Publications by authors named "Sarah F Hamm-Alvarez"

84 Publications

Rab27a Contributes to Cathepsin S Secretion in Lacrimal Gland Acinar Cells.

Int J Mol Sci 2021 Feb 5;22(4). Epub 2021 Feb 5.

Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90033, USA.

Altered lacrimal gland (LG) secretion is a feature of autoimmune dacryoadenitis in Sjögren's syndrome (SS). Cathepsin S (CTSS) is increased in tears of SS patients, which may contribute to disease. Rab3D and Rab27a/b isoforms are effectors of exocytosis in LG, but Rab27a is poorly studied. To investigate whether Rab27a mediates CTSS secretion, we utilized quantitative confocal fluorescence microscopy of LG from SS-model male NOD and control male BALB/c mice, showing that Rab27a-enriched vesicles containing CTSS were increased in NOD mouse LG. Live-cell imaging of cultured lacrimal gland acinar cells (LGAC) transduced with adenovirus encoding wild-type (WT) mCFP-Rab27a revealed carbachol-stimulated fusion and depletion of mCFP-Rab27a-enriched vesicles. LGAC transduced with dominant-negative (DN) mCFP-Rab27a exhibited significantly reduced carbachol-stimulated CTSS secretion by 0.5-fold and β-hexosaminidase by 0.3-fold, relative to stimulated LGAC transduced with WT mCFP-Rab27a. Colocalization of Rab27a and endolysosomal markers (Rab7, Lamp2) with the apical membrane was increased in both stimulated BALB/c and NOD mouse LG, but the extent of colocalization was much greater in NOD mouse LG. Following stimulation, Rab27a colocalization with endolysosomal membranes was decreased. In conclusion, Rab27a participates in CTSS secretion in LGAC though the major regulated pathway, and through a novel endolysosomal pathway that is increased in SS.
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http://dx.doi.org/10.3390/ijms22041630DOI Listing
February 2021

Intralacrimal Sustained Delivery of Rapamycin Shows Therapeutic Effects without Systemic Toxicity in a Mouse Model of Autoimmune Dacryoadenitis Characteristic of Sjögren's Syndrome.

Biomacromolecules 2020 Dec 27. Epub 2020 Dec 27.

Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California 90033, United States.

Sjögren's syndrome (SS) is an autoimmune disease associated with severe exocrinopathy, which is characterized by profound lymphocytic infiltration (dacryoadenitis) and loss of function of the tear-producing lacrimal glands (LGs). Systemic administration of Rapamycin (Rapa) significantly reduces LG inflammation in the male Nonobese Diabetic (NOD) model of SS-associated autoimmune dacryoadenitis. However, the systemic toxicity of this potent immunosuppressant limits its application. As an alternative, this paper reports an intra-LG delivery method using a depot formulation comprised of a thermoresponsive elastin-like polypeptide (ELP) and FKBP, the cognate receptor for Rapa (5FV). Depot formation was confirmed in excised whole LG using cleared tissue and observation by both laser-scanning confocal and lightsheet microscopy. The LG depot was evaluated for safety, efficacy, and intra-LG pharmacokinetics in the NOD mouse disease model. Intra-LG injection with the depot formulation (5FV) retained Rapa in the LG for a mean residence time (MRT) of 75.6 h compared to Rapa delivery complexed with a soluble carrier control (5FA), which had a MRT of 11.7 h in the LG. Compared to systemic delivery of Rapa every other day for 2 weeks (seven doses), a single intra-LG depot of Rapa representing 16-fold less total drug was sufficient to inhibit LG inflammation and improve tear production. This treatment modality further reduced markers of hyperglycemia and hyperlipidemia while showing no evidence of necrosis or fibrosis in the LG. This approach represents a potential new therapy for SS-related autoimmune dacryoadenitis, which may be adapted for local delivery at other sites of inflammation; furthermore, these findings reveal the utility of optical imaging for monitoring the disposition of locally administered therapeutics.
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http://dx.doi.org/10.1021/acs.biomac.0c01468DOI Listing
December 2020

Biosynthesized Multivalent Lacritin Peptides Stimulate Exosome Production in Human Corneal Epithelium.

Int J Mol Sci 2020 Aug 26;21(17). Epub 2020 Aug 26.

Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90033, USA.

Lacripep is a therapeutic peptide derived from the human tear protein, Lacritin. Lacripep interacts with syndecan-1 and induces mitogenesis upon the removal of heparan sulfates (HS) that are attached at the extracellular domain of syndecan-1. The presence of HS is a prerequisite for the syndecan-1 clustering that stimulates exosome biogenesis and release. Therefore, syndecan-1-mediated mitogenesis versus HS-mediated exosome biogenesis are assumed to be mutually exclusive. This study introduces a biosynthesized fusion between Lacripep and an elastin-like polypeptide named LP-A96, and evaluates its activity on cell motility enhancement versus exosome biogenesis. LP-A96 activates both downstream pathways in a dose-dependent manner. HCE-T cells at high confluence treated with 1 μM LP-A96 enhanced cell motility equipotent to Lacripep. However, cells at low density treated with 1 μM LP-A96 generated a 210-fold higher number of exosomes compared to those treated at low density with Lacripep. As monovalent Lacripep is capable of enhancing cell motility but not exosome biogenesis, activation of exosome biogenesis by LP-A96 not only suggests its utility as a novel molecular tool to study the Lacritin biology in the corneal epithelium but also implies activity as a potential therapeutic peptide that can further improve ocular surface health through the induction of exosomes.
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http://dx.doi.org/10.3390/ijms21176157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7504496PMC
August 2020

Reduced Expression of VEGF-A in Human Retinal Pigment Epithelial Cells and Human Muller Cells Following CRISPR-Cas9 Ribonucleoprotein-Mediated Gene Disruption.

Transl Vis Sci Technol 2020 07 14;9(8):23. Epub 2020 Jul 14.

USC Roski Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA.

Purpose: To evaluate the effects of vascular endothelial growth factor-A (VEGF-A) gene editing in human retinal pigment epithelial (RPE) cells and human Muller cells, which are the main VEGF-A producing cells in the eye.

Methods: CRISPR-Cas9 ribonucleoprotein was used to target exon 1 in VEGF-A gene. Lipofectamine CRISPRMAX was used as a vehicle. In vitro gene editing efficiency was assessed on oligonucleotides and genomic DNAs. Sanger sequencing was performed to detect indels. VEGF-A messenger RNA and protein expressions were assessed using quantitative polymerase chain reaction and enzyme-linked immunosorbent assay.

Results: In vitro cleavage assay on a 60-nucleotide DNA duplex showed 88% cleavage of the precursor. The cleavage efficiency was 40% in RPE cells and 32% in Muller cells. Sanger sequencing in the CRISPR-Cas9 treated RPE and Muller cells showed indels at the predicted cut site in both cells. After the VEGF-A gene disruption, VEGF-A protein levels decreased 43% in RPE cells ( < 0.0001) and 38% in Muller cells ( < 0.0001).

Conclusions: CRISPR-Cas9-mediated gene disruption resulted in a significant decrease in the VEGF-A gene protein expression in human RPE and Muller cells. CRISPR-Cas9 ribonucleoprotein may allow simultaneous targeting of multiple VEGF-A producing cells.

Translational Relevance: VEGF-A gene disruption using CRISPR-Cas9 ribonucleoprotein has a potential in treating retinal vascular diseases.
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http://dx.doi.org/10.1167/tvst.9.8.23DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7422915PMC
July 2020

Small RNA Deep Sequencing Identifies a Unique miRNA Signature Released in Serum Exosomes in a Mouse Model of Sjögren's Syndrome.

Front Immunol 2020 17;11:1475. Epub 2020 Jul 17.

Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA, United States.

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 < 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.
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http://dx.doi.org/10.3389/fimmu.2020.01475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7396589PMC
July 2020

Application of advances in endocytosis and membrane trafficking to drug delivery.

Adv Drug Deliv Rev 2020 3;157:118-141. Epub 2020 Aug 3.

Department of Pharmacology and Pharmaceutical Sciences, USC School of Pharmacy, USA; Department of Ophthalmology, Roski Eye Institute, Keck School of Medicine, University of Southern California, USA. Electronic address:

Multidisciplinary research efforts in the field of drug delivery have led to the development of a variety of drug delivery systems (DDS) designed for site-specific delivery of diagnostic and therapeutic agents. Since efficient uptake of drug carriers into target cells is central to effective drug delivery, a comprehensive understanding of the biological pathways for cellular internalization of DDS can facilitate the development of DDS capable of precise tissue targeting and enhanced therapeutic outcomes. Diverse methods have been applied to study the internalization mechanisms responsible for endocytotic uptake of extracellular materials, which are also the principal pathways exploited by many DDS. Chemical inhibitors remain the most commonly used method to explore endocytotic internalization mechanisms, although genetic methods are increasingly accessible and may constitute more specific approaches. This review highlights the molecular basis of internalization pathways most relevant to internalization of DDS, and the principal methods used to study each route. This review also showcases examples of DDS that are internalized by each route, and reviews the general effects of biophysical properties of DDS on the internalization efficiency. Finally, options for intracellular trafficking and targeting of internalized DDS are briefly reviewed, representing an additional opportunity for multi-level targeting to achieve further specificity and therapeutic efficacy.
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http://dx.doi.org/10.1016/j.addr.2020.07.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7853512PMC
August 2020

Caveolin elastin-like polypeptide fusions mediate temperature-dependent assembly of caveolar microdomains.

ACS Biomater Sci Eng 2020 Jan 22;6(1):198-204. Epub 2019 Nov 22.

Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy of the University of Southern California, 1985 Zonal Ave, Los Angeles, CA, USA 90089.

Caveolae are membrane organelles formed by submicron 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 (GEPMs)' 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). By attaching 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.
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http://dx.doi.org/10.1021/acsbiomaterials.9b01331DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7295141PMC
January 2020

A Multivalent ICAM-1 Binding Nanoparticle which Inhibits ICAM-1 and LFA-1 Interaction Represents a New Tool for the Investigation of Autoimmune-Mediated Dry Eye.

Int J Mol Sci 2020 Apr 15;21(8). Epub 2020 Apr 15.

Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90033, USA.

The autoimmune disorder, Sjögren's syndrome (SS), is characterized by lymphocytic infiltration and loss of function of exocrine glands such as the lacrimal gland (LG) and salivary gland. SS-associated changes in the LG are associated with the development of autoimmune-mediated dry eye disease. We have previously reported the accumulation of intercellular adhesion molecule 1 (ICAM-1) in the LG of Non-Obese Diabetic (NOD) mice, a murine model of autoimmune-mediated dry eye in SS, in both LG acinar cells and infiltrating lymphocytes. ICAM-1 initiates T-cell activation and can trigger T-cell migration through binding to lymphocyte function-associated 1 antigen (LFA). To modulate this interaction, this study introduces a new tool, a multivalent biopolymeric nanoparticle assembled from a diblock elastin-like polypeptide (ELP) using the S48I48 (SI) ELP scaffold fused with a mouse ICAM-1 targeting peptide to form IBP-SI. IBP-SI forms a multivalent, monodisperse nanoparticle with a radius of 21.9 nm. Unlike the parent SI, IBP-SI binds mouse ICAM-1 and is internalized by endocytosis into transfected HeLa cells before it accumulates in lysosomes. In vitro assays measuring lymphocyte adhesion to Tumor Necrosis Factor TNF-α-treated bEnd.3 cells, which express high levels of ICAM-1, show that adhesion is inhibited by IBP-SI but not by SI, with IC values of 62.7 μM and 81.2 μM, respectively, in two different assay formats. IBP-SI, but not SI, also blocked T-cell proliferation in a mixed lymphocyte reaction by 74% relative to proliferation in an untreated mixed cell reaction. These data suggest that a biopolymeric nanoparticle with affinity for ICAM-1 can disrupt ICAM-1 and LFA interactions in vitro and may have further utility as an in vivo tool or potential therapeutic.
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http://dx.doi.org/10.3390/ijms21082758DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7216292PMC
April 2020

Tears - more to them than meets the eye: why tears are a good source of biomarkers in Parkinson's disease.

Biomark Med 2020 02 17;14(2):151-163. Epub 2020 Feb 17.

Department of Ophthalmology and Roski Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.

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.
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http://dx.doi.org/10.2217/bmm-2019-0364DOI Listing
February 2020

Cathepsin S activation contributes to elevated CX3CL1 (fractalkine) levels in tears of a Sjögren's syndrome murine model.

Sci Rep 2020 01 29;10(1):1455. Epub 2020 Jan 29.

Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, Roski Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA, 90033, USA.

Autoimmune dacryoadenitis and altered lacrimal gland (LG) secretion are features of Sjögren's syndrome (SS). Activity of cathepsin S (CTSS), a cysteine protease, is significantly and specifically increased in SS patient tears. The soluble chemokine, CX3CL1 (fractalkine), is cleaved from membrane-bound CX3CL1 by proteases including CTSS. We show that CX3CL1 is significantly elevated by 2.5-fold in tears (p = 0.0116) and 1.4-fold in LG acinar cells (LGAC)(p = 0.0026) from male NOD mice, a model of autoimmune dacryoadenitis in SS, relative to BALB/c controls. Primary mouse LGAC and human corneal epithelial cells (HCE-T cells) exposed to interferon-gamma, a cytokine elevated in SS, showed up to 9.6-fold (p ≤ 0.0001) and 25-fold (p ≤ 0.0001) increases in CX3CL1 gene expression, and 1.9-fold (p = 0.0005) and 196-fold (p ≤ 0.0001) increases in CX3CL1 protein expression, respectively. Moreover, exposure of HCE-T cells to recombinant human CTSS at activity equivalent to that in SS patient tears increased cellular CX3CL1 gene and protein expression by 2.8-fold (p = 0.0021) and 5.1-fold (p ≤ 0.0001), while increasing CX3CL1 in culture medium by 5.8-fold (p ≤ 0.0001). Flow cytometry demonstrated a 4.5-fold increase in CX3CR1-expressing immune cells (p ≤ 0.0001), including increased T-cells and macrophages, in LG from NOD mice relative to BALB/c. CTSS-mediated induction/cleavage of CX3CL1 may contribute to ocular surface and LG inflammation in SS.
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http://dx.doi.org/10.1038/s41598-020-58337-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6989636PMC
January 2020

Levels of oligomeric α-Synuclein in reflex tears distinguish Parkinson's disease patients from healthy controls.

Biomark Med 2019 12 25;13(17):1447-1457. Epub 2019 Sep 25.

Department of Neurology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.

Due to active engagement of sensory and afferent nerve fibers in reflex tearing which could be affected in Parkinson's disease (PD), we tested reflex tears as a source of potential PD biomarkers. Reflex tears collected from 84 PD and 84 age- and sex-equivalent healthy controls (HC) were used to measure levels of oligomeric α-Syn (α-Syn), total α-Syn (α-Syn), CCL2, DJ-1, lactoferrin and MMP9. α-syn (p < 0.0001), CCL2 (p = 0.003) and lactoferrin (p = 0.002) were significantly elevated in PD patient tears relative to HC tears. Tear flow was significantly lower in PD relative to HC (p = 0.001). Reflex tears are a potential source for detection of characteristic changes in PD patients.
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http://dx.doi.org/10.2217/bmm-2019-0315DOI Listing
December 2019

Molecular Targeting of Immunosuppressants Using a Bifunctional Elastin-Like Polypeptide.

Bioconjug Chem 2019 09 23;30(9):2358-2372. Epub 2019 Aug 23.

Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy , University of Southern California , Los Angeles , California 90089 , United States.

Elastin-Like Polypeptides (ELP) are environmentally responsive protein polymers which are easy to engineer and biocompatible, making them ideal candidates as drug carriers. Our team has recently utilized ELPs fused to FKBP12 to carry Rapamycin (Rapa), a potent immunosuppressant. Through high affinity binding to Rapa, FKBP carriers can yield beneficial therapeutic effects and reduce the off-site toxicity of Rapa. Since ICAM-1 is significantly elevated at sites of inflammation in diverse diseases, we hypothesized that a molecularly targeted ELP carrier capable of binding ICAM-1 might have advantageous properties. Here we report on the design, characterization, pharmacokinetics, and biodistribution of a new ICAM-1-targeted ELP Rapa carrier (IBPAF) and its preliminary characterization in a murine model exhibiting elevated ICAM-1. Lacrimal glands (LG) of male NOD mice, a disease model recapitulating the autoimmune dacryoadenitis seen in Sjögren's Syndrome patients, were analyzed to confirm that ICAM-1 was significantly elevated in the LG relative to control male BALB/c mice (3.5-fold, < 0.05, = 6). In vitro studies showed that IBPAF had significantly higher binding to TNF-α-stimulated bEnd.3 cells which overexpress surface ICAM-1, relative to nontargeted control ELP (AF)(4.0-fold, < 0.05). A pharmacokinetics study in male NOD mice showed no significant differences between AF and IBPAF for plasma half-life, clearance, and volume of distribution. However, both constructs maintained a higher level of Rapa in systemic circulation compared to free Rapa. Interestingly, in the male NOD mouse, the accumulation of IBPAF was significantly higher in homogenized LG extracts compared to AF at 2 h (8.6 ± 6.6% versus 1.3 ± 1.3%, respectively, = 5, < 0.05). This accumulation was transient with no differences detected at 8 or 24 h. This study describes the first ICAM-1 targeted protein-polymer carrier for Rapa that specifically binds to ICAM-1 in vitro and accumulates in ICAM-1 overexpressing tissue in vivo, which may be useful for molecular targeting in diverse inflammatory diseases where ICAM-1 is elevated.
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http://dx.doi.org/10.1021/acs.bioconjchem.9b00462DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6980866PMC
September 2019

Tear Proteases and Protease Inhibitors: Potential Biomarkers and Disease Drivers in Ocular Surface Disease.

Eye Contact Lens 2020 Mar;46 Suppl 2:S70-S83

Department of Pharmacology and Pharmaceutical Sciences (R.F., W.K., S.F.H.-A.), School of Pharmacy, Los Angeles, CA; and Department of Ophthalmology (R.F., W.K., M.H., M.C.E., S.F.H.-A.), Roski Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA.

Tears are highly concentrated in proteins relative to other biofluids, and a notable fraction of tear proteins are proteases and protease inhibitors. These components are present in a delicate equilibrium that maintains ocular surface homeostasis in response to physiological and temporal cues. Dysregulation of the activity of protease and protease inhibitors in tears occurs in ocular surface diseases including dry eye and infection, and ocular surface conditions including wound healing after refractive surgery and contact lens (CL) wear. Measurement of these changes can provide general information regarding ocular surface health and, increasingly, has the potential to give specific clues regarding disease diagnosis and guidance for treatment. Here, we review three major categories of tear proteases (matrix metalloproteinases, cathepsins, and plasminogen activators [PAs]) and their endogenous inhibitors (tissue inhibitors of metalloproteinases, cystatins, and PA inhibitors), and the changes in these factors associated with dry eye, infection and allergy, refractive surgery, and CLs. We highlight suggestions for development of these and other protease/protease inhibitor biomarkers in this promising field.
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http://dx.doi.org/10.1097/ICL.0000000000000641DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6992486PMC
March 2020

Inhibition of Cathepsin S Reduces Lacrimal Gland Inflammation and Increases Tear Flow in a Mouse Model of Sjögren's Syndrome.

Sci Rep 2019 07 2;9(1):9559. Epub 2019 Jul 2.

Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA, 90033, USA.

Cathepsin S (CTSS) is highly increased in Sjögren's syndrome (SS) patients tears and in tears and lacrimal glands (LG) of male non-obese diabetic (NOD) mice, a murine model of SS. To explore CTSS's utility as a therapeutic target for mitigating ocular manifestations of SS in sites where CTSS is increased in disease, the tears and the LG (systemically), the peptide-based inhibitor, Z-FL-COCHO (Z-FL), was administered to 14-15 week male NOD mice. Systemic intraperitoneal (i.p.) injection for 2 weeks significantly reduced CTSS activity in tears, LG and spleen, significantly reduced total lymphocytic infiltration into LG, reduced CD3+ and CD68+ cell abundance within lymphocytic infiltrates, and significantly increased stimulated tear secretion. Topical administration of Z-FL to a different cohort of 14-15 week male NOD mice for 6 weeks significantly reduced only tear CTSS while not affecting LG and spleen CTSS and attenuated the disease-progression related reduction of basal tear secretion, while not significantly impacting lymphocytic infiltration of the LG. These findings suggest that CTSS inhibitors administered either topically or systemically can mitigate aspects of the ocular manifestations of SS.
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http://dx.doi.org/10.1038/s41598-019-45966-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6606642PMC
July 2019

Oligomeric α-synuclein is increased in basal tears of Parkinson's patients.

Biomark Med 2019 08 2;13(11):941-952. Epub 2019 Jul 2.

Department of Neurology, Keck School of Medicine, Los Angeles, CA 90033-6103, USA.

Secretion of proteins into basal tears of Parkinson's disease (PD) patients may be altered by changes in nerve function. Oligomeric α-Syn and total α-Syn, 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. α-Syn was decreased (p = 0.0043), whereas α-Syn (p < 0.0001) and the ratio of α-Syn/α-Syn (p < 0.0001) were increased in basal tears from PD patients compared with healthy controls. Area under receiver-operating curves of α-Syn and α-Syn/α-Syn contents were 0.70 (95% confidence limits: 0.621-0.774) and 0.72 (95% confidence limits: 0.642-0.792). PD patient basal tears may contain biomarkers that can be assayed noninvasively and inexpensively.
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http://dx.doi.org/10.2217/bmm-2019-0167DOI Listing
August 2019

Berunda Polypeptides: Biheaded Rapamycin Carriers for Subcutaneous Treatment of Autoimmune Dry Eye Disease.

Mol Pharm 2019 07 30;16(7):3024-3039. Epub 2019 May 30.

Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy , University of Southern California , Los Angeles , California 90089 , United States.

The USFDA-approved immunosuppressive drug rapamycin (Rapa), despite its potency, is limited by poor bioavailability and a narrow therapeutic index. In this study, we sought to improve bioavailability of Rapa with subcutaneous (SC) administration and to test its therapeutic feasibility and practicality in a murine model of Sjögren's syndrome (SS), a systemic autoimmune disease with no approved therapies. To improve its therapeutic index, we formulated Rapa with a carrier termed FAF, a fusion of the human cytosolic FK506-binding protein 12 (FKBP12) and an elastin-like polypeptide (ELP). The resulting 97 kDa FAF (i) has minimal burst release, (ii) is "humanized", (iii) is biodegradable, (iv) solubilizes two Rapa per FAF, and (v) avoids organic solvents or amphiphilic carriers. Demonstrating high stability, FAF remained soluble and monodisperse with a hydrodynamic radius of 8 nm at physiological temperature. A complete pharmacokinetic (PK) analysis of FAF revealed that the bioavailability of SC FAF was 60%, with significantly higher blood concentration during the elimination phase compared to IV FAF. The plasma concentration of Rapa delivered by FAF was 8-fold higher with a significantly increased plasma-to-whole blood ratio relative to free Rapa, 24 h after injection. To evaluate therapeutic effects, FAF-Rapa was administered SC every other day for 2 weeks to male non-obese diabetic (NOD) mice, which develop an SS-like autoimmune-mediated lacrimal gland (LG) inflammation and other characteristic features of SS. Both FAF-Rapa and free Rapa exhibited immunomodulatory effects by significantly suppressing lymphocytic infiltration, gene expression of IFN-γ, MHC II, type I collagen and IL-12a, and cathepsin S (CTSS) activity in LG compared to controls. Serum chemistry and histopathological analyses in major organs revealed no apparent toxicity of FAF-Rapa. Given its improved PK and equipotent therapeutic efficacy compared to free Rapa, FAF-Rapa is of further interest for systemic treatments for autoimmune diseases like SS.
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http://dx.doi.org/10.1021/acs.molpharmaceut.9b00263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6957305PMC
July 2019

PP2A: A Novel Target to Prevent Cathepsin S-mediated Damage in Smoking-induced Chronic Obstructive Pulmonary Disease.

Am J Respir Crit Care Med 2019 Jul;200(1):6-8

1 University of Southern California Los Angeles, California.

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http://dx.doi.org/10.1164/rccm.201901-0219EDDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6603066PMC
July 2019

Cathepsin S Alters the Expression of Pro-Inflammatory Cytokines and MMP-9, Partially through Protease-Activated Receptor-2, in Human Corneal Epithelial Cells.

Int J Mol Sci 2018 Nov 9;19(11). Epub 2018 Nov 9.

Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90007, USA.

Cathepsin S (CTSS) activity is increased in tears of Sjögren's syndrome (SS) patients. This elevated CTSS may contribute to ocular surface inflammation. Human corneal epithelial cells (HCE-T cells) were treated with recombinant human CTSS at activity comparable to that in SS patient tears for 2, 4, 8, and 24 h. Acute CTSS significantly increased HCE-T cell gene and protein expression of interleukin 6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) from 2 to 4 h, while matrix metalloproteinase 9 (MMP-9), CTSS, and protease-activated receptor-2 (PAR-2) were increased by chronic CTSS (24 h). To investigate whether the increased pro-inflammatory cytokines and proteases were induced by CTSS activation of PAR-2, HCE-T cells were transfected with PAR-2 siRNA, reducing cellular PAR-2 by 45%. Cells with reduced PAR-2 expression showed significantly reduced release of IL-6, TNF-α, IL-1β, and MMP-9 into culture medium in response to acute CTSS, while IL-6, TNF-α, and MMP-9 were reduced in culture medium, and IL-6 and MMP-9 in cell lysates, after chronic CTSS. Moreover, cells with reduced PAR-2 expression showed reduced ability of chronic CTSS to induce gene expression of pro-inflammatory cytokines and proteases. CTSS activation of PAR-2 may represent a potential therapeutic target for amelioration of ocular surface inflammation in SS patients.
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http://dx.doi.org/10.3390/ijms19113530DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6274678PMC
November 2018

A novel elastin-like polypeptide drug carrier for cyclosporine A improves tear flow in a mouse model of Sjögren's syndrome.

J Control Release 2018 12 23;292:183-195. Epub 2018 Oct 23.

Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA, United States; Department of Ophthalmology, Roski Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States; Department of Biomedical Engineering, Viterbi School of Engineering, University of Southern California, Los Angeles, CA, United States. Electronic address:

As a potent macrolide immunosuppressant, cyclosporine A (CsA) is used to treat multiple autoimmune diseases, including non-autoimmune and autoimmune-mediated dry eye disease, rheumatoid arthritis and psoriasis. Despite its potency, CsA has poor solubility, poor bioavailability, and can cause serious adverse reactions such as nephrotoxicity and neurotoxicity. To overcome these limitations, we invented a new strategy to carry CsA by fusing its cognate human receptor, cyclophilin A (CypA), to a 73 kDa elastin-like polypeptide (ELP) termed A192 using recombinant protein expression. Derived from human tropoelastin, ELPs are characterized by the ability to phase separate above a temperature that is a function of variables including concentration, molecular weight, and hydrophobicity. The resultant fusion protein, termed CA192, which assembles into a dimeric species in solution, effectively binds and solubilizes CsA with a K of 189 nM, comparable to that of endogenous CypA with a K of 35.5 nM. The release profile of CsA from CA192 follows a one phase decay model with a half-life of 957.3 h without a burst release stage. Moreover, CA192-CsA inhibited IL-2 expression induced in Jurkat cells through the calcineurin-NFAT signaling pathway with an IC of 1.2 nM, comparable to that of free CsA with an IC of 0.5 nM. The intravenous pharmacokinetics of CA192 followed a two-compartment model with a mean residence time of 7.3 h. Subcutaneous administration revealed a bioavailability of 30% and a mean residence time of 15.9 h. When given subcutaneously for 2 weeks starting at 14 weeks in male non-obese diabetic (NOD) mice, a model of autoimmune dacryoadenitis used to study Sjögren's syndrome (SS), CA192-CsA (2.5 mg/kg, every other day) significantly (p = 0.014) increased tear production relative to CA192 alone. Moreover, CA192 delivery reduced indications of CsA nephrotoxicity relative to free CsA. CA192 represents a viable new approach to deliver this effective but nephrotoxic agent in a modality that preserves therapeutic efficacy but suppresses drug toxicity.
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http://dx.doi.org/10.1016/j.jconrel.2018.10.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6294338PMC
December 2018

NOD and NOR mice exhibit comparable development of lacrimal gland secretory dysfunction but NOD mice have more severe autoimmune dacryoadenitis.

Exp Eye Res 2018 11 8;176:243-251. Epub 2018 Sep 8.

Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA, United States; Department of Ophthalmology, Roski Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States. Electronic address:

The male Non-Obese Diabetic (NOD) mouse is an established model of autoimmune dacryoadenitis characteristic of Sjögren's Syndrome (SS), but development of diabetes may complicate studies. The Non-Obese Diabetes Resistant (NOR) mouse is a MHC-II matched diabetes-resistant alternative, but development of autoimmune dacryoadenitis is not well-characterized. We compare features of SS in male NOD and NOR mice at 12 and 20 weeks. Stimulated tear secretion was decreased in 12 week NOD relative to BALB/c mice (p < 0.05), while by 20 weeks both NOD and NOR showed decreased stimulated tear secretion relative to BALB/c mice (p < 0.001). Tear CTSS activity was elevated in NOD and NOR relative to BALB/c mice (p < 0.05) at 12 and 20 weeks. While NOD and NOR lacrimal glands (LG) showed increased LG lymphocytic infiltration at 12 and 20 weeks relative to BALB/c mouse LG (p < 0.05), the percentage in NOD was higher relative to NOR at each age (p < 0.05). Gene expression of CTSS, MHC II and IFN-γ in LG were significantly increased in NOD but not NOR relative to BALB/c at 12 and 20 weeks. Redistribution of the secretory effector, Rab3D in acinar cells was observed at both time points in NOD and NOR, but thinning of myoepithelial cells at 12 weeks in NOD and NOR mice was restored by 20 weeks in NOR mice. NOD and NOR mice share features of SS-like autoimmune dacryoadenitis, suggesting common disease etiology. Other findings suggest more pronounced lymphocytic infiltration in NOD mouse LG including increased pro-inflammatory factors that may be unique to this model.
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http://dx.doi.org/10.1016/j.exer.2018.09.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6215720PMC
November 2018

Longitudinal analysis of tear cathepsin S activity levels in male non-obese diabetic mice suggests its potential as an early stage biomarker of Sjögren's Syndrome.

Biomarkers 2019 Feb 12;24(1):91-102. Epub 2018 Sep 12.

a Department of Ophthalmology, USC Keck School of Medicine , Roski Eye Institute , Los Angeles , CA , USA.

Context: Cathepsin S (CTSS) activity is elevated in Sjögren's Syndrome (SS) patient tears.

Objective: To evaluate longitudinal expression of tear and tissue CTSS activity relative to other disease indicators in Non-Obese Diabetic (NOD) mice.

Methods: CTSS activity was measured in tears and lacrimal glands (LG) from male 1-6 month (M) NOD and 1 and 6 M BALB/c mice. Lymphocytic infiltration was quantified by histopathology, while disease-related proteins (Rab3D, CTSS, collagen 1) were quantified using q-PCR and immunofluorescence.

Results: In NOD LG, lymphocytic infiltration was noted by 2 M and established by 3 M (p < 0.01). IFN-ɣ, TNF-α, and MHC II expression were increased by 2 M (p < 0.01). Tear CTSS activity was significantly elevated at 2 M (p < 0.001) to a maximum of 10.1-fold by 6 M (p < 0.001). CTSS activity in LG lysates was significantly elevated by 2 M (p < 0.001) to a maximum of 14-fold by 3 M (p < 0.001). CTSS and Rab3D immunofluorescence were significantly increased and decreased maximally in LG acini by 3 M and 2 M, respectively. Comparable changes were not detected between 1 and 6 M BALB/c mouse LG, although Collagen 1 was decreased by 6 M in LG of both strains.

Conclusion: Tear CTSS activity is elevated with other early disease indicators, suggesting potential as an early stage biomarker for SS.
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http://dx.doi.org/10.1080/1354750X.2018.1514656DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6414277PMC
February 2019

Increased Cathepsin S activity associated with decreased protease inhibitory capacity contributes to altered tear proteins in Sjögren's Syndrome patients.

Sci Rep 2018 07 23;8(1):11044. Epub 2018 Jul 23.

Department of Ophthalmology, USC Roski Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA.

Cathepsin S (CTSS) activity is elevated in Sjögren's Syndrome (SS) patient tears. Here we tested whether protease inhibition and cystatin C (Cys C) levels are reduced in SS tears, which could lead to enhanced CTSS-driven degradation of tear proteins. CTSS activity against Cys C, LF and sIgA was tested in SS or healthy control tears. Tears from 156 female subjects (33, SS; 33, rheumatoid arthritis; 31, other autoimmune diseases; 35, non-autoimmune dry eye (DE); 24, healthy controls) were analyzed for CTSS activity and Cys C, LF, and sIgA levels. Cys C and LF showed enhanced degradation in SS tears supplemented with recombinant CTSS, but not supplemented healthy control tears. CTSS activity was significantly increased, while Cys C, LF and sIgA levels were significantly decreased, in SS tears compared to other groups. While tear CTSS activity remained the strongest discriminator of SS in autoimmune populations, combining LF and CTSS improved discrimination of SS beyond CTSS in DE patients. Reductions in Cys C and other endogenous proteases may enhance CTSS activity in SS tears. Tear CTSS activity is reconfirmed as a putative biomarker of SS in an independent patient cohort while combined LF and CTSS measurements may distinguish SS from DE patients.
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http://dx.doi.org/10.1038/s41598-018-29411-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6056496PMC
July 2018

Interferon-γ treatment in vitro elicits some of the changes in cathepsin S and antigen presentation characteristic of lacrimal glands and corneas from the NOD mouse model of Sjögren's Syndrome.

PLoS One 2017 13;12(9):e0184781. Epub 2017 Sep 13.

Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California, United States of America.

Inflammation and impaired secretion by lacrimal and salivary glands are hallmarks of the autoimmune disease, Sjögren's Syndrome. These changes in the lacrimal gland promote dryness and inflammation of the ocular surface, causing pain, irritation and corneal damage. The changes that initiate and sustain autoimmune inflammation in the lacrimal gland are not well-established. Here we demonstrate that interferon-γ (IFN-γ) is significantly elevated in lacrimal gland and tears of the male NOD mouse, a model of autoimmune dacryoadenitis which exhibits many ocular characteristics of Sjögren's Syndrome, by 12 weeks of age early in lacrimal gland inflammation. Working either with primary cultured lacrimal gland acinar cells from BALB/c mice and/or rabbits, in vitro IFN-γ treatment for 48 hr decreased expression of Rab3D concurrent with increased expression of cathepsin S. Although total cellular cathepsin S activity was not commensurately increased, IFN-γ treated lacrimal gland acinar cells showed a significant increase in carbachol-stimulated secretion of cathepsin S similar to the lacrimal gland in disease. In vitro IFN-γ treatment did not increase the expression of most components of major histocompatibility complex (MHC) class II-mediated antigen presentation although antigen presentation was slightly but significantly stimulated in primary cultured lacrimal gland acinar cells. However, exposure of cultured human corneal epithelial cells to IFN-γ more robustly increased expression and activity of cathepsin S in parallel with increased expression and function of MHC class II-mediated antigen presentation. We propose that early elevations in IFN-γ contribute to specific features of ocular disease pathology in Sjögren's Syndrome.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0184781PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5597228PMC
October 2017

Delivery of Bone Marrow-Derived Mesenchymal Stem Cells Improves Tear Production in a Mouse Model of Sjögren's Syndrome.

Stem Cells Int 2017 2;2017:3134543. Epub 2017 Mar 2.

Department of Comprehensive Care, Tufts University School of Dental Medicine, Boston, MA, USA; Department of Ophthalmology, Tufts University School of Medicine, Boston, MA, USA.

The purpose of the present study was to test the potential of mouse bone marrow-derived mesenchymal stem cells (BD-MSCs) in improving tear production in a mouse model of Sjögren's syndrome dry eye and to investigate the underlying mechanisms involved. NOD mice ( = 20) were randomized to receive i.p. injection of sterile phosphate buffered saline (PBS, control) or murine BD-MSCs (1 × 10 cells). Tears production was measured at baseline and once a week after treatment using phenol red impregnated threads. Cathepsin S activity in the tears was measured at the end of treatment. After 4 weeks, animals were sacrificed and the lacrimal glands were excised and processed for histopathology, immunohistochemistry, and RNA analysis. Following BD-MSC injection, tears production increased over time when compared to both baseline and PBS injected mice. Although the number of lymphocytic foci in the lacrimal glands of treated animals did not change, the size of the foci decreased by 40.5% when compared to control animals. The mRNA level of the water channel aquaporin 5 was significantly increased following delivery of BD-MSCs. We conclude that treatment with BD-MSCs increases tear production in the NOD mouse model of Sjögren's syndrome. This is likely due to decreased inflammation and increased expression of aquaporin 5.
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http://dx.doi.org/10.1155/2017/3134543DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5352970PMC
March 2017

Rapamycin Eye Drops Suppress Lacrimal Gland Inflammation In a Murine Model of Sjögren's Syndrome.

Invest Ophthalmol Vis Sci 2017 01;58(1):372-385

Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California, United States 2Department of Ophthalmology, Roski Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, California, United States.

Purpose: To evaluate the efficacy of topical rapamycin in treating autoimmune dacryoadenitis in a mouse model of Sjögren's syndrome.

Methods: We developed rapamycin in a poly(ethylene glycol)-distearoyl phosphatidylethanolamine (PEG-DSPE) micelle formulation to maintain solubility. Rapamycin or PEG-DSPE eye drops (vehicle) were administered in a well-established Sjögren's syndrome disease model, the male nonobese diabetic (NOD) mice, twice daily for 12 weeks starting at 8 weeks of age. Mouse tear fluid was collected and tear Cathepsin S, a putative tear biomarker for Sjögren's syndrome, was measured. Lacrimal glands were retrieved for histological evaluation, and quantitative real-time PCR of genes associated with Sjögren's syndrome pathogenesis. Tear secretion was measured using phenol red threads, and corneal fluorescein staining was used to assess corneal integrity.

Results: Lymphocytic infiltration of lacrimal glands from rapamycin-treated mice was significantly (P = 0.0001) reduced by 3.8-fold relative to vehicle-treated mice after 12 weeks of treatment. Rapamycin, but not vehicle, treatment increased tear secretion and decreased corneal fluorescein staining after 12 weeks. In rapamycin-treated mice, Cathepsin S activity was significantly reduced by 3.75-fold in tears (P < 0.0001) and 1.68-fold in lacrimal gland lysates (P = 0.003) relative to vehicle-treated mice. Rapamycin significantly altered the expression of several genes linked to Sjögren's syndrome pathogenesis, including major histocompatibility complex II, TNF-α, IFN-γ, and IL-12a, as well as Akt3, an effector of autophagy.

Conclusions: Our findings suggest that topical rapamycin reduces autoimmune-mediated lacrimal gland inflammation while improving ocular surface integrity and tear secretion, and thus has potential for treating Sjögren's syndrome-associated dry eye.
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http://dx.doi.org/10.1167/iovs.16-19159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5270623PMC
January 2017

Imbalanced Rab3D versus Rab27 increases cathepsin S secretion from lacrimal acini in a mouse model of Sjögren's Syndrome.

Am J Physiol Cell Physiol 2016 06 13;310(11):C942-54. Epub 2016 Apr 13.

Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California; Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, California;

The mechanism responsible for the altered spectrum of tear proteins secreted by lacrimal gland acinar cells (LGAC) in patients with Sjögren's Syndrome (SS) remains unknown. We have previously identified increased cathepsin S (CTSS) activity as a unique characteristic of tears of patients with SS. Here, we investigated the role of Rab3D, Rab27a, and Rab27b proteins in the enhanced release of CTSS from LGAC. Similar to patients with SS and to the male nonobese diabetic (NOD) mouse model of SS, CTSS activity was elevated in tears of mice lacking Rab3D. Findings of lower gene expression and altered localization of Rab3D in NOD LGAC reinforce a role for Rab3D in suppressing excess CTSS release under physiological conditions. However, CTSS activity was significantly reduced in tears of mice lacking Rab27 isoforms. The reliance of CTSS secretion on Rab27 activity was supported by in vitro findings that newly synthesized CTSS was detected in and secreted from Rab27-enriched secretory vesicles and that expression of dominant negative Rab27b reduced carbachol-stimulated secretion of CTSS in cultured LGAC. High-resolution 3D-structured illumination microscopy revealed microdomains of Rab3D and Rab27 isoforms on the same secretory vesicles but present in different proportions on different vesicles, suggesting that changes in their relative association with secretory vesicles may tailor the vesicle contents. We propose that a loss of Rab3D from secretory vesicles, leading to disproportionate Rab27-to-Rab3D activity, may contribute to the enhanced release of CTSS in tears of patients with SS.
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http://dx.doi.org/10.1152/ajpcell.00275.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4935202PMC
June 2016

Multidimensional Separation Using HILIC and SCX Pre-fractionation for RP LC-MS/MS Platform with Automated Exclusion List-based MS Data Acquisition with Increased Protein Quantification.

J Proteomics Bioinform 2015 Nov 28;8(11):260-265. Epub 2015 Nov 28.

USC Research Center for Liver Diseases, Keck School of Medicine, University of Southern California, Los Angeles, California, USA; Norris Comprehensive Cancer Center, Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.

Liquid chromatography-mass spectrometry (LC-MS) based proteomics is one of the most widely used analytical platforms for global protein discovery and quantification. One of the challenges is the difficulty of identifying low abundance biomarker proteins from limited biological samples. Extensive fractionation could expand proteomics dynamic range, however, at the cost of high sample and time consumption. Extensive fractionation would increase the sample need and the labeling cost. Also quantitative proteomics depending on high resolution MS have the limitation of spectral acquisition speed. Those practical problems hinder the in-depth quantitative proteomics analysis such as tandem mass tag (TMT) experiments. We found the joint use of hydrophilic interaction liquid chromatography (HILIC) and strong cation exchange Chromatography (SCX) prefractionation at medium level could improve MS/MS efficiency, increase proteome coverage, shorten analysis time and save valuable samples. In addition, we scripted a program, Exclusion List Convertor (ELC), which automates and streamlines data acquisition workflow using the precursor ion exclusion (PIE) method. PIE reduces redundancy of high abundance MS/MS analyses by running replicates of the sample. The precursor ions detected in the initial run(s) are excluded for MS/MS in the subsequent run. We compared PIE methods with standard data dependent acquisition (DDA) methods running replicates without PIE for their effectiveness in quantifying TMT-tagged peptides and proteins in mouse tears. We quantified a total of 845 proteins and 1401 peptides using the PIE workflow, while the DDA method only resulted in 347 proteins and 731 peptides. This represents a 144% increase of protein identifications as a result of PIE analysis.
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http://dx.doi.org/10.4172/jpb.1000378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4720167PMC
November 2015

Elastin-like polypeptides: Therapeutic applications for an emerging class of nanomedicines.

J Control Release 2016 10 11;240:93-108. Epub 2015 Nov 11.

Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90033-9121, USA; Department of Biomedical Engineering, University of Southern California, Los Angeles, CA 90089, USA. Electronic address:

Elastin-like polypeptides (ELPs) constitute a genetically engineered class of 'protein polymers' derived from human tropoelastin. They exhibit a reversible phase separation whereby samples remain soluble below a transition temperature (T) but form amorphous coacervates above T. Their phase behavior has many possible applications in purification, sensing, activation, and nanoassembly. As humanized polypeptides, they are non-immunogenic, substrates for proteolytic biodegradation, and can be decorated with pharmacologically active peptides, proteins, and small molecules. Recombinant synthesis additionally allows precise control over ELP architecture and molecular weight, resulting in protein polymers with uniform physicochemical properties suited to the design of multifunctional biologics. As such, ELPs have been employed for various uses including as anti-cancer agents, ocular drug delivery vehicles, and protein trafficking modulators. This review aims to offer the reader a catalogue of ELPs, their various applications, and potential for commercialization across a broad spectrum of fields.
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http://dx.doi.org/10.1016/j.jconrel.2015.11.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5767577PMC
October 2016

Lacritin-mediated regeneration of the corneal epithelia by protein polymer nanoparticles.

J Mater Chem B 2014 Dec;2(46):8131-8141

Department of Pharmacology and Pharmaceutical Sciences, University of Southern California Los Angeles, CA; 90033-9121 ; Department of Biomedical Engineering, University of Southern California, Los Angeles, CA; 90033.

The avascular corneal epithelium plays an important role in maintaining normal vision and protecting the corneal interior from environmental infections. Delayed recovery of ocular wounds caused by trauma or refractive surgery strengthens the need to accelerate corneal wound healing and better restore the ocular surface. To address this need, we fused elastin-like polypeptide (ELP) based nanoparticles SI with a model mitogenic protein called lacritin. Lacritin fused at the N-terminus of the SI diblock copolymer is called LSI. This LSI fusion protein undergoes thermo-responsive assembly of nanoparticles at physiologically relevant temperatures. In comparison to ELP nanoparticles without lacritin, LSI showed potent signs of lacritin specific effects on a human corneal epithelial cell line (HCE-T), which included enhancement of cellular uptake, calcium-mediated signaling, and closure of a scratch. , the corneas of non-obese diabetic mice (NOD) were found to be highly responsive to LSI. Fluorescein imaging and corneal histology suggested that topical administration of LSI onto the ocular surface significantly promoted corneal wound healing and epithelial integrity compared to mice treated with or without plain ELP. Most interestingly, it appears that ELP-mediated assembly of LSI is essential to produce this potent activity. This was confirmed by comparison to a control lacritin ELP fusion called LS96, which does not undergo thermally-mediated assembly at relevant temperatures. In summary, fusion of a mitogenic protein to ELP nanoparticles appears to be a promising new strategy to bioengineer more potent biopharmaceuticals with potential applications in corneal wound healing.
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http://dx.doi.org/10.1039/C4TB00979GDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4270104PMC
December 2014

Tear-mediated delivery of nanoparticles through transcytosis of the lacrimal gland.

J Control Release 2015 Jun 16;208:2-13. Epub 2014 Dec 16.

Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90033, USA; Department of Biomedical Engineering, University of Southern California, Los Angeles, CA 90089, USA. Electronic address:

Rapid clearance from the tears presents a formidable obstacle to the delivery of peptide drugs to the eye surface. This impedes therapies for ocular infections, wound healing, and dry-eye disease that affect the vision of millions worldwide. To overcome this challenge, this manuscript explores a novel strategy to reach the ocular surface via receptor-mediated transcytosis across the lacrimal gland (LG), which produces the bulk of human tears. The LG abundantly expresses the coxsackievirus and adenovirus receptor (CAR); furthermore, we recently reported a peptide-based nanoparticle (KSI) that targets CAR on liver cells. This manuscript reports the unexpected finding that KSI both targets and transcytoses into the LG acinar lumen, which drains to tear ducts. When followed using ex vivo live cell imaging KSI rapidly accumulates in lumen formed by LG acinar cells. LG transduction with a myosin Vb tail, which is dominant negative towards transcytosis, inhibits lumenal accumulation. Transcytosis of KSI was confirmed in vivo by confocal and TEM imaging of LG tissue following administration of KSI nanoparticles. These findings suggest that it is possible to target nanomaterials to the tears by targeting certain receptors on the LG. This design strategy represents a new opportunity to overcome barriers to ocular delivery.
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http://dx.doi.org/10.1016/j.jconrel.2014.12.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4456098PMC
June 2015