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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.

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.

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.

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.

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

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.

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.