Insights into Selectin Inhibitor Design from Endogenous Isomeric Ligands of SLea and SLex.

Selectins interact with cell-surface glycans to promote the initial tethering and rolling of leukocytes, and these interactions are targets for designs of inhibitors to neutralize diseases related to excessive inflammatory responses in many cardiovascular and immune dysfunctions, as well as tumor markers in different cancers. The isomeric endogenous tetrasaccharides, sialyl Lewis X (sLex) and sialyl Lewis A (sLea), are minimal sugar structures required for selectin binding. Understanding their subtle structural variances and significant advanced binding strengths of sLea over sLex could benefit the rational designs for selectin inhibitors. Modeling based on the E-selectin-sLex crystal structure in the present study demonstrated that the N-acetyl group of GlcNAc in sLex could form steric hindrances in the E-selectin-sLex complex, but the hydroxy methylene group of GlcNAc in sLea at the same position allows for stronger binding interactions. The subsequent designed inhibitor with a synthetic accessible linker molecule that has no exo-cyclic moieties replacing GlcNAc displayed comparable dynamic and energetic binding features to sLea. The present study deciphered the clues from endogenous isomeric sLea and sLex and provided insights into designing selectin inhibitors with simplified synthesis.

Siglec-6 is a target for chimeric antigen receptor T-cell treatment of chronic lymphocytic leukemia.

The only current curative treatment for chronic lymphocytic leukemia (CLL) is allogenic hematopoietic stem cell transplantation. Chimeric antigen receptor treatment targeting CD19 for CLL achieved some complete responses, suggesting the need for alternative or combinational therapies to achieve a more robust response. In this work, we evaluated CAR-T cells specific for Siglec-6, an antigen expressed in CLL, as a novel CAR-T cell treatment for CLL. We found that detection of SIGLEC6 mRNA and Siglec-6 protein is highly restricted to placenta and immune cells in other tissues and it is not expressed in hematopoietic stem cells. We generated CAR-T cells specific for Siglec-6 based on the sequence of the fully human anti-Siglec-6 antibody (JML1), which was identified in a CLL patient that was cured after allo-hematopoietic stem cell transplantation (alloHSCT), and observed that it specifically targeted CLL cells in vitro and in a xenograft mouse model. Interestingly, a short hinge region increased the activity of CAR-T cells to target cells expressing higher Siglec-6 levels but similarly targeted CLL cells expressing lower Siglec-6 levels in vitro and in vivo. Our results identify a novel CAR-T cell therapy for CLL and establish Siglec-6 as a possible target for immunotherapy.

Directing Drugs to Bugs: Antibiotic-Carbohydrate Conjugates Targeting Biofilm-Associated Lectins of Pseudomonas aeruginosa.

Chronic infections by Pseudomonas aeruginosa are characterized by biofilm formation, which effectively enhances resistance toward antibiotics. Biofilm-specific antibiotic delivery could locally increase drug concentration to break antimicrobial resistance and reduce the drug's peripheral side effects. Two extracellular P. aeruginosa lectins, LecA and LecB, are essential structural components for biofilm formation and thus render a possible anchor for biofilm-targeted drug delivery. The standard-of-care drug ciprofloxacin suffers from severe systemic side effects and was therefore chosen for this approach. We synthesized several ciprofloxacin-carbohydrate conjugates and established a structure-activity relationship. Conjugation of ciprofloxacin to lectin probes enabled biofilm accumulation in vitro, reduced the antibiotic's cytotoxicity, but also reduced its antibiotic activity against planktonic cells due to a reduced cell permeability and on target activity. This work defines the starting point for new biofilm/lectin-targeted drugs to modulate antibiotic properties and ultimately break antimicrobial resistance.

CD24 signalling through macrophage Siglec-10 is a target for cancer immunotherapy.

Ovarian cancer and triple-negative breast cancer are among the most lethal diseases affecting women, with few targeted therapies and high rates of metastasis. Cancer cells are capable of evading clearance by macrophages through the overexpression of anti-phagocytic surface proteins called 'don't eat me' signals-including CD471, programmed cell death ligand 1 (PD-L1)2 and the beta-2 microglobulin subunit of the major histocompatibility class I complex (B2M)3. Monoclonal antibodies that antagonize the interaction of 'don't eat me' signals with their macrophage-expressed receptors have demonstrated therapeutic potential in several cancers4,5. However, variability in the magnitude and durability of the response to these agents has suggested the presence of additional, as yet unknown 'don't eat me' signals. Here we show that CD24 can be the dominant innate immune checkpoint in ovarian cancer and breast cancer, and is a promising target for cancer immunotherapy. We demonstrate a role for tumour-expressed CD24 in promoting immune evasion through its interaction with the inhibitory receptor sialic-acid-binding Ig-like lectin 10 (Siglec-10), which is expressed by tumour-associated macrophages. We find that many tumours overexpress CD24 and that tumour-associated macrophages express high levels of Siglec-10. Genetic ablation of either CD24 or Siglec-10, as well as blockade of the CD24-Siglec-10 interaction using monoclonal antibodies, robustly augment the phagocytosis of all CD24-expressing human tumours that we tested. Genetic ablation and therapeutic blockade of CD24 resulted in a macrophage-dependent reduction of tumour growth in vivo and an increase in survival time. These data reveal CD24 as a highly expressed, anti-phagocytic signal in several cancers and demonstrate the therapeutic potential for CD24 blockade in cancer immunotherapy.

Marine bromophenols as an effective inhibitor of virulent proteins (peptidyl arginine deiminase, gingipain R and hemagglutinin A) in Porphyromas gingivalis.

OBJECTIVES: Porphyromonas gingivalis, is one of the major oral pathogen that produce virulent proteins which mediate periodontal tissue inflammation and infection. Marine algae have recently gained popularity for its bioactive molecules and their oral applications. Marine bromophenols (MBs) is abundant in red algae which are reported to have wide medicinal properties. The current research primarily focuses to elucidate the bioactivity of MBs against the virulent proteins produced by P. gingivalis. MATERIALS AND METHODS: Potent MBs which effectively binds and inhibits the virulent proteins peptidyl arginine deiminase (PPAD), gingipain R (Rgp) and hemagglutinin A (HgA) was identified through molecular docking and molecular simulation approach. MBs were extracted from Kappaphycus alvarezii (KAB), marine red algae found in India. The efficacy of this MB was studied against P. gingivalis by employing antibacterial activity assays, gingipain assay, hemagglutination inhibition assay (HIA) and mRNA expression analysis (q RT PCR). RESULTS: MBs with benzene, methyl and glycosyl substitutions demonstrated significant docking score, with good stability and pharmacokinetic properties. In addition to the antibacterial activities against P. gingivalis, KAB was also found to inhibit the gingipain and hemagglutination activities. Exposure of KAB to the virulent genes in P. gingivalis resulted in low mRNA levels of these genes, which suggested the down regulation functions induced by the MBs. CONCLUSION: Biochemical investigations revealed that KAB is a potent natural metabolite that can inhibit and control the virulent proteins produced by P. gingivalis. This study recommends future research to direct towards applicability of MBs in commercial dental products.

Calcitonin gene-related peptide decreases IL-1beta, IL-6 as well as Ym1, Arg1, CD163 expression in a brain tissue context-dependent manner while ameliorating experimental autoimmune encephalomyelitis.

Activation states of immune cells (among them, the so-called pro- or anti-inflammatory states) influence the pathogenesis of multiple sclerosis (MS). The neuropeptide calcitonin gene-related peptide (CGRP) can exert a pro- or anti-inflammatory role in a context-dependent manner. In mice CGRP was found to attenuate the development of experimental autoimmune encephalomyelitis (EAE, a common MS animal model). We analyzed CGRP effects on the expression of cytokines and markers of activation states, as well as its intracellular cascade, following intrathecal administration during EAE immunization. Real Time quantitative-PCR (RT-PCR) showed that IL-1beta and IL-6 (associated to a pro-inflammatory state in EAE), but also Ym1 (also known as Chil3), Arg1 and CD163 (associated to an anti-inflammatory state in EAE) were decreased in the encephalon (devoid of cerebellum). In the cerebellum itself, IL-1beta and Ym1 were decreased. TNF-alpha (associated to a pro-inflammatory state in EAE), but also IL-10 (associated to another type of anti-inflammatory state) and BDNF were unchanged in these two regions. No changes were detected in the spinal cord. Additional tendencies toward a change (as revealed by RT-PCR) were again decreases: IL-10 in the encephalon and Arg1 in the spinal cord. CGRP decreased percentage of Ym1+/CD68+ immunoreactive cells and cell density of infiltrates in the cervical spinal cord pia mater. Instead, Ym1 in the underlying parenchyma and at thoracic and lumbar levels, as well as Arg1, were unchanged. In cultured microglia the neuropeptide decreased Ym1, but not Arg1, immunoreactivity. Inducible NOS (iNOS) was unchanged in spinal cord microglia and astrocytes. The neuropeptide increased the activation of ERK1/2 in the astrocytes of the spinal cord and in culture, but did not influence the activation of ERK1/2 or p38 in the spinal cord microglia. Finally, in areas adjacent to infiltration sites CGRP-treated microglia showed a larger ramification radius. In conclusion, CGRP-induced EAE amelioration was associated to a concomitant, context-dependent decrease in the expression of markers belonging to both pro- or anti-inflammatory activation states of immune cells. It can be hypothesized that CGRP-induced EAE attenuation is obtained through a novel mechanism that promotes down-regulation of immune cell activation that facilitates the establishment of a beneficial environment in EAE provided possibly also by other factors.

Glycomimetic, Orally Bioavailable LecB Inhibitors Block Biofilm Formation of Pseudomonas aeruginosa.

The opportunistic Gram-negative bacterium Pseudomonas aeruginosa is a leading pathogen for infections of immuno-compromised patients and those suffering from cystic fibrosis. Its ability to switch from planktonic life to aggregates, forming the so-called biofilms, is a front-line mechanism of antimicrobial resistance. The bacterial carbohydrate-binding protein LecB is an integral component and necessary for biofilm formation. Here, we report a new class of drug-like low molecular weight inhibitors of the lectin LecB with nanomolar affinities and excellent receptor binding kinetics and thermodynamics. This class of glycomimetic inhibitors efficiently blocked biofilm formation of P. aeruginosa in vitro while the natural monovalent carbohydrate ligands failed. Furthermore, excellent selectivity and pharmacokinetic properties were achieved. Notably, two compounds showed good oral bioavailability, and high compound concentrations in plasma and urine were achieved in vivo.

DCPP1 is the mouse ortholog of human PAUF that possesses functional analogy in pancreatic cancer.

Pancreatic adenocarcinoma upregulated factor (PAUF) overexpressed in pancreatic ductal adenocarcinoma (PDAC) plays a major role in tumor progression and metastasis by autocrine and paracrine manners. However, underlying molecular mechanism of PAUF functioning in pancreatic cancer are not fully understood yet. The objective of this study was to evaluate the potential of demilune cell and parotid protein 1 (DCPP1) as a putative mouse ortholog of human PAUF by sequence alignment and functional studies. Overexpression of mouse DCPP1 in Chinese hamster ovary (CHO) cells or pancreatic cancer cells increased cell proliferation, migration, invasion, and adhesion ability in vitro. Treatment of human pancreatic cancer cells with recombinant mouse DCPP1 elevated cell growth, motility, invasiveness, and adhesiveness. Mouse DCPP1 exerted its function on pancreatic cancer cells by activating intracellular signaling pathways involved in aggressive cancer phenotype of human pancreatic cancer cells. Moreover, subcutaneous injection of mice with DCPP1-overexpressing CHO cells increased tumor sizes. Taken together, we conclude that mouse DCPP1 is a multifunctional promoter of tumor growth through functional activation of pancreatic cancer cells, suggesting it to be an ortholog of human PAUF.

Bivalent O-glycoside mimetics with S/disulfide/Se substitutions and aromatic core: Synthesis, molecular modeling and inhibitory activity on biomedically relevant lectins in assays of increasing physiological relevance.

The emerging significance of recognition of cellular glycans by lectins for diverse aspects of pathophysiology is a strong incentive for considering development of bioactive and non-hydrolyzable glycoside derivatives, for example by introducing S/Se atoms and the disulfide group instead of oxygen into the glycosidic linkage. We report the synthesis of 12 bivalent thio-, disulfido- and selenoglycosides attached to benzene/naphthalene cores. They present galactose, for blocking a plant toxin, or lactose, the canonical ligand of adhesion/growth-regulatory galectins. Modeling reveals unrestrained flexibility and inter-headgroup distances too small to bridge two sites in the same lectin. Inhibitory activity was first detected by solid-phase assays using a surface-presented glycoprotein, with relative activity enhancements per sugar unit relative to free cognate sugar up to nearly 10fold. Inhibitory activity was also seen on lectin binding to surfaces of human carcinoma cells. In order to proceed to characterize this capacity in the tissue context monitoring of lectin binding in the presence of inhibitors was extended to sections of three types of murine organs as models. This procedure proved to be well-suited to determine relative activity levels of the glycocompounds to block binding of the toxin and different human galectins to natural glycoconjugates at different sites in sections. The results on most effective inhibition by two naphthalene-based disulfides and a selenide raise the perspective for broad applicability of the histochemical assay in testing glycoclusters that target biomedically relevant lectins.

Heparin-binding Hemagglutinin of Mycobacterium tuberculosis Is an Inhibitor of Autophagy.

Airway epithelial cell is often the initial site of attack by pathogens, and cell death is commonly caused by internalization of Mycobacterium tuberculosis (Mtb). However, the mechanism of interaction between epithelial cells and Mtb is not well understood. In this study, we investigated the role of the heparin-binding hemagglutinin (HBHA) protein of Mtb in the function of epithelial cells. In particular, the autophagy of A549 cells was determined based on microtubule-associated protein 1 light chain 3 alpha (LC3) activity. Autophagosome formation was detected by Monodansylcadaverine (MDC) staining and immune fluorescence staining of LC3. Autophagy could be significantly suppressed by HBHA protein. In addition, the LDH assay results showed that HBHA treatment could induce death on A549 cells. To explore the form of cell death, we detected the activity of caspase-3 and LDH release of A549 cells in the presence or absence of caspase inhibitor Z-VAD-FMK. Results demonstrated that HBHA treatment could induce apoptosis of A549 cells. To further confirm these results, we constructed the recombinant Mycobacterium smegmatis (MS) expressing HBHA (rMS-HBHA) and explored the influence of rMS-HBHA on the function of A549 cells. rMS-HBHA infection significantly inhibited LC3 expression and the maturation of autophagosomes in A549 cells. Subsequently, we infected A549 cells with MS and detected the viability of intracellular MS by CFU counts. rMS-HBHA showed higher survival and replication capacity in A549 cells than those of the wild-type MS. Finally, infection of A549 cells with rMS-HBHA caused further apoptosis. These findings suggested that rMS-HBHA could inhibit autophagy, promote its survival and replication within A549 cells, and subsequently induce apoptosis on infected cells to facilitate infection.

Synthesis of end-functionalized glycopolymers containing α(2,8) disialic acids via π-allyl nickel catalyzed coordinating polymerization and their interaction with Siglec-7.

An allene monomer containing an α(2,8) disialic acid was polymerized by a π-allyl nickel complex with an azido group to produce end-functional glycopolymers with an excellent polydispersity index. The polymers allowed terminal modification with a fluorescent dye by click chemistry. The glycopolymers can dissociate Siglec-7-GD3 interactions at low concentrations.

OS9 Protein Interacts with Na-K-2Cl Co-transporter (NKCC2) and Targets Its Immature Form for the Endoplasmic Reticulum-associated Degradation Pathway.

Mutations in the renal specific Na-K-2Cl co-transporter (NKCC2) lead to type I Bartter syndrome, a life-threatening kidney disease featuring arterial hypotension along with electrolyte abnormalities. We have previously shown that NKCC2 and its disease-causing mutants are subject to regulation by endoplasmic reticulum-associated degradation (ERAD). The aim of the present study was to identify the protein partners specifically involved in ERAD of NKCC2. To this end, we screened a kidney cDNA library through a yeast two-hybrid assay using NKCC2 C terminus as bait. We identified OS9 (amplified in osteosarcomas) as a novel and specific binding partner of NKCC2. Co-immunoprecipitation assays in renal cells revealed that OS9 association involves mainly the immature form of NKCC2. Accordingly, immunocytochemistry analysis showed that NKCC2 and OS9 co-localize at the endoplasmic reticulum. In cells overexpressing OS9, total cellular NKCC2 protein levels were markedly decreased, an effect blocked by the proteasome inhibitor MG132. Pulse-chase and cycloheximide-chase assays demonstrated that the marked reduction in the co-transporter protein levels was essentially due to increased protein degradation of the immature form of NKCC2. Conversely, knockdown of OS9 by small interfering RNA increased NKCC2 expression by increasing the co-transporter stability. Inactivation of the mannose 6-phosphate receptor homology domain of OS9 had no effect on its action on NKCC2. In contrast, mutations of NKCC2 N-glycosylation sites abolished the effects of OS9, indicating that OS9-induced protein degradation is N-glycan-dependent. In summary, our results demonstrate the presence of an OS9-mediated ERAD pathway in renal cells that degrades immature NKCC2 proteins. The identification and selective modulation of ERAD components specific to NKCC2 and its disease-causing mutants might provide novel therapeutic strategies for the treatment of type I Bartter syndrome.

Silencing pancreatic adenocarcinoma upregulated factor (PAUF) increases the sensitivity of pancreatic cancer cells to gemcitabine.

Pancreatic adenocarcinoma upregulated factor (PAUF) is a new oncogene that activates signaling pathways that play a critical role in resistance to gemcitabine. We thus speculated that PAUF also plays a role in resistance to gemcitabine of pancreatic cancer cells. We established BxPC-3 cell lines with stable PAUF knockdown (BxPC-3_shPAUF) and controls (BxPC-3_shCtrl) and evaluated sensitivity to gemcitabine in vitro by MTT and flow cytometry. We established a xenograft model of human pancreatic cancer to examine PAUF function in gemcitabine resistance in vivo. Gene chip microarrays were performed to identify differentially expressed genes in BxPC-3_shPAUF and BxPC-3_shCtrl cells. Silencing PAUF increased the sensitivity of BxPC-3 cells to gemcitabine in vitro and in vivo. PAUF-knockdown BxPC-3 cell lines treated with gemcitabine showed increased proliferation inhibition and apoptosis compared with controls. Gemcitabine exhibited a more pronounced effect on reduction of BxPC-3_shPAUF tumors than BxPC-3_shCtrl tumors. Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assays confirmed a significantly higher apoptotic rate of BXPC-3_shPAUF tumors compared with BXPC-3_shCtrl tumors. Gene array showed that PAUF function in gemcitabine sensitivity might involve MRP2, MRP3, MDR1, PIK3R1, and NFkB2 genes. PAUF could be considered as a key molecular target for sensitizing pancreatic cancer cells to gemcitabine.

Intelectin 1 suppresses tumor progression and is associated with improved survival in gastric cancer.

Recent evidence shows the emerging roles of intelectin 1 (ITLN1), a secretory lectin, in human cancers. Our previous studies have implicated the potential roles of ITLN1 in the aggressiveness of gastric cancer. Herein, we investigated the functions, downstream targets, and clinical significance of ITLN1 in the progression of gastric cancer. We demonstrated that ITLN1 increased the levels of hepatocyte nuclear factor 4 alpha (HNF4α), resulting in suppression of nuclear translocation and transcriptional activity of ß-catenin in gastric cancer cells. Mechanistically, ITLN1 attenuated the activity of nuclear factor-kappa B, a transcription factor repressing the HNF4α expression, in gastric cancer cells through inactivating the phosphoinositide 3-kinase/AKT/Ikappa B kinase signaling. Gain- and loss-of-function studies demonstrated that ITLN1 suppressed the growth, invasion, and metastasis of gastric cancer cells in vitro and in vivo. In addition, restoration of HNF4α expression prevented the gastric cancer cells from ITLN1-mediated changes in these biological features. In clinical gastric cancer tissues, HNF4α expression was positively correlated with that of ITLN1. Patients with high ITLN1 or HNF4α expression had greater survival probability. Taken together, these data indicate that ITLN1 suppresses the progression of gastric cancer through up-regulation of HNF4α, and is associated with improved survival in patients with gastric cancer.

Pancreatic adenocarcinoma upregulated factor (PAUF) confers resistance to pancreatic cancer cells against oncolytic parvovirus H-1 infection through IFNA receptor-mediated signaling.

Pancreatic adenocarcinoma upregulated factor (PAUF), a novel oncogene, plays a crucial role in the development of pancreatic cancer, including its metastasis and proliferation. Therefore, PAUF-expressing pancreatic cancer cells could be important targets for oncolytic virus-mediated treatment. Panc-1 cells expressing PAUF (Panc-PAUF) showed relative resistance to parvovirus H-1 infection compared with Panc-1 cells expressing an empty vector (Panc-Vec). Of interest, expression of type I IFN-α receptor (IFNAR) was higher in Panc-PAUF cells than in Panc-Vec cells. Increased expression of IFNAR in turn increased the activation of Stat1 and Tyk2 in Panc-PAUF cells compared with that in Panc-Vec cells. Suppression of Tyk2 and Stat1, which are important downstream molecules for IFN-α signaling, sensitized pancreatic cancer cells to parvovirus H-1-mediated apoptosis. Further, constitutive suppression of PAUF sensitized Bxpc3 pancreatic cancer cells to parvovirus H-1 infection. Taken together, these results suggested that PAUF conferred resistance to pancreatic cancer cells against oncolytic parvovirus H-1 infection through IFNAR-mediated signaling.

A PAUF-neutralizing antibody targets both carcinoma and endothelial cells to impede pancreatic tumor progression and metastasis.

Pancreatic adenocarcinoma up-regulated factor (PAUF) is expressed in pancreatic ductal adenocarcinoma (PDAC) and plays an important role in tumor progression and metastasis. Here we evaluate the anti-tumor efficacy of a human monoclonal antibody against PAUF, PMAb83, to provide a therapeutic intervention to treat the disease. PMAb83 reduced tumor growth and distant metastasis in orthotopically xenografted mice of human PDAC cells. PMAb83 treatment retarded proliferation along with weakened aggressiveness traits of the carcinoma cells. AKT/ß-catenin signaling played a role in the carcinoma cell proliferation and the treated xenograft tumors exhibited reduced levels of ß-catenin and cyclin D1. Moreover PMAb83 abrogated the PAUF-induced angiogenic responses of endothelial cells, reducing the density of CD31(+) vessels in the treated tumors. In combination with gemcitabine, PMAb83 conferred enhanced survival of xenografted mice by about twofold compared to gemcitabine alone. Taken together, our findings show that PMAb83 treatment decreases the aggressiveness of carcinoma cells and suppresses tumor vascularization, which culminates in mitigated tumor growth and metastasis with improved survival in PDAC mouse models.

YKL-40 downregulation is a key factor to overcome temozolomide resistance in a glioblastoma cell line.

The frequent recurrence of glioblastoma multiforme (GBM) after standard treatment with temozolomide (TMZ) is a crucial issue to be solved in the clinical field. O6­methylguanine­DNA methyltransferase (MGMT) is considered one of the major mechanisms involved in TMZ resistance. However, some important mechanisms for TMZ resistance other than MGMT have recently been identified. In the present study, we established a TMZ-resistant (TMZ-R) U87 glioblastoma cell line in vitro and in vivo and investigated novel targeting molecules other than MGMT in those cells. The TMZ-R U87 glioblastoma cell line was established in vitro and in vivo. TMZ-R U87 cells showed a more invasive activity and a shorter survival time in vivo. Gene expression analysis using DNA microarray and quantitative PCR (qPCR) demonstrated that YKL­40, MAGEC1 and MGMT mRNA expression was upregulated 100-, 83- and 6-fold, respectively in the TMZ-R U87 cell line. Western blot analysis and qPCR demonstrated that STAT3 phosphorylation, STAT3 target genes and stem cell and mesenchymal marker genes were upregulated to a greater extent in the TMZ­resistant cell line. Notably, short hairpin (sh)RNA­based inhibition against the YKL­40 gene resulted in moderate growth inhibition in the resistant cells in vitro and in vivo. Additionally, YKL­40 gene inhibition exhibited significant suppression of the invasive activity and particularly partially restored the sensitivity to TMZ. Therefore, YKL­40 may be a novel key molecule in addition to MGMT, that is responsible for TMZ resistance in glioblastoma cell lines and could be a new target to overcome TMZ resistance in recurrent glioblastomas in the future.

Novel methylxanthine derivative-mediated anti-inflammatory effects in inflammatory bowel disease.

Family 18 chitinases have a binding capacity with chitin, a polymer of N-acetylglucosamine. Recent studies strongly suggested that chitinase 3-like 1 (CHI3L1, also known as YKL-40) and acidic mammalian chitinase, the two major members of family 18 chitinases, play a pivotal role in the pathogenesis of inflammatory bowel disease (IBD), bronchial asthma and several other inflammatory disorders. Based on the data from high-throughput screening, it has been found that three methylxanthine derivatives, caffeine, theophylline, and pentoxifylline, have competitive inhibitory effects against a fungal family 18 chitinase by specifically interacting with conserved tryptophans in the active site of this protein. Methylxanthine derivatives are also known as adenosine receptor antagonists, phosphodiesterase inhibitors and histone deacetylase inducers. Anti-inflammatory effects of methylxanthine derivatives have been well-documented in the literature. For example, a beneficial link between coffee or caffeine consumption and type 2 diabetes as well as liver cirrhosis has been reported. Furthermore, theophylline has a long history of being used as a bronchodilator in asthma therapy, and pentoxifylline has an immuno-modulating effect for peripheral vascular disease. However, it is still largely unknown whether these methylxanthine derivative-mediated anti-inflammatory effects are associated with the inhibition of CHI3L1-induced cytoplasmic signaling cascades in epithelial cells. In this review article we will examine the above possibility and summarize the biological significance of methylxanthine derivatives in intestinal epithelial cells. We hope that this study will provide a rationale for the development of methylxanthine derivatives, in particular caffeine, -based anti-inflammatory therapeutics in the field of IBD and IBD-associated carcinogenesis.

The lectin pathway of the complement system is downregulated in Crohn's disease patients who respond to anti-TNF-α therapy.

BACKGROUND AND AIMS: The lectin pathway of the complement system is initiated through the recognition of pathogens or altered self-structures by mannan-binding lectin (MBL) or ficolins and subsequent activation of MBL-associated serine proteases (MASPs). Altered ficolin levels may contribute to a dysregulated immune response in Crohn's disease (CD). A complete analysis of the lectin pathway has not been performed in patients with CD. We hypothesised that the lectin pathway proteins exacerbate inflammation in CD. METHODS: We assessed the lectin pathway proteins in 43 patients with active CD & 350 blood donors by measuring the serum levels of MBL; M-, H-, and L-ficolin; MASP-2; MASP-3; and MAp44. In patients with CD, the blood samples were obtained during induction treatment with infliximab or adalimumab. RESULTS: Of 43 patients with CD, 32 (74%) were classified as responders. We observed a nearly 50% decrease in median M-ficolin levels between day 0 and weeks 1/7 in the responders (p<0.001), whereas there was no decrease in the non-responders. MASP-2 levels decreased from baseline to week 1 in both the responders (37%, p<0.0001) and the non-responders (29%, p=0.02). In the responders only, the level of the inhibitory serine protease MASP-3 increased by 26% from baseline to week 1 (p<0.001) and remained high at week 7. CONCLUSIONS: Our findings indicated that M-ficolin, MASP-2, and MASP-3 may act in concert to reduce the activity of the lectin pathway, in patients with CD who respond to biological therapy.

CHI3L1 plays a role in cancer through enhanced production of pro-inflammatory/pro-tumorigenic and angiogenic factors.

Elevated serum levels of a glycoprotein known as chitinase-3-like protein 1 (CHI3L1) have been correlated with poor prognosis and shorter survival of patients with cancer and inflammatory diseases. The biological and physiological functions of CHI3L1 in cancer have not yet been completely elucidated. In this review, we describe the role of CHI3L1 in inducing pro-inflammatory/pro-tumorigenic and angiogenic factors that could promote tumor growth and metastasis.