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PDF file - 690K, The effect of S1P on expression of VEGF-A and VEGF-C. mRNA was isolated from 4T1-luc2 cells treated with 100 nM of S1P or 0.1% BSA. Expression of VEGF-A and VEGF-C was determined by qPCR and normalized to GAPDH mRNA. Data are expressed as fold increases � S.D.

PDF file - 7.4MB, SK1-I reduces recruitment of macrophages to peritumoral regions. Immunofluorescent staining of macrophages in the peritumoral region with F4/80. LYVE-1+ staining revealed only very little co-staining with F4/80.

PDF file - 3.7MB, Effect of knockdown of SphK1 on S1P secretion from 4T1-luc2 cells and on in vitro hemangiogenesis and lymphangiogenesis. (A-C) 4T1-luc2 cells were stably transfected with control-shRNA or SphK1-shRNA. (A) Expression of SphK1 was determined by qPCR and normalized to GAPDH mRNA. *, P < 0.001. (B) SphK1 proteins were determined by western blot. (C) S1P in the culture medium from wild type (WT) 4T1-luc2 cells, 4T1-luc2 cells transfected with control-shRNA and SphK1-shRNA were determined by LC-ESI-MS/MS. *, P

Syndecans (SDCs) are a family of heparan sulfate proteoglycans (HSPGs) glycoproteins ubiquitously expressed on the cell surfaces and extracellular matrix of all mammalian tissues. There are four mammalian syndecans, SDC-1 thorough 4, which play a critical role in cell adhesion, migration, proliferation, differentiation, and angiogenesis through independent and growth factor mediated signaling. An altered expression of SDCs is often observed in autoimmune disorders, cancer, HIV infection, and many other pathological conditions. SDCs modulate disease progression by interacting with a diverse array of ligands, receptors, and other proteins, including extracellular matrix, glycoproteins, integrins, morphogens, and various growth factors and chemokines, along with their receptors and kinases. Specifically, SDCs present on cell surface can bind directly to chemokines to enhance their binding to receptors, downstream signaling, and migration. Alternatively, SDCs can be cleaved and shed to mediate negative regulation of chemokine and growth factor signaling pathways and ligand sequestration. Importantly, SDC shedding may be a biomarker of inflammation, especially in chronic inflammatory diseases. While the current therapies for cancer and several autoimmune disorders have revolutionized treatment outcomes, understanding the pathophysiological role of SDCs and the use of HSPG mimetic or antagonists on cytokine signaling networks may uncover potentially novel targeted therapeutic approaches. This review mainly summarizes the current findings on the role of individual SDCs in disease processes, mechanisms through which SDCs mediate their biological functions, and the possibility of targeting SDCs as future potential therapeutic approaches.

Activating transcription factor 5 (ATF5) is a member of the ATF/cAMP response element–binding protein family of transcription factors. ATF5 regulates stress responses and cell survival, proliferation, and differentiation and also plays a role in viral infections, cancer, diabetes, schizophrenia, and the olfactory system. Moreover, it was found to also have a critical cell cycle–dependent structural function at the centrosome. However, the mechanism that controls the localization of ATF5 at the centrosome is unclear. Here we report that ATF5 is small ubiquitin-like modifier (SUMO) 2/3–modified at a conserved SUMO-targeting consensus site in various types of mammalian cells. We found that SUMOylation of ATF5 is elevated in the G(1) phase of the cell cycle and diminished in the G(2)/M phase. ATF5 SUMOylation disrupted the interaction of ATF5 with several centrosomal proteins and dislodged ATF5 from the centrosome at the end of the M phase. Of note, blockade of ATF5 SUMOylation deregulated the centrosome cycle, impeded ATF5 translocation from the centrosome, and caused genomic instability and G(2)/M arrest in HeLa cells. Our results indicate that ATF5 SUMOylation is an essential mechanism that regulates ATF5 localization and function at the centrosome.

Pro-inflammatory cytokines promote autoimmune inflammation and tissue damage, while anti-inflammatory cytokines help resolve inflammation and facilitate tissue repair. Over the past few decades, this general feature of cytokine-mediated events has offered a broad framework to comprehend the pathogenesis of autoimmune and other immune-mediated diseases, and to successfully develop therapeutic approaches for diseases such as rheumatoid arthritis (RA). Anti-tumor necrosis factor-α (TNF-α) therapy is a testimony in support of this endeavor. However, many patients with RA fail to respond to this or other biologics, and some patients may suffer unexpected aggravation of arthritic inflammation or other autoimmune effects. These observations combined with rapid advancements in immunology in regard to newer cytokines and T cell subsets have enforced a re-evaluation of the perceived pathogenic attribute of the pro-inflammatory cytokines. Studies conducted by others and us in experimental models of arthritis involving direct administration of IFN-γ or TNF-α; in vivo neutralization of the cytokine; the use of animals deficient in the cytokine or its receptor; and the impact of the cytokine or anti-cytokine therapy on defined T cell subsets have revealed a paradoxical anti-inflammatory and immunoregulatory attributes of these two cytokines. Similar studies in other models of autoimmunity as well as limited studies in arthritis patients have also unveiled the disease-protective effects of these pro-inflammatory cytokines. A major mechanism in this regard is the altered balance between the pathogenic T helper 17 (Th17) and protective T regulatory (Treg) cells in favor of the latter. However, it is essential to consider that this aspect of the pro-inflammatory cytokines is context-dependent such that the dose and timing of intervention, the experimental model of the disease under study, and the differences in individual responsiveness can influence the final outcomes. Nevertheless, the realization that pro-inflammatory cytokines can also be immunoregulatory offers a new perspective in fully understanding the pathogenesis of autoimmune diseases and in designing better therapies for controlling them.

Objectives Emergency departments (EDs) commonly analyze cases of patients returning within 72 hours of initial ED discharge as potential opportunities for quality improvement. In this study, we tested the use of a health information exchange (HIE) to improve identification of 72-hour return visits compared to individual hospitals' site-specific data. Methods We collected deidentified patient data over a 5-year study period from Healthix, an HIE in the New York metropolitan area. We measured site-specific 72-hour ED returns and compared these data to those obtained from a regional 31-site HIE (Healthix) and to those from a smaller, antecedent 11-site HIE. Although only ED visits were counted as index visits, either ED or inpatient revisits within 72 hours of the index visit were considered as early returns. Results A total of 12,669,657 patient encounters were analyzed across the 31 HIE EDs, including 6,352,829 encounters from the antecedent 11-site HIE. Site-specific 72-hour return visit rates ranged from 1.1% to 15.2% (median = 5.8%) among the individual 31 sites. When the larger HIE was used to identify return visits to any site, individual EDs had a 72-hour return frequency of 1.8% to 15.5% (median = 6.8%). HIE increased the identification ability of 72-hour ED return analyses by a mean of 11.16% (95% confidence interval = 11.10% to 11.22%) compared with site-specific (no HIE) analyses. Conclusion This analysis demonstrates incremental improvements in our ability to identify early ED returns using increasing levels of HIE data aggregation. Although intuitive, this has not been previously described using HIE. ED quality measurement and patient safety efforts may be aided by using HIE in 72-hour return analyses.

Observations in patients with autoimmune diseases and studies in animal models of autoimmunity have revealed that external environmental factors including exposure to microbes and the state of the host gut microbiota can influence susceptibility to autoimmunity and subsequent disease development. Mechanisms underlying these outcomes continue to be elucidated. These include deviation of the cytokine response and imbalance between pathogenic versus regulatory T cell subsets. Furthermore, specific commensal organisms are associated with enhanced severity of arthritis in susceptible individuals, while exposure to certain microbes or helminths can afford protection against this disease. In addition, the role of metabolites (e.g., short-chain fatty acids, tryptophan catabolites), produced either by the microbes themselves or from their action on dietary products, in modulation of arthritis is increasingly being realized. In this context, re-setting of the microbial dysbiosis in RA using prebiotics, probiotics, or fecal microbial transplant is emerging as a promising approach for the prevention and treatment of arthritis. It is hoped that advances in defining the interplay between gut microbiota, dietary products, and bioactive metabolites would help in the development of therapeutic regimen customized for the needs of individual patients in the near future.

The landscape of mobile devices is changing and their present use by patients for healthcare purposes is unknown. An understanding of current attitudes and usage may help increase patient engagement through mobile applications. This study sought to determine characteristics of mobile device ownership among Emergency Department patients, patients' feelings regarding their use in healthcare, and desired functionality in mobile applications.A cross-sectional survey was undertaken at a single urban tertiary care academic center. A convenience sample of adult English-speaking patients in the Emergency Department were surveyed from June 21 st, 2017 to December 30th, 2017. A secondary analysis of the data was performed based on demographic and socioeconomic factors.260 patients were approached for participation, 11 patients declined, and one patient was excluded. The 248 participants had a median age of 49 (interquartile range 28-62) and 54% were female. 91% of those surveyed own smartphones, 58% owned tablets, and 77% of these patients were comfortable using mobile devices. Those without mobile devices were older (p 0.001) and held less commercial insurance (p = 0.01). A majority of patients were interested in using applications to enter information, track their visit, view results, and communicate with providers during their visit. Following care, there is interest in viewing information about their visit and receiving reminders for appointments and medications. Patients are also interested in using applications for learning about medical conditions and managing medications. Though there are mixed feelings regarding the protection of privacy by apps, they are felt to be safe, effective, useful, and not difficult to use.Ownership of smartphones is high across the Emergency Department population and patients are enthusiastic about using mobile devices as part of their care. Further study can elucidate opportunities to further integrate mobile device applications into patient care.

Rheumatoid arthritis (RA) is characterized by hyperplastic pannus formation mediated by activated synovial fibroblasts that cause joint destruction. We have shown earlier that RA synovial fibroblasts (RASFs) exhibit resistance to apoptosis, primarily as a result of enhanced expression of myeloid cell leukemia-1 (Mcl-1). In this study, we discovered that ursolic acid (UA), a plant-derived pentacyclic triterpenoid, selectively induces B-cell lymphoma 2 homology 3-only protein Noxa in human RASFs. We observed that UA-induced Noxa expression was followed by a consequent decrease in Mcl-1 expression in a dose-dependent manner. Subsequent evaluation of the signaling pathways showed that UA-induced Noxa is primarily mediated by the JNK pathway in human RASFs. Chromatin immunoprecipitation (IP) studies into the promoter region of Noxa indicated the role of transcription factor specificity protein 1 in JNK-mediated Noxa expression. Furthermore, the results from IP studies and proximity ligation assays indicated that UA-induced Noxa colocalizes and associates with Mcl-1 to prime it for proteasomal degradation through K(48)-linked ubiquitination by the selective recruitment of Mcl-1 ubiquitin ligase E3, a homologous to E6-associated protein C terminus domain-containing E3 ubiquitin ligase. These findings unveil a novel mechanism of inducing apoptosis in RASFs and a potential adjunct therapeutic strategy of regulating synovial hyperplasia in RA.—Kim, E. Y., Sudini, K., Singh, A. K., Haque, M., Leaman, D., Khuder, S., Ahmed, S. Ursolic acid facilitates apoptosis in rheumatoid arthritis synovial fibroblasts by inducing SP1-mediated Noxa expression and proteasomal degradation of Mcl-1.

This online presentation reviews the mechanisms of traumatic urinary bladder injury, with case illustrations of the types of urinary bladder injury according to the commonly used radiologic classification system, discusses optimal CT cystographic protocol and technique, and highlights potential diagnostic and interpretive pitfalls to aid in accurate diagnosis of traumatic urinary bladder injury.

SummaryInflammatory bowel disease is an important risk factor for colorectal cancer. We show that sphingosine-1-phosphate (S1P) produced by upregulation of sphingosine kinase 1 (SphK1) links chronic intestinal inflammation to colitis-associated cancer (CAC) and both are exacerbated by deletion of Sphk2. S1P is essential for production of the multifunctional NF-κB-regulated cytokine IL-6, persistent activation of the transcription factor STAT3, and consequent upregulation of the S1P receptor, S1PR1. The prodrug FTY720 decreased SphK1 and S1PR1 expression and eliminated the NF-κB/IL-6/STAT3 amplification cascade and development of CAC, even in Sphk2−/− mice, and may be useful in treating colon cancer in individuals with ulcerative colitis. Thus, the SphK1/S1P/S1PR1 axis is at the nexus between NF-κB and STAT3 and connects chronic inflammation and CAC.

Heat shock proteins (Hsps) are highly conserved, and their expression is upregulated in cells by heat and other stressful stimuli. These proteins play a vital role in preserving the structural and functional integrity of cells under stress. Despite the ubiquitous expression of Hsps in an individual, the immune system is not fully tolerant to them. In fact, Hsps are highly immunogenic in nature, and immune response to these proteins is observed in various inflammatory and autoimmune diseases. Studies on the immunopathogenesis of autoimmune arthritis in the rat adjuvant arthritis (AA) model of human rheumatoid arthritis (RA) as well as observations in patients with RA and juvenile idiopathic arthritis (JIA) have unraveled immunoregulatory attributes of self-Hsp65-directed immunity. Notable features of Hsp65 immunity in AA include protection rather than disease induction following immunization of Lewis rats with self (rat)-Hsp65; the diversification of T cell response to mycobacterial Hsp65 during the course of AA and its association with spontaneous induction of response to self-Hsp65; the cross-reactive T cells recognizing foreign and self homologs of Hsp65 and their role in disease suppression in rats; the suppressive effect of antibodies to Hsp65 in AA; and the use of Hsp65, its peptides, or altered peptide ligands in controlling autoimmune pathology. The results of studies in the AA model have relevance to RA and JIA. We believe that these insights into Hsp65 immunity would not only advance our understanding of the disease process in RA/JIA, but also lead to the development of novel therapeutic approaches for autoimmune arthritis.

Sphingosine-1-phosphate (S1P), a ligand for 5 specific receptors, is a potent lipid mediator that plays important roles in lymphocyte trafficking and immune responses. S1P is produced inside cells and therefore must be secreted to exert its effects through these receptors. Spinster 2 (Spns2) is one of the cell surface transporters thought to secrete S1P. We have shown that Spns2 can export endogenous S1P from cells and also dihydro-S1P, which is active at all cell surface S1P receptors. Moreover, Spns2−/− mice have decreased levels of both of these phosphorylated sphingoid bases in blood, accompanied by increases in very long chain ceramide species, and have defective lymphocyte trafficking. Surprisingly, levels of S1P and dihydro-S1P were increased in lymph from Spns2−/− mice as well as in specific tissues, including lymph nodes, and interstitial fluid. Moreover, lymph nodes from Spns2−/− mice have aberrant lymphatic sinus that appeared collapsed, with reduced numbers of lymphocytes. Our data suggest that Spns2 is an S1P transporter in vivo that plays a role in regulation not only of blood S1P but also lymph node and lymph S1P levels and consequently influences lymphocyte trafficking and lymphatic vessel network organization.—Nagahashi, M., Kim, E. Y., Yamada, A., Ramachandran, S., Allegood, J. C., Hait, N. C., Maceyka, M., Milstien, S., Takabe, K., Spiegel, S. Spns2, a transporter of phosphorylated sphingoid bases, regulates their blood and lymph levels and the lymphatic network.

Agents with selective toxicity to hypoxic cells have shown promise as adjuncts to radiotherapy. Our previous studies showed that the bioreductive alkylating agent KS119 had an extremely large differential toxicity to severely hypoxic and aerobic cells in cell culture, and was effective in killing the hypoxic cells of EMT6 mouse mammary tumors in vivo. However, the limited solubility of that compound precluded its development as an anticancer drug. Here we report our initial studies with KS119W, a water-soluble analog of KS119. The cytotoxicity of KS119W to EMT6 cells in vitro was similar to that of KS119, with both agents producing only minimal cytotoxicity to aerobic cells even after intensive treatments, while producing pronounced cytotoxicity to oxygen-deficient cells. This resulted in large differentials in the toxicities to hypoxic and aerobic cells (1,000-fold at 10 μM). Low pH had only minimal effects on the cytotoxicity of KS119W. Under hypoxic conditions, EMT6 cells transfected to express high levels of either human or mouse versions of the repair protein O(6)-alkylguanine-DNA alkyltransferase, which is also known as O(6)-methylguanine DNA-methyltransferase, were much more resistant to KS119W than parental EMT6 cells lacking O(6)-alkylguanine-DNA alkyltransferase, confirming the importance of DNA O-6-alkylation to the cytotoxicity of this agent. Studies with EMT6 tumors in BALB/c Rw mice using both tumor cell survival and tumor growth delay assays showed that KS119W was effective as an adjunct to irradiation for the treatment of solid tumors in vivo, producing additive or supra-additive effects in most combination regimens for which the interactions could be evaluated. Our findings encourage additional preclinical studies to examine further the antineoplastic effects of KS119W alone and in combination with radiation, and to examine the pharmacology and toxicology of this new bioreductive alkylating agent so that its potential for clinical use as an adjuvant to radiotherapy can be evaluated.

Background. The purpose of this study was to examine trends in neck dissection and regionalization. Methods. This cross-sectional and longitudinal study used the years 2000, 2004, and 2006 data from the Nationwide Inpatient Sample. Chi-square tests compared trends for total neck dissections and specific subsites. To test regionalization, we examined the distribution of procedures across hospital and procedure volume quartiles. Results. From 2000 to 2006, the number of neck dissections increased from 18,112 to 22,918. Three-fourths of the total increase was associated with thyroid and parathyroid gland or skin neoplasms. There was an increase in neck dissections for upper aerodigestive tract (UADT) subsites and no decline for the oropharynx and tongue base. Regionalization occurred as high-volume hospitals and providers performed a greater proportion of neck dissections over time. Conclusion. Neck dissections increased from the year 2000 to 2006. There were no decreases in neck dissections for certain subsites with a greater role for primary chemoradiotherapy. Regionalization has occurred. © 2010 Wiley Periodicals, Inc. Head Neck, 2011

Study objective Analyses of 72-hour emergency department (ED) return visits are frequently used for quality assurance purposes and have been proposed as a means of measuring provider performance. These analyses have traditionally examined only patients returning to the same hospital as the initial visit. We use a health information exchange network to describe differences between ED visits resulting in 72-hour revisits to the same hospital and those resulting in revisits to a different site. Methods We examined data from a 31-hospital health information exchange of all ED visits during a 5-year period to identify 72-hour return visits and collected available encounter, patient, and hospital variables. Next, we used multilevel analysis of encounter-level, patient-level, and hospital-level data to describe differences between initial ED visits resulting in different-site and same-site return visits. Results We identified 12,621,159 patient visits to the 31 study EDs, including 841,259 same-site and 107,713 different-site return visits within 72 hours of initial ED presentation. We calculated odds ratios (ORs) and 95% confidence intervals (CIs) for the initial-visit characteristics' predictive relationship that any return visit would be at a different site: daytime visit (OR 1.10; 95% CI 1.07 to 1.12), patient-hospital county concordance (OR 1.40; 95% CI 1.36 to 1.44), male sex (OR 1.27; 95% CI 1.24 to 1.30), aged 65 years or older (OR 0.55; 95% CI 0.53 to 0.57), sites with an ED residency (OR 0.41; 95% CI 0.40 to 0.43), sites at an academic hospital (OR 1.12; 95% CI 1.08 to 1.15), sites with high density of surrounding EDs (OR 1.73; 95% CI 1.68 to 1.77), and sites with a high frequency of same-site return visits (OR 0.10; 95% CI 0.10 to 0.11). Conclusion This analysis describes how ED encounters with early revisits to the same hospital differ from those with revisits to a second hospital. These findings challenge the use of single-site return-visit frequency as a quality measure, and, more constructively, describe how hospitals can use health information exchange to more accurately identify early ED return visits and to support programs related to these revisits.

Tumor hypoxia influences the outcome of treatment with radiotherapy, chemotherapy and even surgery, not only for the treatment of large bulky tumors with extensive necrosis, but also in the treatment of very small primary tumors and recurrences, micrometastases, and surgical margins with microscopic tumor involvement. Because hypoxic tumor cells are resistant to radiation and to many anticancer drugs, many approaches to circumventing the therapeutic resistance induced by hypoxia have been examined in laboratory studies and clinical trials. In this review, these approaches and the results of past laboratory and clinical studies are described and the limitations of the past agents and their testing are discussed. We describe the importance of new technologies for measuring hypoxia in human tumors, which allow assessment of pretreatment tumor oxygen levels and changes in hypoxia over the course of prolonged treatment regimens. These offer the possibility of improving the design of clinical trials and the selection of patients who will benefit from hypoxia-directed therapies, as well as the possibility of facilitating the development of better agents and regimens for use in hypoxia-directed therapy. We also discuss how the improved understanding of the abnormal vascular beds in solid tumors and of the effects of hypoxia and related microenvironmental insults, resulting from recent and ongoing research, offers the potential for finding new therapeutic targets, that may lead to the development of new agents and novel therapeutic approaches for selectively targeting cells in the adverse microenvironments within solid tumors.

Based on solid-phase binding assays with enzyme-linked immunosorbent assay detection, previous investigators suggested that intracellular carbonic anhydrase II (CA II) interacts at high affinity with the C-terminal (Ct) domains of SLC4 bicarbonate-transport proteins, expressed as glutathione S-transferase (GST) fusion proteins, to form functional HCO3- metabolons. Here we re-evaluated this protein-protein interaction using two solid-phase binding assays. We first compared the ability of the Ct domain of three SLC4 transporters, SLC4-A1 (AE1), SLC4-A4 (NBCe1), and SLC4-A8 (NDCBE), to bind immobilized CA II, using enzyme-linked immunosorbent assay detection. We found that when expressed as GST fusion proteins, all three bind to CA II (Kd 300-600 nM) better than does pure GST. However, we detected no binding of pure SLC4-Ct peptides to immobilized CA II. Second, we reversed assay orientation by immobilizing the SLC4-Ct fusion proteins or peptides. We found that more CA II binds to GST than to any of the three GST-SLC4-Ct fusion proteins. Furthermore, we detected no binding of CA II to any of the immobilized pure SLC4-Ct peptides. Finally, we used surface plasmon resonance to detect possible rapid interactions between CA II and the pure peptides. Although we detected acetazolamide binding to immobilized CA II and specific antibodies binding to immobilized SLC4-Ct peptides, we detected no binding of CA II to immobilized SLC4-Ct or vice versa. Thus, although an HCO3 metabolon may exist, CA II cannot bind directly to pure SLC4-Ct peptides and can bind to GST-SLC4-Ct fusion proteins only when the CA II is immobilized and the fusion protein is soluble, and not vice versa.

The centrosome is the major microtubule (MT)-organizing center (MTOC) in animal cells. It comprises a pair of centrioles and the surrounding pericentriolar materials (PCM), and is duplicated once per cell cycle in a highly spatiotemporally regulated manner. Because of the intricate links between centrosomal functions, mitotic spindle assembly and cell division, defects in the centrosome structure and function often lead to malformation of mitotic spindles and genomic instabilities, which result in a range of human diseases including tumorigenesis, ciliopathy, microcephaly, and dwarfism.1 Previous studies indicated that the proximal end of the mother centriole plays instructive roles in the biogenesis of the PCM and the procentriole and that the PCM is essential for centriole stability. However, the molecules and the mechanism that control centriole-PCM interaction are poorly understood. In our recent study, published in the current issue of Cell,2 we provided evidence showing that the activating transcription factor 5 (ATF5) acts unexpectedly as an essential structural PCM protein that bridges the PCM and the proximal end of the mother centriole. By interacting with both pericentrin (PCNT) in the PCM and the polyglutamylated tubulin (PGT) at the proximal end of the mother centriole, ATF5 controls the formation of the PCM and the integrity of the centrioles in a cell cycle- and centriole age-dependent manner.(Fig. 1)

Many autoimmune diseases are believed to involve primarily T cell-mediated effector mechanisms. There is increasing realization, however, that Abs may also play a vital role in the propagation of T cell-driven disorders. In this study, on the rat adjuvant-induced arthritis (AA) model of human rheumatoid arthritis, we examined the characteristics of serum Ab response to mycobacterial heat shock protein (hsp) 65 (Bhsp65), self (rat) hsp65 (Rhsp65), and linear peptides spanning these two molecules. The AA-resistant WKY (RT.1l) rat responded to the heat-killed Mycobacterium tuberculosis immunization with a rapid burst of Abs to both Bhsp65 and Rhsp65. These Abs reacted with numerous peptide epitopes; however, this response was reduced to a few epitopes with time. On the contrary, the susceptible Lewis (RT.1l) rat developed a relatively lower Ab response to Bhsp65, and Abs to Rhsp65 did not appear until the recovery from the disease. The Ab response in Lewis rats diversified with progression of AA, and there was an intriguing overlap between the repertoire of Bhsp65-reactive B and T cells during the recovery phase of AA. Nonetheless, subsets of the repertoire of the late Abs in both rat strains became focused on the same epitope regions of Bhsp65 and Rhsp65. The functional relevance of these Abs was evident from the results showing that sera from recovery phase Lewis or WKY rats, but not that of naive rats, afforded protection against subsequent AA. These results are of significance in further understanding of the role of humoral immunity in the pathogenesis of autoimmune arthritis.

The tumor necrosis factor (TNF) receptor family member CD40 plays an essential role in the activation of antigen-presenting cells, B cell maturation, and immunoglobulin (Ig) class switching critical for adaptive immunity. Although the bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P) and the kinase that produces it, sphingosine kinase 1 (SphK1), have long been implicated in the actions of TNF mediated by engagement of TNFR1, nothing is yet known of their role in CD40-mediated events. We have now found that ligation of CD40 activates and translocates SphK1 to the plasma membrane, leading to generation of S1P. SphK1 inhibition in human tonsil B cells, as well as inhibition or deletion of SphK1 in mouse splenic B cells, significantly reduced CD40-mediated Ig class switching and plasma cell differentiation ex vivo. Optimal activation of downstream CD40 signaling pathways, including NF-κB, p38, and JNK, also required SphK1. In mice treated with a SphK1 inhibitor or in SphK1−/− mice, isotype switching to antigen-specific IgE was decreased in vivo by 70 and 55%, respectively. Our results indicate that SphK1 is important for CD40-mediated B cell activation and regulation of humoral responses and suggest that targeting SphK1 might be a useful therapeutic approach to control antigen-specific IgE production.—Kim, E. Y., Sturgill, J. L., Hait, N. C., Avni, D., Valencia, E. C., Maceyka, M., Lima, S., Allegood, J., Huang, W.-C., Zhang, S., Milstien, S., Conrad, D., Spiegel, S. Role of sphingosine kinase 1 and sphingosine-1-phosphate in CD40 signaling and IgE class switching.

Both genetic and environmental factors are believed to be involved in the induction of autoimmune diseases. Adjuvant arthritis (AA) is inducible in susceptible rat strains by injection of Mycobacterium tuberculosis, and arthritic rats raise T cell responses to the 65-kDa mycobacterial heat-shock protein (Bhsp65). We observed that Fischer 344 (F344) rats raised in a barrier facility (BF-F344) are susceptible to AA, whereas F344 rats maintained in a conventional facility (CV-F344) show significantly reduced incidence and severity of AA, despite responding well to the arthritogenic determinant within Bhsp65. The acquisition of protection from AA can be circumvented if rats are maintained on neomycin/acidified water. Strikingly, naive unimmunized CV-F344 rats but not BF-F344 rats raised T cell responses to Bhsp65 C-terminal determinants (BCTD) (we have previously shown that BCTD are involved in regulation of acute AA in the Lewis rat); however, T cells of naive CV-F344 and BF-F344 gave a comparable level of proliferative response to a mitogen, but no response at all to an irrelevant Ag. Furthermore, adoptive transfer into naive BF-F344 rats of splenic cells of naive CV-F344 rats (restimulated with BCTD in vitro) before induction of AA resulted in a considerably reduced severity of AA. These results suggest that spontaneous (inadvertent) priming of BCTD-reactive T cells, owing to determinant mimicry between Bhsp65 and its homologues in microbial agents in the conventional environment, is involved in modulating the severity of AA in CV-F344 rats. These results have important implications in broadening understanding of the host-microbe interaction in human autoimmune diseases.

Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid mediator that promotes breast cancer progression by diverse mechanisms that remain somewhat unclear. Here we report pharmacologic evidence of a critical role for sphingosine kinase 1 (SphK1) in producing S1P and mediating tumor-induced hemangiogenesis and lymphangiogenesis in a murine model of breast cancer metastasis. S1P levels increased both in the tumor and the circulation. In agreement, serum S1P levels were significantly elevated in stage IIIA human breast cancer patients, compared with age/ethnicity-matched healthy volunteers. However, treatment with the specific SphK1 inhibitor SK1-I suppressed S1P levels, reduced metastases to lymph nodes and lungs, and decreased overall tumor burden of our murine model. Both S1P and angiopoietin 2 (Ang2) stimulated hemangiogenesis and lymphangiogenesis in vitro, whereas SK1-I inhibited each process. We quantified both processes in vivo from the same specimen by combining directed in vivo angiogenesis assays with fluorescence-activated cell sorting, thereby confirming the results obtained in vitro. Notably, SK1-I decreased both processes not only at the primary tumor but also in lymph nodes, with peritumoral lymphatic vessel density reduced in SK1-I–treated animals. Taken together, our findings show that SphK1-produced S1P is a crucial mediator of breast cancer–induced hemangiogenesis and lymphangiogenesis. Our results implicate SphK1 along with S1P as therapeutic targets in breast cancer. Cancer Res; 72(3); 726–35. ©2012 AACR.

Tumour-necrosis factor (TNF) receptor-associated factor 2 (TRAF2) is a key component in NF-κB signalling triggered by TNF-α1, 2. Genetic evidence indicates that TRAF2 is necessary for the polyubiquitination of receptor interacting protein 1 (RIP1)3 that then serves as a platform for recruitment and stimulation of IκB kinase, leading to activation of the transcription factor NF-κB. Although TRAF2 is a RING domain ubiquitin ligase, direct evidence that TRAF2 catalyses the ubiquitination of RIP1 is lacking. TRAF2 binds to sphingosine kinase 1 (SphK1)4, one of the isoenzymes that generates the pro-survival lipid mediator sphingosine-1-phosphate (S1P) inside cells. Here we show that SphK1 and the production of S1P is necessary for lysine-63-linked polyubiquitination of RIP1, phosphorylation of IκB kinase and IκBα, and IκBα degradation, leading to NF-κB activation. These responses were mediated by intracellular S1P independently of its cell surface G-protein-coupled receptors. S1P specifically binds to TRAF2 at the amino-terminal RING domain and stimulates its E3 ligase activity. S1P, but not dihydro-S1P, markedly increased recombinant TRAF2-catalysed lysine-63-linked, but not lysine-48-linked, polyubiquitination of RIP1 in vitro in the presence of the ubiquitin conjugating enzymes (E2) UbcH13 or UbcH5a. Our data show that TRAF2 is a novel intracellular target of S1P, and that S1P is the missing cofactor for TRAF2 E3 ubiquitin ligase activity, indicating a new paradigm for the regulation of lysine-63-linked polyubiquitination. These results also highlight the key role of SphK1 and its product S1P in TNF-α signalling and the canonical NF-κB activation pathway important in inflammatory, antiapoptotic and immune processes. Fil: Alvarez, Sergio Eduardo. Virginia Commonwealth University. School of Medicine; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentina Fil: Harikumar, Kuzhuvelil B.. Virginia Commonwealth University. School of Medicine; Estados Unidos Fil: Hait, Nitai C.. Virginia Commonwealth University. School of Medicine; Estados Unidos Fil: Allegood, Jeremy. Virginia Commonwealth University. School of Medicine; Estados Unidos Fil: Strub, Graham M.. Virginia Commonwealth University. School of Medicine; Estados Unidos Fil: Kim, Eugene Y.. Virginia Commonwealth University. School of Medicine; Estados Unidos Fil: Maceycka, Michael. Virginia Commonwealth University. School of Medicine; Estados Unidos Fil: Jiang, Hualiang. Chinese Academy of Sciences. Shangai Institute of Materia Medica. State Key Laboratory of Drug Research; China Fil: Lu, Cheng. Chinese Academy of Sciences. Shangai Institute of Materia Medica. State Key Laboratory of Drug Research; China Fil: Kordula, Tomasz. Virginia Commonwealth University. School of Medicine; Estados Unidos Fil: Milstien, Sheldon. Virginia Commonwealth University. School of Medicine; Estados Unidos Fil: Spiegel, Sarah. Virginia Commonwealth University. School of Medicine; Estados Unidos

Adjuvant arthritis (AA) shares several features with human rheumatoid arthritis, and it can be induced in the Lewis (LEW) rat but not the Wistar Kyoto (WKY) rat (both RT.1(l)) by immunization with heat-killed Mycobacterium tuberculosis (Mtb). We set out to unravel the mechanisms underlying the differential susceptibility to AA of these MHC-compatible rat strains.We compared the levels of T-cell proliferative and cytokine response to the immunoregulatory self (rat) hsp65 (Rhsp65) after an arthritogenic (Mtb) challenge and the kinetics of migration of adoptively transferred, (111)Indium-labeled, Mtb-primed leukocytes into the hind paw joints of recipient rats.The WKY rats raised a significantly higher level of T-cell proliferative response coupled with a temporally opposite cytokine profile against the disease-regulating Rhsp65 compared to that of LEW rats. Moreover, the arthritogenic leukocytes accumulated into the joints of WKY rats at significantly lower numbers than that in LEW rats.These results offer novel insights into the immune events influencing the pathogenesis of autoimmune arthritis.

The pathogenesis of T cell-mediated diseases like rheumatoid arthritis (RA) has typically been explained in the context of the Th1-Th2 paradigm: the initiation/propagation by pro-inflammatory cytokines, and downregulation by Th2 cytokines. However, in our study based on the adjuvant-induced arthritis (AA) model of RA, we observed that Lewis (LEW) (RT.1(l)) rats at the recovery phase of AA showed the highest level of IFN-gamma in recall response to mycobacterial heat-shock protein 65 (Bhsp65), whereas AA-resistant Wistar-Kyoto (WKY) (RT.1(l)) rats secreted high levels of IFN-gamma much earlier following disease induction. However, no significant secretion of IL-10 or TGF-beta was observed in either strain. Furthermore, pre-treatment of LEW rats with a peptide of self (rat) hsp65 (R465), which induced T cells secreting predominantly IFN-gamma, afforded protection against AA and decreased IL-17 expression by the arthritogenic epitope-restimulated T cells. These results provide a novel perspective on the pathogenesis of autoimmune arthritis.

The heat shock proteins (HSPs) are molecular chaperones that are emerging as biochemical regulators of cell growth, apoptosis, protein homeostasis and intracellular targeting of peptides. The immunological function of the HSPs are imparted by tissue specific peptides associated with the HSPs and as such autologous cancer derived HSP-peptide complexes are unique therapeutic agents. Since a majority of the intracellular peptides are generated by the proteasome, we examined the consequence of abrogation of proteasome function by proteasome inhibitors (PIs) such as Lactacystin, MG-132 and LLM on the growth and induction profile of HSP70 and gp96 using hematopoietic, lymphoid, and epithelial derived cancer cell lines. The effect on growth was measured by the XTT assay and induction of the heat shock proteins by western blot analyses using HSP70 and gp96 specific antibodies. Of the PIs tested, cancer cells, were most sensitive to MG-132 and least sensitive to LLM. MG-132 also showed a 10-fold differential sensitivity between estrogen receptor positive, (ER+) MCF-7 cells and negative cells, (ER-) MDA-MB-231. Induction of heat shock proteins, gp96 and HSP70 was, however, noted in response to LLM. Since LLM exhibited minimal cytotoxic effect, metabolic stress that results in induction of HSPs may not be translated in cell growth inhibition and that there may exist a cell-type specific phenomenon in the HSP response to PI mediated metabolic stress.

Inflammatory bowel disease is an important risk factor for colorectal cancer. Sphingosine-1-phosphate (S1P), a pleiotropic bioactive sphingolipid metabolite formed by two sphingosine kinases, SphK1 and SphK2, is involved in inflammation and cancer. Enhanced tumor number and size, with a concomitant increase in tumor load was observed in mice overexpressing SphK1 treated with the colonotropic mutagen azoxymethane (AOM) followed by three rounds of oral administration of the luminal toxin dextran sodium sulfate (DSS) to induce colitis-associated cancer (CAC). There was also a large increase in adenomas with a high grade of dysplasia associated with increased SphK1 expression and formation of S1P. We demonstrated that the SphK1/S1P axis is important for production of IL-6 and is involved in persistent activation of STAT3 and consequent upregulation of one of its target genes, the S1P receptor S1PR1. Because the pro-drug FTY720 modulates the immune system by acting as a functional antagonist of S1PR1, inducing its internalization and degradation, we examined its effect on CAC progression. FTY720 drastically reduced colitis-associated tumorigenesis, when it was administered during CAC induction. Remarkably, FTY720 blocked CAC progression even when it was administered later after the last DSS treatment when tumors were already established. Moreover, it reduced SphK1 expression, production of IL-6, and persistent activation of STAT3. These findings suggest that FTY720 may be beneficial for preventing tumor formation in individuals with inflammatory bowel disease. This work was supported by grants from the National Institute of Health (R37GM043880, R01CA61774, U19AI077435 to S.S. and R01CA160688 to K.T.). M.N. is a Japan Society for the Promotion of Science Postdoctoral Fellow. Citation Format: Akimitsu Yamada, Jie Liang, Masayuki Nagahashi, Eugene Y. Kim, Kuzhuvelil B. Harikumar, Wei-Ching Huang, Nitai C. Hait, Jeremy C. Allegood, Megan M. Price, Dorit Avni, Tomasz Kordula, Milstien Sheldon, Kazuaki Takabe, Sarah Spiegel. FTY720 prevent tumorigenesis and suppress the progression of colitis-associated cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4887. doi:10.1158/1538-7445.AM2013-4887

Inflammatory bowel disease is an important risk factor for colorectal cancer. The pro-inflammatory cytokines TNF-alpha and IL-6 and their downstream master transcription factors NF-kappaB and Stat3 are critical regulators of chronic inflammation and cancer. There is growing evidence that sphingosine-1-phosphage (S1P), a pleiotropic bioactive sphingolipid metabolite formed inside cells by two closely related sphingosine kinases, SphK1 and SphK2, is involved in inflammation and cancer. In this work, we have shown that S1P produced by upregulation of SphK1 links chronic intestinal inflammation to CAC and both are exacerbated by deletion of SphK2. S1P is essential for production of the multifunctional NF-kappaB-regulated cytokine IL-6, persistent activation of the transcription factor Stat3, and consequent upregulation of the S1P receptor, S1PR1. Adoptive transfer demonstrated that SphK2 deficiency in hematopoietic cells contributed to colitis severity, IL-6 production, and activation of Stat3 and NF-kappaB. The pro-drug FTY720 decreased SphK1 and S1PR1 and eliminated the NF-kappaB-IL-6-Stat3 amplification cascade and development of CAC even in SphK2-/- mice, suggesting that FTY720 may be useful in treating colon cancer in individuals with ulcerative colitis. Thus, the SphK1-S1P-S1PR1 axis is at the nexus between NF-kappaB and Stat3 and connects chronic inflammation and CAC. This work was supported by NIH grants R37GM043880, R01CA61774 (to S.S.) and K12HD055881 and the Susan G. Komen for the Cure Research Foundation grant (to K.T.). M.N. is a Japan Society for the Promotion of Science Postdoctoral Fellow. E.Y.K. and J.C.A. were supported by NIH training grant T32HL094290. Citation Format: Masayuki Nagahashi, Jie Liang, Eugene Y. Kim, Kuzhuvelil B. Harikumar, Akimitsu Yamada, Wei-Ching Huang, Nitai C. Hait, Jeremy C. Allegood, Megan M. Price, Dorit Avni, Kazuaki Takabe, Tomasz Kordula, Sheldon Milstien, Sarah Spiegel. Sphingosine-1-phosphate links chronic intestinal inflammation to development of colitis-associated cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-325. doi:10.1158/1538-7445.AM2013-LB-325

Sphingosine-1-phosphate (S1P), a ligand for five specific receptors, is a potent lipid mediator that plays important roles in cancer cell proliferation, migration, and cancer-induced angiogenesis/lymphangiogenesis. S1P is produced inside cells and therefore must be secreted in order to exert its effects through these receptors. We have reported that E2-induced export of S1P mediated by ABCC1 and ABCG2 transporters and consequent activation of S1P receptors may contribute to nongenomic signaling of E2 important for breast cancer pathophysiology. Spinster 2 (Spns2) is one of the cell surface transporters thought to secrete S1P, thus it is important to elucidate its physiological role. Although lymphatic endothelial cells contribute to regulation of S1P levels in lymph, little is known how S1P levels are maintained in the lymphatic system. Spinster 2 knockout mice were used, and the lymphatic system was examined by flow cytometry and immunofluorescent staining. Sphingolipids in the blood, lymph, and organs were determined by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). We found that S1P secreted by breast cancer cells promotes tumor progression in a syngeneic model by stimulating angiogenesis and lymphangiogenesis. Interestingly, suppression of tumor S1P production resulted in less lymph node metastasis. Further, we found that serum S1P levels are elevated in breast cancer patients with lymph node metastasis. We confirmed that Spns2 can export endogenous S1P from cells and also dihydro-S1P, which is active at all cell surface S1P receptors. Moreover, Spns2-/- mice have decreased levels of both of these phosphorylated sphingoid bases in blood, accompanied by increases in very long chain ceramide species, and have defective lymphocyte trafficking. Surprisingly, levels of S1P and dihydro-S1P were increased in lymph from Spns2-/- mice as well as in specific tissues, including lymph nodes, and interstitial fluid. Moreover, lymph nodes from Spns2-/- mice have aberrant lymphatic sinuses, which appear collapsed, with reduced numbers of lymphocytes. Our data suggest that Spns2 is a S1P transporter in vivo that regulate not only of blood S1P but also lymph node and lymph S1P levels, which may implicate a role in lymph node metastasis. M.N. is a Japan Society for the Promotion of Science Postdoctoral Fellow. S.S. is supported by NIH R37GM043880, and K.T. by NIH R01CA160688 and Susan G. Komen for the Cure Investigator Initiated Research Grant. Citation Format: Masayuki Nagahashi, Eugene Y. Kim, Akimitsu Yamada, Subramaniam Ramachandran, Jeremy C. Allegood, Nitai Hait, Michael Maceyka, Sheldon Milstien, Sarah Spiegel, Kazuaki Takabe. Spinster 2 exports S1P, an important player in lymph node metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5000. doi:10.1158/1538-7445.AM2013-5000

INTRODUCTION: Sphingosine kinase 1 (SphK1) is one of the two isoenzymes that generate the potent bioactive sphingolipid mediator, sphingosine-1-phosphate (S1P). S1P promotes cancer cell survival, proliferation and hemangiogenesis. The angiopoietin2 (Ang2)-Tie2 axis also plays a significant role in hemangiogenesis and lymphangiogenesis. The aim of this study was to investigate the involvement of SphK1 in Ang2-induced hem- and lymphangiogenesis. METHODS: Human umbilical vein endothelial cells (HUVECs) and human lymphatic endothelial cells (HLECs) were treated without or with an isozyme-specific inhibitor of SphK1 (SK1-I) and qPCR, western blot, ELISA, and tube formation assays carried out. We also used a new method we developed to quantify both hem- and lymphangiogenesis in the same sample in vivo by combining Directed In Vivo Angiogenesis Assays (DIVAA) with Fluorescence Activated Cell Sorting (DIVAA/FACS). RESULTS: qPCR revealed that human HLECs like HUVECs express high levels of SphK1 and the S1PR1 receptor. S1P rapidly stimulated xocytosis of Ang2 from HLEC storage granules determined by ELISA. Ang2 activated Tie2 in HLECs as demonstrated by its rapid phosphorylation on Tyr992, a major site responsible for its activation. Similar to Ang2, S1P also stimulated phosphorylation of Tie2, which may be due to secretion of Ang2. S1P is a potent hem- and lymphangiogenic factor for HUVECs and HLECs, respectively, as demonstrated by enhanced tube formation. Ang2-induced hem- and lymphangiogenesis was mediated at least in part by SphK1 as shown by the inhibitory effect of SK1-I. Furthermore, both S1P and Ang2 significantly induced hem- and lymphangiogenesis in vivo determined by DIVAA/FACS. Similar to its effect on in vitro hem- and lymphangiogenesis, inclusion of SK1-I in the angioreactors together with Ang2 completely abolished its ability to stimulate both of these processes. CONCLUSION: Activation of SphK1 is necessary for Ang2-induced hemangiogenesis and lymphangiogenesis through the SphK1/S1P and Ang2/Tie2 axes. This work was supported by NIH (K12HD055881) and Susan G. Komen for the Cure (KG090510) to KT, NCI (R01CA61774) to SS, and Sumitomo Life Social Welfare Services Foundation grant to MN. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5280. doi:1538-7445.AM2012-5280

INTRODUCTION: Sphingosine-1-phosphate (S1P), a pleiotropic bioactive lipid mediator, regulates many cellular processes important for breast cancer progression. The aim of this study is to investigate a role for S1P produced by sphingosine kinase 1 (SphK1) in breast cancer progression and tumor-induced hemangiogenesis and lymphangiogenesis. METHODS: An isozyme-specific inhibitor of SphK1 (SK1-I) was used. Tumor burden were quantified using an in vivo imaging system (IVIS 2000). S1P and SK1-I levels were measured by LC-ESI-MS/MS. Hemangiogenesis and lymphangiogenesis are determined by morphological analysis of microvessel density as well as by flow cytometric analysis of endothelial cells. RESULTS: A significant dose dependent effect of SK1-I on inhibition of 4T1-luc2 murine breast cancer cell growth was observed. We confirmed that SK1-I decreased the enzymatic activity of SphK1 and that downregulation of SphK1 with specific siRNA also suppressed growth of these cells. We found that S1P levels increased in the tumor and in the circulation after orthotopic implantation of 4T1-luc2 cells. Similarly, serum S1P levels were significantly elevated in stage IIIA breast cancer patients who have lymph node metastases, compared to age/ethnicity-matched healthy volunteers. SK1-I blocked serum S1P increases in in vivo model and reduced lymph node and lung metastases and overall tumor burden in vivo as well. Importantly, SK1-I also decreased hem- and lymphangiogenesis not only in the primary tumor, but also in lymph nodes, the host tumor microenvironment. Whereas supernatants from 4T1-luc2 cells significantly stimulated tube formation of HUVECs and HLECs, shRNA knockdown of SphK1 markedly reduced them, indicating that S1P produced by SphK1 in 4T1-luc2 breast cancer cells is an important contributor to hem- and lymphangiogenesis. CONCLUSIONS: S1P generated by SphK1 is important for tumor progression, tumor-induced hemangiogenesis and lymphangiogenesis, and lymph node metastais. Therefore targeting SphK1 and its product S1P would be a multi-pronged attack against breast cancer. This work was supported by Sumitomo Life Social Welfare Services Foundation grant to MN, NIH (K12HD055881) and Susan G. Komen for the Cure (KG090510) to KT, and NCI (R01CA61774) to SS. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4364. doi:1538-7445.AM2012-4364