Catherine A. St. Hill

ADAM17 activity during human neutrophil activation and apoptosis

Substrates of the metalloprotease ADAM17 (also known as TNF‐α converting enzyme or TACE) undergo ectodomain shedding and include various inflammatory modulators. Though polymorphonuclear leukocytes contribute significantly to inflammation, direct analyses of ADAM17 on human neutrophils are very limited. In addition, the current understanding of the processes regulating ADAM17 activity primarily relate to its rapid activation. Therefore, to extend insights into the mechanisms of ADAM17 activity, we examined its surface expression and the shedding of its substrates during extended periods of neutrophil activation and apoptosis. Contrary to studies with immortalized hematopoietic cell lines, we report that surface expression of ADAM17 is maintained by human neutrophils activated with formyl peptides or by FcR/complement receptor‐mediated phagocytosis. Interestingly, bacterial phagocytosis resulted in a significant increase in ADAM17 expression several hours after pathogen engulfment. We provide novel evidence that ADAM17 surface expression is also maintained during spontaneous and anti‐Fas‐induced neutrophil apoptosis. The well‐validated ADAM17 substrates L‐selectin and proTNF‐α were shed efficiently by neutrophils under each of the conditions tested. Our data thus indicate prolonged ADAM17 expression during neutrophil effector functions. The implications of this may be a role by ADAM17 in both the induction and down‐regulation of neutrophil activity.

ADAM-17-independent shedding of L-selectin

L‐selectin is expressed by leukocytes and facilitates their adhesion under flow along the walls of blood vessels. As do a variety of membrane proteins, L‐selectin undergoes ectodomain shedding. Using approaches that monitor full‐length L‐selectin in short‐term assays, it has been determined that L‐selectin shedding is defective in tumor necrosis factor α‐converting enzyme (ADAM‐17)‐deficient cells. In this study, we examined the steady‐state levels of L‐selectin on ADAM‐17‐deficient cells using a monoclonal antibody to the cytoplasmic region of L‐selectin, which allows for the detection of total L‐selectin (full‐length and the membrane‐associated cleavage fragment). We demonstrate that ADAM‐17‐deficient cells generate a 6‐kDa transmembrane fragment of L‐selectin. Although inducible L‐selctin shedding by phorbol 12‐myristate 13‐acetate stimulation was not observed by these cells in short‐term assays, basal turnover did occur, resulting in the production of soluble L‐selectin, as determined by enzyme‐linked immunosorbent assay. L‐selectin turnover was greatly increased upon ADAM‐17 reconstitution. Truncating the juxtamembrane region of L‐selectin blocked ADAM‐17‐independent shedding as did a hydroxymate metalloprotease inhibitor. Together, these findings demonstrate that a metalloprotease activity separate from ADAM‐17 can use the cleavage domain of L‐selectin. We speculate that separate proteolytic mechanisms of L‐selectin shedding may regulate distinct antiadhesive mechanisms, such as inducible shedding for the rapid dissociation of cell–cell interactions and constitutive shedding for the homeostatic maintenance of high serum levels of soluble L‐selectin, a potential adhesion buffer.

The high affinity selectin glycan ligand C2-O-sLex and mRNA transcripts of the core 2 β-1,6-N-acetylglusaminyltransferase (C2GnT1) gene are highly expressed in human colorectal adenocarcinomas

BackgroundThe metastasis of cancer cells and leukocyte extravasation into inflamed tissues share common features. Specialized carbohydrates modified with sialyl Lewis x (sLex) antigens on leukocyte membranes are ligands for selectin adhesion molecules on activated vascular endothelial cells at inflammatory sites. The activity of the enzyme core 2 β1,6 N-acetylglucosaminyltransferase (C2GnT1) in leukocytes greatly increases their ability to bind to endothelial selectins. C2GnT1 is essential for the synthesis of core 2-branched O-linked carbohydrates terminated with sLex (C2-O-sLex). Our goal was to determine the expression profiles of C2-O-sLex in the malignant progression and metastasis of colorectal adenocarcinomas. The well characterized CHO-131 monoclonal antibody (mAb) specifically recognizes C2-O-sLex present in human leukocytes and carcinoma cells. Using CHO-131 mAb, we investigated whether C2-O-sLex was present in 113 human primary colorectal adenocarcinomas, 10 colorectal adenomas, 46 metastatic liver tumors, 28 normal colorectal tissues, and 5 normal liver tissues by immunohistochemistry. We also examined mRNA levels of the enzyme core 2 β1,6-N-acetylglucosaminyltransferase (C2GnT1) in 20 well, 15 moderately, and 2 poorly differentiated colorectal adenocarcinomas, and in 5 normal colorectal tissues by using quantitative real-time polymerase chain reactions (RT-PCR).ResultsWe observed high reactivity with CHO-131 mAb in approximately 70% of colorectal carcinomas and 87% of metastatic liver tumors but a lack of reactivity in colorectal adenomas and normal colonic and liver tissues. Positive reactivity with CHO-131 mAb was very prominent in neoplastic colorectal glands of well to moderately differentiated adenocarcinomas. The most intense staining with CHO-131 mAb was observed at the advancing edge of tumors with the deepest invasive components.Finally, we analyzed C2GnT1 mRNA levels in 37 colorectal adenocarcinomas and 5 normal colorectal tissues by RT-PCR. Significantly, we observed a greater than 15-fold increase in C2GnT1 mRNA levels in colorectal adenocarcinomas compared to normal colorectal tissues.ConclusionC2-O-sLex, detected by the CHO-131 mAb, is a tumor associated antigen whose expression is highly upregulated in colorectal adenocarcinomas and metastatic liver tumors compared to normal tissues. C2-O-sLex is a potentially useful early predictor of metastasis.

Indirect capture augments leukocyte accumulation on P‐selectin in flowing whole blood

Leukocytes are captured directly by E‐ and P‐selectin on activated endothelium and by indirect means, which includes attached leukocytes capturing free‐flowing leukocytes. However, controversy exists as to whether the latter mechanism occurs in the presence of red blood cells. We analyzed leukocyte capture mechanisms on P‐selectin under circulatory hydrodynamics using whole blood. The selective disruption of leukocyte–leukocyte interactions with an L‐selectin monoclonal antibody reduced leukocyte accumulation by >50% under various stringencies (substrate concentrations and shear stresses). In addition, a direct analysis of leukocyte capture events revealed that 69% were indirect. Our data indicate that in the presence of red blood cells, P‐selectin‐attached leukocytes, individually and as a monolayer, augment leukocyte accumulation by indirect capture. This mechanism may contribute to increasing the density of leukocytes on discrete areas of activated endothelial cells at sites of inflammation. These findings are significant since L‐selectin accounts for the majority of the leukocyte rolling flux in small venules at diverse inflammatory settings. Yet, the primary mechanism by which L‐selectin mediates leukocyte accumulation remains unresolved.

Opioid prescribing patterns for non-malignant chronic pain for rural versus non-rural US adults: a population-based study using 2010 NAMCS data

BackgroundNon-malignant chronic pain (NMCP) is one of the most common reasons for primary care visits. Pain management health care disparities have been documented in relation to patient gender, race, and socioeconomic status. Although not studied in relation to chronic pain management, studies have found that living in a rural community in the US is associated with health care disparities. Rurality as a social determinant of health may influence opioid prescribing. We examined rural and non-rural differences in opioid prescribing patterns for NMCP management, hypothesizing that distinct from education, income, racial or gender differences, rural residency is a significant and independent factor in opioid prescribing patterns.Methods2010 National Ambulatory Medical Care Survey (NAMCS) data were examined using bivariate and multivariate techniques. NAMCS data were collected using a multi-stage sampling strategy. For the multivariate analysis performed the SPSS complex samples algorithm for logistic regression was used.ResultsIn 2010 an estimated 9,325,603 US adults (weighted from a sample of 2745) seen in primary care clinics had a diagnosis of NMCP; 36.4% were prescribed an opioid. For US adults with a NMCP diagnosis bivariate analysis revealed rural residents had higher odds of having an opioid prescription than similar non-rural adults (OR = 1.515, 95% CI 1.513-1.518). Complex samples logistic regression analysis confirmed the importance of rurality and yielded that US adults with NMCP who were prescribed an opioid had higher odds of: being non-Caucasian (AOR =2.459, 95% CI 1.194-5.066), and living in a rural area (AOR =2.935, 95% CI 1.416-6.083).ConclusionsOur results clearly indicated that rurality is an important factor in opioid prescribing patterns that cannot be ignored and bears further investigation. Further research on the growing concern about the over-prescribing of opioids in the US should now include rurality as a variable in data generation and analysis. Future research should also attempt to document the ecological, sociological and political factors impacting opioid prescribing and care in rural communities. Prescribers and health care policy makers need to critically evaluate the implications of our findings and their relationship to patient needs, best practices in a rural setting, and the overall consequences of increased opioid prescribing on rural communities.

C2-O-sLeX Glycoproteins Are E-Selectin Ligands that Regulate Invasion of Human Colon and Hepatic Carcinoma Cells

Similar to mechanisms of recruitment of activated leukocytes to inflamed tissues, selectins mediate adhesion and extravasation of circulating cancer cells. Our objective was to determine whether sialyl Lewis X modified core 2 O-glycans (C2-O-sLeX) present on colon and hepatic carcinoma cells promote their adhesion and invasion. We examined membrane expression of C2-O-sLeX, selectin binding, invasion of human colon and hepatic carcinoma cell lines, and mRNA levels of alpha-2,3 fucosyltransferase (FucT-III) and core 2 beta-1,6 N-acetylglucosaminyltransferase (C2GnT1) genes, necessary for C2-O-sLeX synthesis, by quantitative reverse-transcriptase (RT) PCR. Synthesis of core 2 branched O-glycans decorated by sLeX is dependent on C2GnT1 function and thus we determined enzyme activity of C2GnT1. The cell lines that expressed C2GnT1 and FucT-III mRNA by quantitative RT-PCR were highly positive for C2-O-sLeX by flow cytometry, and colon carcinoma cells possessed highly active C2GnT1 enzyme. Cells bound avidly to E-selection but not to P- and L-selectin. Gene knock-down of C2GnT1 in colon and hepatic carcinoma cells using short hairpin RNAs (shRNA) resulted in a 40–90% decrease in C2-O-sLeX and a 30–50% decrease in E-selectin binding compared to control cells. Invasion of hepatic and colon carcinoma cells containing C2GnT1 shRNA was significantly reduced compared to control cells in Matrigel assays and C2GnT1 activity was down-regulated in the latter cells. The sLeX epitope was predominantly distributed on core 2 O-glycans on colon and hepatic carcinoma cells. Our findings indicate that C2GnT1 gene expression and the resulting C2-O-sLeX carbohydrates produced mediate the adhesive and invasive behaviors of human carcinomas which may influence their metastatic potential.

Increased expression of GCNT1 is associated with altered O‐glycosylation of PSA, PAP, and MUC1 in human prostate cancers

Protein glycosylation is a common posttranslational modification and glycan structural changes have been observed in several malignancies including prostate cancer. We hypothesized that altered glycosylation could be related to differences in gene expression levels of glycoprotein synthetic enzymes between normal and malignant prostate tissues.

Neutrophil interactions with sialyl Lewis X on human nonsmall cell lung carcinoma cells regulate invasive behavior

The carbohydrate sialyl Lewis X (sLeX) is expressed on leukocytes and carcinoma cells and binds to selectins during inflammatory processes and early metastasis. Synthesis of sLeX depends on activity of enzymes, including α(1,3/1,4) fucosyltransferase (FucT‐III). Tumor necrosis factor‐α (TNF‐α) up‐regulates FucT‐III, resulting in increased sLeX in the airways of patients with respiratory disease; however, the mechanisms that regulate sLeX in the inflammatory tumor microenvironment are not well understood.

Team science as interprofessional collaborative research practice: a systematic review of the science of team science literature

The National Institute of Healths concept of team science is a means of addressing complex clinical problems by applying conceptual and methodological approaches from multiple disciplines and health professions. The ultimate goal is the improved quality of care of patients with an emphasis on better population health outcomes. Collaborative research practice occurs when researchers from >1 health-related profession engage in scientific inquiry to jointly create and disseminate new knowledge to clinical and research health professionals in order to provide the highest quality of patient care to improve population health outcomes. Training of clinicians and researchers is necessary to produce clinically relevant evidence upon which to base patient care for disease management and empirically guided team-based patient care. In this study, we hypothesized that team science is an example of effective and impactful interprofessional collaborative research practice. To assess this hypothesis, we examined the contemporary literature on the science of team science (SciTS) produced in the past 10 years (2005–2015) and related the SciTS to the overall field of interprofessional collaborative practice, of which collaborative research practice is a subset. A modified preferred reporting items for systematic reviews and meta-analyses (PRISMA) approach was employed to analyze the SciTS literature in light of the general question: Is team science an example of interprofessional collaborative research practice? After completing a systematic review of the SciTS literature, the posed hypothesis was accepted, concluding that team science is a dimension of interprofessional collaborative practice.

Development of an interprofessional and interdisciplinary collaborative research practice for clinical faculty

ABSTRACT This article describes an interprofessional collaborative research practice fellowship designed to foster the research skills of clinical faculty. The year-long fellowship was grounded in big data analysis and the triangle of informatics—knowledge, information, and data. Fellows were selected to include diverse perspectives, training, and knowledge but had limited experience in team science or being a member of an interprofessional research team. The underlying philosophy of the fellowship was experiential learning. Protected time and formal mentorship were necessary factors for developing the interprofessional research practice and the skills to participate in an interprofessional research team. We believe that this innovative interprofessional faculty research fellowship is a viable option for supporting scholarly activity and research collaboration. The findings could inform interprofessional clinical practice and be implemented for patient care. Engagement in interprofessional collaborative research and incorporation of the perspectives, knowledge and expertise of multiple professions, is a model to de silo knowledge creation.