Abstract Hepatocellular carcinoma (HCC) is hallmarked by inflammatory cell infiltration. Chemokines secreted during inflammation are crucial at directing immune cell infiltration during cancer development. We screened patient dataset from different etiologies including HBV, HCV, alcohol and non-alcoholic steatohepatitis (NASH) and identified CXCL5 as the only chemokine consistently upregulated in HCC. Immunohistochemistry (IHC) of patient liver tumor tissues further showed the production of CXCL5 by tumor surrounding non parenchymal cells. Using a mouse model (Pte loxP/loxP; Alb-Cre+, Pten null) which recapitulates human liver disease progression from non-alcoholic fatty liver disease (NAFLD) to NASH to liver cancer, we found that CXCL5 was expression was gradually increased during disease progression. Further analysis using IHC and flow cytometry analysis showed that CXCL5 is predominantly expressed by Kupffer cells in Pten null livers bearing tumors. Furthermore, we found that LPS stimulated the expression and secretion of CXCL5 in Kupffer cells, but not other macrophages including peritoneal macrophage and macrophage cell line Raw264.7 cells. We hypothesized that the Kupffer cell-secreted CXCL5 in response to LPS promotes tumor progression by promoting HCC cell proliferation and regulating HCC associated immune cell infiltration. To investigate whether CXCL5 increases tumor cell proliferation, we treated wild type and Pten null hepatocytes with various concentrations of CXCL5. Our results showed that CXCL5 increased hepatocytes viability in a dose dependent manner. BrdU incorporation assay further confirmed that CXCL5 treatments induces HCC cell proliferation through its receptor CXCR2. We further demonstrated that CXCR2 knockdown led to reduced xenograft tumor growth using HepG2 cells. Additionally, we injected Pten null mice with CXCR2 inhibitor to block CXCL5 signal and observed partial response. We analyzed the immune cell profile of the responder mice vs. non-responder mice and showed that tumor inhibition is correlated with T cell infiltration. To test whether Kupffer cells regulate T cells through CXCL5, we isolated primary Kupffer cells from murine livers. We performed transwell migration assay using immune cells from murine spleens. Our results indicate that LPS stimulation in Kupffer cells reduced the ratio of migrated CD4+ T cells, while blocking CXCL5 using a CXCL5 antibody significantly increased CD4+ T cell recruitment to the LPS-stimulated Kupffer cells and moderately decreased neutrophil recruitment. Consistently, we found that recombinant CXCL5 protein reduces CD4+ T cell migration independent of neutrophils. In summary, our study suggests that Kupffer cell secreted CXCL5 reduces liver CD4+ T cell recruitment and increases liver cancer cell proliferation. Citation Format: Taojian Tu, Handan Hong, Lina He, Mario Alba, Tang Qi, Curtis T. Okamoto, Bangyan L. Stiles. Dual role of Kupffer cell secreted CXCL5 in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5328.

A major component of the extracellular matrix (ECM), laminins, modulates cells via diverse receptors. Their fragments have emerging utility as components of "ECM-mimetics" optimized to promote cell-based therapies. Recently, we reported that a bioactive laminin peptide known as A99 enhanced cell binding and spreading via fusion to an elastin-like polypeptide (ELP). The ELP "handle" serves as a rapid, noncovalent strategy to concentrate bioactive peptide mixtures onto a surface. We now report that this strategy can be further generalized across an expanded panel of additional laminin-derived elastin-like polypeptides (LELPs). A99 (AGTFALRGDNPQG), A2G80 (VQLRNGFPYFSY), AG73 (RKRLQVQLSIRT), and EF1m (LQLQEGRLHFMFD) all promote cell spreading while showing morphologically distinct F-actin formation. Equimolar mixtures of A99:A2G80-LELPs have synergistic effects on adhesion and spreading. Finally, three of these ECM-mimetics promote the neurite outgrowth of PC-12 cells. The evidence presented here demonstrates the potential of ELPs to deposit ECM-mimetics with applications in regenerative medicine, cell therapy, and tissue engineering.

Liver carcinoma is the 6th most prevalent cancer worldwide in 2020. Moreover, it is the 3rd leading cause of cancer related deaths. In addition to the genomic and transcriptomic heterogeneity of liver tumor cells which is recognized as a major driver in liver cancer progression, the liver immune system is also fundamental to liver carcinogenesis and presents a promising target for therapy. The liver immune response is orchestrated by cytokines and chemokines. Recent studies suggest that chemokines not only recruit immune cells but also regulate various liver functions. In partial hepatectomy, CXCL2 has been shown to promote hepatocyte proliferation. CXCL1, 2, 5 and 8 can induce endothelial cells chemotaxis to promote angiogenesis through binding to CXCR2. These diverse functions suggest that chemokines could play multifaceted roles in liver cancer development. However, chemokines that are commonly associated with liver cancer is still unknown.We analyzed HCC patient data from the GEO database, and we categorized the datasets based on HCC etiologies including HBV, HCV, alcoholic and NASH. We identified CXCL5 as the only chemokine consistently upregulated in HCC with different etiologies compared to healthy or cirrhotic livers. Immunohistochemistry (IHC) analysis reveals that CXCL5 was produced by immune cells but not tumor cells in human HCC tissues. To further study HCC associated CXCL5 expression, the liver-specific Pten deletion mouse model (PM mice) that recapitulates NAFLD-NASH-HCC progression was used. A gradual increase of hepatic CXCL5 expression is observed during HCC development, reaching nearly 100-fold upregulation of CXCL5 mRNA expression in 12-month-old PM mice livers carrying tumors. Examination of liver immune cell populations showed that macrophages were significantly enriched in Pten deleted livers bearing tumors than wild type livers without tumors. Flow cytometry and IHC analysis further identified Kupffer cells (KCs), the liver resident macrophages as the source of CXCL5 in tumor bearing livers using these mice. Since increased LPS is a prominent feature in most chronic liver diseases, we isolated and treated mouse KCs with LPS and found that LPS treatment robustly increased CXCL5 expression by nearly 20-fold. Interestingly, neither murine macrophage cell lines nor primary peritoneal macrophages displayed induced CXCL5 expression in response to LPS. These data suggest that induction of CXCL5 in KCs is likely a unique function of the KCs but not of other macrophages. To explore the function of CXCL5 in HCC development, we treated mouse hepatocytes and HCC cells with CXCL5 and showed that CXCL5 induces the proliferation of these cells. This effect is further blocked by the inhibition of CXCR2, the receptor of CXCL5, demonstrating the specificity for CXCL5 mediated effects. Together we show here for the first time that CXCL5 expression is a unique property of Kupffer cells and the induction of CXCL5 promotes HCC progression. Citation Format: Taojian Tu, Handan Hong, Lina He, Mario Alba, Curtis T. Okamoto, Bangyan L. Stiles. Kupffer cells secrete CXCL5 to promote liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1350.

Monday, April 27April 14, 2020Free AccessBiomarkers for Parkinson’s Disease with Reflex Tears Correlate with Disease Duration (4417)Mark Lew, Srikanth Janga, Danielle Feigenbaum, Dan Freire, Maria Edman, Wendy Mack, Curtis Okamoto, and Sarah Hamm-AlvarezAuthors Info & AffiliationsApril 14, 2020 issue94 (15_supplement)https://doi.org/10.1212/WNL.94.15_supplement.4417 Letters to the Editor

Purpose: To analyze the changes in the lacrimal gland (LG) miRNAome from male nonobese diabetic (NOD) mice with autoimmune dacryoadenitis compared with LG from healthy male BALB/c and dacryoadenitis-free female NOD mice. Methods: LG from these mice were collected for small RNA sequencing to identify dysregulated miRNAs; hits were validated by RT-qPCR in male NOD and BALB/c LG. Dysregulation of validated species within immune cell–enriched cell fractions and epithelial-enriched cell fractions from LG was probed by RT-qPCR. Ingenuity pathway analysis identified putative miRNA targets, which were examined in publicly available mRNA-seq datasets. Western blotting and confocal imaging of immunofluorescence enabled validation of some molecular changes at the protein level. Results: Male NOD LG exhibited 15 and 13 significantly up- and downregulated miRNAs, respectively. Dysregulated expression of 14 of these miRNAs (9 upregulated, 5 downregulated) was validated in male NOD versus BALB/c LG by RT-qPCR. Seven of the upregulated miRNAs were increased owing to their abundance in immune cell–enriched cell fractions, whereas four downregulated miRNAs were largely expressed in epithelial-enriched cell fractions. Ingenuity pathway analysis predicted the upregulation of IL-6 and IL-6–like pathways as an outcome of miRNA dysregulation. Increased expression of several genes in these pathways was confirmed by mRNA-seq analysis, whereas immunoblotting and immunofluorescence confirmed Ingenuity pathway analysis–predicted changes for IL-6Rα and gp130/IL-6st. Conclusions: Male NOD mouse LG exhibit multiple dysregulated miRNAs owing to the presence of infiltrating immune cells, and decreased acinar cell content. The observed dysregulation may increase IL-6Rα and gp130/IL-6st on acini and IL-6Rα on specific lymphocytes, enhancing IL-6 and IL-6–like cytokine signaling.

Dynamin (DNM) is a family of large GTPases possessing a unique mechanical ability to "pinch" off vesicles entering cells. DNM2 is the most ubiquitously expressed member of the DNM family. We developed a novel tool based on elastin-like polypeptide (ELP) technology to quickly, precisely, and reversibly modulate the structure of DNM2. ELPs are temperature-sensitive biopolymers that self-assemble into microdomains above sharp transition temperatures. When linked together, DNM2 and a temperature-sensitive ELP fusion organize into a range of distinct temperature-dependent structures above a sharp transition temperature, which were not observed with wild-type DNM2 or a temperature-insensitive ELP fusion control. The structures comprised three different morphologies, which were prevalent at different temperature ranges. The size of these structures was influenced by an inhibitor of the DNM2 GTPase activity, dynasore; furthermore, they appear to entrap co-expressed cytosolic ELPs. Having demonstrated an unexpected diversity of morphologically distinct structures, DNM2-ELP fusions may have applications in the exploration of dynamin-dependent biology.

Objective The tear miRNAome of the male NOD mouse, a model of ocular symptoms of Sjögren’s syndrome (SS), was analyzed to identify unique miRNAs. Methods Male NOD mice, aged 12-14 weeks, were used to identify tear miRNAs associated with development of autoimmune dacryoadenitis. Age- and sex-matched male BALB/c mice served as healthy controls while age-matched female NOD mice that do not develop the autoimmune dacryoadenitis characteristic of SS were used as additional controls. Total RNA was isolated from stimulated tears pooled from 5 mice per sample and tear miRNAs were sequenced and analyzed. Putative miRNA hits were validated in additional mouse cohorts as well as in tears of SS patients versus patients with another form of dry eye disease, meibomian gland disease (MGD) using qRT-PCR. The pathways influenced by the validated hits were identified using Ingenuity Pathway Analysis. Results In comparison to tears from both healthy (male BALB/c) and additional control (female NOD) mice, initial analy1sis identified 7 upregulated and 7 downregulated miRNAs in male NOD mouse tears. Of these, 8 were validated by RT-qPCR in tears from additional mouse cohorts. miRNAs previously implicated in SS pathology included mmu-miR-146a/b-5p, which were significantly downregulated, as well as mmu-miR-150-5p and mmu-miR-181a-5p, which were upregulated in male NOD mouse tears. All other validated hits including the upregulated miR-181b-5p and mmu-miR-203-3p, as well as the downregulated mmu-miR-322-5p and mmu-miR-503-5p, represent novel putative indicators of autoimmune dacryoadenitis in SS. When compared to tears from patients with MGD, miRNAs hsa-miR-203a-3p, hsa-miR-181a-5p and hsa-miR-181b-5p were also significantly increased in tears of SS patients. Conclusions A panel of differentially expressed miRNAs were identified in tears of male NOD mice, with some preliminary validation in SS patients, including some never previously linked to SS. These may have potential utility as indicators of ocular symptoms of SS; evaluation of the pathways influenced by these dysregulated miRNAs may also provide further insights into SS pathogenesis.

Although natriuretic peptides (NPs) are established as a biomarker of heart failure (HF), NPs have been attracting attention as a mediator in the metabolic sequences recently. On the other hand, metabolic disorders including insulin resistance have been suggested to be involved in left ventricular (LV) concentric remodeling, hinting us to the unexpected relationship among NPs, insulin resistance and LV concentric remodeling. To investigate whether the basal B-type natriuretic peptide (BNP) level is linked to insulin resistance or LV concentric remodeling in the participants independent of HF in the Japanese Arita-cho cohort study. Among 1632 subjects who participated in annual health checks from 2005 to 2008 in Arita-cho, Saga, Japan as a cohort study, we studied 675 subjects without history of cardiovascular disease with LV ejection fraction≥50% and BNP level<35pg/ml (227 men; median 62 years old). Insulin resistance was assessed by homeostatic model assessment of insulin resistance (HOMA-IR) and LV geometry including LV concentric remodeling was classified based on relative wall thickness (RWT) and LV mass index from echocardiographic findings. The tertile levels of BNP were inversely associated with HOMA-IR (the 1st tertile 1.33 (0.76–1.74), the 2nd tertile 1.05 (0.72–1.59), the 3rd tertile 0.95 (0.66–1.58), p=0.005); in the logistic regression analysis, the lower BNP level was related to the prevalence of insulin resistance defined as HOMA-IR≥1.37 after full multivariate adjustment (1 SD increment of BNP: adjusted odds ratio [aOR] 0.740, 95% confidence interval 0.601–0.912, P=0.005. LV concentric remodeling (RWT >0.42 and LV mass index ≤115 g/m2 in men and ≤95 g/m2 in women) was observed in 170 (25%) subjects; both low BNP level and higher insulin resistance were independently linked with LV concentric remodeling after multivariate adjustment (1 SD increment of BNP: aOR 0.714, 95% CI 0.544–0.938, p=0.015, HOMA-IR≥1.37 vs. <1.37: aOR 1.694, 95% CI 1.004–2.857, p=0.048, respectively) (Figure). In the cohort without HF, the low BNP level was linked to insulin resistance and LV concentric remodeling independently, suggesting that the subjects with low NPs levels may cause metabolic disorders and LV morphological abnormalities. Type of funding sources: None.

The extracellular matrix (ECM) is comprised of a large network of proteins that are essential for tissue development and repair. A bioactive RGD-containing peptide from laminin alpha 1 chain, A99 (AGTFALRGDNPQG), promotes strong cell attachment and has demonstrated utility in cell culture and tissue engineering. Various materials can be utilized as a scaffold for bioactive peptides; however, it may be advantageous to design materials that use bioconjugation strategies that do not affect bioactivity, generate homogenous products, and can be produced at scale. This report is the first to compare the methods for preparing chemically conjugated and recombinant A99 to elastin-like polypeptides (ELPs) as the scaffold and characterize the biological and cell attachment activity using human dermal fibroblasts (HDFs). ELPs are biocompatible protein-polymers that are also thermo-responsive. Below a lower critical solution temperature (LCST), they are highly soluble. Above the LCST, ELPs phase separate into a polymer-rich liquid, known as a coacervate. Both chemically conjugated and recombinant fusion between A99 and an ELP (A99-ELP-R) show dose-dependent cell attachment. In addition, coating above the LCST provides better cell spreading compared to coating at 4 degrees C. ELPs provide an excellent structural framework for deposition of bioactive peptides of the ECM, and their intrinsic biophysical properties make laminin peptide-ELPs promising biomaterials for cell culture and tissue engineering.

Abstract Introduction Atrial functional mitral regurgitation (A-FMR) has been under-recognized until recently as a cause of FMR, and the prognostic difference between A-FMR and ventricular FMR (V-FMR) has not been fully elucidated. As there has been different mechanisms of FMR suggested in A-FMR and V-FMR, we hypothesized that prognosis and prognostic predictors of A-FMR may differ from those of V-FMR. Purpose To investigate the prognosis and prognostic predictors of A-FMR in comparison with V-FMR. Methods Among 1312 consecutive patients with grade 3+ (moderate to severe) or 4+ (severe) MR, 378 consecutive FMR patients were identified by excluding patients with degenerative MR, previous cardiac surgery, or concomitant aortic valve disease and/or mitral stenosis. FMR with ejection fraction (EF) <40% or FMR due to regional wall motion abnormalities with leaflet tethering were classified as V-FMR (N=288), and FMR due to left atrial (LA) and/or annular dilatation with preserved or mid-range EF (≥40%) were classified as A-FMR (N=90). All-cause death and heart failure hospitalization were analyzed as cardiovascular (CV) events in this study. Surgical or percutaneous mitral valve intervention without CV events was handled as not reaching an endpoint and these cases were censored. Results A-FMR were significantly older (76 [69–82] vs. 70 [58–77] years), higher rates of female (64 vs. 35%) and atrial fibrillation (88 vs. 42%), and lower B-type natriuretic peptide (BNP) values (169 [101–318] vs. 447 [213–952] pg/ml) compared to V-FMR (all P<0.05). On echocardiography, LV end-diastolic and end-systolic dimensions (52 [48–57] vs. 64 [58–72] mm, 34 [31–37] vs. 55 [48–64] mm), respectively) were smaller, and EF (55 [50–60] vs. 28 [19–35] %) and LA volume (99 [73–137] vs. 73 [57–91] ml/m2) were larger in A-FMR (all P<0.05). Effective regurgitant orifice area (32 [26–40] vs. 31 [24–45] mm2) and regurgitant volume (50±15 vs. 52±16 ml) were similar (both n.s.). During a median follow up of 1407 days, 206 (54%) patients developed CV events. Kaplan-Meier analysis revealed that V-FMR had a significantly higher rates of CV events compared to A-FMR (Figure) with adjusted hazard ratio (HR) of 1.762 [1.168–2.660], P=0.007 after adjusted for variables including age, sex, New York Heart Association functional class, previous heart failure hospitalization, estimated glomerular filtration rate (eGFR) and BNP. Further, stepwise multivariate analysis showed that independent prognostic predictors of A-FMR were LA volume and eGFR, while those for V-FMR were LA volume, age, and LV end-systolic dimension. Conclusions A-FMR had relatively better prognosis compared to V-FMR, and there were different prognostic predictors between A-FMR and V-FMR. Our results suggest that different treatment strategies need to be considered between A-FMR and V-FMR. The Kaplan-Meier life table Funding Acknowledgement Type of funding source: None

Protein-protein interactions are key to define the function of nucleotide binding domain (NBD) and leucine-rich repeat (LRR) family, pyrin domain (PYD)-containing protein 12 (NLRP12).cDNA encoding the human PYD + NBD of NLRP12 was used as bait in a yeast two-hybrid screen with a human leukocyte cDNA library as prey.Hematopoiesis cell kinase (HCK), a member of the c-SRC family of non-receptor tyrosine kinases, was among the top hits.The C-terminal 40 amino acids of HCK selectively bound to NLRP12's PYD + NBD, but not to that of NLRP3 and NLRP8.Amino acids F503, I506, Q507, L510, and D511 of HCK are critical for the binding of HCK's C-terminal 40 amino acids to NLRP12's PYD + NBD.Additionally, the C-terminal 30 amino acids of HCK are sufficient to bind to NLRP12's PYD + NBD, but not to its PYD alone nor to its NBD alone.In cell lines that express HCK endogenously, it was coimmunoprecipitated with stably expressed exogenous NLRP12.Also, NLRP12 co-immunoprecipitated and co-localized with HCK when both were overexpressed in 293T cells.In addition, in this overexpression system, steady-state NLRP12 protein expression levels significantly decreased when HCK was co-expressed.Bioinformatic analysis showed that HCK mRNA co-occurred with NLRP12 mRNA, but not with other NLRP mRNAs, in blood and marrow samples from acute myeloid leukemia (AML) patients.The mRNA of NLRP12 is also co-expressed with HCK in AML patient samples, and the levels of mRNA expression of each are correlated.Together these data suggest that NLRP12, through its binding to HCK, may have an effect on the pathogenesis of AML.

The extracellular matrix (ECM) is comprised of a large network of proteins essential for providing a physical scaffold as well as biochemical and biomechanical signals for tissue development and repair. Laminins are a major component of the ECM and have a diverse set of biological roles such as cell adhesion, migration, proliferation and differentiation. Laminin α1 chain bioactive RGD containing peptide, A99 (AGTFALRGDNPQG), which was previously identified to promote strong cell attachment has demonstrated utility for cell adhesion applications. A wide array of biomaterials has been created to be used as scaffolds for these types of bioactive peptides, but many are not suited for various reasons such as scalability, convenient reaction conditions or lack of homogeneity. Here, we are the first to describe methods for preparing chemically conjugated and recombinant A99 to elastin‐like polypeptides (ELPs) as the scaffold and characterize their behavior as well as evaluate the coating efficiency, cell attachment and spreading activity. These data suggest that the A99‐ELP fusion has great potential for use as a biomaterial in cell culture applications as they greatly promote cell adhesion and spreading. ELPs are biocompatible protein‐polymers that are thermo‐responsive and self‐assemble in response to heat. Below the lower critical solution temperature (LCST), they are highly soluble but above the LCST, ELPs phase separate, forming coacervates. They provide an excellent structural framework for bioactive peptides and their intrinsic biophysical characteristics allow for simple production with high yields and purity making them an ideal biomaterial for cell adhesion applications.Support or Funding InformationThis research is sponsored by the Japan Society for the Promotion of Science (JSPS), PE18045.

Sjögren's Syndrome (SS) is an autoimmune disease characterized by lymphocytic infiltration and loss of function of moisture-producing exocrine glands as well as systemic inflammation. SS diagnosis is cumbersome, subjective and complicated by manifestation of symptoms that overlap with those of other rheumatic and ocular diseases. Definitive diagnosis averages 4-5 years and this delay may lead to irreversible tissue damage. Thus, there is an urgent need for diagnostic biomarkers for earlier detection of SS. Extracellular vesicles called exosomes carry functional small non-coding RNAs which play a critical role in maintaining cellular homeostasis via transcriptional and translational regulation of mRNA. Alterations in levels of specific exosomal miRNAs may be predictive of disease status. Here, we have assessed serum exosomal RNA using next generation sequencing in a discovery cohort of the NOD mouse, a model of early-intermediate SS, to identify dysregulated miRNAs that may be indicative of SS. We found five miRNAs upregulated in serum exosomes of NOD mice with an adjusted p < 0.05-miRNA-127-3p, miRNA-409-3p, miRNA-410-3p, miRNA-541-5p, and miRNA-540-5p. miRNAs 127-3p and 541-5p were also statistically significantly upregulated in a validation cohort of NOD mice. Pathway analysis and existing literature indicates that differential expression of these miRNAs may dysregulate pathways involved in inflammation. Future studies will apply these findings in a human cohort to understand how they are correlated with manifestations of SS as well as understanding their functional role in systemic autoimmunity specific to SS.

Tears are a known source of biomarkers for both ocular and systemic diseases with particular advantages; specifically, the noninvasiveness of sample collection and a unique and increasingly better-defined protein composition. Here, we discuss our rationale for use of tears for discovery of biomarkers for Parkinson's disease (PD). These reasons include literature supporting changes in tear flow and composition in PD, and the interconnections between the ocular surface system and neurons affected in PD. We highlight recent data on the identification of tear biomarkers including oligomeric α-synuclein, associated with neuronal degeneration in PD, in tears of PD patients and discuss possible sources for its release into tears. Challenges and next steps for advancing such biomarkers to clinical usage are highlighted.

Caveolae are membrane organelles formed by submicrometer invaginations in the plasma membrane and are involved in mechanosensing, cell signaling, and endocytosis. Although implicated broadly in physiology and pathophysiology, better tools are required to elucidate the precise role of caveolar processes through selective activation and inactivation of their trafficking. Our group recently reported that thermally responsive elastin-like polypeptides (ELPs) can trigger formation of genetically engineered protein microdomains functionalized with either clathrin-light chain or the epidermal growth factor receptor. This manuscript is the first report of this strategy to modulate Caveolin-1 (CAV1). Through attachment of different ELP sequences to CAV1, mild heating can be used to self-assemble CAV1-ELP microdomains inside of cells. The temperature of self-assembly can be controlled by tuning the ELP sequence. The formation of CAV1-ELP microdomains internalizes cholera toxin subunit B, a commonly used marker of caveolae mediated endocytosis. CAV1-ELPs also colocalize with Cavin-1, an essential component of functional caveolae biogenesis. With the emerging significance of caveolae in health and disease and the lack of specific probes to rapidly and reversibly affect caveolar function, CAV1-ELP microdomains are a new tool to rapidly probe caveolae associated processes in endocytosis, cell signaling, and mechanosensing

Aim: Secretion of proteins into basal tears of Parkinson's disease (PD) patients may be altered by changes in nerve function. Materials & methods: Oligomeric α-SynOligo and total α-SynTotal, CCL-2, DJ-1, LF and MMP-9 were measured in basal tears from 93 PD patients and 82 age- and sex-equivalent healthy controls. Results: α-SynTotal was decreased (p = 0.0043), whereas α-SynOligo (p < 0.0001) and the ratio of α-SynOligo/α-SynTotal (p < 0.0001) were increased in basal tears from PD patients compared with healthy controls. Area under receiver-operating curves of α-SynOligo and α-SynOligo/α-SynTotal contents were 0.70 (95% confidence limits: 0.621-0.774) and 0.72 (95% confidence limits: 0.642-0.792). Conclusion: PD patient basal tears may contain biomarkers that can be assayed noninvasively and inexpensively.

Objective: To evaluate whether the protein composition of reflex tears differs in individuals with Parkinson’s disease (PD) versus Healthy Controls (HC) Background: Lacrimal glands are highly innervated by cholinergic and other neurons, and tear fluid secreted by lacrimal glands is greatly stimulated by parasympathetic and sympathetic neural pathways. The production, packaging and secretion of specific proteins into tears may change in response to alterations in nerve function to lacrimal glands and cornea, which may be tested by collection of reflex tears. Reflex tears are stimulated tears collected with an unanesthetized Schirmer’s test. We have previously reported results with basal tears/anesthetized Schirmer’s test. Design/Methods: Reflex tears were collected from 85 PD patients and 80 age-and sex-matched HC using an unanesthetized Schirmer’s test. Samples were pooled from both eyes for analysis of alpha-synuclein, CC chemokine ligand 2 (CCL-2) and total protein. Values were measured by ELISA or multiplex ELISA. Results: Oligomeric alpha-synuclein was significantly increased by 4.7-fold in PD tears (4.21 ± 0.50 ng/mg tear protein) relative to HC (0.90 ± 0.13 ng/mg tear protein) (p Conclusions: Oligomeric synuclein levels enable discrimination between reflex tears of PD patients and HC. CCL-2 may represent an additional biomarker. The origin of sex differences in biomarker values requires further study. Disclosure: Dr. Lew has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Teva, US World Meds, UCB, Lundbeck, AbbVie, Adamas Pharmaceuticals, Cynapsus, Revance, Acadia Pharmaceuticals, Neurocrine Biosciences, and Acorda Therapeutics. Dr. Lew has received research support from Parkinson’s Study Group, Michael J. Fox Foundation, Biotie, Neuroderm, Enterin Inc., Pharm2B Fellowship Grants, Allergan, and Medtronic. Dr. Feigenbaum has received research support from Intec, Revance, Acorda. Dr. Freire has nothing to disclose. Dr. Janga has nothing to disclose. Dr. Mack has nothing to disclose. Dr. Edman has nothing to disclose. Dr. Okamoto has nothing to disclose. Dr. Hamm-Alvarez has nothing to disclose.