Non-SARS Coronaviruses in Individuals with Psychiatric Disorders.

The pandemic caused by severe acute respiratory syndrome-Coronavirus-2 (SARS-CoV-2) has highlighted the importance of coronaviruses in human health. Several seasonal, non-SARS Coronaviruses are endemic in most areas of the world. In a previous study, we found that the level of antibodies to these seasonal Coronaviruses was elevated in persons with a recent onset of psychosis. In the current study, the level of antibodies to seasonal Coronaviruses was compared between individuals with psychiatric disorders and a non-psychiatric comparison group. Participants (N=195) were persons with a diagnosis of schizophrenia, bipolar disorder, major depressive disorder, or without a psychiatric disorder. Each participant had a blood sample drawn from which were measured IgG antibodies to the spike proteins in four non-SARS Coronaviruses, 229E, HKU1, NL63, and OC43, using a multiplex electrochemiluminescence assay. Linear regression models were employed to compare the levels of antibodies between each psychiatric group and the comparison group adjusting for demographic variables. Logistic regression models were employed to calculate the odds ratios associated with increased levels of antibodies to each seasonal Coronavirus based on the 50th percentile level of the comparison group. The schizophrenia group had significantly increased levels of antibodies to the seasonal Coronaviruses OC43 and NL63. This group also had increased odds of having elevated antibody levels to OC43. The major depression group showed a significantly lower level of antibodies to Coronavirus 229E. There were no significant differences between any of the psychiatric groups and the comparison group in the levels of antibodies to seasonal Coronaviruses 229E or HKU1. The elevated level of antibodies to OC43 and NL63 in the schizophrenia group indicates increased exposure to these agents and raises the possibility that Coronaviruses may contribute to the etiopathology of this disorder. The cause-and-effect relationship between seasonal Coronaviruses and psychiatric disorders should be the subject of additional investigations focusing on longitudinal cohort studies.

158-LB: A Complete-Panel Islet Autoantibody Multiplex ECL Assay

Background: Type 1 diabetes (T1D), a major chronic autoimmune disease starting mostly from childhood, is now predictable with the measurement of four major islet autoantibodies (IAbs) including IAA, GADA, IA-2A and ZnT8A. Currently, there are approximately 1.4 million people affected with T1D in the United States and the incidence has doubled in the last 20 years, especially in young children. Mass screening for eligible subjects and the general population remains inefficiency and laborious bottleneck. Methods: With the platform of our extensively validated high affinity electrochemiluminescence (ECL) assay, we have developed a simple multiplexed ECL assay to combine all four major IAb assays in a single well. Two cohorts were studied including 80 samples from newly diagnosed patients with T1D and 1,140 from Autoimmunity Screening for Kids (ASK) study. Both multiplex ECL assay and standard radio-binding assay (RBA) were performed in parallel. Results: With &amp;gt;99th percentile of specificity for both assays in over 1,000 samples from general population, sensitivity of multiplex ECL assay in new onset patients with T1D were similar to RBA, GADA 75% vs. 71%; IA-2A 91% vs. 90%; IAA 61% vs. 65%; ZnT8A 90% vs. 87%. In ASK cohort, multiplex ECL assay was concordant with RBA among children with multiple IAbs (42/44, 96%). In contrast, only 39% (21/54, p&amp;lt;0.0001) of children with single IAb by RBA were positive by multiplex ECL assay. Conclusion: Multiplex ECL assay illustrated its high sensitivity/specificity as RBA in T1D patients. In general population screening, multiplex ECL assay, similar to single ECL assay, is capable to discriminate high risk IAbs from low risk IAbs generated by RBA. With a multiplexing feature, it provides a high throughput tool with low cost and low sample volume for large scale of population screening for T1D. Disclosure L. He: None. X. Jia: None. D. Miao: None. C. Geno rasmussen: None. M. Rewers: Advisory Panel; Self; Provention Bio, Inc., Consultant; Self; RTI, Research Support; Self; Janssen Research &amp; Development, LLC, JDRF. L. Yu: None. Funding JDRF (2-SRA-2018-533-S-B, 1-SRA-2016-208-S-B); National Institutes of Health (DK32083); Diabetes Research Center (P30DK116073)

Large-Scale Screening in General Population Children for Celiac Disease with a Multiplex Electrochemiluminescence (ECL) Assay.

Background Autoimmunity Screening for Kids (ASK) study was launched to screen general population children for type 1 diabetes (T1D) and celiac disease (CD). Methods A total of 23,319 children from general population were screened. A high throughput multiplex electrochemiluminescence (ECL) assay to screen multiautoantibodies in a single well was applied, parallel with a standard radiobinding assay (RBA). All children with any positive autoantibodies in screening were revisited within one month for confirmation and followed every 6 months. Results Among 23,319 children, 2.6% (606/23,319) of children were tested positive for TGA. Multiplex ECL assay detected more TGA (584/23,319) in the initial screening than RBA (490/23,319, p = 0.004) and was able to detect TGA earlier than RBA in a subset of children by 0.8 to 34.8 months. Prevalence of TGA by either ECL or RBA in children with islet autoantibodies was found significantly higher than overall prevalence in general population screened. Conclusions A multiplex ECL assay was more sensitive than standard RBA by identifying more TGA positivity and detecting TGA earlier in general population screening. It also provides a high efficient tool with its unique advantage of multiplexing measurements to screen for multiple autoimmune diseases simultaneously in general population.

Altered levels of salivary and plasma pain related markers in temporomandibular disorders

BackgroundDifferent pain syndromes may be characterized by different profiles of mediators reflecting pathophysiological differences, and these alterations may be measured in a simple saliva sample. The aims of the current study were to compare concentration of glutamate, serotonin (5-HT), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and substance P (SP) in saliva and plasma from a well-defined group of patients with chronic temporomandibular disorders myalgia (TMD-myalgia) with a group of pain-free controls, and further investigate the relationship between these markers and clinical characteristics.MethodsPatients diagnosed according to the diagnostic criteria for TMD (n = 39), and matched healthy pain-free controls (n = 39) were included. Stimulated whole saliva and plasma samples were collected in the morning. Glutamate was analysed using a colorimetric assay, and 5-HT and SP were analysed by commercially available ELISA. Levels of NGF and BDNF were determined using multiplex electrochemiluminescence assay panel.ResultsPatients expressed higher salivary and plasma levels of glutamate (saliva: 40.22 ± 13.23 μmol/L; plasma: 30.31 ± 18.73 μmol/L) than controls (saliva: 33.24 ± 11.27 μmol/L; plasma: 20.41 ± 15.96 μmol/L) (p < 0.05). Salivary NGF (0.319 ± 0.261 pg/ml) and BDNF (3.57 ± 1.47 pg/ml) were lower in patients compared to controls (NGF: 0.528 ± 0.477 pg/ml; BDNF 4.62 ± 2.51 pg/ml)(p’s < 0.05). Contrary, plasma BDNF, was higher in patients (263.33 ± 245.13 pg/ml) than controls (151.81 ± 125.90 pg/ml) (p < 0.05). 5-HT was undetectable in saliva. Neither plasma 5-HT, nor SP levels differed between groups. BDNF and NGF concentrations correlated to levels of psychological distress (p < 0.0005).ConclusionThe higher levels of salivary and plasma glutamate in patients with TMD-myalgia compared to controls strengthens its importance in the pathophysiology of TMD-myalgia. However, the lack of correlation to pain levels question its role as a putative biomarker. Patients with TMD-myalgia further had lower levels of salivary NGF and BDNF, but higher plasma BDNF. These results and their correlations to psychological distress warrant further investigations.

A High-Throughput Electrochemiluminescence 7-Plex Assay Simultaneously Screening for Type 1 Diabetes and Multiple Autoimmune Diseases.

Islet autoantibodies (IAbs) are widely used in type 1 diabetes (T1D) diagnosis and prediction. Four major IAbs to insulin (IAA), glutamate decarboxylase-65 (GADA), insulinoma antigen-2 (IA-2A), and zinc transporter-8 (ZnT8A) are equally important in disease prediction. Presently, up to 40% of patients diagnosed with T1D go on to develop other autoimmune disorders. Unfortunately, current screening methods using a single autoantibody for measurement are laborious and inefficient for large scale screening studies. We recently developed a simple multiplexed electrochemiluminescence (ECL) assay to address these current issues. The assay combines all 7 autoantibody tests into one well. Each well includes three IAbs (IAA, GADA, and IA-2A), autoantibodies to thyroid peroxidase (TPOA) and thyroid globulin (ThGA) to detect autoimmune thyroid disease, autoantibodies to tissue transglutaminase (TGA) for celiac disease, and autoantibodies to interferon alpha (IFNαA) for autoimmune polyglandular syndrome-1 (APS-1); all of which screen for T1D and other relevant autoimmune diseases, simultaneously. The multiplex ECL assay is based on the single ECL assay platform, but instead uses the multiplex plate combining multiple autoantibody assays, up to 10, into a single well. The main difference from the single ECL assay is that each antibody-antigen complex formed in the fluid-phase is restrained onto a specific spot on each well through a linker system on the multiplex plate. The 7-Plex ECL assay, in the present study, is validated against standard radio-binding assays (RBA) and single ECL assays, using a large cohort of newly diagnosed T1D patients and age-matched healthy controls, resulting in excellent assay sensitivity and specificity.

Comparison between a new multiplex electrochemiluminescence assay and the WHO reference enzyme-linked immunosorbent assay to measure serum antibodies against pneumococcal serotype-specific polysaccharides

BackgroundTwo electrochemiluminescence (ECL) assays were developed which, together, can simultaneously measure serum antibodies against pneumococcal capsular polysaccharides (PnPS) for 17 serotypes. The assays were validated for the 13 PnPS included in the 13-valent pneumococcal conjugate vaccine (PCV13). As recommended by the World Health Organization (WHO), we compared the ECL assays with the WHO reference enzyme-linked immunosorbent assay (ELISA) and derived a threshold corresponding to the 0.35 µg/mL threshold established for the WHO reference ELISA for the non-inferiority comparison and licensure of new PCVs against invasive pneumococcal disease. MethodsA panel of 452 serum samples from children vaccinated with one of the three licensed PCVs was assessed with the ECL assays and the WHO reference ELISA. The ECL assay threshold for the aggregated seven PnPS included in the 7-valent PCV (PCV7) and serotype-specific thresholds were determined using a receiver operating characteristics (ROC) curve-based approach and Deming regression. To evaluate concordance between the ECL assays and the WHO reference ELISA, serostatus agreement rates between both assays and geometric means of the ratios (GMRs) of concentrations obtained with both assays were calculated. ResultsThe thresholds for the seven aggregated PCV7 serotypes obtained with the ROC curve-based approach and Deming regression approximated 0.35 µg/mL (0.38 and 0.34 µg/mL, respectively). Individual thresholds for the PCV13 serotypes ranged between 0.24 and 0.51 µg/mL across both approaches. Serostatus agreement rates using a 0.35 µg/mL threshold for both assays were ≥86.9% for all PCV13 serotypes. GMRs ranged between 0.85 and 1.25 for 11/13 serotypes and were <1.29 for the two remaining serotypes. ConclusionThe ECL assays were comparable to the WHO reference ELISA and offer a sensitive, time- and serum volume-saving method to quantify serotype-specific anti-PnPS antibodies in pediatric sera. A 0.35 µg/mL threshold will be used for each PCV13 serotype to assess PCV immunogenicity in clinical trials.

Decreased IL-8 levels in CSF and serum of AD patients and negative correlation of MMSE and IL-1β.

BackgroundIt is widely accepted that neuroinflammatory processes play an important role in the pathogenesis of Alzheimer’s disease (AD) and high levels of cytokines and chemokines are detected around Aβ plaques.MethodsAs neuroinflammation is involved in the development and progression of AD, we measured the pro-inflammatory cytokines interleukin 1β (IL-1β), IL-8 and tumor necrosis factor α (TNF-α) in serum and cerebrospinal fluid (CSF) samples from 45 AD patients and 53 age-matched control subjects using a highly sensitive multiplex electrochemiluminescence assay. To address the association with disease progression we correlated cognitive status with cytokine levels.ResultsCSF as well as serum IL-8 levels were found to be significantly lower in AD patients than in controls (p = 0.02). A statistically significant inverse correlation was observed between the CSF level of IL-1β and the MMSE score (rs = -0.03, p = 0.02). We therefore stratified the AD patients by their MMSE scores into three equal groups and found that in the AD group with the most severe cognitive impairment CSF-IL-1β was significantly increased compared to age-matched controls (p < 0.05), whereas in the other investigated groups the increase was not statistically significant.ConclusionOur results confirm data suggesting that cytokine alterations are involved in AD pathogenesis and may be helpful as a biomarker for monitoring disease progression.Electronic supplementary materialThe online version of this article (doi:10.1186/s12883-016-0707-z) contains supplementary material, which is available to authorized users.

Multidimensional endotypes of asthma: topological data analysis of cross-sectional clinical, pathological, and immunological data

BackgroundIncomplete understanding of mechanisms and clinicopathobiological heterogeneity in asthma hinders research progress. Pathogenic roles for T-helper-type 17 (Th17) cells and invariant T cells implied by murine data have yet to be assessed in man. We aimed to investigate the role of Th17 and mucosal associated invariant T (MAIT) cells in airway inflammation; to characterise associations between diverse clinical and immunological features of asthma; and to identify novel multidimensional asthma endotypes. MethodsIn this single-centre, cross-sectional observational study in the UK, we assessed volunteers with mild-to-severe asthma and healthy non-atopic controls using clinical and physiological assessment and immunological sampling of blood, induced sputum, endobronchial biopsy, and bronchoalveolar lavage for flow cytometry and multiplex-electrochemiluminescence assays. Primary outcomes were changes in frequencies of Th17 and MAIT cells between health and asthma using Mann-Whitney U tests and the Jonckheere-Terpstra test (linear trend across ranked groups). The study had 80% power to detect 60% differences in T-cell frequencies at p<0·05. Bayesian Network Analysis (BNA) was used to explore associations between parameters. Topological Data Analysis (TDA) was used to identify multidimensional endotypes. The study had local research ethics approval. All participants provided informed consent. FindingsParticipants were 84 male and female volunteers (60 with mild-to-severe asthma and 24 healthy, non-atopic controls) aged 18–70 years recruited from clinics and research cohorts. Th17 cells and γδ17 cells were not associated with asthma, even in severe neutrophilic forms. MAIT-cell frequencies were strikingly reduced in asthma compared with health (median frequency in blood 0·9% of CD3+ cells [IQR 0·3–1·8] in asthma vs 1·6 [1·2–2·6] in health, p=0·005; in sputum 1·1 [0·7–2·0] vs 1·8 [1·6–2·3], p=0·002; and in biopsy samples 1·3 [0·7–2·3] vs 3·9% [1·3–5·3%], p=0·02), especially in severe asthma where BAL regulatory T cells were also reduced compared with those in health (4·4, 3·1–6·1, vs 8·1, 5·6–10; p=0·02). BNA and TDA identified six novel clinicopathobiological clusters of underlying disease mechanisms, with elevated mast cell mediators tryptase (p<0·0001), chymase (p=0·02), and carboxypeptidase A3 (p=0·02) in severe asthma. InterpretationThis study suggests that Th17 cells do not have a major pathogenic role in human asthma. We describe a novel deficiency of MAIT cells in severe asthma. We also provide proof of concept for application of TDA to identification of multidimensional clinicopathobiological endotypes. Endotypes will require validation in further cohorts. FundingWellcome Trust.

Complement activation and kidney injury molecule-1-associated proximal tubule injury in severe preeclampsia.

Kidney injury with proteinuria is a characteristic feature of preeclampsia, yet the nature of injury in specific regions of the nephron is incompletely understood. Our study aimed to use existing urinary biomarkers to describe the pattern of kidney injury and proteinuria in pregnancies affected by severe preeclampsia. We performed a case-control study of pregnant women from Brigham and Women's Hospital from 2012 to 2013. We matched cases of severe preeclampsia (n=25) 1:1 by parity and gestational age to 2 control groups with and without chronic hypertension. Urinary levels of kidney injury molecule-1 and complement components (C3a, C5a, and C5b-9) were measured by enzyme-linked immunosorbent assay, and other markers (albumin, β2 microglobulin, cystatin C, epithelial growth factor, neutrophil gelatinase-associated lipocalin, osteopontin, and uromodulin) were measured simultaneously with a multiplex electrochemiluminescence assay. Median values between groups were compared with the Wilcoxon signed-rank test and correlations with Spearman correlation coefficient. Analysis of urinary markers revealed higher excretion of albumin and kidney injury molecule-1 and lower excretion of neutrophil gelatinase-associated lipocalin and epithelial growth factor in severe preeclampsia compared with chronic hypertension and healthy controls. Among subjects with severe preeclampsia, urinary excretion of complement activation products correlated most closely with kidney injury molecule-1, a specific marker of proximal tubule injury (C5a: r=0.60; P=0.001; and C5b-9: r=0.75; P<0.0001). Taken together, we describe a pattern of kidney injury in severe preeclampsia that is characterized by glomerular impairment and complement-mediated inflammation and injury, possibly localized to the proximal tubule in association with kidney injury molecule-1.

Elevated IL‐8, TNF‐α, and MCP‐1 in men with metastatic prostate cancer starting androgen‐deprivation therapy (ADT) are associated with shorter time to castration‐resistance and overall survival

Chemokines and cytokines have been implicated in progression to castration-resistant prostate cancer (CRPC). Retrospective data were accessed from 122 men with serum samples drawn at a median of 0.5 months after starting ADT for metastatic prostate cancer. MCP-1, IL-1-β, IL-2, IL-8, IL-6, and TNF-α levels were measured by multiplex electrochemiluminescence assays. A multivariable Cox model assessed the association of time to CRPC and overall survival by the protein levels and adjusted for clinical variables (age and prostate specific antigen (PSA) levels at start of ADT, race, ECOG status, and extent of metastases). Associations were reported as hazard ratio (HR) with 95% confidence interval (CI). Median follow-up and overall survival were 44 and 42.2 months, respectively. ECOG performance status (≥1 vs. 0) was negatively associated with overall survival [HR = 2.8 (1.1-7.0), P = 0.03], and PSA nadir < 0.2 was predictive of longer time to development of CRPC [HR = 0.3 (0.2-0.5), P < 0.0001]. The HR for time to CRPC by protein above the median was 1.4 (95% CI: 0.9, 2.2, P = 0.13) for IL-8; 1.3 (95% CI: 0.8, 2, P = 0.18) for TNF-α; 1.0 (95% CI: 0.7, 1.6, P = 0.95) for MCP-1. The HR for median overall survival for protein levels above the median was: 1.9 (95% CI: 1.0, 3.5, P = 0.04) for IL-8; 2.0 (95% CI: 1.1, 3.5, P = 0.02) for TNF-α; 1.7 (95% CI: 1.7, 3.0, P = 0.08) for MCP-1. There was no association with IL-1-β, IL-2, or IL-6. Higher levels of inflammation-associated cytokines correlate with poorer prostate cancer outcomes and may guide strategies to improve prostate cancer therapy.

Elevated insulin‐like growth factor binding protein‐1 (IGFBP‐1) in men with metastatic prostate cancer starting androgen deprivation therapy (ADT) is associated with shorter time to castration resistance and overall survival

Insulin-like growth factor (IGF) and adipokines have been implicated in prostate cancer carcinogenesis. Data from 122 men with serum samples drawn within 3 months of starting ADT for metastatic prostate cancer was accessed retrospectively. IGF-1, IGF binding protein (BP)-1, leptin, and adiponectin levels were measured by multiplex electrochemiluminescence assays. A multivariable Cox model assessed the association of time to castration resistant prostate cancer (CRPC) and overall survival by the protein levels, adjusted for clinical variables, age and prostate specific antigen (PSA) levels at start of ADT, race, ECOG status, extent of metastases and were reported as hazard ratio (HR) with 95% confidence interval (CI). Median follow-up and overall survival were 44 and 42.2 months, respectively. ECOG performance status (≥ 1 vs. 0) was negatively associated with overall survival [H = 2.8 (1.1-7.0), P = 0.03], and PSA nadir <0.2 was predictive of longer time to CRPC [HR = 0.3 (0.2-0.5), P < 0.0001]. The median time to CRPC by low, middle, and top IGFBP-1 tertile distribution was 20.7, 18.1, and 12.4 months, respectively, with HR for middle versus low tertile levels 3.1 (1.7-5), P = 0.0003, and for top versus low tertile levels was 2.4 (1.3-4.2), P = 0.003. The median overall survival by low, middle and top tertile IGFBP-1 level was 48.5, 46.4, and 32.8 months, respectively, with HR for top versus low tertile 2.5 (1.2-5.1), P = 0.01. There was no association with IGF-1, adiponectin and leptin. Elevated IGFBP-1 appears to be associated with shorter time to CRPC and lower overall survival in men with metastatic prostate cancer.

Homeostatic properties of Lactobacillus jensenii engineered as a live vaginal anti-HIV microbicide

BackgroundVaginal probiotics are investigated as a binary strategy for prevention of bacterial vaginosis and HIV. We applied an innovative experimental model using primary and immortalized human cervical and vaginal epithelial cells to assess the functional properties of Lactobacillus jensenii, a predominant constituent of the healthy vaginal microbiome, engineered to express the HIV-1 entry inhibitor modified cyanovirin-N (mCV-N). In this model bacteria colonize the epithelial cells over a period of 24-72 h. Staurosporine and the Toll-like receptor 2/6 ligand macrophage-activating lipopeptide-2 (MALP-2) serve as positive controls for apoptosis and proinflammatory activation, respectively. In 24-hour intervals, the colonized epithelium is assessed microscopically, supernatants are collected for measurement of soluble immunoinflammatory mediators and production of CV-N, and cells are lysed for assessment of: 1) apoptosis by cleaved versus total caspase-3 assay; 2) NF-κB activation by a luciferase reporter assay; or 3) epithelia-associated colony forming units (CFU) in Brucella agar.ResultsWild type (WT) L. jensenii 1153 consistently colonized cervical and vaginal cells in the absence of epithelial damage and apoptosis. The bioengineered derivatives expressing mCV-N or control plasmids showed the same stable colonization pattern, which was reproducible between technologists and bacterial batches (CFU coefficient of variation <10% within and between experiments and epithelial cell types). MALP-2 activated NF-κB and caused fold-increased levels of proinflammatory mediators with clinically established significance in the cervicovaginal environment (IL-1α, IL-1β, IL-6, TNF-α, IL-8, RANTES, MIP-3α, and ICAM-1), measured by a multiplex electrochemiluminescence assay. At the same time levels of protective anti-inflammatory mediators interleukin 1 receptor antagonist (IL-1RA) and secretory leukocyte protease inhibitor (SLPI), both measured by ELISA, remained constant (IL-1RA) or moderately increased (SLPI). Similarly to MALP-2, colonization by L. jensenii WT activated NF-κB; however, unlike the synthetic TLR2/6 ligand, the live microorganisms did not induce significant changes in the secreted levels across all inflammation-associated proteins. The mCV-N production and function were confirmed by western blot and a HIV-1 gp120 binding assay, respectively. The bioengineered lactobacilli expressed mCV-N with anti-HIV activity preserved in the epithelial cell context and caused no significant immunoinflammatory changes as compared to the WT L. jensenii.ConclusionsThese results highlight the translational value of the colonization model and justify further clinical investigation of the homeostatic and anti-HIV effectiveness of the L. jensenii derivates.

The identification and internal validation of a preoperative serum biomarker panel to determine extracapsular extension in patients with prostate cancer

Accurate preoperative staging of prostate cancer (PCa) is important but current diagnostic methods cannot accurately determine extracapsular extension (ECE), resulting in the possible triage of patients towards a less appropriate arm of therapy. This has consequences to patient care and better methods of preoperatively determining ECE are required. We followed a biomarker development pathway and compared the preoperative serum expressions of VEGF-D, PEDF, IGF-I, IGFBP3, and CD14 in patients from the Irish Prostate Cancer Research Consortium (PCRC) with radical prostatectomy determined ECE against patients with nonECE. The expression measurements of five proteins were fitted into a logistic regression model and backwards variable elimination methods were applied which resulted in a model with IGFBP3 and CD14 as the best combination biomarker panel. This panel was tested in an independent cohort of patients using an optimized multiplex electrochemiluminescence assay. Receiver operating characteristic curves were generated and the areas under the curve (AUC) were calculated as an estimation of prediction accuracy. The biomarker panel was validated with an AUC of 76.6%, and a sensitivity and specificity of 80% and 75% was obtained. This is the first internally validated, preoperative serum biomarker panel that identifies ECE in patients with Gleason score 7 PCa with AUC 76.6%. The panel surpasses the routinely used diagnostic standards in accuracy and may help to improve preoperative cancer staging, better inform treatment options, and improve the referral patterns of patients with urgently treatable cancers towards more appropriate arms of therapy.

Use of elevated baseline IL-8, CCL-2, and TNFα to predict for shorter overall survival (OS) in men with metastatic prostate cancer initiating androgen-deprivation therapy (ADT).

13 Background: Inflammation associated chemokines and cytokines have been shown in preclinical models to lead to the induction of castration resistance. We assessed whether higher blood cytokines and chemokines were associated with shorter time to CRPC and OS in patients initiating ADT for metastatic prostate cancer. Methods: A retrospective analysis of inflammatory markers including IL1β, IL-6, IL-8, TNFα and CCL-2 was undertaken in men initiating ADT for metastatic prostate cancer. Proteins were measured using multiplex electrochemiluminescence assays. The analytical cohort of 122 patients included a prospective trial (50 patients) and chart review (72 patients). A multivariate COX model was used to investigate the relationship between serum markers of inflammation and time to CRPC and OS. The covariates were age at time of ADT, ECOG score, race, baseline PSA, and extent of disease. Results: At time of initiating ADT, the median age of the cohort was 67 years, median PSA was 27.4, 82% had an ECOG PS of 0, 62% had extensive metastatic disease and 91% were Caucasian. Median OS was 42.2 months. The median (med) baseline levels for IL8 was 9.3pg/ml, TNFα was 2.4 pg/ml and CCL-2 was 507.5 pg/ml. As detailed below, patients with elevated IL-8, TNFα and CCL-2 had a significantly shorter OS and showed a non-significant shorter time to CRPC. Conclusions: In men with metastatic prostate cancer starting ADT, higher serum inflammatory markers are associated with poorer OS. This correlative data suggests that anti-inflammatory strategies which improved ADT efficacy in preclinical models may be relevant to the clinical setting. [Table: see text]

Use of elevated insulin-like growth factor binding protein-1 (IGFBP-1) to predict outcomes in men with metastatic prostate cancer treated with androgen-deprivation therapy (ADT).

191 Background: IGF-1 has been shown to promote cancer growth by activation of PI3 kinase and downregulation of AMPK. IGFBP-1 shuttles IGF-1 across blood vessel membranes. Elevated IGFBP-1 blood levels may be indicative of tissue IGF-1 levels and has been associated with prostate cancer risk. We hypothesized that IGF-1 and IGFBP-1 blood levels might effect ADT efficacy. Methods: A retrospective analysis of IGF-1 and IGFBP-1 was undertaken in men commencing ADT for metastatic prostate cancer. Blood was drawn within one month of initiating ADT and stored. Proteins were measured using multiplex electrochemiluminescence assays. A cohort of 122 patients was compiled from a prospective trial (50 patients) and chart review (72 patients). A multivariate analysis (MVA) using a COX model was performed to identify associations between blood levels of the markers and time to the development of castration resistant prostate cancer (CRPC) and overall survival (OS). The covariates analyzed were age at time of ADT, ECOG score, race, baseline PSA, and extent of disease. Results: At time of initiating ADT the cohort had a median age of 67 years, PSA of 27.4 and 82% had ECOG 0. 91% were Caucasian. 62% had extensive metastatic disease. Median OS was 42.2 months. The median (med) baseline levels for IGF-1 was 75.6 ng/ml and for IGFBP-1 was 1844 pg/ml. As detailed in the table, patients with IGFBP-1 levels above the median had significantly shorter time to development of CRPC and OS on MVA. There was no association between IGF-1 levels and time to CRPC development or OS. Conclusions: Elevated levels of IGFBP-1 are associated with shorter duration of ADT efficacy in men with metastatic prostate cancer. This correlative data supports the ongoing SWOG trial assessing whether IGF signaling blockade improves ADT efficacy, as has been seen in preclinical models. [Table: see text]