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Retinal pigment epithelium (RPE) performs numerous functions critical to retinal health and visual function. RPE senescence is a hallmark of aging and degenerative retinal disease development. Here,we evaluated the temporal expression of key nicotinamide adenine dinucleotide (NAD+)-biosynthetic genes and associated levels of NAD+,a principal regulator of energy metabolism and cellular fate,in mouse RPE. NAD+ levels declined with age and correlated directly with decreased nicotinamide phosphoribosyltransferase (NAMPT) expression,increased expression of senescence markers (p16INK4a,p21Waf/Cip1,ApoJ,CTGF and $\beta$-galactosidase) and significant reductions in SIRT1 expression and activity. We simulated in vitro the age-dependent decline in NAD+ and the related increase in RPE senescence in human (ARPE-19) and mouse primary RPE using the NAMPT inhibitor FK866 and demonstrated the positive impact of NAD+-enhancing therapies on RPE cell viability. This,we confirmed in vivo in the RPE of mice injected sub-retinally with FK866 in the presence or absence of nicotinamide mononucleotide. Our data confirm the importance of NAD+ to RPE cell biology normally and in aging and demonstrate the potential utility of therapies targeting NAMPT and NAD+ biosynthesis to prevent or alleviate consequences of RPE senescence in aging and/or degenerative retinal diseases in which RPE dysfunction is a crucial element. View Publication

Interleukin-4 (IL-4) suppresses the development of multiple sclerosis in a murine model of experimental autoimmune encephalomyelitis (EAE). Here,we show that,in mice with EAE,the accumulation and persistence in the lymph nodes and spleen of a systemically administered serum albumin (SA)-IL-4 fusion protein leads to higher efficacy in preventing disease development than the administration of wild-type IL-4 or of the clinically approved drug fingolimod. We also show that the SA-IL-4 fusion protein prevents immune-cell infiltration in the spinal cord,decreases integrin expression in antigen-specific CD4+ T cells,increases the number of granulocyte-like myeloid-derived suppressor cells (and their expression of programmed-death-ligand-1) in spinal cord-draining lymph nodes and decreases the number of T helper 17 cells,a pathogenic cell population in EAE. In mice with chronic EAE,SA-IL-4 inhibits immune-cell infiltration into the spinal cord and completely abrogates immune responses to myelin antigen in the spleen. The SA-IL-4 fusion protein may be prophylactically and therapeutically advantageous in the treatment of multiple sclerosis. View Publication

The recent emergence of the novel,pathogenic SARS-coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread pose a global health emergency. Cell entry of coronaviruses depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases. Unravelling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutic targets. Here,we demonstrate that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. A TMPRSS2 inhibitor approved for clinical use blocked entry and might constitute a treatment option. Finally,we show that the sera from convalescent SARS patients cross-neutralized SARS-2-S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention. View Publication

Previous study demonstrated that the low survival of human embryonic stem cells (hESC) under conventional slow-cooling cryopreservation protocols is predominantly due to apoptosis rather than cellular necrosis. Hence,this study investigated whether a synthetic broad-spectrum irreversible inhibitor of caspase enzymes,Z-VAD-FMK can be used to enhance the post-thaw survival rate of hESC. About 100 mM Z-VAD-FMK was supplemented into either the freezing solution,the post-thaw culture media or both. Intact and adherent hESC colonies were cryopreserved so as to enable subsequent quantitation of the post-thaw cell survival rate through the MTT assay,which can only be performed with adherent cells. Exposure to 100 mM Z-VAD-FMK in the freezing solution alone did not significantly enhance the post-thaw survival rate (10.2{\%} vs. 9.9{\%},p {\textgreater} 0.05). However,when 100 mM Z-VAD-FMK was added to the post-thaw culture media,there was a significant enhancement in the survival rate from 9.9{\%} to 14.4{\%} (p {\textless} 0.05),which was further increased to 18.7{\%} when Z-VAD-FMK was also added to the freezing solution as well (p {\textless} 0.01). Spontaneous differentiation of hESC after cryopreservation was assessed by morphological observations under bright-field microscopy,and by immunocytochemical staining for the pluripotency markers SSEA-3 and TRA-1-81. The results demonstrated that exposure to Z-VAD-FMK did not significantly enhance the spontaneous differentiation of hESC within post-thaw culture. View Publication

The bleomycin group antitumor antibiotics have long been of interest as a consequence of their efficacy in the treatment of certain tumors,not to mention their unique structures and properties in mediating dioxygen activation and sequence selective degradation of DNA. At a chemical level,the structure originally assigned to bleomycin was subsequently reassigned and the new structure has been confirmed by total synthesis. Through the elaboration of structurally modified bleomycin congeners and fragments,synthetic efforts have also facilitated an understanding of the contribution of individual structural domains in bleomycin to sequence selective DNA binding and cleavage,and have also provided insights into the nature of the chemical processes by which DNA degradation takes place. Within the last several years,it has also become apparent that bleomycin can mediate the oxidative degradation of all major classes of cellular RNAs; it seems entirely plausible that RNA may also represent an important locus of action for this class of antitumor agent. In parallel with ongoing synthetic and mechanistic efforts using classical methods,the study of bleomycins attached to solid supports has been shown to provide important mechanistic insights,and the actual elaboration of modified bleomycins by solid phase synthesis constitutes a logical extension of such efforts. View Publication

Deregulation of apoptosis,the physiological form of cell death,is closely associated with immunological diseases and cancer. Apoptosis is activated either by death receptor-driven or mitochondrial pathways,both of which may provide potential targets for novel anticancer drugs. Although several ligands stimulating death receptors have been described,the actual molecular events triggering the mitochondrial pathway are largely unknown. Here,we show initiation of apoptosis by gradual depletion of the intracellular coenzyme NAD+. We identified the first low molecular weight compound,designated FK866,which induces apoptosis by highly specific,noncompetitive inhibition of nicotinamide phosphoribosyltransferase (NAPRT),a key enzyme in the regulation of NAD+ biosynthesis from the natural precursor nicotinamide. Interference with this enzyme does not primarily intoxicate cells because the mitochondrial respiratory activity and the NAD+ -dependent redox reactions involved remain unaffected as long as NAD+ is not effectively depleted by catabolic reactions. Certain tissues,however,have a high turnover of NAD+ through its cleavage by enzymes like poly(ADP-ribose) polymerase. Such cells often rely on the more readily available nicotinamide pathway for NAD+ synthesis and undergo apoptosis after inhibition of NAPRT,whereas cells effectively using the nicotinic acid pathway for NAD+ synthesis remain unaffected. In support of this concept,FK866 effectively induced delayed cell death by apoptosis in HepG2 human liver carcinoma cells with an IC(50) of approximately 1 nM,did not directly inhibit mitochondrial respiratory activity,but caused gradual NAD+ depletion through specific inhibition of NAPRT. This enzyme,when partially purified from K562 human leukemia cells,was noncompetitively inhibited by FK866,and the inhibitor constants were calculated to be 0.4 nM for the enzyme/substrate complex (K(i)) and 0.3 nM for the free enzyme (K(i)'),respectively. Nicotinic acid and nicotinamide were both found to have antidote potential for the cellular effects of FK866. FK866 may be used for treatment of diseases implicating deregulated apoptosis such as cancer for immunosuppression or as a sensitizer for genotoxic agents. Furthermore,it may provide an important tool for investigation of the molecular triggers of the mitochondrial pathway leading to apoptosis through enabling temporal separation of NAD+ decrease from ATP breakdown and apoptosis by several days. View Publication

BACKGROUND Asthma is a heterogenous disease characterized by chronic inflammation and airway remodeling. An increase in the severity of airway remodeling is associated with a more severe form of asthma. There is increasing interest in the epithelial to mesenchymal transition process and mechanisms involved in the differentiation and repair of the airway epithelium,especially as they apply to severe asthma. Growing evidence suggests that Epithelial-Mesenchymal transition (EMT) could contribute to airway remodeling and fibrosis in asthma. Severe asthmatic patients with remodeled airways have a neutrophil driven inflammation. Neutrophils are an important source of TGF-$\beta$1,which plays a role in recruitment and activation of inflammatory cells,extracellular matrix (ECM) production and fibrosis development,and is a potent inducer of EMT. OBJECTIVE As there is little data examining the contribution of neutrophils and/or their mediators to the induction of EMT in airway epithelial cells,the objective of this study was to better understand the potential role of neutrophils in severe asthma in regards to EMT. METHODS We used an in vitro system to investigate the neutrophil-epithelial cell interaction. We obtained peripheral blood neutrophils from severe asthmatic patients and control subjects and examined for their ability to induce EMT in primary airway epithelial cells. RESULTS Our data indicate that neutrophils from severe asthmatic patients induce changes in morphology and EMT marker expression in bronchial epithelial cells consistent with the EMT process when co-cultured. TGF-$\beta$1 levels in the culture medium of severe asthmatic patients were increased compared to that from co-cultures of non-asthmatic neutrophils and epithelial cells. CONCLUSIONS AND CLINICAL RELEVANCE As an inducer of EMT and an important source of TGF-$\beta$1,neutrophils may play a significant role in the development of airway remodeling and fibrosis in severe asthmatic airways. View Publication

A mysterious feature of Crohn's disease (CD) is the extra-intestinal manifestation of creeping fat" (CrF) defined as expansion of mesenteric adipose tissue around the inflamed and fibrotic intestine. In the current study we explore whether microbial translocation in CD serves as a central cue for CrF development. We discovered a subset of mucosal-associated gut bacteria that consistently translocated and remained viable in CrF in CD ileal surgical resections and identified Clostridium innocuum as a signature of this consortium with strain variation between mucosal and adipose isolates suggesting preference for lipid-rich environments. Single-cell RNA sequencing characterized CrF as both pro-fibrotic and pro-adipogenic with a rich milieu of activated immune cells responding to microbial stimuli which we confirm in gnotobiotic mice colonized with C. innocuum. Ex vivo validation of expression patterns suggests C. innocuum stimulates tissue remodeling via M2 macrophages leading to an adipose tissue barrier that serves to prevent systemic dissemination of bacteria." View Publication

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Currently approved immune checkpoint inhibitor therapies targeting the PD-1 and CTLA-4 receptor pathways are powerful treatment options for certain cancers; however,most patients across cancer types still fail to respond. Consequently,there is interest in discovering and blocking alternative pathways that mediate immune suppression. One such mechanism is an upregulation of sialoglycans in malignancy,which has been recently shown to inhibit immune cell activation through multiple mechanisms and therefore represents a targetable glycoimmune checkpoint. Since these glycans are not canonically druggable,we designed an $\alpha$HER2 antibody-sialidase conjugate that potently and selectively strips diverse sialoglycans from breast cancer cells. In syngeneic breast cancer models,desialylation enhanced immune cell infiltration and activation and prolonged the survival of mice,an effect that was dependent on expression of the Siglec-E checkpoint receptor found on tumor-infiltrating myeloid cells. Thus,antibody-sialidase conjugates represent a promising modality for glycoimmune checkpoint therapy. View Publication

Tumor-associated macrophages (TAM) in the tumor microenvironment (TME) cooperate with cancer stem cells (CSC) to maintain stemness. We recently identified cluster of differentiation 44 (CD44) as a surface marker defining head and neck squamous cell carcinoma (HNSCC) CSC. PI3K-4EBP1-SOX2 activation and signaling regulate CSC properties,yet the upstream molecular control of this pathway and the mechanisms underlying cross-talk between TAM and CSC in HNSCC remain largely unknown. Because CD44 is a molecular mediator in the TME,we propose here that TAM-influenced CD44 signaling could mediate stemness via the PI3K-4EBP1-SOX2 pathway,possibly by modulating availability of hyaluronic acid (HA),the main CD44 ligand. HNSCC IHC was used to identify TAM/CSC relationships,and in vitro coculture spheroid models and in vivo mouse models were used to identify the influence of TAMs on CSC function via CD44. Patient HNSCC-derived TAMs were positively and negatively associated with CSC marker expression at noninvasive and invasive edge regions,respectively. TAMs increased availability of HA and increased cancer cell invasion. HA binding to CD44 increased PI3K-4EBP1-SOX2 signaling and the CSC fraction,whereas CD44-VCAM-1 binding promoted invasive signaling by ezrin/PI3K. In vivo,targeting CD44 decreased PI3K-4EBP1-SOX2 signaling,tumor growth,and CSC. TAM depletion in syngeneic and humanized mouse models also diminished growth and CSC numbers. Finally,a CD44 isoform switch regulated epithelial-to-mesenchymal plasticity as standard form of CD44 and CD44v8-10 determined invasive and tumorigenic phenotypes,respectively. We have established a mechanistic link between TAMs and CSCs in HNSCC that is mediated by CD44 intracellular signaling in response to extracellular signals. SIGNIFICANCE: These findings establish a mechanistic link between tumor cell CD44,TAM,and CSC properties at the tumor-stroma interface that can serve as a vital area of focus for target and drug discovery. View Publication

Camostat mesilate (CM),an oral protease inhibitor,has been used clinically for the treatment of chronic pancreatitis in Japan. However,the mechanism by which it operates has not been fully understood. Our aim was to evaluate the therapeutic efficacy of CM in the experimental pancreatic fibrosis model induced by dibutyltin dichloride (DBTC),and we also determined the effect of CM on isolated monocytes and panceatic stellate cells (PSCs). In vivo,chronic pancreatitis was induced in male Lewis rats by single administration of 7 mg/kg DBTC and a special diet containing 1 mg/g CM was fed to the DBTC+CM-treated group from day 7,while the DBTC-treated group rats were fed a standard diet. At days 0,7,14 and 28,the severity of pancreatitis and fibrosis was examined histologically and enzymologically in both groups. In vitro,monocytes were isolated from the spleen of a Lewis rat,and activated with lipopolysaccharide stimulation. Thereafter,the effect of CM on monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-alpha) production from monocytes was examined. Subsequently,cultured rat PSCs were exposed to CM and tested to see whether their proliferation,MCP-1 production and procollagen alpha1 messenger RNA expression was influenced by CM. In vivo,the oral administration of CM inhibited inflammation,cytokines expression and fibrosis in the pancreas. The in vitro study revealed that CM inhibited both MCP-1 and TNF-alpha production from monocytes,and proliferation and MCP-1 production from PSCs. However,procollagen alpha1 expression in PSCs was not influenced by CM. These results suggest that CM attenuated DBTC-induced rat pancreatic fibrosis via inhibition of monocytes and PSCs activity. View Publication