Whole blood coagulation in an ex vivo thrombus is sufficient to induce clot neutrophils to adopt a myeloid-derived suppressor cell signature and shed soluble Lox-1.

BACKGROUND: Blood clots are living tissues that release inflammatory mediators including IL-8/CXCL8 and MCP-1/CCL2. A deeper understanding of blood clots is needed to develop new therapies for prothrombotic disease states and regenerative medicine. OBJECTIVES: To identify a common transcriptional shift in cultured blood clot leukocytes. METHODS: Differential gene expression of whole blood and cultured clots (4 hours at 37 °C) was assessed by RNA sequencing (RNAseq), reverse transcriptase-polymerase chain reaction, proteomics, and histology (23 diverse healthy human donors). Cultured clot serum bioactivity was tested in endothelial barrier functional assays. RESULTS: All cultured clots developed a polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) signature, including up-regulation of OLR1 (mRNA encoding lectin-like oxidized low-density lipoprotein receptor 1 [Lox-1]), IL-8/CXCL8, CXCL2, CCL2, IL10, IL1A, SPP1, TREM1, and DUSP4/MKP. Lipopolysaccharide enhanced PMN-MDSC gene expression and specifically induced a type II interferon response with IL-6 production. Lox-1 was specifically expressed by cultured clot CD15+ neutrophils. Cultured clot neutrophils, but not activated platelets, shed copious amounts of soluble Lox-1 (sLox-1) with a donor-dependent amplitude. sLox-1 shedding was enhanced by phorbol ester and suppressed by heparin and by beta-glycerol phosphate, a phosphatase inhibitor. Cultured clot serum significantly enhanced endothelial cell monolayer barrier function, consistent with a proresolving bioactivity. CONCLUSION: This study suggests that PMN-MDSC activation is part of the innate immune response to coagulation which may have a protective role in inflammation. The cultured blood clot is an innovative thrombus model that can be used to study both sterile and nonsterile inflammatory states and could be used as a personalized medicine tool for drug screening.

Pilot clinical trial and phenotypic analysis in chemotherapy-pretreated, metastatic triple-negative breast cancer patients treated with oral TAK-228 and TAK-117 (PIKTOR) to increase DNA damage repair deficiency followed by cisplatin and nab paclitaxel.

BACKGROUND: A subset of triple-negative breast cancers (TNBCs) have homologous recombination deficiency with upregulation of compensatory DNA repair pathways. PIKTOR, a combination of TAK-228 (TORC1/2 inhibitor) and TAK-117 (PI3Kα inhibitor), is hypothesized to increase genomic instability and increase DNA damage repair (DDR) deficiency, leading to increased sensitivity to DNA-damaging chemotherapy and to immune checkpoint blockade inhibitors. METHODS: 10 metastatic TNBC patients received 4 mg TAK-228 and 200 mg TAK-117 (PIKTOR) orally each day for 3 days followed by 4 days off, weekly, until disease progression (PD), followed by intravenous cisplatin 75 mg/m2 plus nab paclitaxel 220 mg/m2 every 3 weeks for up to 6 cycles. Patients received subsequent treatment with pembrolizumab and/or chemotherapy. Primary endpoints were objective response rate with cisplatin/nab paclitaxel and safety. Biopsies of a metastatic lesion were collected prior to and at PD on PIKTOR. Whole exome and RNA-sequencing and reverse phase protein arrays (RPPA) were used to phenotype tumors pre- and post-PIKTOR for alterations in DDR, proliferation, and immune response. RESULTS: With cisplatin/nab paclitaxel (cis/nab pac) therapy post PIKTOR, 3 patients had clinical benefit (1 partial response (PR) and 2 stable disease (SD) ≥ 6 months) and continued to have durable benefit in progression-free survival with pembrolizumab post-cis/nab pac for 1.2, 2, and 3.6 years. Their post-PIKTOR metastatic tissue displayed decreased mismatch repair (MMR), increased tumor mutation burden, and significantly lower levels of 53BP1, DAG Lipase ß, GCN2, AKT Ser473, and PKCzeta Thr410/403 compared to pre-PIKTOR tumor tissue. CONCLUSIONS: Priming patients' chemotherapy-pretreated metastatic TNBC with PIKTOR led to very prolonged response/disease control with subsequent cis/nab pac, followed by pembrolizumab, in 3 of 10 treated patients. Our multi-omics approach revealed a higher number of genomic alterations, reductions in MMR, and alterations in immune and stress response pathways post-PIKTOR in patients who had durable responses. TRIAL REGISTRATION: This clinical trial was registered on June 21, 2017, at ClinicalTrials.gov using identifier NCT03193853.

Drug discovery efforts at George Mason University.

With over 39,000 students, and research expenditures in excess of $200 million, George Mason University (GMU) is the largest R1 (Carnegie Classification of very high research activity) university in Virginia. Mason scientists have been involved in the discovery and development of novel diagnostics and therapeutics in areas as diverse as infectious diseases and cancer. Below are highlights of the efforts being led by Mason researchers in the drug discovery arena. To enable targeted cellular delivery, and non-biomedical applications, Veneziano and colleagues have developed a synthesis strategy that enables the design of self-assembling DNA nanoparticles (DNA origami) with prescribed shape and size in the 10 to 100 nm range. The nanoparticles can be loaded with molecules of interest such as drugs, proteins and peptides, and are a promising new addition to the drug delivery platforms currently in use. The investigators also recently used the DNA origami nanoparticles to fine tune the spatial presentation of immunogens to study the impact on B cell activation. These studies are an important step towards the rational design of vaccines for a variety of infectious agents. To elucidate the parameters for optimizing the delivery efficiency of lipid nanoparticles (LNPs), Buschmann, Paige and colleagues have devised methods for predicting and experimentally validating the pKa of LNPs based on the structure of the ionizable lipids used to formulate the LNPs. These studies may pave the way for the development of new LNP delivery vehicles that have reduced systemic distribution and improved endosomal release of their cargo post administration. To better understand protein-protein interactions and identify potential drug targets that disrupt such interactions, Luchini and colleagues have developed a methodology that identifies contact points between proteins using small molecule dyes. The dye molecules noncovalently bind to the accessible surfaces of a protein complex with very high affinity, but are excluded from contact regions. When the complex is denatured and digested with trypsin, the exposed regions covered by the dye do not get cleaved by the enzyme, whereas the contact points are digested. The resulting fragments can then be identified using mass spectrometry. The data generated can serve as the basis for designing small molecules and peptides that can disrupt the formation of protein complexes involved in disease processes. For example, using peptides based on the interleukin 1 receptor accessory protein (IL-1RAcP), Luchini, Liotta, Paige and colleagues disrupted the formation of IL-1/IL-R/IL-1RAcP complex and demonstrated that the inhibition of complex formation reduced the inflammatory response to IL-1B. Working on the discovery of novel antimicrobial agents, Bishop, van Hoek and colleagues have discovered a number of antimicrobial peptides from reptiles and other species. DRGN-1, is a synthetic peptide based on a histone H1-derived peptide that they had identified from Komodo Dragon plasma. DRGN-1 was shown to disrupt bacterial biofilms and promote wound healing in an animal model. The peptide, along with others, is being developed and tested in preclinical studies. Other research by van Hoek and colleagues focuses on in silico antimicrobial peptide discovery, screening of small molecules for antibacterial properties, as well as assessment of diffusible signal factors (DFS) as future therapeutics. The above examples provide insight into the cutting-edge studies undertaken by GMU scientists to develop novel methodologies and platform technologies important to drug discovery.

Laser Capture Proteomics: spatial tissue molecular profiling from the bench to personalized medicine.

INTRODUCTION: Laser Capture Microdissection (LCM) uses a laser to isolate, or capture, specific cells of interest in a complex heterogeneous tissue section, under direct microscopic visualization. Recently, there has been a surge of publications using LCM for tissue spatial molecular profiling relevant to a wide range of research topics. AREAS COVERED: We summarize the many advances in tissue Laser Capture Proteomics (LCP) using mass spectrometry for discovery, and protein arrays for signal pathway network mapping. This review emphasizes: a) transition of LCM phosphoproteomics from the lab to the clinic for individualized cancer therapy, and b) the emerging frontier of LCM single cell molecular analysis combining proteomics with genomic, and transcriptomic analysis. The search strategy was based on the combination of MeSH terms with expert refinement. EXPERT OPINION: LCM is complemented by a rich set of instruments, methodology protocols, and analytical A.I. (artificial intelligence) software for basic and translational research. Resolution is advancing to the tissue single cell level. A vision for the future evolution of LCM is presented. Emerging LCM technology is combining digital and AI guided remote imaging with automation, and telepathology, to a achieve multi-omic profiling that was not previously possible.

Encouraging long-term survival following autophagy inhibition using neoadjuvant hydroxychloroquine and gemcitabine for high-risk patients with resectable pancreatic carcinoma.

INTRODUCTION: Preoperative autophagy inhibition with hydroxychloroquine (HCQ) in combination with gemcitabine in pancreatic adenocarcinoma (PDAC) has been shown to be safe and effective in inducing a serum biomarker response and increase resection rates in a previous phase I/II clinical trial. We aimed to analyze the long-term outcomes of preoperative HCQ with gemcitabine for this cohort. METHODS: A review of patients enrolled between July 2010 and February 2013 in the completed phase I/II single arm (two doses of fixed-dose gemcitabine (1500 mg/m2 ) in combination with oral hydroxychloroquine administered for 31 consecutive days until the day of surgery for high-risk pancreatic cancer) was undertaken. Progression-free survival (PFS) and overall survival analysis (OS) using Kaplan-Meier estimates were performed. RESULTS: Of 35 patients initially enrolled, 29 patients underwent surgical resection (median age at diagnosis: 62 years, 45% females). Median duration of follow-up was 7.5 years. There was a median 15% decrease in the serum CA19-9 levels following completion of neoadjuvant therapy and 83% of the cohort underwent a pancreaticoduodenectomy, 7 (24%) patients had a concomitant venous resection. On histopathology, 14 (48%) patients had at least a partial treatment response. The median PFS and OS were 11 months (95% Confidence interval [CI]: 7-28) and 31 months (95% CI: 13-47), respectively, while 9 (31%) patients survived beyond 5 years from diagnosis; a rate that compares very favorably with contemporaneous series. CONCLUSION: Compared to historical data, neoadjuvant autophagy inhibition with HCQ plus gemcitabine is associated with encouraging long-term survival for patients with PDAC.

Local production of lactate, ribose phosphate, and amino acids within human triple-negative breast cancer.

BACKGROUND: Upregulated glucose metabolism is a common feature of tumors. Glucose can be broken down by either glycolysis or the oxidative pentose phosphate pathway (oxPPP). The relative usage within tumors of these catabolic pathways remains unclear. Similarly, the extent to which tumors make biomass precursors from glucose, versus take them up from the circulation, is incompletely defined. METHODS: We explore human triple negative breast cancer (TNBC) metabolism by isotope tracing with [1,2-13C]glucose, a tracer that differentiates glycolytic versus oxPPP catabolism and reveals glucose-driven anabolism. Patients enrolled in clinical trial NCT03457779 and received IV infusion of [1,2-13C]glucose during core biopsy of their primary TNBC. Tumor samples were analyzed for metabolite labeling by liquid chromatography-mass spectrometry (LC-MS). Genomic and proteomic analyses were performed and related to observed metabolic fluxes. FINDINGS: TNBC ferments glucose to lactate, with glycolysis dominant over the oxPPP. Most ribose phosphate is nevertheless produced by oxPPP. Glucose also feeds amino acid synthesis, including of serine, glycine, aspartate, glutamate, proline and glutamine (but not asparagine). Downstream in glycolysis, tumor pyruvate and lactate labeling exceeds that found in serum, indicating that lactate exchange via monocarboxylic transporters is less prevalent in human TNBC compared with most normal tissues or non-small cell lung cancer. CONCLUSIONS: Glucose directly feeds ribose phosphate, amino acid synthesis, lactate, and the TCA cycle locally within human breast tumors.

Protocol for the Mason: Health Starts Here prospective cohort study of young adult college students.

BACKGROUND: Young adulthood is a period of increasing independence for the 40% of young adults enrolled in U.S. colleges. Previous research indicates differences in how students' health behaviors develop and vary by gender, race, ethnicity, and socioeconomic status. George Mason University is a state institution that enrolls a highly diverse student population, making it an ideal setting to launch a longitudinal cohort study using multiple research methods to evaluate the effects of health behaviors on physical and psychological functioning, especially during the COVID-19 pandemic. RESULTS: Mason: Health Starts Here was developed as a longitudinal cohort study of successive waves of first year students that aims to improve understanding of the natural history and determinants of young adults' physical health, mental health, and their role in college completion. The study recruits first year students who are 18 to 24 years old and able to read and understand English. All incoming first year students are recruited through various methods to participate in a longitudinal cohort for 4 years. Data collection occurs in fall and spring semesters, with online surveys conducted in both semesters and in-person clinic visits conducted in the fall. Students receive physical examinations during clinic visits and provide biospecimens (blood and saliva). CONCLUSIONS: The study will produce new knowledge to help understand the development of health-related behaviors during young adulthood. A long-term goal of the cohort study is to support the design of effective, low-cost interventions to encourage young adults' consistent performance of healthful behaviors, improve their mental health, and improve academic performance.

Proteomic Analysis of Cardioembolic and Large Artery Atherosclerotic Clots Using Reverse Phase Protein Array Technology Reveals Key Cellular Interactions Within Clot Microenvironments.

Thrombus characteristics are dependent on clot composition, but identification of the etiology based on histological analysis has proved inconclusive. Identification of proteomic signatures may help to differentiate between clots of different etiologies such as cardioembolic, large artery atherosclerotic, and other known etiologies, information that could enhance an individualized medicine approach to secondary stroke prevention. In this study, total protein extracts from cardioembolic (n=25) and large artery atherosclerotic (n=23) thrombus specimens were arrayed in quadruplicate on nitrocellulose slides and immunostained for 31 proteins using a Dako Autostainer (Agilent Technologies, Inc., Santa Clara, USA). We quantified 31 proteins involved in platelet and/or endothelial function, inflammation, oxidative stress, and metabolism. Pathway analysis showed more heterogeneity and protein network interactions in the cardioembolic clots but no specific correlations with clot etiology. Reverse-phase protein arrays are a powerful tool for assessing cellular interactions within the clot microenvironment and may enhance understanding of clot formation and origination. This tool could be further explored to help in identifying stroke etiology in large vessel occlusion patients with embolic stroke of an undetermined source.

Reverse Phase Protein Arrays.

Reverse phase protein arrays (RPPA) are used to quantify proteins and protein posttranslational modifications in cellular lysates and body fluids. RPPA technology is suitable for biomarker discovery, protein pathway profiling, functional phenotype analysis, and drug discovery mechanism of action. The principles of RPPA technology are (a) immobilizing protein-containing specimens on a coated slide in discrete spots, (b) antibody recognition of proteins, (c) amplification chemistries to detect the protein-antibody complex, and (d) quantifying spot intensity. Construction of a RPPA begins with the robotic liquid transfer of protein-containing specimens from microtiter plates onto nitrocellulose-coated slides. The robotic arrayer deposits each sample as discrete spots in an array format. Specimens, controls, and calibrators are printed on each array, thus providing a complete calibrated assay on a single slide. Each RPPA slide is subsequently probed with catalyzed signal amplification chemistries and a single primary antibody, a secondary antibody, and either fluorescent or colorimetric dyes. The focus of this chapter is to describe RPPA detection and imaging using a colorimetric (diaminobenzidine (DAB)) detection strategy.

Effects of HER Family-targeting Tyrosine Kinase Inhibitors on Antibody-dependent Cell-mediated Cytotoxicity in HER2-expressing Breast Cancer.

PURPOSE: Antibody-dependent cell-mediated cytotoxicity (ADCC) is one mechanism of action of the monoclonal antibody (mAb) therapies trastuzumab and pertuzumab. Tyrosine kinase inhibitors (TKIs), like lapatinib, may have added therapeutic value in combination with mAbs through enhanced ADCC activity. Using clinical data, we examined the impact of lapatinib on HER2/EGFR expression levels and natural killer (NK) cell gene signatures. We investigated the ability of three TKIs (lapatinib, afatinib, and neratinib) to alter HER2/immune-related protein levels in preclinical models of HER2-positive (HER2+) and HER2-low breast cancer, and the subsequent effects on trastuzumab/pertuzumab-mediated ADCC. EXPERIMENTAL DESIGN: Preclinical studies (proliferation assays, Western blotting, high content analysis, and flow cytometry) employed HER2+ (SKBR3 and HCC1954) and HER2-low (MCF-7, T47D, CAMA-1, and CAL-51) breast cancer cell lines. NCT00524303 provided reverse phase protein array-determined protein levels of HER2/pHER2/EGFR/pEGFR. RNA-based NK cell gene signatures (CIBERSORT/MCP-counter) post-neoadjuvant anti-HER2 therapy were assessed (NCT00769470/NCT01485926). ADCC assays utilized flow cytometry-based protocols. RESULTS: Lapatinib significantly increased membrane HER2 levels, while afatinib and neratinib significantly decreased levels in all preclinical models. Single-agent lapatinib increased HER2 or EGFR levels in 10 of 11 (91%) tumor samples. NK cell signatures increased posttherapy (P = 0.03) and associated with trastuzumab response (P = 0.01). TKI treatment altered mAb-induced NK cell-mediated ADCC in vitro, but it did not consistently correlate with HER2 expression in HER2+ or HER2-low models. The ADCC response to trastuzumab and pertuzumab combined did not exceed either mAb alone. CONCLUSIONS: TKIs differentially alter tumor cell phenotype which can impact NK cell-mediated response to coadministered antibody therapies. mAb-induced ADCC response is relevant when rationalizing combinations for clinical investigation.

Evaluation of pathogen specific urinary peptides in tick-borne illnesses.

Mass spectrometry enhanced by nanotechnology can achieve previously unattainable sensitivity for characterizing urinary pathogen-derived peptides. We utilized mass spectrometry enhanced by affinity hydrogel particles (analytical sensitivity = 2.5 pg/mL) to study tick pathogen-specific proteins shed in the urine of patients with (1) erythema migrans rash and acute symptoms, (2) post treatment Lyme disease syndrome (PTLDS), and (3) clinical suspicion of tick-borne illnesses (TBI). Targeted pathogens were Borrelia, Babesia, Anaplasma, Rickettsia, Ehrlichia, Bartonella, Francisella, Powassan virus, tick-borne encephalitis virus, and Colorado tick fever virus. Specificity was defined by 100% amino acid sequence identity with tick-borne pathogen proteins, evolutionary taxonomic verification for related pathogens, and no identity with human or other organisms. Using a cut off of two pathogen peptides, 9/10 acute Lyme Borreliosis patients resulted positive, while we identified zero false positive in 250 controls. Two or more pathogen peptides were identified in 40% of samples from PTLDS and TBI patients (categories 2 and 3 above, n = 59/148). Collectively, 279 distinct unique tick-borne pathogen derived peptides were identified. The number of pathogen specific peptides was directly correlated with presence or absence of symptoms reported by patients (ordinal regression pseudo-R2 = 0.392, p = 0.010). Enhanced mass spectrometry is a new tool for studying tick-borne pathogen infections.

Lipoarabinomannan antigenic epitope differences in tuberculosis disease subtypes.

An accurate urine test for diverse populations with active tuberculosis could be transformative for preventing TB deaths. Urinary liporabinomannan (LAM) testing has been previously restricted to HIV co-infected TB patients. In this study we evaluate urinary LAM in HIV negative, pediatric and adult, pulmonary and extrapulmonary tuberculosis patients. We measured 430 microbiologically confirmed pretreatment tuberculosis patients and controls from Peru, Guinea Bissau, Venezuela, Uganda and the United States using three monoclonal antibodies, MoAb1, CS35, and A194, which recognize distinct LAM epitopes, a one-sided immunoassay, and blinded cohorts. We evaluated sources of assay variability and comorbidities (HIV and diabetes). All antibodies successfully discriminated TB positive from TB negative patients. ROAUC from the average of three antibodies' responses was 0.90; 95% CI 0.87-0.93, 90% sensitivity, 73.5% specificity (80 pg/mL). MoAb1, recognizing the 5-methylthio-D-xylofuranose(MTX)-mannose(Man) cap epitope, performed the best, was less influenced by glycosuria and identified culture positive pediatric (N = 19) and extrapulmonary (N = 24) patients with high accuracy (ROAUC 0.87, 95% CI 0.77-0.98, 0.90 sensitivity 0.80 specificity at 80 pg/mL; ROAUC = 0.96, 95% CI 0.92-0.99, 96% sensitivity, 80% specificity at 82 pg/mL, respectively). The MoAb1 antibody, recognizing the MTX-Man cap epitope, is a novel analyte for active TB detection in pediatric and extrapulmonary disease.

Tumor-Draining Lymph Secretome En Route to the Regional Lymph Node in Breast Cancer Metastasis.

BACKGROUND: During metastasis, tumor cells metastasize from primary tumors to distant organs via the circulatory and the lymphatic systems. There is a plethora of information about metastasis through the circulatory system, however not much information is available about the tumor cells dissemination through the lymphatic system or the lymphatic microenvironment that aids in this process in breast cancer metastasis. PURPOSE: The study designed to examine the tumor-derived secretome in lymph before reaching the draining lymph nodes. METHODS: Using a microsurgical technique, we have collected the lymph in transit from the primary tumor en route to the regional lymph node in animals with metastatic and non-metastatic mammary carcinoma and healthy controls. The lymph samples were subjected to LC-MS/MS analysis, bioinformatics, and pathway analysis. RESULTS: The metastatic tumor-draining lymph before its entry into the closest regional lymph node contain 26 proteins with >175-folds in abundance compared to lymph from non-metastatic tumor-bearing animals. Among these proteins were biliverdin reductase B, heat shock protein, coagulation factor XIII, lymphocytes cytosol protein 1, and aldose reductase. These proteins were not identified in the lymph from healthy animals. Pathways analysis revealed that cadherin-mediated endocytosis, acute phase response, junction signaling, gap junction, VEGF singling, and PI3K/AKT singling pathways are overrepresented in the lymph from metastatic tumor-bearing compared to the lymph from non-metastatic tumor-bearing animals. Among the significantly up-regulated proteins in the lymph from metastatic tumor-bearing animals were proteins that identified in exosomes include heat shock protein, enolase 1 alpha, S100, and biliverdin reductase B. One of the proteins significantly down-regulated in lymph from animals with metastasis is Kininogen, a known metastasis inhibitor protein. CONCLUSION: Proteins and exosomal proteins in lymph draining a metastatic tumor are different from those in lymph draining non-metastatic tumors, and these proteins involved in pathways that regulate tumor cells migration and invasion.

A Randomized Phase II Preoperative Study of Autophagy Inhibition with High-Dose Hydroxychloroquine and Gemcitabine/Nab-Paclitaxel in Pancreatic Cancer Patients.

PURPOSE: We hypothesized that autophagy inhibition would increase response to chemotherapy in the preoperative setting for patients with pancreatic adenocarcinoma. We performed a randomized controlled trial to assess the autophagy inhibitor hydroxychloroquine in combination with gemcitabine and nab-paclitaxel. PATIENTS AND METHODS: Participants with potentially resectable tumors were randomized to two cycles of nab-paclitaxel and gemcitabine (PG) alone or with hydroxychloroquine (PGH), followed by resection. The primary endpoint was histopathologic response in the resected specimen. Secondary clinical endpoints included serum CA 19-9 biomarker response and margin negative R0 resection. Exploratory endpoints included markers of autophagy, immune infiltrate, and serum cytokines. RESULTS: Thirty-four patients in the PGH arm and 30 in the PG arm were evaluable for the primary endpoint. The PGH arm demonstrated statistically improved Evans grade histopathologic responses (P = 0.00016), compared with control. In patients with elevated CA 19-9, a return to normal was associated with improved overall and recurrence-free survival (P < 0.0001). There were no differences in serious adverse events between arms and chemotherapy dose number was equivalent. The PGH arm had greater evidence of autophagy inhibition in their resected specimens (increased SQSTM1, P = 0.027, as well as increased immune cell tumor infiltration, P = 0.033). Overall survival (P = 0.59) and relapse-free survival (P = 0.55) did not differ between the two arms. CONCLUSIONS: The addition of hydroxychloroquine to preoperative gemcitabine and nab-paclitaxel chemotherapy in patients with resectable pancreatic adenocarcinoma resulted in greater pathologic tumor response, improved serum biomarker response, and evidence of autophagy inhibition and immune activity.

Characterization and Validation of Arg286 Residue of IL-1RAcP as a Potential Drug Target for Osteoarthritis.

Osteoarthritis (OA) is the most common form of arthritis and the fastest growing cause of chronic disability in the world. Formation of the ternary IL-1ß /IL-1R1/IL-1RAcP protein complex and its downstream signaling has been implicated in osteoarthritis pathology. Current OA therapeutic approaches target either the cytokine IL-1ß or the primary receptor IL-1RI but do not exploit the potential of the secondary receptor IL-1RAcP. Our previous work implicated the Arg286 residue of IL-1RAcP as a key mediator of complex formation. Molecular modeling confirmed Arg286 as a high-energy mediator of the ternary IL-1ß complex architecture and interaction network. Anti-IL-1RAcP monoclonal antibodies (mAb) targeting the Arg286 residue were created and were shown to effectively reduce the influx of inflammatory cells to damaged joints in a mouse model of osteoarthritis. Inhibitory peptides based on the native sequence of IL-1RAcP were prepared and examined for efficacy at disrupting the complex formation. The most potent peptide inhibitor had an IC50 value of 304 pM in a pull-down model of complex formation, and reduced IL-1ß signaling in a cell model by 90% at 2 µM. Overall, therapies that target the Arg286 region surface of IL-1RAcP, and disrupt subsequent interactions with subunits, have the potential to serve as next generation treatments for osteoarthritis.

RPPA: Origins, Transition to a Validated Clinical Research Tool, and Next Generations of the Technology.

RPPA technology has graduated from a research tool to an essential component of clinical drug discovery research and personalized medicine. Next generations of RPPA technology will be a single clinical instrument that integrates all the steps of the workflow.

Solid Pin Protein Array Printing Platforms.

Reverse phase protein arrays (RPPA) are miniature dot blots constructed using robotic arrayers to deposit protein containing samples onto nitrocellulose-coated glass slides. Reverse phase protein arrays address the challenge of quantifying low-abundance proteins and posttranslationally modified proteins in cellular lysates and body fluids. RPPA technology is ideally suited to biomarker discovery, signal pathway profiling, functional phenotype analysis, and mechanism of action studies for drug discovery. Each array is fabricated with specimens, controls, and calibrators, thus providing a complete assay on each slide. Constructing a reverse phase protein array initially consists of selecting an arrayer, pin type, print head configuration, and nitrocellulose slide that is optimized for the particular specimen type and protein detection method. Herein we present the nuances of RPPA fabrication and study design using a solid pin arrayer and nitrocellulose-coated slides.

Different measures of HMGB1 location in cancer immunology.

HMGB1 is the most abundant non-histone nuclear protein. It regulates transcriptional access to open areas of chromatin and limits release of DNA with apoptotic death, serving to both inhibit apoptosis and promote DNA repair. When HMGB1 is translocated to the cytosol with many types of cellular stress, it is a powerful inducer of autophagy. It can also be released by activated immune cells and damaged or dying cells into the extracellular space, where it acts as a damage associated molecular pattern (DAMP) molecule, contributing to the pathogenesis and progression of cancer. Here, the most common methodologies to not only measure HMGB1 but also to effectively determine its subcellular localization, which dictates many of HMGB1's different functions, are reviewed.

A new model isolates glioblastoma clonal interactions and reveals unexpected modes for regulating motility, proliferation, and drug resistance.

Tumor clonal heterogeneity drives treatment resistance. But robust models are lacking that permit eavesdropping on the basic interaction network of tumor clones. We developed an in vitro, functional model of clonal cooperation using U87MG glioblastoma cells, which isolates fundamental clonal interactions. In this model pre-labeled clones are co-cultured to track changes in their individual motility, growth, and drug resistance behavior while mixed. This highly reproducible system allowed us to address a new class of fundamental questions about clonal interactions. We demonstrate that (i) a single clone can switch off the motility of the entire multiclonal U87MG cell line in 3D culture, (ii) maintenance of clonal heterogeneity is an intrinsic and influential cancer cell property, where clones coordinate growth rates to protect slow growing clones, and (iii) two drug sensitive clones can develop resistance de novo when cooperating. Furthermore, clonal communication for these specific types of interaction did not require diffusible factors, but appears to depend on cell-cell contact. This model constitutes a straightforward but highly reliable tool for isolating the complex clonal interactions that make up the fundamental "hive mind" of the tumor. It uniquely exposes clonal interactions for future pharmacological and biochemical studies.