The fatty liver disease-causing protein PNPLA3-I148M alters lipid droplet-Golgi dynamics.

Nonalcoholic fatty liver disease, recently renamed metabolic dysfunction-associated steatotic liver disease (MASLD), is a progressive metabolic disorder that begins with aberrant triglyceride accumulation in the liver and can lead to cirrhosis and cancer. A common variant in the gene , encoding the protein PNPLA3-I148M, is the strongest known genetic risk factor for MASLD. Despite its discovery 20 y ago, the function of PNPLA3, and now the role of PNPLA3-I148M, remain unclear.

The fatty liver disease-causing protein PNPLA3-I148M alters lipid droplet-Golgi dynamics.

Non-alcoholic fatty liver disease (NAFLD), recently renamed metabolic dysfunction-associated steatotic liver disease (MASLD), is a progressive metabolic disorder that begins with aberrant triglyceride accumulation in the liver and can lead to cirrhosis and cancer. A common variant in the gene , encoding the protein PNPLA3-I148M, is the strongest known genetic risk factor for MASLD to date. Despite its discovery twenty years ago, the function of PNPLA3, and now the role of PNPLA3-I148M, remain unclear.

Use of In Vitro Human Skin Models to Assess Potential Immune Activation In Response to Biotherapeutic Attributes and Process-related Impurities.

Subcutaneous (SubQ) injection is a common administration route for biotherapeutics. However, limited tools are available for understanding the dynamic relationships between drug products and resident cells following injection. Advances in tissue engineering have enabled the production of in vitro skin models that recapitulate the morphological structure and functional activity of human skin.

Impact of Precipitation of Antibody Therapeutics After Subcutaneous Injection on Pharmacokinetics and Immunogenicity.

Antibody therapeutics with poor solubility in the subcutaneous matrix may carry unintended risks when administered to patients. The objective of this work was to estimate the risk of antibodies that precipitate in vitro at neutral pH by determining the impact of poor solubility on distribution of the drug from the injection site as well as immunogenicity in vivo. Using fluorescence imaging in a mouse model, we show that one such precipitation-prone antibody is retained at the injection site in the subcutaneous space longer than a control antibody.

Novel protein therapeutic joint retention strategy based on collagen-binding Avimers.

Designing drugs to treat diseases associated with articular joints, particularly those targeting chondrocytes, is challenging due to unique local environmental constraints including the avascular nature of cartilage, the absence of a closed joint compartment, and a highly cross-linked extracellular matrix. In an effort to address these challenges, we developed a novel strategy to prolong residence time of intra-articularly administered protein therapeutics. Avimer domains are naturally found in membrane polypeptides and mediate diverse protein-protein interactions.

Three-dimensional second-harmonic generation imaging of fibrillar collagen in biological tissues.

Multiphoton-induced second-harmonic generation (SHG) has developed into a very powerful approach for in depth visualization of some biological structures with high specificity. In this unit, we describe the basic principles of three-dimensional SHG microscopy. In addition, we illustrate how SHG imaging can be utilized to assess collagen fibrils in biological tissues.

Intracellular uptake and trafficking of difluoroboron dibenzoylmethane-polylactide nanoparticles in HeLa cells.

In this study, nanoparticles based on difluoroboron dibenzoylmethane-poly(lactic acid) (BF(2)dbmPLA) are prepared. Polylactic acid or polylactide is a commonly used degradable polymer, while the boron dye possesses a large extinction coefficient, high emission quantum yield, two-photon absorption, and sensitivity to the surrounding environment. BF(2)dbmPLA exhibits molecular-weight-dependent emission properties and can be formulated as stable nanoparticles, suggesting that its unique optical properties may be useful in multiple contexts for probing intracellular environments.

Lymphocytic infiltration leads to degradation of lacrimal gland extracellular matrix structures in NOD mice exhibiting a Sjögren's syndrome-like exocrinopathy.

We previously reported that lacrimal glands (LGs) of male non-obese diabetic (NOD) mice, an established mouse model of autoimmune inflammatory LG disease that displays many features of human LGs in patients afflicted with Sjögren's syndrome (SjS), exhibit significant degradation of extracellular matrix (ECM) structures as well as increased expression of matrix metalloproteinases (MMPs). The purpose of the current study was to expand the spectrum of proteases identified, to clarify their probable origin as well as to identify the contribution of these changes to disease pathogenesis. We explored in depth the changes in ECM structures and ECM protease expression at the onset of disease (6 weeks) versus late stage disease (18 weeks) in male NOD mouse LGs, relative to LGs of age-matched male NODscid, a severely immunocompromised congenic strain, and healthy BALB/c mice.

Cardiomyopathy is associated with structural remodelling of heart valve extracellular matrix.

Aims: To increase the supply, many countries harvest allograft valves from explanted hearts of transplant recipients with ischaemic (ICM) or dilated cardiomyopathy (DCM). This study determines the structural integrity of valves from cardiomyopathic hearts.

Methods And Results: Extracellular matrix (ECM) was examined in human valves obtained from normal, ICM, and DCM hearts.

Site-specific labeling of enveloped viruses with quantum dots for single virus tracking.

This study reports a general method of labeling enveloped viruses with semiconductor quantum dots (QDs) for use in single virus trafficking studies. Retroviruses, including human immunodeficiency virus (HIV), could be successfully tagged with QDs through the membrane incorporation of a short acceptor peptide (AP) that is susceptible to site-specific biotinylation and attachment of streptavidin-conjugated QDs. It was found that this AP tag-based QD labeling had little effect on the viral infectivity and allowed for the study of the kinetics of the internalization of the recombinant lentivirus enveloped with vesicular stomatitis virus glycoprotein (VSVG) into the early endosomes.

Transduced viral IL-10 is exocytosed from lacrimal acinar secretory vesicles in a myosin-dependent manner in response to carbachol.

The purpose of this study was to determine the intracellular trafficking and release pathways for the therapeutic protein, viral IL-10 (vIL-10), from transduced acinar epithelial cells from rabbit lacrimal gland. Primary cultured rabbit lacrimal gland acinar cells (LGACs) were transduced with adenovirus serotype 5 containing viral interleukin-10 (AdvIL-10). The distribution of vIL-10 was assessed by confocal fluorescence microscopy.

Quantitative second harmonic generation imaging of cartilage damage.

Cartilage damage was studied using non-invasive multiphoton-excited autofluorescence and quantitative second harmonic generation (SHG) microscopy. Two cryopreservation techniques based upon freezing and vitrification methods, respectively, were employed to determine whether or not the collagen fiber structure of full thickness porcine articular cartilage was affected by cryopreservation and whether the level of collagen damage could be determined quantitatively in non-processed (non-fixed, non-sliced, non-stained) tissues. Multiphoton-induced autofluorescence imaging revealed the presence of chondrocytes, as well as collagenous structures in all fresh, vitrified and frozen cryopreserved cartilage samples.

Polystyrene nanoparticle trafficking across alveolar epithelium.

We investigated trafficking of polystyrene nanoparticles (PNP; 20 and 100 nm; carboxylate, sulfate, or aldehyde-sulfate modified [negatively charged] and amidine-modified [positively charged]) across rat alveolar epithelial cell monolayers (RAECM). Apical-to-basolateral fluxes of nanoparticles were estimated as functions of apical PNP concentration ([PNP]) and temperature. Uptake of nanoparticles into RAECM was determined using confocal microscopy.

Increased degradation of extracellular matrix structures of lacrimal glands implicated in the pathogenesis of Sjögren's syndrome.

Lacrimal glands (LGs) of male non-obese diabetic (NOD) mice display many features of human LGs in patients afflicted with the autoimmune disease Sjögren's syndrome (SS), including the loss of secretory functions and a lymphocytic infiltration into the glands by 4 months of age. So far, research has mainly focused on the intracellular events that are involved in initiating LG dysfunction; however, the impact of SS on extracellular matrix (ECM) structures of the diseased LGs has not yet been determined. In this study we identified and compared LG ECM formation and integrity of age-matched male healthy (BALB/c) and diseased (NOD) mice.

Optimized preservation of extracellular matrix in cardiac tissues: implications for long-term graft durability.

Background: Cryopreservation of human tissues, particularly heart valves, is widespread in clinical practice although the effects of this process on underlying tissue structures and its potential impact on valve durability have been poorly studied. Multiphoton imaging and second-harmonic generation (SHG) microscopy permit high-resolution, noninvasive analysis of living tissues at a subcellular level. In the present study we used these novel imaging modalities to compare the effects of vitreous and frozen cryopreservation on the extracellular matrix (ECM) of cardiac tissues.

Novel fiber-dependent entry mechanism for adenovirus serotype 5 in lacrimal acini.

The established mechanism for infection of most cells with adenovirus serotype 5 (Ad5) involves fiber capsid protein binding to coxsackievirus-adenovirus receptor (CAR) at the cell surface, followed by penton base capsid protein binding to alpha(v) integrins, which triggers clathrin-mediated endocytosis of the virus. Here we determined the identity of the capsid proteins responsible for mediating Ad5 entry into the acinar epithelial cells of the lacrimal gland. Ad5 transduction of primary rabbit lacrimal acinar cells was inhibited by excess Ad5 fiber or knob (terminal region of the fiber) but not excess penton base.

Altered traffic to the lysosome in an ex vivo lacrimal acinar cell model for chronic muscarinic receptor stimulation.

Evidence suggests that lacrimal and salivary epithelial cells constitutively expose potentially pathogenic autoantigens, but that active regulatory networks normally suppress pathological autoimmune responses . Events that potentially disrupt the regulatory networks include increased exposure of constitutive autoantigens and induced exposure of previously cryptic autoantigen epitopes. Chronic muscarinic receptor (MAChR) stimulation in an ex vivo rabbit lacrimal acinar cell model induces functional and biochemical alterations reminiscent of the functional quiescence associated with Sjogren's syndrome .

Role of the microtubule cytoskeleton in traffic of EGF through the lacrimal acinar cell endomembrane network.

We have previously documented a novel biphasic traffic pattern for epidermal growth factor (EGF) in the acinar epithelial cell of the lacrimal gland. Different from the typical paradigm observed in many other cell types, EGF initially accumulates in the acinar basal-lateral recycling endosome, then is re-directed to the prelysosomes and lysosomes and degraded. While the cellular content of intact EGF decreases by 40% between 20 and 120 m of continuous incubation at 37 degrees C, the EGF receptor (EGFR) content decreases only modestly [J.

Novel biphasic traffic of endocytosed EGF to recycling and degradative compartments in lacrimal gland acinar cells.

The purpose of this study was to delineate the traffic patterns of EGF and EGF receptors (EGFR) in primary cultured acinar epithelial cells from rabbit lacrimal glands. Uptake of [(125)I]-EGF exhibited saturable and non-saturable, temperature-dependent components, suggesting both receptor-mediated and fluid phase endocytosis. Accumulation of [(125)I] was time-dependent over a 120-min period, but the content of intact [(125)I]-EGF decreased after reaching a maximum at 20 min.

Modulation of secretory functions in epithelia by adenovirus capsid proteins.

To evaluate the safety of adenovirus-derived capsid proteins for ocular gene delivery, we have investigated their effects on the morphology and function of the acinar epithelial cells of the lacrimal gland. These cells are responsible for basal and stimulated release of proteins and electrolytes into ocular fluid, a process essential in maintaining the health of the ocular surface. Acinar epithelial cells from rabbit lacrimal gland were exposed to one of two adenovirus serotype 5 capsid proteins, penton or knob (the carboxy-terminal fragment of the fiber capsid protein).

Impairing actin filament or syndapin functions promotes accumulation of clathrin-coated vesicles at the apical plasma membrane of acinar epithelial cells.

In this article, we investigate the contributions of actin filaments and accessory proteins to apical clathrin-mediated endocytosis in primary rabbit lacrimal acini. Confocal fluorescence and electron microscopy revealed that cytochalasin D promoted apical accumulation of clathrin, alpha-adaptin, dynamin, and F-actin and increased the amounts of coated pits and vesicles at the apical plasma membrane. Sorbitol density gradient analysis of membrane compartments showed that cytochalasin D increased [14C]dextran association with apical membranes from stimulated acini, consistent with functional inhibition of apical endocytosis.

Cytoplasmic dynein participates in apically targeted stimulated secretory traffic in primary rabbit lacrimal acinar epithelial cells.

A major function of the acinar cells of the lacrimal gland is the production and stimulated release of tear proteins into ocular surface fluid. We investigate the participation of cytoplasmic dynein in carbachol-stimulated traffic to the apical plasma membrane in primary rabbit lacrimal acinar epithelial cells. Confocal fluorescence microscopy revealed a major carbachol-induced, microtubule-dependent recruitment of cytoplasmic dynein and the dynactin complex into the subapical region.