Biological Effects of Intravenous Vitamin C on Neutrophil Extracellular Traps and the Endothelial Glycocalyx in Patients with Sepsis-Induced ARDS.

(1) Background: The disease-modifying mechanisms of high-dose intravenous vitamin C (HDIVC) in sepsis induced acute respiratory distress syndrome (ARDS) is unclear. (2) Methods: We performed a post hoc study of plasma biomarkers from subjects enrolled in the randomized placebo-controlled trial CITRIS-ALI. We explored the effects of HDIVC on cell-free DNA (cfDNA) and syndecan-1, surrogates for neutrophil extracellular trap (NET) formation and degradation of the endothelial glycocalyx, respectively. (3) Results: In 167 study subjects, baseline cfDNA levels in HDIVC (84 subjects) and placebo (83 subjects) were 2.18 ng/µL (SD 4.20 ng/µL) and 2.65 ng/µL (SD 3.87 ng/µL), respectively, p = 0.45. At 48-h, the cfDNA reduction was 1.02 ng/µL greater in HDIVC than placebo, p = 0.05. Mean baseline syndecan-1 levels in HDIVC and placebo were 9.49 ng/mL (SD 5.57 ng/mL) and 10.83 ng/mL (SD 5.95 ng/mL), respectively, p = 0.14. At 48 h, placebo subjects exhibited a 1.53 ng/mL (95% CI, 0.96 to 2.11) increase in syndecan-1 vs. 0.75 ng/mL (95% CI, 0.21 to 1.29, p = 0.05), in HDIVC subjects. (4) Conclusions: HDIVC infusion attenuated cell-free DNA and syndecan-1, biomarkers associated with sepsis-induced ARDS. Improvement of these biomarkers suggests amelioration of NETosis and shedding of the vascular endothelial glycocalyx, respectively.

Hyperphagia in offsprings of in utero hyperglycemic mothers is associated with increased expression of heparan sulfate proteoglycans in hypothalamus.

In utero hyperglycemia has consequences on future outcomes in the offsprings. We had earlier shown that in utero hyperglycemia impacts proteoglycans/glycosaminoglycans, one of the key molecules involved in brain development. Hypothalamic HSPGs such as syndecan-1 and syndecan-3 are well known for their involvement in feeding behavior. Therefore, studies were carried out to determine the effect of maternal hyperglycemia on the expression of HSPGs in the hypothalamus of offspring brain. Results revealed increased protein abundance of Syndecan-1 and -3 as well as glypican-1 in postnatal adults from hyperglycemic mothers. This was associated with increased hyperphagia and increased expression of Neuropeptide Y. These results indicate the likely consequences on offsprings exposed to in utero hyperglycemia on its growth.

Ageing alters the lipid sensing process in the hypothalamus of Wistar rats. Effect of food restriction.

INTRODUCTION: Lipids regulate a wide range of biological processes. The mechanisms by which fatty acids (FA) and its metabolites influence the hypothalamic regulation of energy homeostasis have been highly studied. However, the effect of ageing and food restriction (FR) on this process is unknown. METHODS: Herein, we analyzed the gene expression, protein and phosphorylation levels of hypothalamic enzymes and transcription factors related to lipid metabolism. Experiments were performed in male Wistar rats of 3-, 8- and 24-month-old Wistar rats fed ad libitum (AL), as ageing model. Besides, 5- and 21-month-old rats were subjected to a moderate FR protocol (equivalent to ≈ 80% of normal food intake) for three months before the sacrifice. RESULTS: Aged Wistar rats showed a situation of chronic lipid excess as a result of an increase in de novo FA synthesis and FA levels that reach the brain, contributing likely to the development of central leptin and insulin resistance. We observe a hypothalamic downregulation of AMP-activated protein kinase (AMPK) and stearoyl-CoA desaturase (SCD1) and an increase of carnitine palmitoyltransferase-1c (CPT1c) expression. DISCUSSION: Our results suggest an impairment in the physiological lipid sensing system of aged Wistar rats, which would alter the balance of the intracellular mobilization and trafficking of lipids between the mitochondria and the Endoplasmic Reticulum (ER) in the hypothalamus, leading probably to the development of neurolipotoxicity in aged rats. Lastly, FR can only partially restore this imbalance.Schematic representation of the fate of LCFA-CoA in the hypothalamus of young and old rats. Blood circulating LCFAs in young Wistar rats reach the hypothalamus, where they are esterified to LCFA-CoA. Into glial cells or neurons, LCFA-CoA are driven to mitochondria (CPT1a) or ER (CPT1c) where could be desaturated by SDC1 and, thereby, converted into structural and signaling unsaturated lipids as oleic acid, related with neuronal myelinization and differentiation. However, the excess of LCFA that reach to the hypothalamus in old animals, could generate an increase in LCFA-CoA, which together with an increase in CPT1c levels, could favor the capture of LCFA-CoA to the ER. The decrease in the levels of SCD1 in old rats would decrease FA unsaturation degree that could trigger lipotoxicity process and neurodegeneration, both related to the development of neurodegenerative diseases linked to age.

Salvianolic acid B attenuates renal interstitial fibrosis by regulating the HPSE/SDC1 axis.

Salvianolic acid B (Sal B) is one of the main water­soluble components of Salvia miltiorrhiza Bge. Numerous reports have demonstrated that it could exert significant renal­protective effects, but the underlying mechanism remains unclear. The present study demonstrated that Sal B could alleviate renal injury by regulating the heparanase/syndecan­1 (HPSE/SDC1) axis. In vivo, the serum creatinine, blood urea nitrogen, transforming growth factor­ß1 (TGF­ß1) and fibroblast growth factor­2 (FGF­2) levels, and the histopathological changes of mice kidneys were examined. Sal B could notably reduce the renal injury caused by left ureteral ligation. In vitro, Sal B downregulated the expression levels of HPSE/FGF­2/TGF­ß1/α­smooth muscle actin and upregulated the expression levels of SDC1/E­cadherin in angiotensin II­stimulated HK­2 cells in a dose­dependent manner. In summary, to the best of the authors' knowledge, the present study provided evidence for the first time that Sal B could exert renal­protective effects via the inhibition of the HPSE/SDC1 axis, and these results suggest that the administration of Sal B may be a novel therapeutic strategy in treating renal interstitial fibrosis.

Inhibitors of elastase stimulate murine B lymphocyte differentiation into IgG- and IgA-producing cells.

It is well established that dendritic cells and macrophages play a role in antigen presentation to B and T cells and in shaping B and T cell responses via cytokines they produce. We have previously reported that depletion of neutrophils improves the production of mucosal IgA after sublingual immunization with Bacillus anthracis edema toxin as adjuvant. These past studies also demonstrated that an inverse correlation exists between the number of neutrophils and production of IgA by B cells. Using specific inhibitors of elastase, we addressed whether the elastase activity of neutrophil could be the factor that interferes with production of IgA and possibly other immunoglobulin isotypes. We found that murine splenocytes and mesenteric lymph node cells cultured for 5 days in the presence of neutrophil elastase inhibitors secreted higher levels of IgG and IgA than cells cultured in the absence of inhibitors. The effect of the inhibitors was dose-dependent and was consistent with increased frequency of CD138+ cells expressing IgG or IgA. Finally, neutrophil elastase inhibitors increased transcription of mRNA for AID, IL-10, BAFF and APRIL, factors involved in B cell differentiation. These findings identify inhibitors of elastase as potential adjuvants for increasing production of antibodies.

Multiple Myeloma Tumor Cells are Selectively Killed by Pharmacologically-dosed Ascorbic Acid.

High-dose chemotherapies to treat multiple myeloma (MM) can be life-threatening due to toxicities to normal cells and there is a need to target only tumor cells and/or lower standard drug dosage without losing efficacy. We show that pharmacologically-dosed ascorbic acid (PAA), in the presence of iron, leads to the formation of highly reactive oxygen species (ROS) resulting in cell death. PAA selectively kills CD138+ MM tumor cells derived from MM and smoldering MM (SMM) but not from monoclonal gammopathy undetermined significance (MGUS) patients. PAA alone or in combination with melphalan inhibits tumor formation in MM xenograft mice. This study shows PAA efficacy on primary cancer cells and cell lines in vitro and in vivo.

Mechanisms of heparanase inhibitors in cancer therapy.

Heparanase is an endo-ß-D-glucuronidase capable of cleaving heparan sulfate side chains contributing to breakdown of the extracellular matrix. Increased expression of heparanase has been observed in numerous malignancies and is associated with a poor prognosis. It has generated significant interest as a potential antineoplastic target because of the multiple roles it plays in tumor growth and metastasis. The protumorigenic effects of heparanase are enhanced by the release of heparan sulfate side chains, with subsequent increase in bioactive fragments and cytokine levels that promote tumor invasion, angiogenesis, and metastasis. Preclinical experiments have found heparanase inhibitors to substantially reduce tumor growth and metastasis, leading to clinical trials with heparan sulfate mimetics. In this review, we examine the role of heparanase in tumor biology and its interaction with heparan surface proteoglycans, specifically syndecan-1, as well as the mechanism of action for heparanase inhibitors developed as antineoplastic therapeutics.

Morphometric analysis of inflammation in bronchial biopsies following exposure to inhaled diesel exhaust and allergen challenge in atopic subjects.

BACKGROUND: Allergen exposure and air pollution are two risk factors for asthma development and airway inflammation that have been examined extensively in isolation. The impact of combined allergen and diesel exhaust exposure has received considerably less attention. Diesel exhaust (DE) is a major contributor to ambient particulate matter (PM) air pollution, which can act as an adjuvant to immune responses and augment allergic inflammation. We aimed to clarify whether DE increases allergen-induced inflammation and cellular immune response in the airways of atopic human subjects. METHODS: Twelve atopic subjects were exposed to DE 300 µg.m(-3) or filtered air for 2 h in a blinded crossover study design with a four-week washout period between arms. One hour following either filtered air or DE exposure, subjects were exposed to allergen or saline (vehicle control) via segmental challenge. Forty-eight hours post-allergen or control exposure, bronchial biopsies were collected. The study design generated 4 different conditions: filtered air + saline (FAS), DE + saline (DES), filtered air + allergen (FAA) and DE + allergen (DEA). Biopsies sections were immunostained for tryptase, eosinophil cationic protein (ECP), neutrophil elastase (NE), CD138, CD4 and interleukin (IL)-4. The percent positivity of positive cells were quantified in the bronchial submucosa. RESULTS: The percent positivity for tryptase expression and ECP expression remained unchanged in the bronchial submucosa in all conditions. CD4 % positive staining in DEA (0.311 ± 0.060) was elevated relative to FAS (0.087 ± 0.018; p = 0.035). IL-4% positive staining in DEA (0.548 ± 0.143) was elevated relative to FAS (0.127 ± 0.062; p = 0.034). CD138 % positive staining in DEA (0.120 ± 0.031) was elevated relative to FAS (0.017 ± 0.006; p = 0.015), DES (0.044 ± 0.024; p = 0.040), and FAA (0.044 ± 0.008; p = 0.037). CD138% positive staining in FAA (0.044 ± 0.008) was elevated relative to FAS (0.017 ± 0.006; p = 0.049). NE percent positive staining in DEA (0.224 ± 0.047) was elevated relative to FAS (0.045 ± 0.014; p = 0.031). CONCLUSIONS: In vivo allergen and DE co-exposure results in elevated CD4, IL-4, CD138 and NE in the respiratory submucosa of atopic subjects, while eosinophils and mast cells are not changed. TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01792232.

Chemopreventive and hepatoprotective effects of Epigallocatechin-gallate against hepatocellular carcinoma: role of heparan sulfate proteoglycans pathway.

OBJECTIVE: Epigallocatechin-gallate (EGCG) claims a plethora of health benefits including protection against neoplastic diseases. Meanwhile, heparan-sulfate proteoglycans (HSPGs) have defensive role against tumour cell invasion. Therefore, the chemopreventive and hepatoprotective effects of EGCG were studied in hepatocellular carcinoma (HCC) in vivo and in vitro and compared with strong water soluble antioxidant, sodium ascorbate. METHODS: HCC was induced in SD rats by thioacetamide (200 mg/Kg). Some rats were treated with EGCG (20 mg/Kg) or sodium ascorbate (100 mg/Kg). Liver impairment was assessed by measuring serum α-fetoprotein and investigating liver sections stained with H/E. Hepatic HSPGs, syndecan-1 and matrix metalloproteinase-9 (MMP-9) were measured by ELISA. Gene expression of fibroblast growth factor (FGF)-2 was measured. Cell death was assessed by caspase-3 activity. In addition, all markers were measured in human hepatocellular carcinoma cell line (HepG2). KEY FINDINGS: EGCG increased the animal survival and decreased both α-fetoprotein and HepG2 viability. In addition, EGCG ameliorated fibrosis and massive hepatic tissue breakdown. EGCG restored HSPGs and reduced expression of MMP-9, syndecan-1 and FGF-2 in-vivo and in-vitro. Sodium ascorbate showed significantly lower results than EGCG. CONCLUSIONS: Besides antioxidant activity, other mechanisms are involved in the chemopreventive and hepatoprotective effects of EGCG including restoration of HSPGs receptors and inhibition of vascular invasion.

Strychnos nux-vomica root extract induces apoptosis in the human multiple myeloma cell line-U266B1.

Multiple myeloma (MM) is an incurable B cell neoplasm causing significant morbidity and mortality. Despite recent advances in the MM-treatment, the disease still remains incurable; necessitating a search for new therapeutic agents. Therefore, Strychnos nux-vomica L. root extract (SN) was screened using the human MM-cell line, U266B1. SN-extract demonstrated anti-proliferative effect in a time- and dose-dependent manner. Morphological studies, cell cycle analysis by FACS indicate apoptosis. Furthermore, assessment of signaling molecules like IL-6 (interleukin-6) and CD-138 (Sydencan-1) confirmed apoptosis. The anti-proliferative activity of SN-extract is associated with apoptosis, which is likely due to the presence of alkaloids, strychnine and brucine, which have been identified by MALDI-TOF-MS and RP-HPLC analysis.

Multiple myeloma cells expressing low levels of CD138 have an immature phenotype and reduced sensitivity to lenalidomide.

CD138 expression is a hallmark of plasma cells and multiple myeloma cells. However, decreased expression of CD138 is frequently observed in plasma cells of myeloma patients, although the clinical significance of this is unclear. To evaluate the significance of low expression of CD138 in MM, we examined the phenotypes of MM cells expressing low levels of CD138. Flow cytometric analysis of primary MM cells revealed a significant decrease in CD138 expression in patients with relapsed/progressive disease compared with untreated MM patients. Patients with low levels of CD138 had a worse overall survival compared with patients with high levels of CD138, in newly diagnosed patients and patients receiving high-dose chemotherapy followed by autologous stem-cell transplantation. Two MM cell lines, KYMM-1 (CD138- low) and KYMM-2 (CD138- high), were established from a single MM patient with decreased CD138 expression. High expression of BCL6 and PAX5, and downregulation of IRF4, PRDM1 and XBP1 was observed in KYMM-1 compared with KYMM-2 cells, indicative of the immature phenotype of KYMM-1. KYMM-1 was less sensitive to lenalidomide than KYMM-2, while no difference in sensitivity to bortezomib was observed. KYMM-2 cells were further divided in CD138+ and CD138- fractions using anti-CD138-coated magnetic beads. CD138- cells sorted from the KYMM-2 cell line also showed high BCL6, low IRF4 expression and decreased sensitivity to lenalidomide compared with CD138+ cells. Our observations suggest that low CD138 expression relates to i) poor prognosis, ii) immature phenotype and iii) low sensitivity to lenalidomide. The observed distinct characteristics of CD138 low MM cells, suggest this should be recognized as a new clinical entity. Establishment of a treatment strategy for MM cells expressing low levels of CD138 is needed to improve their poor outcome.

Syndecan 1 plays a novel role in enteral glutamine's gut-protective effects of the postischemic gut.

Syndecan 1 is the predominant heparan sulfate proteoglycan found on the surface of epithelial cells and, like glutamine, is essential in maintaining the intestinal epithelial barrier. We therefore hypothesized that loss of epithelial syndecan 1 would abrogate the gut-protective effects of enteral glutamine. Both an in vitro and in vivo model of gut ischemia-reperfusion (IR) was utilized. In vitro, intestinal epithelial cells underwent hypoxia-reoxygenation to mimic gut IR with 2 mM (physiologic) or 10 mM glutamine supplementation. Permeability, caspase activity, cell growth, and cell surface and shed syndecan 1 were assessed. In vivo, wild-type and syndecan 1 knockout (KO) mice received ± enteral glutamine followed by gut IR. Intestinal injury was assessed by fluorescent dye clearance and histopathology, permeability as mucosal-to-serosal clearance ex vivo in everted sacs, and inflammation by myeloperoxidase (MPO) activity. In an in vitro model of gut IR, glutamine supplementation reduced epithelial cell permeability and apoptosis and enhanced cell growth. Shed syndecan 1 was reduced by glutamine without an increase in syndecan 1 mRNA. In vivo, intestinal permeability, inflammation, and injury were increased after gut IR in wild-type mice and further increased in syndecan 1 KO mice. Glutamine's attenuation of IR-induced intestinal hyperpermeability, inflammation, and injury was abolished in syndecan 1 KO mice. These results suggest that syndecan 1 plays a novel role in the protective effects of enteral glutamine in the postischemic gut.

Immunohistochemical study identifying prognostic biomolecular markers in nasopharyngeal carcinoma treated by radiotherapy.

BACKGROUND: We evaluated the predictive significance of 14 reported markers using immunohistochemical study in nasopharyngeal carcinoma. METHODS: Immunohistochemical stainings were done in 38 patients for Met, cyclooxygenase-2 (COX-2), nm23-H1, epidermal growth factor receptor (EGFR), p63, early growth response factor 1 (Egr1), chromosome segregation 1-like (CSE1L), cathepsin-D (aspartyl protease), C-erbB2, p53, signal transducers and activators of transcription (STAT3/STAT5), CD138 (Syndecan-1), and LIN28 with the usual methods. RESULTS: The median follow-up time was 30 months (11-83 months). High Met and CD138 expression were statistically significant negative prognostic factors on survival. The expression of Egr1 had a positive prognostic effect on survival. The combined score of these 3 markers, Met plus CD138 minus Egr1, was a strong prognostic factor. The median survival curve was distinctly separated in accord with this combined score. No prognostic value was revealed in COX-2, nm23-H1, EGFR, p63, CSE1L, cathepsin-D, C-erbB2, p53, STAT3, STAT5, and LIN28. CONCLUSIONS: The combined score of these markers could be used to stratify biomolecular risk groups.

Immunohistological profiling by B-cell differentiation status of primary central nervous system lymphoma treated by high-dose methotrexate chemotherapy.

Primary central nervous system lymphoma (PCNSL) remains a devastating disease with poor prognosis, despite the improvement offered by methotrexate (MTX)-based chemotherapy. Several studies have attempted to identify biomarkers predictive of prognosis, which are expected to be both clinically useful and biologically important for understanding PCNSL. The present study attempts to classify human immunodeficiency virus (HIV)-unrelated PCNSL patients treated with radiation combined with rapid high-dose MTX chemotherapy according to B-cell differentiation status, and retrospectively examines the prognostic impact. Initial response to MTX was a strong predictor of favorable prognosis in terms of both progression-free survival (PFS) and overall survival (OS). Thirteen out of 29 cases were CD10(-)/BCL-6(+)/MUM-1(+), being more frequent compared with systemic peripheral nodal lymphoma. Although post-germinal-center B-cell-originating PCNSLs (CD10(-)/BCL-6(-)/MUM-1(+)) showed a trend towards better response to MTX and progression-free survival than did germinal-center-related B-cell-originating PCNSLs (CD10(+) OR CD10(-)/BCL-6(+)/MUM-1(+)), the difference was only marginal (P = 0.04 Gehan-Breslow-Wilcoxon, P = 0.17 log-rank). Our results imply that different B-cell stages in PCNSL have significant relevance in terms of biological behavior. However, clinical use as a prognostic marker requires further investigation.

Deficient TACI expression on B lymphocytes of newborn mice leads to defective Ig secretion in response to BAFF or APRIL.

Capsular polysaccharides of encapsulated bacteria do not induce immune response in newborns and the mechanism for this unresponsiveness is not clear. In adults, transmembrane activator and calcium-modulator and cyclophilin [corrected] ligand interactor (TACI) is a TNFR family member molecule with a pivotal role in Ab responses against polysaccharide vaccines. We investigated the expression and the functions of the TNF family cytokines, B cell-activating factor of the TNF family (BAFF) and a proliferation-inducing ligand (APRIL), and their receptors in newborn mice and found that TACI expression on B lymphocytes was dramatically reduced (p < 0.0001) in newborns as compared with adults. More importantly, TACI ligands BAFF or APRIL were unable to induce IgA/IgG/IgM secretion from newborn B lymphocytes. Additionally, TACI expression seems to be important in plasma cell development. Indeed, in contrast to adults, stimulation of newborn B lymphocytes with BAFF or APRIL did not result in up-regulation of CD138 expression. In vitro or in vivo exposure of newborn B lymphocytes to oligodeoxynucleotides (CpG ODN) led to up-regulation of TACI expression on newly formed, follicular, and marginal zone as well as B1 B lymphocyte populations, and rendered them responsive to BAFF- or APRIL-mediated CD138 expression and IgA/IgG secretion. Finally, immunization of newborn BALB/c mice but not TACI knockout mice with CpG ODN containing (4-hydroxy-3-nitrophenyl)acetyl-Ficoll led to development of IgG Abs against (4-hydroxy-3-nitrophenyl)acetyl. These findings demonstrate that low TACI expression may be a critical factor that determines the susceptibility of newborns to infections with encapsulated bacteria and the impaired immunogenicity of polysaccharide vaccines. Finally, CpG ODNs may correct deficient newborn response to polysaccharide vaccines by up-regulating TACI.