Plasma Samples Research Articles

Abstract A045: Pan-viral serological characterization in African Americans and European Americans with Hepatocellular Carcinoma

Abstract Hepatocellular carcinoma (HCC) is often diagnosed at late-stages, particularly among African Americans (AA). Poor screening and diagnosis are key factors that contribute to worse prognoses and disproportionately high mortality rates. We propose investigating viral exposure history as a potential useful screening method. Additionally, by accounting for neighborhood deprivation index (NDI), a metric for differences in socio-economic statuses, we can potentially mitigate this disparity. With NDI and viral exposure history, our study aims to assess HCC prognoses in racially disparate populations. We used a technique called VirScan, which conjugates Phage-Immunoprecipitation (PhIP) and DNA-sequencing, to profile the antibody repertoire against a viral epitope library. Analyzing serum and plasma samples from population controls (PC), individuals at high-risk for developing HCC (HR), and HCC cases, we performed VirScan on 524 AA (272 PC, 205 HR, and 47 HCC) and 342 European Americans (EA) (141 PC, 130 HR, and 71 HCC) participants from the NCI-UMD cohort between 2009-2020 from the greater Baltimore area (NCT00913757; clinicaltrials.gov). We used XGBoost, a machine- learning model, to classify viral features that can discriminate HCC cases from PC group. XGBoost predicted 31 important viral features which distinguish HCC from PC. Of the 31 viral features, 6 viruses were different between AA and EA (FDR < 0.05). To determine the extent to which socio-economic factors contribute to the cancer disparities, we gathered variables from the 1999 census tract as contributors to deprivation. We used Principal Component Analysis (PCA) to determine loadings of these contributor variables; composite of the final variables is derived as NDI. Comparing across groups, we find that average NDI is highest in HR group (x = 0.33), followed by HCC group (x = 0.21), then PC group (x = 0.19). We find that NDI for AA is significantly higher in HCC group (p = 1.3e-09), HR group (p < 2.2e-16), and PC group (p = 5.0e-10) when compared to that of EA. Thus, we demonstrate that both the viral exposure history and NDI of an individual might be useful in prognoses of HCC. This approach could be used to examine the disparities we see in both incidence and mortality of HCC between AA and EA. Citation Format: Theressa Ewa, Whitney Leet, Limin Wang, Marshonna Forgues, Xin Wei Wang. Pan-viral serological characterization in African Americans and European Americans with Hepatocellular Carcinoma [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr A045.

Type 2-like polarization and elevated CXCL4 secretion of monocyte derived macrophages upon internalization of plasma-derived exosomes from head and neck cancer patients

BackgroundExosomes are closely associated with different aspects of tumor-progression in patients with head and neck squamous cell carcinoma (HNSCC), such as angiogenesis or immune regulation. As extracellular vesicles they are involved in the intercellular communication by transferring their cargo such as proteins and nucleic acids from one cell to another. However, the influence of tumor related plasma-derived exosomes on the polarization and characteristics of monocyte derived macrophages is not fully understood.MethodsExosomes were isolated from plasma samples of healthy donors (HD) and HNSCC patients and further evaluated with regard to morphology, size and protein composition via transmission electron microscopy, nanoparticle tracking, western blot analysis and cytokine assays. Differentiation and characteristics of monocyte derived macrophages upon exosome internalization were analyzed using flow cytometry and fluorescence microscopy. Macrophage cytokine secretion patterns were analyzed by human cytokine antibody arrays and ELISA measurements.ResultsOur data revealed elevated overall plasma levels of CTLA-4, PD-L1, and TIM-3 as well as elevated exosome-associated CTLA-4, PD-L2, TIM-3, and LAG-3 levels in HNSCC patients compared to HD. Furthermore, we observed a significant type 2-like polarization and elevated CXCL4 secretion of monocyte derived macrophages upon internalization of plasma-derived exosomes from HNSCC patients, which could be visualized by fluorescence microcopy of membrane stained exosomes.ConclusionsThe study provides new insights regarding exosome driven pro-tumorigenic immune regulation in the circulation of patients with head and neck cancer and could help to better understand the individual immunologic situation.

Integrated Ultrasound-Enrichment and Machine Learning in Colorimetric Lateral Flow Assay for Accurate and Sensitive Clinical Alzheimer's Biomarker Diagnosis.

The colloidal gold nanoparticle (AuNP)-based colorimetric lateral flow assay (LFA) is one of the most promising analytical tools for point-of-care disease diagnosis. However, the low sensitivity and insufficient accuracy still limit its clinical application. In this work, a machine learning (ML)-optimized colorimetric LFA with ultrasound enrichment is developed to achieve the sensitive and accurate detection of tau proteins for early screening of Alzheimer's disease (AD). The LFA device is integrated with a portable ultrasonic actuator to rapidly enrich microparticles using ultrasound, which is essential for sample pre-enrichment to improve the sensitivity, followed by ML algorithms to classify and predict the enhanced colorimetric signals. The results of the undiluted serum sample testing show that the protocol enables efficient classification and accurate quantification of the AD biomarker tau protein concentration with an average classification accuracy of 98.11% and an average prediction accuracy of 99.99%, achieving a limit of detection (LOD) as sensitive as 10.30pgmL-1. Further point-of-care testing (POCT) of human plasma samples demonstrates the potential use of LFA in clinical trials. Such a reliable lateral flow immunosensor with high precision and superb sensing performance is expected to put LFA in perspective as an AD clinical diagnostic platform.

The plasma miRNAome in ADNI: Signatures to aid the detection of at-risk individuals.

MicroRNAs are short non-coding RNAs that control proteostasis at the systems level and are emerging as potential prognostic and diagnostic biomarkers for Alzheimer's disease (AD). We performed small RNA sequencing on plasma samples from 847 Alzheimer's Disease Neuroimaging Initiative (ADNI) participants. We identified microRNA signatures that correlate with AD diagnoses and help predict the conversion from mild cognitive impairment (MCI) to AD. Our data demonstrate that plasma microRNA signatures can be used to not only diagnose MCI, but also, critically, predict the conversion from MCI to AD. Moreover, combined with neuropsychological testing, plasma microRNAome evaluation helps predict MCI to AD conversion. These findings are of considerable public interest because they provide a path toward reducing indiscriminate utilization of costly and invasive testing by defining the at-risk segment of the aging population. We provide the first analysis of the plasma microRNAome for the ADNI study. The levels of several microRNAs can be used as biomarkers for the prediction of conversion from MCI to AD. Adding the evaluation of plasma microRNA levels to neuropsychological testing in a clinical setting increases the accuracy of MCI to AD conversion prediction.

Location and function of TDP-43 in platelets, alterations in neurodegenerative diseases and arising considerations for current plasma biobank protocols

The TAR DNA Binding Protein 43 (TDP-43) has been implicated in the pathogenesis of human neurodegenerative diseases and exhibits hallmark neuropathology in amyotrophic lateral sclerosis (ALS). Here, we explore its tractability as a plasma biomarker of disease and describe its localization and possible functions in the cytosol of platelets. Novel TDP-43 immunoassays were developed on three different technical platforms and qualified for specificity, signal-to-noise ratio, detection range, variation, spike recovery and dilution linearity in human plasma samples. Surprisingly, implementation of these assays demonstrated that biobank-archived plasma samples yielded considerable heterogeneity in TDP-43 levels. Importantly, subsequent investigation attributed these differences to variable platelet recovery. Fractionations of fresh blood revealed that ≥ 95% of the TDP-43 in platelet-containing plasma was compartmentalized within the platelet cytosol. We reasoned that this highly concentrated source of TDP-43 comprised an interesting substrate for biochemical analyses. Additional characterization of platelets revealed the presence of the disease-associated phosphoserine 409/410 TDP-43 proteoform and many neuron- and astrocyte-expressed TDP-43 mRNA targets. Considering these striking similarities, we propose that TDP-43 may serve analogous functional roles in platelets and synapses, and that the study of platelet TDP-43 might provide a window into disease-related TDP-43 dyshomeostasis in the central nervous system.

A Randomized, Double-blind, Placebo-controlled, Short-term Monotherapy Study of MK-6186, an HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitor (NNRTI), in Treatment-Naïve HIV-Infected Participants.

To assess the antiviral activity, pharmacokinetics, and safety of MK-6186 in NNRTI-naïve, HIV-1-infected male participants. Double-blind, randomized, two-panel study. In 2 sequential panels, 18 participants received MK-6186 (40 mg [Panel A] or 150 mg [Panel B]) or matching placebo once daily for 7 days. Plasma samples were collected for measurement of HIV-1 RNA levels and MK-6186 pharmacokinetics. For the mean change from baseline in HIV-1 RNA (log10 copies/mL) at 24 hours post Day 7 dose, the mean difference (90% confidence interval) between MK-6186 and placebo was 1.54 (-1.73, -1.34) in the 40-mg group and -1.28 (-1.81, -0.75) in the 150-mg group. One participant in the 150-mg group had viral rebound at 24 hours after Day 6 dosing (Day 7 predose) associated with outgrowth of the V106A minority variant. Ultra-deep sequencing confirmed expansion of this predose minority variant from 0.26% to 63.67%. No outgrowth and rebound was seen in another participant in whom a V106A minority variant was also detected. MK-6186 was generally well tolerated. MK-6186 was rapidly absorbed with peak concentrations at 2 hours followed by a biphasic decline. The effective t½ of MK-6186 was 43.9 to 48.7 hrs. Steady state was not achieved. Daily monotherapy with MK-6186 demonstrated robust antiviral activity with maximal antiviral activity at a dose of 40 mg. One participant in the 150-mg group exhibited viral rebound with outgrowth of the resistant V106A minority variant, demonstrating a risk of resistance development typical of NNRTIs. The reason for this outgrowth remains unclear as no outgrowth occurred in a participant in the 40-mg group in whom the V106A minority variant was also detected. MK-6186 may be an alternative next-generation NNRTI in combination therapy, in that combination antiretroviral therapy could prevent outgrowth of resistant minority variants.

Novel Metabolites Associated with Decreased GFR in Finnish Men: A 12-Year Follow-Up of the METSIM Cohort

Identification of the individuals having impaired kidney function is essential in preventing the complications of this disease. We measured 1009 metabolites at the baseline study in 10,159 Finnish men of the METSIM cohort and associated the metabolites with an estimated glomerular filtration rate (eGFR). A total of 7090 men participated in the 12-year follow-up study. Non-targeted metabolomics profiling was performed at Metabolon, Inc. (Morrisville, NC, USA) on EDTA plasma samples obtained after overnight fasting. We applied liquid chromatography mass spectrometry (LC-MS/MS) to identify the metabolites (the Metabolon DiscoveryHD4 platform). We performed association analyses between the eGFR and metabolites using linear regression adjusted for confounding factors. We found 108 metabolites significantly associated with a decrease in eGFR, and 28 of them were novel, including 12 amino acids, 8 xenobiotics, 5 lipids, 1 nucleotide, 1 peptide, and 1 partially characterized molecule. The most significant associations were with five amino acids, N-acetylmethionine, N-acetylvaline, gamma-carboxyglutamate, 3-methylglutaryl-carnitine, and pro-line. We identified 28 novel metabolites associated with decreased eGFR in the 12-year follow-up study of the METSIM cohort. These findings provide novel insights into the role of metabolites and metabolic pathways involved in the decline of kidney function.

Constitutive innate immune defenses in relation to urbanization and population density in an urban bird, the feral pigeon Columba livia domestica.

Urbanization processes modulate the immunological challenges faced by animals. Urban habitat transformations reshape pathogen diversity and abundance, while high population density-common in urban exploiter species-promotes disease transmission. Responses to urbanization may include adaptive adjustments of constitutive innate immune defenses (e.g. complement system and natural antibodies [NAbs]), which serve as first-line protection against infections. Here, we investigated associations of habitat urbanization and host population density with complement and NAbs in an urban bird, the feral pigeon Columba livia domestica. To do so, we employed the hemolysis-hemagglutination assay to analyze nearly 200 plasma samples collected across urbanization and pigeon population density gradients in five major cities in Poland. We found a negative association between urbanization score and hemagglutination (i.e. NAbs activity), but not hemolysis (i.e. complement activity), indicating either immunosuppression or adaptive downregulation of this immune defense in highly transformed urban landscape. Population density was not significantly related to either immune parameter, providing no evidence for density-dependent modulation of immune defenses. At the same time, there was a negative association of hemolysis with condition (scaled mass index), suggesting resource allocation trade-offs or contrasting effects of the urban environment on immune defenses and body condition. The results demonstrate that habitat structure can be an important factor shaping the immune defenses of the feral pigeon, although these associations were not mediated by variation in population density. Our study highlights the complexity of the links between immune defenses in wildlife and urbanization and reinforces the need for comprehensive ecoimmunological studies on urban animals.

Bioinformatics-driven untargeted metabolomic profiling for clinical screening of methamphetamine abuse.

Amphetamine-type stimulants are very common, and their usage is becoming a very big social problem all over the world. Thousands of addicts encounter several health problems including mental, metabolic, behavioral and neurological disorders. In addition to these, there are several reports about the elevated risk of tendency on committing criminal cases by addicted persons. Hence, methamphetamine addiction is not only an individual health problem but also a social problem. In our study, we aimed to investigate the pathogenesis of chronic usage of methamphetamine via untargeted metabolomics approach. 38 plasma samples were carefully collected and extracted for untargeted metabolomics assay. A liquid-liquid extraction was performed to get as much metabolite as possible from the samples. After the extraction procedure, samples were transferred into vials and they were evaluated via time of flight mass spectrometry instrument. Significantly, altered metabolites were identified by the fold analysis and Welch's test between the groups. 42 different compounds were annotated regarding to data-dependent acquisition method. Pathway analysis were also performed to understand the hazardous effect of methamphetamine on human body. It has been reported that drug exposure may affect several metabolic pathways for amino acids, fats, energy metabolism and vitamins. An alternative bioinformatic model was also developed and validated in order to predict the chronic methamphetamine drug users in any criminal cases. This generated model passes the ROC curve analysis and permutation test and classify the controls and drug users correctly by evaluating the metabolic alterations between the groups.

Comparative evaluation of plasma biomarkers of Schistosoma haematobium infection in endemic populations from Burkina Faso.

Infection with Schistosoma haematobium causes urogenital disease associated with organ disfunction, bleeding, pain, and higher susceptibility to infections and cancer. Timely and accurate diagnosis is crucial for prompt and appropriate treatment as well as surveillance efforts, and the use of plasma biomarkers offers important advantages over parasitological examination of urine, including increased sensitivity and the possibility to use the same specimen for multiple investigations. The present study aims to evaluate the diagnostic performance of different plasma biomarkers in endemic populations from Burkina Faso, West Africa. Schistosoma spp. Circulating Anodic Antigen (CAA), cell free S. haematobium DNA (cfDNA), class M and G antibodies against S. haematobium Soluble Worm Antigen Preparation (SWAP) and Soluble Egg Antigen (SEA) were measured in 406 plasma samples. Results of each biomarker test were compared to those of CAA, a Composite Reference Standard (CRS) and Latent Class Analysis (LCA). An identical proportion of positive samples (29%) was observed as a result of CAA and cfDNA testing, with a substantial agreement (84%, Cohen k = 0.62) between the results of the two tests, and a comparable agreement with the results of CRS and LCA. A higher positivity was observed, as expected, as a result of specific antibody testing (47%-72%), with IgG showing a higher agreement than IgM with the three references. Also, higher IgG levels were observed in current vs past infection, and ROC analysis identified optimal cutoff values for improved testing accuracy. This study provides compelling evidence that can inform the choice of the most appropriate diagnostic plasma biomarker for urogenital schistosomiasis in endemic areas, depending on the purpose, context, and available resources for testing. Either CAA or cfDNA testing can be used for the diagnosis of patients and for epidemiological investigations, even in absence of urine filtration microscopy, whereas anti-SWAP or anti-SEA IgG can be employed for surveillance and integrated monitoring of control interventions against poverty-associated diseases.

Comparison of manual titrations to automated microplate and gel titration assays used in the screening of blood donors for production of Low-Titer O Whole Blood.

The popularity of Low-Titer O Whole Blood (LTOWB) for treating trauma patients requires that donor centers and transfusion services make decisions on what titer testing capabilities to institute and an appropriate titer level threshold. This study compared the titer results determined by four methods to find a rate of agreement. Isohemagglutinin titers were tested on 300 plasma samples utilizing various methods, each determining IgM antibody levels by direct hemagglutination with A1 and B reference cells. The methods used were the Beckman Coulter's PK7300, Immucor's NEO Iris microplate technology, Ortho Clinical Diagnostics (OCD) Vision, and manual titrations. Only 42.7% of the samples tested showed agreement across all methods on ABO isohemaglutinin titer levels and only 32.5% demonstrated "High titer" agreement. Sample agreement was close to 90% if the Immucor method was excluded. At a <1:256 titer level threshold, the pass rate was 94.3% for Immucor, 89.7% for the PK7300, 87.3% for manual testing, and 75.7% for OCD's gel method. Sensitivity and specificity rates at a ± 1 titer level were respectively 100% and 95.4% for OCD's gel, 73.7% and 100% for Immucor, and 100% and 99.6% for the PK7300. Overall method accuracy was 91.7% for Immucor, 90.3% for the PK7300, and 86.7% for OCD's gel method as compared to manual titration. All three automated methods perform comparably to the manual method at a ± 1 titer tolerance level. Based on these comparisons, a titer level of <1:256 would maximize LTOWB production regardless of the method used.

A Multiplexed Quantitative Proteomics Approach to the Human Plasma Protein Signature

Despite the plasma proteome being able to provide a unique insight into the health and disease status of individuals, holding singular promise as a source of protein biomarkers that could be pivotal in the context of personalized medicine, only around 100 proteins covering a few human conditions have been approved as biomarkers by the US Food and Drug Administration (FDA) so far. Mass spectrometry (MS) currently has enormous potential for high-throughput analysis in clinical research; however, plasma proteomics remains challenging mainly due to the wide dynamic range of plasma protein abundances and the time-consuming procedures required. We applied a new MS-based multiplexed proteomics workflow to quantitate proteins, encompassing 67 FDA-approved biomarkers, in &gt;1300 human plasma samples from a clinical cohort. Our results indicate that this workflow is suitable for large-scale clinical studies, showing good accuracy and reproducibility (coefficient of variation (CV) &lt; 20 for 90% of the proteins). Furthermore, we identified plasma signature proteins (stable in time on an individual basis), stable proteins (exhibiting low biological variability and high temporal stability), and highly variable proteins (with low temporal stability) that can be used for personalized health monitoring and medicine.

Autoantibodies to ADAMTS13 in human immunodeficiency virus-associated thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is a potentially fatal thrombotic microangiopathic disorder that can result from human immunodeficiency virus (HIV) infection. The pathogenesis involves a deficiency of the von Willebrand factor (vWF) cleaving protease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin motifs member 13) and the presence of anti-ADAMTS13 autoantibodies. However, there is insufficient information regarding the epitope specificity and reactivity of these autoantibodies. This study aimed to perform epitope-mapping analysis to provide novel insights into the specific epitopes on ADAMTS13 domains affected by autoantibodies. The study analysed 59 frozen citrate plasma samples from HIV-associated TTP patients in South Africa, measuring ADAMTS13 activity using Technozyme® ADAMTS13 activity test, total immunoglobulin (Ig) M and IgA antibodies levels using ELISA kit and purifying IgG antibodies using NAb™ Protein G spin columns. A synthetic ADAMTS13 peptide library was used for epitope mapping. Overall, 90% of samples showed anti-ADAMTS13 IgG autoantibodies, with 64% of these antibodies being inhibitory, as revealed by mixing studies. Samples with ADAMTS13 antigen levels below 5% showed high anti-ADAMTS13 IgG autoantibody titres (≥50 IU/mL), whereas those with 5%-10% levels had low autoantibody titres (<50 IU/mL).The metalloprotease, cysteine-rich and spacer domains were 100% involved in binding anti-ADAMTS13 IgG antibodies, with 58% of samples containing antibodies binding to the C-terminal part of the ADAMTS13 disintegrin-like domain, indicating different pathogenic mechanisms. The metalloprotease, cysteine-rich and spacer domains are the primary targets for anti-ADAMTS13 IgG autoantibodies in patients with HIV-associated TTP. These findings suggest potential effects on the proteolytic activity of ADAMTS13, highlighting the complex nature of the pathogenic mechanisms involved.

Effects of short-term oral letrozole on fresh semen parameters, endocrine balance, and prostate gland dimensions in domestic dogs

BackgroundAromatase inhibitors improve male fertility by modifying the hormonal control of spermatogenesis. The present study aimed to investigate the effects of oral administration of letrozole on testosterone and estradiol concentrations and their ratios in blood serum, seminal plasma, prostatic fluid, sperm quality in fresh semen, and prostate gland dimensions. Seven adult male intact mixed-breed dogs were selected. The animals received letrozole (72 µg/kg, PO) daily for four weeks. Blood samplings and semen collections were carried out on days 0 (control), 14 (treatment), 28 (treatment), and 42 (post-treatment).ResultsOur results showed that letrozole administration resulted in a 4.3 fold significant increase in serum, seminal plasma, and prostatic fluid testosterone levels after 14 days. This remained high until the end of the study. Serum and prostatic fluid estradiol levels did not change significantly over the study period. However, the seminal plasma estradiol level showed a significant increase on day 14. The estradiol: testosterone ratio was significantly reduced on day 14 in serum, seminal plasma, and prostatic fluid samples. Letrozole significantly improved the ejaculated spermatozoa viability and concentration after 28 days of oral administration. However, the sperm plasma membrane functional integrity and kinematic parameters were not significantly affected by the treatment. Transabdominal ultrasound examination revealed a significant increase in the height, width, and volume of the prostate gland after 28 days of treatment.ConclusionsAccording to the present research, oral administration of letrozole for 28 days affects local and systemic sex hormone balance leading to an improvement of the ejaculated canine spermatozoa viability and concentration concurrent with an increase in the prostate gland dimensions.

Physiological responses of Lizard, Toad and Pigeon during rainy season in tropical savanna climate

BackgroundThis study investigated the adaptation mechanisms of Pigeons, Toads and Lizards during the rainy season in tropical savanna climates. Male Agama Lizards (Agama agama), Afep Pigeons (Columba unicincta) and Toads (Sclerophrys perreti) were captured at a location in Ibadan, Oyo state. Five (5) Lizards marked: L1 to L5, Afep Pigeons marked: B1 to B5 and Toads marked: T1 to T5. Each of the animals was manually constrained within six to eight hours of capture, and a 1.0-ml syringe was used to obtain blood from the heart or coccygeal vein. Blood samples were collected in an EDTA and plain bottle, respectively. Plasma and sera samples were separated by centrifugation at − 4 °C using a cold centrifuge and then analysed for creatinine, urea, glucose, Mg+, k+, Cl−, Na+, albumin, TP, ALP, ALT, AST, GGT, MDA, SOD, CAT, GSH and haematological parameters. Cervical dislocation was performed on the animals before organs such as kidneys and liver were collected from each of the animals. Each organ collected from each animal was placed in separate plain tubes (filled with phosphate buffer).ResultThe Pigeon had higher body temperature, urea, glucose, ALP, PCV, haemoglobin concentration, neutrophil and triglyceride compared to the Lizard and Toad, while the Toad had higher Na+, Mg+ and Cl−, basophil and monocytes compared to Pigeon and Lizard and the Lizard has higher creatinine, lymphocyte and cholesterol compared to the Pigeon and Toad. On the liver oxidative stress markers, the Pigeon has higher superoxide dismutase and reduced glutathione compared to the Lizard and Toad, while the Toad has higher MDA and catalase compared to the Pigeon and Lizard. On the kidney oxidative stress markers, the Pigeon has higher MDA compared to the Lizard and Toad, while the Toad has higher catalase than the Pigeon and Lizard.ConclusionsFrom this study, the elevated level of lymphocytes in Lizard and eosinophil, basophil and monocytes in Toad suggests that Lizards and Toad are more vulnerable to inflammation. The high value of cholesterol in Lizard and triglyceride in Pigeon as observed in this study may relate to the degree of stress. Also, the activation of antioxidant systems under comparative study is a part of the survival strategy of animals like amphibians, reptiles and aves when facing environmental problems.

Profiling of metabolic dysregulation in ovarian cancer tissues and biofluids

Ovarian cancer (OC) is the most lethal gynecologic cancer, mainly due to late diagnosis with widespread peritoneal spread at first presentation. We performed metabolomic analyses of ovarian and paired control tissues using capillary electrophoresis-mass spectrometry and liquid chromatography-mass spectrometry to understand its metabolomic dysregulation. Of the 130 quantified metabolites, 96 metabolites of glycometabolism, including glycolysis, tricarboxylic acid cycles, urea cycles, and one-carbon metabolites, showed significant differences between the samples. To evaluate the local and systemic metabolomic differences in OC, we also analyzed low or non-invasively available biofluids, including plasma, urine, and saliva collected from patients with OC and benign gynecological diseases. All biofluids and tissue samples showed consistently elevated concentrations of N1,N12-diacetylspermine compared to controls. Four metabolites, polyamines, and betaine, were significantly and consistently elevated in both plasma and tissue samples. These data indicate that plasma metabolic dysregulation, which the most reflected by those of OC tissues. Our metabolomic profiles contribute to our understanding of metabolomic abnormalities in OC and their effects on biofluids.

Characterizing the Cell-Free Transcriptome in a Humanized Diffuse Large B-Cell Lymphoma Patient-Derived Tumor Xenograft Model for RNA-Based Liquid Biopsy in a Preclinical Setting

The potential of RNA-based liquid biopsy is increasingly being recognized in diffuse large B-cell lymphoma (DLBCL), the most common subtype of non-Hodgkin’s lymphoma. This study explores the cell-free transcriptome in a humanized DLBCL patient-derived tumor xenograft (PDTX) model. Blood plasma samples (n = 171) derived from a DLBCL PDTX model, including 27 humanized (HIS) PDTX, 8 HIS non-PDTX, and 21 non-HIS PDTX non-obese diabetic (NOD)-scid IL2Rgnull (NSG) mice were collected during humanization, xenografting, treatment, and sacrifice. The mice were treated with either rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), CD20-targeted human IFNα2-based AcTaferon combined with CHOP (huCD20-Fc-AFN-CHOP), or phosphate-buffered saline (PBS). RNA was extracted using the miRNeasy serum/plasma kit and sequenced on the NovaSeq 6000 platform. RNA sequencing data of the formalin-fixed paraffin-embedded (FFPE) tissue and blood plasma samples of the original patient were included. Flow cytometry was performed on immune cells isolated from whole blood, spleen, and bone marrow. Bulk deconvolution was performed using the Tabula Sapiens v1 basis matrix. Both R-CHOP and huCD20-Fc-AFN-CHOP were able to control tumor growth in most mice. Xenograft tumor volume was strongly associated with circulating tumor RNA (ctRNA) concentration (p &lt; 0.001, R = 0.89), as well as with the number of detected human genes (p &lt; 0.001, R = 0.79). Abundance analysis identified tumor-specific biomarkers that were dynamically tracked during tumor growth or treatment. An 8-gene signature demonstrated high accuracy for assessing therapy response (AUC 0.92). The tumoral gene detectability in the ctRNA of the PDTX-derived plasma was associated with RNA abundance levels in the patient’s tumor tissue and blood plasma (p &lt; 0.001), confirming that tumoral gene abundance contributes to the cell-free RNA (cfRNA) profile. Decomposing the transcriptome, however, revealed high inter- and intra-mouse variability, which was lower in the HIS PDTX mice, indicating an impact of human engraftment on the stability and profile of cfRNA. Immunochemotherapy resulted in B cell depletion, and tumor clearance was reflected by a decrease in the fraction of human CD45+ cells. Lastly, bulk deconvolution provided complementary biological insights into the composition of the tumor and circulating immune system. In conclusion, the blood plasma-derived transcriptome serves as a biomarker source in a preclinical PDTX model, enables the assessment of biological pathways, and enhances the understanding of cfRNA dynamics.

Rapid assembly of mixed thiols for toll-like receptor-based electrochemical pathogen sensing.

Herein, we describe a rapid and facile fabrication of electrochemical sensors utilizing two different toll-like receptor (TLR) proteins as biorecognition elements to detect bacterial pathogen associated molecular patterns (PAMPs). Using potential-assisted self-assembly, binary mixtures of 11-mercaptoundecanoic acid (MUA) and 6-mercapto-1-hexanol (MCH), or MUA and an in-house synthesized zwitterionic sulfobetaine thiol (DPS) were assembled on a gold working electrode within 5 minutes, which is >200 times shorter than other TLR sensors' preparation time. Electrochemical methods and X-ray photoelectron microscopy were used to characterize the SAM layers. SAMs composed of the betaine terminated thiol exhibited superior resistance to nonspecific interactions, and were used to develop the TLR sensors. Biosensors containing two individually immobilized TLRs (TLR4 and TLR9) were fabricated on separate MUA-DPS SAM modified Au electrodes (MUA-DPS/Au) and tested for their response towards their respective PAMPs. The changes to electron transfer resistance in EIS of the TLR4/MUA-DPS/Au sensor showed a detection limit of 4 ng mL-1 for E. coli 0157:H7 endotoxin (lipopolysaccharide, LPS) and a dynamic range of up to 1000 ng mL-1. The TLR4-based sensor showed negligible response when tested with LPS spiked human plasma samples, showing no interference from the plasma matrix. The TLR9/MUA-DPS/Au sensor responded linearly up to 350 μg mL-1 bacterial DNA, with a detection limit of 7 μg mL-1. The rapid assembly of the TLR sensors, excellent antifouling properties of the mixed SAM assembly, small size and ease of operation of EIS hold great promise for the development of a portable and automated broad-spectrum pathogen detection and classification tool.

Abstract B054: Protease activity as a biomarker and potential target among pancreatic cancer patients

Abstract Background: The pancreas produces digestive proteases, which are often elevated in diseased states and capable of indiscriminate cell surface receptor cleavage. High levels of digestive protease activity have been implicated in the pathophysiology of pancreatic ductal adenocarcinoma (PDAC). We hypothesize that elevated digestive protease activity might downregulate or cleave immune cell receptors from the surface of cancer cells and contribute to the resistance of PDAC to immunotherapy. We have focused initially on major histocompatibility complex (MHC)-1, a ubiquitous receptor essential for antigen presentation, and CD155, an adhesion molecule over-expressed on cancer cells that regulates immune surveillance through binding to the TIGIT and CD226 receptors on leukocytes. Methods: The Rapid Assay for Protease Detection (RAPD) assay is a novel assay developed at our institution that utilizes fluorescent charge-changing peptide substrates to produce a positively charged fluorescent product fragment upon cleavage by the target protease. The RAPD assay was used to analyze protease activity levels in PDAC patients (N=50) and healthy (N=25) plasma samples. We also analyzed plasma samples obtained from an orthotopic KPC-derived mouse model for PDAC. We investigated the effects of specific proteases as well as plasma samples from PDAC and healthy subjects on MHC-1 and CD-155 receptor cleavage in cultured cells in vitro. Utilizing flow cytometry and immunofluorescent imaging, receptor loss was quantified for different proteases and plasma samples. Results: We observed elevated activity levels of digestive protease chymotrypsin as well as cathepsin-S and MMP-2 in the plasma of PDAC patients compared to healthy subjects. Similar patterns of protease activity were seen in plasma from tumor-bearing mice compared to non-tumor-bearing mice. Additionally, our results suggest loss of CD155 and MHC-1 from pancreatic cancer cells when incubated with trypsin and cathepsin-S. Incubation of cells with PDAC plasma also decreases the number of cells expressing MHC-1 and CD155 compared to control plasma, with greater differences observed in plasma with higher protease levels. Higher levels of cleaved CD155 are also detectable by ELISA in human and mouse PDAC plasma than in control plasma. Also, the protease activity (MMP2 and trypsin) correlates with plasma levels of CD155 and PD-L1. Conclusions: These studies begin to elucidate a possible role of digestive proteases in the ability of PDAC to evade the immune system. Ongoing experiments are exploring other immune receptors (including checkpoints) and whether specific protease inhibitors can block immune receptor cleavage. These experiments will inform subsequent experiments combining protease inhibitors with immunotherapy in mouse models. Citation Format: Utsav Joshi, Heather Farris, Jorge De La Torre De La Torre, kim Nguyen-Ta1, Michael Heller, Geert Schmid-Schonbein, Rebekah White. Protease activity as a biomarker and potential target among pancreatic cancer patients [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B054.

Pharmacokinetics and Plasma Protein Binding of Flunixin in Rainbow Trout (Oncorhynchus mykiss).

Flunixin's pharmacokinetics, bioavailability, and plasma protein binding were examined in rainbow trout. The experiment involved 252 rainbow trout (Oncorhynchus mykiss) maintained at 12 ± 0.6°C. Flunixin was administered to rainbow trout via intravascular (IV), intramuscular (IM), and oral routes at a dosage of 2.2 mg/kg. Plasma samples were collected at times 0 (control), 0.25, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 48, 72, and 96 h. High-pressure liquid chromatography-ultraviolet was employed to quantify flunixin concentrations. The elimination half-life (t1/2ʎz) for flunixin was 8.37 h for IV, 8.68 h for IM, and 8.76 h for oral. The t1/2ʎz was similar between administration groups. The volume of distribution at a steady state and total body clearance were 55.81 mL/kg and 6.83 mL/h/kg, respectively, after IV administration. The mean peak plasma concentration was 6.24 ± 0.41 μg/mL at 4 h for oral administration and 13.98 ± 0.86 μg/mL at 2 h for IM administration. The invitro protein binding ratio of flunixin in rainbow trout plasma was 96.34 ± 2.29%. The bioavailability of flunixin after oral (25.74%) administration was lower than that after IM (66.70%) administration. Thus, developing an oral pharmaceutical formulation that can be administered with feed and has high bioavailability could enhance the therapeutic effect.