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Nacre is generally regarded as tough body armor, but it was often smashed by predators with a certain striking speed. Nacre-like architectures have been demonstrated to dissipate abundant energy by tablets sliding at static or specific low-speed loads, but whether they’re still impact-resistant templates in a wide range of impact velocities remains unclear. Here, we find an anomalous phenomenon that nacre-like structures show superior energy-dissipation ability only in a narrow range of low impact velocities, while they exhibit lower impact resistance than laminated structures when impact velocity exceeds a critical value. This is because the tablets sliding in nacre-like structure occurs earlier and wider at low impact velocities, while it becomes localized at excessive impact velocities. Such anomalous phenomenon remains under different structural sizes and boundary conditions. It further inspires us to propose a hybrid architecture design strategy that achieves optimal impact resistance in a wide range of impact velocities.

The application of prestresses is a valuable strategy for enhancing the overall mechanical performances of structural materials. Residual stresses, acting as prestresses, exist naturally in biological structural materials, such as the nacre with the 3D "brick-and-mortar" arrangement. Although regulation of the tablets sliding has recently been demonstrated to be vital to improve toughness in synthetic nacre-like structures, the effects of prestresses on the tablets-sliding mechanism in these nacre-like structures remain unclear. Here, by a combination of simulation, additive manufacturing, and drop tower testing the authors reveal that, at a critical prestress, synergistic effects between the prestress-enhanced tablets sliding and prestress-weakened structural integrality result in optimized impact resistance of nacre-like structures. Furthermore, the prestressing strategy is easily implemented to a designed nacre-inspired separator to enhance the impact resistance of lithium batteries. The findings demonstrate that the prestressing strategy combined with bioinspired architectures can be exploited for enhancing the impact resistance of engineering structural materials and energy storage devices.

Summary Inspired by microstructures of polar bear hair, herein, we describe a simple solution-based strategy to fabricate a macroscopic-scale and lightweight carbon tube aerogel with super-elasticity and excellent thermal insulation. The microstructure-derived thermal conductivity and super-elasticity are strongly dependent on the shell thickness of the interconnected tubes, as well as the aperture of the aerogel. Remarkably, the optimized aerogel can maintain structural integrity after more than one million compress-release cycles at 30% strain and 10,000 cycles at 90% strain. Moreover, this biomimetic aerogel offers a fast and accurate dynamic piezoresistive response to broad bandwidth frequency forces. Particularly, the super-elasticity is further confirmed by its fastest rebounding speed of 1,434 mm s −1 among the traditional elastic materials measured by a standard falling steel ball. Furthermore, the optimized minimum thermal conductivity is as low as 23 mW m −1 K −1 which performs better than the thermal conductivity of dry air.

Summary Low density, high strength and toughness, together with good environmental stability are always desirable but hardly to achieve simultaneously for man-made structural materials. Replicating the design motifs of natural nacre clearly provides one promising route to obtain such kind of materials, but fundamental challenges remain. Herein, by choosing aramid nanofibers and mica microplatelets as building blocks, we produce a nacreous aramid-mica bulk material with a favorable combination of low density (∼1.7 g cm−3), high strength (∼387 MPa) and toughness (∼14.3 MPa m1/2), and impressive mechanical stability in some harsh environments, including acid/alkali solutions, strong ultraviolet radiation, boiling water, and liquid nitrogen, standing out from previously reported biomimetic bulk composites. Moreover, the obtained material outperforms other bulk nacre-mimetics and most engineering structural materials in terms of its specific strength (227 MPa/[Mg m−3]) and specific toughness (8.4 MPa m1/2/[Mg m−3]), making it a new promising engineering structural material for different technical fields., Graphical abstract, Highlights • Spray, protonation, evaporation, and lamination techniques are combined • Aramid nanofibers and mica microplatelets are assembled into nacreous bulk • Nacre-inspired design imparts the nacreous bulk with high mechanical performance • Nacreous bulk displays favorable mechanical stability in some harsh environments, Biomimetics; Mechanical Property; Biomaterials

Background: Multiple myeloma (MM) is an incurable cancer characterized by clonal plasma cell proliferation in the bone marrow, accounting for approximately 10% of all hematologic malignancies. Recently, patients with relapsed or refractory disease have been treated with a combination of the oncolytic reovirus Pelareorep, bortezomib, and dexamethasone, which was well-tolerated and led to prolonged progression free survival of over 3 years in a subset of patients. To understand the complex tumor immune microenvironment (TiME) and immune responses in patients before and after this treatment, we used imaging mass cytometry (IMC) to perform single cell, highly multiplexed, analysis of these patients’ bone marrow samples. Methods: We comprehensively characterized the changes in the MM TIME in pre and post bone marrow biopsy specimens taken from patients treated on a Phase 1b study with a combination of Pelareorep, bortezomib, and dexamethasone. For analysis with IMC, a marker panel of 35 antibodies was assembled to interrogate the various immune subsets of the bone marrow biopsies; each of these antibodies were conjugated to a unique metal isotope. After validation, the antibody cocktail was used to stain the biopsies. Pixel-based classification was performed in Ilastik to generate cell probability masks and processed in Cellprofiler. PhenoGraph was run in HistoCAT to identify the unique phenotypes. Rstudio was used for t-stochastic neighborhood embedding (tSNE) plot generation, and nearest neighbor analyses. ImaCytE was used for image visualization and spatial analysis. Results: Initial visualization of the raw, unsegmented data showed increased infiltration of natural killer cells and T cells in the post-treatment samples when compared against the pre-treatment samples. These changes correlated with immunohistochemical findings, clinical response to treatment, and changes in T cell clonality. After segmentation, the marker expression heatmaps for each of the clusters identified by PhenoGraph and the further subphenotyping in Rstudio showed complex ecosystems of cell-cell interactions. Nearest neighbor spatial analysis of the post-treatment samples revealed that NK cells (NKG2D+ and NKG2A+ subsets), monocytes (CD14+), macrophages (CD68+), cytotoxic T cells (CD3+, CD8+), and T helper cells (CD3+, CD4+) were significantly closer to the Pelareorep-primed MM than the non-primed MM. Further analysis in ImaCytE highlighted specific instances of these immune neighborhoods. Conclusions: IMC allows us to analyze the potent immune response and cellular interactions in the tumor microenvironment in multiple myeloma treated with Pelareorep and Bortezomib. Characterization of these complex interactions allows for a deeper understanding of the key mechanisms of action of these treatments and planning of future combination studies. Citation Format: Julian Olea, Kaijin Wu, Anthony Colombo, Claudia Villa Celi, Thomas Heineman, Matt Coffey, Steffan T. Nawrocki, Akil Merchant, Kevin R. Kelly. Using imaging mass cytometry to visualize the multiple myeloma tumor microenvironment post immune priming [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6354.

Smart contact lenses attract extensive interests due to their capability of directly monitoring physiological and ambient information. However, previous demonstrations usually lacked efficient sensor modalities, facile fabrication process, mechanical stability, or biocompatibility. Here, we demonstrate a flexible approach for fabrication of multifunctional smart contact lenses with an ultrathin MoS2 transistors-based serpentine mesh sensor system. The integrated sensor systems contain a photodetector for receiving optical information, a glucose sensor for monitoring glucose level directly from tear fluid, and a temperature sensor for diagnosing potential corneal disease. Unlike traditional sensors and circuit chips sandwiched in the lens substrate, this serpentine mesh sensor system can be directly mounted onto the lenses and maintain direct contact with tears, delivering high detection sensitivity, while being mechanically robust and not interfering with either blinking or vision. Furthermore, the in vitro cytotoxicity tests reveal good biocompatibility, thus holding promise as next-generation soft electronics for healthcare and medical applications., Graphical abstract, Highlights • Smart contact lenses integrate photodetectors, glucose sensors, and temperature sensors • Ultrathin serpentine mesh sensor layer can be directly mounted onto the contact lens • Sensor layer directly contacts with tears and does not interfere with blinking or vision • It shows high detection sensitivity, good biocompatibility, and mechanical robustness, Progress and potential Wearable smart contact lenses have attracted extensive interests because of their ability to monitor physiological information and ambient information directly from eyeball and body fluids. However, conventional smart contact lens systems lack efficient sensor modalities, facile fabrication process, mechanical stability, or biocompatibility. This study develops a multifunctional, high-transparency and easy-access smart contact lens system. Specifically, the serpentine mesh sensor system can be directly mounted onto the lens substrate and maintain direct contact with tears, delivering high detection sensitivity, while being mechanically robust and not interfering with either blinking or vision. This integrated contact lens sensor system and fabrication strategy allows for easy incorporation of other functional components, such as an electrode array for electroretinography, antennas for wireless communication, and power modules for future in vivo exploration., A multifunctional smart contact lens with an ultrathin MoS2 transistors-based serpentine mesh sensor system was developed, featuring easy assembly, good detection sensitivity, strong robustness, high stretchability, transparency, and full biocompatibility. The integrated sensor systems contain a photodetector for receiving optical information, imaging and vision assistance, a temperature sensor for diagnosing potential corneal disease, and a glucose sensor for monitoring glucose levels directly from the tear fluid.

Bioinspired architectural design for composites with much higher fracture resistance than that of individual constituent remains a major challenge for engineers and scientists. Inspired by the survival war between the mantis shrimps and abalones, we design a discontinuous fibrous Bouligand (DFB) architecture, a combination of Bouligand and nacreous staggered structures. Systematic bending experiments for 3D-printed single-edge notched specimens with such architecture indicate that total energy dissipations are insensitive to initial crack orientations and show optimized values at critical pitch angles. Fracture mechanics analyses demonstrate that the hybrid toughening mechanisms of crack twisting and crack bridging mode arising from DFB architecture enable excellent fracture resistance with crack orientation insensitivity. The compromise in competition of energy dissipations between crack twisting and crack bridging is identified as the origin of maximum fracture energy at a critical pitch angle. We further illustrate that the optimized fracture energy can be achieved by tuning fracture energy of crack bridging, pitch angles, fiber lengths, and twist angles distribution in DFB composites.

The activated carbons with well-developed microporosity were prepared from fir wood (Cunninghamia lanceolata) impregnated with small amounts of phosphoric acid (impregnation ratios, 1.5 – 4.5 wt.%). For comparison purpose, a parallel study of fir wood without impregnation was carried out in the same conditions. The results showed that the addition of small amounts of H3PO4 could lower the thermal decomposition temperature of fir wood and significantly promote the adsorption capacity and the yield of activated carbon. The yield without phosphoric acid impregnation was 6.55 wt.%, which rose to 19.82 wt.% in the case of 3.0 wt.% H3PO4 impregnation. Using small amounts of phosphoric acid as additive was beneficial to improve specific surface area (SBET) and micropore volume (Vmic), and could produce activated carbons with well-developed microporous structure. With 3.0 wt.% H3PO4 impregnation, SBET and Vmic of the activated carbon reached as high as 1281.6 m2/g and 0.535 mL/g, the ratio of Vmic/Vtot (total pore volume) was higher than 80 %. DOI: http://dx.doi.org/10.5755/j01.ms.24.4.18999

Exploration of graphene-based artificial nacres with excellent mechanical properties demonstrates the potential to surpass natural nacre. Recent experimental studies report that optimal crosslink density defined as concentration of the surface functional groups is usually observed in these artificial nacres towards superb mechanical performance. A hybrid model integrating a nonlinear shear-lag model and atomistic simulations reveals the emergence of an optimal crosslink density at which the maximum strength and toughness are achieved. The origin is due to the balance among the reduction of in-plane tensile properties of the graphene sheets, the enhancement of the shear strength of the interlayer and the reduction of interface plasticity. In addition, our results also reveal that the size insensitivity of the graphene sheet appears when the shear stress of the interlayer is highly localized, the increase of the crosslink density intensifies the nonuniformity of the shear stress and the optimal mechanical properties of the artificial nacre cannot be further enhanced by tuning the size of the graphene sheets. Three kinds of interface molecular interactions with their optimal crosslink densities are also proposed to simultaneously maximize the strength and toughness of graphene-based artificial nacres.

Background: Murine double minute 2 (MDM2) is the primary negative regulator of the tumor suppressor protein, p53. Navtemadlin (KRT-232), a potent, selective, orally available MDM2 inhibitor, restores p53 activity to drive apoptosis of cancer cells in TP53 WT malignancies through the intrinsic (mitochondrial) pathway. Navtemadlin is being evaluated in a phase 3 trial in relapsed or refractory myelofibrosis, and in phase 1b/2 trials in various hematologic malignancies and solid tumors. Because p53 is infrequently mutated and MDM2 is often overexpressed in acute myeloid leukemia (AML), navtemadlin represents a rational therapeutic candidate (Prokocimer et al. Blood. 2017). Synergy between navtemadlin and the hypomethylating agent decitabine has been demonstrated in preclinical AML models (Canon et al. AACR. 2016), although the mechanisms mediating this effect remain unclear. The present study aims to identify mechanisms of navtemadlin-mediated apoptosis within AML cell lines, timing of pathway activation, and effects of combination treatment with navtemadlin + decitabine. Methods: MOLM13 or MV-4-11 AML cell lines were treated with a vehicle control, 0.5-1.0 µM navtemadlin, 1.0 µM decitabine, or with a combination of navtemadlin + decitabine. Cells were pretreated with decitabine for 42-48 hours followed by navtemadlin + fresh decitabine for 0, 5, 10, or 24 hours, and were then harvested for RNA-Seq and immunoblotting analyses of apoptotic pathway proteins. Gene set enrichment analysis (GSEA) and signaling pathway impact analysis (SPIA) were performed on RNA-Seq data to determine the most differentially regulated pathways (using KEGG curated signaling pathways) between controls, single-agent navtemadlin or decitabine, or combination treatment. Protein expression analyses were conducted to validate RNA-Seq data. Results: GSEA of MOLM13 and MV-4-11 cells at 10 hours showed that, relative to controls, several signaling pathways were differentially regulated to a statistically significant level (P≤0.05) with navtemadlin alone, including activation of apoptosis and p53 signaling pathways, and suppression of DNA replication and cell cycle pathways, in line with previous reports (Figure 1a). In MOLM13 cells treated with single-agent decitabine, lipid and proteoglycan-related signaling pathways were activated, whereas several metabolic pathways were suppressed. With combination treatment in both cell lines, unique activated pathways relative to controls included immune-related and cancer-signaling pathways; and unique suppressed pathways included base excision repair and cellular senescence pathways. SPIA analysis of both cell lines at 10 hours produced higher perturbation scores for apoptosis and p53 signaling pathways with combination treatment vs navtemadlin alone (Figure 1b), indicating more robust pathway activation. Additionally, tumor microenvironment (TME) and extracellular matrix (ECM) remodeling pathways showed more robust perturbation when combined with navtemadlin vs decitabine alone. At 24 hours, only SPIA was conducted, which showed less apoptotic pathway induction relative to 10 hours in both cell lines, suggesting time-dependent signaling effects. Cellular senescence pathways were suppressed, whereas central carbon metabolism and HIF-1 signaling were induced by single-agent navtemadlin relative to controls in both cell lines, with combination treatment further enhancing these effects. Combination treatment also induced significant changes in the expression of ECM remodeling genes including MMP9, MMP2, and ITGB7. Protein expression analyses in both cell lines confirmed early upregulation of key apoptosis regulators including p53, p21, PUMA, cleaved caspase-3 and cleaved-PARP with navtemadlin alone, and the DNA-damage response protein, yH2AX, with decitabine alone. The combination enhanced the induction of p53, p21, and yH2AX vs controls or single agents alone. Conclusion: Initial results from this study suggest that navtemadlin + decitabine enhanced apoptosis and p53 signaling pathways to a greater degree than either single agent in AML cell lines. Combination treatment also induced several unique DNA damage response, ECM-remodeling, and TME-related pathways. Activation of both proapoptotic and tumor-stromal interaction pathways suggest a unique mechanism of navtemadlin + decitabine for inhibiting AML cell growth. Figure 1 Figure 1. Disclosures Valmeekam: Telios Pharmaceuticals: Current Employment, Current holder of stock options in a privately-held company, Other: travel, accommodations, expenses. Canon: Oncovalent Therapeutics: Current Employment; Amgen, Inc.: Current holder of individual stocks in a privately-held company, Ended employment in the past 24 months. Krejsa: Acerta Pharma: Current holder of individual stocks in a privately-held company; AstraZeneca: Current equity holder in publicly-traded company; Seattle Genetics: Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months; Kartos Therapeutics: Current Employment, Current holder of stock options in a privately-held company, Other: travel, accommodations, expenses. Kelly: AstraZeneca: Consultancy; Bayer: Speakers Bureau; Verastem: Consultancy; Amgen: Consultancy; Berkley Lights: Current equity holder in publicly-traded company; Denovo Biopharma: Consultancy; Takeda: Consultancy; Sanofi-Aventis: Consultancy; Novartis: Speakers Bureau; Janssen: Speakers Bureau; Agios: Current equity holder in publicly-traded company; Celgene: Speakers Bureau; Epizyme: Speakers Bureau; Pharmacyclics: Speakers Bureau; Karyopharm: Speakers Bureau; Gilead: Speakers Bureau. OffLabel Disclosure: Yes, navtemadlin (KRT-232) is an investigational small molecule inhibitor.

Adaptive resistance mediated by inhibitory ligands such as programmed death 1 ligand (PD-L1) has emerged as an important mechanism of malignant cell survival. This has spurred the development of new agents that disrupt the PD-L1/PD-1 immune checkpoint. Analysis of patient specimens from clinical trials of novel immune checkpoint inhibitors indicates that high basal expression of PD-L1 on tumor cells may predict sensitivity to and be necessary to elicit significant clinical benefit from this drug class. Notably, many multiple myeloma (MM) cell lines and primary CD138+ cells from MM patients do not overexpress PD-L1 compared to normal plasma cells and this may preclude patients with MM from optimally benefitting from immune checkpoint inhibitor therapy. These data suggest that strategies that transiently increase PD-L1 levels could potentially sensitize malignant cells with low PD-L1 expression to anti-PD-1/PD-L1 blockade. The oncolytic reovirus-based anticancer agent Reolysin is known to have significant immunomodulatory effects and has demonstrated promising preclinical efficacy in MM models and favorable safety and tolerability in early MM clinical trials. We demonstrated that Reolysin selectively replicates in MM cells and possesses significant activity in preclinical in vitro and in vivo MM models. These findings established the framework for an ongoing investigator-initiated phase 1b clinical study of Reolysin in combination with bortezomib and dexamethasone in patients with relapsed/refractory MM. Recent gene ontology analyses of RPMI-8226 and U266 MM cells treated with Reolysin revealed that reovirus exposure triggers a highly significant transient increase in CD274 (PD-L1) in MM cell lines. Reolysin-mediated PD-L1 upregulation was confirmed by immunoblotting, qRT-PCR, and flow cytometric analyses in MM cell lines and primary patient specimens treated with Reolysin. Increased PD-L1 expression was also detected by immunohistochemistry in MM tumor samples collected from mice treated with Reolysin. A comparison of the anti-MM effects achieved by live reovirus versus UV-inactivated reovirus demonstrated that live reovirus is required to decrease MM cell viability and upregulate PD-L1 expression. Our data demonstrate proof of concept that reovirus infection and replication in MM cells can efficiently and selectively upregulate PD-L1 levels in malignant cells with low target expression. We hypothesized that Reolysin treatment could be used as a precision priming strategy to potentiate the anti-MM efficacy of PD-1/PD-L1 targeted therapy by promoting myeloma immune recognition and PD-L1 upregulation. To investigate this therapeutic approach, 5TGM1-luc murine MM cells were injected IV into immunocompetent mice to generate MM bone disease. After disease was established, mice were randomized into groups and treated with vehicle, Reolysin (5 x 108 TCID50, Q7D), murine anti-PD-L1 antibody (200 mg/mouse, Q2D) or the combination for 5 weeks. Mice treated with the combination demonstrated decreased disease burden as measured by bioluminescent imaging and also showed reduced IgG2bk levels (specific IgG secreted by 5TGM1 cells) by ELISA. Importantly, the combination also led to increased overall animal survival compared to vehicle control and either single agent treatment (P Disclosures Kelly: Amgen: Honoraria; Abbvie: Honoraria; Pharmacyclics: Honoraria; Jannsen: Honoraria.

The commercial ceramic nanoparticle coated microporous polyolefin separators used in lithium batteries are still vulnerable under external impact, which may cause short circuits and consequently severe safety threats, because the protective ceramic nanoparticle coating layers on the separators are intrinsically brittle. Here, a nacre-inspired coating on the separator to improve the impact tolerance of lithium batteries is reported. Instead of a random structured ceramic nanoparticle layer, ion-conductive porous multilayers consisting of highly oriented aragonite platelets are coated on the separator. The nacre-inspired coating can sustain external impact by turning the violent localized stress into lower and more uniform stress due to the platelet sliding. A lithium-metal pouch cell using the aragonite platelet coated separator exhibits good cycling stability under external shock, which is in sharp contrast to the fast short circuit of a lithium-metal pouch cell using a commercial ceramic nanoparticle coated separator.

Endoplasmic reticulum chaperone protein GRP78 interacts with CD44v and regulates F-actin integrity, cell adhesion, and cell spreading in breast cancer cells. Targeting cell surface GRP78 by antibodies can reduce CD44v expression., GRP78 conducts protein folding and quality control in the ER and shows elevated expression and cell surface translocation in advanced tumors. However, the underlying mechanisms enabling GRP78 to exert novel signaling functions at cell surface are just emerging. CD44 is a transmembrane protein and an important regulator of cancer metastasis, and isoform switch of CD44 through incorporating additional variable exons to the extracellular juxtamembrane region is frequently observed during cancer progression. Using super-resolution dual-color single-particle tracking, we report that GRP78 interacts with CD44v in plasma membrane nanodomains of breast cancer cells. We further show that targeting cell surface GRP78 by the antibodies can effectively reduce cell surface expression of CD44v and cell spreading of tamoxifen-resistant breast cancer cells. Our results uncover new functions of GRP78 as an interacting partner of CD44v and as a regulator of CD44v membrane homeostasis and cell spreading. This study also provides new insights into anti-CD44 therapy in tamoxifen-resistant breast cancer.

Metastasis is the major cause of death among cancer patients, yet early detection and intervention of metastasis could significantly improve their clinical outcomes. We have sequenced and analyzed RNA (Expression) and DNA (Mutations) from the primary tumor (PT), tumor extension (TE) and lymphatic metastatic (LM) sites of patients with clear cell renal cell carcinoma (CCRCC) before treatment. Here, we report a three-nucleotide deletion near the C-region of Plk5 that is specifically associated with the lymphatic metastasis. This mutation is un-detectable in the PT, becomes detectable in the TE and dominates the LM tissue. So while only a few primary cancer cells carry this mutation, the majority of metastatic cells have this mutation. The increasing frequency of this mutation in metastatic tissue suggests that this Plk5 deletion could be used as an early indicator of CCRCC metastasis, and be identified by low cost PCR assay. A large scale clinical trial could reveal whether a simple PCR assay for this mutation at the time of nephrectomy could identify and stratify high-risk CCRCC patients for treatments.

Based on the temperature dependent behaviors of the Young's modulus and the strength of high temperature ceramics, temperature dependent failure characteristics of high temperature ceramic lattice truss materials were analyzed to build temperature-dependent failure theory. In compression, compression fracture and buckling are the competing failure modes. In shearing, tensile fracture and buckling are the competing failure modes. High temperature softening of the ceramic strut induces fatal buckling collapse for the lightweight lattice truss structure at ultra-high temperature. Collapse criteria under different planar stress states were built and are supported by high temperature experiments. Instructed by the criteria, collapse surfaces consisting of fracture surface and buckling surface were plotted and give helpful suggestions to design high temperature resistant as well as lightweight lattice truss ceramic composites.

Background and objective The development of the tyrosine kinase inhibitor Imatinib (IM) represents a milestone in CML (Chronic Myeloid Leukemia) treatment. However, it is not curative and patients develop IM resistance. IM resistance has been previously correlated with the emergence of drug-resistant LIC/LSC (Leukemia Initiating Cell/Leukemia Stem Cell) and increased nuclear catenin levels and enhanced Wnt signaling. It has been demonstrated previously that drug resistant CML LIC/LSC can be safely eliminated both in vitro and in vivo via disruption of the CBP/catenin interaction, utilizing the highly biochemically selective small molecule CBP/catenin antagonist ICG- 001. Methods Here, we utilized an in vitro IM selection of primary CML patients' samples to identify drug-resistant LIC/LSC populations. In this report, we characterized the drug-resistant CML LIC/LSC population using FACS, Smartchip qPCR and colony assays to analyze cell surface markers, transcriptomics and function. Results As opposed to previous characterization of the CML leukemic stem cell population as being either CD34+CD38- or CD34+CD38+, the in vitro selected Imatinib resistant (IM-R) CML LSC population was consistently CD34-CD38-. In Long-Term Culture Initiating Cell assay (LTC-IC, a surrogate assay for long term repopulating stem cells), our results suggest that the CBP/catenin antagonist ICG- 001 sensitizes LIC/LSC to IM treatment by forced differentiative elimination of the CML LIC/LSC population. Conclusion In vitro selected IM resistant cells are negative for both CD34 and CD38 by FACS analysis. These cells acquire CD34/CD38 expression after co-culture with stromal cells. CBP/catenin antagonist ICG-001 facilitates IM function in eliminating these cells.

There is currently no FDA approved therapeutic agent for ARS mitigation post radiation exposure. Here we report that the small molecule YH250, which specifically antagonizes p300/catenin interaction, stimulates hematopoiesis in lethally or sublethally irradiated mice. A single administration of YH250 24 hours post irradiation can significantly stimulate HSC proliferation, improve survival and accelerate peripheral blood count recovery. Our studies suggest that promotion of the expansion of the remaining HSC population via stimulation of symmetric non-differentiative proliferation is at least part of the mechanism of action.

Nacre with a combination of superior strength and toughness is one of the best natural body armors due to its unique brick-and-mortar structure, which is recently regarded as the model system for the lightweight and high impact performance composite design. However, most of the studies of nacreous structure focused on static loadings or simple one-dimensional dynamic load. Our study presents 3D finite element simulation, 3D printing and drop-tower impact testing to reveal the role of interfacial strength and impact velocity in impact resistance of the nacreous structure. An optimal interfacial strength is observed at which the impact resistance reaches a maximum. Sorting of various evolving damage patterns demonstrates that the simultaneous intralayer radial cracks propagation and interlayer delamination lead to a maximal energy dissipation in the presence of the optimal interfacial strength. The phase diagram of damage patterns depending on interfacial strength and impact velocity reveals that the optimal interfacial strength decreases with increasing impact velocity. This study demonstrates that the impact-resistance of nacreous structure under different impact velocities can be enhanced by tuning the interfacial strength, and gives some guidance for advanced protective materials designs. Keywords: Nacreous structure, Impact resistance, Interfacial strength, Finite element analysis, 3D-printing, Drop-tower impact experiment

Over 400 million years ago, the evolution of vertebrates gave rise to a life cycle in which the organism began to live longer particularly as an adult. To accommodate such a longer lifespan, the organism underwent adaptation, developing a mechanism for long-lived cellular homeostasis. This adaptation required a population of long-lived relatively quiescent somatic stem cells (SSCs) along with a more proliferative differentiated daughter cell population, and was necessary to safeguard the genetic attributes with which SSCs were endowed. Intriguingly, cAMP response element binding protein (CREB)-binding protein (CBP) and E1A-binding protein, 300 kDa (p300), the highly homologous Kat3 coactivators had diverged, through duplication of ancestral Kat3, immediately preceding the evolution of vertebrates, given that both CBP and p300 have been detected in nearly all vertebrates versus non-vertebrates. We now demonstrate that a relatively small, highly evolutionarily conserved, amino terminal 9 amino acid deletion in CBP versus p300, plays a critical role in allowing for both robust maintenance of genomic integrity in stem cells and the initiation of a feed-forward differentiation mechanism by tightly controlling the interaction of the nuclear receptor family with the Wnt signaling cascade in either an antagonistic or synergistic manner.

Background: Although canonical Wnt signaling is known to promote tumorigenesis in pancreatic ductal adenocarcinoma (PDAC), a cancer driven principally by mutant K-Ras, the detailed molecular mechanisms by which the Wnt effector β-catenin regulates such tumorigenesis are largely unknown. We have previously demonstrated that β-catenin’s differential usage of the Kat3 transcriptional coactivator cyclic AMP-response element binding protein-binding protein (CBP) over its highly homologous coactivator p300 increases self-renewal and suppresses differentiation in other types of cancer. Aim/methods: To investigate Wnt-mediated carcinogenesis in PDAC, we have used the specific small molecule CBP/β-catenin antagonist, ICG-001, which our lab identified and has extensively characterized, to examine its effects in human pancreatic cancer cells and in both an orthotopic mouse model and a human patient-derived xenograft (PDX) model of PDAC. Results/conclusion: We report for the first time that K-Ras activation increases the CBP/β-catenin interaction in pancreatic cancer; and that ICG-001 specific antagonism of the CBP/β-catenin interaction sensitizes pancreatic cancer cells and tumors to gemcitabine treatment. These effects were associated with increases in the expression of let-7a microRNA; suppression of K-Ras and survivin; and the elimination of drug-resistant cancer stem/tumor-initiating cells.

The acinar epithelial cells of the lacrimal gland are responsible for the production, packaging and regulated exocytosis of tear proteins into ocular surface fluid. This review summarizes new findings on the mechanisms of exocytosis in these cells. Participating proteins are discussed within the context of different categories of trafficking effectors including targeting and specificity factors (rabs, SNAREs) and transport factors (microtubules, actin filaments and motor proteins). Recent information describing fundamental changes in basic exocytotic mechanisms in the NOD mouse, an animal model of Sjögren's syndrome, is presented.

Lacrimal glands of male NOD and BALB/c mice have very small, pleomorphic acinar lumens. Acini contain isolated zones of highly complex cell surface interdigitations at the basal surface, sometimes occurring between acinar and myoepithelial cells. In NOD mice, cytological abnormalities, including mitochondrial deterioration, pleomorphic and heterogeneous cytoplasmic vacuoles, and lipid accumulation are evident within acinar cells at 1 month. Accumulation of lipid is further increased as the animal ages, accompanied by lymphocytic infiltration and destruction of acini. These results demonstrate alterations from normal cytology as early as 1 month in NOD mice, well before detection of clinical signs of Sjögren syndrome.

The lacrimal glands of male NOD mice exhibit many of the features of the human lacrimal gland in patients afflicted with the autoimmune disease, Sjögren's syndrome, including loss of secretory functions and lymphocytic infiltration into the lacrimal gland. To elucidate the early changes in the secretory pathway associated with development of Sjögren's syndrome, we investigated the organization of the exocytotic pathway in lacrimal glands of age-matched male BALB/c and NOD mice. Cryosections from lacrimal glands from 1 and 4 month male BALB/c and NOD mice were processed for confocal fluorescence and electron microscopic evaluation of different participants in exocytosis. No changes in apical actin filaments were noted in glands from NOD mice, but these glands exhibited thickening of basolateral actin relative to that seen in the BALB/c mice. Rab3D immunofluorescence associated with mature secretory vesicles was distributed abundantly in a continuous vesicular network concentrated beneath the apical plasma membrane in glands from 1 and 4 month BALB/c mice. In glands from 1 month NOD mice, rab3D immunofluorescence exhibited marked discontinuity and irregularity in the vesicular labeling pattern. While this change was also detected in glands from 4 month NOD mice, many of these glands exhibited an additional extension of rab3D labeling through the cell to the basolateral membrane. Electron microscopic analysis confirmed the formation of irregularly shaped, unusually large secretory vesicles in lacrimal glands from NOD mice. Quantitation of multiple secretory vesicles from electron micrographs revealed a significant (por =0.05) increase in the percentage of secretory vesicles incorporated into multivesicular aggregates in lacrimal glands from 1 and 4 month NOD mice compared to BALB/c mice. The M3 muscarinic receptor, a key signaling effector of exocytosis, was redistributed away from its normally basolateral locale in glands from BALB/c mice, with concomitant enrichment in intracellular aggregates in glands from NOD mice. These findings show that lacrimal glands in NOD mice as young as 1 month contain aberrant secretory vesicles with altered effector composition that undergo premature cytoplasmic fusion, and that changes in the distribution of the M3 muscarinic receptor occur within the same time frame.

Background: While novel agents have improved the outcome for multiple myeloma (MM), the disease remains incurable. Our preclinical work has shown that MM cells from relapsed/refractory patients are very sensitive to the combination of Reolysin (a proprietary formulation of an oncolytic reovirus) and bortezomib (BZ), resulting in synergistic levels of endoplasmic reticulum (ER) stress. A pilot phase 1 study showed that Reolysin was well tolerated in relapsed/refractory MM patients and was associated with prolonged stable disease. Methods: Relapsed/refractory MM patients including patients refractory to BZ were included. This is a phase 1b study of 3 escalating doses of Reolysin (cohort 1; 3 x1010 TCID50, cohort 2; 4.5 x1010 TCID50, and cohort 3; 9 x1010 TCID50). Reolysin is given on days 1, 2, 8, 9, 15, and 16. Patients receive 40 mg dexamethasone and 1.5 mg/m2bortezomib on days 1, 8, and 15. Cycles are repeated every 28 days in the absence of disease progression or unacceptable toxicity. Results: Eight patients have been enrolled, seven were male and the median age was 55 (range, 33 - 66). The median number of prior therapies was 4 (range 1 to 6). All patients were previously exposed to BZ, and 6 patients were previously exposed to both an immunomodulatory agent and carfilzomib. Most patients had ISS stage I disease at study entry (n=5), 2 had stage II and 1 had stage III. The combination was well tolerated and most treatment-related toxicities were transient and easily managed with supportive care. The most common treatment-related toxicities were grade 1 diarrhea (n=4), grade 1 fatigue (n=4), grade 1 flu-like symptoms (n=5) and grade 1 headache (n=4). No dose limiting toxicities occurred in cohort 1 or 2. Three patients completed 1 cycle of treatment only, 2 completed 3 cycles, and 1 patient completed 4 cycles. One patient remains on treatment. Reasons for treatment discontinuation included disease progression (n=4), clinical deterioration (n=1) and patient withdrawal (n=2). Six patients were evaluable for response, 3 patients had stable disease lasting at least 3 cycles whereas 3 patients had progressive disease at the end of cycle 1. Ex vivo treatment of primary MM cells and MM cell lines (U266 and RPMI-8226) with Reolysin revealed a dramatic induction of PD-L1 expression as measured by qRT-PCR and flow cytometry. Furthermore, ex vivo treatment of MM patient mononuclear cells with Reolysin resulted in NK and T cell activation. These results suggest that the addition of an anti-PD-1 or anti-PD-L1 agent may augment the anti-MM activity of Reolysin. Conclusions: The combination of Reolysin, BZ and dexamethasone is well tolerated in a heavily pretreated MM patient population. Cohort 3 is currently enrolling at the 9 x1010 TCID50 dose level of Reolysin. The potential anti-MM effects of immune activation following Reolysin infusion may be mitigated by MM expression of PD-L1. Additional cohorts exploring the tolerability, efficacy and pharmacodynamics of the combination of Reolysin, BZ or carfilzomib, dexamethasone and an immune checkpoint inhibitor will be added following completion of cohort 3. Disclosures Kelly: Pharmacyclics: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees. Coffey:Oncolytics Biotech: Employment. Gill:Oncolytics Biotech: Employment.

Apolipoprotein F (apoF) is 29 kilodalton secreted sialoglycoprotein that resides on the HDL and LDL fractions of human plasma. Human ApoF is also known as Lipid Transfer Inhibitor protein (LTIP) based on its ability to inhibit cholesteryl ester transfer protein (CETP)-mediated transfer events between lipoproteins. In contrast to other apolipoproteins, ApoF is predicted to lack strong amphipathic alpha helices and its true physiological function remains unknown. We previously showed that overexpression of Apolipoprotein F in mice reduced HDL cholesterol levels by 20–25% by accelerating clearance from the circulation. In order to investigate the effect of physiological levels of ApoF expression on HDL cholesterol metabolism, we generated ApoF deficient mice. Unexpectedly, deletion of ApoF had no substantial impact on plasma lipid concentrations, HDL size, lipid or protein composition. Sex-specific differences were observed in hepatic cholesterol content as well as serum cholesterol efflux capacity. Female ApoF KO mice had increased liver cholesteryl ester content relative to wild type controls on a chow diet (KO: 3.4+/−0.9 mg/dl vs. WT: 1.2+/−0.3 mg/dl, p

To develop a nomenclature for the lacrimal duct system in the rabbit, based on the anatomic and structural characteristics of each duct segment, and to provide RT-PCR and immunofluorescence data to support the notion that the duct system plays important roles in lacrimal function.Paraffin-embedded lacrimal glands (LGs) were stained with hematoxylin and eosin (HE) and evaluated with a stereomicroscope. Cryosections of LG were stained with cresyl violet, and acinar cells and ductal epithelial cells were isolated from each duct segment by laser capture microdissection (LCM). mRNA levels from these cells were analyzed by real-time RT-PCR. Standard protocol was followed for immunofluorescence detection of ionic transporters.The lacrimal duct system was divided into six segments on the basis of morphologic characteristics: the intercalated, intralobular, interlobular, intralobar, interlobar, and main excretory ducts. Although the morphologic features change incrementally along the entire duct system, the gene expression of ionic transporters and aquaporins, including AE3, AQP4, AQP5, CFTR, ClC2gamma, KCC1, NHE1, NKAalpha1, NKAbeta1, NKAbeta2, NKAbeta3, and NKCC1 varied greatly among duct segments. Immunofluorescence results were generally in accordance with the abundance of mRNAs along the acinus-duct axis.Most LG research has focused on the acinar cells, with relatively little attention being paid to the lacrimal ducts. The lack of knowledge regarding the lacrimal ducts was so profound that a precise nomenclature had not been established for the duct system. The present data establish a nomenclature for each segment of the lacrimal duct system and provide evidence that ducts play critical roles in lacrimal secretion.

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 identify their probable origin as well as to clarify 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 age-matched male NOD-scid, a severely immunocompromised congenic strain, and healthy BALB/c mice. LG tissues were examined using routine histological, immunohistochemical, Western Blot and gene expression analyses, as well as with novel multiphoton imaging technologies. We further characterized the profile of infiltrating immune cells under each condition using flow cytometry. Our results show that the initial infiltrating cells at 6 weeks of age are responsible for increased MMP and cathepsin H expression and therefore initiate the LG ECM degradation in NOD mice. More importantly, NOD-scid mice exhibited normal LG ECM structures, indicating the lymphocytes seen in the LGs of NOD mice are responsible for the degradation of the LG ECM. The disease-related remodeling of LG ECM structures may play a crucial role in altering the acinar signaling environment, disrupting the signaling scaffolds within the cells, which are required to mobilize the exocytotic trafficking machinery, ultimately leading to a loss of LG function in patients afflicted with SjS.

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. LG tissues were examined using routine histological, biochemical, immunohistochemical and gene expression analysis. Multiphoton imaging and second-harmonic generation (SHG) microscopy permitted the non-invasive analysis of major LG ECM structures including collagen- and elastin-containing fibers. Biochemical testing demonstrated a significant loss of collagen, glycosaminoglycans and desmosine in NOD-LGs when compared to healthy BALB/c-LGs. Immunohistochemical staining and gene expression analysis confirmed this disease-related alteration of LG ECM structures. Furthermore, laser-induced autofluorescence and SHG microscopy revealed dramatic changes in the structural organization of most collagenous and elastic fibers of the diseased LG tissues that were more pronounced than those displayed by histological analysis. Our results clearly show an enhanced degradation of ECM proteins accompanied by the severe disorganization and deformation of ECM structures of diseased LG tissues. These new insights into the involvement of ECM degradation in SS may lead to novel therapies for patients suffering from dry eye disease.

The integrity of the ocular surface is dependent upon maintenance of tear fluid of appropriate composition and volume. There has been considerable interest in the application of gene therapy to the anterior segment of the eye for applications including enhancement of corneal and conjunctival wound healing (9), correction of allograft rejection after corneal transplantation (5), and suppression of lacrimal gland autoimmunity associated with Sjogren's syndrome (61, 62). Many delivery strategies for introduction of foreign genes into ocular and other tissues have utilized subgroup C adenovirus serotype 5 (Ad5)-derived vectors. The application of traditional Ad5-derived vectors has been limited by host immunogenicity and toxicity. As alternatives, we and others have focused on the use of viral constituents that are able to interact with host factors to facilitate entry of associated DNA. Many next-generation Ad5-derived nonviral vectors use viral capsid proteins to exploit endogenous pathways used by the intact virus for cell entry. The capsid fiber protein has been thought to be responsible for initial cell attachment of Ad5 to the plasma membranes (PM) of many cells, including the well-studied HeLa cell model, through high-affinity binding of its carboxy (C)-terminal globular domain, or “knob” region, to the extracellular N-terminal D1 domain of the coxsackievirus-adenovirus receptor (CAR). This receptor is a ubiquitous integral membrane glycoprotein of the immunoglobulin superfamily which also plays a role in cell adhesion (10, 27, 53). Following the initial attachment, the low-affinity interaction of RGD and LDV motifs exposed in a protruding loop of the penton base with the cell surface integrins is thought to trigger virus uptake by clathrin-dependent receptor-mediated endocytosis. Once internalized, Ad5 is delivered to acidic endosomal compartments and undergoes endosomolysis by escaping to the cytosol upon cell signaling, a process that has been proposed to involve the penton base (42, 43, 57, 58). Ad5 capsid proteins (possibly penton base) then interact with microtubules and dynein/dynactin during transit to the nucleus (31, 36, 60). In addition to this established internalization pathway in cultured cells, Meier et al. recently demonstrated that Ad2, which is similar to Ad5 in its capsid protein composition, can elicit macropinocytosis in epithelial cells (35). Ad2-induced macropinocytosis could enhance its acid-activated penetration into endosomes even though the virion particles were internalized into clathrin-coated vesicles. Although these and other studies have shed insight into the general mechanisms of Ad internalization, use of cell lines such as HeLa and A549 cells that no longer retain specialized epithelial morphology may not reveal the exact mechanism that Ad5 uses to infect its in vivo target cells, most of which are epithelial in origin. The acinar epithelial cells of the lacrimal gland (LGAC) are secretory epithelial cells responsible for the basal and stimulated release of a variety of essential proteins into tear fluid (13, 19, 37, 52). Changes in LGAC secretory functions are associated with diseases ranging in severity from keratoconjunctivitis sicca (dry eye) to the autoimmune disease Sjogren's syndrome (18, 39). Despite real prospects for the rational design of advanced macromolecular therapeutics for treatment of severe dry eye disorders, therapy remains a remote possibility from a drug delivery standpoint. The only methods utilized successfully in animal models for gene delivery to the lacrimal gland have used replication-defective Ad5-derived vectors, which are sufficiently immunogenic to preclude their use in clinical trials (61, 62). To our knowledge, there are no reports regarding delivery of other therapeutic molecules such as proteins, peptides, or antisense oligonucleotides into the lacrimal gland. Ad5 capsid proteins therefore represent promising drug carriers for delivery of macromolecular conjugates to the gland. We have previously reported that exposure of reconstituted rabbit LGAC to replication-deficient Ad5 at a multiplicity of infection (MOI) of 5 PFU/cell for time intervals as short as 1 to 2 h resulted in transduction of 85 to 90% of acinar cells (30, 50). We have also reported that the recombinant penton base protein of Ad5 appears unable to enter LGAC (25), although it can recapitulate the Ad5 entry pathway in HeLa cells (41). Since Ad5 infects LGAC at high efficiency in an apparently penton-independent manner, we hypothesized that Ad5 might be internalized in LGAC through a unique mechanism. In the studies presented here, as a first step in testing this hypothesis we sought to identify the capsid proteins responsible for mediating Ad5 entry into LGAC. Competition studies showed that fiber, knob, or CAR-specific small interfering RNA (siRNA) significantly and substantially inhibited Ad5 infectivity, while penton base had no effect. Surprisingly, incubation of LGAC with capsid proteins revealed that recombinant penton base protein remained associated with the PM, while recombinant knob was internalized. Inhibition of heparan sulfate-glycosaminoglycan (HS-GAG) interactions by pretreating Ad5 with heparin and by cleavage of cellular HS-GAGs using a heparinase cocktail had a modest but significant additive inhibitory effect on Ad5 infectivity. Analysis of fluorescein isothiocyanate (FITC)-dextran uptake and time-lapse video microscopy of green fluorescent protein (GFP)-actin ruffling showed that Ad5 could stimulate macropinocytosis in LGAC, a process that could also be elicited by fiber and knob but not penton base. Further studies suggested that macropinocytosis is important for efficient Ad5 transduction but that it does not participate directly in Ad5 entry. Altogether, these findings suggest that Ad5 internalization into LGAC is through a unique fiber-dependent pathway that may involve CAR and HS-GAGs rather than the penton base-integrin-mediated endocytotic mechanism seen in other cell models so far investigated.

The acinar epithelial cells of the lacrimal gland exocytose the contents of mature secretory vesicles containing tear proteins at their apical membranes in response to secretagogues. Here we use time-lapse confocal fluorescence microscopy and fluorescence recovery after photobleaching to investigate the changes in actin filaments located beneath the apical membrane during exocytosis evoked by the muscarinic agonist, carbachol (100 μM). Time-lapse confocal fluorescence microscopy of apical actin filaments in reconstituted rabbit lacrimal acini transduced with replication-deficient adenovirus containing GFP-actin revealed a relatively quiescent apical actin array in resting acini. Carbachol markedly increased apical actin filament turnover and also promoted transient actin assembly around apparent fusion intermediates. Fluorescence recovery after photobleaching measurements revealed significant (P≤0.05) increases and decreases, respectively, in mobile fraction (Mf) and turnover times (t½) for apical actin filaments in carbachol-stimulated acini relative to untreated acini. The myosin inhibitors, 2,3-butanedione monoxime (BDM, 10 mM, 15 minutes) and ML-7 (40 μM, 15 minutes), significantly decreased carbachol-stimulated secretion of bulk protein and the exogenous secretory vesicle marker, syncollin-GFP; these agents also promoted accumulation of actin-coated structures which were enriched, in transduced acini, in syncollin-GFP, confirming their identity as fusion intermediates. Actin-coated fusion intermediates were sized consistent with incorporation of multiple rather than single secretory vesicles; moreover, BDM and ML-7 caused a shift towards formation of multiple secretory vesicle aggregates while significantly increasing the diameter of actin-coated fusion intermediates. Our findings suggest that the increased turnover of apical actin filaments and the interaction of actin with non-muscle myosin II assembled around aggregates of secretory vesicles facilitate exocytosis in lacrimal acinar epithelial cells.

Recent evidence from a double-blind, randomized study showed that treatment with apolipoprotein A-I Milano (ApoA-I Milano ) in a complex with phospholipids produced significant regression of the coronary atheroma burden in patients with acute coronary syndromes. We previously showed similar regression of atherosclerosis in an animal model. Here, we examined a viral vector-based gene delivery system as a basis for ApoA-I Milano gene therapy. Comparing levels of expression using combinations of the cytomegalovirus (CMV) promoter in a recombinant serotype 2 adeno-associated virus (rAAV2) linked to ApoA-I Milano or the enhanced green fluorescent protein (EGFP) genes, we found that a promoter construct of two CMV core promoters sharing a CMV enhancer was more active than other combinations or a single CMV promoter. In vivo assessment of this optimal CMV construct using rAAV2 virus particles for intravenous (IV) or intramuscular (IM) routes of delivery produced high circulating levels of ApoA-I Milano protein for extended periods (up to 220 ng/ml at 22 weeks p.i.) by IV delivery while the IM route resulted in a relatively short period of very low-level ApoA-I Milano expression. Since there was no difference in the immune response between the two routes of delivery, we reasoned that tissue tropism might be responsible for this differential gene expression. To explore this possibility, we investigated the effect of different AAV serotypes on ApoA-I Milano gene expression in vivo. It found that rAAV1-mediated expression of ApoA-I Milano was approximately 15- and 9-fold higher than rAAV2 and rAAV5, respectively when IM injection routes were compared while all three AAV serotypes produced substantial levels of ApoA-I Milano expression from IV injection. These studies demonstrate that by modifying the promoter and serotype, increases in the efficiency of AAV-directed transgene expression could be achieved and support the potential of AAV-mediated gene therapy.

Sjogren's syndrome (SjS) is a chronic autoimmune inflammatory disease characterized by lymphocytic infiltration and destruction of lacrimal glands (LGs) and salivary glands (SGs). The male nonobese diabetic (NOD) mouse is a well-established animal model in which to evaluate the processes of the dacryoadenitis and sialoadenitis characteristic of the human disease. This mouse strain spontaneously develops insulin-dependent diabetes mellitus (IDDM) as well as SjS-like disease.1–3 Dacryoadenitis, which is more severe than sialoadenitis in this model, is fully manifested by 12 to 20 weeks.1,4 The NOD severe combined immunodeficiency (SCID) mouse strain is an immune-incompetent NOD mouse Prkdc congenic strain that can be compared to the NOD mouse, to distinguish events associated with inflammation from events characteristic of the strain that are independent of T- and B-cell-mediated inflammatory responses.5 The NOD SCID strain is significantly depleted in functional T, B, and natural killer (NK) cells and is free of exocrine tissue destruction.6 The early pathologic events associated with dacryoadenitis in the NOD mouse and other disease models include the development of functional quiescence (e.g., inability of acinar cells to secrete tear proteins from preformed secretory vesicles) in regions of the LGs, with otherwise normal-appearing and intact acinar cells, the infiltration of inflammatory cells, and formation of foci in the periductular regions and then in widespread foci throughout the LGs and the damage of extracellular matrix and acinar cells by factors released from these infiltrating immune cells. Over time, the healthy acinar cell mass in the LGs is replaced by lymphocytic foci and regions of necrotic and apoptotic cell debris. We have recently reported that lipid droplets, primarily containing cholesterol esters, start accumulating in the cytoplasm of acinar cells in the LGs of the male NOD mouse between 5 and 6 weeks and that this event correlates directly with the immune cell infiltration detected within the same course.4,7 Changes in apolipoprotein expression and sorting may partially underlie this particular change in NOD mouse LGs.4 The observation of significant lipid deposition in NOD mouse LGs is consistent with findings in SjS patients that progressive lipid deposition occurs in both the SGs and the LGs.8,9 We considered the possibility that accumulation of lipids triggers or potentiates the subsequent dacryoadenitis and damage of the LGs. The lipid accumulation characteristic of the male NOD mouse LG was also observed in the male NOD SCID mouse LG,4 indicating that it is genetically determined, not inflammation induced. The commonly accepted view of obesity, a consequence of lipid metabolic malfunction with excessive storage of lipids in adipocytes, defines this disease as a chronic inflammatory disorder associated with induction of a group of proinflammatory factors, including TNFα, IL-6, IL-1β, and cathepsin S (CATS) in human visceral adipose tissues.10 Another cytokine, IL-15, has been reported as a negative regulator of adipose tissue mass in humans and rats, besides its regulation of T and NK cells.11,12 More detailed analysis has defined the major source of proinflammatory factors as macrophages,13 and the major source of CATS as adipose cells.10 It has been demonstrated that this condition promotes the development and progression of atherosclerosis.14 In addition, studies in recent years have unraveled deleterious roles of CATS and other family members in diseases such as cancer, viral infection, osteoporosis, and arthritis.15,16 Furthermore, T lymphocytes and autoantibodies have been detected that are directed against lipids or lipid adducts in multiple sclerosis.17–22 On the other hand, inactivation of CATS results in diminished collagen-induced arthritis, and application of CATS inhibitor prevents autoantigen presentation and autoimmunity in mouse models.23,24 Based on these observations and the knowledge acquired from previous studies, we hypothesized that key factors contributing to disease development and progress in the LGs of the NOD mouse may be induced or upregulated in response to an imbalance in acinar cell lipid homeostasis. These factors would be proinflammatory and could be produced in either epithelial or nonepithelial cells. Our results showed that several gene products related to lipid accumulation in obesity—cathepsins and cytokines—were also collectively upregulated in diseased LGs from NOD and NOD SCID mice.