Endogenous insulin clearance occurs primarily in hepatocytes and to a lower extent in kidney's proximal tubule cells (KPTCs). CEACAM1 promotes receptor-mediated insulin uptake to be degraded in hepatocytes in a phosphorylation-dependent manner. Its deletion/inactivation causes hyperinsulinemia-driven insulin resistance, steatohepatitis and liver fibrosis. CEACAM2, the dominant CEACAM protein in murine KPTCs, shares a high homology with CEACAM1. Thus, we examined whether it regulates renal insulin disposal to maintain renal homeostasis. KPTCs derived from Ceacam2 null mice ( Cc2 -/- ) exhibited lower receptor-mediated insulin uptake. Combined with the gradual decline in CEACAM1-dependent hepatic insulin clearance, impaired renal insulin clearance contributed to chronic hyperinsulinemia and insulin resistance starting at 10 months of age in Cc2 -/- males. This was followed by proteinuria and reduced glomerular filtration rate in association with glomerulosclerosis and tubulointerstitial damage. Increased collagen deposition in Cc2 -/- kidneys could be mediated in part, by hyperinsulinemia-driven activation of the α5β1 integrin-focal adhesion kinase (FAK) signaling pathways. Together, the data demonstrated that loss of CEACAM2 impaired renal insulin clearance that contributed to hyperinsulinemia and resultant insulin resistance, followed by kidney dysfunction and renal fibrosis. This study provided an in vivo demonstration of the regulation of kidney function by insulin clearance along the liver-kidney axis.

Wnt ligands belong to a family of secreted glycoproteins in which binding to a range of receptors/co-receptors activates several intracellular pathways. WNT5A, a member of the Wnt family, is classified as a non-canonical Wnt whose activation triggers planar cell polarity (PCP) and Ca+2 downstream pathways. Aberrant expression of WNT5A has been shown to play both protective and harmful roles in an array of conditions, such as inflammatory disease and cancer. In the present study, using histological, immunohistochemical, and molecular methods, we investigated the expression of two isoforms of WNT5A, WNT5A-Short (WNT5A-S) and WNT5A-Long (WNT5A-L) in bladder urothelial carcinoma (UC). Three UC cell lines (RT4, J82, and T24), as well as a normal urothelial cell line, and formalin-fixed, paraffin-embedded (FFPE) transurethral resection (TUR) tissue samples from 17 patients diagnosed with UC were included in the study. WNT5A-L was the predominantly expressed isoform in urothelial cells, although WNT5A-S was also detectable. Further, although no statistically significant difference was found between the percentage of WNT5A-S transcripts in low-grade versus high-grade tumors, we did find a difference between the percentage of WNT5A-S transcripts found in non-invasion versus invasion of the lamina propria, subgroups of non-muscle-invasive tumors. In conclusion, both WNT5A-S and WNT5A-L isoforms are expressed in UC, and the percentage of their expression levels suggests that a higher proportion of WNT5A-S transcription may be associated with lamina propria invasion, a process preceding muscle invasion.

Despite the 2019 Executive Order on Advancing American Kidney Health Initiative, kidney disease has moved up in rank from the 9th to the 8th leading cause of death in the United States. A recent push in the field of nephrology has been to identify molecular markers and/or molecular profiles involved in kidney disease process or injury that can help identify the cause of injury and predict patient outcomes. While these studies have had moderate success, they have not yet considered that many of the health conditions that cause kidney disease (diabetes, hypertension, etc.) can also be caused by environmental factors (such as viruses), which in and of themselves can cause kidney disease. Thus, the goal of this study was to identify molecular and phenotypic profiles that can differentiate kidney injury caused by diabetes (a health condition resulting in kidney disease) and coxsackievirus B4 (CVB4) exposure (which can cause diabetes and/or kidney disease), both alone and together. Non-obese diabetic (NOD) mice were used for this study due to their susceptibility to both type 1 diabetes (T1D)- and CVB4-mediated kidney injury, in order to glean a better understanding of how hyperglycemia and viral exposure, when occurring on their own and in combination, may alter the kidneys’ molecular and phenotypic profiles. While no changes in kidney function were observed, molecular biomarkers of kidney injury were significantly up- and downregulated based on T1D and CVB4 exposure, both alone and together, but not in a predictable pattern. By combining individual biomarkers with function and phenotypic measurements (i.e., urinary albumin creatinine ratio, serum creatinine, kidney weight, and body weight), we were able to perform an unbiased separation of injury group based on the type of injury. This study provides evidence that unique kidney injury profiles within a kidney disease health condition are identifiable, and will help us to identify the causes of kidney injury in the future.

Urothelial carcinoma (UC), the most common form of bladder cancer, has a high rate of recurrence and a low five‐year survival rate for high grade and metastatic tumors. Expression of Wnt5a, a protein that plays a role in many critical processes in cells and is involved in many different molecular signaling pathways, has been shown to correlate with UC development and pathological stage. Recently the Wnt5a gene has been shown to encode two different isoforms (Wnt5a‐L and Wnt5a‐S). Correlation of isoform expression with tumor cell phenotype suggests opposing roles, either as tumor‐promoting or tumor‐suppressing, depending on the cancer type. The effects of each Wnt5a isoform in UC, however, are not well understood.In order to understand how the Wnt5a isoforms function in the development of UC, it is important to understand which signaling pathways are being activated or inhibited by each isoform. This project aims to identify Wnt5a receptors that are differentially upregulated or downregulated in response to treatment with either Wnt5a‐L or Wnt5a‐S in several different UC cell lines derived from different stages of tumors, as well as in a primary human bladder cell line. The project will include identification and characterization of the Wnt5a receptors normally expressed in the untreated, basal state, information which is currently unknown. Wnt5a isoform treatment will be conducted using conditioned media collected from Chinese Hamster Ovary (CHO) cells that have been genetically modified to express either Wnt5a‐L or Wnt5a‐S. Using western blot analysis, we have already quantified the amount of each isoform present in the conditioned media so that equal amounts of each isoform can be used for cell treatments. We have also used western blot analysis to demonstrate that we can isolate membrane proteins from cell lysates. We will now be treating the cell lines with equal concentrations of Wnt5a‐L, Wnt5a‐S or commercially available Wnt5a as a positive control; isolating the membrane proteins from each cell line after treatment; and applying the isolated membrane proteins to custom antibody arrays in order to examine the expression of 17 different Wnt5a receptors. Results from these experiments will show what receptors are upregulated or downregulated in response to each Wnt5a isoform, which will help elucidate the signaling pathways regulated by each isoform in the pathogenesis of UC.Support or Funding InformationThis project was supported in part by funding from the Honors Tutorial College, Student Enhancement Award, and Heritage College of Osteopathic Medicine Research and Scholarly Activities Committee, all at Ohio University. Special thanks is given to Dr. Karl Willert, who provided the CHO cell lines expressing each Wnt5a isoform.

In 2018, cancer was the second leading cause of death in the United States. Past research has demonstrated the importance of macrophages in both the tumor microenvironment and in cancer progression. Macrophages are specialized immune cells that are differentiated into either the M1 phenotype (pro‐inflammatory/tumor‐suppressing) or M2 phenotype (anti‐inflammatory/tumor‐promoting), depending on the signals in the tumor microenvironment. Research has demonstrated that the expression of Wnt5a, a secreted glycoprotein, is correlated with a cancer’s level of aggression. Additionally, evidence shows that the treatment of M1 macrophages with Wnt5a increased the expression of IL‐10, a molecule expressed by M2 macrophages and associated with the progression of cancer. The objective of this study is to investigate the effect of exogenous Wnt5a on the expression levels of M1/M2 phenotypic markers. The hypothesis is that Wnt5a facilitates the switching of macrophages from the M1 to M2 phenotype through the induction of IL‐10. This would support the idea that Wnt5a functions as a tumor promoter and potentially contribute in the progression of cancer.To test this hypothesis, THP‐1 and human peripheral blood monocyte derived macrophages were evaluated for expression of the M1 markers CD80 and IL‐6 and the M2 markers CD163 and IL‐10 using flow cytometry and RT‐qPCR either with or without Wnt5a treatment.Our preliminary results confirm that, without Wnt5a treatment, M1 macrophages strongly expressed Wnt5a and, further, demonstrate that, after treatment with exogenous recombinant Wnt5a, the expression of M2 markers, including IL‐10, increased, while the expression of M1 markers decreased. Results from this study lead us to speculate that Wnt5a could play a role in switching macrophage phenotype from M1 to M2 and thus determining the overall tumor behavior. Furthermore, the manipulation of Wnt5a signaling could potentially be an attractive strategy in the development of more effective cancer treatments.Support or Funding InformationThe John J. Kopchick MCB/TBS Undergraduate Student Support Fund (Ohio University) andThe Provost Undergraduate Research Fund (Ohio University)

The pathogenesis of atherosclerosis is complex, evolves, and involves many cell types. Macrophages and vascular smooth muscle cells (VSMCs) are critically involved in atherosclerosis development and progression. Several studies have shown that WNT5A protein is abundantly expressed in human atherosclerotic lesions; however, the mechanism and role of WNT signaling pathway activation is not clearly known. Using THP-1 derived macrophages, and human aortic VSMC cells, we evaluated in vitro how oxidized low-density lipoprotein (oxLDL) and WNT5A signaling interact in these two cell lines. We used western blot, scratch assay, metabolic proliferation assay, as well as immunostaining to analyze the effect of Wnt signaling activation. The results demonstrated that oxLDL, as well as WNT5A (control), induced Disheveled-2 (DVL2) activation and Kif26b degradation, indicating activation of non-canonical Wnt signaling. We found that oxLDL and WNT5A induced FZD5-ROR2 co-localization at the cellular membrane in vitro in THP-1 derived macrophages. Box5 (FZD5 receptor antagonist) inhibited oxLDL-induced DVL2/JNK activation secondary to newly secreted WNT protein from THP-1 derived macrophages. We found that WNT3A (canonical Wnt) and WNT5A showed different roles in this VSMC cell line. These findings indicate that WNT5A is upregulated by oxLDL, promotes foam cell formation, and affects VSMC phenotype and migration in these two cell lines. Also, in these cell lines FZD5 signaling seems to be necessary for lipid accumulation and, through this mechanism, WNT5A could modulate foam cell formation. Thus, our results suggest that WNT5A may contribute to the pathogenesis of vascular disease through modulating macrophage and VSMC behavior.

End-stage renal disease (ESRD) is described by four primary diagnoses, diabetes, hypertension, glomerulonephritis, and cystic kidney disease, all of which have viruses implicated as causative agents. Enteroviruses, such as coxsackievirus (CV), are a common genus of viruses that have been implicated in both diabetes and cystic kidney disease; however, little is known about how CVs cause kidney injury and ESRD or predispose individuals with a genetic susceptibility to type 1 diabetes (T1D) to kidney injury. This study evaluated kidney injury resulting from coxsackievirus B4 (CVB4) inoculation of non-obese diabetic (NOD) mice to glean a better understanding of how viral exposure may predispose individuals with a genetic susceptibility to T1D to kidney injury. The objectives were to assess acute and chronic kidney damage in CVB4-inoculated NOD mice without diabetes. Results indicated the presence of CVB4 RNA in the kidney for at least 14 days post-CVB4 inoculation and a coordinated pattern recognition receptor response, but the absence of an immune response or cytotoxicity. CVB4-inoculated NOD mice also had a higher propensity to develop an increase in mesangial area 17 weeks post-CVB4 inoculation. These studies identified initial gene expression changes in the kidney resulting from CVB4 exposure that may predispose to ESRD. Thus, this study provides an initial characterization of kidney injury resulting from CVB4 inoculation of mice that are genetically susceptible to developing T1D that may one day provide better therapeutic options and predictive measures for patients who are at risk for developing kidney disease from T1D.

Atherosclerosis (ATH), the build up of fat in the arteries, is a principal cause of heart attack and stroke. Drug instability and lack of target specificity are major drawbacks of current clinical therapeutics. These undesirable effects can be eliminated by site-specific drug delivery. The endothelial surface over ATH lesions has been shown to overexpress vascular cell adhesion molecule1 (VCAM1), which can be used for targeted therapy. Here, we report the synthesis, characterization, and development of anti VCAM1-functionalized liposomes to target cells overexpressing VCAM1 under static and flow conditions. Liposomes were composed of dioleoyl-phosphatidylcholine, sphingomyelin, cholesterol, and distearoyl-phosphatidylethanolamine-polyethylene glycol-cyanur (31.67:31.67:31.67:5 mol%). VCAM1 expression in endothelial cells was induced by lipopolysaccharide (LPS) treatment. Characterization study revealed that liposomes were negatively charged (− 7.7 ± 2.6 mV) with an average diameter of 201.3 ± 3.3 nm. Liposomes showed no toxicity toward THP-1 derived macrophages and endothelial cells. Liposomes were able to target both fixed and non-fixed endothelial cells, in vitro, with significantly higher localization observed in non-fixed conditions. To mimic biological and physiologically-relevant conditions, liposome targeting was also examined under flow (4 dyn/cm2) with or without erythrocytes (40% v/v hematocrit). Liposomes were able to target LPS-treated endothelial cells under dynamic culture, in the presence or absence of erythrocytes, although targeting efficiency was five-fold lower in flow compared to static conditions. This liposomal delivery system showed a significant improvement in localization on dysfunctional endothelium after surface functionalization. We conclude that VCAM1-functionalized liposomes can target and potentially deliver therapeutic compounds to ATH regions.

It is projected that there will be over 81,000 new bladder cancer diagnoses in 2018 in the United States alone. In fact, 1 in 27 men will develop bladder cancer in their lifetime. Despite its prevalence, little is known about this cancer's molecular mechanism. Research performed both at Ohio University and by other scientists has linked a protein, Wnt5a, to urothelial carcinoma (UC), which is the most common form of bladder cancer. This protein has been shown to be involved in an array of diseases, including several types of cancer. Recent research has suggested that two Wnt5a isoforms, Wnt5a‐Short (S) and Wnt5a‐Long (L), may play differing roles in a variety of cancers, with Wnt5a‐S promoting cellular proliferation and Wnt5a‐L suppressing cellular proliferation. Based on this and the research linking Wnt5a to UC, we want to investigate the roles of the Wnt5a isoforms in UC cell lines and human UC biopsies. We extracted RNA from human UC cell lines derived from low and high grade tumors to quantify the amount of each Wnt5a isoform and total Wnt5a expression using real‐time reverse transcription / polymerase chain reaction (RT‐RT/PCR). Trends in expression for Wnt5a‐L and Wnt5a‐Total were extremely similar, suggesting that Wnt5a‐L is the predominantly expressed isoform in urothelial cells. Wnt5a‐L RNA expression was highest in a cell line derived from a high grade tumor but also lowest in a cell line derived from a second high grade tumor. In contrast, expression of Wnt5a‐S RNA was highest in cell lines derived from high grade tumors in comparison to low grade tumors. Linking this to previous research published by our lab, the pattern of Wnt5a‐S expression was similar to that of vimentin, a marker of epithelial mesenchymal transition (EMT) linked to metastasis. We are currently performing immunohistochemistry (IHC) and laser microdissection (LMD) to visualize the location of Wnt5a isoform expression in human UC biopsies from both low grade and high grade tumors. Our hypothesis is that high grade tumors will express higher levels of the Wnt5a‐S isoform than low grade tumors; in contrast, high grade tumors will express lower levels of Wnt5a‐L than low grade tumors. An affirmative outcome of our hypothesis would suggest that the Wnt5a‐S isoform promotes oncogenic properties in UC, while the Wnt5a‐L isoform promotes tumor suppressive properties. Future experiments will be aimed at determining the molecular pathway(s) through which each isoform acts in order to advance diagnostic, prognostic, and therapeutic approaches to UC. Support or Funding Information Funding and support were provided in part by Ohio University Heritage College of Osteopathic Medicine and its Research and Scholarly Advancement Fellowship (RSAF), the Diabetes Institute at Ohio University, as well as by an Ohio University Student Enhancement Award (SEA). This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Cell adhesion mediated by selectins (expressed by activated endothelium, activated platelets, and leukocytes) binding to their resepective selectin ligands (expressed by cancer cells) may be involved in metastasis. Therefore, methods of characterizing selectin ligands expressed on human tissue may serve as valuable assays. Presented herein is an innovative method for detecting functional selectin ligands expressed on human tissue that uses a dynamic approach, which allows for control over the force applied to the bonds between the probe and target molecules. This new method of tissue interrogation, known as dynamic biochemical tissue analysis (DBTA), involves the perfusion of molecular probe-coated microspheres over tissues. DBTA using selectin-coated probes is able to detect functional selectin ligands expressed on tissue from multiple cancer types at both primary and metastatic sites.

Background and aims: Wnt5a is a highly studied member of the Wnt family and recently has been implicated in the pathogenesis of atherosclerosis, but its precise role is unknown. Foam cell development is a critical process to atherosclerotic plaque formation. In the present study, we investigated the role of noncanonical Wnt5a signaling in the development of foam cells. Methods: Human carotid atherosclerotic tissue and THP-1-derived macrophages were used to investigate the contribution of Wnt5a signaling in the formation of foam cells. Immunohistochemistry was used to evaluate protein expression of scavenger receptors and noncanonical Wnt5a receptors [frizzled 5 (Fz5) and receptor tyrosine kinase-like orphan receptor 2 Ror2)] in human atherosclerotic macrophages/foam cells. Changes in protein expression in response to Wnt5a stimulation/inhibition were determined by Western blot, and lipid accumulation was evaluated by fluorescent lipid droplet staining. Results: Wnt5a (P<.05), Fz5 (P < 0.1) and Ror2 (P<.01) were significantly expressed in advanced atherosclerotic lesions compared to less advanced lesions (N=10).Wnt5a,Fz5, and Ror2 were expressed in macrophages/foam cells within the plaque. In vitro studies revealed that Wnt5a significantly increased the expression of the lipid uptake receptorCD36 (P<.05) but not the lipid efflux receptor ATP-binding cassette transporter (P>.05). rWnt5a also significantly increased lipid accumulation in MP-1 macrophages (P<.05). Furthermore, inhibition of Wnt5a signaling with Boxy prevented lipid accumulation (P<.01) and prevented CD36 up-regulation (P<.01). Conclusions: These results suggest a direct role for Wnt5a signaling in the pathogenesis of atherosclerosis, specifically the accumulation of lipid in macrophages and the formation of foam cells. (C) 2018 Elsevier Inc. All rights reserved

Chronic diseases account for approximately 45% of all deaths in developed countries and are particularly prevalent in countries with the most sophisticated and robust public health systems. Chronic metabolic diseases, specifically lifestyle-related diseases pertaining to diet and exercise, continue to be difficult to treat clinically. The most prevalent of these chronic metabolic diseases include obesity, diabetes, non-alcoholic fatty liver disease, chronic kidney disease and cardiovascular disease and will be the focus of this review. Wnt proteins are highly conserved glycoproteins best known for their role in development and homeostasis of tissues. Given the importance of Wnt signalling in homeostasis, aberrant Wnt signalling likely regulates metabolic processes and may contribute to the development of chronic metabolic diseases. Expression of Wnt proteins and dysfunctional Wnt signalling has been reported in multiple chronic diseases. It is interesting to speculate about an interrelationship between the Wnt signalling pathways as a potential pathological mechanism in chronic metabolic diseases. The aim of this review is to summarize reported findings on the contrasting roles of Wnt signalling in lifestyle-related chronic metabolic diseases; specifically, the contribution of Wnt signalling to lipid accumulation, fibrosis and chronic low-grade inflammation.

Bladder cancer is the fourth most common cancer in men and the most common malignancy of the urinary tract. Bladder cancers detected at an early stage have a very high five-year survival rate, but when detected after local metastasis the rate is only about 50%. Our group recently reported a positive correlation between the expression of Wnt5a, a member of the Wnt proteins family, and histopathological grade and stage of urothelial carcinoma (UC). The objective of this study was to analyze UC cases reported in Athens, Ohio and investigate the major components of Wnt5a / planar cell polarity (PCP) signaling pathway in UC human tissue samples and UC cell lines. Formalin fixed and paraffin embedded transurethral resection tissues were immunostained for Wnt5a, Ror-2, CTHRC1 and E-cadherin. In addition, in vitro studies using UC cell lines were investigated for Wnt5a/PCP signaling and epithelial mesenchymal transition (EMT) gene expression. The IHC results showed a correlation between the expression of Wnt5a, Ror2 and CTHRC1 with high histological grade of the tumor, while E-cadherin showed an opposite trend of expression. Real time RT-PCR results showed that RNA expression of the Wnt5a/ PCP pathway genes vary in low and high grade UC cell lines and that the high grade cell lines exhibited signs of EMT. These findings support that Wnt5a-Ror2 signaling plays a role in UC, support the potential use of Wnt5a as a prognostic marker and provide evidence that Wnt5a signaling may be used as an effective molecular target for novel therapeutic tools.

Heightened co-expression and dysregulated signaling associated with Tolllike receptor 3 (TLR3) and Wnt5a is an integral component of solid tumors and hematological malignancies. Our previous findings in pancreatic cancer and melanoma suggest that inhibition of these pathways by a TLR3 signaling inhibitor, phenylmethimazole (C10), results in significantly decreased IL-6 levels, STAT3 phosphorylation, minimal cancer cell migration and reduced cancer cell growth in vitro and in vivo. In this study, we extended our earlier observations by performing studies in human breast cancer cells. We found that human MCF-7 breast cancer cells express high basal levels of TLR3 and Wnt5a RNA. C10 treatment resulted in significantly decreased TLR3 and Wnt5a expression levels. This functionally translated into significantly reduced IL-6 levels and STAT3 phosphorylation in vitro. In addition, the inhibition of this signaling cascade by C10 further resulted in decreased cell viability and migration of MCF-7 cells. Strikingly, the combination of C10 and tamoxifen, the standard of care therapy for breast cancer, further decrease cancer cell growth better than either agent alone. These data support the novel finding that inhibition of TLR3 signaling in combination with tamoxifen, may increase the effectiveness of current treatments of breast cancer.

A growing body of evidence suggests that L-selectin ligands presented on circulating tumor cells facilitate metastasis by binding L-selectin presented on leukocytes. Commonly used methods for detecting L-selectin ligands on tissues, e.g., immunostaining, are performed under static, no-flow conditions. However, such analysis does not assay for functional L-selectin ligands, specifically those ligands that promote adhesion under shear flow conditions. Recently our lab developed a method, termed dynamic biochemical tissue analysis (DBTA), to detect functional selectin ligands in situ by probing tissues with L-selectin-coated microspheres under hemodynamic flow conditions. In this investigation, DBTA was used to probe human colon tissues for L-selectin ligand activity. The detection of L-selectin ligands using DBTA was highly specific. Furthermore, DBTA reproducibly detected functional L-selectin ligands on diseased, e.g., cancerous or inflamed, tissues but not on noncancerous tissues. In addition, DBTA revealed a heterogeneous distribution of functional L-selectin ligands on colon cancer tissues. Most notably, detection of L-selectin ligands by immunostaining using HECA-452 antibody only partially correlated with functional L-selectin ligands detected by DBTA. In summation, the results of this study demonstrate that DBTA detects functional selectin ligands to provide a unique characterization of pathological tissue.

The presence of functional P-selectin ligands is well-documented for human colon cancer cell lines, but not in situ on human colon carcinoma tissue. Presently, immunostaining with antibodies is used to detect critical components of selectin ligands, e.g., sialofucosylated moieties. However, this static biochemical tissue analysis (SBTA) cannot ascertain if a potential selectin ligand is able to mediate (rolling) adhesion. Due to the immense difficulty in detecting functional selectin ligands using traditional methods, we have developed a flow-based assay known as dynamic biochemical tissue analysis (DBTA) for detecting functional selectin ligands expressed on human tissue. DBTA using P-selectin microspheres was performed on colon cancer tissue sections from multiple cases, in conjunction with SBTA using P-selectin, antibodies against purported selectin ligand carbohydrate moieties sLeX and sLeA (HECA-452, CSLEX-1, and KM-231), and antibodies against peptide structures of putative P-selectin ligands (CD24, CD44, and PSGL-1). Examination of serial sections, in the same regions of tissue displaying DBTA probe adhesion, revealed significant detection inconsistencies with SBTA. Subsequently, due to the well-documented force-dependency of selectin ligands, DBTA was conducted with microspheres coated with either HECA-452, CSLEX-1, or KM-231 to determine the effect of applied force on the detection capabilities of these antibodies. Analysis of signet ring cell colon carcinoma tissue revealed microspheres coated with HECA-452, CSLEX-1, or KM-231 antibodies all displayed significantly lower amounts of adhesion than the DBTA P-selectin microspheres. Interestingly, although microspheres coated with these antibodies adhered to signet ring cell carcinoma tissue, these DBTA probes did not interact with all regions of tissue that displayed adhesion with P-selectin microspheres. Specificity of interaction was validated using corresponding isotype control coated microspheres. Taken together, these results show DBTA with P-selectin coated microspheres is able to unequivocally detect functional P-selectin ligands, in contrast to SBTA (immunostaining) and DBTA using microspheres coated with antibodies. In summary, DBTA using P-selectin coated microspheres is able to detect functional P-selectin ligands expressed on colon cancer tissue, data that may provide valuable diagnostic and prognostic information for malignant tumors. Citation Format: Eric W. Martin, Ramiro Malgor, Vicente A. Resto, Douglas J. Goetz, Monica M. Burdick. Detection of functional P-selectin ligands expressed on colon cancer tissue using a novel flow-based assay. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4944.

Oxidative stress contributes substantially to the pathophysiology of diabetic nephropathy (DN). Consumption of an antioxidant-fortified (AO) diet from an early age prevents or delays later development of DN in the Zucker rat female with type 2 diabetes. We hypothesize this is due to effects on mesangial matrix and renal nitric oxide synthase (NOS) distribution and to sex-specific differences in NOS responses in the diabetic kidney. Total glomerular tuft area (GTA) and PAS-positive tuft area (PTA), endothelial (e), neuronal (n) and inducible (i) NOS were quantified in males and females on AO or regular (REG) diet at 6 and 20 weeks of age. eNOS was observed in glomeruli and tubules. nNOS predominantly localized to tubular epithelium in both cortex and medulla. iNOS was expressed in proximal and distal tubules and collecting ducts. Sex, diabetes duration and AO diet affected the distribution of the three isoforms. GTA and PTA increased with duration of hyperglycemia and showed a negative correlation with renal levels of all NOS isoforms. AO diet in both genders was associated with less PAS-positive staining and less mesangial expansion than the REG diet, an early increase in cortical iNOS in males, and sex-specific changes in cortical eNOS at 20 weeks. These effects of AO diet may contribute to sex-specific preservation of renal function in females.