Matched Serum Research Articles

Is the synovial fluid cobalt-to-chromium ratio related to the serum partitioning of metal debris following metal-on-metal hip arthroplasty?

ObjectivesWe investigated the reliability of the cobalt-chromium (CoCr) synovial joint fluid ratio (JFR) in identifying the presence of a severe aseptic lymphocyte-dominated vasculitis-associated lesion (ALVAL) response and/or suboptimal taper performance (SOTP) following metal-on-metal (MoM) hip arthroplasty. We then examined the possibility that the CoCr JFR may influence the serum partitioning of Co and Cr.MethodsFor part A, we included all revision surgeries carried out at our unit with the relevant data, including volumetric wear analysis, joint fluid (JF) Co and Cr concentrations, and ALVAL grade (n = 315). Receiver operating characteristic curves were constructed to assess the reliability of the CoCr JFR in identifying severe ALVAL and/or SOTP. For part B, we included only patients with unilateral prostheses who had given matched serum and whole blood samples for Co and Cr analysis (n = 155). Multiple regression was used to examine the influence of JF concentrations on the serum partitioning of Co and Cr in the blood.ResultsA CoCr JFR > 1 showed a specificity of 83% (77% to 88%) and sensitivity of 63% (55% to 70%) for the detection of severe ALVAL and/or SOTP. In patients with CoCr JFRs > 1, the median blood Cr to serum Cr ratio was 0.99, compared with 0.71 in patients with CoCr JFRs < 1 (p < 0.001). Regression analysis demonstrated that the blood Cr to serum Cr value was positively associated with the JF Co concentration (p = 0.011) and inversely related to the JF Cr concentration (p < 0.001).ConclusionElevations in CoCr JFRs are associated with adverse biological (severe ALVAL) or tribocorrosive processes (SOTP). Comparison of serum Cr with blood Cr concentrations may be a useful additional clinical tool to help to identify these conditions.Cite this article: D. J. Langton, S. Natu, C. F. Harrington, J. G. Bowsher, A. V. F. Nargol. Is the synovial fluid cobalt-to-chromium ratio related to the serum partitioning of metal debris following metal-on-metal hip arthroplasty? Bone Joint Res 2019;8:146–155. DOI: 10.1302/2046-3758.83.BJR-2018-0049.R1.

Abstract P5-12-07: Expression of adipocyte fatty acid binding protein promotes obesity-associated mammary tumor growth

Abstract Background: The underlying mechanisms that drive obesity-related breast cancer remain unclear. Adipocyte/macrophage fatty acid binding protein (A-FABP) is linked to obesity and breast cancer, while transforming growth factor (TGF)β appears to play a pleiotropic role in breast cancer, suppressing its development but promoting its progression. Whether the proteins work together to drive breast cancer progression is not known. We evaluated the expression of these two markers in matched serum from healthy women and women with breast cancer. Hypothesis: A-FABP and TGFβ drive breast cancer development and progression. Methods: Serum was collected under an institutional review board approved protocol. A-FABP was measured in serum collected from 275 women (92 with breast cancer and 183 without) and TGFβ from 245 matched women (92 with breast cancer and 153 without). A-FABP levels were measured using a human A-FABP4 ELISA kit while TGFβ was measured using human TGFβ ELISA kit. The Kruskal-Wallis test was used to determine if a difference in marker expression existed between women with and without breast cancer, as well as in women with early vs. more advanced breast cancer. Linear mixed effect models were used to examine the relationship between A-FABP and BMI, as well as between TGFβ and BMI, controlling for age, menopause status and a diagnosis of breast cancer. Results:A-FABP expression was higher in obese than lean postmenopausal women, both those with breast cancer (mean : 44.9 vs. 25.1 ng/mL, p=0.002) and without breast cancer (39.4 vs. 26.9 ng/mL, p=0.003). A-FABP expression was also higher in premenopausal obese vs. lean women with cancer (28.9 vs. 12.7 ng/mL, p=0.027), but not in premenopausal healthy women. A-FABP expression was higher in postmenopausal obese vs. lean women with early stage (0-2A) breast cancer (45.6 vs. 21.9 ng/mL, p=0.013) and was inversely associated with HER-2 expression, though being of borderline significance (p=0.060). This was most notable among triple negative vs. ER/PR negative HER2 positive breast cancers. Considering both early and advanced breast cancer, TGFβ expression trended higher in post- than in pre-menopausal obese women with breast cancer (138.9 vs. 68.7 pg/mL, p=0.061), however among premenopausal women with advanced (Stages 2B-3C) disease, TGFβ expression was 5 fold higher in lean than obese individuals (251.7 vs. 48.2 pg/mL, p=0.029) but trended higher in obese vs lean postmenopausal women. TGFβ and A-FABP were found to be significantly associated (ρ=0.14, p=0.024). Impact: Both A-FABP and TGFβ expression are associated with postmenopausal breast cancer among obese women, and their expression in matched samples is significantly associated.. TGFβ is associated with premenopausal advanced breast cancer in premenopausal women. TGFβ is known to induce epithelial mesenchymal transition and may play a role in pregnancy associated breast cancer. Further studies are needed to determine if A-FABP and TGFβ work together in postmenopausal breast cancer. Citation Format: Sauter E, Hao J, Yan X, Kong M, Li B. Expression of adipocyte fatty acid binding protein promotes obesity-associated mammary tumor growth [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-12-07.

HLA-G is upregulated in advanced endometriosis

ObjectiveTo assess whether the HLA-G immunomodulatory protein is potentially involved in the pathophysiology of endometriosis or disease progression. Study designCross-sectional observational study of 227 women who underwent laparoscopy, being 146 for endometriosis excision and 81 for elective tubal ligation (control group). Soluble HLA-G (sHLA-G) levels in the serum and peritoneal fluid (PF), as well as the HLA-G protein expression in matched eutopic and ectopic endometrium of women with and without endometriosis were evaluated by ELISA and immunohistochemistry assays, respectively. Women with endometriosis were separated into groups according to the initial (I/II, n = 60) and advanced (III/IV, n = 86) stages of disease. sHLA-G measurement was performed only in women with matched serum and PF samples in both the control (CTRL; n = 77) and endometriosis (EDT; I–II, n = 60; III–IV, n = 83) groups. HLA-G protein expression was evaluated in 26 women with deep endometriosis (I–II, n = 12; III–IV, n = 14) and 22 controls. ResultsHigher concentrations of sHLA-G (P = 0.013) in the serum but not in the PF were observed in women with advanced endometriosis compared to the control group. In situ expression of HLA-G protein was also higher in ectopic (P = 0.018) but not in eutopic endometrium of women with advanced endometriosis compared to control group. ConclusionOur findings suggest that HLA-G upregulation in advanced stages may contribute to the state of immunosuppression in endometriosis as disease progresses.

Anti-Mycobacterial Antibodies in Paired Cerebrospinal Fluid and Serum Samples from Japanese Patients with Multiple Sclerosis or Neuromyelitis Optica Spectrum Disorder.

Local synthesis of antibodies and presence of oligoclonal bands in the cerebrospinal fluid (CSF) are hallmarks of multiple sclerosis (MS). We investigated the frequency of antibodies against mycobacterial and relevant human epitopes in the CSF of patients with MS or neuromyelitis optica spectrum disorder (NMOSD) and whether these antibodies differed from those present in the serum. Matched serum and CSF samples from 46 patients with MS, 42 patients with NMOSD, and 29 age-matched and sex-matched control subjects were screened retrospectively for the presence of antibodies against Mycobacterium avium subsp. paratuberculosis (MAP) pentapeptide (MAP_5p), MAP_2694295–303, and myelin basic protein (MBP)85–98 peptides by using indirect ELISA. Serum levels of anti-MAP_5p and anti-MAP_2694295–303 antibodies were highly prevalent in patients with MS when compared to patients with NMOSD and controls. Several patients with MS had detectable anti-MAP_5p and anti-MAP_2694295–303 antibodies in the CSF. Furthermore, a group of patients with MS showed intrathecally restricted production of antibodies against these peptides. Women appeared to mount a stronger humoral response to mycobacterial peptides than men. No significant difference in the frequency of anti-MBP85–98 antibodies was found between patients with MS and those with NMOSD. These data highlight the zoonotic potential of MAP, which suggests its involvement in MS etiopathogenesis.

Steroid profiles by liquid chromatography-mass spectrometry of matched serum and single dominant ovarian follicular fluid from women undergoing IVF.

Can IVF outcomes be predicted from the steroid profile generated by liquid chromatography-mass spectrometry (LC-MS/MS) from follicular fluid collected from a single dominant follicle and serum after ovarian stimulation. Prospective observational cohort study in which serum and follicular fluid were collected from women and used to generate steroid profiles by LC-MS/MS. A total of 93 consecutive women enrolled for IVF treatment were recruited at the Fertility Unit, Royal Prince Alfred Women and Babies Hospital, Sydney between September 2014 and July 2015. Baseline and serum levels at oocyte retrieval, as well as follicular fluid samples from the largest single antral follicle, were collected. All samples underwent steroid analysis within a single batch to measure progesterone (P4), oestradiol (E2), oestrone (E1), dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T), dihydrotestosterone (DHT), and 3 α, 5α androstanediol (3α-diol) and 3β, 5α androstanediol (3β-diol). P4, E2, E1, A4, T, DHEA and A4 were detectable in all baseline serum levels, at oocyte retrieval and in follicular fluid samples, whereas DHT, 3α-diol and 3β-diol were only detectable in a minority of samples. The most consistent predictor of pre-transfer (number of follicles >14mm in diameter, oocytes retrieved or fertilized, day-5 blastocysts) outcomes was baseline serum anti-Müllerian hormone. In follicular fluid, E2 was a negative predictor of the number of oocytes retrieved and the number of day-5 blastocysts but no follicular fluid steroids predicted pregnancy outcome. None of the nine steroids measured in follicular fluid predicted pregnancy outcome in women undergoing IVF.

Serum retinyl esters are positively correlated with analyzed total liver vitamin A reserves collected from US adults at time of death

Minimal human data exist on liver vitamin A (VA) compared with serum biomarkers. Cutoffs of 5% and 10% total serum VA as retinyl esters (REs) suggest a VA intoxication diagnosis. We compared total liver VA reserves (TLRs) with the percentage of total serum VA as REs to evaluate hypervitaminosis with the use of US adult autopsy samples. Secondary objectives evaluated serum retinol sensitivity, TLRs among lobes, and hepatic α-retinol concentrations, an α-carotene cleavage product. Matched serum and liver samples were procured from cadavers (n=27; mean ±SD age: 70.7±14.9 y; range: 49-101 y). TLRs and α-REs were quantified by ultra-performance liquid chromatography. Pearson correlations showed liver and serum associations. Sensitivity and specificity were calculated for >5%, 7.5%, and 10% total serum VA as REs to predict TLRs and for serum retinol <0.7 and 1 μmol/L to predict deficiency. Serum RE concentrations were correlated with TLRs (r=0.497, P<0.001). Nine subjects (33%) had hypervitaminosis A (≥1.0 μmol VA/g liver), 2 of whom had >7.5% total serum VA as REs; histologic indicators corroborated toxicity at 3 μmol/g liver. No subject had >10% total serum VA as REs. Serum retinol sensitivity to determine deficiency (TLRs <0.1 μmol VA/g) was 83% at 0.7 and 1 μmol/L. Hepatic α-retinol was positively correlated with age (P=0.047), but removing an outlier nullified significance. This study evaluated serum REs as a biomarker of VA status against TLRs (gold standard), and abnormal histology suggested that 7.5% total serum VA as REs is diagnostic for toxicity at the individual level in adults. The long-term impact of VA supplements and fortificants on VA status is currently unknown. Considering the high prevalence of hypervitaminotic TLRs in this cohort, and given that many countries are adding preformed VA to processed products, population biomarkers diagnosing hypervitaminosis before toxicity are urgently needed. This trial was registered at clinicaltrials.govas NCT03305042.

Validity and prognostic significance of sperm protein 17 as a tumor biomarker for epithelial ovarian cancer: a retrospective study

BackgroundPrior small studies have shown increased expression of sperm protein 17 (Sp17) in epithelial ovarian cancer (EOC) tissue and suggest Sp17 as a potential biomarker for EOC. However, how Sp17 expression varies with histology, grade, and stage of EOC and its expression in other ovarian neoplasms has not been defined. It is unknown whether patients with EOC have elevated serum Sp17 levels or if Sp17 expression is associated with survival outcomes.MethodsThe study included 982 patients with benign, borderline, and malignant ovarian neoplasms and normal ovary. There were 878 patients with tissue only, 39 with serum only, and 65 with matching serum and tissue. Immunohistochemical (IHC) staining with anti-Sp17 antibody was performed on tissue specimens and the intensity scored as weak, moderate, or strong. A sandwich enzyme-linked immunosorbent assay (ELISA) was performed to measure Sp17 sera concentrations.ResultsSp17 expression was most commonly seen in serous cystadenomas (83%) and serous borderline tumors (100%). Of the 773 EOC specimens, 223 (30%) expressed Sp17. Grade and histology were significantly associated with Sp17 expression among EOC specimens (p < 0.001) on both univariate and multivariable analysis, with grade 1 serous adenocarcinomas showing the highest expression (51%). Sp17 expression was limited in other benign and non-epithelial malignant neoplasms. Neither Sp17 tissue expression nor serum concentration correlated with survival outcomes. Serum concentrations were higher in patients with Sp17 tissue expression, and the highest concentrations were noted among patients with serous and clear cell adenocarcinomas.ConclusionsSp17 is highly expressed in benign, borderline, and low grade malignant serous ovarian neoplasms and can be quantified in serum. Sp17 expression may have diagnostic significance in this subset of patients.

Correlation of Lyme Disease-Associated IgG4 Autoantibodies With Synovial Pathology in Antibiotic-Refractory Lyme Arthritis.

To determine whether IgG subclasses of Borrelia burgdorferi antibodies differ from those of 3 Lyme disease (LD)-associated autoantibodies. IgG antibody subclasses were determined by enzyme-linked immunosorbent assay in serum samples from 215 patients with features representative of each of the 3 stages of LD. Antibody and cytokine profiles were measured in matched serum and synovial fluid (SF) samples from patients with Lyme arthritis. Synovial tissue from patients with antibiotic-refractory arthritis was examined for histologic features, IgG subclasses of plasma cells, and messenger RNA (mRNA) subclass expression. B burgdorferi antibodies were primarily of the IgG1 and IgG3 subclasses, and the levels increased as the infection progressed. In contrast, LD-associated autoantibodies were mainly of the IgG2 and IgG4 subclasses, and these responses were found primarily in patients with either antibiotic-refractory or antibiotic-responsive arthritis, particularly in SF. However, compared with the responsive group, the inflammatory milieu in SF in the refractory group was enriched for cytokines representative of innate, Th1, Th2, and Th17 responses. Synovial tissue in a subgroup of patients with refractory arthritis showed marked expression of mRNA for IgG4 antibodies and large numbers of IgG4-staining plasma cells. IgG4 autoantibodies in SF to each of the 3 LD-associated autoantigens correlated with the magnitude of obliterative microvascular lesions and fibrosis in the tissue. Our findings indicate that the subclasses of IgG antibodies to B burgdorferi differ from those of LD-associated autoantibodies. Furthermore, the correlation of IgG4 autoantibodies with specific synovial pathology in the refractory group suggests a role for these autoantibodies, either protective or pathologic, in antibiotic-refractory Lyme arthritis.

Abstract A74: Identification of novel targetable resistance mechanisms and candidate clinical response biomarkers in drug-resistant ovarian cancer, following single-agent and combination chemotherapy

Abstract The aim of our translational research program in Dundee is to develop innovative experimental models to understand the molecular basis of adaptive drug resistance in ovarian cancer patients, extending our findings to the development of novel drug targets, combination chemotherapy approaches, and predictive clinical response biomarkers. Ovarian cancer is a deadly gynecologic malignancy, frequently diagnosed when the disease is already advanced. First-line treatment options in the UK mandate combination carboplatin and paclitaxel chemotherapy, with olaparib and additional PARP inhibitors under evaluation in patients with specific molecular signatures. While treatment is initially effective in many patients, development of drug resistance frequently limits clinical response to chemotherapy. Our understanding of the underlying molecular mechanisms is limited by lack of cell line models that appropriately mimic typical drug choices and commonly prescribed drug combinations in ovarian cancer patients. To address this issue, we have developed a series of novel drug-resistant cell lines, following treatment of A2780 and additional chemotherapy-naïve parental cells with clinically relevant doses of cisplatin (A2780cisR), carboplatin (A2780carboR), paclitaxel (A2780pacR), olaparib (A2780olapR), and combination carboplatin and paclitaxel chemotherapy (A2780cpR), and have used these cell lines to describe a common ABCB1 (MDR1)-mediated drug resistance mechanism common to A2780pacR and A2780olapR cells (Vaidyanathan et al., Br J Cancer 2016;115:431-41). More recently, we have used RNASeq (Ion Ampliseq Targeted Sequencing Technology) and Reverse Phase Protein Array (RPPA) analysis to compare the expression of whole-genome mRNA and more than 300 protein and phosphoprotein targets in pairwise comparisons of drug-sensitive and drug-resistant cells—unsupervised hierarchical clustering of these datasets revealed marked differences in expression between paclitaxel, olaparib, and platinum drug-resistant cells, and significant differences in cisplatin and carboplatin-selected cells. Consistent with improved clinical response, our combination A2780cpR line has a unique phenotype, which is distinct from both A2780carboR and A2780pacR cells. Importantly, we have identified significant increases in the expression of targetable phosphoproteins, for example, pATM (Ser1981), pAKT (Tyr308 and Ser473), pCHK1 (Ser296), and pGSK3b (Ser9/Ser21) in A2780cpR cells. In complementary experiments, we have used Taqman Low Density Arrays (TLDAs) to compare the expression of 377 miRNAs in our cell line panel, and have identified multiple differentially expressed miRNAs, both common to all resistant cells and unique to individual drug-resistant phenotypes. To validate our prioritized candidate phosphoprotein targets and miRNA biomarkers, we have created a unique clinical study, the Dundee Ovarian Cancer Study (DOCS), in which we are creating serial ascites-derived primary cell lines from initially drug-sensitive and subsequently drug-resistant ovarian cancer patients. We are currently using MTT cytotoxicity and colony formation assays in these cell lines to evaluate combination chemotherapy approaches with pathway specific inhibitors to each of our targetable resistance mechanisms, and developing quantitative PCR and immunohistochemical-based methods for evaluation of candidate drug resistance biomarkers in matched serum and plasma samples from DOCS patients. Our combined laboratory and clinical studies therefore provide a “bench to bedside” approach to molecular characterization and prioritization of novel strategies to combat drug resistance, the major treatment-limiting complication in the clinical management of ovarian cancer patients. Citation Format: Aparajitha Vaidyanathan, Lynne Sawers, Probir Chakravarty, Susan E. Bray, K. Wendy McMullen, Michelle J. Ferguson, Gillian Smith. Identification of novel targetable resistance mechanisms and candidate clinical response biomarkers in drug-resistant ovarian cancer, following single-agent and combination chemotherapy. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A74.

In Situ Humoral Immunity to Vimentin in HLA-DRB1*03+ Patients With Pulmonary Sarcoidosis.

Vimentin has been implicated in pulmonary sarcoidosis as a T-cell autoantigen, particularly in the context of HLA-DRB1*03, the Vα2.3/Vβ22 T-cell receptor (TCR), and Löfgren’s syndrome. As vimentin is a known antigenic target in B-cell-mediated autoimmunity, we investigated in situ humoral anti-vimentin responses in pulmonary sarcoidosis and their relationship with HLA-DRB1*03. Sarcoid and healthy control (HC) lung biopsies were analyzed by multi-color confocal microscopy for B-cells, T-cells, proliferation, and vimentin, and compared to tonsillectomy tissue. Bronchoalveolar lavage fluid (BALF) and serum from 48 sarcoidosis patients and 15 healthy volunteers were typed for HLA-DRB1*03 and titrated for antibodies to full-length vimentin, vimentin truncations, and total IgG and IgA by ELISA. Presence of extracellular vimentin in BALF was determined by mass spectrometry and T-cell populations measured by flow cytometry. Sarcoid lung samples, especially from HLA-DRB1*03+ patients, contained vimentin-rich tertiary lymphoid structures and corresponding BALF was highly enriched for both IgG and IgA anti-vimentin antibody (AVA) titers. Furthermore, sarcoidosis patient BALF AVA concentrations (expressed as arbitrary units per milligram of total immunoglobulin isotype) correlated with the percentage of CD4+ T-cells expressing the Vα2.3/Vβ22 TCR. BALF antibody reactivity to the vimentin N-terminus was most prominent in HCs, whereas reactivity to the C-terminus (VimC-term) was enriched in the sarcoid lung. Specifically, HLA-DRB1*03+ patient BALF contained higher concentrations of anti-VimC-term antibodies than BALF from both HCs and HLA-DRB1*03− patients. Consistent with the lung as a site of AVA production, the concentration of AVAs in BALF was dramatically higher than in matched serum samples. Overall, there was a poor correlation between BALF and serum AVA concentrations. Together, these studies reveal the presence of linked in situ recognition of vimentin by both T- and B-cells in HLA-DRB1*03+ sarcoidosis patients, associated with a selective humoral immune response to the vimentin C-terminus.

A fluid transition into the field

Diagnostics Many point-of-care diagnostics rely on lateral flow assays or microfluidics; however, these methods generally cannot test multiple samples simultaneously. Ng et al. optimized inkjet-printed digital microfluidic cartridges and a portable control system to perform serological immunoassays in remote settings. Digital microfluidics use electrostatic forces to mix and separate reagents and samples in small droplets of fluids. The system measured immunoglobulin G (IgG) antibodies for measles and rubella in human blood samples obtained from adults and children on site in a refugee camp in Kenya. Four samples could be tested simultaneously, although digital microfluidic IgG detection was less sensitive and specific than laboratory-based ELISA (enzyme-linked immunosorbent assay) testing of matched serum samples. The emergence of this field-compatible technology brings with it tools for advancing global health. Sci. Transl. Med. 10 , aar6076 (2018).

Perfluoroalkyl and polyfluoroalkyl substances in matched parental and cord serum in Shandong, China

BackgroundPerfluoroalkyl and polyfluoroalkyl substances (PFASs) have been widely detected in different populations. However, limited data is available about exposure among family members. ObjectivesTo investigate the PFASs levels in parents and their newborns and to understand their correlation and health implications of in utero exposure to PFASs. MethodsTen PFASs were measured in matched parental and cord serum (N = 369 families) from a birth cohort in Shandong, one of the regions seriously polluted by PFASs in China. The correlation of PFASs levels within families was examined. A questionnaire survey on maternal factors and risk assessment using the hazard quotients (HQs) approach based on maternal PFASs levels was conducted. ResultsWithin a family, the father had the highest levels of all PFASs. Among the 10 PFASs, perfluorooctanoic acid (PFOA) was the highest, with 103.38, 42.83, and 34.67 ng/ml in paternal, maternal and cord serum, respectively. PFASs levels were positively correlated among family members (r = 0.14–0.91, p < 0.01). Maternal age, body mass index (BMI); smoking history; and intake of fish, milk, poultry, vegetables and tap water were significantly related to PFASs concentrations in cord serum. Twenty-seven (7.3%) HQ values exceeded 1 for both PFOA and the sum of PFOA and perfluorooctane sulfonate (PFOS), indicating potential concern for developmental toxicity in the local newborns. ConclusionsPFASs, and especially PFOA levels were extremely high and positively correlated between parents, indicating heavy pollution in this region and common sources of exposure. In utero exposure to PFASs might pose potential concern for developmental toxicity in the local newborns.

Isolation and characterization of pathogenic leptospires associated with cattle

Pathogenic leptospires colonize the renal tubules of reservoir hosts of infection, including cattle, and are excreted via urine. In order to identify circulating serovars of pathogenic leptospires in beef cattle, and their associated rates of urinary excretion, a cross sectional study was performed. Fifty urine samples were collected one day each month over 12 consecutive months (N = 600), directly from the bladder of beef cattle at a single slaughter facility and assessed for the presence of leptospires by culture and the fluorescent antibody test (FAT). Where possible, a matched serum sample was also collected for the microscopic agglutination test (MAT). Forty-three urine samples were either culture positive or FAT positive, indicating that 7.2% of sampled beef cattle were actively excreting leptospires in urine. Twenty-three urine samples were culture positive. Sequence analysis of 16S ribosomal DNA and secY indicated that all isolates were Leptospira borgpetersenii. Typing by serology indicated that all isolates were serogroup Sejroe. An overall seroprevalence of 20% (MAT ≥ 1:25) was determined; positive bovine sera was most reactive to serogroup Sejroe (serovar Hardjo) (8.1%), and serogroup Australis (serovar Bratislava) (6.7%). There was poor correlation between seroprevalence and excretion of leptospires since 18/43 (41.9%) cattle, which were positive by culture or FAT, were seronegative. The virulence of two selected isolates of L. borgpetersenii was confirmed by experimental infection in small animal models of infection. Results confirm that L. borgpetesenii continues to circulate in beef cattle and that multiple diagnostic assays are required to detect active shedding. These findings also highlight beef cattle as a reservoir host for the potential zoonotic transmission of leptospires.

Validation of a novel, rapid, high precision sclerostin assay not confounded by sclerostin fragments.

Sclerostin is a 190 amino acid protein secreted primarily by osteocytes. It was initially identified due to mutations in the SOST gene associated with high bone mass phenotypes. Much recent work has sought to determine the importance of sclerostin across an array of conditions which affect the human skeleton. However, accurate measurement of sclerostin from serum and plasma sources remains a significant impediment, with currently available commercial assays showing marked differences in measured sclerostin values. Accordingly, sclerostin assay standardization remains an important but unmet need before sclerostin measurements can be used for the clinical management of bone disease. Here we characterize a novel automated chemiluminescent sclerostin assay (LIAISON®, DiaSorin) which overcomes many of these limitations. Important assay characteristics include: a wide dynamic range (50-6500pg/mL); high intra- (<2.5%) and inter- (<5%) assay precision; matched serum and plasma equivalence (<10% difference); specificity for the intact sclerostin molecule; and rapid assay results. Serum sclerostin levels measured with the LIAISON® assay in a population-based sample of adult men (n=278) and women (n=348) demonstrated that sclerostin levels were significantly higher in men as compared to women and were positively associated with age in both sexes, consistent with previously published work. In postmenopausal women, serum sclerostin levels measured with the LIAISON® assay were reduced in response to treatment with either estrogen or teriparatide, again consistent with previous findings. Collectively, the above data demonstrate that the LIAISON® sclerostin assay provides a reliable tool for more confident assessment of emergent mechanisms wherein sclerostin may impact a number of bone related pathologies.

Proteomic Characterization of Dermal Interstitial Fluid Extracted Using a Novel Microneedle-Assisted Technique.

As wearable fitness devices have gained commercial acceptance, interest in real-time monitoring of an individual's physiological status using noninvasive techniques has grown. Microneedles have been proposed as a minimally invasive technique for sampling the dermal interstitial fluid (ISF) for clinical monitoring and diagnosis, but little is known about its composition. In this study, a novel microneedle array was used to collect dermal ISF from three healthy human donors and compared with matching serum and plasma samples. Using a shotgun quantitative proteomic approach, 407 proteins were quantified with at least one unique peptide, and of those, 135 proteins were differently expressed at least 2-fold. Collectively, these proteins tended to originate from the cytoplasm, membrane bound vesicles, and extracellular vesicular exosomes. Proteomic analysis confirmed previously published work that indicates that ISF is highly similar to both plasma and serum. In this study, less than one percent of proteins were uniquely identified in ISF. Taken together, ISF could serve as a minimally invasive alternative for blood-derived fluids with potential for real-time monitoring applications.

Profiling microRNAs in individuals at risk of progression to rheumatoid arthritis

BackgroundIndividuals at risk of rheumatoid arthritis (RA) demonstrate systemic autoimmunity in the form of anti-citrullinated peptide antibodies (ACPA). MicroRNAs (miRNAs) are implicated in established RA. This study aimed to (1) compare miRNA expression between healthy individuals and those at risk of and those that develop RA, (2) evaluate the change in expression of miRNA from “at-risk” to early RA and (3) explore whether these miRNAs could inform a signature predictive of progression from “at-risk” to RA.MethodsWe performed global profiling of 754 miRNAs per patient on a matched serum sample cohort of 12 anti-cyclic citrullinated peptide (CCP) + “at-risk” individuals that progressed to RA. Each individual had a serum sample from baseline and at time of detection of synovitis, forming the matched element. Healthy controls were also studied. miRNAs with a fold difference/fold change of four in expression level met our primary criterion for selection as candidate miRNAs. Validation of the miRNAs of interest was conducted using custom miRNA array cards on matched samples (baseline and follow up) in 24 CCP+ individuals; 12 RA progressors and 12 RA non-progressors.ResultsWe report on the first study to use matched serum samples and a comprehensive miRNA array approach to identify in particular, three miRNAs (miR-22, miR-486-3p, and miR-382) associated with progression from systemic autoimmunity to RA inflammation. MiR-22 demonstrated significant fold difference between progressors and non-progressors indicating a potential biomarker role for at-risk individuals.ConclusionsThis first study using a cohort with matched serum samples provides important mechanistic insights in the transition from systemic autoimmunity to inflammatory disease for future investigation, and with further evaluation, might also serve as a predictive biomarker.

Abstract 3504: High expression of lncRNA-ATB and miR-21 as a biomarker in human hepatocellular carcinoma

Abstract Non-coding RNAs (ncRNAs) including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) have emerged as novel gene-regulators. The aim of this study was to determine the clinical relevancies of 2 lncRNAs (lncRNA-ATB and MEG3) and 2 miRNAs (miR-21 and miR-31) in hepatocellular carcinoma (HCC). We analyzed clinical specimens from 100 pairs of HCC and matched normal liver (NL) tissues. MiRNAs and lncRNAs expression levels were evaluated by quantitative real-time PCR. In addition, we determined the clinical significance of the ncRNAs expression in matching tissue and serum exosome samples from HCC patients. MiR-21 significantly increased in HCC (P&amp;lt;0.01) compared to corresponding NL tissues. LncRNA-ATB was significantly up-regulated in HCC (P&amp;lt;0.05); however, lncRNA-MEG3 was down-regulated in HCC (P&amp;lt;0.05) tissues. In addition, expression of miR-21 and lncRNA-ATB was significantly associated with the tumor size and poor prognosis of HCC patients. More importantly, expression of serum exosomal lncRNA-ATB and miR-21 was more significantly associated with HCC progression and poor patients survival compared to its’ tissue expression. We conclude that non-coding RNAs (lncRNA-ATB and miR-21) expression has the potential to serve as biomarkers for prognosis and targeted therapy in HCC patients. Citation Format: Eunhye Lee, Gyeonghwa Kim, Se Young Jang, Yong-Hun Choi, Yu Rim Lee, Won Young Tak, Young Oh Kweon, Soo Young Park, Keun Hur. High expression of lncRNA-ATB and miR-21 as a biomarker in human hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3504. doi:10.1158/1538-7445.AM2017-3504

KRAS and BRAF mutations in serum exosomes from patients with colorectal cancer in a Chinese population.

The efficacy of epidermal growth factor receptor- targeted therapy is significantly associated with Kirsten rat sarcoma viral oncogene homolog (KRAS) and B-raf serine/threonine kinase proto-oncogene (BRAF) mutation in patients with colorectal cancer (CRC), for which the standard gene testing is currently performed using tumor tissue DNA. The aim of the present study was to compare the presence of KRAS and BRAF mutations in the serum exosome and primary tumor tissue from patients with CRC. Genomic DNA were extracted from the tumor tissues of 35 patients with histologically-confirmed CRC and exosomal mRNA were obtained from peripheral blood, which were collected from the corresponding patients prior to surgery. Three mutations in the KRAS gene (codons 12, 13 and 61) and a mutation in the BRAF gene (codon 600) were detected using a polymerase chain reaction-based sequencing method and their presence were compared between tumor tissues and the matched serum exosomes. The KRAS mutation rates in tumor tissues and the matched serum exosomes were 57.6 and 42.4%, respectively, which was not significantly different (P=0.063). The detection rate of the BRAF mutation was 24.2 and 18.2% in tumor tissues and the matched serum exosomes, respectively, and there was no significant difference (P=0.500). The patients with CRC that had a KRAS mutation of codon 12 in exon 2 in their tumor tissues and serum exosomes were significantly older compared with those without this mutation (tumor tissue, P=0.002; serum exosome, P=0.022). The sensitivity of KRAS and BRAF mutation detection using exosomal mRNA was 73.7 and 75%, respectively. The specificity of the detected mutations exhibited an efficiency of 100%, and the total consistency rate was 94.9 and 93.9% for KRAS and BRAF mutations, respectively. These results suggested that serum exosomal mRNA may be used as a novel source for the rapid and non-invasive genotyping of patients with CRC.

Immunoassay for Detection of Infliximab in Whole Blood Using a Fiber-Optic Surface Plasmon Resonance Biosensor.

Monitoring the concentration of a therapeutic drug antibody, infliximab (IFX), is recommended for enhancing its efficacy in patients with inflammatory bowel disease (IBD). However, IFX concentrations are currently determined in patients' serum/plasma, which requires sample preparation from blood, hence hampering the turnaround time. In this paper, we present a short immunoassay (10 min) using a fiber-optic surface plasmon resonance (FO-SPR) biosensor for detection of IFX spiked in 100-fold diluted serum, plasma, and whole blood. The calculated limits of detection (LOD) based on calibration curves were 1.42, 1.00, and 1.34 ng/mL, respectively, which coincides with expected IFX concentrations in diluted samples from IBD patients. A linear correlation was established among different matrixes, indicating that the matrix effect was insignificant. The established point-of-care (POC) FO-SPR bioassay was also used to measure IFX in 100-fold diluted extracts of dried blood spots (DBS), and LOD achieved was below 2 ng/mL. Although DBS might be ideal for POC, this is the first report of using an SPR biosensor for measuring DBS samples. Finally, the POC FO-SPR immunoassay was validated by using matching serum and plasma samples from five IBD patients. A Pearson correlation of 0.968 was obtained between serum and plasma samples. IFX concentrations determined with FO-SPR were compared to a clinically validated enzyme-linked immunosorbent assay (ELISA), resulting in excellent Pearson correlation and intraclass correlation coefficient, both being 0.99 for serum and plasma samples. In conclusion, this paper demonstrates that our FO-SPR biosensor can be used as a true POC diagnostic tool for determining IFX concentrations in a variety of matrixes.

Comparing identified and statistically significant lipids and polar metabolites in 15-year old serum and dried blood spot samples for longitudinal studies.

The use of dried blood spots (DBS) has many advantages over traditional plasma and serum samples such as the smaller blood volume required, storage at room temperature, and ability to sample in remote locations. However, understanding the robustness of different analytes in DBS samples is essential, especially in older samples collected for longitudinal studies. Here we analyzed the stability of polar metabolites and lipids in DBS samples collected in 2000-2001 and stored at room temperature. The identified and statistically significant molecules were then compared to matched serum samples stored at -80°C to determine if the DBS samples could be effectively used in a longitudinal study following metabolic disease. A total of 400 polar metabolites and lipids were identified in the serum and DBS samples using gas chromatograph/mass spectrometry (GC/MS), liquid chromatography (LC)/MS, and LC/ion mobility spectrometry-MS (LC/IMS-MS). The identified polar metabolites overlapped well between the sample types, though only one statistically significant metabolite was conserved in a case-control study of older diabetic males with low amounts of high-density lipoproteins and high body mass indices, triacylglycerides and glucose levels when compared to non-diabetic patients with normal levels, indicating that degradation in the DBS samples affects polar metabolite quantitation. Differences in the lipid identifications indicated that some oxidation occurs in the DBS samples. However, 36 statistically significant lipids correlated in both sample types. The difference in the number of statistically significant polar metabolites and lipids indicated that the lipids did not degrade to as great of a degree as the polar metabolites in the DBS samples and lipid quantitation was still possible. Copyright © 2016 John Wiley & Sons, Ltd.